印度老船長 癌症自然食療

多西環素Doxycycline+ 維生素C…消滅癌症幹細胞

癌友的新生力軍

 

摘錄一些文獻重點如下:

1. 癌症幹細胞（幹細胞）被認為是腫瘤復發，遠處轉移和治療耐藥性的“根本原因”
2. 根據美國Pubmed 的論文以及台中廖醫師所給的建議是相同的，也就是在施打高Ｃ之前至少一個小時左右可以加上服用Doxycycline 抗生素，在施打完後再加上硫辛酸。高Ｃ本身會產生比化療殺害癌幹細胞的能力多十倍。所以建議正在規律施打高Ｃ的朋友可以加上Doxycycline抗生素。Doxycycline 本身沒啥毒性與副作用，一般醫師可以開此處方簽，很便宜的藥，一顆（100mg) 才五元台幣，但需要醫師處方。
3. 我們證明了幾種無毒的抗生素可以用來阻止幹細胞的繁殖
4. 四環素抗生素（例如強力黴素Doxycycline）可被重新用於根除多種類型的癌症幹細胞。 這八種不同的癌症類型包括：DCIS，乳腺癌（ER（+）和ER（-）），卵巢癌，前列腺癌，肺癌和胰腺癌，以及黑素瘤和成膠質細胞瘤。強力黴素還可以有效地阻止患者晚期的轉移，包含乳腺癌患者幹細胞的繁殖。
5. 強力黴素具有很強的放射增敏作用，可以成功克服乳腺癌幹細胞中的抗輻射性。
6. 我們在這裡證實DoxyR 幹細胞對維生素C的影響更敏感，可以提高療效4至10倍之間
7. 強力黴素和維生素C可能代表了一種新型的癌腫瘤幹細胞致死藥，可用於消除幹細胞。
8. 在多種癌症類型中，強力黴素誘導的代謝僵化可能是避免治療失敗的實用解決方案。
9. 在9週的時間內，服用強力黴素的濃度（12.5至50μM）。
10. 我們測試了糖酵解抑製劑（例如2-脫氧葡萄糖（2-DG）和維生素C（抗壞血酸））的功效，維生素C比2-DG更有效。 它在250μM時抑制DoxyR 幹細胞傳播大於 90％，在500μM時抑制100％。(註:大約是吃脂質C 40克  240CC能達到血液濃度500μM)
11. 與口服相比，靜脈注射維生素C可使維生素C的血漿濃度提高30到70倍。此外，每天食用5至9份水果和蔬菜可使維生素C的血漿水平在穩態時達到80μM，峰值為220μM。值得注意的是，靜脈注射維生素C可以達到15,000μM（即15 mM）的血漿水平。有趣的是，每天最多50克的劑量緩慢注入，對癌症患者沒有任何毒的副作用。這些觀察結果表明，靜脈注射維生素C可能在癌症治療中起很大作用，因為這種途徑允許的血漿濃度高於最大耐受口服劑量所能達到的血漿濃度。
12. 多西環素是FDA批准的抗生素，可作為線粒體蛋白的抑製劑，因此在癌細胞中線粒體的特異性標靶指向治療中的病患病灶。可能具有治療價值。因此，了解強力黴素有助於我們開發新的合成癌細胞致死策略，以更有效地靶向幹細胞
13. 多西環素是線粒體生物的抑製劑，可有效減少早期乳腺癌患者的癌症幹細胞。臨床前實驗證明強力黴素可以選擇性地根除乳腺癌患者體內的癌幹細胞。
14. 方法：在手術前口服強力黴素14天，每日劑量為200 mg。其中有9例是用強力黴素治療，6例作為對照（未治療）。結果證實：與強力黴素前腫瘤樣品相比，強力黴素後腫瘤樣品顯示出乾性標記CD44的統計學顯著降低（p值<0.005）。 更具體地說，在用強力黴素治療的9位患者中，有8位的CD44水平降低了17.65至66.67％。 相反，只有一名患者的CD44升高了15％。 總體而言，這代表了近90％的效果。兩組之間的線粒體，腫瘤增殖，凋亡和新血管生成等均獲得相似效果。
15. 這能調理  最難搞的  胰臟癌以及腦瘤 那藥調理   未列出來的癌症如  鼻咽癌、淋巴癌、肝癌...可能都有  能力
16. 群友吃Doxycycline 抗生素已有5個月（每吃一個月休息一個月，在休息的一個月吃Mebendazole 驅蟲藥，兩者交替吃）沒有感到任何副作用！

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下面是文獻與翻譯

Vitamin C and Doxycycline: A synthetic lethal combination therapy targeting metabolic flexibility in cancer stem cells (CSCs)

維生素C和強力黴素：一種針對癌症幹細胞（CSC）致死性聯合療法

重點都有翻譯 並以紅色字體

Ernestina Marianna De Francesco,1,2 Gloria Bonuccelli,3 Marcello Maggiolini,1 Federica Sotgia,3 and Michael P. Lisanti3

Ernestina Marianna De Francesco

1 Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, Italy

2 The Paterson Institute, University of Manchester, Withington, United Kingdom

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Gloria Bonuccelli

3 Translational Medicine, School of Environment and Life Sciences, Biomedical Research Centre (BRC), University of Salford, Greater Manchester, United Kingdom

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Marcello Maggiolini

1 Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, Italy

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Federica Sotgia

3 Translational Medicine, School of Environment and Life Sciences, Biomedical Research Centre (BRC), University of Salford, Greater Manchester, United Kingdom

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Michael P. Lisanti

3 Translational Medicine, School of Environment and Life Sciences, Biomedical Research Centre (BRC), University of Salford, Greater Manchester, United Kingdom

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Author information Article notes Copyright and License information Disclaimer

1 Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende, Italy

2 The Paterson Institute, University of Manchester, Withington, United Kingdom

3 Translational Medicine, School of Environment and Life Sciences, Biomedical Research Centre (BRC), University of Salford, Greater Manchester, United Kingdom

Correspondence to:Michael P. Lisanti,Email: moc.liamg@itnasil.pleahcim

Federica Sotgia,Email: moc.liamg@aigtosf

Received 2017 May 5; Accepted 2017 May 17.

Copyright : © 2017 De Francesco et al.

This article is distributed under the terms of the Creative Commons Attribution License (CC-BY), which permits unrestricted use and redistribution provided that the original author and source are credited.

This article has been cited by other articles in PMC.

 

Abstract

Here, we developed a new synthetic lethal strategy for further optimizing the eradication of cancer stem cells (CSCs). Briefly, we show that chronic treatment with the FDA-approved antibiotic Doxycycline effectively reduces cellular respiration, by targeting mitochondrial protein translation. The expression of four mitochondrial DNA encoded proteins (MT-ND3, MT-CO2, MT-ATP6 and MT-ATP8) is suppressed, by up to 35-fold. This high selection pressure metabolically synchronizes the surviving cancer cell sub-population towards a predominantly glycolytic phenotype, resulting in metabolic inflexibility. We directly validated this Doxycycline-induced glycolytic phenotype, by using metabolic flux analysis and label-free unbiased proteomics.

Next, we identified two natural products (Vitamin C and Berberine) and six clinically-approved drugs, for metabolically targeting the Doxycycline-resistant CSC population (Atovaquone, Irinotecan, Sorafenib, Niclosamide, Chloroquine, and Stiripentol). This new combination strategy allows for the more efficacious eradication of CSCs with Doxycycline, and provides a simple pragmatic solution to the possible development of Doxycycline-resistance in cancer cells. In summary, we propose the combined use of i) Doxycycline (Hit-1: targeting mitochondria) and ii) Vitamin C (Hit-2: targeting glycolysis), which represents a new synthetic-lethal metabolic strategy for eradicating CSCs.

This type of metabolic Achilles’ heel will allow us and others to more effectively “starve” the CSC population.

摘要

在這裡，我們開發了一種新的合成致死策略，用於進一步優化根除癌症幹細胞（CSC）的能力。簡而言之，我們表明，通過靶向線粒體蛋白，用FDA批准的抗生素多西環素進行的長期治療有效地降低了細胞呼吸。四種線粒體DNA編碼蛋白（MT-ND3，MT-CO2，MT-ATP6和MT-ATP8）被抑制多達35倍。這種高選擇壓力在代謝上將存活的癌細胞亞群同步化為主要為糖酵解表型，從而導致代謝僵化。通過使用代謝通量分析和無標記的無偏蛋白質組學，我們直接驗證了強力黴素誘導的糖酵解功能。

接下來，我們確定了兩種天然產物（維生素C和黃連素）和六種經臨床批准的藥物（阿托伐醌，伊立替康，索拉非尼，尼氯酰胺，氯喹和斯替戊噴醇），這些藥物可代謝靶向強力黴素的CSC幹細胞。這種新的聯合策略允許用強力黴素更有效地根除CSC， 並為癌細胞中強力黴素抗性的可能發展提供了簡單實用的解決方案。總而言之，我們建議結合使用i）強力黴素（Hit-1：靶向線粒體）和ii）維生素C（Hit-2：靶向糖酵解），這是消除CSCs的新的合成致死代謝策略。

這種新陳代謝的致命弱點將使我們能更有效地“餓死” CSC幹細胞。

INTRODUCTION

Cancer stem cells (CSCs) are thought to be the “root cause” of tumor recurrence, distant metastasis and therapy-resistance, driving poor clinical outcome in advanced cancer patients [1–3]. Therefore, new therapeutic strategies are necessary to identify and eradicate CSCs [4–7]. As such, this goal remains an unmet medical need.

Recently, we identified that high mitochondrial mass is a new common and characteristic feature of CSCs, based on high-resolution proteomics analysis [8–10]. Importantly, high mitochondrial mass is a surrogate marker for increased mitochondrial biogenesis and/or elevated mitochondrial protein translation. Thus, this simple metabolic observation provides a new means for both i) CSC identification [9–13] and ii) CSC eradication [9, 10, 14–19].

Specifically, we showed that a mitochondrial fluorescent dye (MitoTracker) could be effectively used for the enrichment and purification of CSC activity from a heterogeneous population of living cells [11–13]. In this context, cancer cells with the highest mitochondrial mass had the strongest functional ability to undergo anchorage-independent growth, a characteristic normally associated with metastatic potential [11–13]. The ‘Mito-high’ cell sub-population also had the highest tumor-initiating activity in vivo, as shown using pre-clinical models. High mitochondrial mass was strictly correlated with i) increased hTERT activity and ii) the ability to undergo cell proliferation, which was sensitive to CDK4/6 inhibitors, such as palbociclib [16]. Complementary results were obtained with other fluorescent mitochondrial probes for ROS and hydrogen peroxide, as well as NADH auto-fluorescence, an established marker of mitochondrial “power”/high OXPHOS activity [13].

Moreover, we demonstrated that several classes of non-toxic antibiotics could be used to halt CSC propagation [14–19]. Because of the conserved evolutionary similarities between aerobic bacteria and mitochondria, certain classes of antibiotics inhibit mitochondrial protein translation, as an off-target side-effect [14]. One such group of antibiotics is the tetracyclines, the prototypic family member being Doxycycline.

介紹

癌症幹細胞（CSC）被認為是腫瘤復發，遠處轉移和治療耐藥性的“根本原因”，導致晚期癌症患者的臨床預後較差[1-3]。因此，有必要採用新的治療策略來鑑定和根除CSCs [4-7]。因此，該目標仍然是目前的醫療需求。

最近，基於高分辨率蛋白質組學分析，我們發現高線粒體質量是CSC的一個新的共同特徵，[8-10]。重要的是，高線粒體質量是增加線粒體生物發生和/或提高線粒體蛋白質繁殖的替代標誌。因此，這種簡單的代謝觀察為i）CSC鑑定[9-13]和ii）根除CSC [9，10，14-19]提供了新的手段。

具體而言，我們表明線粒體熒光染料（MitoTracker）可有效地用於從異質活細胞群體中集中和純化CSC活性[11-13]。在這種情況下，線粒體質量最高的癌細胞具有最強的功能與能力，可以不依賴錨定地生長，這通常與轉移潛能有關[11-13]。如臨床前模型所示，“ Mito-high”細胞亞群在體內也具有最高的腫瘤啟動活性。高線粒體質量與i）hTERT活性增加和ii）對CDK4 / 6抑製劑（如palbociclib）敏感的細胞增殖能力密切相關[16]。與其他用於ROS和過氧化氫的熒光線粒體探針以及NADH自發熒光（線粒體“能力” /高OXPHOS活性的既定標誌物）一起獲得的補充結果[13]。

此外，我們證明了幾種無毒的抗生素可以用來阻止CSC的繁殖[14-19]。由於有氧細菌和線粒體之間保守的進化相似性，某些種類的抗生素會抑制線粒體蛋白轉移，這是一種脫靶副作用[14]。這樣的抗生素是四環素，原型則是強力黴素。

 

Through this analysis, it became apparent that tetracycline antibiotics, such as Doxycycline, could be re-purposed to eradicate CSCs, in multiple cancer types [14, 20, 21]. These eight distinct cancer types included: DCIS, breast (ER(+) and ER(−)), ovarian, prostate, lung, and pancreatic carcinomas, as well as melanoma and glioblastoma. Doxycycline was also effective in halting the propagation of primary cultures of CSCs from breast cancer patients, with advanced metastatic disease (isolated from ascites fluid and/or pleural effusions) [20].

