Digitoxin inhibits HeLa cell growth through the induction of G2/M cell cycle arrest and apoptosis in vitro and in vivo

Gan,Hua; Qi,Ming; Chan,Chakpiu; Leung,Pan; Ye,Geni; Lei,Yuhe; Liu,Aiai; Xue,Feifei; Liu,Dongdong; Ye,Wencai; Zhang,Dongmei; Deng,Lijuan; Chen,Jiaxu

doi:10.3892/ijo.2020.5070

Digitoxin inhibits HeLa cell growth through the induction of G2/M cell cycle arrest and apoptosis in vitro and in vivo

Authors:
- Hua Gan
- Ming Qi
- Chakpiu Chan
- Pan Leung
- Geni Ye
- Yuhe Lei
- Aiai Liu
- Feifei Xue
- Dongdong Liu
- Wencai Ye
- Dongmei Zhang
- Lijuan Deng
- Jiaxu Chen
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Affiliations: Formula‑pattern Research Center, School of Traditional Chinese Medicine, Jinan University, Guangzhou, Guangdong 510632, P.R. China, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine and New Drugs Research, Jinan University, Guangzhou, Guangdong 510632, P.R. China, Department of Pharmacy, Shenzhen Hospital of Guangzhou University of Chinese Medicine, Shenzhen, Guangdong 518034, P.R. China
Published online on: May 25, 2020 https://doi.org/10.3892/ijo.2020.5070
Pages: 562-573

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Abstract

Cervical cancer is the fourth most common gynecological malignancy affecting the health of women worldwide and the second most common cause of cancer‑related mortality among women in developing regions. Thus, the development of effective chemotherapeutic drugs for the treatment of cervical cancer has become an important issue in the medical field. The application of natural products for the prevention and treatment of various diseases, particularly cancer, has always attracted widespread attention. In the present study, a library of natural products composed of 78 single compounds was screened and it was found that digitoxin exhibited the highest cytotoxicity against HeLa cervical cancer cells with an IC50 value of 28 nM at 48 h. Furthermore, digitoxin exhibited extensive antitumor activities in a variety of malignant cell lines, including the lung cancer cell line, A549, the hepatoma cell line, MHCC97H, and the colon cancer cell line, HCT116. Mechanistically, digitoxin caused DNA double‑stranded breaks (DSBs), inhibited the cell cycle at the G2/M phase via the ataxia telangiectasia mutated serine/threonine kinase (ATM)/ATM and Rad3‑related serine/threonine kinase (ATR)‑checkpoint kinase (CHK1)/checkpoint kinase 2 (CHK2)‑Cdc25C pathway and ultimately triggered mitochondrial apoptosis, which was characterized by the disruption of Bax/Bcl‑2, the release of cytochrome c and the sequential activation of caspases and poly(ADP‑ribose) polymerase (PARP). In addition, the in vivo anticancer effect of digitoxin was confirmed in HeLa cell xenotransplantation models. On the whole, the findings of the present study demonstrate the efficacy of digitoxin against cervical cancer in vivo and elucidate its molecular mechanisms, including DSBs, cell cycle arrest and mitochondrial apoptosis. These results will contribute to the development of digitoxin as a chemotherapeutic agent in the treatment of cervical cancer.

