一篇2019年的博士論文,對 乳癌 肺癌與攝護腺癌做實驗

當使用高濃度苦瓜皂甘...對乳癌 肺癌能產生高達100%的凋亡效果...

學術界  似乎還沒見過。

苦瓜皂苷,用途非常廣泛幾乎對所有癌腫瘤,都有效果,其效果與服用劑量成正比,也就是吃得越多,抗癌效果越好,而苦瓜皂苷對人體無毒,安全性很高。

能調理的癌症包含頭頸部癌腫瘤、胰臟癌,肺癌,結腸癌,子宮頸癌、胃癌、卵巢癌,肺癌,白血病(血癌),膀胱癌,肝癌,乳癌和攝護腺癌,包含幹細胞抑制)

苦瓜皂苷20%  團購價 1kg-1250元台幣。(20%定義是 10KG青苦瓜提取出來2KG有效物質)

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THE EFFECTS OF BITTER MELON EXTRACT ON THREE HUMAN
CANCER C

ELL LINES: BT549, A549 and PC3

苦瓜提取物對三種人類的癌細胞之影響

癌細胞株:乳腺癌(BT549),肺癌(A549)和攝護腺癌(PC3

一份2019年年底發表的博士論文

文章很長  閱讀紅字即可

(原稿是PDF檔案 轉換過程格式有些變)

A Dissertation
Submitted to the Graduate School

Of
Tennessee State University
In
Partial Fulfillment of the Requirements
for the Degree of
Doctor of Philosophy in Biological Sciences

Christopher Anthony Davis Sr.
December 2019

Keywords: Natural phytochemicals, breast, lung and prostate cancer, bitter
melon extract, apoptosis, proliferation and lipid peroxidation

ProQuest Number: 27740737

All rights reserved

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The quality of this reproduction is dependent on the quality of the copy submitted.

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and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.

 

ProQuest 27740737

Published by ProQuest LLC ( 2020). Copyright of the Dissertation is held by the Author.

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ii

COPYRIGHT

 

THE EFFECTS OF BITTER MELON EXTRACT ON THREE HUMAN
CANCER CELL LINES: BT549, A549 and PC3
December 2019

Christopher Anthony Davis Sr.

2019
All rights reserved.

Keywords: Natural phytochemicals, prostate, lung and breast cancer, bitter
melon extract, apoptosis proliferation and lipid peroxidation

iii

To the Graduate School:

We are submitting a dissertation written by Christopher Anthony Davis, entitled “The Effects of Bitter Melon Extract on Three Cancer Cell lines”: BT549, A549 and PC3. We recommend that it be accepted in partial fulfillment of the requirements for the degree,” Doctor of Philosophy” in Biological Science.

致研究院:

我們正在提交由克里斯托弗·安東尼·戴維斯(Christopher Anthony Davis)撰寫的論文,題為“苦瓜提取物對三種癌細胞系的影響”:BT549A549PC3。 我們建議您以部分滿足該學位要求的要求接受該學位,即生物科學的“哲學博士”。

Elbert Lewis Myles Jr.Ph.D.

Chairperson

Terrance L. Johnson, Ph.D.  Committee Member

William Boadi, Ph.D.  Committee Member

Anthony Ejiofor, Ph.D.  Committee Member

Ahmad Naseer Aziz, Ph.D.  Committee Member

Accepted for the Graduate School:

Robbie Melton, Ph.D.  Dean of the Graduate School

DEDICATION

This dissertation is dedicated to my parents William Henry and Marietta Davis, for always supporting me and who made me understand that to achieve anything requires faith and belief in yourself, hard work, determination, and dedication. I also dedicate it to my siblings: Henrietta Davis, William Davis, Cedric Davis, and my sister Bridgette Becker and my first wife Felecia Davis, who both suggested I pursue this journey and to my fiancé Sherlyn Harrison for all her support. Thank you all so much for all you have done for me. I also dedicate it to my children and all my future generations to let them know that they can reach their goals and dreams with hard work and dedication.

 

這篇論文是我的父母威廉·亨利和瑪麗埃塔·戴維斯,一直以來都支持我,使我明白,要實現任何目標都需要有對自己的信念和信念,勤奮,決心和奉獻精神。 我也將其獻給我的兄弟姐妹:亨里埃塔·戴維斯(Henrietta Davis),威廉·戴維斯(William Davis),塞德里克·戴維斯(Cedric Davis)和我的妹妹布里奇特·貝克爾(Bridgette Becker)和我的第一任妻子費萊西亞·戴維斯(Felecia Davis),他們都建議我繼續這一旅程,並向未婚夫謝琳·哈里森(Sherlyn Harrison)表示支持。 非常感謝你們為我所做的一切。 我也將其獻給我的孩子們以及我們的後代,讓他們知道他們可以通過努力和奉獻來實現自己的目標和夢想。

ACKNOWLEDGEMENTS

First and foremost, I would like to thank God for walking with me every step of the way during this journey and for bringing all the right people into my life for me to complete my lifelong dream.

This dissertation would not be possible without the Tennessee Doctoral Program and Dr. Alex Sekwat who gave me the financial support I needed. I am incredibly grateful for their assistance.

The completion of this Doctoral Degree could not have been accomplished without the combined efforts of many people who were involved at various stages of this process. Therefore, I want to express my most profound appreciation and gratitude for their help, support, and encouragement. Firstly, to my committee members, Dr. Terrance Johnson for granting me the opportunity and accepting me into the Doctoral Program and for always encouraging me with his kind words. A special thank you to Dr. William Boadi for his friendship and encouragement, Dr. Anthony Ejiofor, for his guidance and Dr. Ahmad Naseer Aziz for his offering of help, as well as Dr. John Robinson for his support and assistance. I truly appreciate all their invaluable support. Secondly, my utmost gratitude to the person most responsible for the completion of this degree: Dr. Elbert Myles. Dr. Myles was my research advisor, mentor, and friend. Thank you, Dr. Myles, for always being available seven days a week, for your guidance and for having the patience to answer a myriad of questions while at the same time allowing me the opportunity to learn, grow, and figure out certain aspects of this journey. It was a great pleasure and absolute honor for me to have a chance to work with him. Thirdly, I would like to express my appreciation to Dr. Quincy A. Quick, Dr. Venkataswarup, and Dr. Bharat Pokharel and as well as the research administrative coordinator Michael Doster for their invaluable help.

Moreover, I want to thank The Agriculture Department and Dr. Arvazena Clardy for supplying me with the fruit needed for my research.

Additionally, I would like to acknowledge a host of associates and colleagues who assisted me along this journey; namely, Dr. Tamara Martin, Dr. Heba Alhamdi, Dr. Jawaher Albaqami, Alexzander Garcia, Duaa Ali Babaer, Chanel Alfred and Mailene King, I thank you all immensely.

 

致謝

首先,我要感謝上帝,在旅途中的每一步都與我同行,並感謝所有進入我的生活的人,使我完成了我一生的夢想。

如果沒有田納西州博士計劃和亞歷克斯·塞克瓦特博士給我所需的經濟支持,這篇論文將是不可能的。我非常感謝他們的協助。

沒有許多參與此過程各個階段的人員的共同努力,就不可能完成該博士學位。因此,我要對他們的幫助,支持和鼓勵表示最深切的謝意。首先,對於我的委員會成員,Terrance Johnson博士給予了我機會並接受了我加入博士學位課程,並總是以他的客氣話語鼓勵我。特別感謝William Boadi博士的友誼和鼓勵,Anthony Ejiofor博士的指導,Ahmad Naseer Aziz博士的幫助,以及John Robinson博士的支持和協助。我非常感謝他們的寶貴支持。其次,我最感謝完成此學位的最負責人:埃爾伯特·邁爾斯(Elbert Myles)博士。邁爾斯博士是我的研究顧問,導師和朋友。 Myles博士,謝謝您每週一整天的工作,每天為您提供指導,並耐心回答無數問題,同時也讓我有機會學習,成長並弄清某些方面的問題,謝謝您。這個旅程。我有幸與他合作,這是我的榮幸和榮幸。第三,我要感謝Quincy A. Quick博士,Venkataswarup博士和Bharat Pokharel博士,以及研究行政協調員Michael Doster的寶貴幫助。

此外,我還要感謝農業部和Arvazen​​a Clardy博士為我提供了研究所需的水果。

另外,我還要感謝在這段旅程中為我提供幫助的許多同事和同事;分別是Tamara Martin博士,Heba Alhamdi博士,Jawaher Albaqami博士,Alexzander GarciaDuaa Ali BabaerChanel AlfredMailene King,非常感謝大家。

THE EFFECTS OF BITTER MELON EXTRACT ON THREE HUMAN

CANCER CELL LINES: BT549, A549 AND PC3

苦瓜提取物對三類人的影響

癌細胞株:BT549A549PC3

ABSTRACT

CHRISTOPHER A. DAVIS. The Effects of Bitter Melon Extract on Three Human

Cancer Cell Lines: BT549, A549 and PC3(under the direction of Dr. E. Lewis Myles).

Cancer remains a significant risk factor in public health globally, causing approximately 9.8 million deaths worldwide in 2018. Despite advances in conventional treatment modalities for cancer treatment, there are still few effective therapies available due to the lack of selectivity, adverse side effects, non-specific toxicities, and tumor recurrence. Therefore, there is an immediate need for essential alternative therapeutics, which can prove to be beneficial and safe against cancer. Various phytochemicals from natural sources have been found to exhibit beneficial medicinal properties against multiple human diseases (Girsa et al., 2019). Bitter Melon is one such natural source that has been found to exhibit beneficial medicinal properties against human diseases. Numerous studies have revealed that plant extracts are capable of not only reducing the lipid peroxidase but also inducing apoptosis in several cancer cell lines. The present study aims to evaluate the anticancer properties of bitter melon (Momordica charantia) extract (苦瓜提取物) in a dose-dependent manner against human breast cancer BT549, lung cancer (A549), and prostate cancer (PC3). The exposure of cancer cells to serially diluted concentrations of 苦瓜提取物 for 24 hours demonstrated anti-cancer effects. Alamar Blue is used to determine the cell viability, and 苦瓜提取物 inhibited proliferation at high levels in all three cancer cell lines. ELISA (Enzyme-Linked Immunosorbent Assay) assay forcaspase-3 activity identified the induction of apoptosis in the three cell lines. 苦瓜提取物 induced apoptosis in all three cell lines. Moreover, a Lipid Peroxidation assay was done to measure the malondialdehyde after exposure to 苦瓜提取物, and we found that less reactive oxygen species (ROS) accumulated compared to that of the control.

摘要

苦瓜提取物對三種人類癌細胞的影響 癌細胞系:BT549A549PC3(在Dr. E. Lewis Myles的指導下)。癌症仍然是全球公共衛生的重要風險因素,2018年在全球造成約980萬人死亡。儘管癌症治療的常規治療方法有所進步,但由於缺乏選擇性,不良副作用,非選擇性治療,有效的療法仍然很少。特異性毒性和腫瘤復發。因此,迫切需要基本的替代療法,它可以證明是有益且安全的抗癌藥。已發現來自自然資源的各種植物化學物質對多種人類疾病均顯示出有益的藥用特性。苦瓜是一種天然來源,已發現對人類疾病顯示出有益的藥用特性。大量研究表明,植物提取物不僅能夠減少脂質過氧化物酶,而且還可以誘導幾種癌細胞系的凋亡。本研究旨在評估苦瓜提取物(苦瓜提取物)對人乳腺癌BT549,肺癌(A549)和攝護腺癌(PC3)的抗癌特性。癌細胞在連續稀釋濃度的苦瓜提取物中暴露24小時顯示出抗癌作用。 Alamar Blue用於確定細胞活力,而苦瓜提取物在所有三種癌細胞系中均能抑制增殖。 ELISA(酶聯免疫吸附測定)測定 caspase-3活性確定了這三種細胞系中凋亡的誘導。 苦瓜提取物誘導所有三個細胞系的凋亡。此外,在接觸苦瓜提取物後進行了脂質過氧化測定以測量丙二醛,我們發現與對照相比,其積累的活性氧(ROS)較少。

The results indicated that 苦瓜提取物 stimulated cell proliferation at lower concentrations of .079 and .157 µg/ml in the cell lines A549 and BT549. In the PC3 cells, growth increased only in the lowest concentration of .079 µg/ml, however, in the higher levels, there was a gradual decrease in cell proliferation, and there was a significant decrease in the highest level of 5.03 µg/ml compared to the cells to DMSO (the control). Moreover, the lipid peroxidase assay revealed a significant decrease in the MDA level in the cell line A549 in concentrations 2.51 and 5.03 µg/ml, however, in BT549 and PC3 cell line only concentration 5.03 µg/ml showed a significant decrease in the MDA level, compared to the cells treated with DMSO (the control). Plus, the caspase-3 activity revealed a gradual increase in apoptosis in all three cell lines. However, there was a significant increase in the highest level of 5.03µg/ml in all three cell lines. These findings suggest that 苦瓜提取物 has anti-tumor activity, and further studies are needed to determine if it may be beneficial in the fight against cancer.

 

結果表明,苦瓜提取物在A549BT549細胞系中以較低的.079.157 µg / ml刺激細胞增殖。在PC3細胞中,僅在最低濃度.079 µg / ml時生長增加,但是在較高濃度下,細胞增殖逐漸降低,而最高濃度5.03 µg / ml則明顯降低。與DMSO(對照)相比。此外,脂質過氧化物酶測定顯示濃度為2.515.03 µg / ml的細胞系A549中的MDA水平顯著降低,但是,僅濃度為5.03 µg / mlBT549PC3細胞系中的MDA水平顯著降低。與用DMSO處理的細胞(對照)相比。另外,caspase-3活性揭示了所有三種細胞系中凋亡的逐漸增加。然而,在所有三種細胞系中,最高水平5.03µg / ml都有顯著增加。這些發現表明苦瓜提取物具有抗腫瘤活性,還需要進一步的研究來確定苦瓜提取物在對抗癌症中是否有益。

Table of Contents

 

CHAPTER I

INTRODUCTION

第一章

介紹

Cancer is a major health problem in the world, which refers to a set of diseases triggered by irregular cell growth with evasive capacities (Devi, et al., 2015). A great amount of cancer occurrence and deaths are caused by different environmental and hereditary factors like obesity, little fruit and vegetable intake, absence of physical activity, tobacco use, alcohol ingesting, exposure to radiation, chronic infections, and genetics (Ahmadi & Shadboorestan 2016). Presenting new bioactive components with natural origins, chiefly from plant bases, may be considered as a distinctive and dependable therapeutic element to treat various types of human cancers based on their discerning molecular targets (Rengarajan, et al., 2014, Montaser & Luesch 2011).

Moreover, oxidative stress has a crucial role in the pathophysiology of different kinds of cancer. Consequently, a lot of consideration has been paid to antioxidants as a novel therapeutic line of attack for cancer (Devi, et al. 2015). Throughout the last two decades, plant-derived bioactive combinations were reported as novel health-giving agents for prevention or mitigation of various human diseases like cancer, inflammation, cardiovascular, and neurodegenerative diseases (Ahmad & Shadboorestan & Nabavi & Setzer & Nabavi 2015).

 

癌症是世界上主要的健康問題,指的是由具有逃避能力的不規則細胞生長引發的一系列疾病(Devi等,2015)。多數癌症的發生和死亡是由不同的環境和遺傳因素引起的,例如肥胖,水果和蔬菜攝入少,缺乏體育鍛煉,吸煙,飲酒,暴露於輻射,慢性感染和遺傳學。提出具有天然起源的新生物活性成分(主要來自植物基地),可以根據其敏銳的分子靶標將其視為治療各種類型人類癌症的獨特且可靠的治療手段。

此外,氧化應激在不同種類癌症的病理生理中起著至關重要的作用。因此,抗氧化劑已被廣泛考慮作為一種新型的癌症治療手段(Devi等,2015)。在過去的二十年中,據報導植物來源的生物活性組合物是預防或減輕各種人類疾病(如癌症,炎症,心血管疾病和神經退行性疾​​病)的新型保健劑。

 

Oldest Description of Cancer

According to the American Cancer Society: Humans and other animals have had cancer during noted history. Therefore, it’s no wonder that from the beginning of time people have written about cancer. Fossilized bone tumors, human mummies in ancient Egypt, and ancient manuscripts reveal some of the initial confirmation of disease. Mummies have been found to have growths indicative of the bone cancer called osteosarcoma. Also, mummies have bony skull damage that happened because of malignancy.

Our oldest account of cancer was found in Egypt and dates back to around 3000 BC. Its refered to as the Edwin Smith Papyrus and is a duplicate of part of an ancient Egyptian textbook on trauma surgery. It refer to 8 cases of tumor or ulcers of the breast that were detached by cauterization with a instrument called the fire drill. The inscription states about the disease, “There is no treatment.”

癌症的最古老的描述

根據美國癌症協會的說法:在著名的歷史中,人類和其他動物都患有癌症。 因此,從一開始人們就寫關於癌症的文章就不足為奇了。僵化的骨腫瘤,古埃及的人類木乃伊和古代手稿揭示了疾病的一些初步證據。 已發現木乃伊具有指示稱為骨肉瘤的骨癌的生長。此外,木乃伊還會因惡性腫瘤而發生骨質頭骨損傷。

我們最古老的癌症記載是在埃及發現的,其歷史可追溯到公元前3000年。它被稱為“埃德溫·史密斯紙莎草紙(Edwin Smith Papyrus)”,是古埃及創傷外科教科書一部分的副本。它寫的是8例通過稱為“消防演習”的工具經燒灼而分離出來的乳房腫瘤或潰瘍。銘文指出該疾病:“沒有治療方法。”

Origin of the Word Cancer

The origin of the word cancer is accredited to the Greek physician Hippocrates (460-370 BC), who is said to be the “Father of Medicine.” Hippocrates used the terms carcinos and carcinoma to define non-ulcer forming and ulcer forming tumors. In Greek these words denote a crab, probable applied to the disease due to the finger-like spreading projections from the cancer called was remindful of the shape of a crab. The Greek expression was later translated into cancer, which is the latin term for crab, by the Roman doctor, Celsus (28-50 BC). Galen (130-200 AD), another Greek physician, used the term oncos, (Greek for swelling) to define tumors. Even though the crab analogy of Hippocrates and Celsus is used today to define malignant tumors, Galen’s word is part of the name for cancer specialist-oncologists.

Also, according to the American Cancer Society, though there has been substantial development in the war on cancer, a lot of the improvements that have helped the developed world are still absent in low- and middle-income countries. In addition the three top challenges for the war on cancer in the 21st century are rapid increase in obesity rates, tobacco use esculating in developing countries and access to Cancer Detection and Treatment in lower income countries. “Overweight is a danger factor for breast (post­menopausal), colorectal, endometrial, kidney, esophageal and pancreatic cancers, “according to the Cancer Atlas. Recent research also reveals that people who are overweight or are obese may be at higher risk for cancer reappearance and death. Regrettably, obesity and overweight rates have increased in countries in all income ranks.

癌症一詞的由來

癌症一詞的起源由希臘醫師希波克拉底(Hippocrates,公元前460-370年)認可,他被稱為“醫學之父”。希波克拉底使用術語癌來定義非潰瘍形成和潰瘍形成腫瘤。在希臘語中,這些詞表示螃蟹,可能是由於螃蟹的形狀使這種疾病適用於這種病,原因是癌症中的手指狀突起物被稱為癌。後來,羅馬醫生塞爾蘇斯(Celsus)將希臘語的表達翻譯成癌症,這是螃蟹的拉丁語。另一位希臘醫生Galen130-200 AD)使用術語oncos(希臘語為腫脹)來定義腫瘤。即使螃蟹類比

 如今,希波克拉底和凱爾索斯(Hippocrates and Celsus)被用於定義惡性腫瘤。

同樣,據美國癌症協會稱,儘管抗癌戰爭已取得實質性進展,但在中低收入國家中,仍缺乏如同發達國家的進步。此外,21世紀抗癌戰爭面臨的三大挑戰是肥胖率的迅速上升,發展中國家煙草的使用禁忌,和低收入國家獲得癌症檢測和治療的機會很缺乏。 “超重是乳腺癌(絕經後),大腸癌,子宮內膜癌,腎癌,食道癌和胰腺癌的危險因素,”根據癌症地圖集。最近的研究還顯示,超重或肥胖的人可能會更容易出現癌症和死亡。遺憾的是,所有收入階層的國家中的肥胖和超重率都有所上升。

According to the Cancer Atlas “about 1.3 billion people throughout the world presently smoke tobacco.” Smoking causes numerous kinds of cancer. It is liable for 1 in 5 deaths from cancer throughout the world. Although developed countries, like the United States, have made advancement to limit tobacco consumption by a tax increase on tobacco merchandises, prohibiting certain types of publicity, and the implementation of laws to make smoke-free work and public places, the tobacco epidemic is getting worse in a lot of developing countries. For instance, cigarette consumption increased by 57% in the Middle East and Africa from 1990 to2009. Moreover, few smokers in developing nations give up smoking by middle age. “Stop smoking by middle age can avoid more than 60% of the danger of lung cancer,” according to the Canc Tax and price guidelines are frequently cited as the best effected demand-sided policy,” according to the Cancer Atlas, which means they are the best policies for reducing consumer demand. Nevertheless, cigarettes have become cheaper in low- and middle-income countries” due to the fact that taxes and prices have been rising slower than increases in income.er Atlas.

根據《癌症地圖集》的報導,“目前全世界大約有13億人吸煙。”吸煙會導致多種癌症。全世界有五分之一的人死於癌症。儘管像美國這樣的發達國家已經通過提高煙草製品的稅收,禁止某些類型的宣傳以及實施法律以實現無菸工作和公共場所的方式來限製菸草的消費,但煙草流行還在逐步蔓延。在許多發展中國家更糟。例如,從1990年到2009年,中東和非洲的捲煙消費量增長了57%。此外,發展中國家很少有吸煙者在中年之前戒菸。 《癌症地圖集》稱:“中年停止吸煙可以避免60%以上的肺癌危險。”《癌症地圖集》表示,稅收和價格指南經常被認為是效果最好的需求方面的政策,這意味著它們是減少消費者需求的最佳政策。儘管如此,“由於稅收和價格的增長慢於收入的增長,捲菸在中低收入國家變得更便宜了。”

“In low- and middle-income countries, restricted access to reasonable and worthy cancer diagnosis and treatment has added to mortality-to-incidence ratios around 20% higher than those of developed countries,” according to the Cancer Atlas. This deficiency of access is caused by both the high cost of cancer care and limited healthcare infrastructure as well as workers to carry it out.

Macronutrients (protein, fats and carbohydrates) and micronutrients (vitamins and minerals) are not the only benefits in foods: also contained in food is large amounts of non-nutritional chemicals that are very beneficial to our health. These substances are commonly called “phytochemicals”.

