Bufalin induced apoptosis in SCC‑4 human tongue cancer cells by decreasing Bcl‑2 and increasing Bax expression via the mitochondria‑dependent pathway

Chou,Han‑Yu; Chueh,Fu‑Shin; Ma,Yi‑Shih; Wu,Rick  Sai‑Chuen; Liao,Ching‑Lung; Chu,Yung‑Lin; Fan,Ming‑Jen; Huang,Wen‑Wen; Chung,Jing‑Gung

doi:10.3892/mmr.2017.7651

Bufalin induced apoptosis in SCC‑4 human tongue cancer cells by decreasing Bcl‑2 and increasing Bax expression via the mitochondria‑dependent pathway

Authors:
- Han‑Yu Chou
- Fu‑Shin Chueh
- Yi‑Shih Ma
- Rick Sai‑Chuen Wu
- Ching‑Lung Liao
- Yung‑Lin Chu
- Ming‑Jen Fan
- Wen‑Wen Huang
- Jing‑Gung Chung
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Affiliations: Department of Biological Science and Technology, China Medical University, Taichung 40402, Taiwan, R.O.C., Department of Food Nutrition and Health Biotechnology, Asia University, Taichung 41354, Taiwan, R.O.C., School of Chinese Medicine for Post‑Baccalaureate, I‑Shou University, Kaohsiung 84001, Taiwan, R.O.C., Department of Anesthesiology, China Medical University Hospital, Taichung 40402, Taiwan, R.O.C., College of Chinese Medicine, School of Post‑Baccalaureate Chinese Medicine, China Medical University, Taichung 40402, Taiwan, R.O.C., Department of Food Science, International College, National Pingtung University of Science and Technology, Pingtung 912, Taiwan, R.O.C., Department of Biotechnology, Asia University, Taichung 41354, Taiwan, R.O.C.
Published online on: September 28, 2017 https://doi.org/10.3892/mmr.2017.7651
Pages: 7959-7966
Copyright: © Chou et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The aim of the present study was to investigate the cytotoxic effects of bufalin on SCC‑4 human tongue cancer cells. Cell morphological changes and viability were examined using phase contrast microscopy and flow cytometry, respectively. The results indicated that bufalin induced morphological changes and reduced total viable cells. Apoptotic cell death was analyzed by DAPI staining and DNA gel electrophoresis; the results revealed that bufalin induced cell apoptosis. Levels of reactive oxygen species (ROS), Ca2+, nitric oxide (NO) and mitochondrial membrane potential (ΔΨm) were measured by flow cytometry, and bufalin was observed to increase Ca2+ and NO production, decrease the ΔΨm and reduce ROS production in SCC‑4 cells. In addition, western blotting was performed to detect apoptosis‑associated protein expression. The results demonstrated that bufalin reduced the expression of the anti‑apoptotic protein B‑cell lymphoma 2 (Bcl‑2) and increased the expression of the pro‑apoptotic protein, Bcl‑2‑associated X protein. However, bufalin treatment also increased the expression of other apoptosis‑associated proteins such as apoptosis‑inducing factor and endonuclease G in SCC‑4 cells. Based on these findings, bufalin may induce apoptotic cell death via mitochondria‑dependent pathways in human tongue cancer SCC‑4 cells.

