Efficacy of atovaquone on EpCAM<sup>+</sup>CD44<sup>+</sup> HCT‑116 human colon cancer stem cells under hypoxia

Fu,Changhao; Xiao,Xu; Xu,Hao; Lu,Weifei; Wang,Yi

doi:10.3892/etm.2020.9416

December-2020 Volume 20 Issue 6

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December-2020 Volume 20 Issue 6

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Article Open Access

Efficacy of atovaquone on EpCAM⁺CD44⁺ HCT‑116 human colon cancer stem cells under hypoxia

Authors:
- Changhao Fu
- Xu Xiao
- Hao Xu
- Weifei Lu
- Yi Wang
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Affiliations: Department of Regenerative Medicine, School of Pharmaceutical Sciences, Jilin University, Changchun, Jilin 130021, P.R. China, Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education, Cancer Center, The First Hospital of Jilin University, Changchun, Jilin 130021, P.R. China, Department of Animal Biotechnology, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan 450002, P.R. China

Copyright: © Fu et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Article Number: 286
|
Published online on: October 29, 2020

https://doi.org/10.3892/etm.2020.9416
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Abstract

Tumor hypoxia contributes to the development of resistance to chemotherapeutic drugs in several human cancer cell lines. Atovaquone, an anti‑malaria drug approved by the US Food and Drug Administration, has recently demonstrated anti‑cancer effects in vitro and in vivo in several cancer models. To assess the potential of atovaquone as an anti‑cancer agent under hypoxia in colorectal carcinoma, EpCAM⁺CD44⁺ colon cancer stem cells were isolated from HCT‑116 human colon cancer cells through magnetic‑activated cell sorting. The efficacy of atovaquone on cytotoxicity, tumorsphere formation, apoptosis, invasion and cell‑cycle progression under hypoxic conditions were evaluated. MTS assays indicated that atovaquone inhibited the proliferation of EpCAM⁺CD44⁺ HCT‑116 cells with a half‑maximal inhibitory concentration of 15 µM. Atovaquone inhibited tumorsphere formation and cell proliferation by causing cell‑cycle arrest in S‑phase, which induced apoptosis of EpCAM⁺CD44⁺ HCT‑116 cells, as detected by Annexin V‑FITC/PI double staining assays, and caused mitochondrial membrane potential depolarization, as determined by a JC‑1 staining assay. Reverse transcription‑quantitative PCR demonstrated increased expression of Bax and downregulation of Bcl‑2. Transwell invasion assays indicated that atovaquone inhibited the invasiveness of EpCAM⁺CD44⁺ HCT‑116 cells under hypoxia, which was associated with upregulation of MMP‑2 and ‑9 and increased expression of tissue inhibitor of MMPs (TIMP)‑1. Taken together, atovaquone reduced the tumorsphere formation and invasion ability of EpCAM⁺CD44⁺ HCT‑116 cells, at least in part by increasing the expression of TIMP‑1 and downregulating the expression of MMP‑2 and ‑9, as well as the cells' viability by inducing cell‑cycle arrest in S‑phase and induction of apoptosis via the Bcl‑2/Bax pathway under hypoxic conditions. Further studies are warranted to explore the mechanisms of action of atovaquone as a promising anticancer agent in the treatment of colorectal carcinoma.