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1	Bonde JH, Sandri MT, Gary DS and Andrews JC: Clinical utility of human papillomavirus genotyping in cervical cancer screening: A systematic review. J Low Genit Tract Dis. 24:1–13. 2020. View Article : Google Scholar :
2	Wang R, Pan W, Jin L, Huang W, Li Y, Wu D, Gao C, Ma D and Liao S: Human papillomavirus vaccine against cervical cancer: Opportunity and challenge. Cancer Lett. 471:88–102. 2020. View Article : Google Scholar
3	Eskander RN and Tewari KS: Chemotherapy in the treatment of metastatic, persistent, and recurrent cervical cancer. Curr Opin Obstet Gynecol. 26:314–321. 2014. View Article : Google Scholar : PubMed/NCBI
4	Monk BJ and Tewari KS: Evidence-based therapy for recurrent cervical cancer. J Clin Oncol. 32:2687–2690. 2014. View Article : Google Scholar : PubMed/NCBI
5	Liontos M, Kyriazoglou A, Dimitriadis I, Dimopoulos MA and Bamias A: Systemic therapy in cervical cancer: 30 years in review. Crit Rev Oncol Hematol. 137:9–17. 2019. View Article : Google Scholar : PubMed/NCBI
6	Li H, Wu X and Cheng X: Advances in diagnosis and treatment of metastatic cervical cancer. J Gynecol Oncol. 27:e432016. View Article : Google Scholar : PubMed/NCBI
7	Gokduman K: Strategies targeting DNA topoisomerase I in cancer chemotherapy: Camptothecins, nanocarriers for camp-tothecins, organic non-camptothecin compounds and metal complexes. Curr Drug Targets. 17:1928–1939. 2016. View Article : Google Scholar
8	Khanna C, Rosenberg M and Vail DM: A review of paclitaxel and novel formulations including those suitable for use in dogs. J Vet Intern Med. 29:1006–1012. 2015. View Article : Google Scholar : PubMed/NCBI
9	Newman DJ and Cragg GM: Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod. 75:311–335. 2012. View Article : Google Scholar : PubMed/NCBI
10	Carter GT: Natural products and Pharma 2011: Strategic changes spur new opportunities. Nat Prod Rep. 28:1783–1789. 2011. View Article : Google Scholar : PubMed/NCBI
11	Cragg GM and Newman DJ: Natural products: A continuing source of novel drug leads. Biochim Biophys Acta. 1830:3670–3695. 2013. View Article : Google Scholar : PubMed/NCBI
12	Harvey AL, Edrada-Ebel R and Quinn RJ: The re-emergence of natural products for drug discovery in the genomics era. Nat Rev Drug Discov. 14:111–129. 2015. View Article : Google Scholar : PubMed/NCBI
13	Mishra BB and Tiwari VK: Natural products: An evolving role in future drug discovery. Eur J Med Chem. 46:4769–4807. 2011. View Article : Google Scholar : PubMed/NCBI
14	Newman DJ and Cragg GM: Natural products as sources of new drugs from 1981 to 2014. J Nat Prod. 79:629–661. 2016. View Article : Google Scholar : PubMed/NCBI
15	Patel S: Plant-derived cardiac glycosides: Role in heart ailments and cancer management. Biomed Pharmacother. 84:1036–1041. 2016. View Article : Google Scholar : PubMed/NCBI
16	López-Lázaro M, Pastor N, Azrak SS, Ayuso MJ, Austin CA and Cortés F: Digitoxin inhibits the growth of cancer cell lines at concentrations commonly found in cardiac patients. J Nat Prod. 68:1642–1645. 2005. View Article : Google Scholar : PubMed/NCBI
17	Zhang YZ, Chen X, Fan XX, He JX, Huang J, Xiao DK, Zhou YL, Zheng SY, Xu JH, Yao XJ, et al: Compound library screening identified cardiac glycoside digitoxin as an effective growth inhibitor of gefitinib‑resistant non‑small cell lung cancer via downregulation of α-tubulin and inhibition of microtubule formation. Molecules. 21:3742016. View Article : Google Scholar