通過這一分析，很明顯，四環素抗生素（例如強力黴素）可被重新用於根除多種癌症類型的CSC [14，20，21]。 這八種不同的癌症類型包括：原位乳癌，乳癌（ER（+）和ER（-）），卵巢癌，前列腺癌，肺癌和胰腺癌，以及黑素瘤和成膠質細胞瘤。 強力黴素還可以有效地阻止患有晚期轉移性疾病（從腹水和/或胸腔積液中分離出來）的乳腺癌患者的原始培養的CSC的繁殖[20]。

Remarkably, Doxycycline behaves as a strong radio-sensitizer, successfully overcoming radio-resistance in breast CSCs [20]. This has important clinical implications, as the majority of ER(+) breast cancer patients are currently treated with breast-conserving surgery (lumpectomy) plus radiation therapy and hormonal therapy with an anti-estrogen.

引人注目的是，強力黴素具有很強的放射增敏作用，可以成功克服乳腺癌CSC中的抗輻射性[20]。這具有重要的臨床意義，因為目前大多數ER（+）乳腺癌患者都接受了手術（腫塊切除術），放療和抗雌激素激素治療。

Doxycycline is an FDA-approved drug, which first became available in 1967, ∼50 years ago now. It has excellent pharmacokinetic properties, with absorption of nearly 100% and a half-life of 18 to 24 hours. However, as with any new potential therapy, there is always a concern regarding the possible development of drug-resistance.

Here, we show that cancer cells can indeed escape the effects of Doxycycline, by reverting to a purely glycolytic phenotype. Fortunately, the metabolic inflexibility conferred by this escape mechanism allows Doxycycline-resistant (DoxyR) CSCs to be more effectively targeted with many other metabolic inhibitors, including Vitamin C, which functionally blocks aerobic glycolysis.

強力黴素是一種FDA批准的藥物，大約在50年前的1967年首次上市。 它具有出色的藥代動力學特性，吸收率接近100％，半衰期為18至24小時。 但是，與任何新的潛在療法一樣，人們始終擔心可能會產生耐藥性。

在這裡，我們顯示癌細胞確實可以通過恢復為純糖酵解表型而逃脫強力黴素的作用。 幸運的是，這種逃逸機制反而使強力黴素抗性（DoxyR）對CSC幹細胞更有效地靶向它們，加上維生素C，能阻止需氧糖的酵解。

Interestingly, previous studies have shown that Vitamin C inhibits GAPDH (a glycolytic enzyme) and depletes the cellular pool of glutathione, resulting in high ROS production and oxidative stress [22]. We show here that DoxyR CSCs are between 4- to 10-fold more susceptible to the effects of Vitamin C, inhibiting their propagation in the range of 100 to 250 μM. Therefore, Doxycycline and Vitamin C may represent a new synthetic lethal drug combination for eradicating CSCs, by ultimately targeting both mitochondrial and glycolytic metabolism.

有趣的是，以前的研究表明，維生素C抑制GAPDH（一種糖酵解酶）並耗盡穀胱甘肽的細胞群，從而導致高ROS產生和氧化應激[22]。 我們在這裡顯示DoxyR 強力黴素能幫助維生素C的抗癌功能更敏感，介於4至10倍之間，從而在100至250μM範圍內抑制了癌細胞的繁殖(註:單次攝取36g劑量的"脂質體維生素C的血漿濃度。其峰值血漿濃度，可達400um / L)。 因此，通過最終靶向線粒體和糖酵解代謝，強力黴素和維生素C可能代表了一種新型的合成致死藥，可用於消滅CSCs。

 

RESULTS

Metabolic flexibility is the intrinsic ability of a cell to change from one carbon fuel source to another; conversely, metabolic inflexibility is the exact opposite: the lack of ability (or dramatically reduced ability) to change fuel sources. It is believed that metabolic flexibility in cancer cells allows them to escape therapeutic eradication, leading to chemo- and radio-resistance. Here, we used doxycycline to pharmacologically induce metabolic inflexibility in CSCs, by chronically inhibiting mitochondrial biogenesis. This treatment resulted in a purely glycolytic population of surviving cancer cells. Then, we identified six other clinically-approved therapeutics, two natural products and one experimental drug, that all successfully eradicate the remaining glycolytic CSCs. Therefore, Doxycycline-induced metabolic inflexibility may be a practical solution to avoiding treatment failure, in a variety of cancer types.

Generating a Doxycycline-resistant MCF7 cell line, to study the potential mechanism(s) underlying drug resistance

結果

代謝柔韌性是細胞從一種碳燃料源轉變為另一種碳燃料源的內在能力。相反，新陳代謝的剛性恰好相反：缺乏改變燃料來源的能力（或顯著降低的能力）。據信，癌細胞的代謝靈活性使它們能夠逃避治療性根除，從而導致化學和放射抗性。在這裡，我們用強力黴素通過長期抑制線粒體的成長，在藥理學上誘導了CSCs的代謝僵化。這種治療導致存活的癌細胞發生了純粹的糖酵解。然後，我們確定了其他六種臨床批准的療法，兩種天然產物和一種實驗藥物，它們均能成功根除剩餘的糖酵解CSC幹細胞。因此，在多種類型的癌症中，強力黴素誘導的代謝僵化可能是避免治療失敗的切實可行的解決方案。

產生強力黴素抗性MCF7細胞系，以研究潛在耐藥性的潛在機制

To study the potential role of Doxycycline-resistance as an escape mechanism during Doxycycline treatment, we created a Doxycycline-resistant MCF7 cell line by serially passaging Doxycycline-sensitive MCF7 cells, in the presence of increasing concentrations of Doxycycline (from 12.5 to 50 μM), over a period of 9 weeks. The experimental procedure we utilized is briefly outlined in Figure ​Figure11 and is detailed in the Materials and Methods section.

為了研究強力黴素抗性在強力黴素治療過程中作為逃逸機制的潛在作用，我們通過在強力黴素濃度不斷增加（從12.5至50μM）的情況下，通過對強力黴素敏感的MCF7細胞進行連續傳代來創建強力黴素抗性MCF7細胞系。為期9個星期。 圖11簡要概述了我們的實驗程序，在“材料和方法”章節，則對此進行了詳細說明。

 

 

 

^{Figure 1}

^{Generating MCF7 DoxyR cells}

^{Doxycycline-resistant (DoxyR) MCF7 cells were generated by serially passaging MCF7 cells, in the presence of increasing step-wise concentrations of Doxycycline (12.5, 25 and 50 μM), over a period of 9 weeks. See the Materials and Methods section for further details. Unless stated otherwise, MCF7 cells resistant to 25 μM Doxycycline were utilized for experiments, such as unbiased proteomics analysis.}

^{Doxycycline-treated MCF7 cells were analyzed at each stage for mitochondrial mass. As shown in Figure 2A-2D, Doxycycline-resistant (DoxyR) MCF7 cells show a significant increase in mitochondrial mass (by ∼1.3- to 1.7-fold), as compared to acute treatment with Doxycycline, at the same drug concentration. This overall increase in mitochondrial mass was confirmed by immuno-blot analysis with specific antibodies directed against TOMM20, a well-established marker of mitochondrial mass (Figure ​(Figure2E2E).}

圖1

產生MCF7 DoxyR細胞

在9週的時間內，在不斷增加的強力黴素濃度（12.5、25和50μM）的條件下，通過連續傳代MCF7細胞來產生強力黴素抗性（DoxyR）MCF7細胞。 有關更多詳細信息，請參見“材料和方法”部分。 除非另有說明，否則將對25μM強力黴素具有抗性的MCF7細胞用於實驗，例如無偏蛋白組學分析。

在每個階段分析強力黴素處理的MCF7細胞的線粒體質量。 如圖2A-2D所示，與強力黴素的急性治療相比，在相同藥物濃度下，強力黴素（DoxyR）MCF7細胞的線粒體質量顯著增加（約1.3倍至1.7倍）。 通過針對TOMM20的特異性抗體的免疫印跡分析證實了線粒體質量的總體增加，TOMM20是線粒體質量的成熟標記物（圖2E2E）。

 

 

Figure 2

MCF7 DoxyR cells exhibit an increase in mitochondrial mass

A.-D. MCF7 cells were treated with DMSO or Doxycycline for acute (48 h) and chronic stimulation (3 weeks), as specified in Materials and Methods, and then mitochondrial mass was quantitated by FACS analysis using the probe MitoTracker Deep-Red (640-nm). Note that MCF7 cells chronically treated with 12.5 μM (A., fold change 1.33), 25 μM (B., fold change 1.68) and 50 μM (C., fold change 1.36) Doxycycline show a significant increase in mitochondrial mass compared to MCF7 cells treated with vehicle. Data shown are the mean ± SEM of at least 3 independent experiments performed in triplicate. (**) p < 0.01; (***) p < 0.001. D. Representative plots showing increased mitochondrial mass in MCF7 DoxyR cells as compared to MCF7 cells. E. Evaluation of the mitochondrial protein TOMM20 in MCF7 and MCF7 DoxyR cells by western blotting. Side panel shows densitometric analysis of the blots normalized to β-actin. Data shown are the mean ± SEM of 3 independent experiments. (**) p < 0.01.

圖2

MCF7 DoxyR細胞線粒體質量增加

按照材料和方法中的規定，用DMSO或強力黴素對MCF7細胞進行急性（48小時）和慢性刺激（3週）處理，然後使用探針MitoTracker Deep-Red（640 nm）通過FACS分析定量線粒體質量。注意長期用12.5μM（A.，倍數變化1.33），25μM（B.，倍數變化1.68）和50μM（C.，倍數變化1.36）長期治療的MCF7細胞與MCF7相比線粒體質量顯著增加用載體處理的細胞。所示數據為一式三份進行的至少3次獨立實驗的平均值±SEM。 （**）p <0.01； （***）p <0.001。 D.代表性圖顯示與MCF7細胞相比，MCF7 DoxyR細胞中線粒體質量增加。 E.通過蛋白質印跡法評估MCF7和MCF7 DoxyR細胞中線粒體蛋白TOMM20。側面圖顯示了歸一化為β-肌動蛋白的印蹟的光密度分析。顯示的數據是3個獨立實驗的平均值±SEM。

To understand the effects of chronic Doxycycline treatment on cell metabolism, we next performed metabolic flux analysis with the Seahorse XFe96. Interestingly, Figure ​Figure33 illustrates that MCF7-DoxyR cells show a dramatic reduction in oxygen consumption rates (OCR), as compared to matched control MCF7 cells, processed in parallel. As a consequence, ATP levels were severely depleted. Conversely, glycolysis was substantially increased, as measured by the ECAR (extracellular acidification rate) (Figure ​4). Therefore, DoxyR cells are mainly glycolytic. As such, a sub-population of MCF7 cells survive and develop Doxycycline-resistance, by adopting a purely glycolytic phenotype.

為了了解慢性強力黴素治療對細胞代謝的影響，我們接下來使用Seahorse XFe96進行了代謝通量分析。 有趣的是，圖33顯示，與並行處理的匹配對照MCF7細胞相比，MCF7-DoxyR細胞的耗氧率（OCR）顯著降低。 結果，ATP水平被嚴重消耗。 相反，通過ECAR（細胞外酸化率）測量，糖酵解作用顯著增加（圖4）。 因此，DoxyR細胞主要是糖酵解的。 這樣，通過採用純糖酵解表型，MCF7細胞亞群得以存活並發展出對多西環素的抗性。

 

 

Figure 3

Mitochondrial respiration is inhibited in MCF7 DoxyR cells

The metabolic profile of MCF7 DoxyR cells monolayers chronically treated with increasing concentrations of Doxycycline (12.5 μM ÷ 50 μM), as described in Materials and Methods, was assessed using the Seahorse XF-e96 analyzer. A. Representative tracing of metabolic flux. Dose-dependent significant reduction in basal respiration, proton leak, maximal respiration, ATP levels and spare respiratory capacity were observed B. Data shown are the mean ± SEM of 3 independent experiments performed in sextuplicate. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

圖3

線粒體呼吸在MCF7 DoxyR細胞中受到抑制

如材料和方法中所述，使用Seahorse XF-e96分析儀評估了用濃度遞增的多西環素（12.5μM÷50μM）慢性處理的MCF7 DoxyR細胞單層的代謝譜。 A.代謝通量的代表性示踪。 觀察到劑量依賴性顯著降低基礎呼吸，質子洩漏，最大呼吸，ATP水平和備用呼吸能力。B.顯示的數據是六次重複進行的3個獨立實驗的平均值±SEM。 （*）p <0.05； （**）p <0.01； （***）p <0.001。

 

 

Figure 4

Glycolysis is increased in MCF7 DoxyR cells

The metabolic profile of MCF7 DoxyR cells monolayers chronically treated with increasing concentrations of Doxycycline (12.5 μM ÷ 50 μM), as described in Materials and Methods, was assessed using the Seahorse XF-e96 analyzer. A. Representative tracing of metabolic flux. B. Dose-dependent significant increase in glycolysis and decrease in glycolytic reserve as well as glycolytic reserve capacity were observed. Data shown are the mean ± SEM of 3 independent experiments performed in sextuplicate. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001.

