The Apoptosis Mechanism of Epirubicin Combined with BCG on Human Bladder Cancer Cells

Author(s): Yang Luo, Xiaoyi Fu, Bin Han, Fafu Zhang, Lihong Yuan, Hongsheng Men, Shulin Zhang, Sujuan Tian, Bin Dong*, Minjie Meng*

Journal Name: Anti-Cancer Agents in Medicinal Chemistry
(Formerly Current Medicinal Chemistry - Anti-Cancer Agents)

Volume 20 , Issue 13 , 2020

Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Abstract:

Aims: The purpose of our study was to explore the combination effect of epirubicin and Bacillus Calmette Guerin (BCG) and its mechanism.

Background: Bladder cancer is a threat to human health worldwide. Commonly used chemotherapy drugs and biotherapy have significant therapeutic effects on bladder cancer, but the mechanism and combined effects are still unclear.

Objective: To evaluate the anti-cancer effect of epirubicin combined with BCG on human bladder cancer cells, our studies were carried out.

Methods: The viability of human bladder cancer cells with epirubicin and/or BCG treatments was examined by Cell Counting Kit-8 (CCK-8) assay. Apoptosis and cell cycle phase were determined by flow cytometry analysis. Pre-apoptosis factors of caspase-3, p53, B-cell lymphoma 2 associated X protein (Bax) and anti-apoptosis factor of B-cell lymphoma 2 (Bcl-2) were detected by western blot.

Results: The viability of human bladder cancer with epirubicin or BCG treatment was decreased and the viability with epirubicin combined with BCG treatment was decreased more, which were determined by CCK-8 assay. Both epirubicin and BCG increased the apoptosis rate of human bladder cancer and arrested more cells into G0/G1 phase, which were tested by flow cytometry. The expression of caspase-3, p53 and Bax was increased and the expression of Bcl-2 was decreased with epirubicin treatment on human bladder cells, which were analyzed by western blot. The expression of caspase-3 and p53 was increased with BCG treatment, which was examined by western blot.

Conclusion: Epirubicin induced apoptosis in human bladder cancer cells by up-regulating the expression of proapoptotic factors (caspase-3, p53 and Bax) and down-regulating the expression of anti-apoptotic factor (Bcl-2). BCG promoted apoptosis of human bladder cancer cells by up-regulating the expression of caspase-3 and p53. BCG plays a potential role at the time of the combination of epirubicin and BCG on bladder cancer cells in early stage. Both epirubicin and BCG affected cell cycle distribution via arresting more bladder cancer cells at G0/G1 phase, which ultimately led bladder cancer proliferation in vitro and promoted apoptosis.

Keywords: Human bladder cancer, epirubicin, BCG, apoptosis, CCK-8 assay, flow cytometry.