The war on cancer is not limited to research on conventional chemotherapeutic agents. For example, scientific studies of phytochemicals are revealing an esculation in the understanding of the mechanisms of action, and in specific of their effects on cell signaling pathways (Baena Ruiz R et al. 2015). Epidemiological and pre-clinical statistics put forward that numerous natural phytochemicals and dietary compounds hold chemopreventive properties, and in-vitro and in-vivo studies reveal that these compounds may modulate signaling pathways involved in cell proliferation and apoptosis in transformed cells, improve the host immune system and sensitize malignant cells to cytotoxic mediators. In spite of promising results from experimental studies, only a small amount of these compounds have been verified in clinical trials and have shown variable outcomes (Kotesha & Takami & Espinoza 2016).

《癌症地圖集》稱:“在中低收入國家,對合理,有價值的癌症診斷和治療的限制獲得了成功,死亡率/發病率比發達國家高了約20%。”這種獲取不足的原因是癌症護理費用高昂,醫療基礎設施有限以及實施該技術的工人所致。

大量營養素(蛋白質,脂肪和碳水化合物)和微量營養素(維生素和礦物質)並不是食物的唯一好處:食物中還含有大量對我們的健康非常有益的非營養化學物質。這些物質通常被稱為“植物化學物質”

抗癌戰爭不僅限於傳統化學治療劑的研究。例如,對植物化學物質的科學研究揭示了一種對作用機理的理解,尤其是它們對細胞信號傳導途徑的影響的保護作用。流行病學和臨床前統計數據表明,許多天然植物化學物質和膳食化合物具有化學預防特性,體外和體內研究表明,這些化合物可調節轉化細胞中細胞增殖和凋亡所涉及的信號通路,提高宿主免疫力。系統並使惡性細胞對5種細胞毒性介質。儘管實驗研究取得了令人鼓舞的結果,但只有少量這些化合物在臨床試驗中得到了驗證,並顯示出不同的結果。

Purpose

This study aims to find a compound in Bitter Melon extract that inhibits the growth of cancer. The present study is aimed at investigating the effects of Bitter Melon extract on breast (BT549), lung (A549) and prostate (PC3) cancer cell lines, and to understand the underlying molecular pathways.

Research Hypothesis

We hypothesize that Bitter Melon extract will reduce lipid peroxidase levels and

induce apoptosis, as well as inhibit cancer growth in the three cancer cell lines. The following aims are used to test the hypothesis.

目的

這項研究旨在在苦瓜提取物中找到一種化合物,該化合物可以抑制

癌症的成長。 本研究旨在研究苦瓜提取物對乳腺癌(BT549),肺癌(A549)和攝護腺癌(PC3)癌細胞系的作用,並了解其潛在的分子途徑。

研究假說

我們先假設苦瓜提取物會降低脂質過氧化物酶水平,誘導凋亡,並抑制三種癌細胞系中的癌細胞生長。 以下目標用於檢驗假設。

Aims of Study

Specific aim l.

Cell viability to determine if Bitter Melon extract inhibits the growth of cancer cells in the three cancer cell lines, BT549 (breast), A549 (lung) and PC3 (prostate). Specific aim 2.

ROS (Reactive Oxygen Species) Study the effect of Bitter Melon extract on the lipid peroxidase levels on the three cancer cell lines, BT549 (breast), A549 (lung) and PC3 (prostate).Specific aim 3.

Apoptosis, look at the effect of Bitter Melon extract on the caspase-3 activation in the three cancer cell lines, BT549 (breast), A549 (lung and PC3 (prostate).

Hypothesis: Bitter melon extract will inhibit proliferation of the cancer cell and reduce the MDA levels as well as induce apoptosis in breast cancer cell line BT549, lung cancer cell line A549 and prostate cancer cell line PC3.

The facts and statistics collected on phytochemicals against cancer is not sufficient and additional investigation is required in order to provide additional comprehension for developing potential anticancer drugs.

 

研究的目的

目標l

確定苦瓜提取物是否抑制三種癌細胞系BT549(乳腺癌),A549(肺)和PC3(攝護腺)中癌細胞的生長的細胞活力。

目標2

ROS(活性氧)研究苦瓜提取物對三種癌細胞系BT549(乳腺),A549(肺)和PC3(攝護腺)的脂質過氧化物酶水平的影響。

目標3

凋亡,研究苦瓜提取物對三種癌細胞系BT549(乳腺),A549(肺和PC3(攝護腺))中caspase-3活化的影響。

 

假設:苦瓜提取物將抑制癌細胞增殖並降低MDA水平,並誘導乳腺癌細胞BT549,肺癌細胞A549和攝護腺癌細胞PC3凋亡。

關於癌症的植物化學物質收集的事實和統計資料還不夠,還需要進行進一步的調查,以便為開發潛在的抗癌藥物提供更多的理解。

以下目標用於檢驗假設。

 

CHAPTER II

LITERATURE REVIEW

第二章

文獻評論

Plant resources have been a major part of human culture throughout history. World Health Organization (WHO) projected that roughly 80% of the developing world’s populace use traditional herbal medicines (Alves & Rosa, 2005). In developing countries, customary medicines provide a low-priced and substitute source for major health care (Bannerman & Burton & Chen, 1983, Manandhar, 1998, Svarstad & Dhillion, 2000) due to absence of modern health accommodations, their helpfulness, culture priorities, and choices (Plotkin & Famolare, 1992, Taylor & Manandhar, 1995, Balick & Elisabetsky & Laird, 1996). In advanced nations, the use of conventional herbal medicines is also a phenomenon that is rapidly growing. For example in China conventional herbal provisions account for 30 to 50% of the over-all drug ingesting. Whereas at the same time, in countries like Nigeria Ghana, Zambia and Mali, the first choice for 60% children in distress with elevated malarial fever is herbal medicines. In Ethiopia, nearly 80% of the populace use traditional medicines because of the cultural satisfactoriness of healers and local pharmacopeias, reasonably little cost of traditional medicines and absence of access to contemporary drugs (Kebede & Alemayehu & Binyam & Yunis, 2006). The certification of ancestral knowledge in ethnobotanical surveys could cove the present gap to uncover operative drugs (Henrich & Kufer & Leonti & Pardo-de-Santayana, 2006)

Pakistan has a variety of climate zones with exclusive biodiversity and contains 6,000 plant types, of which about 400-600 species are thought to be medically important

 

在歷史中,植物資源一直是人類文化的重要組成部分。世界衛生組織(WHO)預測,發展中國家約80%的民眾使用傳統草藥(AlvesRosa2005年)。在發展中國家,由於缺乏現代化的衛生設施,植物性藥物為主要衛生保健提供了廉價的替代來源。在發達國家,常規草藥的使用也出現一種迅速增長的現象。例如,在中國,常規草藥的攝入量佔全部藥物攝入量的30%至50%。同時,在像尼日利亞加納,贊比亞和馬里這樣的國家,患有瘧疾高發的60%兒童的首選藥物是草藥。在埃塞俄比亞,由於治療師和當地藥典的文化滿意度,傳統藥物的成本相當低以及無法獲得現代藥物,近80%的民眾使用傳統藥物。

巴基斯坦擁有專屬生物多樣性的各種氣候區,包含6,000種植物,其中約400-600種被認為具有重要的醫學意義。

(Hamayun & Khan & Begum, 2003, Ali, 2008). In the country, numerous findings have related the therapeutic uses of plant properties (Akhtai & Rashid & Murad & Bergmeier, 2013, Abbasi & Khan & Shah & Shah & Perez,2013, Khan et al.. 2013, Ahmed et al.. 2014, Amjad & Arshad, 2014, Bano & Ahmad & Hadda, 2014, Aziz et al.. 2016, Aziz, 2017). The folk information on old-fashioned herbal remedies typically travels from one generation to another generation verbolly (Aziz, et al.. 2016, Aziz, et al.. 2017, Mahmood & Mahmood, 2011, Mahmood & Mahmood,2011). In vertical transfer, likelihoods of removal of knowledge are going in parallel, and that poses a huge threat and essential to be focused on for stoppage. In the past few decades, a substantial tendency in scientific and commercial interest has been detected because of the cultural tolerability and economic strength of plant based herbal products throughout the country (Mahmood et al.. Sher & Aldosari & Ali & de Boar, 2015). The country has assorted cultures and a many languages spoken mainly in rural and distant areas. People from rural areas have the least access to health care facilities, that’s one of the key causes for the application of traditional herbal medicines in these cultures (Claudhry & Malik & Ashraf, 1995)

Deprived eating customs constitute one of the highest substantial factors in the occurrence of both cancer and other conditions, including cardiovascular, endocrine, and inflammatory diseases. Extensively fluctuating epidemiological reports have proven the profits of embracing a wholesome lifestyle and diet, involving, notably, a overall decrease in cancer risk. That is the reason, several international organizations, like the World Cancer Research Fund (WCRF) and the American Institute for Cancer Research (AICR), advocates an escalation in the consumption of specific vegetables, fruits, and grains due to their link with a lesser risk for the growth of various tumors and cancers (Lawrence et al.. 2012, Perera & Thompson & Weseman, 2012).

在該國,許多發現與植物特性的治療用途相關。關於老式草藥的民間信息通常從一代到另一代都非常冗長。在垂直轉移中,知識轉移的可能性並行進行,這構成了巨大的威脅,因此必須集中精力進行停工。在過去的幾十年中,由於在全國范圍內以植物為基礎的草藥產品具有文化上的耐受性和經濟實力,人們已經發現科學和商業上的重大趨勢。該國的文化多種多樣,主要在農村和偏遠地區講多種語言。農村地區的人們獲得醫療保健設施的機會最少,這是在這些文化中使用傳統草藥的主要原因之一,在癌症和其他疾病(包括心血管疾病,內分泌疾病和炎性疾病)的發生中,缺乏飲食習慣是最高的實質性因素之一。廣泛波動的流行病學報告已證明,擁護有益健康的生活方式和飲食習慣尤其可從總體上降低癌症風險。因此,一些國際組織,例如世界癌症研究基金會(WCRF)和美國癌症研究所(AICR),都提倡增加特定蔬菜,水果和堅果的消費量。

 穀物由於它們與各種腫瘤和癌症的生長風險,產生較小的聯繫。

Macronutrients like proteins fats and carbohydrates, and micronutrients like vitamins and minerals are not the only benefits contained in food: there is also enormous amounts of non-nutritional chemicals that have favorable results on our health. These ingredients are usually termed “phytochemicals”. Plants produce phytochemicals in order to defend them against outside dangers and environmental vehicles such as ultraviolent rays and generators of damaging free radicals. The incorporation of these kinds of foods into our nutrition could give us the protection that the phytochemicals offer for the plants (e.g., the ability to nullify free radicals in the body).

In vitro and in vivo findings have revealed further assets of phytochemicals, outside their antioxidant activities. These vehicles may also effect the proliferation, growth, and metastasis of tumors (Vauzour et al..2010). These and other plant-derived properties may be a source natural anti-cancer drugs.

Escaping exposure to dangers that aid in the development of tumors would be the best direct cancer strategy that could be embraced (Anand, et al.2008). However, the weight of environmental factors and diet makes this protective route unrealistic. Inherited elements add only around 5-10% of the cancer risk, whereas environmental influences causes 90-95% (10-15% is the result of chemical and industrial carcinogens, 15-20% from infections, 25-30% from tobacco, and 30-35% from diet) (Irigaray & Newby & Clapp & Hardell & Howard, 2007).

 

蛋白質,脂肪和碳水化合物等常量營養素以及維生素和礦物質等微量營養素並不是食物中唯一的益處:還有大量非營養性化學物質,對我們的健康有積極作用。這些成分通常被稱為“植物化學物質”。植物生產植物化學物質是為了保護植物免受外界危險和環境媒介物的侵害,例如紫外線和有害自由基的產生者。將這些食物摻入我們的營養中可以為我們提供植物化學物質為植物提供的保護(例如,消除體內自由基的能力)。

體外和體內發現揭示了除抗氧化劑活性外的其他植物化學物質。這些媒介物也可能影響腫瘤的增殖,生長和轉移。這些和其他植物來源的特性可能是天然抗癌藥的來源。

避免暴露於有助於腫瘤發展的危險中,這將是可以接受的最佳直接癌症策略。然而,環境因素和飲食使得這種保護途徑不切實際。遺傳元素僅增加大約5-10%的癌症風險,而環境影響則佔90-95%(10-15%是化學和工業致癌物的結果,15-20%來自感染,25-30%來自煙草,和飲食中的30%至35%)

 

Early Theories of Cancer Origin and Cause There were numerous theories that address the origin and cause of cancer, it was thought that cancer was the result of parasites, trauma, or injury, circulating lymph fluids and could spread through the body like a liquid, and plus there were ideas that cancer was contagious (Lee 2000, Modlin 2001, Raju 1998). Later those concepts were doubtful, and additionally, scientists were clever enough to prove that cancer consist of cells and not lymph. Nevertheless, it was proven in the late 19th century that cancer spread by malignant cells (Lee, 2000). The best development in cancer research and development of treatment strategies came about in the 20th century (Papac 2001). During this period it was revealed that some chemical elements or particles (carcinogens) like coal tar (tobacco) and radiation were capable of inducing, though, exact mechanisms of how the disease was began continued to be unclear. After that, an exact and extremely sophisticated hypothesis was made signifying that cancer cells made it to all the other parts of the body by way of the bloodstream, but were only capable of growth in a few organs (Ribatti,2006).

Historical Overview of the Development of Cancer Treatments

Surgery.

The search for treatments and upgraded ways of diagnosis of this fatal disease continued vigorously. In1700’s it was proposed that some cancers could be healed by surgery if the tumor had not attacked neighboring tissue and was “movable” (Harvey 1974). It was well-known that surgery could only work if it was done at the early stages of tumor growth, it was known that beyond that surgery might have minor benefits once tumor cells have metastasized. Surgery then was extremely primitive with a lot of difficulties, which included the loss of blood. Then in the 19th and early 20th century major advances were accomplished in general surgery and precisely in cancer surgery (Harvey, 1974). This was brought about because anesthesia became accessible in 1846, a group of accoomplished surgeons arose whose work quickly progressed the field and the classic cancer procedures like radical mastectomy was established. Additionally, the invention and usage of the contemporary microscope led to the growth of scientific oncology and the scientific source for the modern pathologic study of cancer (Lee, 2000).

癌症起源與成因的早期理論

有許多理論探討了癌症的起源和原因,人們認為癌症是寄生蟲,創傷或損傷,淋巴液循環的結果,並可能像液體一樣擴散到全身,此外,還有一些想法。後來這些概念令人懷疑,此外,科學家已證明癌症由細胞而不是淋巴組成。然而,在19世紀後期,已經證明惡性細胞會擴散癌症。癌症研究和治療策略的最佳發展出現在20世紀。在此期間,人們發現,一些化學元素或顆粒(致癌物),例如煤焦油(煙草)和輻射能夠誘導,儘管這種疾病開始的確切機制仍不清楚。此後,提出了一個精確而極其複雜的假說,表明癌細胞通過血液將其傳播到身體的所有其他部位,但只能在少數器官中生長。

癌症治療發展的歷史概述

手術。

繼續積極尋找這種致命疾病的治療方法和升級診斷方法。 1700年,有人提出,如果腫瘤沒有侵襲鄰近組織並且是“可移動的”,則可以通過手術治愈某些癌症。眾所周知,手術只能在早期階段進行,對於腫瘤的生長,一旦腫瘤細胞轉移,該手術可能僅會帶來較小的益處。那時的外科手術非常原始,有很多困難,包括失血。然後在19世紀和20世紀初期,在普外科和精確地在癌症外科方面取得了重大進展。之所以出現這種情況,是因為1846年開始可以使用麻醉方法,一群經驗豐富的外科醫生出現了,他們迅速地推動了該領域的發展,並建立了經典的癌症手術方法,例如根治性乳腺切除術。。

The assumption that “cells from the principal tumor spread but could grow only in certain, and every organs” was a concept that was later established by the procedures of contemporary cellular and molecular biology nearly a hundred years later (Lee, 2000). Knowing how metastasis work became a crucial element in identifying the limitation of cancer surgery. It was noted early by ancient physicians and surgeons documented that after surgical removal, cancer would most likely return. Knowing how metastasis work eventually permitted doctors to advance systemic treatments used pre- or post- surgery to abolish cells that had traveled through the body and use less invasive and harming procedures in treating various kinds of cancers.

Radiation and hormone therapy.

The accidental finding that some hormones caused stimulating effects on cancer happened when a surgeon revealed that the elimination of the overies (oophorectomy) often caused the improvement of breast cancer patients, this was prior to the hormone estrogen itself was revealed (Beaston 1896). This work made a foundation for the contemporary use of hormone therapy, such as tamoxifen, (Figure 1) for the treatment and prevention of breast cancer.

“腫瘤的細胞會擴散,但只能在某些器官中生長”這一假設是一個概念,後來在近一百年後的當代細胞和分子生物學程序中得以確立。知道轉移的工作方式已成為確定癌症手術是局限性。古代醫師和外科醫生早已指出,手術切除後,癌症很可能會復發。知道轉移的最終作用方式後,醫生可以進行外科手術前或手術後的全身性治療,消除已經穿過人體的細胞,並採用侵入性和傷害性較小的方法來治療各種癌症。

放射和激素療法。

偶然發現某些激素對癌症有刺激作用,這是在外科醫生髮現切除卵巢(卵巢切除術)通常可以改善乳腺癌患者的情況時發生的,這是在激素之前。雌激素本身被發現。這項工作為當代使用激素療法(例如他莫昔芬)治療和預防乳腺癌奠定了基礎。

 

Figure 1: Anticancer Drug: Tamoxifen

1:抗癌藥:他莫昔芬

The basic principels of radiation therapy began with the use of radium and moderately low-voltage diagnostic machines (Kaplan, 1979). Later it was revealed that daily quantities of radiation over numerous weeks would significantly improve therapeutic response (Kaplan, 1979). While today radiation therapy has progressed to where it is distributed with good precision straight to the target tissue, this was not the situation during its initial usage and abolishing malignant tumors also produced damage to adjacent regular tissue. Ultimately, it was revealed that radiation could be the source of cancer as well. Subsequently several early radiologist developed leukemia basically from using the skin on their arms to test the strength of radiation from their radiotherapy apparatuses, while attempting to figure out the “erythema dose,” which was a well-thought-out estimate of the correct daily fraction of radiation (Bottger, 1956). Chemotherapy.

The first time nitrogen mustards and folic acid antagonist was used as drugs was in the 1940’s and this began the era of cancer chemotherapy (Goodman, 1984, Farber, 1948). The detection of nitrogen mustard, a chemical warfare agent used during World War II, was later found to be effective in treating cancer. Subsequent, investigations of the potential therapeutic applications of mustard gas revealed substantial activity against lymphoma, a cancer of the lymph nodes. This led in 1943, to the first documented treatment of a patient with lymphoma (Gilman1963). Substantial decrease in the patient’s tumor masses was detected. Even though this effect persisted only a few weeks, this was the beginning of the realization that cancer could be treated by pharmacological mediators (Goodman, 1946). Studies of the poisonous properties of mustard gas served as the model for a lengthy series of comparable but more effective go-betweens called “alkylating” agents that destroyed swiftly proliferating cancer cells by harming their DNA.

放射治療的基本原理始於使用鐳和中等低壓的診斷儀。後來發現,連續數週每天的輻射量將顯著改善治療反應。雖然如今放射治療已經發展到可以高精度地直接分佈到目標組織的位置,但在最初使用時就沒有這種情況,消除惡性腫瘤同時也對相鄰的正常組織產生了損害。最終,人們發現輻射也可能是癌症的來源。隨後,幾位早期放射科醫生基本上是通過使用手臂上的皮膚來測試其放射治療設備的放射強度,同時試圖找出“紅斑劑量”而發展為白血病。

 

化學療法。

氮芥子和葉酸拮抗劑首次在1940年代被用作藥物,這開始了癌症化學療法的時代。後來發現發現第二次世界大戰期間使用的化學戰劑氮芥菜可以有效治療癌症。隨後,對芥子氣的潛在治療應用的研究表明,它具有抵抗淋巴瘤(淋巴結癌)的顯著活性。這導致了1943年,第一個有記錄的淋巴瘤患者的治療方法。發現患者的腫瘤塊明顯減少。即使這種作用僅持續了幾週,這還是人們開始認識到可以通過藥理介質治療癌症的方法。對芥子氣有毒特性的研究成為了一系列可比但更有效的過渡劑的模型,這些過渡劑通過損害其DNA破壞了迅速增殖的癌細胞。

Soon after the detection of nitrogen mustard, it was verified that aminopterin, an antagonist to the vitamin, folic acid, formed remission in acute leukemia in children (Farber, 1948). Aminopterin (Figure 2) blocked a major chemical reaction needed for DNA replication (Nichol, 1950). While remissions were brief, it was plainly evident that aminopterin, an antagonist to the vitamin antifolates could overpower proliferation of malignant cells and could thereby reinstate regular bone marrow function (Goldin, 1955). Aminopterin was the forerunner of methotrexate, (Warkany, 1978). a cancer treatment drug that is frequently used today. Now quite a few drugs have been made that blocked various roles involved in cell growth and replication.

在氮芥末後不久,已證實氨基蝶呤是維生素葉酸的拮抗劑,可導致兒童急性白血病的緩解。 氨基蝶呤(圖2)阻斷了DNA複製所需的主要化學反應。 儘管緩解期很短,但顯而易見的是,氨蝶呤是維生素類抗葉酸的拮抗劑,可以抑制惡性細胞的增殖,從而恢復正常的骨髓功能。 氨基蝶呤是甲氨蝶呤的先驅。

癌症治療

今天經常使用的藥物。 現在,已經製造出許多藥物來阻止細胞生長和復制中的各種作用。

 

Figure 2: Aminopterin

2:氨基蝶呤

Combination therapy was a major achivement in cancer therapy that happened founded on the idea that an assortment of chemotherapy drugs, individually with a different mechanism of action would be much better when administered in combination than given separately. Cancer cells could possibly become unaffected to a single agent, but allegedly it would be additionally difficult for the tumor to grow resistance to the mixture. A number of treatments became broadly used for their substantial effects in treating lymphomas. The tactic was stretched to many different cancers and now it is a typical practice to order combination therapy (Bonadonna, 1976).

Chemotherapy did not work on all tumors, it was well-known though that if the tumor burden could be condensed first by surgery, then chemotherapy would work better in eliminating residual malignant cells. This tactic gave rise to the approach known as “adjuvant therapy” which is given along with primary treatment (Jaffe, 1981). Adjuvant therapy was verified initially in breast cancer and found to be operative, it was later established that high amounts of methotrexate inhibited reappearance of osteosarcoma following surgical elimination of the primary tumor and 5-fluorouracil, an inhibitor of DNA synthesis, also revealed to improve survival when used as an adjuvant to surgery in treating patients with colon cancer. The efficiency of adjuvant drugs was verified in many additional cases (Bellamy, 1991).