Doxycycline-resistant MCF7 cells show an increase in CSC markers, but not in functional CSC activity, as measured using mammosphere assays, proliferation and cell migration

圖4

糖酵解在MCF7 DoxyR細胞中增加

如材料和方法中所述，使用Seahorse XF-e96分析儀評估了用濃度遞增的多西環素（12.5μM÷50μM）長期處理的MCF7 DoxyR細胞單層的代謝譜。 A.代謝通量的代表性示踪。 B.觀察到糖酵解的劑量依賴性顯著增加和糖酵解儲備以及糖酵解儲備能力的降低。 顯示的數據是六次重複進行的3個獨立實驗的平均值±SEM。

耐強力黴素的MCF7細胞的CSC標記物增加，但功能性CSC活性卻沒有增加，如使用乳球測定，增殖和細胞遷移所測

ALDH activity and CD44/CD24 levels are routinely used as typical markers to identify breast CSCs [1–7]. Interestingly, MCF7-DoxyR cells show a substantial increase in these two CSC markers, as revealed by FACS analysis (Figure ​(Figure5).5). However, these markers do not reflect CSC activity. To more directly assess functional CSC activity, we used the mammosphere assay. Remarkably, MCF7-DoxyR cells show a > 60% reduction in CSC activity using the mammosphere assay as a readout (Figure ​(Figure6).6). Therefore, the increases in CSC markers that we observed do not actually reflect a functional increase in CSC propagation.

通常將ALDH活性和CD44 / CD24水平用作識別乳腺CSC的典型標誌[1-7]。 有趣的是，如通過FACS分析所揭示的，MCF7-DoxyR細胞在這兩個CSC標記中顯示出實質性的增加（圖5）。 但是，這些標記不能反映CSC活性。 為了更直接地評估功能性CSC活性，我們使用了乳球測定法。 值得注意的是，使用乳球測定作為讀數，MCF7-DoxyR細胞的CSC活性降低了60％以上（圖6）。 因此，我們觀察到的CSC標記的增加實際上並未反映CSC傳播的功能性增加。

 

 

 

Figure 5

MCF7 DoxyR cells show increased CSC markers

48h after seeding, MCF7 and MCF7 DoxyR cells were processed for the evaluation of ALDEFLUOR activity, an independent marker of CSCs. Each sample was normalized using diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor, as negative control A. The tracing of representative samples is shown B. 48h after seeding, MCF7 and MCF7 DoxyR cells were re-plated on low-attachment plates, for anoikis assay for 10 hours. Expression of CSC markers (CD24 and CD44) was analysed by FACS C. Representative dot plot for the the CD44+/CD24low cell population is shown D. This represents an ∼10-fold increase in ALDH functional activity and a ∼3-fold induction of the CD44+/CD24low population. Data are the mean ± SEM of 3 independent experiments performed in triplicate. (***) p < 0.001.

圖5

MCF7 DoxyR細胞顯示出增加的CSC標記

接種後48小時，處理MCF7和MCF7 DoxyR細胞以評估ALDEFLUOR活性，這是CSC的獨立標記。 每個樣品均使用特定的ALDH抑製劑二乙氨基苯甲醛（DEAB）進行標準化，作為陰性對照A。B顯示了代表性樣品的踪影。播種後48小時，將MCF7和MCF7 DoxyR細胞重新鋪板在低附著平板上以進行陽極氧化 化驗10小時。 通過FACS C分析CSC標誌物（CD24和CD44）的表達。CD44+ / CD24low細胞群的代表性點圖顯示為D。這表示ALDH功能活性增加約10倍，並且誘導了約3倍。 CD44 + / CD24low人口。 數據是一式三份同時進行的3個獨立實驗的平均值±SEM。

 

 

Figure 6

Mammosphere formation is inhibited in MCF7 DoxyR cells: Targeting DoxyR cells with Atovaquone and Chloroquine

Evaluation of mammosphere formation in MCF7 and MCF7 DoxyR cells cultured in low attachment plates and treated with vehicle or the selective OXPHOS inhibitor Atovaquone (ATO) A. or Chloroquine B. (which has been shown to impair mitochondrial metabolism), for 5 days before counting. Note that sphere formation is inhibited in MCF7 DoxyR cells as compared to MCF7 cells. In addition, mitochondrial-targeting agents like atovaquone and Chloroquine were effective in reducing the number of spheres in both MCF7 and MCF7 DoxyR cells. Data shown are the mean ± SEM of 3 independent experiments performed in triplicate. (***) p < 0.001.

Consistent with our above findings using the mammosphere assay, MCF7-DoxyR cells appear to be relatively quiescent, as they show dramatic reductions in their ability to proliferate by > 60%, as measured using EdU-incorporation, which reflects reduced DNA-synthesis (Figure 7A, 7B). Similarly, MCF7-DoxyR cells also show a clear defect in cell migration, with a > 50% reduction, as observed using the standard “scratch assay” (Figure 7C, 7D).

圖6

在MCF7 DoxyR細胞中乳腺球形成受到抑制：以Atovaquone和氯喹靶向DoxyR細胞

在計數前5天，評估在低附著板中培養並用媒介物或選擇性OXPHOS抑製劑Atovaquone（ATO）A.或氯喹B.（已證明會損害線粒體代謝）處理的MCF7和MCF7 DoxyR細胞中的乳球形成情況。注意，與MCF7細胞相比，MCF7 DoxyR細胞中的球形成受到抑制。此外，線粒體靶向劑（如atovaquone和氯喹）可有效減少MCF7和MCF7 DoxyR細胞中球體的數量。所示數據是一式三份進行的3個獨立實驗的平均值±SEM。

與我們使用乳球測定法得出的上述發現一致，MCF7-DoxyR細胞似乎相對靜止，因為它們的增殖能力顯著降低（如使用EdU摻入法測量的> 60％），這反映了DNA合成的減少（圖7A，7B）。同樣，MCF7-DoxyR細胞也顯示出明顯的細胞遷移缺陷，減少幅度超過50％，如使用標準“划痕分析”所觀察到的（圖7C，7D）。

 

 

Figure 7

MCF7 DoxyR cells show a quiescent phenotype, with significantly reduced proliferation and cell migration, as well as suppression of ERK- and AKT-signaling

Evaluation of cell proliferation by EdU incorporation assay using FACS analysis in MCF7 and MCF7 DoxyR cells 48h after seeding A. Note the reduction in EdU positive population in MCF7 DoxyR cells as compared to MCF7 cells. The tracing of a representative sample is shown B. Data shown are the mean ± SEM of 4 independent experiments performed in triplicate. (***) p < 0.001. Evaluation of cell migration by wound healing assay in MCF7 and MCF7 DoxyR cells which were seeded in 6 well plate to create a confluent monolayer. 24h after seeding a wound was created, then cells were washed and incubated at 37°C for 24 h. Images were acquired at 0 h and 24 h using Incucyte Zoom (Essen Bioscience). Quantification of cell migration was performed using ImageJ software and was expressed as % of wound closure C. Note the low migratory capacity of MCF7 DoxyR cells as compared to MCF7 cells. Representative images showing scratch assay D. Bar scale 100 μm. Data shown are the mean ± SEM of 3 independent experiments performed in triplicate. (***) p < 0.001. Evaluation of ERK1/2 E. and AKT Ser 473 F. phosphorylation in MCF7 and MCF7 DoxyR cells by western blotting. Side panels show densitometric analysis of the blots normalized to ERK2 and AKT respectively. Data shown are the mean ± SEM of 3 independent experiments. (*) p < 0.05; (**) p < 0.01.

圖7

MCF7 DoxyR細胞表現出靜止的表型，增殖和細​​胞遷移明顯減少，並抑制ERK和AKT信號傳導

在接種A後48h，通過FACS分析使用EdU摻入法評估細胞增殖，在MCF7和MCF7 DoxyR細胞中進行。注意，與MCF7細胞相比，MCF7 DoxyR細胞中EdU陽性種群的減少。代表性樣品的示踪示於B。所示數據為一式三份進行的4次獨立實驗的平均值±SEM。通過傷口癒合試驗評估MCF7和MCF7 DoxyR細胞的細胞遷移，這些細胞接種在6孔板中以形成匯合的單層。播種傷口後24小時，然後洗滌細胞並在37℃下孵育24小時。使用Incucyte Zoom（Essen Bioscience）在0小時和24小時採集圖像。使用ImageJ軟件進行細胞遷移的定量，並表示為傷口閉合C的百分比。請注意，與MCF7細胞相比，MCF7 DoxyR細胞的遷移能力低。代表性圖像顯示了刮擦試驗D.柵尺為100μm。所示數據是一式三份進行的3個獨立實驗的平均值±SEM。 （***）p <0.001。通過蛋白質印跡評估MCF7和MCF7 DoxyR細胞中ERK1 / 2 E.和AKT Ser 473 F.的磷酸化。側板顯示分別歸一化為ERK2和AKT的印蹟的光密度分析。顯示的數據是3個獨立實驗的平均值±SEM。

Taken together, these findings are consistent with an overall tendency towards a quiescent glycolytic cell phenotype. Consistent with this assertion, DoxyR cells also show dramatic reductions in ERK-activation and AKT-activation, as revealed by immuno-blot analysis, with phospho-specific antibody probes (Figure 7E, 7F).

Proteomics analysis of MCF7-DoxyR cells provides validating evidence for a predominantly glycolytic phenotype, due to a loss of mitochondrial function

In order to further validate our functional observations from metabolic flux analysis, we also performed unbiased label-free proteomics analysis [8]. These results are summarized and presented in Tables ​Tables11–5. Based on this comprehensive proteomics analysis, MCF7-DoxyR cells show severe reductions in mitochondrial proteins, both those encoded by mitochondrial DNA (mt-DNA) and those encoded by nuclear DNA (nuc-DNA).

綜上所述，這些發現與靜止糖酵解細胞表型的總體趨勢一致。 與此主張一致，DoxyR細胞也顯示出磷酸化特異性抗體探針的免疫印跡分析所揭示的ERK激活和AKT激活的顯著降低（圖7E，7F）。

由於線粒體功能的喪失，MCF7-DoxyR細胞的蛋白質組學分析為主要糖酵解表型提供了有效的證據

為了進一步從代謝通量分析中驗證我們的功能觀察結果，我們還進行了無偏態的無標記蛋白質組學分析[8]。 這些結果已匯總並列在表表11-5中。 基於這種全面的蛋白質組學分析，MCF7-DoxyR細胞顯示線粒體蛋白質的嚴重減少，線粒體蛋白質既由線粒體DNA（mt-DNA）編碼，又由核DNA（nuc-DNA）編碼。

Table 1

Key Mitochondrial-related Proteins are Down-regulated in Doxy-Resistant MCF7 Cells