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1	Siegel RL, Miller KD and Jemal A: Cancer statistics, 2020. CA Cancer J Clin. 70:7–30. 2020.PubMed/NCBI View Article : Google Scholar
2	Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, Hoey T, Gurney A, Huang EH, Simeone DM, et al: Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci USA. 104:10158–10163. 2007.PubMed/NCBI View Article : Google Scholar
3	Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C and De Maria R: Identification and expansion of human colon-cancer-initiating cells. Nature. 445:111–115. 2007.PubMed/NCBI View Article : Google Scholar
4	Nguyen LV, Vanner R, Dirks P and Eaves CJ: Cancer stem cells: An evolving concept. Nat Rev Cancer. 12:133–143. 2012.PubMed/NCBI View Article : Google Scholar
5	Nandy SB and Lakshmanaswamy R: Cancer stem cells and metastasis. Prog Mol Biol Transl Sci. 151:137–176. 2017.PubMed/NCBI View Article : Google Scholar
6	Todaro M, Alea MP, Di Stefano AB, Cammareri P, Vermeulen L, Iovino F, Tripodo C, Russo A, Gulotta G, Medema JP and Stassi G: Colon cancer stem cells dictate tumor growth and resist cell death by production of interleukin-4. Cell Stem Cell. 1:389–402. 2007.PubMed/NCBI View Article : Google Scholar
7	Was H, Czarnecka J, Kominek A, Barszcz K, Bernas T, Piwocka K and Kaminska B: Some chemotherapeutics-treated colon cancer cells display a specific phenotype being a combination of stem-like and senescent cell features. Cancer Biol Ther. 19:63–75. 2018.PubMed/NCBI View Article : Google Scholar
8	Szarynska M, Olejniczak A and Kmieć Z: The role of cancer stem cells in pathogenesis of colorectal cancer. Postepy Hig Med Dosw (Online). 70:1469–1482. 2016.PubMed/NCBI View Article : Google Scholar
9	Dawood S, Austin L and Cristofanilli M: Cancer stem cells: Implications for cancer therapy. Oncology (Williston Park). 28:1101–1107, 1110. 2014.PubMed/NCBI
10	Muz B, de la Puente P, Azab F and Azab AK: The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy. Hypoxia (Auckl). 3:83–92. 2015.PubMed/NCBI View Article : Google Scholar
11	Vaupel P, Mayer A and Höckel M: Tumor hypoxia and malignant progression. Methods Enzymol. 381:335–354. 2004.PubMed/NCBI View Article : Google Scholar
12	Zhou J, Schmid T, Schnitzer S and Brune B: Tumor hypoxia and cancer progression. Cancer Lett. 237:10–21. 2006.PubMed/NCBI View Article : Google Scholar
13	Stamenkovic I: Matrix metalloproteinases in tumor invasion and metastasis. Semin Cancer Biol. 10:415–433. 2000.PubMed/NCBI View Article : Google Scholar
14	Hicklin DJ and Ellis LM: Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol. 23:1011–1027. 2005.PubMed/NCBI View Article : Google Scholar
15	Graham K and Unger E: Overcoming tumor hypoxia as a barrier to radiotherapy, chemotherapy and immunotherapy in cancer treatment. Int J Nanomedicine. 13:6049–6058. 2018.PubMed/NCBI View Article : Google Scholar
16	Strese S, Fryknas M, Larsson R and Gullbo J: Effects of hypoxia on human cancer cell line chemosensitivity. BMC Cancer. 13(331)2013.PubMed/NCBI View Article : Google Scholar
17	Srivastava IK, Rottenberg H and Vaidya AB: Atovaquone, a broad spectrum antiparasitic drug, collapses mitochondrial membrane potential in a malarial parasite. J Biol Chem. 272:3961–3966. 1997.PubMed/NCBI View Article : Google Scholar
18	Takabe H, Warnken ZN, Zhang Y, Davis DA, Smyth HDC, Kuhn JG, Weitman S and Williams Iii RO: A Repurposed drug for brain cancer: Enhanced atovaquone amorphous solid dispersion by combining a spontaneously emulsifying component with a polymer carrier. Pharmaceutics. 10(60)2018.PubMed/NCBI View Article : Google Scholar
19	Fiorillo M, Lamb R, Tanowitz HB, Mutti L, Krstic-Demonacos M, Cappello AR, Martinez-Outschoorn UE, Sotgia F and Lisanti MP: Repurposing atovaquone: Targeting mitochondrial complex III and OXPHOS to eradicate cancer stem cells. Oncotarget. 7:34084–34099. 2016.PubMed/NCBI View Article : Google Scholar
20	Tian S, Chen H and Tan W: Targeting mitochondrial respiration as a therapeutic strategy for cervical cancer. Biochem Biophys Res Commun. 499:1019–1024. 2018.PubMed/NCBI View Article : Google Scholar
21	Gao X, Liu X, Shan W, Liu Q, Wang C and Zheng J, Yao H, Tang R and Zheng J: Anti-malarial atovaquone exhibits anti-tumor effects by inducing DNA damage in hepatocellular carcinoma. Am J Cancer Res. 8:1697–1711. 2018.PubMed/NCBI
22	Ke F, Yu J, Chen W, Si X, Li X, Yang F, Liao Y and Zuo Z: The anti-malarial atovaquone selectively increases chemosensitivity in retinoblastoma via mitochondrial dysfunction-dependent oxidative damage and Akt/AMPK/mTOR inhibition. Biochem Biophys Res Commun. 504:374–379. 2018.PubMed/NCBI View Article : Google Scholar