[1]
Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics, 2018. CA Cancer J. Clin., 2018, 68(1), 7-30.
[http://dx.doi.org/10.3322/caac.21442] [PMID: 29313949]
[2]
Matsuda, Y.; Seki, A.; Nonaka, K.; Kakizaki, M.; Wang, T.; Aida, J.; Ishikawa, N.; Nakano, Y.; Kaneda, D.; Takata, T.; Takahashi-Fujigasaki, J.; Murayama, S.; Takubo, K.; Ishiwata, T.; Sawabe, M.; Arai, T. Clinicopathological characteristics of distant metastases of adenocarcinoma, squamous cell carcinoma and urothelial carcinoma: An autopsy study of older Japanese patients. Geriatr. Gerontol. Int., 2018, 18(2), 211-215.
[http://dx.doi.org/10.1111/ggi.13165] [PMID: 28914998]
[3]
Morales, J.; Malles, A.; Kimble, M.; Rodriguez de la Vega, P.; Castro, G.; Nieder, A.M.; Barengo, N.C. Does health insurance modify the association between race and cancer-specific survival in patients with urinary bladder malignancy in the U.S.? Int. J. Environ. Res. Public Health, 2019, 16(18) E3393
[http://dx.doi.org/10.3390/ijerph16183393] [PMID: 31540198]
[4]
Cheng, L.; Neumann, R.M.; Nehra, A.; Spotts, B.E.; Weaver, A.L.; Bostwick, D.G. Cancer heterogeneity and its biologic implications in the grading of urothelial carcinoma. Cancer, 2000, 88(7), 1663-1670.
[http://dx.doi.org/10.1002/(SICI)1097-0142(20000401)88:7<1663: AID-CNCR21>3.0.CO;2-8] [PMID: 10738225]
[5]
Hodgson, A.; Xu, B.; Downes, M.R. p53 immunohistochemistry in high-grade urothelial carcinoma of the bladder is prognostically significant. Histopathology, 2017, 71(2), 296-304.
[http://dx.doi.org/10.1111/his.13225] [PMID: 28342221]
[6]
Kaufman, D.S.; Shipley, W.U.; Feldman, A.S. Bladder cancer. Lancet, 2009, 374(9685), 239-249.
[http://dx.doi.org/10.1016/S0140-6736(09)60491-8] [PMID: 19520422]
[7]
Suo, N.; Wang, M.; Jin, Y.; Ding, J.; Gao, X.; Sun, X.; Zhang, H.; Cui, M.; Zheng, J.; Li, N.; Jin, X.; Jiang, S. Magnetic multiwalled carbon nanotubes with controlled release of epirubicin: An intravesical instillation system for bladder cancer. Int. J. Nanomedicine, 2019, 14, 1241-1254.
[http://dx.doi.org/10.2147/IJN.S189688] [PMID: 30863057]
[8]
Jiang, H.P.; Zhu, J.X.; Peng, C.; Gao, J.; Zheng, F.; Xiao, Y.X.; Feng, Y.Q.; Yuan, B.F. Facile one-pot synthesis of a aptamer-based organic-silica hybrid monolithic capillary column by “thiol-ene” click chemistry for detection of enantiomers of chemotherapeutic anthracyclines. Analyst (Lond.), 2014, 139(19), 4940-4946.
[http://dx.doi.org/10.1039/C4AN00767K] [PMID: 25072056]
[9]
Smyth, E.; Zhang, S.; Cunningham, D.; Wotherspoon, A.; Soong, R.; Peckitt, C.; Valeri, N.; Fassan, M.; Rugge, M.; Okines, A.; Allum, W.; Stenning, S.; Nankivell, M.; Langley, R.; Tan, P. Pharmacogenetic Analysis of the UK MRC (Medical Research Council) MAGIC Trial: Association of polymorphisms with toxicity and survival in patients treated with perioperative epirubicin, cisplatin, and 5-fluorouracil (ECF) chemotherapy. Clin. Cancer Res., 2017, 23(24), 7543-7549.
[http://dx.doi.org/10.1158/1078-0432.CCR-16-3142] [PMID: 28972045]
[10]
Librenjak, D.; Situm, M.; Vrdoljak, E.; Milostić, K.; Gotovac, J. Results of long-term follow-up of patients with superficial bladder carcinoma treated with intravesically applied bacillus Calmette-Guerin vaccine according to the schedule of 6 weekly + 6 monthly instillations. Urol. Oncol., 2012, 30(3), 259-265.
[http://dx.doi.org/10.1016/j.urolonc.2010.02.007] [PMID: 20843705]
[11]
Alexandroff, A.B.; Jackson, A.M.; O’Donnell, M.A.; James, K. BCG immunotherapy of bladder cancer: 20 years on. Lancet, 1999, 353(9165), 1689-1694.
[http://dx.doi.org/10.1016/S0140-6736(98)07422-4] [PMID: 10335805]
[12]
Azadi, D.; Motallebirad, T.; Ghaffari, K.; Shojaei, H. Mycobacteriosis and Tuberculosis: Laboratory diagnosis. Open Microbiol. J., 2018, 12, 41-58.
[http://dx.doi.org/10.2174/1874285801812010041] [PMID: 29785215]
[13]