關鍵的線粒體相關蛋白在抗氧化MCF7細胞中被下調

Symbol	Description		Fold-reduction (Down-regulation)
Mitochondrial proteins encoded by mitochondrial DNA
MT-ND3	NADH-ubiquinone oxidoreductase chain 3	(Complex I)	35.07
MT-CO2	Cytochrome c oxidase subunit 2	(Complex IV)	19.26
MT-ATP8	ATP synthase protein 8	(Complex V)	6.42
MT-ATP6	ATP synthase subunit 6	(Complex V)	5.08
Mitochondrial proteins encoded by nuclear DNA
NDUFS1	NADH-ubiquinone oxidoreductase 75 kDa subunit, mitochondrial		12.53
NNT	NAD(P) transhydrogenase, mitochondrial		10.49
SSBP1	Single-stranded DNA-binding protein, mitochondrial		9.27
NDUFB8	NADH dehydrogenase 1 beta subcomplex subunit 8, mitochondrial		8.50
CKMT1A	Creatine kinase U-type, mitochondrial		7.49
TFAM	Transcription factor A, mitochondrial		6.89
COX7C	Cytochrome c oxidase subunit 7C, mitochondrial		5.40
COX7A2	Cytochrome c oxidase subunit 7A2, mitochondrial		5.34
SDHB	Succinate dehydrogenase iron-sulfur subunit, mitochondrial		4.86
COX5B	Cytochrome c oxidase subunit 5B, mitochondrial		4.83
CKMT2	Creatine kinase S-type, mitochondrial		4.78
COQ6	Ubiquinone biosynthesis monooxygenase COQ6, mitochondrial		4.71
HYOU1	Hypoxia up-regulated protein 1		4.55
CHDH	Choline dehydrogenase, mitochondrial		4.42
NDUFV1	NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial		4.31
PUS1	tRNA pseudouridine synthase A, mitochondrial		4.28
OXCT1	Succinyl-CoA:3-ketoacid coenzyme A transferase 1, mitochondrial		4.17
TOMM6	Mitochondrial import receptor subunit TOM6		4.15
ACAA2	3-ketoacyl-CoA thiolase, mitochondrial		4.04
NFU1	NFU1 iron-sulfur cluster scaffold homolog, mitochondrial		3.96
CPT1A	Carnitine O-palmitoyltransferase 1, liver isoform		3.52
UQCRC1	Cytochrome b-c1 complex subunit 1, mitochondrial		3.51
PRKDC	DNA-dependent protein kinase catalytic subunit		3.43
MDH2	Malate dehydrogenase, mitochondrial		3.30
ACSF3	Acyl-CoA synthetase family member 3, mitochondrial		3.29
FH	Fumarate hydratase, mitochondrial		3.27
PDHX	Pyruvate dehydrogenase protein X component, mitochondrial		3.23
BDH1	D-beta-hydroxybutyrate dehydrogenase, mitochondrial		3.16
NDUFS3	NADH dehydrogenase iron-sulfur protein 3, mitochondrial		3.16
MMAB	Cob(I)yrinic acid a,c-diamide adenosyltransferase, mitochondrial		3.12
DARS2	Aspartate--tRNA ligase, mitochondrial		3.00
SUCLA2	Succinyl-CoA ligase [ADP-forming] subunit beta, mitochondrial		2.91
ABAT	4-aminobutyrate aminotransferase, mitochondrial		2.83
LACTB	Serine beta-lactamase-like protein LACTB, mitochondrial		2.81
CHDH	Choline dehydrogenase, mitochondrial		2.78
GLS	Glutaminase kidney isoform, mitochondrial		2.77
TOMM34	Mitochondrial import receptor subunit TOM34		2.76
NDUFA10	NADH dehydrogenase 1 alpha subcomplex subunit 10, mitochondrial		2.70
MUL1	Mitochondrial ubiquitin ligase activator of NFKB 1		2.60
UQCRC2	Cytochrome b-c1 complex subunit 2, mitochondrial		2.54
COX7A2L	Cytochrome c oxidase subunit 7A-related protein, mitochondrial		2.54
SLC25A24	Calcium-binding mitochondrial carrier protein SCaMC-1		2.51
NDUFA9	NADH dehydrogenase 1 alpha subcomplex subunit 9, mitochondrial		2.50
GLUL	Glutamine synthetase		2.50
PDHA1	Pyruvate dehydrogenase E1 subunit alpha, somatic, mitochondrial		2.50
SDHA	Succinate dehydrogenase flavoprotein subunit, mitochondrial		2.48
NDUFS8	NADH dehydrogenase iron-sulfur protein 8, mitochondrial		2.42

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Table 5

A Subset of Cellular Ribosomal Proteins are Decreased in Doxy-Resistant MCF7 Cells

Symbol	Description	Fold-reduction (Down-regulation)
Small subunit
RPS15	40S ribosomal protein S15	2.12
RPS21	40S ribosomal protein S21	2.08
RPS4X	40S ribosomal protein S4, X isoform	2.06
RPS23	40S ribosomal protein S23	1.82
Large subunit
RPL34	60S ribosomal protein L34	9.85
RPL3	60S ribosomal protein L3	6.39
RPLP2	60S acidic ribosomal protein P2	3.68
RPL10A	60S ribosomal protein L10a	2.28
RPL27A	60S ribosomal protein L27a	2.06
RPL8	60S ribosomal protein L8	1.93
RPL22L1	60S ribosomal protein L22-like 1	1.82
Other
RSL1D1	Ribosomal L1 domain-containing protein 1	3.08

A loss of mt-DNA-encoded proteins is characteristic hallmark of the inhibition of mitochondrial protein translation. Therefore, MCF7-DoxyR cells should be expected to metabolically phenocopy a mt-DNA-deficient genotype (rho(0) cells). For example, the cellular levels of MT-ND3, MT-CO2, MT-ATP6 and MT-ATP8 are all reduced between 5- to 35-fold (Table ​(Table1),1), which should inactivate or impair Complex I, IV and V. Similarly, > 45 nuclear-encoded mitochondrial proteins, such as NDUFS1, NDUFB8 and COX7C, are all decreased between 2- and 12-fold (Table ​(Table11).

In striking contrast, the levels of 10 glycolytic enzymes were all increased between 2- and 7-fold, including PGM1, LDHA, ALDOC and GAPDH (Table ​2). Similarly, enzymes associated with glycogen metabolism were also increased, between 3- and 4-fold (GYS1, PYGM, PYGL); markers of hypoxia were also elevated (myoglobin and hemoglobin (alpha/delta)) (Table ​(Table3),3), supporting a predominant glycolytic phenotype. Consistent with an increase in Aldefluor activity, several ALDH gene products were increased, especially ALDH1A3. This increased ALDH activity may reflect their tendency towards glycolysis, as ALDH isoforms contribute significantly to the glycolytic pathway.

mt-DNA編碼蛋白的丟失是抑制線粒體蛋白繁殖的特徵。因此，應該期望MCF7-DoxyR細胞通過代謝表型分析mt-DNA缺乏的基因型（rho（0）細胞）。例如，MT-ND3，MT-CO2，MT-ATP6和MT-ATP8的細胞水平都降低了5到35倍（表1），這會失活或損害複合物I，IV類似地，> 45種核編碼的線粒體蛋白（例如NDUFS1，NDUFB8和COX7C）都減少了2到12倍（表2）。

與之形成鮮明對比的是，包括PGM1，LDHA，ALDOC和GAPDH在內的10種糖酵解酶的水平都增加了2到7倍（表2）。同樣，與糖原代謝相關的酶也增加了3到4倍（GYS1，PYGM，PYGL）；缺氧的標誌物也升高（肌紅蛋白和血紅蛋白（α/δ））（表3），支持主要的糖酵解表型。與Aldefluor活性的增加一致，增加了幾種ALDH基因產物，特別是ALDH1A3。由於ALDH同工型對糖酵解途徑有顯著貢獻，因此增加的ALDH活性可能反映了它們的糖酵解趨勢。

Table 2

Enzymes Related to Glycolysis and Glycogen Metabolism are Up-regulated in Doxy-Resistant MCF7 Cells

表2

與糖酵解和糖原代謝相關的酶在抗氧性MCF7細胞中上調

Symbol	Description	Fold-Increase (Up-regulation)
Glycolytic enzymes
PGM1	Phosphoglucomutase-1	7.16
LDHA	L-lactate dehydrogenase A	7.09
ALDOC	Fructose-bisphosphate aldolase C	3.44
GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	3.06
GPD1L	Glycerol-3-phosphate dehydrogenase 1-like protein	2.72
ALDOA	Fructose-bisphosphate aldolase A	2.71
PFKP	ATP-dependent 6-phosphofructokinase, platelet type	2.69
PGK1	Phosphoglycerate kinase 1	2.64
GPI	Glucose-6-phosphate isomerase	2.46
PKM	Pyruvate kinase	2.10
Glycogen metabolism
GYS1	Glycogen [starch] synthase, muscle	4.11
PYGM	Glycogen phosphorylase, muscle form	3.45
PYGL	Glycogen phosphorylase, liver form	3.39

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Table 3

Markers of Hypoxia and Cancer Stem Cells are Up-regulated in Doxy-Resistant MCF7 Cells

Symbol	Description	Fold-Increase (Up-regulation)
Hypoxia markers
MB	Myoglobin	5.86
HBA1	Hemoglobin subunit alpha	3.46
HBD	Hemoglobin subunit delta	1.81
ALDH gene isoforms
ALDH1A3	Aldehyde dehydrogenase family 1 member A3	1,681.32
ALDH1A2	Retinal dehydrogenase 2	5.22
ALDH5A1	Succinate-semialdehyde dehydrogenase, mitochondrial	3.87
ALDH18A1	Delta-1-pyrroline-5-carboxylate synthase	2.75
ALDH16A1	Aldehyde dehydrogenase family 16 member A1	2.04
Other cancer stem cell (CSC) markers
RGAP2	SLIT-ROBO Rho GTPase-activating protein 2	2.80
CD44	CD44 antigen	2.09

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Table ​Table44 shows that 10 mitochondrial ribosomal proteins (MRPs) were increased, between 1.5- to 3-fold. This would mechanistically explain the compensatory increase in mitochondrial mass observed in Figure ​Figure22.

Table 4

A Subset of Mitochondrial Ribosomal Proteins (MRPs) are Increased in Doxy-Resistant MCF7 Cells

Symbol	Description	Fold-Increase (Up-regulation)
Small subunit
MRPS25	28S ribosomal protein S25, mitochondrial	3.02
MRPS9	28S ribosomal protein S9, mitochondrial	1.69
MRPS18C	28S ribosomal protein S18c, mitochondrial	1.58
Large subunit
MRPL10	39S ribosomal protein L10, mitochondrial	2.90
MRPL12	39S ribosomal protein L12, mitochondrial	2.21
MRPL46	39S ribosomal protein L46, mitochondrial	2.13
MRPL53	39S ribosomal protein L53, mitochondrial	2.13
MRPL37	39S ribosomal protein L37, mitochondrial	2.05
MRPL19	39S ribosomal protein L19, mitochondrial	1.95
MRPL15	39S ribosomal protein L15, mitochondrial	1.94

Finally, Table ​Table55 illustrates that 12 cellular ribosomal proteins were clearly down-regulated, between 1.8- and 9-fold, which may drive a severe decrease in cellular protein synthesis, due to mitochondrial energy deficits, resulting in a relatively quiescent metabolic phenotype.

A synthetic lethal strategy for eradicating DoxyR CSCs, using atovaquone or chloroquine

最後，表Table5說明了12種細胞核醣體蛋白明顯下調，介於1.8倍和9倍之間，這可能由於線粒體能量不足而導致細胞蛋白合成的嚴重降低，從而導致相對靜止的代謝表型。

使用atovaquone阿托伐醌或chloroquine氯奎寧消除DoxyR CSC的合成的一種致命策略

Thus far, our experimental results indicate that DoxyR cells acquire a predominantly glycolytic phenotype, to escape the anti-mitochondrial effects of Doxycycline. This means that DoxyR cells have been inadvertently metabolically synchronized and suffer from a type of functional metabolic inflexibility. As such, they should be extremely sensitive to additional metabolic stressors or perturbations, allowing them to be eliminated completely. This immediately suggests a new synthetic lethal strategy for the metabolic eradication of CSCs, to avoid any resistance to Doxycycline.

More specifically, if we consider DoxyR as the first metabolic Hit in a two-Hit scheme, then DoxyR cells should be extremely susceptible to a second metabolic Hit. This second metabolic Hit could be achieved by using virtually any other “safe” metabolic inhibitors, targeting either glycolysis, OXPHOS or autophagy. This two-Hit metabolic scheme is illustrated schematically in Figure ​Figure88.

到目前為止，我們的實驗結果表明DoxyR細胞獲得了主要的糖酵解表型，從而逃脫了強力黴素的抗線粒體作用。 這意味著DoxyR細胞已經在無意間進行了代謝同步，並產生某種類型的功能性新陳代謝僵化。 因此，它們應對其他代謝應激源或攝動非常敏感，以使其完全消除。 這顯示了一種新的合成殺傷性策略，用於消除CSC的代謝，以避免對強力黴素產生任何抗藥性。

更具體地說，如果我們將DoxyR視為兩次命中方案中的第一個代謝命中物，則DoxyR細胞應該極易受到第二個代謝命中物的影響。 可以通過使用幾乎任何其他針對糖酵解，OXPHOS或自噬的“安全”代謝抑製劑來實現第二次代謝命中。 圖88示意性地說明了這種兩次命中的代謝方案。

 

 

 

Figure 8

A two-hit synthetic lethal strategy for eradicating DoxyR CSCs

Here, we outline a new therapeutic strategy for targeting CSCs. Our experimental results indicate that DoxyR cells acquire a predominantly glycolytic phenotype, to escape the anti-mitochondrial effects of Doxycycline. As such, they should be extremely sensitive to additional metabolic stressors, allowing them to be eliminated completely. This immediately suggests a new synthetic lethal strategy for the metabolic eradication of CSCs, to avoid resistance to Doxycycline. Specifically, if we consider DoxyR as the first metabolic Hit in a two-Hit scheme, then DoxyR cells should be extremely susceptible to a second metabolic Hit. This second metabolic Hit could be achieved by using virtually any other “safe” metabolic inhibitors, targeting either glycolysis, OXPHOS or autophagy.