組合療法是發生在癌症療法中的一項主要成就,其基於這樣的想法,即各種具有不同作用機理的化療藥物聯合使用時比單獨使用要好得多癌細胞可能不受單一藥物的影響,但是據稱,腫瘤對混合物的耐藥性將進一步增加。由於其在治療淋巴瘤中的實質作用,許多方法被廣泛使用。該策略已擴展到許多不同的癌症,現在是聯合療法的一種典型做法。化學療法並非對所有腫瘤都有效,但眾所周知,如果可以通過手術首先減輕腫瘤負擔,那麼化學療法在消除殘留的惡性細胞方面會更好。這種策略產生了一種稱為“輔助治療”的方法,該方法與主要治療方法同時使用。輔助療法最初在乳腺癌中得到證實,並且可以行之有效,後來證實,甲氨蝶呤可抑制外科手術切除原發腫瘤後的骨肉瘤重現,而5-氟尿嘧啶是DNA合成的抑製劑,也顯示可提高生存率用作為治療結腸癌患者的手術佐劑。在許多其他案例中也證實了輔助藥物的有效性。

State of Cancer Treatments and Cytotoxicity

Unfavorable data and rising projections of worldwide deaths at an disturbing rate reveal a gap in present strategies. Regardless of the accessible of a large number of anticancer drugs and numerous therapeutic choices, there is still a serious need for drugs that are not as toxic and extra potent cancer fighting agents and remains to be the concern. Consequently, other sources of novel molecules that are physically unique and effective are essential in the development of cancer drugs.

“All ingredients_____________________ are poisons; there not any which is not poison. The correct dose

differentiates a venomous and remedy”

-Parzcelsus (description of what could be an actual anticancer agent. That is to say, while cytotoxicity is an action that is consistent with antitumor activity, amount and concentration are the main standards in shaping the potential of a compound as drug applicant. Though the main objective requires that the compound be cytotoxic to tumor cells, preferably the cytotoxic properties should be within a bearable dosage and elicited through a mechanism that selectively marks tumor cells while allowing the endurance of regular cells. A lot of chemotherapeutic agents today are cytotoxic to all cells, triggering a lot of side effects. (Cheng, 1973)

癌症治療和細胞毒性

不利的數據和對世界上死亡人數的不斷上升的令人不安表明,當前戰略存問題。不管可獲得大量的抗癌藥物和眾多治療選擇,仍然迫切需要不是毒性和超強抗癌劑的藥物,這仍然是令人關注的問題。因此,在物理上獨特且有效的新穎分子的其他來源對於開發抗癌藥物至關重要。

可能是真正的抗癌藥。也就是說,雖然細胞毒性是與抗腫瘤活性相一致的作用,但是數量和濃度是塑造化合物作為藥物申請人的潛力的主要標準。儘管主要目的是要求該化合物對腫瘤細胞具有細胞毒性,但優選細胞毒性應在可承受的劑量範圍內,並通過選擇性標記腫瘤細胞同時允許常規細胞耐受的機制引發。如今,許多化學治療劑對所有細胞都具有細胞毒性,從而引發許多副作用

History of Natural Product Drug Discovery

Primary and secondary metabolites are the two types of chemical products produced by biological organisms. Secondary metabolites are usually organic compounds that are not directly involved in the ordinary growth and development of organisms, unlike primary metabolites like protein and sugar which are directly involved in the procedures. However, Secondary metabolites are needed for the communication of an organism with its environment, like the area of defense against pathogens or competitors. (Swerdlow, 2000) The normal plasticity of secondary metabolism triggers the ease with which microorganisms develop resistance to antibiotics and also parasites and insects resist long-term chemical control. The manifold endurance tactics of organisms have given rise to an arsenal of ordinary products with pharmacological, antibiotic, herbicidal and insecticidal activities. (Swerdlow, 2000) These compounds frequently have comercial relevance in direct use or serve as lead compounds for the expansion of new industrial products. For centuries people have referred to Nature for cures to the multitude of diseases that afflict us. The ancient civilizations of the Chinese, Indians and North Africans provide written evidence for the use of natural sources for curing various diseases. (Phillipsoms,2001) Roots of the endive plant were used for treatment of gallbladder disorders, and raw garlic was prescribed for circulatory disorders and mandrake was prescribed for pain relief, all which are still utilized as alternative medicines.

天然藥物發現的歷史

初級和次級代謝產物是生物有機體產生的兩種化學產物。次生代謝物通常是不直接參與生物體正常生長和發育的有機化合物,與直接參與程序的主要代謝物(如蛋白質和糖)不同。然而,有機體與環境之間的交流需要次級代謝產物,例如抵抗病原體或競爭者的區域。 次生代謝的正常可塑性觸發了微生物對抗生素產生抗藥性,並且寄生蟲和昆蟲也產生抵抗長期的化學控制。

這些化合物通常具有商業意義,或用作擴展新工業產品的先導化合物。幾個世紀以來,人們一直指望大自然來治愈困擾我們的多種疾病。中國,印度人和北非人的古代文明為利用自然資源治療各種疾病提供了書面證據。菊科植物的根被用來治療膽囊疾病,生大蒜被指定用於循環系統疾病,而曼陀羅被指定用於緩解疼痛,所有這些仍被用作替代藥物。

While sciences of medicine and chemistry ripened in the nineteenth century the first drugs were purified compounds from plants, Friedrich Sertürner isolated morphine ( Figure 3) from Papaver somniferum in 1806, French researchers Pierre Joseph Pelletier and Joseph Bienaimé Caventou in 1817 isolated quinine (Figure 4) which played a significant role in the colonization of Africa by Europeans, since then natural products have been extensively screened for their medicinal purposes.

當醫學和化學科學在19世紀成熟時,第一批藥物是從植物中提煉的化合物,Friedrich Sertürner1806年從罌粟中分離出嗎啡(圖3),法國研究人員Pierre Joseph PelletierJosephBienaimé Caventou1817年分離了奎寧(圖4)。在歐洲人對非洲的殖民統治中發揮了重要作用,從那時起,就針對其藥用目的對天然產物進行了廣泛的篩選。

Figure 4: Quinine

4:奎寧

Other samples of active components derived from natural sources are CNS stimulant strychnine (Figure 5), penicillin (Figure 6) a β-lactam antibiotic used in the treatment of bacterial infections and popular anti-cancer agent Taxol (Figure 7) attained from the bark of the Pacific yew tree.

其他來自自然來源的活性成分樣品包括中樞神經系統刺激性士的寧(圖5),青黴素(圖6)和從樹皮中獲得的流行的抗癌藥紫杉醇(圖7)。

 

 

Figure 6: Penicillin

6:青黴素

 

Figure 7: Taxol

7:紫杉醇

Later, it was found that some natural products could, in some cases be made better through the use of chemistry to produce semi-synthetic compounds (i.e. aspirin and later penicillin’s). (Newman & Butler, 2004) As synthetic methods got better it became conceivable, in some cases, to generate new medicinal compounds built on natural products that were better and/or cost less than the original natural product template (Newman & Crag & Koehn, 2005).

A Summary of Carcinogenesis

Carcinogenesis is a process that involves several stages that are categorized by a development of discrete molecular changes that eventually reprogram and alter a cell to undergo uncontainable cellular division (Loeb & Harris, 2008). In last 50 years, research has exposed countless serious molecular players and targeted pathways and highlighted the underlying equilibrium of aberrant activation of proto-oncogenes and inactivation of tumor suppressor genes. In every disturbance, cells undergo changes basically represented by tumor initiation, promotion, and progression (Tokarz & Blasiak, 2014, Barcellos-Hoff & Leden & Wang, 2013).

後來發現,在某些情況下,可以通過使用化學方法生產半合成化合物(例如阿司匹林和後來的青黴素)來改善某些天然產物。隨著合成方法的改進,在某些情況下,可以在天然產品上生產出比原始天然產品更好和成本更低的新藥用化合物。

 

致癌概述

致癌作用是一個涉及多個階段的過程,該階段根據離散分子變化的發展進行分類,這些變化最終會重新編程並改變細胞,使其經歷無法控制的細胞分裂。在過去的50年中,研究已經揭露了無數嚴重的分子分子和靶向途徑,並強調了原癌基因異常激活和腫瘤抑制基因失活的潛在平衡。在每種干擾中,細胞都會發生變化,這些變化基本上以腫瘤的發生,促進和發展為代表。

Tumor commencement is a swift and irrevocable process that begins with an contact to a carcinogenic agent, trailed by its circulation and transportation to tissues causing non-lethal mutations in cellular DNA. These “initiated cells” start to collect extra irreversible genetic changes that continue with every new cycle of proliferation (Barcellos-Hoff & Leden & Wang, 2013). Functionally, initiated cells are further immune to inhibitory indicators mediated by cell differentiation inducers and negative growth regulators (Ashkenazi & Gentry & Jackson, 2008, Quail & Joyce, 2013).

Tumor promotion includes the discerning clonal expansion and proliferation of initiated cells permitting for additional mutations to gather. While on the other hand, initiation, tumor promotion is a comparatively lengthy and reversible procedure that actively proliferating pre-neoplastic cells start dividing and propagate. Tumor progression, the last stage of neoplastic transformation, happens after these mutations

ends in an invasive cellular phenotype with metastatic potential (Barcellos-Hoff et al. 2013, Kim & Emi & Tanabe, 2007).

腫瘤的發生是一個迅速且不可撤銷的過程,始於與致癌劑的接觸,然後通過其循環和運輸進入組織,從而引起細胞DNA的非致命突變。這些“起始細胞”開始收集額外的不可逆的遺傳變化,這些變化隨著每個新的增殖週期而持續。從功能上講,起始細胞可進一步免疫細胞分化誘導劑和負性生長調節劑介導的抑制指標。

腫瘤成長包括啟動細胞的克隆擴增和增殖,從而允許其他突變聚集。另一方面,啟動腫瘤的促進是一個相對較長且可逆的過程,該過程會積極增殖腫瘤前細胞開始分裂並增殖。腫瘤進展是腫瘤轉化的最後階段,發生在這些突變之後終止於具有轉移潛能的侵入性細胞表型。

Developments in our understanding of tumor development reveal that each step is comprised of exceedingly variable and complexed systems. For example, epigenetic changes of tumor suppressor genes through DNA methylation in pre-neoplastic tissues could bring about an accelerated carcinogenesis (Aio et al., 2014, Letelier & Brebi & Tapia & Roa, 2012). The active regulation of proteins tangled in cellular apoptosis by micro-RNAs may substantially influence the tumor promotion as well as progression (Suzuki & Katsura & Matsuyama & Miyazono 2015). Lastly, current evidence has emphasized the serious role of the tumor microenvironment on the existence and mutation of pre-neoplastic cells (Quail et al.,.2013).

Cancer chemoprevention centers on the identification of agents that precisely influence the initial stages of cellular transformation (Sapienza & Issa, 2016; Collins & Azqueta & Langue 2012). Naturally occurring phytochemicals have been known to have a extensive range of cellular effects (figure 1). For example, phytochemicals might stop carcinogens from attainment of targeted sites and supports detoxification of extremely reactive molecules (Royston & Tollefsbol, 2015). Furthermore, Select phytochemicals improve innate immune surveillance and improve the removal transferred cells (Espinoza & Takami & Trung & Nakao, 2013). To conclude, phytochemicals have several impacts on intrinsic DNA repair mechanisms and may influence tumor suppression and prevent cellular proliferation pathways (Sapienza et al., 2016).

 

我們對腫瘤發展的了解表明,每個步驟都包括極其可變和復雜的系統。例如,通過腫瘤前組織中的DNA甲基化來抑制腫瘤抑制基因的表觀遺傳學變化可能會加速致癌作用。微小RNA糾纏在細胞凋亡中的蛋白質的主動調節可能會極大地影響腫瘤的生長和進展。最後,目前的證據強調了腫瘤微環境在腫瘤前細胞的存在和突變中的重要作用。

癌症化學預防的重點是精確識別可影響細胞轉化初始階段的藥物。已知天然存在的植物化學物質具有廣泛的細胞效應(圖1)。例如,植物化學物質可能會阻止致癌物到達目標部位,並支持極活潑分子的解毒作用。此外,精選植物化學物質可改善先天免疫監測並改善轉移的細胞的清除。總而言之,植物化學物質會對內在的DNA修復機制產生若干影響,並可能影響腫瘤抑制並阻止細胞增殖途徑

 

Figure 8: Carcinogenesis

8:致癌作用

Carcinogenesis involves several stages that eventually reprogram a regular cell into a cancer cell. Phytochemicals may employ their chemopreventive effects by blocking crucial events of tumor initiation and promotion there by reversing the premalignant stage. Also these compounds may stop tumorigenesis by hindering or retarding tumor progression or by promoting cell differentiation (Kotecha, Ritesh., Takami, Akiyoshi., Espinoza, Luis., 2016)

Natural Phytochemicals

Phytochemicals are (non-nutritional) bioactive ingredients found in a variety of

vegetables and fruits and grains. Some helpful properties on health are credited to phytochemicals. A huge number of phytochemicals have been recognized, but for most of them the possible helpful effects remain unknown and necessitate elucidation (Weng & Yin, 2012)

Phytochemicals can be classified by their chemical structure, their function, or the signaling pathway that they act (Nivya & Raja & Kumaravel, 2012). As far as their chemical structure, they can be classified schematically in the following way:

  • Carotenoids (lycopene, alpha- and beta-carotene, lutein, beta-cryptoxanthin, zeaxanthin, and astaxanthin).
  • Phenolic compounds (Phenolic acids, flavonoids, stilbenes, coumarins, and tannins).
  • Alkaloids.
  • Nitrogenated compounds
  • Organosulfurous compounds (isothiocyanates, allyl sulfur compounds, and indoles).

致癌作用涉及幾個階段,這些階段最終將細胞重編程為癌細胞。植物化學物質可以通過逆轉癌前期階段來阻止腫瘤發生和促進的關鍵事件,從而發揮其化學預防作用。這些化合物也可以通過阻礙或延遲腫瘤進展或促進細胞分化來停止腫瘤發生

天然植物化學

植物化學物質是多種生物(非營養)成分。

蔬菜,水果和穀物。一些有益於健康的特性歸功於植物化學物質。但是對於大多數植物化學物質而言,可能的有益作用仍然未知,需要闡明。

植物化學物質可以按其化學結構,功能或它們起作用的信號途徑進行分類。至於它們的化學結構,可以按以下方式示意性地分類:

類胡蘿蔔素(番茄紅素,α-和β-胡蘿蔔素,葉黃素,β-隱黃質,玉米黃質和蝦青素)。

酚類化合物(酚酸,類黃酮,芪,香豆素和丹寧酸)。

生物鹼。

含氮化合物

有機亞硫化合物(異硫氰酸酯,烯丙基硫化合物和吲哚)。

Moreover, an extensive diversity of phytochemicals exist and they differ in their possible activity, however, functionally, they can be categorized in the following way:

  • Antioxidants
  • Anti-carcinogens
  • Anti-oestrogens
  • Anti-inflammatory agents
  • Immunomodulatory agents

Phytochemicals are able to intermingle with the metabolic pathways and signaling cascades that regulate growth, proliferation, differentiation, and cell survival or death. The amount of signaling pathways that are acknowledged and are associated with cancer is increasing. The greatest characterized pathways include the mitogen-activated protein kinase (MAPK), Phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (P13K/Akt/mTOR), and nuclear factor kappa-beta (NF-kB) pathways (Vauzour, 2010 & Weng & Dorai 2004).

此外,植物化學物質種類繁多,它們的可能活性也不同,但是從功能上講,它們可以按以下方式分類:

抗氧化劑

抗致癌物

抗雌激素

抗炎藥

免疫調節劑

 植物化學物質能夠與代謝途徑和信號級聯反應混合,從而調節生長,增殖,分化以及細胞存活或死亡。 公認的與癌症相關的信號通路數量正在增加。 表徵最大的途徑包括促分裂原活化蛋白激酶(MAPK),磷脂酰肌醇3-激酶/ Akt /哺乳動物雷帕黴素靶標(P13K / Akt / mTOR)和核因子kappa-betaNF-kB)途徑(Vauzour2010) &WengDorai 2004)。

Relevant Phytochemicals

Carotenoids.

Lycopene, alpha-carotene, and beta-carotene are carotenoids that have encountered a lot of attention because of their likely activity in decreasing the danger of tumors like breast and prostate (Tan, 2010 & Howard, 2010 & Donaldson, 2004 &Abar, 2016 & Chen, 2016). The molecular mechanism that are fundamental to the association amongst carotenoids and cancer prevention includes their vigorous antioxidant activity, antiproliferative activity and modulation of immune function (Liu, 2004 & Elliot, 2005). These phytochemicals may originate in numerous fruits and vegetables, but predominantly high levels are in carrots (alpha- and beta-carotenes) and tomatoes (lycopene). A meta analysis of epidemiological studies (11 case-control and 6 cohort studies) exposed an inverse association among the intake of tomatoes and tomato-containing foods and the danger of gastric cancer. A 27% decrease in hazard was found when comparing those with the highest intake, those with the lowest ingesting (lycopene decreased the danger by 12%, though the reduction was not statistically significant) (Yang, 2013). Lycopene was connected with a 30-40% decrease in the danger of prostate cancer (specially in advance disease) when analyzing those with the highest quintile of intake to those with the least ingesting (Donaldson, 2004). Reliable with these results, positive statistics were also found from the investigation of 8 prospective studies of breast cancer, where a 22% danger reduction was detected for lycopene, 13% for alpha-carotene, 17% for beta-carotene, and 19% for total carotenoids, once more this is in comparison among the quintile with the uppermost levels and the group with the bottommost levels of intake of these carotenoids (Eliassen, 2015) An original matched case-control study yielded alike outcomes. Carotenoid levels in plasma were found in 18,743 women during a 20 years period. Women that have the high plasma levels of carotenoids showed a substantial 18-28% decrease in the danger of breast cancer (the most concentration quintile versus the fewest)(eliassen, 2015). Moreover, there was a solid inverse relationship among carotenoid concentrations and recurrence of breast cancer (for instance, beta-carotene substantially decreased the danger of reappearance by 68% (when comparing the quintiles that had the most and fewest levels).

相關植物化學物質

類胡蘿蔔素。

番茄紅素,α-胡蘿蔔素和β-胡蘿蔔素是類胡蘿蔔素,由於它們可能會減少乳腺和攝護腺癌等腫瘤的危險而備受關注。類胡蘿蔔素與癌症預防之間關係的根本分子機制包括其強大的抗氧化活性,抗增殖活性和免疫功能的調節。這些植物化學物質可能起源於許多水果和蔬菜,但其中的高含量主要來自胡蘿蔔(α-和β-胡蘿蔔素)和番茄(番茄紅素)。一項流行病學研究(11個病例對照研究和6個隊列研究)的薈萃分析揭示了番茄和含番茄食物的攝入與胃癌危險之間的負相關關係。與攝入量最高的人和攝入量最低的人相比,發現危險降低了27%。

當分析攝入量最高的人群與攝入量最少的人群時,番茄紅素可將攝護腺癌(特別是疾病晚期)的危險性降低30-40%。根據這些結果,從對8項乳腺癌的前瞻性研究的調查中也發現了積極的統計數據,其中番茄紅素的危險性降低了22%,α-胡蘿蔔素降低了13%,β-胡蘿蔔素降低了17%,而β-胡蘿蔔素降低了19%。總類胡蘿蔔素的攝入量再一次高於最高水平的五分位數和最低攝入量的人群之間的比較。原始的匹配病例對照研究得出了相似的結果。在20年的時間裡,有18,743名婦女的血漿中類胡蘿蔔素水平被發現。血漿中類胡蘿蔔素水平高的女性患乳腺癌的風險顯著降低了18-28%。此外,類胡蘿蔔素的濃度與乳腺癌的複發之間存在牢固的反比關係(例如,當比較具有最高和最低水平的五分位數時,β-胡蘿蔔素可將重現的危險顯著降低68%)。

Experments have revealed that lycopene lowers intracellular generation of reactive oxygen species (ROS) by perhaps augmenting proteins that are in antioxidant reactions that involve superoxide dismutase-1 (SOD-1) and glutathione-S-transferase­omega-1. Lycopene could decrease oxidative stress as well by down-regulating expression of FROS producing proteins like ERO-1 like protein-α and CLIC-1 (Palozza & Parrone & Catalano & Simone, 2010). Additionally, it has been revealed that lycopene has the ability to inhibit cell proliferation, induce apoptosis, and in prostate cancer models, has proven to weaken the metastatic volume of cancer cells (Palozza & Parrone & Catalano & Simone, 2010, Ono & Takeshima & Nakano, 2015).

Siems et al. (2003) experimented on the effects of water-soluble carotenoids and carotenoid breakdown products on neutrophil activation and apoptosis. They discovered that most carotenoids that are now in the nanomolar and the micromolar concentration range improve superoxide generation by activated cells but prevent superoxide generation and help apoptosis at advanced concentrations.

實驗表明,番茄紅素可能通過增強涉及超氧化物歧化酶-1SOD-1)和穀胱甘肽-S-轉移酶-ω-1的抗氧化反應中的蛋白質來降低細胞內活性氧(ROS)的產生。番茄紅素還可以通過下調產生FROS的蛋白ERO-1之類的表達來降低氧化應激,這些蛋白如ERO-1(如α-蛋白和CLIC-1)。另外,已經發現番茄紅素具有抑制細胞增殖,誘導細胞凋亡以及在攝護腺癌中的能力

 事實證明,這些模型可以減弱癌細胞的轉移能力

Siems等人2003年實驗了水溶性類胡蘿蔔素和類胡蘿蔔素分解產物對中性粒細胞活化和凋亡的影響。他們發現,現在處於納摩爾濃度和微摩爾濃度範圍內的大多數類胡蘿蔔素可改善活化細胞產生的超氧化物,但可防止超氧化物的產生,並在較高濃度時幫助凋亡。

Gloria et al. (2014) determined the effect of lycopene and beta-carotene on cell cycle and cell viability in three different human breast cancer cell lines at different time points. The results from their study showed that lycopene presented distinct properties when joined to different kinds of cells, however, the decrease in cell viability was detected in all cancer cells. The MCF-7 cell line offered a reduction in the cell number only at the most concentrated level of lycopene (2.5-10 μM). Additionally, the cells that were treated with 10 μM, a 20% decrease was observed. An seeming effect was detected on the MDA-MB-235 cell line only when lycopene had the most advanced doses. A significant reduction in the number of viable cells was observed after 48 hours of treatment in the MDA-MB-231 cell line, making a 30% decrease compared to untreated cells. After 96 hours lycopene substantially changed the cell viability in all cell lines. Lycopene treatment repressed cell viability of MCF-7 and MDA-MB-235 cells by 30% and 20%, respectively.