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1	Khalili J: Oral cancer: Risk factors, prevention and diagnostic. Exp Oncol. 30:259–264. 2008.PubMed/NCBI
2	Lee CH, Shih YL, Lee MH, Au MK, Chen YL, Lu HF and Chung JG: Bufalin induces apoptosis of human osteosarcoma U-2 os cells through endoplasmic reticulum stress, caspase- and mitochondria-dependent signaling pathways. Molecules. 22:E4372017. View Article : Google Scholar : PubMed/NCBI
3	Gomez DR, Zhung JE, Gomez J, Chan K, Wu AJ, Wolden SL, Pfister DG, Shaha A, Shah JP, Kraus DH, et al: Intensity-modulated radiotherapy in postoperative treatment of oral cavity cancers. Int J Radiat Oncol Biol Phys. 73:1096–1103. 2009. View Article : Google Scholar : PubMed/NCBI
4	Major AG, Pitty LP and Farah CS: Cancer stem cell markers in head and neck squamous cell carcinoma. Stem Cells Int. 2013:3194892013. View Article : Google Scholar : PubMed/NCBI
5	Urban D, Corry J and Rischin D: What is the best treatment for patients with human papillomavirus-positive and -negative oropharyngeal cancer? Cancer. 120:1462–1470. 2014. View Article : Google Scholar : PubMed/NCBI
6	Krenn L and Kopp B: Bufadienolides from animal and plant sources. Phytochemistry. 48:1–29. 1998. View Article : Google Scholar : PubMed/NCBI
7	Meng Z, Yang P, Shen Y, Bei W, Zhang Y, Ge Y, Newman RA, Cohen L, Liu L, Thornton B, et al: Pilot study of huachansu in patients with hepatocellular carcinoma, nonsmall-cell lung cancer, or pancreatic cancer. Cancer. 115:5309–5318. 2009. View Article : Google Scholar : PubMed/NCBI
8	Han KQ, Huang G, Gu W, Su YH, Huang XQ and Ling CQ: Anti-tumor activities and apoptosis-regulated mechanisms of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice. World J Gastroenterol. 13:3374–3379. 2007. View Article : Google Scholar : PubMed/NCBI
9	Hashimoto S, Jing Y, Kawazoe N, Masuda Y, Nakajo S, Yoshida T, Kuroiwa Y and Nakaya K: Bufalin reduces the level of topoisomerase II in human leukemia cells and affects the cytotoxicity of anticancer drugs. Leuk Res. 21:875–883. 1997. View Article : Google Scholar : PubMed/NCBI
10	Watabe M, Ito K, Masuda Y, Nakajo S and Nakaya K: Activation of AP-1 is required for bufalin-induced apoptosis in human leukemia U937 cells. Oncogene. 16:779–787. 1998. View Article : Google Scholar : PubMed/NCBI
11	Li D, Qu X, Hou K, Zhang Y, Dong Q, Teng Y, Zhang J and Liu Y: PI3 K/Akt is involved in bufalin-induced apoptosis in gastric cancer cells. Anticancer Drugs. 20:59–64. 2009. View Article : Google Scholar : PubMed/NCBI
12	Yu CH, Kan SF, Pu HF, Jea Chien E and Wang PS: Apoptotic signaling in bufalin- and cinobufagin-treated androgen-dependent and -independent human prostate cancer cells. Cancer Sci. 99:2467–2476. 2008. View Article : Google Scholar : PubMed/NCBI
13	Shen S, Zhang Y, Wang Z, Liu R and Gong X: Bufalin induces the interplay between apoptosis and autophagy in glioma cells through endoplasmic reticulum stress. Int J Biol Sci. 10:212–224. 2014. View Article : Google Scholar : PubMed/NCBI
14	Huang WW, Yang JS, Pai SJ, Wu PP, Chang SJ, Chueh FS, Fan MJ, Chiou SM, Kuo HM, Yeh CC, et al: Bufalin induces G0/G1 phase arrest through inhibiting the levels of cyclin D cyclin E, CDK2 and CDK4 and triggers apoptosis via mitochondrial signaling pathway in T24 human bladder cancer cells. Mutat Res. 732:26–33. 2012. View Article : Google Scholar : PubMed/NCBI
15	Jiang Y, Zhang Y, Luan J, Duan H, Zhang F, Yagasaki K and Zhang G: Effects of bufalin on the proliferation of human lung cancer cells and its molecular mechanisms of action. Cytotechnology. 62:573–583. 2010. View Article : Google Scholar : PubMed/NCBI