23	Ashton TM, Fokas E, Kunz-Schughart LA, Folkes LK, Anbalagan S, Huether M, Kelly CJ, Pirovano G, Buffa FM, Hammond EM, et al: The anti-malarial atovaquone increases radiosensitivity by alleviating tumour hypoxia. Nat Commun. 7(12308)2016.PubMed/NCBI View Article : Google Scholar
24	Fu C, Zhou N, Zhao Y, Duan J, Xu H and Wang Y: Dendritic cells loaded with CD44 CT-26 colon cell lysate evoke potent antitumor immune responses. Oncol Lett. 18:5897–5904. 2019.PubMed/NCBI View Article : Google Scholar
25	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001.PubMed/NCBI View Article : Google Scholar
26	Phi LTH, Sari IN, Yang YG, Lee SH, Jun N, Kim KS, Lee YK and Kwon HY: Cancer stem cells (CSCs) in drug resistance and their therapeutic implications in cancer treatment. Stem Cells Int. 2018(5416923)2018.PubMed/NCBI View Article : Google Scholar
27	Falloon J, Sargent S, Piscitelli SC, Bechtel C, LaFon SW, Sadler B, Walker RE, Kovacs JA, Polis MA, Davey RT Jr, et al: Atovaquone suspension in HIV-infected volunteers: Pharmacokinetics, pharmacodynamics, and TMP-SMX interaction study. Pharmacotherapy. 19:1050–1056. 1999.PubMed/NCBI View Article : Google Scholar
28	Cosse JP and Michiels C: Tumour hypoxia affects the responsiveness of cancer cells to chemotherapy and promotes cancer progression. Anticancer Agents Med Chem. 8:790–797. 2008.PubMed/NCBI View Article : Google Scholar
29	Zhang C, Tian Y, Song F, Fu C, Han B and Wang Y: Salinomycin inhibits the growth of colorectal carcinoma by targeting tumor stem cells. Oncol Rep. 34:2469–2476. 2015.PubMed/NCBI View Article : Google Scholar
30	Xiong B, Ma L, Hu X, Zhang C and Cheng Y: Characterization of side population cells isolated from the colon cancer cell line SW480. Int J Oncol. 45:1175–1183. 2014.PubMed/NCBI View Article : Google Scholar
31	Müller M, Hermann PC, Liebau S, Weidgang C, Seufferlein T, Kleger A and Perkhofer L: The role of pluripotency factors to drive stemness in gastrointestinal cancer. Stem Cell Res. 16:349–357. 2016.PubMed/NCBI View Article : Google Scholar
32	Larsson P, Engqvist H, Biermann J, Werner Rönnerman E, Forssell-Aronsson E, Kovács A, Karlsson P, Helou K and Parris TZ: Optimization of cell viability assays to improve replicability and reproducibility of cancer drug sensitivity screens. Sci Rep. 10(5798)2020.PubMed/NCBI View Article : Google Scholar
33	Chen D, Sun X, Zhang X and Cao J: Targeting mitochondria by anthelmintic drug atovaquone sensitizes renal cell carcinoma to chemotherapy and immunotherapy. J Bioch Mol Toxicol. 32(e22195)2018.PubMed/NCBI View Article : Google Scholar
34	Baggish AL and Hill DR: Antiparasitic agent atovaquone. Antimicrob Agents Chemother. 46:1163–1173. 2002.PubMed/NCBI View Article : Google Scholar
35	Nixon GL, Moss DM, Shone AE, Lalloo DG, Fisher N, O'Neill PM, Ward SA and Biagini GA: Antimalarial pharmacology and therapeutics of atovaquone. J Antimicrob Chemother. 68:977–985. 2013.PubMed/NCBI View Article : Google Scholar
36	Zhou J, Duan L, Chen H, Ren X, Zhang Z, Zhou F, Liu J, Pei D and Ding K: Atovaquone derivatives as potent cytotoxic and apoptosis inducing agents. Bioorg Med Chem Lett. 19:5091–5094. 2009.PubMed/NCBI View Article : Google Scholar
37	Yu L, Wan F, Dutta S, Welsh S, Liu Z, Freundt E, Baehrecke EH and Lenardo M: Autophagic programmed cell death by selective catalase degradation. Proc Natl Acad Sci USA. 103:4952–4957. 2006.PubMed/NCBI View Article : Google Scholar
38	Rambold AS and Lippincott-Schwartz J: Mechanisms of mitochondria and autophagy crosstalk. Cell Cycle. 10:4032–4038. 2011.PubMed/NCBI View Article : Google Scholar
39	Kinoshita M, Johnson DL, Shatney CH, Lee YL and Mochizuki H: Cancer cells surviving hypoxia obtain hypoxia resistance and maintain anti-apoptotic potential under reoxygenation. Int J Cancer. 91:322–326. 2001.PubMed/NCBI View Article : Google Scholar
40	Zhou H and Huang S: Role of mTOR signaling in tumor cell motility, invasion and metastasis. Curr Protein Pept Sci. 12:30–42. 2011.PubMed/NCBI View Article : Google Scholar
41	Stevens AM, Xiang M, Heppler LN, Tošić I, Jiang K, Munoz JO, Gaikwad AS, Horton TM, Long X, Narayanan P, et al: Atovaquone is active against AML by upregulating the integrated stress pathway and suppressing oxidative phosphorylation. Blood Adv. 3:4215–4227. 2019.PubMed/NCBI View Article : Google Scholar
42	Fang Z, Tang Y, Fang J, Zhou Z, Xing Z, Guo Z, Guo X, Wang W, Jiao W, Xu Z and Liu Z: Simvastatin inhibits renal cancer cell growth and metastasis via AKT/mTOR, ERK and JAK2/STAT3 pathway. PLoS One. 8(e62823)2013.PubMed/NCBI View Article : Google Scholar
43	Xiang M, Kim H, Ho VT, Walker SR, Bar-Natan M, Anahtar M, Liu S, Toniolo PA, Kroll Y, Jones N, et al: Gene expression-based discovery of atovaquone as a STAT3 inhibitor and anticancer agent. Blood. 128:1845–1853. 2016.PubMed/NCBI View Article : Google Scholar

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