All cell lines treated with beta-carotene offered dissimilar behaviors when related to lycopene-treated cells. After 48 hours of treatment, beta-carotene produced substantialchanges in the cell viability of the three cell lines. The decrease was in order of 40% in the MCF-7 cell lines, 30% in MDA-MB-235 cells and 70% in MDA-MB-231 cells when treated with 10 µM.

Apoptosis induction was examined by annexin and PI biomarkers. Basically lycopene and beta-carotene stimulated apoptosis in all cell lines after 48 hours. Phenolic Compounds.

Gloria等人2014確定了番茄紅素和β-胡蘿蔔素在不同時間點對三種不同的人類乳腺癌細胞系的細胞週期和細胞活力的影響。他們的研究結果表明,番茄紅素與不同類型的細胞結合時具有不同的性質,但是,在所有癌細胞中都發現了細胞活力的下降。 MCF-7細胞系僅在番茄紅素的最高濃度(2.5-10μM)時,細胞數量減少。此外,用10μM處理的細胞觀察到減少了20%。僅當番茄紅素具有最先進的劑量時,才在MDA-MB-235細胞系上檢測到明顯的作用。在MDA-MB-231細胞系中處理48小時後,觀察到存活細胞數量顯著減少,與未處理細胞相比,減少了30%。 96小時後,番茄紅素顯著改變了所有細胞系的細胞活力。番茄紅素處理將MCF-7MDA-MB-235細胞的細胞活力分別抑制了30%和20%。

當與番茄紅素處理過的細胞相關時,所有用β-胡蘿蔔素處理過的細胞系均表現出不同的行為。治療48小時後,β-胡蘿蔔素產生大量改變三種細胞系的細胞活力。當用10 µM處理時,MCF-7細胞系的下降順序為40%,MDA-MB-235細胞為30%,MDA-MB-231細胞為70%。

通過膜聯蛋白和PI生物標誌物檢查凋亡誘導。 48小時後,基本上番茄紅素和β-胡蘿蔔素刺激了所有細胞系的凋亡

Polyphenols are in many kinds of foods, like green tea, red wine, cocoa, berries, cranberries, pomegranate, papaya, and more. There is substantial proof that they employ effects on tumor development and carcinogenesis at the cellular level (Donaldson, 2004 & Suh 2012 &Adhami, 2009). Their favorable impact on the action of some metabolic enzymes, like glutathione peroxidase, catalase, NADPH, quinone oxidoreductase, glutathione S-transferase, and/or cytochrome P450, that contribute in the physiological methods of detoxification could avoid, or at least decrease the incidence of, tumor processes. Polyphenols also control signaling cascades (e.g., MAPK, PI3K, and NF-Kb) involved in the development and proliferation of tumor cells (Vauzour, 2010 & Weng, 2012 & Nivya, 2012 & Vasilis, 2010).

According to Weiguang Yi et al. (2006), who studied the possible anticancer activities of polyphenols in rabbiteye blueberries and muscadine grapes using a HepG2 cell model. They revealed that phenolic compounds in blueberries and muscadine grapes could prevent HepG2 liver cancer cell population growth as well as induce apoptosis. Albaqami (2019) studied the anticancer effects of onosma bracteatum against breast cancer cell line BT549 and lung cancer cell line A549 and human prostate cancer cell line PC3 and reported that O. bracteatum had anti-proliferation activity and induced apoptosis, as well as decreased the concentration of MDA formation.

酚類化合物。

多酚存在於多種食物中,例如綠茶,紅酒,可可,漿果,酸果蔓,石榴,木瓜等。有大量證據表明它們在細胞水平上對腫瘤的發展和致癌作用有影響。它們對某些有助於解毒的生理學方法的代謝酶(如穀胱甘肽過氧化物酶,過氧化氫酶,NADPH,醌氧化還原酶,穀胱甘肽S-轉移酶和/或細胞色素P450)的作用產生有利影響,可以避免或至少降低發病率的腫瘤過程。多酚還控制涉及腫瘤細胞發育和增殖的信號級聯反應(例如MAPKPI3KNF-Kb)。

據魏光義等人,他使用HepG2細胞模型研究了多酚在兔眼藍莓和麝香葡萄中的可能抗癌活性。他們發現藍莓和麝香葡萄中的酚類化合物可以阻止HepG2肝癌細胞的生長並誘導細胞凋亡Albaqami2019)研究了小os對乳腺癌細胞BT549和肺癌細胞A549和人類攝護腺癌的抗癌作用細胞株PC3並報導了其具有抗增殖活性並誘導了細胞凋亡,並降低了MDA形成的濃度。

Flavonoids.

Epigallocatechin gallate (EGCG).

Contained in green tea is flavonoid called EGCG and it has been extensively studied. The widespread scientific literature, both in vitro and in vivo studies, has confirmed the activity of EGCG in the prevention of tumor cells proliferation and angiogenesis inhibition (promoted by VEGE) in a many kinds of tumors, like colon, breast, head and neck, esophagus, and prostate (Nivya, 2012 & Yuan, 2011 & Henning, 2011) Camellia sinensis, or tea, is one of the most ancient and popular beverages used worldwide. While the specific composition differs broadly, tea typically has a mixture of polyphenols, alkaloids, minerals, and additional volatile organic compounds (Seeran N.P., 2006). Moreover, there is a large amount of catechin polyphenols like Epigallocatechin-3-gallate (EGCG) and Epicatechin-3-gallate(ECG). Remarkably, green tea has a higher amount of these precise catechins when related with other black teas (Wu, 2009). Catechin polyphenols, particularly EGCG and EBCG have strong antioxidant action and are believed to apply their role as antioxidants by stopping precise DNA injury by reactive oxygen species, thereby avoiding tumor mutagenesis of healthy cells (Sutandyo, 2010 & Wu, 2009). In pre-clinical experiments, tea polyphenols have proven to prevent tumor cell growth by inducing apoptosis through multiple pathways connected to cancer development (Wu, 2013). Tea polyphenols have also been associated in several carcinogenesis pathway counting inhibiting angiogenesis modulating the glutathione S-transferase and quinone reductase pathways (Sutandyo 2010).

Many studies have revealed complete conflicting statistics about the possible decrease in cancer danger due to ingredients of green tea in several populations (Sun, 2006). This discrepancy in the findings may be due to multiple kinds of drinks that are often used in trials and have multiple tea preparations, unknown concentrations of various kinds of antioxidants, and the bioavailability of a lot of these compounds after consumption is varied worldwide. Additionally, a lot of these experiments are many times confounded by other intakes that could eventually lead to the development of cancers counting tobacco and alcohol use, and it is hard to differentiate these confounding factors. (Wu, 2013).

類黃酮。

兒茶素沒食子酸酯(EGCG)。

綠茶中含有黃酮類化合物,稱為EGCG,已經得到了廣泛的研究。廣泛的科學文獻(無論是體內還是體外研究)都證實了EGCG在預防多種腫瘤(如結腸癌,乳癌,頭頸癌,肝癌,食道和攝護腺)中的腫瘤細胞增殖和抑制血管生成(由VEGE促進)中的活性。綠茶花茶是世界上使用最古老和最受歡迎的飲料之一。儘管具體成分差異很大,但茶通常含有多酚,生物鹼,礦物質和其他揮發性有機化合物的混合物。此外,存在大量的兒茶素多酚,例如表沒食子兒茶素-3-沒食子酸酯(EGCG)和表兒茶素-3-沒食子酸酯(ECG)。值得注意的是,綠茶中的這些兒茶素含量較高,與其他黑茶有關。兒茶素多酚,特別是EGCGEBCG具有很強的抗氧化作用,據信通過阻止活性氧對DNA的精確損傷來發揮其抗氧化劑的作用,從而避免了健康細胞的腫瘤誘變。在臨床前實驗中,茶多酚已被證明可通過與癌症發展有關的多種途徑誘導細胞凋亡來預防腫瘤細胞的生長。茶多酚也有關聯

 在幾種致癌途徑中計數抑制血管生成調節穀胱甘肽S-轉移酶和醌還原酶途徑。

許多研究已經揭示出,在一些人群中,由於綠茶成分可能降低患癌症的風險的統計數據相互矛盾。研究結果之間的差異可能是由於多種飲料經常在試驗中使用,並且具有多種茶製劑,各種抗氧化劑的濃度未知,而且這些化合物在世界範圍內消費後的生物利用度也不盡相同。此外,許多這些實驗常常與其他攝入混淆,這些攝入最終可能導致發展為計算吸煙和飲酒的癌症,並且很難區分這些混雜因素。

As of today there have been numerous clinical trials that have set out to classify the possible clinical role these tea polyphenols may have in cancer prevention. Two randomized clinical trials assessed the effects of tea extracts on premalignant oral lesions called leukoplakia. In a double-blinded interventional trial, subjects were given either three grams of mixed tea products, both orally and topically, or placebo. After six months of treatment, 38% of patients in the treatment group had partial deterioration of their oral lesions compared to 10% in the placebo group (Li, 1999).

The antioxidant and anticancer properties of flavonoid glycosides, that naturally occurring substance contained in medicinal plants were examined by Bae et al. (2017). They studied glycosides extracted from Limonium tetragonum (myricetin 3-galactoside and quercetin 3-o-beta-galactopyranoside) and the effects they had on Matrix metalloproteinases (MMP’s) which are endopeptidases accountable for extracellular matrix degradation which leads to the spreading of malignant tumor cells. They found that myricetin 3-galactoside and quercetin3-o-beta-galactopyranoside can prevent MMP activity and restrain MAPK-linked MMP upregulation. Subsequently, the two enzymes responsible invasion and metastatic spread of tumors. These results verifies that tetragonum could be applied as a basis of anti-MMP in the fight against cancer.

Daidzein, genistein, and glycitein.

迄今為止,已有許多臨床試驗對這些茶多酚在預防癌症中可能發揮的臨床作用進行了分類。兩項隨機臨床試驗評估了茶提取物對癌前口腔病變白斑的影響。在一項雙盲干預試驗中,給受試者口服或局部給予三克混合茶的飲料或安慰劑。經過六個月的治療,治療組中38%的患者口腔病變部分惡化,而安慰劑組為10

Bae等人研究了黃酮苷的抗氧化和抗癌特性,黃酮苷是藥用植物中天然存在的物質。。他們研究了從四味子檸檬提取的糖苷(楊梅素3-半乳糖苷和槲皮素3-o-β-半乳糖吡喃糖苷)及其對基質的影響

內肽酶金屬蛋白酶(MMP)負責細胞外基質降解,導致惡性腫瘤細胞擴散。他們發現,楊梅素3-半乳糖苷和槲皮素3-o-β-吡喃半乳糖苷可以阻止MMP活性並抑制MAPK相關的MMP上調。隨後,這兩種酶負責腫瘤的侵襲和轉移擴散。這些結果證明四方黃可以作為抗MMP的基礎來對抗癌症。

黃豆苷元,染料木黃酮和糖精。

The soybean (Glycine max) isoflavones have been well categorized as well as the subject of many studies, both in vitro and in vivo. Due to their chemical structure, that is comparable to that of endogenous estrogen, they perform as competitive agonists against the estrogen receptor (ER)(albeit with less power). Plus their ability to interaction with ER, other anticancer aspects have been described, like their antiangiogenic and antiproliferative effects and inhibition of tyrosine kinases (Adlercreutz, 1997). The outcomes described in systematic reviews and meta-analysis display an association between soybean intake and the danger of breast cancer, however with substantial heterogeneity in the studies as far as the amount of soybean foods injested, the period of exposure, population type (Asian or Western), menopausal status, hormonal outline, and even the oestrogen receptor standing of the tumor (Nagata, 2010 & Zhong, 2012 & Chen, 2014 & Dong, 2011).

Nevertheless, a current meta-analysis presented that soy food intake after diagnosis of breast cancer was related to decreased mortality and recurrence (14% and 26%, relating to the groups that have the most versus the least consumption) (Chi, 2013).

ER has two isoforms, ER-alpha and ER-beta, that show contrary effects as far as proliferation and differentiation in breast cancer. ER-alpha mediates mitogenic effects, whereas ER-beta mediates tumors suppressing activity. The ERalpha/ERbeta ratio is changed in breast cancer cells, preferring the expression of the ER-alpha isoform (Rizza, 2014). A new prospective learning with a cohort of 70,578 Chinese women, who were observed for a median of 13.2 years, reveal an inverse association between soybean consumption and breast cancer danger in older age. Precisely, the quintile with the uppermost intake of soy protein (16.4 g/day) had a danger decrease of 22% equated with that for the lowest quintile. This decrease was even more substantial for premenopausal women: 54% (Baglia, 2016).

大豆(Glycine max)異黃酮已經被很好地分類,並且是許多體內外研究的主題。由於它們的化學結構與內源性雌激素相當,因此它們可作為對抗雌激素受體(ER)的競爭性激動劑。加上與他們互動的能力

ER,其他抗癌方面也已有描述,例如它們的抗血管生成和抗增殖作用以及酪氨酸激酶的抑製作用。在系統評價和薈萃分析中描述的結果顯示,大豆攝入量與乳腺癌危險性之間存在關聯,但是在研究中,大豆食品的攝入量,接觸時間,人群類型(亞洲人或西方),更年期狀態,荷爾蒙輪廓,甚至腫瘤的雌激素受體水平。儘管如此,目前的一項薈萃分析顯示,乳腺癌診斷後的大豆食品攝入量與死亡率和復發率降低相關(分別為14%和26%,與消費量最大而消費量最小的人群有關)。

ER具有兩種同工型,即ER-α和ER-β,它們在乳腺癌的增殖和分化方面表現出相反的作用。 ER-α介導有絲分裂作用,而ER-β介導腫瘤抑制活性。乳腺癌細胞中的ERalpha / ERbeta比值發生了變化,更傾向於表達ER-alpha異構體。一項新的前瞻性研究對70,578名中國婦女進行了研究,平均年齡為13.2歲,該研究揭示了大豆消費與老年乳腺癌風險之間的反比關係。準確地說,攝入大豆蛋白最多的五分之一(16.4/天)的危險降低了22%,相當於最低的五分之一。對於絕經前女性而言,這一下降幅度更大:54

Additionally soybean consumption has been connected to a decrease in the danger of other tumors, like the colon (21% decrease only in females) (Yan, 2010) and prostate (Souza, 2010 & Die, 2014) tumors. Meta-analyses of epidemiological studies have revealed decreases in the hazard of lung cancer of either 23% (8 case-control) studies and 3 prospective cohorts) (Yang, 2011) or 41% when looking at the group with the highest consumption of soybean or isoflavones and comparing them to the group with the fewest consumption (Yang, 2011) or 41% when comparing the group with the most consumption of soybeans or isoflavones to the group with the most moderate use (Yang, 2012).

Organosulfurous Compounds

Isothiocyanates and indoles.Scientific findings proposes that implementing a diet that has a lot of vegetables like the Cruciferae (e.g., broccoli, cabbage cauliflower, mustard greens, Brussels sprouts, and kale) could decrease the danger of some kinds of cancers, like colorectal, prostate, breast, renal, and gastric cancers (Sutandyo, 2010 & Wu, 2009 & Yang, 2013 & Wu, 2013 & Liu,2012 & Liu, 2013 & Liu, 2013). Several meta-analysis of epidemiological have revealed decreases in the danger of cancer: 19% for gastric cancer ( 16 case-control evaluations and 6 prospective) (Wu, 2013) and 18% for colorectal cancer when equating the group with most intake to the group with the fewest (24 case-control studies and 11 prospective (Wu, 2013), 10% for prostate cancer (7 cohort studies and 6 case-control, even though the decrease was substantial only for the case-control studies) (Liu, B. Mao, Q., Xie, L., 2012), 15% for breast cancer (11 case-control and 2 cohort) (Liu, X., Lv, K., 2013), 28% for kidney cancer (3 cohort studies and 7 case-control, even though the decrease was statistically substantial only for the case-control experiments) (Liu, 2013), and 20% for bladder cancer (5 cohort studies and 5 case-control, though the decrease was only statistically substantial for the case-control studies (Liu, 2013).

此外,食用大豆與減少其他腫瘤的危險性有關,例如結腸癌(僅女性中減少了21%)和攝護腺癌。流行病學研究的薈萃分析顯示,在大豆消費量最高的人群中,肺癌的風險降低了23%(8個病例對照)和3個前瞻性隊列或41%。

有機硫化合物

異硫氰酸鹽和吲哚。

 科學發現建議,飲食中多加十字花科的蔬菜(例如西蘭花,甘藍菜花,芥菜,抱子甘藍和羽衣甘藍),可以降低某些癌症的風險,例如結直腸癌,攝護腺癌,乳腺癌,腎癌和胃癌。流行病學的一些薈萃分析顯示,癌症的危險性降低了:當將攝入量最多的人群等同於胃癌時,胃癌的比例為19%(16個病例對照評估,6個前瞻性),結直腸癌為18%。組最少(24個病例對照研究和11個前瞻性研究,攝護腺癌的發生率為10%(7個隊列研究和6個病例對照,儘管只有病例對照研究的減少幅度很大),乳腺癌佔15%(11個病例對照和2個隊列),腎癌佔28%( 3項隊列研究和7項病例對照,儘管僅在病例對照實驗中統計學下降顯著(Liu2013年),膀胱癌的比例為20%(5項隊列研究和5項病例對照,儘管僅在病例對照研究中具有統計學意義。

The defensive effects resulting from the digestion of the Cruciferae are based on the high levels of phytochemicals, like isothiocyanates (sulforaphane, phenethyl isothiocyanate, and benzyl isothiocyanate) and indole-3-carbinol. These phytochemicals have revealed activity in modulating both cellular signaling pathways, like PI3k/Akt/Mtor and carcinogen detoxification pathways. These phytochemicals prevent the carcinogen activation in phase 1 metabolization by hindering enzymes of the

cytochrome P450 family (CYP) and inducing phase 2 enzymes, like glutathione S­transferases, therefore hastening the clearance of carcinogens from the body (Wu, 2009).

Allyl Sulfur Compounds

The possible defensive effect of the consumption of vegetables of the Allium family (onion, garlic, shallot, chives, and leeks) has been examined for several kinds of cancer. Several meta-analysis of epidemiological findings have revealed decreases in the danger of diseases of the upper aerodigestive tract (seemingly sturdier in case-control studies and more for oesophageal compared to the head and neck cancers): 26% for garlic intake and 28% for onion (comparing those with highest versus the fewest ingestion)(Guercio, 2016). For gastric cancer, use of huge quantities of Allium vegetables decreased the danger by 46% (when comparing the highest and lowest ingesting groups) (Zhou, 2011). Current findings from a series of case-control studies presented a substantial protective effect of garlic and onion against cancer of the oral cavity ( 39% and 86% risk reduction), oesophagus (57% and 88%), colorectum (26% and 56%), larynx (44% and 83%), endometrium (38% and 60%) and ovary (22% and 73%) (in comparison to the highest versus the lowest category of intake) (Turati, 2015). The most demonstrative phytochemical available in vegetables of the Allium family is a sulfur compound named diallyl trisulfide (DTS). The instrument that DTS exerts its antitumor activity is not completely established, even though studies have revealed an inhibitory effect on the process of oncogenes and angiogenesis and an upgrading in pathways linked

to the detoxification of carcinogens and cellular apoptosis (Antony, 2011 &Gianni, 2015).

十字花科植物的消化產生的防禦作用是基於高水平的植物化學物質,如異硫氰酸酯(蘿蔔硫烷,異硫氰酸苯乙基酯和異硫氰酸芐酯)和吲哚-3-甲醇。這些植物化學物質已揭示出在調節細胞信號傳導途徑(如PI3k / Akt / Mtor)和致癌物解毒途徑中的活性。這些植物化學物質通過阻礙植物體內的酶而阻止1期代謝中的致癌物活化。細胞色素P450家族(CYP)和誘導2期酶(如穀胱甘肽S-轉移酶),從而加速了體內致癌物的清除

烯丙基硫化合物

已經研究了食用蔥屬植物的蔬菜(洋蔥,大蒜,蔥,韭菜和韭菜)可能產生的防禦作用,可預防多種癌症。幾項流行病學調查結果的薈萃分析表明,上消化道疾病的危險性降低了(病例對照研究似乎更堅固,與頭頸癌相比,食道疾病的危害更大):大蒜攝入量為26%,食道癌為28%洋蔥(比較攝入量最高與攝入最少的洋蔥)(Guercio2016)。對於胃癌,使用大量的蔥屬蔬菜可將危險性降低46%(比較最高和最低攝入量人群時)。一系列病例對照研究的最新發現表明,大蒜和洋蔥對口腔癌(降低39%和86%的風險),食道(57%和88%),結直腸癌(26%和56)具有實質性的保護作用%),喉部(44%和83%),子宮內膜(38%和60%)和卵巢(22%和73%)(與最高攝入量類別和最低攝入量類別相比)。蔥屬蔬菜中最具代表性的植物化學物質是一種名為二烯丙基三硫化物(DTS)的硫化合物。 DTS發揮其抗腫瘤活性的手段尚未完全確立,儘管研究表明對癌基因和血管生成過程具有抑製作用,並且相關通路的升級

致癌物和細胞凋亡的解毒作用

Antony2011Gianni2015)。

Phenolic Acid Derivatives

Curcumin is the most plentiful phytochemical available in turmeric (Curcuma longa), a native herb of south-western India. Several studies have exposed the different instruments of action of this polyphenol and its effectiveness in decreasing the danger of cancer (Kumar, 2016). Its anti-inflammatory activity implies that its effectiveness is not limited to chemoprevention: curcumin might also assist in decreasing the threat of chronic, inflammatory, and metabolic diseases, like diabetes, arteriosclerosis, and metabolic syndrome (Mesa, 2000).

Curcumin performances on multiple pathways or molecules related to humoral processes: Through MAP kinases, curcumin decreases the stimulation of NF-kB and subsequently the manufacturing of TNF alpha, COX-2, and IL-6, thus motivating anti-inflammatory activity (Camacho-Barquero, 2007). Curcumin improves the activity of detoxifying enzymes, like glutathione-S- transferases and p21, causing the prevention of cell proliferation (Ye,2007). Curcumin provokes the expression of pro-apoptotic proteins (Bax, Bim, Bak, and Noxa) enabling the anti-apoptotic proteins (Bcl-2, Bcl-Xl) (Chen, 2011) Curcumin decreases the expression of VEGF and matrix metalloproteases, stopping the development of metastases (Durko, 2014).

The scientific proof from in vivo experiments supports the possibility of curcumin in chemoprevention tactics against several kinds of cancer, counting breast, colorectal,pancreatic, prostate, and lung cancers (Gupta, 2013 & Kanai, 2014 & Johnson, 2007 & Gupta, 2012 & Park, 2013). Notwithstanding the low occurrence of adverse effects (e.g., diarrhea) in human experiments, statistics in animals propose it might have some carcinogenic effects (hepatic adenomas and interstitial intestinal carcinoma). Consequently, more statistics must be acquired from safety experiments.