18	Iyer AKV, Zhou M, Azad N, Elbaz H, Wang L, Rogalsky DK, Rojanasakul Y, O'Doherty GA and Langenhan JM: A direct comparison of the anticancer activities of digitoxin MeON-Neoglycosides and O-Glycosides: Oligosaccharide chain length-dependent induction of caspase-9-mediated apoptosis. ACS Med Chem Lett. 1:326–330. 2010. View Article : Google Scholar : PubMed/NCBI
19	Trenti A, Boscaro C, Tedesco S, Cignarella A, Trevisi L and Bolego C: Effects of digitoxin on cell migration in ovarian cancer inflammatory microenvironment. Biochem Pharmacol. 154:414–423. 2018. View Article : Google Scholar : PubMed/NCBI
20	Hsu IL, Chou CY, Wu YY, Wu JE, Liang CH, Tsai YT, Ke JY, Chen YL, Hsu KF and Hong TM: Targeting FXYD2 by cardiac glycosides potently blocks tumor growth in ovarian clear cell carcinoma. Oncotarget. 7:62925–62938. 2016. View Article : Google Scholar : PubMed/NCBI
21	Prassas I, Karagiannis GS, Batruch I, Dimitromanolakis A, Datti A and Diamandis EP: Digitoxin-induced cytotoxicity in cancer cells is mediated through distinct kinase and interferon signaling networks. Mol Cancer Ther. 10:2083–2093. 2011. View Article : Google Scholar : PubMed/NCBI
22	Shang Z and Zhang L: Digitoxin increases sensitivity of glioma stem cells to TRAIL-mediated apoptosis. Neurosci Lett. 653:19–24. 2017. View Article : Google Scholar : PubMed/NCBI
23	Lee DH, Cheul Oh S, Giles AJ, Jung J, Gilbert MR and Park DM: Cardiac glycosides suppress the maintenance of stemness and malignancy via inhibiting HIF-1α in human glioma stem cells. Oncotarget. 8:40233–40245. 2017. View Article : Google Scholar : PubMed/NCBI
24	Pollard BS, Suckow MA, Wolter WR, Starr JM, Eidelman O, Dalgard CL, Kumar P, Battacharyya S, Srivastava M, Biswas R, et al: Digitoxin Inhibits epithelial-to-mesenchymal-transition in hereditary castration resistant prostate cancer. Front Oncol. 9:6302019. View Article : Google Scholar : PubMed/NCBI
25	Liu M, Feng LX, Sun P, Liu W, Mi T, Lei M, Wu W, Jiang B, Yang M, Hu L, et al: Knockdown of apolipoprotein E enhanced sensitivity of Hep3B cells to cardiac steroids via regulating Na+/K+-ATPase signalosome. Mol Cancer Ther. 15:2955–2965. 2016. View Article : Google Scholar : PubMed/NCBI
26	Felth J, Rickardson L, Rosén J, Wickström M, Fryknäs M, Lindskog M, Bohlin L and Gullbo J: Cytotoxic effects of cardiac glycosides in colon cancer cells, alone and in combination with standard chemotherapeutic drugs. J Nat Prod. 72:1969–1974. 2009. View Article : Google Scholar : PubMed/NCBI
27	Xiao Y, Yan W, Guo L, Meng C, Li B, Neves H, Chen PC, Li L, Huang Y, Kwok HF, et al: Digitoxin synergizes with sorafenib to inhibit hepatocelluar carcinoma cell growth without inhibiting cell migration. Mol Med Rep. 15:941–947. 2017. View Article : Google Scholar
28	Einbond LS, Shimizu M, Ma H, Wu HA, Goldsberry S, Sicular S, Panjikaran M, Genovese G and Cruz E: Actein inhibits the Na+-K+-ATPase and enhances the growth inhibitory effect of digitoxin on human breast cancer cells. Biochem Biophys Res Commun. 375:608–613. 2008. View Article : Google Scholar : PubMed/NCBI
29	Einbond LS, Wu HA, Sandu C, Ford M, Mighty J, Antonetti V, Redenti S and Ma H: Digitoxin enhances the growth inhibitory effects of thapsigargin and simvastatin on ER negative human breast cancer cells. Fitoterapia. 109:146–154. 2016. View Article : Google Scholar
30	Kulkarni YM, Kaushik V, Azad N, Wright C, Rojanasakul Y, O'Doherty G and Iyer AK: Autophagy-induced apoptosis in lung cancer cells by a novel digitoxin analog. J Cell Physiol. 231:817–828. 2016. View Article : Google Scholar :