圖8

消除DoxyR CSC的兩擊式合成殺傷性策略

在這裡，我們概述了針對CSC的新治療策略。我們的實驗結果表明，DoxyR細胞獲得了主要的糖酵解表型，從而逃脫了強力黴素的抗線粒體作用。 因此，它們應對其他代謝應激源極為敏感，為使其完全消除。 這立即提出了一種新的合成殺傷性策略，用於消除CSC的代謝，從而避免了對強力黴素的耐藥性。具體來說，如果我們將DoxyR視為兩次命中方案中的第一個代謝命中物，則DoxyR細胞應該極易遭受第二個代謝命中。可以通過使用幾乎任何其他針對糖酵解，OXPHOS氧化磷酸化或自噬的“安全”代謝抑製劑來實現第二次代謝命中。

To test this hypothesis, we first used Atovaquone, an FDA-approved OXPHOS inhibitor, which targets mitochondrial Complex III. Similarly, we examined the effects of Chloroquine, a well-known autophagy inhibitor [17]. Both Atovaquone and Chloroquine are normally used clinically for the treatment and prevention of malaria, a parasitic infection. A list summarizing these metabolic inhibitors is presented in Figure ​Figure99.

為了驗證該假設，我們首先使用了FDA批准的OXPHOS抑製劑Atovaquone阿托伐醌，其靶向線粒體複合物III。同樣，我們測試了氯喹（一種眾所周知的自噬抑製劑）的作用[17]。 臨床上通常使用Atovaquone和氯喹治療和預防瘧疾（一種寄生蟲感染）。 這些代謝抑製劑的清單如圖99所示。

 

 

Figure 9

Metabolic inhibitors successfully employed for the eradication of DoxyR CSCs

Briefly, a list of small molecules that we successfully used in conjunction with Doxycycline is shown. These include 9 known inhibitors of OXPHOS, glycolysis and autophagy. Two natural products (Vitamin C and Berberine), six clinically-approved drugs (Atovaquone, Chloroquine, Irinotecan, Sorafenib, Niclosamide, and Stiripentol) and one experimental drug (2-DG), are all highlighted.

圖9

代謝抑製劑已成功用於根除DoxyR CSC

簡要地，顯示了我們成功與多西環素結合使用的小分子列表。 這些包括9種已知的OXPHOS抑製劑，糖酵解和自噬。突出顯示了兩種天然產物（維生素C和黃連素），六種臨床批准的藥物（阿托伐醌，氯喹，伊立替康，索拉非尼，尼氯酰胺和斯替戊噴醇）和一種實驗藥物（2-DG）。

Importantly, Figure ​Figure66 shows that DoxyR CSC propagation is clearly more sensitive to Atovaquone, as compared with control MCF7 CSCs. More specifically, treatment with Atovaquone (1 μM) inhibited the CSC propagation of the DoxyR cells by > 85%. Previously, we showed that the IC-50 for Atovaquone was 1 μM for MCF7 CSC propagation [17]. Similarly, Chloroquine inhibited their propagation by > 75% at 25 μM and by > 90% at 50 μM. Thus, it is possible to target the propagation of DoxyR CSCs, using existing FDA-approved OXPHOS and autophagy inhibitors.

A synthetic lethal strategy for eradicating DoxyR CSCs, using natural products (Vitamin C and Berberine) and other FDA-approved drugs

Next, we tested the efficacy of glycolysis inhibitors, such as 2-deoxy-glucose (2-DG) and Vitamin C (ascorbic acid). Treatment with 2-DG inhibited the propagation of DoxyR CSCs by > 90% at 10 mM and 100% at 20 mM (Figure ​(Figure10).10). In addition, Vitamin C was more potent than 2-DG; it inhibited DoxyR CSC propagation by > 90% at 250 μM and 100% at 500 μM (Figure ​(Figure10).10). As such, the IC-50 for Vitamin C in this context was between 100 to 250 μM, which are within the known achievable blood levels, when Vitamin C is taken orally. Previously, we showed that the IC-50 for Vitamin C was 1 mM for MCF7 CSC propagation [13]. Therefore, DoxyR CSCs are between 4- to 10-fold more sensitive to Vitamin C than control MCF7 CSCs, under identical assay conditions.

重要的是，圖66顯示，與對照MCF7 CSC相比，DoxyR CSC繁殖對Atovaquone明顯更敏感。更具體地，用阿托伐醌（1μM）處理將DoxyR細胞的CSC增殖抑制了＞ 85％。以前，我們顯示Atovaquone的IC-50對於MCF7 CSC傳播為1μM[17]。同樣，氯喹在25μM時抑制其傳播> 75％，在50μM時抑制> 90％。因此，使用現有的FDA批准的OXPHOS和自噬抑製劑可以靶向DoxyR CSC的繁殖。

 

使用天然產物（維生素C和黃連素）和其他FDA批准的藥物消除DoxyR CSC的合成致命策略

接下來，我們測試了糖酵解抑製劑（例如2-脫氧葡萄糖（2-DG）和維生素C（抗壞血酸））的功效。用2-DG處理可抑制DoxyR CSC在10 mM時的傳播> 90％，在20 mM時的100％（圖10）。此外，維生素C比2-DG更有效。它在250μM時抑制DoxyR CSC傳播> 90％，在500μM時抑制100％（圖10）。因此，在這種情況下，口服維生素C時，維生素C的IC-50為100至250μM(註:一次空腹服用脂C 20克(約120 CC)，2.5小時後開始，血液C的濃度達到300um/L，能維持3.5小時左右)，處於已知可達到的血液水平之內。以前，我們顯示維生素C的IC-50對於MCF7 CSC繁殖而言為1 mM [13]。因此，在相同的測定條件下，DoxyR CSC對維生素C的敏感性比MCF7 CSC高4至10倍。

 

 

Figure 10

Glycolysis inhibitors reduce mammosphere formation in MCF7 DoxyR cells

Evaluation of mammosphere formation in MCF7 and MCF7 DoxyR cells cultured in low attachment plates and treated with Vehicle or increasing concentrations of the glycoysis inhibitor 2-deoxy-glucose (2 DG) (10 mM to 20 mM) for 5 days before counting A. Mammosphere formation is inhibited in MCF7 DoxyR cells cultured in low attachment plates and treated with increasing concentrations of the glycoysis inhibitor Ascorbic Acid (100 μM to 500 μM) for 5 days before counting B. Data shown are the mean ± SEM of 3 independent experiments performed in triplicate. (***) p < 0.001.

圖10

糖酵解抑製劑可減少MCF7 DoxyR細胞中的乳球形成

在計數A.乳腺球之前，評估在低附著板中培養並經溶媒或濃度增加的糖酵解抑製劑2-脫氧葡萄糖（2 DG）（10 mM至20 mM）處理的MCF7和MCF7 DoxyR細胞中乳球形成的情況。 在低黏附板中培養的MCF7 DoxyR細胞中，其形成受到抑制，並在計數B之前，用濃度增加的糖基化抑製劑抗壞血酸（100μM至500μM）處理5天。顯示的數據是在 一式三份。

We also examined the efficacy of 4 other clinically-approved drugs that functionally behave as either OXPHOS inhibitors (Irinotecan, Sorafenib, Niclosamide) or glycolysis inhibitors (Stiripentol) (Figure ​(Figure11).11). In this context, Stiripentol functions as an LDH inhibitor. Their rank order potency for inhibiting DoxyR CSC propagation is: Niclosamide (IC-50 ∼ 100 nM) > Irinotecan (IC-50 ∼ 500 nM) > Sorafenib (IC-50 ∼ 0.5 to 1 μM) > Stiripentol (IC-50 ∼ 10 to 50 μM).

我們還檢查了其他4種經臨床批准的藥物的功效，這些藥物在功能上起OXPHOS抑製劑（伊立替康，索拉非尼，尼氯酰胺）或糖酵解抑製劑（Stiripentol）的作用（圖（圖11）.11）。 在這種情況下，斯替戊噴醇起LDH抑製劑的作用。 它們抑制DoxyR CSC繁殖的等級效力為：尼氯酰胺（IC-50〜100 nM）>伊立替康（IC-50〜500 nM）>索拉非尼（IC-50〜0.5至1μM）>斯替戊噴醇（IC-50〜10） 至50μM）。

 

 

Figure 11

A panel of clinically-approved drugs inhibits mammosphere formation in MCF7 DoxyR cells

圖11

一組臨床批准的藥物可抑制MCF7 DoxyR細胞中的乳球形成

Evaluation of mammosphere formation in MCF7 DoxyR cells cultured in low attachment plates and treated with Vehicle or increasing concentrations of the LDH inhibitor Stiripentol (2 μM to 100 μM) A. or the OXPHOS inhibitors Irinotecan (500 nM to 80 μM) B., Sorafenib (500 nM to 40 μM) C., Berberine Chloride (500 nM to 10 μM) D. and Niclosamide E.-F. for 5 days before counting. Data shown are the mean ± SEM of 3 independent experiments performed in triplicate. (**) p < 0.01: (***) p < 0.001.

評估在低附著板中培養並用媒介物或濃度增加的LDH抑製劑Stiripentol（2μM至100μM）A.或OXPHOS抑製劑Irinotecan（500 nM至80μM）B. （500 nM至40μM）C.，黃連素氯化物（500 nM至10μM）D.和Niclosamide E.-F. 計數前5天。 所示數據是一式三份進行的3個獨立實驗的平均值±SEM。

Finally, we tested the efficacy of Berberine, which is a naturally occurring antibiotic that also behaves as an OXPHOS inhibitor. It was used as early as 3,000 B.C. in China, for medicinal purposes. Figure ​Figure1111 shows that treatment with Berberine effectively inhibited the propagation of the DoxyR CSCs by > 50% at 1 μM and > 80% at 10 μM.

最後，我們測試了黃連素的功效，黃連素是一種天然存在的抗生素，也可以作為OXPHOS抑製劑。 它早在公元前3,000年就被使用了。 在中國用於藥用。 如圖11所示，黃連素處理有效地抑制了DoxyR CSCs，在1μM時> 50％的擴散，在10μM時> 80％的擴散。

 

DISCUSSION

In this report, we present new functional evidence to support a novel synthetic lethal strategy to eradicate CSCs. More specifically, we demonstrate that the use of Doxycycline, a clinically approved antibiotic, induces metabolic stress in cancer cells. This allows the remaining cancer cells to be synchronized towards a purely glycolytic phenotype, driving a form of metabolic inflexibility. This Doxycycline-driven aerobic glycolysis was further confirmed and validated, by employing high-resolution proteomics analysis and metabolic phenotyping. In addition, we discovered that both natural products and FDA-approved drugs could be re-purposed to eradicate the Doxycycline-resistant CSC population. These 9 small molecules included: Vitamin C, Berberine, 2-DG, Atovaquone, Irinotecan, Sorafenib, Niclosamide, Chloroquine, and Stiripentol.

討論

在本報告中，我們提出了新的證據，以支持根除CSC的新型致死策略。 更具體地說，我們證明了多西環素（一種臨床批准的抗生素）的使用可誘導癌細胞的代謝應激。 這使剩餘的癌細胞與純糖酵解表型同步，從而驅動新陳代謝缺乏彈性的形式。通過採用高分辨率蛋白質組學分析和代謝表型分析，進一步證實並驗證了強力黴素驅動的有氧糖酵解。 此外，我們發現天然產物和FDA批准的藥物都可以重新用於根除強力黴素耐藥的CSC幹細胞。這9個小分子包括：維生素C，黃連素，2-DG，阿托伐醌，伊立替康，索拉非尼，尼氯胺，氯喹和斯替戊噴醇。

Our new therapeutic strategy should provide for the more efficient eradication of CSCs using Doxycycline, as well as a practical means for solving the potential problem of Doxycycline resistance in CSCs. As such, we suggest a new synthetic lethal strategy for eradicating CSCs, by employing i) Doxycycline (to target mitochondria) and ii) Vitamin C (to target glycolysis) (Figure ​(Figure12).12). Use of this combined metabolic strategy should help prevent CSCs from exploiting the essential nutrients that they normally derive from the tumor microenvironment.

我們的新治療策略應提供使用多西環素更有效地根除CSC幹細胞的方法，並為解決CSC中對多西環素耐藥性的潛在問題提供一種實用手段。 因此，我們建議採用…1強力黴素（靶向線粒體）和2.維生素C（靶向糖酵解）來消除CSC的新的合成致死策略（圖12）。 使用這種聯合代謝策略應有助於防止CSC利用它們通常從腫瘤微環境中獲得的必需營養素。

 

 

Figure 12

Vitamin C and Doxycycline: A synthetic lethal combination therapy for eradicating CSCs

圖12

維生素C和強力黴素：消除CSC的致死性聯合療法

Note that both OXPHOS and the glycolytic pathway jointly contribute to ATP production. Doxycycline inhibits mitochondrial biogenesis and OXPHOS, by acting via mitochondrial ribosomal proteins (MRPs); Vitamin C inhibits glycolytic metabolism by targeting and inhibiting the enzyme GAPDH. Therefore, their use together, as a sequential drug combination, will more severely target cell metabolism and energy production, thereby preventing or blocking the propagation of CSCs.