16	Zhu Z, Li E, Liu Y, Gao Y, Sun H, Ma G, Wang Z, Liu X, Wang Q, Qu X, et al: Inhibition of Jak-STAT3 pathway enhances bufalin-induced apoptosis in colon cancer SW620 cells. World J Surg Oncol. 10:2282012. View Article : Google Scholar : PubMed/NCBI
17	Wu SH, Wu TY, Hsiao YT, Lin JH, Hsu SC, Hsia TC, Yang ST, Hsu WH and Chung JG: Bufalin induces cell death in human lung cancer cells through disruption of DNA damage response pathways. Am J Chin Med. 42:729–742. 2014. View Article : Google Scholar : PubMed/NCBI
18	Chang Y-M, Velmurugan BK, Kuo W-W, Chen Y-S, Ho T-J, Tsai C-T, Ye C-X, Tsai C-H, Tsai F-J and Huang C-Y: Inhibitory effect of alpinate Oxyphyllae fructus extracts on Ang II-induced cardiac pathological remodeling-related pathways in H9c2 cardiomyoblast cells. Biomedicine. 3:148–152. 2013. View Article : Google Scholar
19	Yu FS, Huang AC, Yang JS, Yu CS, Lu CC, Chiang JH, Chiu CF and Chung JG: Safrole induces cell death in human tongue squamous cancer SCC-4 cells through mitochondria-dependent caspase activation cascade apoptotic signaling pathways. Environ Toxicol. 27:433–444. 2012. View Article : Google Scholar : PubMed/NCBI
20	Chueh FS, Chen YL, Hsu SC, Yang JS, Hsueh SC, Ji BC, Lu HF and Chung JG: Triptolide induced DNA damage in A375.S2 human malignant melanoma cells is mediated via reduction of DNA repair genes. Oncol Rep. 29:613–618. 2013. View Article : Google Scholar : PubMed/NCBI
21	Liu KC, Yen CY, Wu RS, Yang JS, Lu HF, Lu KW, Lo C, Chen HY, Tang NY, Wu CC and Chung JG: The roles of endoplasmic reticulum stress and mitochondrial apoptotic signaling pathway in quercetin-mediated cell death of human prostate cancer PC-3 cells. Environ Toxicol. 29:428–439. 2014. View Article : Google Scholar : PubMed/NCBI
22	Lin M-C, Tsai S-Y, Wang F-Y, Liu F-H, Syu J-N and Tang F-Y: Leptin induces cell invasion and the upregulation of matrilysin in human colon cancer cells. Biomedicine. 3:174–180. 2013. View Article : Google Scholar
23	Iglesias-Guimarais V, Gil-Guinon E, Sanchez-Osuna M, Casanelles E, Garcia-Belinchon M, Comella JX and Yuste VJ: Chromatin collapse during caspase-dependent apoptotic cell death requires DNA fragmentation factor, 40-kDa subunit-/caspase-activated deoxyribonuclease-mediated 3′-OH single-strand DNA breaks. J Biol Chem. 288:9200–9215. 2013. View Article : Google Scholar : PubMed/NCBI
24	Jiang L, Zhao MN, Liu TY, Wu XS, Weng H, Ding Q, Shu YJ, Bao RF, Li ML, Mu JS, et al: Bufalin induces cell cycle arrest and apoptosis in gallbladder carcinoma cells. Tumour Biol. 35:10931–10941. 2014. View Article : Google Scholar : PubMed/NCBI
25	Zhang DM, Liu JS, Tang MK, Yiu A, Cao HH, Jiang L, Chan JY, Tian HY, Fung KP and Ye WC: Bufotalin from venenum bufonis inhibits growth of multidrug resistant HepG2 cells through G2/M cell cycle arrest and apoptosis. Eur J Pharmacol. 692:19–28. 2012. View Article : Google Scholar : PubMed/NCBI
26	Wu SH, Bau DT, Hsiao YT, Lu KW, Hsia TC, Lien JC, Ko YC, Hsu WH, Yang ST, Huang YP and Chung JG: Bufalin induces apoptosis in vitro and has Antitumor activity against human lung cancer xenografts in vivo. Environ Toxicol. 32:1305–1317. 2017. View Article : Google Scholar : PubMed/NCBI
27	Yin PH, Liu X, Qiu YY, Cai JF, Qin JM, Zhu HR and Li Q: Anti-tumor activity and apoptosis-regulation mechanisms of bufalin in various cancers: new hope for cancer patients. Asian Pac J Cancer Prev. 13:5339–5343. 2012. View Article : Google Scholar : PubMed/NCBI
28	Zhao H, Zhao D, Tan G, Liu Y, Zhuang L and Liu T: Bufalin promotes apoptosis of gastric cancer by down-regulation of miR-298 targeting bax. Int J Clin Exp Med. 8:3420–3428. 2015.PubMed/NCBI