31	Trenti A, Zulato E, Pasqualini L, Indraccolo S, Bolego C and Trevisi L: Therapeutic concentrations of digitoxin inhibit endothelial focal adhesion kinase and angiogenesis induced by different growth factors. Br J Pharmacol. 174:3094–3106. 2017. View Article : Google Scholar : PubMed/NCBI
32	Deng LJ, Peng QL, Wang LH, Xu J, Liu JS, Li YJ, Zhuo ZJ, Bai LL, Hu LP, Chen WM, et al: Arenobufagin intercalates with DNA leading to G2 cell cycle arrest via ATM/ATR pathway. Oncotarget. 6:34258–34275. 2015. View Article : Google Scholar : PubMed/NCBI
33	Silva IT, Munkert J, Nolte E, Zanchett Schneider NF, Carvalho Rocha S, Pacheco Ramos AC, Kreis W, Castro Braga F, de Pádua RM, et al: Cytotoxicity of AMANTADIG - a semi-synthetic digitoxigenin derivative - alone and in combination with docetaxel in human hormone-refractory prostate cancer cells and its effect on Na⁺/K⁺-ATPase inhibition. Biomed Pharmacother. 107:464–474. 2018. View Article : Google Scholar : PubMed/NCBI
34	Johansson S, Lindholm P, Gullbo J, Larsson R, Bohlin L and Claeson P: Cytotoxicity of digitoxin and related cardiac glycosides in human tumor cells. Anticancer Drugs. 12:475–483. 2001. View Article : Google Scholar : PubMed/NCBI
35	Marzo-Mas A, Barbier P, Breuzard G, Allegro D, Falomir E, Murga J, Carda M, Peyrot V and Marco JA: Interactions of long-chain homologues of colchicine with tubulin. Eur J Med Chem. 126:526–535. 2017. View Article : Google Scholar
36	Stark GR and Taylor WR: Control of the G2/M transition. Mol Biotechnol. 32:227–248. 2006. View Article : Google Scholar : PubMed/NCBI
37	Kastan MB and Bartek J: Cell-cycle checkpoints and cancer. Nature. 432:316–323. 2004. View Article : Google Scholar : PubMed/NCBI
38	Löbrich M, Shibata A, Beucher A, Fisher A, Ensminger M, Goodarzi AA, Barton O and Jeggo PA: gammaH2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization. Cell Cycle. 9:662–669. 2010. View Article : Google Scholar : PubMed/NCBI
39	Matsuoka S, Rotman G, Ogawa A, Shiloh Y, Tamai K and Elledge SJ: Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro. Proc Natl Acad Sci USA. 97:10389–10394. 2000. View Article : Google Scholar : PubMed/NCBI
40	Zhao H and Piwnica-Worms H: ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1. Mol Cell Biol. 21:4129–4139. 2001. View Article : Google Scholar : PubMed/NCBI
41	Perdiguero E and Nebreda AR: Regulation of Cdc25C activity during the meiotic G2/M transition. Cell Cycle. 3:733–737. 2004. View Article : Google Scholar : PubMed/NCBI
42	Gupta S and Bhattacharyya B: Antimicrotubular drugs binding to vinca domain of tubulin. Mol Cell Biochem. 253:41–47. 2003. View Article : Google Scholar : PubMed/NCBI
43	Jordan MA, Thrower D and Wilson L, Jordan MA, Thrower D and Wilson L: Mechanism of inhibition of cell proliferation by Vinca alkaloids. Cancer Res. 51:2212–2222. 1991.PubMed/NCBI
44	Johnson IS, Armstrong JG, Gorman M and Burnett JP Jr: The vinca alkaloids, a new class of oncolytic agents. Cancer Res. 23:1390–1427. 1963.PubMed/NCBI
45	Sadeghi-Aliabadi H, Minaiyan M and Dabestan A: Cytotoxic evaluation of doxorubicin in combination with simvastatin against human cancer cells. Res Pharm Sci. 5:127–133. 2010.PubMed/NCBI
46	Yin Y, Lian BP, Xia YZ, Shao YY and Kong LY: Design, synthesis and biological evaluation of resveratrol-cinnamoyl derivates as tubulin polymerization inhibitors targeting the colchicine binding site. Bioorg Chem. 93:1033192019. View Article : Google Scholar : PubMed/NCBI
47	Hosseini M, Taherkhani M and Ghorbani Nohooji M: Introduction of Adonis aestivalis as a new source of effective cytotoxic cardiac glycoside. Nat Prod Res. 33:915–920. 2019. View Article : Google Scholar