酚酸衍生物

薑黃素是薑黃(Curcuma longa)中提取的植物化學物質,薑黃是印度西南部的一種天然草藥。多項研究揭示了該多酚的不同作用手段及其在降低癌症風險中的有效性。它的抗炎活性意味著其有效性不僅限於化學預防:薑黃素還可以幫助減少慢性,炎性和代謝性疾病的威脅,例如糖尿病,動脈硬化和代謝綜合症

薑黃素在與體液過程有關的多種途徑或分子上的表現:薑黃素通過MAP激酶減少了對NF-kB的刺激,進而減少了TNF-αCOX-2IL-6的產生,從而激發了抗炎活性。薑黃素可提高解毒酶(如穀胱甘肽-S-轉移酶和p21)的活性,從而阻止細胞增殖薑黃素激發促凋亡蛋白的表達,從而使抗凋亡蛋白(Bcl-2Bcl-Xl)成為可能。薑黃素可降低VEGF和基質金屬蛋白酶的表達,從而停止轉移的發展。

體內實驗的科學證據支持薑黃素在針對幾種癌症的化學預防策略中的可能性,包括對乳腺癌,結直腸癌,胰腺癌,攝護腺癌和肺癌。儘管在人體實驗中不良反應(例如腹瀉)的發生率很低,但動物統計數據表明它可能具有某些致癌作用(肝腺瘤和間質性腸癌)。因此,必須從安全性實驗中獲取更多統計信息。

Stilbenes.

Resveratrol (trans-3, 5,4-tridydroxystilbene) is contained in grapes (primarily in their skin), red wine, berries, peanuts, and Japanese knotweed roots (Polygonum cuspidatum). Several in vitro and in vivo studies (animal) have assessed the activity of this phytochemical in carcinogenic cells and signaling pathways, and resveratrol has revealed activity through mechanisms related to antioxidation, the modulation of detoxification enzymes of the cytochrome P450 family, the inhibition and downregulation of cyclooxygenase (COX) inflammation mediator enzymes, and effects on transcription factors, such as NF-kB (Weng, 2012 & Gesher, 2013 & Athar, 2007). The facts attained from this kind of study promotes its possible use in chemoprevention strategies. Nevertheless, the proof from both in vivo experiments and epidemiological modes of breast, colon, prostate, liver, and pancreatic cancer is varying (Carter, 2014). The dissimilarity acquired from in vivo models may be somewhat credited to the absence of homogeneity of criteria as far as the dose and the timing of administration, bioavailability, sex, or the cancer study model (initial or further stage,plus other factors) (Carter, 2014).

 

斯蒂芬妮斯。

白藜蘆醇(反式354-三乙氧基二苯乙烯)包含在葡萄(主要在其表皮),紅酒,漿果,花生和虎杖根中。幾項體外和體內研究(動物)已評估了這種植物化學物質在致癌細胞和信號傳導途徑中的活性,白藜蘆醇通過與抗氧化,細胞色素P450家族解毒酶的調節,抑制和下調有關的機制揭示了其活性。氧合酶(COX)炎症介體酶的合成,以及對轉錄因子如NF-kB的影響。通過這種研究獲得的事實促進了其在化學預防策略中的可能用途。然而,乳腺癌,結腸癌,攝護腺癌,肝癌和胰腺癌的體內實驗和流行病學模式的證據都各不相同。從體內模型獲得的差異可能在一定程度上歸因於標準的不均一性,例如劑量和給藥時間,生物利用度,性別或癌症研究模型(初始或進一步的階段,以及其他因素)。

 

The roots of the Polygonum cuspidatum, or Japanese knotweed, holds the most naturally occurring concentrations of resveratrol and has been used in customary Japanese and Chinese medicinal treatments for dermatitis, bacterial infections and inflammation. Plants manufacture resveratrol in answer, normally to mechanical damages, ultraviolet radiation and as a protection for viral and fungal infections (Keylor, 2015 & Espinoza, 2012). Now resveratrol is typically the most phytoalexin presently studied in modern healthcare due to the much publicized “French Paradox,” an opinion that there is a comparatively low occurrence of cardiovascular disease in France even though they have a mostly rich saturated-fat diet from the high intake of red wine (Scott, 2012).

Initial studies recognized that resveratrol has anti-cancer effects against several distinctive kinds of tumors and affects numerous stages of tumor initiation and proliferation. Precisely, resveratrol is able to induce cancer cell apoptosis by interceding with several signaling pathways activated in transformed cells (Espinoza, 2012 & Scott, 2012 & Shukla,2011 & Quoc, 2013 & Trung, 2015 & Kato, 2015). Clinical trials have also described the safety, pharmacokinetics, and metabolism as both a single synthesis agent and constituent of food at adjustable doses (Patel, 2011). These experiments also revealed that opposing effects counting diarrhea, nausea, and abdominal pain happen in those taking more than one gram of resveratrol daily, which has now become a normal dose limit in succeeding clinical work (Petal, 2014).

虎杖的根部具有最天然高濃度的白藜蘆醇,已被用於日本和中國針對皮膚炎,細菌感染和炎症的常規藥物治療。植物生產的白藜蘆醇通常是為了抵抗損傷,紫外線輻射並保護病毒和真菌感染。現在白藜蘆醇通常是現代醫療保健中目前研究最多的植物抗毒素,這是由於廣為流傳的“法國悖論”,一種觀點認為,即使法國人從高脂肪飲食中攝入的脂肪含量較高,其心血管疾病的發病率也相對較低,在於他們攝入紅酒。

最初的研究認識到白藜蘆醇對幾種不同類型的腫瘤具有抗癌作用,並影響腫瘤起始和增殖的多個階段。準確地說,白藜蘆醇能夠通過干擾轉化細胞中激活的幾種信號通路來誘導癌細胞凋亡。臨床試驗還描述了安全性,藥代動力學和新陳代謝既是單一合成劑,又是劑量可調的食物成分。這些實驗還顯示,每天服用超過一克白藜蘆醇的人在計算腹瀉,噁心和腹痛的反制作用,現已成為成功開展臨床工作的正常劑量

Resveratrol supplementation and its possible effects in healthy people have been tested lately in clinical trials. Initially, one study looked at the effects of resveratrol at various high doses (0.5, 1.25, and 5 grams/day for 29 days; n=10-12/dose) on circulating concentrations of insulin-like growth factor (IGF-1) and IGF-binding protein 3 (IGFBP­3) (Brown, 2010), two markers frequently related to increased tumor formation and following metastasis (Renehan, 2004). Surprisingly, these doses were found to be safe, and higher doses of five grams daily were linked with mild gastrointestinal adverse effects. Subjects given 2.5 grams daily of resveratrol exhibited considerably lower levels of circulating IGF-1 and IGFBP-3 plasma levels (Brown, 2010), implying promising chemopreventive potential. Upcoming analyses that use these surrogates will have to be interpreted with attention as levels of IGF-1 and IGFBP-3 fluctuate in different cancer models and are powerfully influenced by other dietary compounds, counting citrus fruits and Vitamin C (Tran, 2006).

Chow and colleagues investigated whether resveratrol could have an effect on drug metabolism and enzymes connected with carcinogenesis. In their investigation, one gram of resveratrol daily for four weeks was proven to have substantial reduced the effects on plasma cytochrome P450 enzymes, counting CYP3A4, 2D6, and 2C9, and heightened CYP1A2 associated with baseline measurements in healthy volunteers (Chow, 2010). These modulatory effects on enzymatic systems used in detoxification and carcinogen activation may be the reason for some chemopreventive effects, and also significantly, may alter metabolism of other agents. As many chemotherapeutic and other medicines are metabolized throughout the cytochrome system, this experiment critically increases safety matters for co-administration with other pharmaceutical agents.

 

白藜蘆醇補充劑及其對健康人的可能作用最近已在臨床試驗中進行了測試。一項研究著眼於白藜蘆醇的作用胰島素樣生長因子(IGF-1)和IGF結合蛋白3IGFBP3)循環濃度的各種高劑量(0.51.255/天,共29 n = 10-12 /劑量),這兩個標誌物經常與腫瘤形成增加和轉移有關。出乎意料的是,發現這些劑量是安全的,每天高劑量五克與輕度胃腸道不良反應有關。每天服用2.5克白藜蘆醇的受試者表現出較低的循環IGF-1IGFBP-3血漿水平,這意味著有希望的化學預防潛力。由於這些IGR-1IGFBP-3的水平在不同的癌症模型中會發生波動,並受到其他飲食化合物的強烈影響(柑橘類水果和維生素C的計數),因此使用這些替代物的近期分析將必須引起注意)。

Chow及其同事研究了白藜蘆醇是否可能對藥物代謝和與癌變有關的酶產生影響。在他們的研究中,每天四克的白藜蘆醇被證實對健康志願者的血漿細胞色素P450酶,CYP3A42D62C9計數有顯著降低的作用,並且與基線測量相關的CYP1A2升高。這些對排毒和致癌物激活中使用的酶系統的調節作用可能是某些化學預防作用的原因,並且也可能顯著改變其他藥物的代謝。由於許多化學療法和其他藥物在整個細胞色素系統中都會代謝,因此該實驗極大地提高了與其他藥物共同給藥的安全性。

 

Furthermore, breast cancer patients have been greatly examined for their response to resveratrol. In a case-control experiment, Levi and colleagues revealed that females with excessive total intake of resveratrol had a lesser chance of breast cancer when related to those with a low level of consumption (OR: 0.39) (Levi, 2005). Lately, a randomized, double-blind placebo trial uncovered that in females at a bigger jeopardy for development of breast cancer, two times a day resveratrol dosing for twelve weeks was connected to a reduction in methylation of four cancer-related genes on mammary tissue biopsies post-treatment. This study signifies the direct in-vivo anti-proliferative effects and mechanism in tissue specimens once treated (Zho, 2012). Other pre-clinical and clinical experiments have suggested that resveratrol could modulate hormonal metabolism as well and effects used in breast cancer and prostate cancer. In a randomized placebo controlled trial, Kjaer and colleagues revealed that even though resveratrol consumption was not connected with prostate size or a decrease in prostate serum antigen levels, it is connected to lesser concentrations of androgen precursors involving androstenolone (DHEA) and hence could have a relevant effect on benign prostatic hyperplasia and cancer growth (Kiaer, 2015). These results are consistent with other pre-clinical studies recording that resveratrol reduces prostate cancer growth in rat models, an effect facilitated largely through downregulation of androgen receptor expression and suppression of androgen responsive glandular kallikrein, an orthologue of the human prostate-specific antigen (PSA) (Seeni, 2008).

Moreover, the possible therapeutic effect of resveratrol could be by promoting immunosurveillance through the innate immune system, thus improving the elimination of spontaneous tumor cells prior to proliferation. Natural killer (NK) cells are mainly effector lymphocytes of this system and can notably recognize transferred or infected cells before being education by antigen process cells. This permits NK cells to effectively remove quickly progressing tumor cells at a far greater rapid rate when related with T lymphocytes, which need antigen recognition and education prior to activation (Bui, 2007). NKG2D, an antigen receptor expressed by cytotoxic lymphocytes, including NK cells, CD8, and T cells, appear to have a substantial part in tumor surveillance as these cells use the NKG2D receptor to identify specific surface ligands expressed on transformed cells for cytotoxicity (Gurra, 2008).

此外,已經對乳腺癌患者對白藜蘆醇的反應進行了大量檢查。在病例對照實驗中,Levi和同事發現,與低食用量女性相比,白藜蘆醇總攝入量過多的女性患乳腺癌的可能性較小。最近,一項隨機,雙盲安慰劑試驗發現,在女性罹患乳腺癌的危險更大的情況下,每天兩次白藜蘆醇給藥十二週與減少乳腺組織活檢中四個癌症相關基因的甲基化有關。後處理。這項研究表明,一旦處理,在組織標本中的直接體內抗增殖作用和機理。其他的臨床前和臨床實驗表明,白藜蘆醇也可以調節激素代謝,並可以用於乳腺癌和攝護腺癌。在一項隨機的安慰劑對照試驗中,Kjaer及其同事發現,即使食用白藜蘆醇與攝護腺素的大小或攝護腺血清抗原水平的降低無關,但它與濃度較低的涉及雄烯醇酮(DHEA)的雄激素前體有關,因此可能具有對良性攝護腺增生和癌症生長的相關影響。這些結果與其他臨床前研究一致,該研究表明白藜蘆醇減少了大鼠模型中攝護腺癌的生長,這種作用在很大程度上通過下調雄激素受體表達和抑制雄激素反應性腺激肽釋放酶(人類攝護腺特異性抗原(PSA)的直向同源物)而得以促進。 )此外,白藜蘆醇可能的治療作用可能是通過先天免疫系統促進免疫監視,從而改善消除自發性腫瘤細胞的增殖。天然殺傷(NK)細胞主要是該系統的效應淋巴細胞,在接受抗原加工細胞進行培養之前,可以明顯識別轉移或感染的細胞。當與T淋巴細胞相關時,這使得NK細胞能夠以更高的快速速率有效去除快速發展的腫瘤細胞,而T淋巴細胞需要在激活之前進行抗原識別和教育。 NKG2D是由細胞毒性淋巴細胞(包括NK細胞,CD8yöT細胞)表達的抗原受體,在腫瘤監測中似乎起著重要的作用,因為這些細胞利用NKG2D受體來識別轉化細胞上表達的特定表面配體以產生細胞毒性。

To describe the possible role of resveratrol in the innate immune system, a clinical study focused on uncovering differences in immune system profiles was done in healthy subjects given one gram of resveratrol for two weeks. The management of this compound was discovered to correlate with heightened expression of the NKG2D receptor on circulating peripheral blood NK cells (Espinoza, 2013). Because pre-clinical findings have established that resveratrol can stimulate the expression of NKG2D ligands in transformed cells and thus make these cells more prone to NK cell lysis via NKG2D cytotoxic pathways (Espinoza, 2013), the findings implied that resveratrol may control this axis to permit improved tumor surveillance by the innate immune system (Espinoza, 2013).

Lately, the thought patterns have changed, that is the whole food-derived components are not “idle onlookers,” but active contributors in regulating aberrant metabolic and signaling pathways in healthy as well as diseased persons. Like the whole food from the Cucurbitaceae family is “bitter melon” (Momordica charantia, also referred to as bitter gourd, balsam apple, etc.), which has gotten a lot of consideration in present years as substitute medicine in developing parts of the world. The improved attention on painful melon ingestion may, in part, be because of several current pre-clinical efficacy studies demonstrating bitter melon probablility to target obesity /type II diabetes-associated metabolic aberrations and its pre-clinical anti-cancer efficacy against some malignancies. Moreover, the bioassay-guided fractionations have categorized the bitter melon chemical constituents established on their antidiabetic or cytotoxic properties. Thus, by definition, these bitter melon constituents are at turning point on the bioactivity parameters; they either have discriminating efficacy for improving metabolic aberrations or targeting cancer cells or have favorable effects in both circumstances. However, given the massive, though dispersed, literature findings on the bioactivity and beneficial attributes of better melon constituents, a comprehensive assessment on the bitter melon components and the overlapping helpful characteristics is absent (Raina et al., 2016). The present study addresses the substantial hole in our understanding of the potential benefits of bitter melon.

為了瞭解白藜蘆醇在先天免疫系統中的可能作用,在健康受試者中給予一克白藜蘆醇兩週,進行了一項旨在揭示免疫系統特徵差異的臨床研究。發現該化合物的處理與循環的外周血NK細胞中NKG2D受體的表達升高相關。由於臨床前發現已經證實白藜蘆醇可以刺激轉化細胞中NKG2D配體的表達,從而使這些細胞更容易通過NKG2D細胞毒性途徑裂解NK細胞,該發現暗示白藜蘆醇可以可以通過先天免疫系統改善對腫瘤的監測

最近,思想模式發生了變化,即整個食物來源的成分並不是“閒置的圍觀者”,而是在健康人和患病者中調節異常代謝和信號傳導途徑的積極貢獻者。

像整個葫蘆科的食物是“苦瓜”(Momordica charantia),近年來已成為世界上發展中地區的替代藥物。對疼痛性甜瓜攝入的關注度提高,部分原因是由於一些當前的臨床前功效研究表明,苦瓜具有針對肥胖症/ II型糖尿病相關代謝異常的概率及其針對某些惡性腫瘤的臨床前抗癌功效。此外,生物測定指導的分級將苦瓜化學成分歸類於其抗糖尿病或細胞毒性特性。因此,根據定義,這些苦瓜成分正處於生物活性參數的轉折點。它們在改善代謝畸變或靶向癌細胞方面具有區分功效,或者在兩種情況下均具有良好的效果。然而,鑑於大量文獻關於更好的甜瓜成分的生物活性和有益屬性的文獻發現,因此缺乏對苦瓜成分和有用的重疊特徵的全面評估。本研究解決了我們對苦瓜潛在好處的認識上的重大漏洞

The information revealed from this study could have clinical implications associated with bitter melon and cancer treatments. We want to investigate bitter melons potential to decrease proliferation, activate apoptosis as well as lower the MDA levels The investigation will be done by three in vitro experiments; cell viability, lipid peroxidation and by caspase-3 activation.

 

本研究揭示的信息可能對苦瓜在癌症治療具有臨床意義。 我們想研究苦瓜減少增殖,激活細胞凋亡以及降低MDA水平的潛力。該研究將通過三個體外實驗進行; 細胞活力,脂質過氧化和caspase-3激活。

An escalation in free radicals produces an overproduction in MDA. MDA is not only indicator for oxidative stress and the antioxidant status in cancerous patients, but also is a potentially significant contributor to DNA injury. Lipid peroxidation is linked to the underlying mechanisms involved in many disorders, such as; heart disease and cancer. Therefore, quantification of lipid peroxidase is essential for accessing the role of oxidative injury in pathophysiological disorders.

Cancer cells display more significant ROS stress than healthy cells mostly because of oncogenic stimulation, improved metabolic activity, and mitochondrial malfunction. ROS has two contradictory aspects. On the one hand, small amounts of ,ROS facilitates cancer cell survival because cell-cycle progression driven by growth factors and receptor tyrosine kinases (RTK) necessitate ROS for activation (Liu, J. & Head, E. & Gharib AM. & Yuan, W & Ingersoll, RT. & Hagen, TM. & Ames BN., 2002 ) and chronic inflammation, a main mediator of cancer, is controlled by ROS. Mean while, an elevated level of ROS can prevent tumor growth through the persistent activation of cell cycle inhibitor (Stadtman, ER., 1992 & Carney, JM., et al. 1991) and induction of cell death and senescence by injuring molecules. Most of the chemotherapeutic and radiotherapeutic agents kill cancer cells by augmenting ROS stress. Modest levels of ROS are needed for the survival of cancer cells, while extreme concentrations eradicate them.

自由基的增加導致MDA的過量生產。 MDA不僅是癌症患者氧化應激和抗氧化劑狀態的指標,而且還是DNA損傷的潛在重要因素。脂質過氧化與許多疾病所涉及的潛在機制有關,例如;心臟病和癌症。因此,脂質過氧化物酶的定量對於獲得氧化損傷在病理生理疾病中的作用至關重要。

細胞顯示出比健康細胞更顯著的ROS應激,主要是由於致癌性刺激,改善的代謝活性和線粒體功能障礙。 ROS有兩個矛盾的方面。一方面,少量的ROS可促進癌細胞的存活,因為生長因子和受體酪氨酸激酶(RTK)驅動的細胞週期進程需要激活ROS,而慢性炎症是癌症的主要介體,受ROS控制。同時,升高水平的ROS可以通過細胞週期抑製劑的持續激活來阻止腫瘤的生長,並通過損傷分子來誘導細胞死亡和衰老。大多數化學治療劑和放射治療劑通過增加ROS應激來殺死癌細胞。癌細胞的生存需要適度的ROS而非常高濃度的ROS則可以根除它們

According to Wong (2011) Understanding the mechanisms of apoptosis is essential and aids in the understanding of the pathogenesis of disorders as a result of chaotic apoptosis. consequently, this may assist in the development of treatments that target precise apoptosis genes or pathways Apoptosis is an ordered and arranged cellular function that happens in physiological and pathological conditions. An understanding of the underlying mechanism of apoptosis is central as it plays a crucial role in the pathogenesis of several diseases. In some, The problem is because too much apoptosis occurs in some, like in the case of degenerative diseases, on the other hand, too little apoptosis is the problem. Cancer is one of the cases that too small apoptosis happens, which leads to malignant cells that stay alive. The method of apoptosis is intricate and involves several pathways, resulting in malignant transformation of the affected cells, tumor metastasis and resistance to anticancer drugs. Although it is the basis of problem, apoptosis plays a vital role in the healing of cancer as it is a standard target of many treatment approaches.

In cancer, there is a loss of balance between cell division and cell death and cells that should have died but did not get the signals to die. The setback can happen in any one step along the way of apoptosis. Caspases are essential to the mechanism of apoptosis as they are both the initiators and executioners. There are three pathways that caspases can be activated. The two frequently described initiation pathways are the intrinsic (or mitochondrial) and extrinsic (or death receptor) pathways of apoptosis. However, the two paths ultimately lead to a mutual path or the execution phase of apoptosis. The third less familiar initiation pathway is the intrinsic endoplasmic reticulum pathway.

根據Wong2011)的研究,了解細胞凋亡的機制是至關重要的,有助於理解混沌細胞凋亡所致疾病的發病機理。因此,這可能有助於治療方法的發展,

 靶向精確的凋亡基因或途徑凋亡是在生理和病理條件下發生的有序的細胞功能。了解細胞凋亡的潛在機制至關重要,因為它在幾種疾病的發病機理中起著至關重要的作用。在某些情況下,問題是因為在某些情況下發生了太多的細胞凋亡,例如在退化性疾病的情況下,問題在於細胞凋亡太少。癌症是發生太小的細胞凋亡的情況之一,其導致惡性細胞存活。凋亡的方法是複雜的,涉及多種途徑,導致受影響細胞的惡性轉化,腫瘤轉移和對抗癌藥的耐藥性。儘管它是問題的基礎,但凋亡是癌症治療中的關鍵作用,因為它是許多治療方法的標準靶標

在癌症中,細胞分裂與細胞死亡之間的平衡喪失,本來應該死亡但沒有死亡信號的細胞也失去了平衡。挫折可能發生在細胞凋亡的任何一步。胱天蛋白酶對於細胞凋亡機制是必不可少的,因為它們既是引髮劑又是執行者。可以激活胱天蛋白酶的三種途徑。經常描述的兩個起始途徑是細胞凋亡的內在途徑(或線粒體途徑)和外在途徑(或死亡受體途徑)。然而,這兩種途徑最終導致細胞凋亡的共同途徑或執行階段。第三個不太熟悉的起始途徑是內在的內質網途徑。

The extrinsic death receptor pathway initiates when death ligands fastens to a death receptor. The most popular death receptor is the type 1 TNF receptor (TNFR1), and a related protein named Fas (CD95) and their ligands are called TNF and Fas ligand (FasL) respectively (Hengartner, MO., 2000). These death receptors have an intracellular death domain that enlist adapter proteins like TNF receptor-associated death domain (TRADD) and Fas-associated death domain (fadd), and cysteine proteases such as caspase 8 (Schneider, P., Tschopp, J., 2000). Attaching of the death ligand to the death receptor results in the formation of a binding site for an adaptor protein, and the whole ligand-receptor-adaptor protein complex is referred to as the death-inducing signaling complex (DISC). DISC then starts the assembly and activation of pro-caspase 8. The activated form of the enzyme, caspase 8 is an initiator caspase, and it initiates apoptosis by cleaving other downstream or executioner caspases (Karp, G., 2008).