Miyake, M.; Tatsumi, Y.; Gotoh, D.; Ohnishi, S.; Owari, T.; Iida, K.; Ohnishi, K.; Hori, S.; Morizawa, Y.; Itami, Y.; Nakai, Y.; Inoue, T.; Anai, S.; Torimoto, K.; Aoki, K.; Shimada, K.; Konishi, N.; Tanaka, N.; Fujimoto, K.; Regulatory, T. Cells and tumor-associated macrophages in the tumor microenvironment in non-muscle invasive bladder cancer treated with intravesical bacille Calmette-Guérin: A long-term follow-up study of a Japanese cohort. Int. J. Mol. Sci., 2017, 18(10), 2186.
[http://dx.doi.org/10.3390/ijms18102186] [PMID: 29048388]
[14]
Lamm, D.L.B.B.; Blumenstein, B.A.; Crawford, E.D.; Montie, J.E.; Scardino, P.; Grossman, H.B.; Stanisic, T.H.; Smith, J.A., Jr; Sullivan, J.; Sarosdy, M.F. A randomized trial of intravesical doxorubicin and immunotherapy with bacille Calmette-Guérin for transitional-cell carcinoma of the bladder. N. Engl. J. Med., 1991, 325(17), 1205-1209.
[http://dx.doi.org/10.1056/NEJM199110243251703] [PMID: 1922207]
[15]
Kawai, K.; Miyazaki, J.; Joraku, A.; Nishiyama, H.; Akaza, H. Bacillus Calmette-Guerin (BCG) immunotherapy for bladder cancer: Current understanding and perspectives on engineered BCG vaccine. Cancer Sci., 2013, 104(1), 22-27.
[http://dx.doi.org/10.1111/cas.12075] [PMID: 23181987]
[16]
Begnini, K.R.; Rizzi, C.; Campos, V.F.; Borsuk, S.; Schultze, E.; Yurgel, V.C.; Nedel, F.; Dellagostin, O.A.; Collares, T.; Seixas, F.K. Auxotrophic recombinant Mycobacterium bovis BCG overexpressing Ag85B enhances cytotoxicity on superficial bladder cancer cells in vitro. Appl. Microbiol. Biotechnol., 2013, 97(4), 1543-1552.
[http://dx.doi.org/10.1007/s00253-012-4416-2] [PMID: 23053076]
[17]
Veeratterapillay, R.; Heer, R.; Johnson, M.I.; Persad, R.; Bach, C. High-risk non-muscle-invasive bladder cancer-therapy options during intravesical BCG shortage. Curr. Urol. Rep., 2016, 17(9), 68.
[http://dx.doi.org/10.1007/s11934-016-0625-z] [PMID: 27492610]
[18]
Redelman-Sidi, G.; Iyer, G.; Solit, D.B.; Glickman, M.S. Oncogenic activation of Pak1-dependent pathway of macropinocytosis determines BCG entry into bladder cancer cells. Cancer Res., 2013, 73(3), 1156-1167.
[http://dx.doi.org/10.1158/0008-5472.CAN-12-1882] [PMID: 23378476]
[19]
Younes, M.; Lechago, J.; Chakraborty, S.; Ostrowski, M.; Bridges, M.; Meriano, F.; Solcher, D.; Barroso, A.; Whitman, D.; Schwartz, J.; Johnson, C.; Schmulen, A.C.; Verm, R.; Balsaver, A.; Carlson, N.; Ertant, A. Relationship between dysplasia, p53 protein accumulation, DNA ploidy, and Glut1 overexpression in Barrett metaplasia. Scand. J. Gastroenterol., 2000, 35(2), 131-137.
[http://dx.doi.org/10.1080/003655200750024281] [PMID: 10720109]
[20]
Sylvester, R.J.; Oosterlinck, W.; Holmang, S.; Sydes, M.R.; Birtle, A.; Gudjonsson, S.; De Nunzio, C.; Okamura, K.; Kaasinen, E.; Solsona, E.; Ali-El-Dein, B.; Tatar, C.A.; Inman, B.A.; N’Dow, J.; Oddens, J.R.; Babjuk, M. Systematic review and individual patient data meta-analysis of randomized trials comparing a single immediate instillation of chemotherapy after transurethral resection with transurethral resection alone in patients with stage pTa-pT1 urothelial carcinoma of the bladder: Which patients benefit from the instillation? Eur. Urol., 2016, 69(2), 231-244.
[http://dx.doi.org/10.1016/j.eururo.2015.05.050] [PMID: 26091833]
[21]
Zhan, Y.X.; Chen, Q.B.; Lg, X.X.; Qin, X.P.; Chen, J.F.; Huang, B.Y.; Huang, J.; Zhuo, Y.M. Berberine promotes epirubicin-induced G0/G1 phase arrest in T24 bladder cancer cells. Chin. J. Pathophysiol., 2017, 33(6), 1048-1052.
[22]
Huang, T.C.; Chiu, P.R.; Chang, W.T.; Hsieh, B.S.; Huang, Y.C.; Cheng, H.L.; Huang, L.W.; Hu, Y.C.; Chang, K.L. Epirubicin induces apoptosis in osteoblasts through death-receptor and mitochondrial pathways. Apoptosis, 2018, 23(3-4), 226-236.
[http://dx.doi.org/10.1007/s10495-018-1450-2] [PMID: 29468482]
[23]