Chronic Doxycycline treatment functionally confers a mitochondrial-deficient metabolic phenotype, actively suppressing CSC activity

請注意，OXPHOS和糖酵解途徑共同促進ATP的產生。強力黴素通過線粒體核醣體蛋白（MRP）發揮作用，從而抑制線粒體的生物發生和OXPHOS。 維生素C通過靶向和抑制GAPDH酶來抑製糖酵解代謝。因此，將它們一起用作順序藥物組合，將更嚴格地靶向細胞代謝和能量產生，從而防止或阻止CSC的繁殖。

慢性強力黴素治療在功能上賦予線粒體缺陷型代謝表型，從而積極抑制CSC活性

Previous studies have shown that human cancer cells lacking mt-DNA [called rho(0) cells] have largely lost their ability to undergo mitochondrial OXPHOS and they fail to initiate tumors in vivo, as determined by using pre-clinical animal models to assess tumorigenicity [3]. Importantly, their ability to undergo OXPHOS and to form tumors was effectively restored by genetic replacement of their mt-DNA [5]. As such, it appears that mitochondrial oxidative function and mt-DNA are required for energetically initiating the process of tumorigenesis, across multiple cancer types [3, 5].

先前的研究表明，缺乏mt-DNA的人類癌細胞（稱為rho（0）細胞）已經喪失了接受線粒體OXPHOS的能力，並且無法通過體內的臨床前動物模型評估其致瘤性來體內啟動腫瘤。 [3]。 重要的是，通過mt-DNA的基因置換有效地恢復了它們經歷OXPHOS和形成腫瘤的能力[5]。 因此，在多種癌症類型中，似乎需要線粒體的氧化功能和mt-DNA來積極啟動腫瘤發生過程[3，5]。

Here, we observed that MCF7-DoxyR cells strongly phenocopy the metabolic behavior of mt-DNA deficient (rho(0)) cells, by exhibiting a purely glycolytic phenotype. Consistent with this hypothesis, MCF7-DoxyR cells lack the expression of four mitochondrial proteins normally encoded by mt-DNA (MT-ND3, MT-CO2, MT-ATP6 and MT-ATP8) and they exhibit a near complete loss of OXPHOS activity, with a strong induction of aerobic glycolysis. MCF7-DoxyR cells also show significant functional reductions in cell proliferation and migration, as well as a loss of CSC propagation - a surrogate marker of tumor-initiating activity.

在這裡，我們觀察到MCF7-DoxyR細胞通過表現出純粹的糖酵解表型，強烈地表型化了mt-DNA缺陷（rho（0））細胞的代謝行為。 與此假設相符，MCF7-DoxyR細胞缺乏通常由mt-DNA編碼的四種線粒體蛋白（MT-ND3，MT-CO2，MT-ATP6和MT-ATP8）的表達，並且它們的OXPHOS活性幾乎完全喪失， 強烈誘導有氧糖酵解。 MCF7-DoxyR細胞還顯示出細胞增殖和遷移的功能顯著降低，以及CSC傳播的喪失-這是腫瘤啟動活性的替代標誌。

Therefore, chronic doxycycline treatment provides a pharmacological means to mimic a rho(0) cell phenotype, to therapeutically reduce tumor growth and to avoid tumor recurrence. However, Doxycycline-resistance still remains a valid concern.

Overcoming resistance to Doxycycline in cancer cells, using metabolic inflexibility, synthetic lethality and Vitamin C

因此，慢性強力黴素的治療提供了一種藥理學手段，可模擬rho（0）細胞表型，治療性減少腫瘤生長並避免腫瘤復發。 但是，強力黴素的耐藥性仍然是一個有效的問題。

利用代謝僵化，合成殺傷力和維生素C克服癌細胞對強力黴素的耐藥性

Resistance to anti-cancer therapy remains as one of the key issues in cancer patient management. Treatment failure is regarded as an alarming outcome of numerous different therapeutic approaches. Indeed, the use of combination strategies aimed at hitting multiple aspects of tumor progression is currently considered as a promising tool to overcome resistance. Mounting evidence suggests that CSCs act as the main promoter of tumor recurrence and patient relapse [1–5]. Thus, a better understanding of the biological and biochemical behavior of CSCs during drug resistance may unveil new vulnerabilities, to be exploited in a therapeutic setting.

In this context, our data indicates that a metabolic shift from oxidative to glycolytic metabolism represents an escape mechanism for breast cancer cells chronically-treated with a mitochondrial stressor like Doxycycline, as mitochondrial dys-function leads to a stronger dependence on glucose.

因此，長期強力黴素治療提供了對抗癌治療的抵抗力，仍然是癌症患者管理中的關鍵問題之一。 治療失敗被認為是許多不同治療方法的令人震驚的結果。 確實，目前已將旨在擊中腫瘤進展多個方面的聯合策略視為克服耐藥性的有前途的工具。 越來越多的證據表明，CSCs是腫瘤復發和患者復發的主要促進因素[1-5]。 因此，對CSCs在耐藥性過程中的生物學和生化行為的更好理解可能揭示了新的漏洞，將在治療環境中加以利用。

在這種情況下，我們的數據表明，從氧化代謝到糖酵解代謝的轉變代表了用線粒體應激物（如強力黴素）長期治療的乳腺癌細胞的逃逸機制，因為線粒體功能障礙導致對葡萄糖的依賴性更大。

Our current findings are in line with previous studies showing the highly plastic nature of CSCs allows them to adjust and adapt their metabolic environment, in order to maintain their distinctive properties, in a hostile tumor microenvironment, often characterized by an inadequate nutrient and oxygen supply (reviewed in [23]). Here, we have taken advantage of the glycolytic shift exhibited by DoxyR CSCs, as we have used several glycolysis inhibitors with the aim to turn their strict metabolic inflexibility, into a lethal phenotype. Among the agents tested in the our study, Vitamin C has been demonstrated to selectively kill cancer cells in vitro and to inhibit tumor growth in experimental mouse models [24, 25]. Remarkably, many of these actions have been attributed to the ability of Vitamin C to act as a glycolysis inhibitor, by targeting GAPDH and depleting the NAD pool [22, 26, 27].

我們目前的發現與以前的研究相吻合，表明CSC的高度可塑性使他們能夠在不利的腫瘤微環境中調節和適應其代謝環境，以維持其獨特的特性，而該環境通常以營養和氧氣供應不足為特徵（ 在[23]中回顧）。 在這裡，我們利用了DoxyR CSC所表現出的糖酵解轉變的優勢，因為我們已經使用了幾種糖酵解抑製劑，旨在將其嚴格的新陳代謝僵化轉變為致命的表型。 在我們的研究中測試的藥物中，維生素C已被證明可以在體外選擇性殺死癌細胞並在實驗性小鼠模型中抑制腫瘤生長[24，25]。 值得注意的是，這些作用中的許多作用都歸因於維生素C通過靶向GAPDH並耗盡NAD庫而充當糖酵解抑製劑的能力[22，26，27]。

In this context, we have previously demonstrated that Vitamin C effectively inhibits 3D breast tumor spheroid formation, with an IC-50 of 1 mM, suggesting that this micronutrient also works as an inhibitor of CSCs [13], whose activity is critically dependent on an active mitochondrial TCA cycle and OXPHOS. In contrast, here we show that DoxyR CSCs are more vulnerable to the inhibitory effects of Vitamin C, at 4- to 10-fold lower concentrations, between 100 to 250 μM. These findings are further supported by clinical studies showing that the concurrent use of Vitamin C, with standard chemotherapy, reduces tumor recurrence and patient mortality [28, 29].

在此背景下，我們先前已證明維生素C有效抑制3D乳腺腫瘤球體的形成，其IC-50為1 mM，這表明這種微量營養素還可以作為CSC的抑製劑[13]，其活性嚴重依賴於CSCs。 活躍的線粒體TCA週期和OXPHOS。 相反，這裡我們顯示DoxyR CSC在100至250μM之間的濃度低4至10倍時更容易受到維生素C的抑製作用。 這些研究結果進一步得到臨床研究的支持，臨床研究表明，同時使用維生素C和標準化學療法可降低腫瘤復發率和患者死亡率[28，29]。

It is worth noting that Vitamin C plasma levels vary considerably with the route of administration. For instance, pharmacokinetic studies performed by different research groups have assessed that, after oral administration, Vitamin C plasma levels reach concentrations of ∼70-220 μM [reviewed in reference [30], which represents the maximum tolerated oral dose. By contrast, Padayatty and co-workers found that, compared to oral intake, intravenous administration results in 30- to 70- fold higher plasma concentrations of Vitamin C [31]. Furthermore, consumption of 5 to 9 servings of fruits and vegetables per day allows plasma levels of Vitamin C to reach up to 80 μM at steady-state, with peak values of 220 μM [31]. Remarkably, an intravenous infusion of Vitamin C can reach plasma levels of 15,000 μM (i.e., 15 mM). Interestingly, doses of up to 50 grams per day, infused slowly, didn't exhibit any toxic side effects on cancer patients [30]. These observations suggest that intravenous administration of Vitamin C may have a role in cancer treatment, as this route allows higher plasma concentrations than those achievable with the maximum tolerated oral dose.

值得注意的是，維生素C血漿水平會隨著給藥途徑的不同而變化。例如，由不同研究小組進行的藥代動力學研究已評估，口服後維生素C血漿水平達到約70-220μM的濃度[參考文獻[30]綜述]，其代表最大耐受口服劑量。相比之下，Padayatty及其同事發現，與口服相比，靜脈內給藥可使維生素C的血漿濃度高30到70倍[31]。此外，每天食用5至9份水果和蔬菜可使維生素C的血漿穩態水平達到80μM，峰值為220μM[31]。值得注意的是，靜脈內註入維生素C可以達到15,000μM（即15 mM）的血漿水平。有趣的是，每天50克的劑量緩慢注入，對癌症患者沒有任何毒副作用[30]。這些觀察結果表明，靜脈內給予維生素C可能在癌症治療中起作用，因為這種途徑所允許的血漿濃度高於最大口服劑量所能達到的血漿濃度。

Previous studies have demonstrated that Vitamin C behaves as a potent dietary antioxidant, as well as a pro-oxidant. This pro-oxidant activity results from Vitamin C's action on metal ions, which generates free radicals and hydrogen peroxide, and is associated with cell toxicity. Of note, it has been shown that high-dose Vitamin C is more cytotoxic to cancer cells than to normal cells [32, 33]. This selectivity appears to be due to the higher catalase content observed in normal cells (∼10-100 fold greater), as compared to tumor cells. Hence, Vitamin C may be regarded as a safe agent that selectively targets cancer cells.

以前的研究表明，維生素C可以作為有效的飲食抗氧化劑，也可以作為前氧化劑。 維生素C對金屬離子的作用導致了這種前氧化活性，維生素C產生自由基和過氧化氫，並與細胞毒性有關。 值得注意的是，已顯示高劑量的維生素C對癌細胞的細胞毒性比對正常細胞的細胞毒性更大[32，33]。 與腫瘤細胞相比，這種選擇性似乎是由於在正常細胞中觀察到的過氧化氫酶含量較高（約高10-100倍）。 因此，維生素C可以被視為選擇性靶向癌細胞的安全劑。

A recent study performed on a panel of cancer cells (A431, Panc-1, HeLa, HT29, and MCF7) showed that Vitamin C only affects cell viability at concentration of ∼3 to 10 mM [34], thus providing more evidence to support a lack of toxicity for low micromolar concentrations of Vitamin C.

Similarly, phase I and II clinical trials, designed to deliver high-dose intravenous Vitamin C, have shown a lack of toxic side effects for concentrations of up to 292 μM [35]. Taken together with these findings, our data suggest that Vitamin C's action as a glycolytic inhibitor may represent a safe and effective strategy to be used in combination therapies, with conventional anticancer drugs, as well as with Doxycycline.

最近對一組癌細胞（A431，Panc-1，HeLa，HT29和MCF7）進行的研究表明，維生素C僅在約3至10 mM的濃度下影響細胞生存力[34]，從而提供了更多證據支持 低濃度的維生素C缺乏毒性。

類似地，旨在提供大劑量靜脈內維生素C的I和II期臨床試驗顯示，對於濃度高達292μM的藥物，無毒副作用[35]。 結合這些發現，我們的數據表明維生素C作為糖酵解抑製劑的作用可能是一種安全有效的策略，可與常規抗癌藥物以及強力黴素聯合使用。

Because of our success with 2-DG and Vitamin C, we explored additional FDA-approved drugs with glycolysis-inhibiting activity that could be repurposed to eradicate CSC propagation, in combination with Doxycycline. For example, we demonstrated that the LDH enzyme inhibitor Stiripentol is also effective at targeting DoxyR CSCs; this drug is currently used clinically as an anti-epileptic in children.