The intrinsic pathway is initiated in the cell. Internal factors like irreparable hereditary damage, hypoxia, very high levels of cytosolic Ca2, + and intense oxidative stress are some activates of the initiation of the intrinsic mitochondria pathway (Karp, G., 2008). Irrespective of the stimuli, this pathway is caused by the enhanced mitochondria permeability and the release of pro-apoptotic molecules like cytochrome-C into the cytoplasm (Danial, NN., Korsmeyer, SJ., 2004). This pathway is tightly controlled by a group of protein belonging to the Bcl-2 family, named after BCL2 gene initially observed at the chromosomal breakpoint of the translocation of chromosome 18 to 14 in follicular non-Hodgkin lymphoma. There are two key groups of the Bcl-2 proteins, namely the pro­apoptotic proteins (e.g. Bax, Bak, Bad, Bcl-Xs, Bid, Bim, and Hrk) and the anti-apoptotic proteins (e.g. Bcl-2, Bcl-X, Bcl-W, Bfl-1 and Mcl-1). While the anti-apoptotic proteins control apoptosis by preventing the mitochondria release of cytochrome-c, the pro‑apoptotic proteins act by helping such release. Cytoplasmic release of cytochrome-c activates capase-3 via formation of a complex referred to as apoptosome which is consist of cytochrome-c, Apaf-1, and caspase 9 (Kroemer, G., Galluzzi, L., Brenner, C., 2006).

當死亡配體固定在死亡受體上時,外部死亡受體途徑開始。最受歡迎的死亡受體是1TNF受體(TNFR1),名為FasCD95)的相關蛋白及其配體分別稱為TNFFas配體(FasL)。這些死亡受體具有胞內死亡結構域,該結構域需要銜接子蛋白,如TNF受體相關死亡結構域(TRADD)和Fas相關死亡結構域(fadd),以及半胱氨酸蛋白酶,例如caspase 8。死亡配體與死亡受體的連接導致銜接子蛋白的結合位點的形成,整個配體-受體-適配子蛋白複合物被稱為誘導死亡的信號複合物(DISC)。然後DISC開始組裝和激活前胱天蛋白酶8。該酶的活化形式,胱天蛋白酶8是啟動子胱天蛋白酶,並且它通過切割其他下游或死刑半胱天冬酶來啟動細胞凋亡。

固有途徑在細胞中起始。內在因素如不可挽回的遺傳性損傷,缺氧,非常高的胞質Ca2 +水平和強烈的氧化應激是內在線粒體途徑啟動的一些激活因素。與刺激無關,該途徑是由增強的線粒體通透性和促凋亡分子如細胞色素C釋放到細胞質中引起的。該通路由一組屬於Bcl-2家族的蛋白嚴格控制,該蛋白以BCL2基因命名,該蛋白最初在濾泡性非霍奇金淋巴瘤的18號至14號染色體易位的染色體斷裂點觀察到。 Bcl-2蛋白有兩個關鍵的組,即促凋亡蛋白和抗凋亡蛋白。抗凋亡蛋白通過阻止線粒體釋放細胞色素c來控制細胞凋亡,凋亡蛋白通過幫助這種釋放而起作用。細胞色素C的胞質釋放通過形成稱為凋亡小體的複合物來激活capase-3,該複合物由細胞色素CApaf-1和半胱天冬酶9組成。

The execution phase of apoptosis encompasses the activation of a series of caspases. The upstream caspase for the intrinsic pathway is caspase 9, and that of the extrinsic pathway is caspase 8. The intrinsic and extrinsic pathways converge to caspase 3. Caspase-3 then cleaves the inhibitor of the caspase-activated deoxyribonuclease, which is in control for nuclear apoptosis. Plus downstream caspases induce cleavage of protein kinases, cytoskeletal proteins, DNA repair proteins and inhibitory subunits of endonucleases family, which together contribute to the usual morphological changes in apoptosis (Ghobrial, IM., Witzig, TE., Adjei, AA., 2005).

This intrinsic endoplasmic reticulum (ER) pathway is the third pathway and not as popular. It is said to be caspase-12 dependent and mitochondria-independent. When the ER is damaged by cellular stresses such as hypoxia, free radicals or glucose starvation, there is unfolding of proteins and less protein synthesis in the cell, and an adaptor protein referred to as TNF receptor-associated factor 2 (TRAF2) separates from procaspase-12, leading to an activation of the procaspase-12 (O’Brien MA., Kirby, R., 2008).

 

凋亡的執行階段包括一系列胱天蛋白酶的激活。內在途徑的上游胱天蛋白酶是caspase 9,外在途徑的上游胱天蛋白酶是caspase8。內在途徑和外在途徑匯聚到caspase3。然後Caspase-3裂解了caspase活化的脫氧核糖核酸酶的抑製劑,該抑製劑可控制核凋亡。加上下游胱天蛋白酶誘導蛋白激酶,細胞骨架蛋白,DNA修復蛋白和核酸內切酶家族的抑制性亞基的裂解,它們共同導致凋亡中通常的形態變化。

這種內在的內質網(ER)途徑是第三種途徑,而不是普遍的。據說是caspase-12依賴性和線粒體依賴性的。當內質網受到缺氧,自由基或葡萄糖不足等細胞應激的損害時,細胞中的蛋白質就會展開,蛋白質的合成會減少,並且稱為TNF受體相關因子2TRAF2)的銜接子蛋白會與蛋白酶分解-12,導致激活procaspase-12

 

CHAPTER III

MATERIALS AND METHODS

第三章

材料和方法

Preparation of Methanolic Bitter Melon Extract

Several Indian bitter melons were obtained from Dr. Zena Clarda, who grew them at Tennessee State University‘s farm. (Shown in Figure 9) To acquire the bitter melons secondary products for the potential use in the battle against cancer the melon was cut into small pieces; seeds were removed. Then the bitter melon pieces were sent to the Agriculture Department at Tennessee State University to be lyophilized then placed in 50ml tubes and stored in the -80 freezer until extraction. The lyophilized samples were refluxed in methanol, to remove the secondary compounds, and distilled for 8 hours in a soxhlet.(Figure 10) The extract was rotary evaporated (Figure 11)to remove the methanol and yield the crude extract and is herein referred to as bitter melon extract (苦瓜提取物). Stock solutions were prepared by reconstituting evaporated crude extract in the proper volume of dimethyl sulphoxide (DMSO). The DMSO-dissolved extract was then deposited in freeze vials and stored at -200 C until later use.

 

甲醇苦瓜提取物的製備

曾在田納西州立大學農場種植的Zena Clarda博士獲得了幾種印度苦瓜。 (圖9所示)為了獲得苦瓜次級產品以用於抗擊癌症,將瓜子切成小塊;種子被去除。然後將苦瓜塊送至田納西州立大學農業部進行凍乾,然後放入50ml試管中,並保存在-80冰箱中直至提取。將凍乾的樣品在甲醇中回流,以除去次要化合物,然後在索氏提取器中蒸餾8小時。(圖10)將提取物旋轉蒸發(圖11)以除去甲醇,得到粗提取物,在本文中稱為作為苦瓜提取物(苦瓜提取物)。通過在適當體積的二甲基亞砜(DMSO)中重構蒸發的粗提物來製備儲備溶液。然後將溶解了DMSO的提取物沉積在冷凍小瓶中,並保存在-200 C直至以後使用。

 

Figure 9: Indian Bitter Melon

 

Figure 11: Rotary Evaporator

Serial Dilution

Before the bitter melon extract being applied to the cancer cell lines, the

extract was aliquoted (serial diluted) into desired concentrations. A serial dilution was

pat

performed by a factor of 50%. This results in a geometric progression of the masses in a

a      c

vaporation

logarithmic fashion. The crude extract was serial diluted by 0.5 ml in DMSO. The dilutions ranged from 1 to 1/64.

Dimethyl Sulfoxide

Dimethyl sulfoxide (DMSO) was purchased from Sigma Aldrich, and it was used as the control in the experiments. DMSO is an organosulfur compound with the molecular formula (CH3) 2SO. DMSO is a colorless clear hygroscopic liquid that is an important polar aprotic solvent that dissolves both polar and nonpolar compounds and is miscible in a wide range of organic solvents as well as water.

 

連續稀釋

在將苦瓜提取物應用於癌細胞系之前,將提取物等分(稀釋)至所需濃度。 連續稀釋為效果提高了50%。將粗提取物在DMSO中系列稀釋0.5ml。 稀釋範圍為11/64

二甲基亞砜

二甲基亞砜(DMSO)購自Sigma Aldrich,用作實驗中的對照。 DMSO是一種有機硫化合物,具有分子式(CH32SODMSO是一種無色透明的吸濕性液體,是一種重要的極性非質子溶劑,可溶解極性和非極性化合物,並且可與多種有機溶劑以及水混溶。

Cell Culture

Cytotoxicity and antiproliferative activity were evaluated by using Human cell based screening systems. American Type Culture Collection (Rockville, MD) provided various types of cancer cell lines; Table 1 represents the three different cell lines used in the experiments. The Cells were preserved in F12K supplemented with 10% heat-inactivated FBS, 2mM L-glutamine, and 1% penicillin-streptomycin, with the exception of a low glucose variant (Gibco) having 2mM L-glutamine, non-essential amino acid, penicillin-streptomycin, 10% fetal calf serum (Atlanta Biologicals) supplemented with 0.01 mg/ml insulin and 1mM sodium pyruvate. Cells were grown at 37 degrees C in a 5% CO2 humidified incubator. After reaching 80% confluence the cells were passed biweekly in 1:2/1:4 ratios for less than 40 passages.

 

細胞培養

通過使用基於人類細胞的篩選系統評估細胞毒性和抗增殖活性。美國典型培養物保藏中心(美國典型培養物保藏中心)提供了多種類型的癌細胞系。 表1表示實驗中使用的三種不同細胞系。 將細胞保存在補充有10%熱滅活的FBS2mM L-谷氨酰胺和1%青黴素-鏈黴素的F12K中,但低糖變體(Gibco)除外,其中含有2mM L-谷氨酰胺,非必需氨基酸, 青黴素-鏈黴素,10%胎牛血清(Atlanta Biologicals)補充0.01 mg / ml胰島素和1mM丙酮酸鈉。 細胞在37°C5CO2濕潤培養箱中生長。 達到80%匯合後,細胞每兩週以12/14的比例通過少於40次傳代。

 

 

Figure 12: Cancer Cell Lines

  • A549 was developed in 1972 by D.J. Giard, it is lung tissue of a 58 year old Caucasian male. The cells produced were adenocarcinomic alveolar basal epithelial cells.
  • BT549 was developed in 1978 by W.G. Coutinho and EY Lasfargues, it is mammary gland, breast tissue from a 72 year old Caucasian female. They are epithelial cells.
  • PC3 (PC-3) human prostate cancer cell line was established in 1979 from bone metastasis of grade 4 of prostate cancer in a 62-year-old Caucasian male.

 

Cell Viability

To measure the cell viability and cytotoxic effects the integrity of the cells membrane has to be evaluated. Viability was decided using the trypan blue exclusion method. Cells were plated in 12 and 24 well tissue culture plates at about 1 × 10 cells per wells and in 2 ml of medium in the 12 well plate and 1 × 10 cells per wells and in 1 ml of medium in the 24 well plate, then incubated at 37 degrees C and 5% CO2. The medium was removed after 24 hours, and phosphate-buffered saline was used to wash the cells. Then the cells were treated for 2-3 minutes at 37 degrees with a solution of 0.25% trypsin-EDTA. The live cells were counted using a hemocytometer after being stained with trypan blue. Then the cells were diluted to ten thousand cells per 20tl.

A549D.J.賈德(Giard),是58歲的白人男性的肺組織。產生的細胞是腺癌的肺泡基底上皮細胞。

BT549W.G. CoutinhoEY Lasfargues1978年開發,它是一種乳腺癌,來自72歲的白人女性的乳房組織。它們是上皮細胞。

PC3PC-3)人攝護腺癌細胞係於1979年從一名62歲的白人男性中4級攝護腺癌的骨轉移中建立。

細胞活力

為了測量細胞活力和細胞毒性作用,必須評估細胞膜的完整性。使用錐蟲藍排除法確定生存力。將細胞以每孔約1×10個細胞,12孔板中2 ml的培養基,每孔1×10細胞和24孔板中的1 ml培養基接種到1224孔組織培養板中。在37°C5CO2下孵育。 24小時後除去培養基,並用磷酸鹽緩衝鹽水洗滌細胞。然後將細胞用0.25%胰蛋白酶-EDTA溶液在37度下處理2-3分鐘。用台盼藍染色後,使用血細胞計數器對活細胞計數。然後將細胞稀釋至每20tl一萬個細胞。

Alamar Blue

Once the cells were treated with bitter melon extract, Alamar Blue is the reagent used to estimate the number of viable cells. Alamar Blue is designed to provide a rapid and sensitive way to measure cell proliferation and cytotoxicity in various human cell lines, bacteria, and fungi. A reducing environment is sustained in the cytosol of cells that are alive. The active ingredient in Alamar Blue is resazurin. Resazurin is a non-toxic, cell-permeable compound that is a weakly fluorescent blue dye until it enters the cells, then resazurin is irreversibly reduced by the metabolic activity of the cell to resorufin, which is pink colored and highly fluorescent.Experimental Procedure

First label 2 sets of nine 1.5ml tubes 0 thru 6 and label one tube B for the blank and another tube D for DMSO the control. Secondly, the tubes labeled B for blank add 180 tL of media and 20 μl of Alamar Blue. Thirdly, add 15 tls of DMSO to the tubes labeled D, and add 15 μl from each of the concentrations to both sets of its corresponding 1.5 ml tube. Vortex the DMSO and each concentration the moment before pipetting the 15 μl. Fourthly, to one tube labeled D and one set of the tubes labeled 0-6 add 465 μl of media and 60 μl of Alamar Blue and 60 μl of the cancer cell line in that order. Then to the second tube labeled D and the second set of tubes labeled 0-6 add 565 μl of media and 60 μl of Alamar Blue. Each 1.5 ml tubes must be individually pipetted up and down 7 times just prior to putting the mixture in the 96 well plate. All of the tubes except the blank are triplicated by adding one tube of mixture to 3 different wells. To wells A1 and F1 add

190 μl from the tubes labeled B, and to the wells B1, B2 and B3 add 190 µl from the tube labeled D with the cancer cells in them. The mixtures are added to the wells so the extract concentrations are added from the weakest to the strongest. Thus, to wells A3, B3 and C3 add 190 µl from the tube labeled 6 with the cancer cells, and to wells A4, B4 and C4 add 190 µl from the tube labeled 5. Then repeat this until all the concentrations with the cancer cells are in the wells in descending order, and continue this same procedure to the bottom three rolls with the tubes that do not have cancer cells in them. This procedure was performed in a sterile environment, (under the hood). After 24 hours in a CO2 incubator at 370 C the sample is read on a fluorescent spectrophotometer. Resazurin is blue and weakly fluorescent. Resorufin is pink and highly fluorescent

 

阿拉瑪藍

用苦瓜提取物處理細胞後,Alamar Blue是用於估計活細胞數量的試劑。 Alamar Blue旨在提供一種快速而靈敏的方法來測量各種人類細胞系,細菌和真菌中的細胞增殖和細胞毒性。還原環境在活細胞的細胞質中得以維持。 Alamar Blue中的活性成分是刃天青。刃天青是無毒的,細胞可滲透的化合物,在進入細胞之前是一種弱熒光的藍色染料,然後細胞的代謝活性將其變成粉紅色且熒光強的刃天青不可逆地還原刃天青。

 

實驗程序

首先標記2組,每組91.5ml的試管06,將一個試管B標記為空白,將另一個試管D標記為DMSO作為對照。其次,標有B的空白管中加入180 tL培養基和20μlAlamar Blue。第三,在標有D的試管中加入15 tls DMSO,並將每種濃度的15μl加入相應的1.5 ml管中。將DMSO和每種濃度渦旋移液至15μl之前。第四,依次向一個標有D的試管和一組標有0-6的試管中添加465μl培養基,60μlAlamar Blue60μl癌細胞系。然後向標有D的第二支試管和標有0-6的第二支試管中添加565μl培養基和60μlAlamar Blue。在將混合物放入96孔板之前,必須分別將每個1.5 ml試管分別上下吸移7次。通過將一管混合物添加到3個不同的孔中,將除空白管外的所有管一式三份。向A1F1井添加

從標有B的管中取出190μl,並向B1B2B3孔中添加標有D的管中含有癌細胞的190 µl。將混合物添加到孔中,以便從最弱到最強添加提取物濃度。因此,向A3B3C3孔中添加190 µl標有癌細胞的管,向A4B4C4孔中添加190 µl標有5的管。然後重複此操作,直到所有濃度的癌細胞按降序排列在孔中,並使用底部沒有癌細胞的試管繼續進行相同的操作,直到底部三卷。該程序在無菌環境下(引擎蓋下)進行。在370°CCO2培養箱中放置24小時後,在熒光分光光度計上讀取樣品。刃天青為藍色且熒光較弱。試鹵靈為粉紅色且高度熒光

 

 

Figure 13: Resazurin is reduced to Resorufin

Statistical Analysis

The results are expressed as the mean ± standard deviation (SD). One-way ANOVA analysis was applied to determine the statistically difference and Stat View 5.0.1. (SAS Institute Inc., Cary, N). P < 0.05 was regarded as statistically significant.

Lipid Peroxidation Assay

Lipid peroxidation assay is used to determine the levels of malondialdehyde (MDA). The formation of lipid peroxidation products leads to the spread of free radical reactions which leads to cell damage.

The cells were plated in 6 well plates for 4-5 days until the plated wells were70- 80% confluent. Then the media was removed, DMSO was added to the first two wells as the control, and concentrations 0 and 2 of the Bitter Melon extract were added to the other four wells in serum free media. After 24 hours of incubation the media was removed and the wells were rinsed in 1ml of 1x PBS. Then 600 µl of trypsin was added and the plate was incubated for 3 minutes. Then scrape all the cells and put in micro centrifuge tubes and add 600 µl of media and centrifuge them for 10 minutes. Discard the supernatant and store the pellet in the -80 freezer until use.

MDA Assay

The kit for the MDA assay and Bradford assay was provided by MD Biosciences inc. Remove the pellets from the freezer and put them in a bucket of ice. Add 110 µl of 1x PBS to the pellets and sonicate the pellets on the sonic dismembrator while on ice in a 100 ml beaker. Divide the solution in half by putting 50 μl of the sample in 1.5 ml tubes for the MDA assay and 50 μl in another set of tubes for the Bradford assay.

First turn the water bath on and set the temperature for 500. Then make the R1 by adding 6 ml of ferric ion/methanol to 18 ml of R1, then add 163 μl of R1 to each sample tube, then add 37.5 μl of HCl to each sample and tape the lid after closing it, and vortex each tube. Then put the tubes in the water bath at 500 for 1 hour. From each sample put 200 μl in 96 well plate and put the 96 well plate in the Microplate Reader (Filter Max) for reading. Set the absorbance wavelength at 586 nm.

統計分析

結果表示為平均值±標準偏差(SD)。應用單因素方差分析確定統計差異,並使用Stat View 5.0.1。 (SAS Institute Inc.,卡里,N)。 P <0.05被認為具有統計學意義。

脂質過氧化測定

脂質過氧化測定法用於測定丙二醛(MDA)的水平。脂質過氧化產物的形成導致自由基反應的擴散,從而導致細胞損傷。

將細胞鋪在6孔板中4-5天,直到鋪板的孔匯合70-80%。然後移出培養基,將DMSO加到前兩個孔中作為對照,然後將濃度為02的苦瓜提取物添加到培養基中。

 其他四孔在無血清培養基中。溫育24小時後,除去培養基,並將孔用1ml 1x PBS沖洗。然後加入600μl的胰蛋白酶,並將板孵育3分鐘。然後刮去所有細胞,放入微量離心管中,加入600 µl培養基,離心10分鐘。丟棄上清液,並將沉澱物儲存在-80冰箱中直至使用。

MDA分析

MDA分析和Bradford分析的試劑盒由MD Biosciences inc提供。從冰箱中取出顆粒,並將其放入冰桶中。向沉澱物中加入110 µl 1x PBS,然後在100 ml燒杯中的冰上用超聲分散器對沉澱進行超聲處理。將50μl樣品放入1.5 ml試管中進行MDA分析,將50μl放入另一組試管中進行Bradford分析,將溶液分成兩半。

首先打開水浴並將溫度設置為500。然後通過向18 ml R1中添加6 ml鐵離子/甲醇來製成R1,然後向每個樣品管中添加163μlR1,然後向其中添加37.5μlHCl。將每個樣品蓋好後蓋上膠帶,並渦旋每個試管。然後將試管在500的水浴中放置1小時。從每個樣品中,將200μl放入96孔板中,然後將96孔板放入微孔板讀取器(Filter Max)中進行讀取。將吸收波長設置為586 nm

Bradford Assay

Add 250 μl of Coomassie Plus (Bradford reagent) to the samples and vortex the tubes. After incubating the sample tubes at for 30 minutes at room temperature (37°) add 200 μl of sample to 96 well plate and read on the Filter Max Microplate Reader. The absorbance wavelength is read at 620 nm. Chemical Preparation R1 preparation. Add 6 ml of ferric ion/methanol to 18 ml of reagent R1 (N-methyl-2-phenylindole)

BSA 5% Preparation.

Add 5 g of bovine serum albumin (BSA) to 100 ml of water and mix well until all the BSA has dissolved.

Washing buffer preparation.

Add 1,680 μl of 20X washing buffer to 31,920 μl of distilled water to make the

1X washing buffer.

Substrate solution.

Add 2,350 of reagent A to 2,350 μl of reagent B and mix well.

Primary antibody (Rabbit).

Dilution of the primary antibody is 1: 1,00, add 4.7 μl of primary antibody to 4,700 μl of PBS.

Secondary antibody (Anti Rabbit).