Nakano, K.; Vousden, K.H. PUMA, a novel proapoptotic gene, is induced by p53. Mol. Cell, 2001, 7(3), 683-694.
[http://dx.doi.org/10.1016/S1097-2765(01)00214-3] [PMID: 11463392]
[24]
Yu, J.; Zhang, L. PUMA, a potent killer with or without p53. Oncogene, 2008, 27(Suppl. 1), S71-S83.
[http://dx.doi.org/10.1038/onc.2009.45] [PMID: 19641508]
[25]
Reshi, L.; Wang, H.V.; Hui, C.F.; Su, Y.C.; Hong, J.R. Anti-apoptotic genes Bcl-2 and Bcl-xL overexpression can block iridovirus serine/threonine kinase-induced Bax/mitochondria-mediated cell death in GF-1 cells. Fish Shellfish Immunol., 2017, 61, 120-129.
[http://dx.doi.org/10.1016/j.fsi.2016.12.026] [PMID: 28025159]
[26]
Sinha, K.; Das, J.; Pal, P.B.; Sil, P.C. Oxidative stress: The mitochondria-dependent and mitochondria-independent pathways of apoptosis. Arch. Toxicol., 2013, 87(7), 1157-1180.
[http://dx.doi.org/10.1007/s00204-013-1034-4] [PMID: 23543009]
[27]
Chaudhary, K.S.; Lu, Q.L.; Abel, P.D.; Khandan-Nia, N.; Shoma, A.M.; el Baz, M.; Stamp, G.W.; Lalani, E.N. Expression of bcl-2 and p53 oncoproteins in schistosomiasis-associated transitional and squamous cell carcinoma of urinary bladder. Br. J. Urol., 1997, 79(1), 78-84.
[http://dx.doi.org/10.1046/j.1464-410X.1997.30717.x] [PMID: 9043502]
[28]
Weng, C.; Li, Y.; Xu, D.; Shi, Y.; Tang, H. Specific cleavage of Mcl-1 by caspase-3 in Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL)-induced apoptosis in Jurkat leukemia T cells. J. Biol. Chem., 2005, 280(11), 10491-10500.
[http://dx.doi.org/10.1074/jbc.M412819200] [PMID: 15637055]
[29]
Cohen, G.M. Caspases: The executioners of apoptosis. Biochem. J., 1997, 326(Pt 1), 1-16.
[http://dx.doi.org/10.1042/bj3260001] [PMID: 9337844]
[30]
Xu, Z.; Zhang, F.; Zhu, Y.; Liu, F.; Chen, X.; Wei, L.; Zhang, N.; Zhou, Q.; Zhong, H.; Yao, C.; Zhu, X.; Gong, C.; Zhu, S.; Zou, C. Traditional Chinese medicine Ze-Qi-Tang formula inhibit growth of non-small-cell lung cancer cells through the p53 pathway. J. Ethnopharmacol., 2019, 234, 180-188.
[http://dx.doi.org/10.1016/j.jep.2019.01.007] [PMID: 30660711]
[31]
Miyashita, T.; Reed, J.C. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell, 1995, 80(2), 293-299.
[http://dx.doi.org/10.1016/0092-8674(95)90412-3] [PMID: 7834749]
[32]
Ahmad, I.; Sansom, O.J.; Leung, H.Y. Exploring molecular genetics of bladder cancer: Lessons learned from mouse models. Dis. Model. Mech., 2012, 5(3), 323-332.
[http://dx.doi.org/10.1242/dmm.008888] [PMID: 22422829]
[33]
Gao, S.B.; Li, K.L.; Qiu, H.; Zhu, L.Y.; Pan, C.B.; Zhao, Y.; Wei, S.H.; Shi, S.; Jin, G.H.; Xue, L.X. Enhancing chemotherapy sensitivity by targeting PcG via the ATM/p53 pathway. Am. J. Cancer Res., 2017, 7(9), 1874-1883.
[PMID: 28979810]
[34]
Florescu, M.; Magda, L.S.; Enescu, O.A.; Jinga, D.; Vinereanu, D. Early detection of epirubicin-induced cardiotoxicity in patients with breast cancer. J. Am. Soc. Echocardiogr., 2014, 27(1), 83-92.
[http://dx.doi.org/10.1016/j.echo.2013.10.008] [PMID: 24268372]
[35]
Babjuk, M.; Böhle, A.; Burger, M.; Capoun, O.; Cohen, D.; Compérat, E.M.; Hernández, V.; Kaasinen, E.; Palou, J.; Rouprêt, M.; van Rhijn, B.W.; Shariat, S.F.; Soukup, V.; Sylvester, R.J.; Zigeuner, R. EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder: Update 2016. Eur. Urol., 2017, 71(3), 447-461.
[http://dx.doi.org/10.1016/j.eururo.2016.05.041] [PMID: 27324428]
[36]
Kumar, P.; John, V.; Gupta, A.; Bhaskar, S. Enhanced survival of BCG-stimulated dendritic cells: Involvement of anti-apoptotic proteins and NF-κB. Biol. Open, 2018, 7(6) bio032045
[http://dx.doi.org/10.1242/bio.032045] [PMID: 29848490]


Rights & PermissionsPrintExport Cite as

Article Details

VOLUME: 20
ISSUE: 13
Year: 2020
Published on: 14 September, 2020
Page: [1571 - 1581]
Pages: 11
DOI: 10.2174/1871520620666200502004002
Price: $65

Article Metrics

PDF: 24
HTML: 2