We also further explored other suitable metabolic approaches to overcome Doxycycline resistance. In this context, we evaluated the efficacy of a panel of compounds that share the ability to impair mitochondrial function (OXPHOS), as a common off-target side-effect. This approach was based on the assumption that DoxyR cells, which exhibit altered oxidative metabolism, are extremely sensitive to the induction of additional mitochondrial dysfunction. These effective compounds included four FDA-approved drugs, such as Atovaquone, Irinotecan, Sorafenib, and Niclosamide, as well as the natural product Berberine.

由於我們在2-DG和維生素C方面的成功，我們與多西環素一起探索了其他具有FDA批准的，具有糖酵解抑制活性的藥物，這些藥物可用於消除CSC繁殖。例如，我們證明了LDH酶抑製劑Stiripentol司替戊醇胶囊對DoxyR CSCs也有效。該藥物目前在臨床上用作兒童的抗癲癇藥。

我們還進一步探索了克服多西環素抗性的其他合適的代謝途徑。在這種情況下，我們評估了一組共同損害線粒體功能（OXPHOS）的化合物作為常見的脫靶副作用的功效。該方法基於這樣的假設，即表現出氧化代謝改變的DoxyR細胞對誘導其他線粒體功能異常極為敏感。這些有效的化合物包括四種FDA批准的藥物，例如Atovaquone，Irinotecan，Sorafenib和Niclosamide，以及天然產品黃連素。

有趣的是，我們觀察到氯喹還降低了DoxyR細胞的球狀形成效率，表明自噬的抑制可能代表了另一種有效的聯合策略，可使乳腺癌細胞對多西環素的作用敏感。

We believe that the emerging functional relationship between metabolism and stemness, also known as “metabo-stemness” [36], holds great promise for the future of anti-cancer therapy. Thus, our novel findings may pave the way for the discovery and validation of more effective therapeutic strategies to fully eradicate CSCs, ultimately preventing treatment failure and minimizing metastatic dissemination.

Synergistic effects of Doxycycline and Vitamin C in the treatment of infectious disease states

我們認為，新陳代謝和幹細胞特性之間的功能關係，也稱為“代謝幹性” [36]，為抗癌治療的未來帶來了廣闊前景。 因此，我們的新發現可能為發現和驗證更有效的治療策略以徹底根除CSCs，最終防止治療失敗和最小化轉移擴散鋪平道路。

強力黴素與維生素C在治療傳染病狀態下的協同作用

Is there any precedent for the use of Doxycycline in combination with Vitamin C, in clinical trials? Interestingly, another group published a report on a randomized clinical trial of the effects of Vitamin C on dyspareunia and vaginal discharge, in women receiving Doxycycline and Triple sulfa for chlamydial cervicitis infections [37]. Importantly, they concluded that the cure rate was nearly 5-fold higher in the patients that received Vitamin C, together with antibiotic therapy [37]. So, the concurrent use of Doxycycline and Vitamin C, in the context of this infectious disease, appeared to be highly synergistic in patients.

在臨床試驗中是否有將強力黴素與維生素C結合使用的先例？ 有趣的是，另一個研究小組發表了一份關於維生素C對衣原體宮頸炎感染接受強力黴素和三聯磺胺治療的婦女的性交困難和白帶排出影響的隨機臨床試驗的報告[37]。 重要的是，他們得出結論，接受維生素C以及抗生素治療的患者治愈率高出近5倍[37]。 因此，在這種傳染病的背景下，同時使用強力黴素和維生素C似乎對患者俱有高度的協同作用。

Similarly, Goc et al., 2016, showed that Doxycycline is synergistic in vitro with certain phytochemicals and micronutrients, including Vitamin C, in the in vitro killing of the vegetative spirochete form of Borrelia spp., the causative agent underlying Lyme disease [38]. Vitamin C has also been shown to be synergistic with Tetracycline and Chloramphenicol, against the pathogenic bacteria, Pseudomonas aeruginosa [39]. However, in the above examples, no follow-up mechanistic studies were conducted to determine exactly why Doxycycline, Tetracycline and Vitamin C were somehow synergistic.

同樣，Goc等人，2016年顯示，強力黴素與某些植物化學物質和微量營養素（包括維生素C）在體外具有協同作用，可在體外殺死萊姆病的病原體螺旋藻形式的營養螺旋體[38] [38] 。 維生素C還被證明與四環素和氯黴素具有協同作用，可對抗病原菌銅綠假單胞菌[39]。 但是，在上述示例中，沒有進行任何後續的機理研究來確切確定為什麼強力黴素，四環素和維生素C具有某種協同作用。

 

CONCLUSIONS

Numerous functional studies have now directly shown that mitochondria are an important new therapeutic target in cancer cells [3, 5, 8–21, 40–53]. Since Doxycycline, an FDA-approved antibiotic, behaves as an inhibitor of mitochondrial protein translation, it may have therapeutic value in the specific targeting of mitochondria in cancer cells. However, in this paper, we have identified a novel metabolic mechanism by which CSCs successfully escape from the anti-mitochondrial effects of Doxycycline, by assuming a purely glycolytic phenotype. Therefore, DoxyR CSCs are then more susceptible to other metabolic perturbations, because of their metabolic inflexibility, allowing for their eradication with natural products and other FDA-approved drugs. Thus, understanding the metabolic basis of Doxycycline-resistance has ultimately helped us to develop a new synthetic lethal strategy, for more effectively targeting CSCs.

結論

現在，許多研究直接表明，線粒體是癌細胞中重要的新治療靶點[3，5，8–21，40–53]。 由於多西環素是FDA批准的抗生素，可作為線粒體蛋白繁殖的抑製劑，因此在癌細胞中線粒體的特異性靶向中可能具有治療價值。 但是，在本文中，我們通過假定純糖酵解表型，確定了一種新的代謝機制，通過該機制，CSC可以成功擺脫強力黴素的抗線粒體作用。 因此，由於DoxyR CSC的新陳代謝缺乏靈活性，因此它們更容易受到其他新陳代謝擾動的影響，從而可以用天然產物和其他FDA批准的藥物根除。 因此，了解強力黴素抗性的代謝基礎最終有助於我們開發新的合成致死策略，以更有效地靶向CSC。

 

MATERIALS AND METHODS

Materials

Doxycycline, Ascorbic Acid, 2-Deoxy-D-glucose (2-DG), Irinotecan, Berberine Chloride, Niclosamide, Chloroquine diphosphate, Stiripentol and Atovaquone were all purchased from Sigma Aldrich. Sorafenib was obtained from Generon. All compounds were dissolved in DMSO, except Ascorbic Acid, 2-deoxy-D-glucose (2-DG) and Chloroquine diphosphate, which were dissolved in cell culture medium.

Cell cultures

材料和方法

用料

強力黴素，抗壞血酸，2-脫氧-D-葡萄糖（2-DG），伊立替康，黃連素氯化物，尼氯酰胺，二磷酸氯喹，苯乙烯基戊醇和阿托伐醌均購自Sigma Aldrich。 索拉非尼獲自Generon。 除抗壞血酸，2-脫氧-D-葡萄糖（2-DG）和二磷酸氯喹外，所有化合物均溶解在DMSO中，它們溶解在細胞培養基中。

細胞培養

MCF7 breast cancer cells were obtained from ATCC and cultured in DMEM (Sigma Aldrich). MCF-7 cells resistant to Doxycycline (MCF7 DoxyR) were selected by a stepwise exposure to increasing concentration of Doxycycline. In particular, wild type MCF7 cells were initially exposed to 12.5 μM Doxycycline and the dose gradually increased to 50 μM over a 3-month period. The population of resistant cells, named MCF7 DoxyR, was selected after 3 weeks of treatment with 12.5 μM Doxycycline, followed by 3 weeks of treatment with 25 μM Doxycycline. MCF7 DoxyR cells were routinely maintained in regular medium supplemented with 25 μM Doxycycline.

Mammosphere formation

MCF7乳腺癌細胞獲自ATCC，並在DMEM（Sigma Aldrich）中培養。 通過逐步暴露於濃度增加的強力黴素來選擇對強力黴素具有抗性的MCF-7細胞（MCF7 DoxyR）。 尤其是，最初將野生型MCF7細胞暴露於12.5μM強力黴素，並在3個月內逐漸增加至50μM劑量。 在用12.5μM強力黴素處理3週後，接著用25μM強力黴素處理3週後，選擇了名為MCF7 DoxyR的抗性細胞群。 MCF7 DoxyR細胞通常維持在補充有25μMDoxycycline的常規培養基中。

乳球形成

A single cell suspension of MCF7 or MCF7 DoxyR cells was prepared using enzymatic (1x Trypsin-EDTA, Sigma Aldrich), and manual disaggregation (25 gauge needle) [54]. Cells were then plated at a density of 500 cells/cm2 in mammosphere medium (DMEM-F12/B27/20-ng/ml EGF/PenStrep) in nonadherent conditions, in culture dishes coated with (2-hydroxyethylmethacrylate) (poly-HEMA, Sigma), in the presence of treatments, were required. Cells were grown for 5 days and maintained in a humidified incubator at 37°C at an atmospheric pressure in 5% (v/v) carbon dioxide/air. After 5 days for culture, spheres > 50 μm were counted using an eye piece graticule, and the percentage of cells plated which formed spheres was calculated and is referred to as percentage mammosphere formation. Mammosphere assays were performed in triplicate and repeated three times independently.

Evaluation of mitochondrial mass and function

使用酶催化（1x胰蛋白酶-EDTA，Sigma Aldrich）和手動分解（25號針頭）製備MCF7或MCF7 DoxyR細胞的單細胞懸液[54]。 然後將細胞以500個細胞/平方厘米的密度在非貼壁條件下舖於乳球狀培養基（DMEM-F12 / B27 / 20-ng / ml EGF / PenStrep）中，塗在（2-甲基丙烯酸羥乙酯）（poly-HEMA， 需要在存在治療的情況下進行。 細胞生長5天，並保持在37℃，5％（v / v）二氧化碳/空氣的大氣壓下的潮濕培養箱中。 培養5天后，使用目鏡刻度尺對大於50μm的球進行計數，併計算形成球的被鍍細胞的百分數，稱為乳球形成百分數。 三次進行乳球測定，並獨立重複三次。

線粒體質量和功能的評估

To measure mitochondrial mass by FACS analysis, cells were stained with MitoTracker Deep Red (Life Technologies), which localizes to mitochondria regardless of mitochondrial membrane potential. Cells were incubated with pre-warmed MitoTracker staining solution (diluted in PBS/CM to a final concentration of 10 nM) for 30-60 min at 37°C. All subsequent steps were performed in the dark. Cells were washed in PBS, harvested, re-suspended in 300 μL of PBS and then analyzed by flow cytometry (Fortessa, BD Bioscience). Data analysis was performed using FlowJo software. Extracellular acidification rates (ECAR) and real-time oxygen consumption rates (OCR) for MCF7 cells were determined using the Seahorse Extracellular Flux (XFe-96) analyzer (Seahorse Bioscience) [15]. Briefly, 15,000 MCF7 and MCF7 DoxyR cells per well were seeded into XFe-96 well cell culture plates for 24h. Then, cells were washed in pre-warmed XF assay media (or for OCR measurement, XF assay media supplemented with 10mM glucose, 1mM Pyruvate, 2mM L-glutamine and adjusted at 7.4 pH). Cells were then maintained in 175 μL/well of XF assay media at 37C, in a non-CO2 incubator for 1 hour. During the incubation time, 5 μL of 80mM glucose, 9 μM oligomycin, and 1 M 2-deoxyglucose (for ECAR measurement) or 10μM oligomycin, 9 μM FCCP, 10 μM Rotenone, 10 μM antimycin A (for OCR measurement), were loaded in XF assay media into the injection ports in the XFe-96 sensor cartridge. Data set was analyzed by XFe-96 software after the measurements were normalized by protein content (SRB). All experiments were performed three times independently.