48	Laiho M and Latonen L: Cell cycle control, DNA damage checkpoints and cancer. Ann Med. 35:391–397. 2003. View Article : Google Scholar : PubMed/NCBI
49	Harper JW and Elledge SJ: The DNA damage response: Ten years after. Mol Cell. 28:739–745. 2007. View Article : Google Scholar : PubMed/NCBI
50	Durocher D and Jackson SP: DNA-PK, ATM and ATR as sensors of DNA damage: variations on a theme? Curr Opin Cell Biol. 13:225–231. 2001. View Article : Google Scholar : PubMed/NCBI
51	Elbaz HA, Stueckle TA, Wang HYL, O'Doherty GA, Lowry DT, Sargent LM, Wang L, Dinu CZ and Rojanasakul Y: Digitoxin and a synthetic monosaccharide analog inhibit cell viability in lung cancer cells. Toxicol Appl Pharmacol. 258:51–60. 2012. View Article : Google Scholar :
52	Nurse P: A long twentieth century of the cell cycle and beyond. Cell. 100:71–78. 2000. View Article : Google Scholar : PubMed/NCBI
53	Bloom J and Cross FR: Multiple levels of cyclin specificity in cell-cycle control. Nat Rev Mol Cell Biol. 8:149–160. 2007. View Article : Google Scholar : PubMed/NCBI
54	Fisher DL and Nurse P: A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins. EMBO J. 15:850–860. 1996. View Article : Google Scholar : PubMed/NCBI
55	Hayles J, Fisher D, Woollard A and Nurse P: Temporal order of S phase and mitosis in fission yeast is determined by the state of the p34cdc2-mitotic B cyclin complex. Cell. 78:813–822. 1994. View Article : Google Scholar : PubMed/NCBI
56	Gould KL and Nurse P: Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature. 342:39–45. 1989. View Article : Google Scholar : PubMed/NCBI
57	Rahimtoola SH: Digitalis therapy for patients in clinical heart failure. Circulation. 109:2942–2946. 2004. View Article : Google Scholar : PubMed/NCBI
58	Lapostolle F, Borron SW, Verdier C, Taboulet P, Guerrier G, Adnet F, Clemessy JL, Bismuth C and Baud FJ: Digoxin‑specific Fab fragments as single first‑line therapy in digitalis poisoning. Crit Care Med. 36:3014–3018. 2008. View Article : Google Scholar : PubMed/NCBI
59	Haux J, Klepp O, Spigset O and Tretli S: Digitoxin medication and cancer; case control and internal dose-response studies. BMC Cancer. 1:112001. View Article : Google Scholar : PubMed/NCBI
60	Eskiocak U, Ramesh V, Gill JG, Zhao Z, Yuan SW, Wang M, Vandergriff T, Shackleton M, Quintana E, Johnson TM, et al: Synergistic effects of ion transporter and MAP kinase pathway inhibitors in melanoma. Nat Commun. 7:123362016. View Article : Google Scholar : PubMed/NCBI
61	Daniel D, Süsal C, Kopp B, Opelz G and Terness P: Apoptosis-mediated selective killing of malignant cells by cardiac steroids: maintenance of cytotoxicity and loss of cardiac activity of chemically modified derivatives. Int Immunopharmacol. 3:1791–1801. 2003. View Article : Google Scholar : PubMed/NCBI
62	López-Lázaro M: Digitoxin as an anticancer agent with selectivity for cancer cells: Possible mechanisms involved. Expert Opin Ther Targets. 11:1043–1053. 2007. View Article : Google Scholar : PubMed/NCBI
63	Zhao B, Kim J, Ye X, Lai ZC and Guan KL: Both TEAD-binding and WW domains are required for the growth stimulation and oncogenic transformation activity of yes-associated protein. Cancer Res. 69:1089–1098. 2009. View Article : Google Scholar : PubMed/NCBI
64	Yakisich JS, Azad N, Venkatadri R, Kulkarni Y, Wright C, Kaushik V, O'Doherty GA and Iyer AK: Digitoxin and its synthetic analog MonoD have potent antiproliferative effects on lung cancer cells and potentiate the effects of hydroxyurea and paclitaxel. Oncol Rep. 35:878–886. 2016. View Article : Google Scholar :