Dilute the secondary 1: 5,000

 

統計分析

結果表示為平均值±標準偏差(SD)。應用單因素方差分析確定統計差異,並使用Stat View 5.0.1。 (SAS Institute Inc.,卡里,N)。 P <0.05被認為具有統計學意義。

脂質過氧化測定

脂質過氧化測定法用於測定丙二醛(MDA)的水平。脂質過氧化產物的形成導致自由基反應的擴散,從而導致細胞損傷。

將細胞鋪在6孔板中4-5天,直到鋪板的孔匯合70-80%。然後移出培養基,將DMSO加到前兩個孔中作為對照,然後將濃度為02的苦瓜提取物添加到培養基中。

 

其他四孔在無血清培養基中。溫育24小時後,除去培養基,並將孔用1ml 1x PBS沖洗。然後加入600μl的胰蛋白酶,並將板孵育3分鐘。然後刮擦所有細胞,並進行微量Bradford分析

向樣品中加入250μlCoomassie PlusBradford試劑)並渦旋離心管。在室溫(37°)下於30℃孵育樣品管30分鐘後,將200μl樣品加到96孔板中,並在Filter Max Microplate Reader上讀取。在620nm處讀取吸收波長。

化學製劑R1製劑。向18 ml試劑R1N-甲基-2-甲基)中加入6 ml鐵離子/甲醇

苯基吲哚)

BSA 5%的製備。

5 g牛血清白蛋白(BSA)加入100 ml水中,並充分混合直至所有BSA溶解。

洗滌緩衝液的製備。

1,680μl20X洗滌緩衝液添加到31,920μl蒸餾水中以使1X洗滌緩衝液。

底物溶液。

2,350試劑A添加到2,350μl試劑B中並充分混合。

一抗(兔)。

一抗的稀釋度為11,將4.7μl一抗添加到4,700μlPBS中。

二抗(Anti Rabbit)。

稀釋次要15,000

Caspase 3 Assay

Apoptotic effect.

Apoptosis is a genetically programmed cell death mechanism that can be activated by various stimuli. We want to see if bitter melon extract will activate apoptosis in the three cancer cell lines. A serial dilution of the bitter melon extract was performed. Label six 15 ml tubes 1-6 and add 150 μl of DMSO to each tube. To tube labeled 1 add 850 μl of Bitter Melon crude extract. Then take 850 μl from the tube labeled 1 and add it to tube labeled 2, then take 850 μl from the tube labeled 2 and add it to the tube labeled 3. Then take 850 μl from the tube labeled 3 and add it to tube labeled 4. Repeat this process for tubes 5 and 6.

The cells were plated in 6 well plates for 4-5 days or until the cells were 70-80% confluent. The media was removed from the 6 well plates and DMSO (The control) was added and the six increasing concentrations were added in serum free media wells. After 48 hours of incubation the wells were rinsed in 1X PBS. Then they were trypsinized by adding 600 μl of trypsin and 600 μl of media, and centrifuged for 10 minutes, and discard the supernatant and save the pellet. Add 60 μl of lysate buffer (cell lysis reagent provided by Sigma-Aldrich, St Louis, MO, USA), and vortex. Then triplicate the sample add 20 μl from each tube of the concentrations to 3 different wells in a 96 well plate. Add 180 μl of 5% BSA (Blocking solution), then wrap in cellophane and incubate at 40 C for 24 hours.

The protein is then probed with a combination of antibodies: one antibody is specific to the protein of interest (primary antibody), and another antibody specific to the host species of the primary antibody (secondary antibody). The secondary antibody is complexed with an enzyme that is combined with a substrate that will produce a detectable signal.

半胱天冬酶3測定

凋亡作用。

凋亡是一種基因編程的細胞死亡機制,可以被各種刺激激活。我們想看看苦瓜提取物是否會激活三種癌細胞系的凋亡。進行苦瓜提取物的系列稀釋。標記六個15 ml試管1-6,然後向每個試管中加入150μlDMSO。向標有1的試管中加入850μl苦瓜粗提取物。然後從標記為1的試管中取出850μl,然後將其添加到標記為2的試管中,然後從標記為2的試管中取出850μl,並將其添加到標有3的試管中。 4.對管56重複此過程。

將細胞鋪在6孔板中4-5天或直至細胞匯合70-80%。從6孔板中移出培養基,並在無血清的培養基孔中加入DMSO(對照),並添加六種遞增濃度。孵育48小時後,將孔用1X PBS沖洗。然後通過添加600μl胰蛋白酶和600μl培養基對它們進行胰蛋白酶消化,離心10分鐘,棄去上清液並保存沉澱。加入60μl裂解液緩衝液(細胞裂解試劑,由美國密蘇里州聖路易斯的Sigma-Aldrich提供),並渦旋振盪。然後將樣品一式三份從每個濃度的管中添加20μl96孔板中的3個不同的孔中。加入180μl5BSA(封閉溶液),然後包裹在玻璃紙中,並在40°C下孵育24小時。

然後用抗體組合探測蛋白質:一種抗體對目標蛋白質具有特異性(一抗),另一種抗體對特定蛋白質具有特異性

一抗的宿主物種(二抗)。二抗與酶複合,酶與會產生可檢測信號的底物結合。

Wash the plates by adding 300 μl of 1X washing buffer and smack the 96 well plate on paper towels to remove all of the washing buffer. The caspase-3 primary was provided by the Cell Signal Company. Add 100 μl of the diluted caspase-3 rabbit primary to each well, and wrap in cellophane and store in 40 C for 24 hours.

Discard and wash the plate by adding 300 μl of 1X washing buffer and smack on paper towels to remove all of the washing solution. The secondary antibody was also provided by the Cell Signaling Company. To each well add 100 μl of the diluted secondary which is anti-rabbit HRP-linked antibody, and incubate for one hour at 370 C or room temperature. Discard and wash with 300 μl of 1X washing buffer. Then add 100 μl of substrate Reagent A and B to each well and wrap in aluminum foil and incubate for 30 minutes in the dark at 370 C. Add 50 μl of stop solution to each well. Read the plate on the microplate reader (Filter Max), the absorbance was read at wavelength 450 nm.

加入300μl1X洗滌緩衝液洗滌平板,然後在紙巾上敲打96孔板以去除所有洗滌緩衝液。 caspase-3的主要成分是由Cell Signal Company提供的。 向每個孔中添加100μl稀釋的caspase-3兔原代培養液,並包裹在玻璃紙中,並在40°C下儲存24小時。

通過加入300μl1X洗滌緩衝液棄去並洗滌板,並在紙巾上敲打以去除所有洗滌液。 二抗也由Cell Signaling Company提供。 向每個孔中添加100μl的稀釋後的抗兔HRP聯結抗體,然後在370℃或室溫下孵育1小時。 丟棄並用300μl1X洗滌緩衝液洗滌。 然後向每個孔中添加100μl底物試劑AB,並包裹在鋁箔中,並在370°C的黑暗環境中孵育30分鐘。向每個孔中添加50μl終止溶液。 在酶標儀上讀取板(最大濾光片),在波長450nm 處讀取吸光度CHAPTER IV

RESULTS

第四章

結果

The Effect of 苦瓜提取物 on the Proliferation of Cancer Cell Line BT549

The cytotoxicity of the Bitter Melon extract was determined on the breast cancer cell line BT549 and the lung cancer cell line A549 as well as the prostate cancer cell line PC3. Using the cell viability assay the proliferation was evaluated. In order to study the effect of cell viability in a dose-response manner, the three cancer cell lines were incubated with various concentrations of BMW (0.079, 0.157, 0.315, 0.629, 1.26, 2.52, and 5.03 μg/ml) for 24 to 36 hours in 96 well plates. The dose response experiments were executed in triplets with cancer cells treated with 苦瓜提取物 and the analyses revealed that Bitter Melon has therapeutic potential in treatment of the three cancer cell lines.

The effect of 苦瓜提取物 on the proliferation of breast cancer cell line BT549

In breast cancer cell line BT549 the lowest concentration of 苦瓜提取物 extract (0.079μg/ml) stimulated proliferation by about 6% (Figure 13) and the second lowest (0.157 μg/ml) increased cell viability nearly 22%, while on the contrary the next three lowest concentrations (0.313, 0.629 and 1.26 μg/ml) resulted in the decrease of cell viability with the increase in concentration approximately 6%, 61% and 90% respectively. In the second highest concentration (2.52 μg/ml) around 98% of the cancer cells died, while the highest concentration (5.03 μg/ml) resulted in 100% cell death. These results were compared to the cells treated with DMSO, which was the control in the experiment (and no 苦瓜提取物).

 

苦瓜提取物對癌細胞系BT549增殖的影響

確定苦瓜提取物對乳腺癌細胞系BT549和肺癌細胞系A549以及攝護腺癌細胞系PC3的細胞毒性。使用細胞活力測定法評估增殖。為了研究細胞存活率的劑量效應,將三種癌細胞係與各種濃度的BMW0.0790.1570.3150.6291.262.525.03μg/ ml)孵育24至在96孔板中放置36小時。劑量反應實驗是在三聯體中用苦瓜提取物處理過的癌細胞進行的,分析表明,苦瓜具有治療三種癌細胞系的治療潛力。

苦瓜提取物對乳腺癌細胞BT549增殖的影響

在乳腺癌細胞BT549中,最低濃度的苦瓜提取物提取物(0.079μg/ ml)刺激了6%的增殖(圖13),第二低濃度的(0.157μg/ ml)則使細胞活力提高了近22%,而相反接下來的三個最低濃度(0.3130.6291.26μg/ ml)導致細胞活力降低,濃度分別升高約6%,61%和90%。在第二高濃度(2.52μg/ ml)中,約98%的癌細胞死亡,而最高濃度(5.03μg/ ml)則導致100%細胞死亡。將這些結果與用DMSO處理的細胞進行比較,後者是實驗中的對照(無苦瓜提取物)。

 

 

 

 

Control 0.079            0.157              0.315              0.629         1.26                 2.52         5.03

Treatment μg/ml

Percent Viability

文字方塊: Percent Viability

160

140

120

100

40

20

80

60

0

Ave

A

A

AB

AB

B

B

_______ A_____

A

Figure 14: Viability Analysis of Breast Cancer Cell Line BT549 After 24 hr Exposure to 苦瓜提取物.

 

14:暴露於苦瓜提取物 24小時後的乳腺癌細胞BT549的生存力分析。

The effect of 苦瓜提取物 on the Proliferation of Lung Cancer Cell Line A549

In the lung cancer cell line A549 proliferation increased approximately 15% and 21% in the two lowest concentrations 0.079 and 0.157 μg/ml respectively (Figure 13). However, in the next concentrations 0.315, 0.629 and 1.26 the proliferation decreased with increasing 苦瓜提取物 concentrations roughly 11%, 51%, 65%, respectively and in the two highest concentrations 2.52 and 5.03 μg/ml there was significant decrease in the proliferation of nearly 98% and 100% respectively. The results were compared to the cells treated with DMSO which was used as the control in the experiments (And no 苦瓜提取物).

 

 

 

 

苦瓜提取物對肺癌細胞A549增殖的影響

在肺癌細胞系A549,兩種最低濃度0.0790.157μg/ ml的增殖分別增加了約15%和21%(圖13)。

然而,在接下來的濃度0.3150.6291.26中,增殖隨著苦瓜提取物濃度的增加而分別降低,分別約為11%,51%,65%,在兩個最高濃度2.525.03μg/ ml中,分別為98%和100%。 將結果與用DMSO處理的細胞進行比較,該細胞在實驗中用作對照(無苦瓜提取物)。


 

 

 

Control              0.079        0.157        0.315               0.629          1.26                  2.52                5.03

Treatmnts (μg/ml)

percent Viability

文字方塊: percent Viability

160

140

120

100

40

80

60

20

0

A_

A

A

AB

BC

C

D           D

Ave

Figure 15: Viability Analysis of Lung Cancer Cell Line A549 After 24 hr Exposure to 苦瓜提取物.

 

15:暴露於苦瓜提取物 24小時後的肺癌細胞A549的生存力分析

The effect of 苦瓜提取物 on the Proliferation of Prostate Cancer Cell Line PC3

In prostate cancer cell line PC3 the lowest three concentrations 0.079, 0.157 and 0.315 μg/ml there was a increase in the proliferation by approximately 41%, 33% and 28% respectively. Conversely, in the higher concentrations 0.629, 1.26, 2.52 and 5.03 μg/ml the proliferation decreased by roughly 5%, 27%, 79% and 94% respectively. The results were compared to the cells treated with DMSO, which was used as the control in the experiments. The control was not treated with 苦瓜提取物.

 

 

 

 

苦瓜提取物對攝護腺癌細胞PC3增殖的影響

在攝護腺癌細胞系PC3中,最低的三個濃度0.0790.1570.315μg/ ml,增殖分別增加了約41%,33%和28%。 相反,在0.6291.262.525.03μg/ ml的較高濃度下,增殖分別降低了約5%,27%,79%和94。 將結果與用DMSO處理的細胞進行比較,該細胞在實驗中用作對照。 對照未用苦瓜提取物治療。

 

 

 

Control 0.079 0.157 0.315 0.629        1.26              2.52            5.03

Treatments (μg/ml)

Percent Viability

文字方塊: Percent Viability

160

140

120

100

40

80

60

20

0

Ave

AB

A_

AB          AB

ABC

ABC

BC__

C

Figure16: Viability Analysis of Prostate cancer cell line PC3 After 24 hr Exposure to 苦瓜提取物.

 

16:暴露於苦瓜提取物 24小時後的攝護腺癌細胞系PC3的活力分析

The effects of 苦瓜提取物 on Lipid Peroxidase on Three Cancer Cell Lines, BT549 (breast), A549 (lung) and PC3 (prostate)

Reactive oxygen species (ROS) are a normal product of cellular function. However, excessive amounts of reactive oxygen species can be harmful and leads to the formation of reactive carbonyl compounds that are most abundant in malondialdehyde. MDA is the end product of lipid peroxidation and it is reported as being the highest in cancer cells. Consequently the measurement of the malondialdehyde (MDA) is used as an indicator of lipid peroxidation and to detect oxidative change. In these experiments we

assessed the effects 苦瓜提取物 has on the cells ability to assemble lipid peroxidase, which is measured by the production of cellular melondialdehyde.

The effect of 苦瓜提取物 on the MDA levels in the BT549 breast cancer cell line.

The cells were plated at 1 X 104 and then treated with 苦瓜提取物 concentrations of 3.43 and 5.03 μg/ml for 24 hours. The measurement of the production of MDA in the cancer cell line BT549 is shown in figure 15. We found that the 苦瓜提取物 reduced the MDA levels in BT549 cancer cell line. The results were compared to the control (DMSO) that had MDA levels of 5.09 μM/mg. The MDA levels somewhat decreased in lower concentration of 3.43 μg/ml to 4.88 μM/mg, however, in the concentration 5.03 μg/ml there was a more meaningful decrease to 1.72 μM/mg.

苦瓜提取物對三種癌細胞系BT549(乳腺),A549(肺)和PC3(攝護腺)的脂質過氧化物酶的影響

活性氧(ROS)是細胞功能的正常產物。但是,過量的活性氧可能是有害的,並導致形成丙二醛中含量最高的活性羰基化合物。 MDA是脂質過氧化作用的終產物,據報導它是癌細胞中最高的。因此,丙二醛(MDA)的測量可用作脂質過氧化的指標並檢測氧化變化。在這些實驗中,我們

 

評估了苦瓜提取物對細胞組裝脂質過氧化物酶能力的影響,脂質過氧化物酶是通過細胞中乙二醛的產生來衡量的。

苦瓜提取物對BT549乳腺癌細胞系中MDA水平的影響。

將細胞以1 X 104鋪板,然後用3.435.03μg/ ml的苦瓜提取物濃度處理24小時。癌細胞系BT549MDA產生的測量結果如圖15所示。我們發現苦瓜提取物降低了BT549癌細胞系中MDA的水平。將結果與MDA水平為5.09μM/ mg的對照(DMSO)進行比較。在3.43μg/ ml的較低濃度下,MDA含量略微降低至4.88μM/ mg,但是,在5.03μg/ ml的濃度下,MDA含量更有意義地降低至1.72μM/ mg

 

 

 

 

4

MD& (IIMkng)

文字方塊: 4
MD& (IIMkng)

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0

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Control                                                                              2.51                                                                              5.03

Treatment (μg/ml)

A

A

B

Series1

Figure 17: The Effect of 苦瓜提取物 on MDA level in BT549 cancer cell line

 

 

17:苦瓜提取物對BT549癌細胞系MDA水平的影響

 

 

The Effect of Bitter Melon Extract on MDA level in A549 cancer cell
line

Control          2.51                                                                                                                       5.03

Treatments (μg/ml)

 

A_

12

MDA (gm/Mg)

文字方塊: MDA (gm/Mg)

10

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A549

Figure 18: The effect of 苦瓜提取物 on the MDA levels in the A549 cancer cell line.

 

18:苦瓜提取物對A549癌細胞系中MDA水平的影響

The Effect of 苦瓜提取物 on the MDA Levels in the A549 Lung Cancer Cell Line.

The cells were plated at 1 X 104, then treated with 苦瓜提取物 concentrations of 3.43 and 5.03 μg/ml for 24 hours. The measurement of the production of MDA in the A549 cancer cell line is shown in figure 16, and we found that the 苦瓜提取物 reduced the MDA levels in the A549 cancer cell line. The results were compared to the control (DMSO) that had MDA levels of 10.63 μM/mg. The MDA levels decreased to 6.90 μM/mg in the lower concentration of 2.51 μg/ml, and reduced even further to 5.82 μM/mg in the highest concentration of 5.03 μg/ml.

苦瓜提取物對A549肺癌細胞系MDA水平的影響。

將細胞以1 X 104鋪板,然後用3.435.03μg/ ml的苦瓜提取物濃度處理24小時。 A549癌細胞系中MDA產生的測量結果如圖16所示,我們發現苦瓜提取物降低了A549癌細胞系中MDA的水平。 將結果與MDA水平為10.63μM/ mg的對照(DMSO)進行比較。 在2.51μg/ ml的較低濃度下,MDA含量降至6.90μM/ mg,在5.03μg/ ml的最高濃度下,MDA含量進一步降至5.82μM/ mg

 

 

The Effect of 苦瓜提取物 on the MDA Levels in the PC3 Prostate Cancer Cell Lines.

The cells were plated at 1 X 104 then the cells were treated with the following concentrations of 苦瓜提取物 (3.43 and 5.03 μg/ml) for 24 hours. The measurement of the production of MDA in the cancer cell line A549 is shown in figure 16. The results were compared to the control (DMSO) which had no 苦瓜提取物 and MDA levels of 5.41 μM/mg. There was a slight decrease in the MDA levels of concentration 2.51 μg/ml to 5.06 μM/mg, however, the concentration 5.03 μg/ml the MDA level decreased significantly to

2. 苦瓜提取物對PC3攝護腺癌細胞系MDA水平的影響。

將細胞以1 X 104鋪板,然後用以下濃度的苦瓜提取物(3.435.03μg/ ml)處理細胞24小時。 癌細胞系A549MDA產生的測量結果如圖16所示。將結果與不含苦瓜提取物和MDA水平為5.41μM/ mg的對照(DMSO)進行了比較。 MDA濃度從2.51μg/ ml輕微降低至5.06μM/ mg,而MDA濃度5.03μg/ ml則顯著降低至2.86μM/ mg86 μM/mg.

 

 

 

 

 

 

7

A_

CONTROL                        2.51                                            5.03

MDA (gm/Mg

4

0

6

5

3

2

1

B

C

Ave

文字方塊: MDA (gm/Mg

 

 

The Effect of Bitter Melon Extract on MDA level in PC3 cancer cell

Line Treatment (μg/ml)

 

The Assessment of the Activation of Caspase-3 by 苦瓜提取物 on the Three Human Cancer Cell Lines of BT549 (breast), A549 (lung) and PC3 (prostate)

In these experiments we determined whether the cancer cells exposed to bitter melon extract would activate Caspase-3, and it was assessed using the enzyme-linked immunosorbent assay (ELISA) using anti-caspase-3. Cancer cells from the three human cancer cell lines, BT549, A549 and PC3, were treated with methanolic extract of bitter melon and DMSO as the control. There was a significant increase in the apoptosis levels in all the analyzed cancer cell lines.

Analysis of the activation of caspase-3 in the BT549 cancer cell line

In the breast cancer cell line BT549 the results from the treatment of 苦瓜提取物 with the cancer cells revealed a substantial gradual increase in the apoptosis levels in the cells with the higher concentrations of 3.52 µg/ml, 4.03 µg/ml, 4.35 µg/ml and 5.03 µg/ml when compared to the cells treated with DMSO which was the control (Figure 18). However, there was not a significant increase observed in apoptosis levels in the lower concentrations of 2.52 µg/ml and 3.02 µg/ml.

Analysis of caspase-3 activation on A549 lung cancer cell line.

In the lung cancer cell line A549 the results from the treatment of 苦瓜提取物 with the cancer cells show a gradual increase in the apoptosis levels in the cells with the highest concentrations of 3.93, 4.53 and 5.03 μg/ml, when compared to the cells treated with DMSO which was the control figure 19. However, in the lowest concentration of 2.42 μg/ml, there was not an increase in the apoptosis levels

 

苦瓜提取物對BT549(乳腺),A549(肺)和PC3(攝護腺)這三種人類癌細胞系激活Caspase-3的評估

在這些實驗中,我們確定了暴露於苦瓜提取物的癌細胞是否會激活Caspase-3,並使用酶聯免疫吸附測定(ELISA)和抗caspase-3對其進行了評估。用苦瓜的甲醇提取物和DMSO作為對照處理來自三種人類癌細胞系BT549A549PC3的癌細胞。在所有分析的癌細胞系中,凋亡水平均顯著增加。

BT549癌細胞系中caspase-3活化的分析

在乳腺癌細胞系BT549中,用癌細胞處理苦瓜提取物的結果表明,較高濃度的3.52 µg / ml4.03 µg / ml4.35 µg / ml0.56 µg / ml的細胞凋亡水平顯著增加。與以DMSO處理的細胞(作為對照)相比,為5.03 µg / ml(圖18)。然而,在較低濃度的2.52 µg / ml3.02 µg / ml下,凋亡水平沒有顯著增加。

caspase-3A549肺癌細胞系中的活化分析。

在肺癌細胞系A549中,用苦瓜提取物處理癌細胞後的結果顯示,與用BMC處理的細胞相比,最高濃度為3.934.535.03μg/ ml的細胞凋亡水平逐漸增加。 DMSO是對照圖19。但是,在最低濃度2.42μg/ ml下,細胞凋亡水平沒有增加

 

 

 

 

Control         2.52                    3.02                    3.52                    4.03                    4.53                  5.03

Treatment (μg/ml)

Caspase-3 Activity

文字方塊: Caspase-3 Activity

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

A

C            C             C

C

B

AVE

B

Figure 20: The Effect of 苦瓜提取物 on the Caspase-3 Activity in BT549 Cancer Cell Line.