為了通過FACS分析測量線粒體質量，將細胞用MitoTracker深紅色（Life Technologies）染色，無論線粒體膜電位如何，該深紅都定位於線粒體。將細胞與預熱的MitoTracker染色溶液（在PBS / CM中稀釋至最終濃度10 nM）在37°C孵育30-60分鐘。所有後續步驟均在黑暗中進行。用PBS洗滌細胞，收集細胞，將其重懸浮於300μLPBS中，然後通過流式細胞術（Fortessa，BD Bioscience）進行分析。使用FlowJo軟件進行數據分析。使用Seahorse細胞外通量（XFe-96）分析儀（Seahorse Bioscience）確定MCF7細胞的細胞外酸化率（ECAR）和實時耗氧率（OCR）[15]。簡而言之，將每孔15,000個MCF7和MCF7 DoxyR細胞接種到XFe-96孔細胞培養板中24小時。然後，將細胞在預熱的XF分析培養基中洗滌（或進行OCR測量，在XF分析培養基中添加10mM葡萄糖，1mM丙酮酸，2mM L-谷氨酰胺並調節至7.4 pH）。然後將細胞在非CO2培養箱中於37°C的175μL/孔XF分析培養基中保存1小時。在孵育期間，裝入了5μL80mM葡萄糖，9μM寡黴素和1 M 2-脫氧葡萄糖（用於ECAR測量）或10μM寡黴素，9μMFCCP，10μM魚烯酮，10μM抗黴素A（用於OCR測量）。在XF分析介質中註入XFe-96傳感器盒中的進樣口。通過蛋白質含量（SRB）對測量值進行歸一化後，通過XFe-96軟件分析數據集。所有實驗均獨立進行三次。

ALDEFLUOR assay and separation of the ALDH positive population

ALDH activity was assessed by FACS analysis (Fortessa, BD Bioscence) in MCF7 cells and MCF7 DoxyR cells. The ALDEFLUOR kit (StemCell Technologies) was used to isolate the population with high ALDH enzymatic activity. Briefly, 1 × 105 MCF7 and MCF7 DoxyR cells were incubated in 1ml ALDEFLUOR assay buffer containing ALDH substrate (5 μl/ml) for 40 minutes at 37°C. In each experiment, a sample of cells was stained under identical conditions with 30 μM of diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor, as a negative control. The ALDEFLUOR-positive population was established in according to the manufacturer's instructions and was evaluated in 3 × 104 cells. Data analysis was performed using FlowJo software.

Anoikis assay

ALDEFLUOR檢測和ALDH陽性人群的分離

通過FACS分析（Fortessa，BD Bioscence）在MCF7細胞和MCF7 DoxyR細胞中評估ALDH活性。 ALDEFLUOR試劑盒（StemCell Technologies）用於分離具有高ALDH酶活性的種群。 簡而言之，將1×105 MCF7和MCF7 DoxyR細胞在含有ALDH底物（5μl/ ml）的1ml ALDEFLUOR分析緩衝液中於37°C孵育40分鐘。 在每個實驗中，將細胞樣品在相同條件下用30μM的特異性ALDH抑製劑二乙氨基苯甲醛（DEAB）染色，作為陰性對照。 根據製造商的說明建立ALDEFLUOR陽性群體，並在3×104個細胞中進行評估。 使用FlowJo軟件進行數據分析。

諾氏測定

MCF7 and MCF7 DoxyR cells were seeded on low-attachment plates to enrich for the CSC population [54]. Under these conditions, the non-CSC population undergoes anoikis (a form of apoptosis induced by a lack of cell-substrate attachment) and CSCs are believed to survive. The surviving CSC fraction was analyzed by FACS analysis. Briefly, 1 × 105 MCF7 and MCF7 DoxyR monolayer cells were seeded for 48h in 6-well plates. Then, cells were trypsinized and seeded in low-attachment plates in mammosphere media. After 10h, cells were spun down and incubated with CD24 (IOTest CD24-PE, Beckman Coulter) and CD44 (APC mouse Anti-Human CD44, BD Pharmingen) antibodies for 15 minutes on ice. Cells were rinsed twice and incubated with LIVE/DEAD dye (Fixable Dead Violet reactive dye; Life Technologies) for 10 minutes. Samples were then analyzed by FACS (Fortessa, BD Bioscence). Only the live population, as identified by the LIVE/DEAD dye staining, was analyzed for CD24/CD44 expression. Data were analyzed using FlowJo software.

將MCF7和MCF7 DoxyR細胞接種在低附著平板上以富集CSC群體[54]。在這些條件下，非CSC群體會發生無神經症（由於缺乏細胞-基質附著而誘導的凋亡形式），並且CSC被認為可以存活。通過FACS分析分析存活的CSC部分。簡而言之，將1×105 MCF7和MCF7 DoxyR單層細胞接種到6孔板中48小時。然後，用胰蛋白酶消化細胞，並將其接種在乳房球狀培養基中的低附著平板中。 10小時後，將細胞離心分離，並與CD24（IOTest CD24-PE，Beckman Coulter）和CD44（APC小鼠抗人類CD44，BD Pharmingen）抗體在冰上孵育15分鐘。將細胞漂洗兩次，並與LIVE / DEAD染料（Fixable Dead Violet活性染料； Life Technologies）一起孵育10分鐘。然後通過FACS（Fortessa，BD Bioscence）分析樣品。僅通過LIVE / DEAD染料染色鑑定的活種群的CD24 / CD44表達被分析。使用FlowJo軟件分析數據。

Label-free semi-quantitative proteomics analysis

Cell lysates were prepared for trypsin digestion by sequential reduction of disulphide bonds with TCEP and alkylation with MMTS. Then, the peptides were extracted and prepared for LC-MS/MS. All LC-MS/MS analyses were performed on an LTQ Orbitrap XL mass spectrometer (Thermo Scientific, San Jose, CA) coupled to an Ultimate 3000 RSLC nano system (Thermo Scientific, formerly Dionex, The Netherlands). Xcalibur raw data files acquired on the LTQ-Orbitrap XL were directly imported into Progenesis LCMS software (Waters Corp., Milford, MA, formerly Non-linear dynamics, Newcastle upon Tyne, UK) for peak detection and alignment. Data were analyzed using the Mascot search engine. Five technical replicates were analyzed for each sample type [8, 12].

無標籤半定量蛋白質組學分析

通過依次用TCEP還原二硫鍵和用MMTS烷基化製備用於胰蛋白酶消化的細胞裂解液。 然後，提取肽並準備用於LC-MS / MS。 所有LC-MS / MS分析均在LTQ Orbitrap XL質譜儀（Thermo Scientific，加利福尼亞州聖何塞）上進行，該質譜儀與Ultimate 3000 RSLC納米系統（Thermo Scientific，前身為荷蘭Dionex）耦合。 將在LTQ-Orbitrap XL上獲取的Xcalibur原始數據文件直接導入Progenesis LCMS軟件（Waters Corp.，Milford，MA，前身為非線性動力學，英國泰恩河畔紐卡斯爾）以進行峰檢測和比對。 使用Mascot搜索引擎分析數據。 對每種樣品類型進行了五次技術重複分析[8，12]。

Immuno-blot analysis

MCF7 and MCF7 DoxyR cells protein lysates were electrophoresed through a reducing SDS/10% (w/v) polyacrylamide gel, electroblotted onto a nitrocellulose membrane and probed with primary antibodies against phosphorylated AKT (Ser 473) and ATK (Cell Signaling), Phopshorylated ERK 1/2 (E-4), ERK2 (C-14), TOMM20 (F-10) and β-actin (C2), all purchased from Santa Cruz Biotechnology. Proteins were detected by horseradish peroxidase-linked secondary antibodies and revealed using the SuperSignal west pico chemiluminescent substrate (Fisher Scientific).

Click-iT EdU proliferation assay

免疫印跡分析

通過還原性SDS / 10％（w / v）聚丙烯酰胺凝膠對MCF7和MCF7 DoxyR細胞蛋白裂解物進行電泳，電印跡到硝酸纖維素膜上，並用抗磷酸化AKT（Ser 473）和ATK（Cell Signaling）的一抗進行探測，磷酸化ERK 1/2（E-4），ERK2（C-14），TOMM20（F-10）和β-肌動蛋白（C2），均購自Santa Cruz Biotechnology。 通過辣根過氧化物酶連接的第二抗體檢測蛋白質，並使用SuperSignal west pico化學發光底物（Fisher Scientific）進行顯示。

Click-iT EdU增殖測定

48h after seeding MCF7 and MCF7 DoxyR were subjected to proliferation assay using Click-iT Plus EdU Pacific Blue Flow Cytometry Assay Kit (Life Technologies), customized for flow cytometry. Briefly, cells were treated with 10 μM EdU for 2 hours and then fixed and permeabilized. EdU was detected after permeabilization by staining cells with Click-iT Plus reaction cocktail containing the Fluorescent dye picolylazide for 30 min at RT. Samples were then washed and analyzed using flow cytometer (Fortessa, BD Bioscence). Background values were estimated by measuring non-EdU labeled, but Click-iT stained cells. Data were analyzed using FlowJo software.

接種後48h，使用為流式細胞儀定制的Click-iT Plus EdU太平洋藍細胞流式細胞分析試劑盒（Life Technologies）對MCF7和MCF7 DoxyR進行增殖分析。 簡而言之，將細胞用10μMEdU處理2小時，然後固定並透化。 透化後，通過在室溫下用含有熒光染料吡啶甲基疊氮化物的Click-iT Plus反應混合物對細胞進行染色30分鐘，檢測到EdU。 然後洗滌樣品並使用流式細胞儀（Fortessa，BD Bioscence）進行分析。 通過測量非EdU標記但Click-iT染色的細胞來估算背景值。 使用FlowJo軟件分析數據。

Migration assay

MCF7 and MCF7 DoxyR cells were allowed to grow in regular growth medium until they were 70-80 % confluent. Next, to create a scratch of the cell monolayer, a p200 pipette tip was used. Cells were washed twice with PBS and then incubated at 37°C in regular medium for 24h. The migration assay was evaluated using Incucyte Zoom (Essen Bioscience) [55]. The rate of migration was measured by quantifying the % of wound closure area, determined using the software ImageJ, according to the formula:

% of wound closure = [(At = 0 h - At = Δ h)/At = 0 h] × 100%

Statistical analysis

Data is represented as the mean ± standard error of the mean (SEM), taken over ≥ 3 independent experiments, with ≥ 3 technical replicates per experiment, unless otherwise stated. Statistical significance was measured using the t-test. P ≤ 0.05 was considered significant.

遷移測定

使MCF7和MCF7 DoxyR細胞在常規生長培養基中生長，直到它們達到70-80％融合為止。 接下來，為了刮擦細胞單層，使用了p200移液器吸頭。 用PBS洗滌細胞兩次，然後在37℃的常規培養基中孵育24小時。 使用Incucyte Zoom（Essen Bioscience）[55]評估遷移測定。 根據以下公式，通過使用ImageJ軟件確定傷口閉合面積的百分比來測量遷移率：

傷口閉合百分比= [（（At = 0 h-At =Δh）/ At = 0 h]×100％

統計分析

除非另有說明，否則數據表示為平均值±平均值標準誤差（SEM），取自≥3個獨立實驗，每個實驗具有≥3個技術重複。 使用t檢驗測量統計學顯著性。 P≤0.05被認為是顯著的。

Acknowledgments

We are grateful to the University of Manchester, which allocated start-up funds and administered a donation, to provide the necessary resources required to start and complete this drug discovery project (to MPL and FS). Dr. Ernestina M. De Francesco was supported by a fellowship from the Associazione Italiana per la Ricerca sul Cancro (AIRC) co-funded by the European Union. The Lisanti and Sotgia Laboratories are currently supported by private donations, and by funds from the Healthy Life Foundation (HLF) and the University of Salford (to MPL and FS). We also wish to thank Dr. Duncan Smith, who performed the proteomics analysis on whole cell lysates, within the CRUK Core Facility. MM was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC, IG 16719).

我們感謝曼徹斯特大學（University of Manchester），該大學分配了資金並進行了捐贈，以提供啟動和完成此藥物開發項目（向MPL和FS）所需的必要資源。 Ernestina M. De Francesco博士得到了由歐盟共同資助的意大利國家研究基金會的研究金（AIRC）的資助。 Lisanti和Sotgia實驗室目前得到私人捐款以及健康生活基金會（HLF）和索爾福德大學（向MPL和FS）提供資金的支持。 我們還要感謝在CRUK核心設施中對全細胞裂解物進行蛋白質組學分析的Duncan Smith博士。 MM受制於Cancro公司的意大利協會（AIRC，IG 16719）。

 

Footnotes

 

Contributed by

Author contributions

Professor Michael Lisanti and Dr. Federica Sotgia conceived and initiated this collaborative project. All the experiments in this paper were performed by Dr. Ernestina M. De Francesco, with minor technical assistance from other lab members; Dr. Ernestina M. De Francesco analyzed all the data and generated the final figures and tables, and she wrote significant portions of the manuscript. Drs. Michael P. Lisanti, Ernestina M. De Francesco, Gloria Bonuccelli, Marcello Maggiolini and Federica Sotgia all contributed to the writing and the editing of the manuscript. Professor Lisanti generated the schematic summary diagrams.

 

CONFLICTS OF INTEREST

 

MPL and FS hold a minority interest in Lunella, Inc.

 

 

 

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