 

 

20:苦瓜提取物對BT549癌細胞系中Caspase-3活性的影響。

 

 

Caspase-3 Activity

文字方塊: Caspase-3 Activity

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0

Control                 2.52                               4.03                                4.53                              5.03

Treatment (μg/ml)

BC

C

C

B

A

Series1

Figure 21: The Effect of 苦瓜提取物 on the Caspase-3 Activity in the A549 cancer cell line.

 

 

21:苦瓜提取物對A549癌細胞系中Caspase-3活性的影響

 

 

Caspase-3 Activity

文字方塊: Caspase-3 Activity

0.8

0.7

0.6

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Control           2.52                       3.02                      3.52                       4.03                      4.53                     5.03

Treatment μg/ml

C

B             B           BC

AB

AB                             A

Figure 22: The Effect of 苦瓜提取物 on the Caspase-3 Activity in PC3 Cancer Cell Line.

 

22:苦瓜提取物對PC3癌細胞系中Caspase-3活性的影響。

Analysis of Caspase-3 Activation on PC3 Prostate Cancer Cell Line.

In the prostate cancer cell line PC3 the results from the treatment of 苦瓜提取物 with the cancer cells show an increase in all of the concentrations. There lower three concentrations (2.52, 3.02 and 3.52) increased about the same, however, in the three highest concentrations 4.03,4.53 and 5.03 μg/ml there was a substantial gradual increase in the apoptosis levels.

 

Caspase-3PC3攝護腺癌細胞系上的激活分析。

在攝護腺癌細胞系PC3中,用苦瓜提取物治療癌細胞的結果顯示所有濃度的增加。 較低的三個濃度(2.523.023.52)幾乎相同地增加,但是,在三個最高濃度4.034.535.03μg/ ml中,凋亡水平卻有逐漸增加的趨勢。

 

CHAPTER V

CONCLUSION

第五章

結論

Cancer remains a significant risk factor to public health worldwide, resulting in approximately 9.8 million deaths globally in 2018. In spite of improvements in conventional treatment modalities for cancer treatment, there continues to be a few effective therapies available because of the absence of selectivity, adverse side effects, non-specific toxicities, and tumor recurrence. Consequently, there is an immediate demand for vital alternative treatments, that can prove to be helpful and harmless against cancer. Countless phytochemicals from natural sources have been found to display helpful medicinal properties against various human diseases Girisa et al.. (2019).

Fruits and vegetables and everyday beverages, and several herbs and plants with expanded pharmacological properties, are excellent sources of micro-chemicals with the possiblility to inhibit human cancers Khan et al., (2010), Thangapazham et al. (2006)

Bitter melon (BM) belongs to the family of Cucurbitaceae. It is cultivated in the tropical regions of Asia, Africa, and South America. For many years BM has been usilyzed as traditional medicines in Asia. It is commonly known to have antidiabetic, antioxidant, antiviral, and anticancer effects. The efficacy of BM for numerous diseases has been confirmed. It has been reported that 苦瓜提取物 can decrease the expression of ER-stress proteins like ATF6, XBP1, PERK, and CHOP in colonic epithelial cells. 苦瓜提取物 is also known for improving high-fat diet (HFD)-induced obesity and hyperlipidemia in animal models Adam MA et al.. (2015) Kunde DA et al.. (2017).

 

癌症仍然是全球公共衛生的重要風險因素,2018年導致全球約980萬人死亡。儘管傳統的癌症治療方法有所改進,但由於缺乏選擇性,不良反應,仍然存在一些有效的治療方法副作用,非特異性毒性和腫瘤復發。因此,迫切需要重要的替代療法,這種療法可證明對癌症無害。已發現無數來自自然資源的植物化學物質對各種人類疾病均顯示出有益的藥用特性Girisa等人。

水果和蔬菜以及日常飲料,以及具有擴展藥理學特性的幾種草藥和植物,都是極好的微化學物質來源,具有抑制人類癌症的能力Khan等人,Thangapazham等人。

苦瓜(BM)屬於葫蘆科。它生長在亞洲,非洲和南美的熱帶地區。多年來,BM在亞洲已被廣泛用作傳統藥物。眾所周知,它具有抗糖尿病,抗氧化,抗病毒和抗癌作用。 BM對於多種疾病的功效已經得到證實。據報導,苦瓜提取物可以降低結腸上皮細胞中ER應激蛋白如ATF6XBP1PERKCHOP的表達。 苦瓜提取物還因改善動物模型中的高脂飲食(HFD)引起的肥胖症和高脂血症而聞名。

Because BM affects several diseases, BM has become a well known plant in the scientific community (10) While effects of BM on many bugs have been studied, studies of 苦瓜提取物 on cancer cell lines are insufficient, and few experiments have been done to evaluate their possible effects on cancer cells. Consequently, the focus of this study was on the impact of 苦瓜提取物 on BT548, A549, and PC3 cancer cell lines.

The finding from this study shows that bitter melon extract stimulates proliferation in BT549 and A549 cancer cell lines in the two lowest concentrations of .079 and .157 µg/ml. The reproduction is also encouraged in the PC3 cancer cell line but only in the lowest level of 0.079 μg/ml. However, starting at levels 0.315 μg/ml there is a gradual increase in cancer cell deaths. The decrease was significant in BT549 and A549 in the two highest levels of 2.52 and 5.03 μg/ml, however, in the PC3 the decrease was significant only in the highest concentration of 5.03 μg/ml. Due to the cytotoxic effect of the bitter melon extract the highest concentration (5.03 μg/ml) had the most significant decrease in cell viability in the BT549, A549 and PC3 cancer cell line. These results were compared to that of the control which was DMSO.

 

由於BM會影響多種疾病,因此BM已成為科學界的知名植物,雖然已經研究了BM對許多蟲子的影響,但對苦瓜提取物對癌細胞系的研究不足,並且很少進行實驗來評估它們對癌細胞的可能影響。因此,這項研究的重點是苦瓜提取物對BT548A549PC3癌細胞系的影響。

這項研究的發現表明,苦瓜提取物以0.079.157 µg / ml的兩個最低濃度刺激BT549A549癌細胞系的增殖。在PC3癌細胞系中也會增加繁殖,但那是僅以最低水平0.079μg/ ml進行。但是,從0.315μg/ ml的水平開始,癌細胞死亡會逐漸增加。在BT549A549中,兩個最高水平2.525.03μg/ ml的下降顯著,但是在PC3中,下降僅在最高濃度5.03μg/ ml時才顯著。由於苦瓜提取物的細胞毒性作用,最高濃度(5.03μg/ ml)在BT549A549PC3癌細胞系中的細胞活力下降最明顯。將這些結果與作為對照的DMSO進行比較。

ther phytochemicals have shown anticancer potential either in these cell lines or in cell viability assays. According to Ajji et al.. who did a study on balsamic a 28 kDa Type I ribosome-inactivating protein is rich in the seeds of Momordica balsamina. In this study, the molecular mechanism and the possible effects of balsamin on the two critical hallmarks of cancer were investigated. The induction of apoptosis in human breast cancer MCF-7 and BT549 cells showed that balsam-induced involved increases in caspase-3 and caspase-8 activity. Furthermore, balsam inhibited the proliferation of breast cancer cells in a dose-dependent manner in MCF-7 and BT549 breast cancer cell lines.

Additionally, Gloria et al. investigated the effects of lycopene and beta-carotene (carotenoids) on the cell cycle and cell viability in human breast cancer cell lines MCF7 and MDA-MB-231. Their statistics revealed a substantial decrease in the amount of viable breast cancer cells on treatment with the carotenoids. Furthermore, an increase in apoptosis was observed in cell lines when cells were treated with carotenoids. The MCF­7 cell line presented a decrease in the cell number only in the highest concentration of lycopene (2.5-10 μM). After 48 hr MCF-7 and MDA-MB-235 presented an increase of apoptotic cells with an average increase of 1.90-and 1.30-fold.

這些植物化學物質在這些細胞系或細胞活力測定中均顯示出抗癌潛力。根據對香脂進行研究的Ajji等人的研究,一種28 kDaI型核醣體失活蛋白富含苦瓜的種子。在這項研究中,研究了香脂素對癌症的兩個關鍵標誌的分子機制和可能的作用。人乳腺癌MCF-7BT549細胞凋亡的誘導表明,香脂誘導的caspase-3caspase-8活性。此外,苦瓜以劑量依賴性方式抑制MCF-7BT549乳腺癌細胞系中乳腺癌細胞的增殖

另外,Gloria等。研究了番茄紅素和β-胡蘿蔔素(類胡蘿蔔素)對人乳腺癌細胞MCF7MDA-MB-231的細胞週期和細胞活力的影響。他們的統計數據顯示,使用類胡蘿蔔素治療後,存活的乳腺癌細胞數量大大減少。此外,當用類胡蘿蔔素處理細胞時,在細胞系中觀察到凋亡的增加。僅在番茄紅素的最高濃度(2.5-10μM)下,MCF7細胞系細胞數減少。 48小時後,MCF-7MDA-MB-235呈現出凋亡細胞的增加,平均增加1.90倍和1.30

Another similar investigation to ours was done by Driggins (2017), he evaluated the effects of extracts of Echinacea pallida, Echinacea purpurea as well as the standard components of Echinacea were observed on breast cancer cell line BT549. The results revealed that the extract of E. Purpurea leaf signigicantly decreased the growth of breast cancer cell line BT549 in comparison to DMSO control cells. He also investigated the anti-proliferation effects of water soluble extracts of E. purpurea root and E. purpurea leaf on BT549 cancer cell line. The results revealed that both water soluble extracts significantly decreased the proliferation of BT549.

Since this lab found that 苦瓜提取物 efficiently inhibited proliferation we further examined the effect of 苦瓜提取物 on the lipid peroxidation levels in the three cancer cell lines. The results from this study indicates that 苦瓜提取物 lowers the MDA levels significantly in A549 in concentrations of 2.51 and 5.03. However, the MDA levels are only significantly reduced in BT549 and PC3 in the highest concentration of 5.03. Due to the cytotoxic effect of 苦瓜提取物 the MDA levels are significantly lowered in BT549, A549 and PC3 cancer cell lines. These results were compared to that of the control (DMSO) Mandal et al.. (2009) evaluated the leaves and stems of Drymaria diandra and their anti-oxidant efficiency to inhibit anti-lipid peroxidation. Their investigation showed that methanol extracts of D. diandra have antiradical activity by inhibiting DPPH radical. DPPH is usually used as a substrate to evaluate anti-oxidative activity of antioxidants.

After this lab found that 苦瓜提取物 efficiently reduced cell viability as well as reduced MDA levels in the three cancer cell lines, the effect of 苦瓜提取物 on the caspase-3 activation in the three cancer cell lines was examined. This research found that 苦瓜提取物 induces apoptosis in the three cancer cell lines. In breast cancer cell line BT549, the lower concentrations had a very litter effect. However, in the highest concentrations 0f 4.03, 4.53, and 5.03 μg/ml, there was a significant gradual increase in the apoptotic levels. Moreover, in A549 in the low concentrations there was no apoptotic effect, but in the two highest concentrations of 4.53 and 5.03 μg/ml there was a significant increase in the apoptotic level.

Driggins2017)與我們進行了另一項類似的研究,他評估了紫錐菊,紫錐菊和紫錐菊提取物對乳腺癌細胞BT549的作用。結果表明,與DMSO對照細胞相比,紫癜葉提取物顯著降低了乳腺癌細胞株BT549的生長。他還研究了紫莖澤蘭根和紫莖澤蘭葉的水溶性提取物對BT549癌細胞系的抗增殖作用。結果表明,兩種水溶性提取物均顯著降低了BT549的增殖。

由於該實驗室發現苦瓜提取物有效抑制了增殖,因此我們進一步檢查了苦瓜提取物對三種癌細胞系中脂質過氧化水平的影響。這項研究的結果表明,苦瓜提取物可以在濃度為2.515.03A549中顯著降低MDA水平。但是,MDA水平僅顯著

 最高濃度為5.03時,BT549PC3的含量降低。由於苦瓜提取物的細胞毒性作用,BT549A549PC3癌細胞系中的MDA水平顯著降低。將這些結果與對照(DMSO)進行比較

Mandal等人(2009年)評估了荷蓮豆草(Drymaria diandra)的葉子和莖及其抗氧化劑的功效,以抑制抗脂質過氧化作用。他們的研究表明,狄氏andra的甲醇提取物具有抑制DPPH自由基的抗自由基活性。 DPPH通常用作評估抗氧化劑的抗氧化活性的底物。

在本實驗室發現苦瓜提取物有效降低了這三種癌細胞系的細胞活力以及降低了MDA含量後,檢查了苦瓜提取物對這三種癌細胞系中caspase-3活化的影響。這項研究發現苦瓜提取物誘導了這三種癌細胞系的凋亡。在乳腺癌細胞系BT549中,較低的濃度具有非常低的效果。但是,在4.034.535.03μg/ ml的最高濃度下,凋亡水平顯著逐漸增加。而且,在低濃度的A549中沒有凋亡作用,但是在兩個最高濃度4.535.03μg/ ml中,凋亡水平顯著增加。

Conversely, in the PC3 cancer cell line all of the frequencies had a substantial increase in the apoptotic levels starting with the lower levels of 2.52, 3.02, 3.52, 4.03, and 4.54 with the highest level of 5.03 μg/ml having the most significant response. The

results were compared to that of the control (DMSO).

There have been several other investigations similar to this study, Elcady (2019) evaluated the anticancer properties of various extracts isolated from C speciosus against human prostate cancer PC3 cells. Extracts derived from C specious were analyzed by chromatography-mass spectrometry, and their effects of the proliferation, migration, invasion, apoptosis and cell cycle distribution of PC3 cells were investigated. Results showed that crude hexane extracts of C speciosus (CHECS) inhibited proliferation, clonogenic and metastatic potential of PC3 cells. It induced apoptosis in PC3 cells associated with generation of reactive oxygen species (ROS), reduction of GHS and permeabilization of mitochondrial and lysosomal membranes, induction of caspase-9 /3 activity and PARP-1 cleavage, DNA damage and an increase in ratio of BAX/Bcl-2 proteins.

相反,PC3癌細胞系中,所有頻率均從較低的2.523.023.524.034.54水平開始,使凋亡水平顯著增加,最高水平為5.03μg/ ml,反應最顯著

將結果與對照(DMSO)進行比較。

還有其他一些與這項研究相似的研究,Elcady2019)評估了從C物種分離出的各種提取物的抗癌特性

 人攝護腺癌PC3細胞。用色譜-質譜法分析了來自Cspecious的提取物,並研究了它們對PC3細胞的增殖,遷移,侵襲,凋亡和細胞週期分佈的影響。結果表明,C物種的粗己烷提取物(CHECS)抑制PC3細胞的增殖,克隆形成和轉移潛能。它誘導PC3細胞凋亡,與活性氧(ROS)產生,GHS減少和線粒體和溶酶體膜通透性相關,誘導caspase-9 / 3活性和PARP-1裂解,DNA損傷以及比例增加。 BAX / Bcl-2蛋白。

 

Likewise, Samarghandian et al.. (2011) investigated the antiproliferativer and apoptotic effect of honey and chrysin, which is a natural product found in honey, on cultured human prostate cancer cells. Their results revealed that both compounds had an antiproliferative effect on PC3 cells in a dose and time-dependent manner. The significant implication was only in the high concentrations. Furthermore, chrysin induced apoptosis in PC3 cells as determined by flow cytometry.

Also, Bhavana J et al.. (2016) evaluated the acetone extract of the Croton bonplandianus Baill., for cytotoxicity, apoptosis, cell cycle arrest in A549 lung cancer cell line. In their study they found that acetone leaf extract of C. bonplandianus showed cytotoxicity and cell proliferation inhibition in lung carcinoma cells A549 in a dose-dependent manner without affecting the healthy cells. Furthermore, the extract showed apoptosis induced cytotoxic effect.Similarly, Choi et al.. (2012) studied the inhibitory effects and underlying mechanisms of a methanol extract of Sanguisorba officinalis (MESO) in PC3 human prostate cancer cell line. The results suggest that MESO inhibits the growth of prostate cancer cells and induces apoptotic cell death by the downregulation of Mcl-1 protein expression and the oligomerization of Bax.

樣,Samarghandian等人(2011年)研究了蜂蜜和蘆薈對人攝護腺癌細胞的抗增殖和凋亡作用,後者是蜂蜜中的天然產物。他們的結果表明,這兩種化合物均以劑量和時間依賴性方式對PC3細胞具有抗增殖作用。僅在高濃度下才有意義。此外,通過流式細胞術測定,Chrysin誘導PC3細胞凋亡。

此外,Bhavana J等人(2016)對波氏巴豆(Croton bonplandianus Baill)的丙酮提取物進行了評估,以了解A549肺癌細胞系中的細胞毒性,凋亡和細胞週期停滯情況。在他們的研究中,他們發現,邦氏梭菌的丙酮葉提取物在肺癌細胞A549中顯示出劑量依賴性的細胞毒性和細胞增殖抑製作用,而不會影響健康細胞。此外,提取物顯示出凋亡誘導的細胞毒性作用。

 同樣,Choi等人(2012年)研究了山茱San甲醇提取物(MESO)的甲醇提取物對PC3人攝護腺癌細胞系的抑製作用及其潛在機制。結果表明,MESO通過下調Mcl-1蛋白表達和Bax寡聚化來抑制攝護腺癌細胞的生長並誘導凋亡性細胞死亡。

Additionally, Fang (2019) investigated the cytotoxic effects of bisindole alkaloid from Melodinus us- Caveolins against BT549 cancer cell line. The results indicate that the bisindole alkaloid exhibited significant cytotoxicity on the breast cancer cells. The study showed that bisindole inhibited BT549 cell proliferation by inducing apoptosis through activation of caspase-3 and p53, and down-regulation of Bcl-2. The authors in the literature also used the same cancer cell lines breast (BT549), lung (A549) and prostate (PC3), and other phytochemical compounds. The findings from this study imply that 苦瓜提取物 has similar potential as the compounds in the literature.

The present study provides clear evidence that all tested cell lines were sensitive to 苦瓜提取物 growth-inhibitory potential. The data showed a significant decrease in the number of viable cancer cells in all three cell lines on treatment with 苦瓜提取物. The maximum effect was observed in the 5.03µg/ml concentration, where 苦瓜提取物 induced over 90% decrease in percentage of cell viability in all tested cancer cell lines. Plus, 苦瓜提取物 also significantly decreases the MDA level in all the established cancer cell lines. Furthermore, a significant increase in apoptosis was observed in all cancer cell lines when cells were treated with 苦瓜提取物. The findings show the capacity of 苦瓜提取物 to inhibit cell proliferation, activate caspase-3 activity and decreased MDA levels in all tested cancer cell lines. Taken together these findings denote that 苦瓜提取物 had the potential to be a useful tool in the battle against cancer. The results might be due to the many phytochemicals present in BM.

According to (Prarthna 2014) quantitative analysis of Momordica charantia confirms the presence of phytochemicals like flavonoids, saponins, terpenoids, coumarins, emodins, alkaloids, proteins, cardiac glycosides, anthraquinones, anthocyanins, steroids, etc.

此外,Fang2019)研究了Melodinus us-Caveolins的雙吲哚生物鹼對BT549癌細胞的細胞毒性作用。結果表明,雙吲哚生物鹼對乳腺癌細胞具有明顯的細胞毒性。研究表明,雙吲哚通過激活caspase-3p53以及下調Bcl-2誘導凋亡,從而抑制BT549細胞增殖。文獻中的作者還使用了相同的癌細胞系乳腺癌(BT549),肺癌(A549)和攝護腺癌(PC3),以及其他植物化學化合物。這項研究的結果表明,苦瓜提取物與文獻中的化合物具有相似的潛力。

本研究提供了明確的證據,表明所有測試的細胞系均對苦瓜提取物的生長抑制潛力敏感。數據顯示,用苦瓜提取物治療後,所有三種細胞系中的存活癌細胞數量均明顯減少。在5.03µg / ml的濃度中觀察到了最大的效果,其中苦瓜提取物誘導了所有測試癌細胞系中細胞活力百分比降低90%以上。另外,苦瓜提取物還可以顯著降低所有已建立的癌細胞系中的MDA水平。此外,當用苦瓜提取物處理細胞時,在所有癌細胞系中觀察到凋亡的顯著增加。研究結果表明,在所有測試的癌症中,苦瓜提取物具有抑制細胞增殖,激活caspase-3活性和降低MDA水平的能力。

這些發現合起來表明苦瓜提取物有潛力成為抗擊癌症的有用工具。結果可能歸因於BM中存在許多植物化學物質。

根據(Prarthna 2014),苦瓜的定量分析證實了植物化學物質的存在,如類黃酮,皂角苷,萜類化合物,香豆素,大黃素,生物鹼,蛋白質,強心苷,蒽醌,花色苷,類固醇等。

The present study is the first to examine the effects of 苦瓜提取物 against breast cancer cell line BT549, lung cancer cell line A549 and the human prostate cancer cell line PC3. In conclusion, the current study demonstrates that BM has anti-cancer potential. The results herein provide essential information on the action of BM for justification as a potential candidate for anticancer drugs.

According to Lindsey et al. (2016), A large proportion of cancers can be prevented through measures including tobacco control, vaccination, early detection, and promotion of healthy lifestyle. Besides, the burden of suffering can be reduced through appropriate treatment and palliative care. To apply these cancer control measures equitably around the world, a concerted effort will be required not only from individual country governments, but also from international agencies, donors, civil society, and the private sector.

Future studies should be aimed at isolating the various metabolites in bitter melon to find out which compound has the most potential to inhibit cancer growth. Application of bitter melon in our diet and pharmaceutical field are still lacking, and perhaps our study will help accelerate the process of bitter melon being used as a cancer preventative and treatment agent.

 

本研究是第一個研究苦瓜提取物對乳腺癌細胞BT549,肺癌細胞A549和人攝護腺癌細胞PC3的作用的研究。總之,當前的研究表明BM具有抗癌潛力。本文的結果提供了有關BM發揮作用作為抗癌藥潛在候選藥物的必要信息。

根據林賽等。,可以通過煙草控制,疫苗接種,及早發現和促進健康生活方式等措施預防大部分癌症。此外,可以通過適當的治療和姑息治療減輕痛苦。為了在世界各地公平地應用這些癌症控制措施,不僅需要各國政府共同努力,而且還需要國際機構,捐助者,民間社會和私營部門共同努力。

未來的研究應旨在分離苦瓜中的各種代謝物,以找出哪種化合物最有可能抑制癌症的生長。苦瓜在我們的飲食和製藥領域仍然缺乏應用,也許我們

 

這項研究將有助於加速苦瓜被用作癌症預防和治療劑的過程。

 

 

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