29	Hong SH and Choi YH: Bufalin induces apoptosis through activation of both the intrinsic and extrinsic pathways in human bladder cancer cells. Oncol Rep. 27:114–120. 2012.PubMed/NCBI
30	Jing Y, Watabe M, Hashimoto S, Nakajo S and Nakaya K: Cell cycle arrest and protein kinase modulating effect of bufalin on human leukemia ML1 cells. Anticancer Res. 14:1193–1198. 1994.PubMed/NCBI
31	Numazawa S, Shinoki MA, Ito H, Yoshida T and Kuroiwa Y: Involvement of Na⁺, K(+)-ATPase inhibition in K562 cell differentiation induced by bufalin. J Cell Physiol. 160:113–120. 1994. View Article : Google Scholar : PubMed/NCBI
32	Liu K, Cang S, Ma Y and Chiao JW: Synergistic effect of paclitaxel and epigenetic agent phenethyl isothiocyanate on growth inhibition, cell cycle arrest and apoptosis in breast cancer cells. Cancer Cell Int. 13:102013. View Article : Google Scholar : PubMed/NCBI
33	Chu YL, Ho CT, Chung JG, Raghu R, Lo YC and Sheen LY: Allicin induces anti-human liver cancer cells through the p53 gene modulating apoptosis and autophagy. J Agric Food Chem. 61:9839–9848. 2013. View Article : Google Scholar : PubMed/NCBI
34	Green DR and Fitzgerald P: Just so stories about the evolution of apoptosis. Curr Biol. 26:R620–R627. 2016. View Article : Google Scholar : PubMed/NCBI
35	Li W, Zhao L, Wei T, Zhao Y and Chen C: The inhibition of death receptor mediated apoptosis through lysosome stabilization following internalization of carboxyfullerene nanoparticles. Biomaterials. 32:4030–4041. 2011. View Article : Google Scholar : PubMed/NCBI
36	Grunnet LG, Aikin R, Tonnesen MF, Paraskevas S, Blaabjerg L, Storling J, Rosenberg L, Billestrup N, Maysinger D and Mandrup-Poulsen T: Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells. Diabetes. 58:1807–1815. 2009. View Article : Google Scholar : PubMed/NCBI
37	Santin I, Moore F, Colli ML, Gurzov EN, Marselli L, Marchetti P and Eizirik DL: PTPN2, a candidate gene for type 1 diabetes, modulates pancreatic beta-cell apoptosis via regulation of the BH3-only protein Bim. Diabetes. 60:3279–3288. 2011. View Article : Google Scholar : PubMed/NCBI
38	Milutinovic S, Kashyap AK, Yanagi T, Wimer C, Zhou S, O'Neil R, Kurtzman AL, Faynboym A, Xu L, Hannum CH, et al: Dual agonist surrobody simultaneously activates death receptors dr4 and dr5 to induce cancer cell death. Mol Cancer Ther. 15:114–124. 2016. View Article : Google Scholar : PubMed/NCBI
39	Lin FL, Hsu JL, Chou CH, Wu WJ, Chang CI and Liu HJ: Activation of p38 MAPK by damnacanthal mediates apoptosis in SKHep 1 cells through the DR5/TRAIL and TNFR1/TNF-alpha and p53 pathways. Eur J Pharmacol. 650:120–129. 2011. View Article : Google Scholar : PubMed/NCBI
40	Chen Y, Azad MB and Gibson SB: Superoxide is the major reactive oxygen species regulating autophagy. Cell Death Differ. 16:1040–1052. 2009. View Article : Google Scholar : PubMed/NCBI
41	Ip SW, Chu YL, Yu CS, Chen PY, Ho HC, Yang JS, Huang HY, Chueh FS, Lai TY and Chung JG: Bee venom induces apoptosis through intracellular Ca2⁺ -modulated intrinsic death pathway in human bladder cancer cells. Int J Urol. 19:61–70. 2012. View Article : Google Scholar : PubMed/NCBI
42	Chiu TH, Lan KY, Yang MD, Lin JJ, Hsia TC, Wu CT, Yang JS, Chueh FS and Chung JG: Diallyl sulfide promotes cell-cycle arrest through the p53 expression and triggers induction of apoptosis via caspase- and mitochondria-dependent signaling pathways in human cervical cancer Ca Ski cells. Nutr Cancer. 65:505–514. 2013. View Article : Google Scholar : PubMed/NCBI
43	Tsai CW, Yang MD, Hsia TC, Chang WS, Hsu CM, Hsieh YH, Chung JG and Bau DT: Dithiothreitol enhanced arsenic-trioxide-induced cell apoptosis in cultured oral cancer cells via mitochondrial dysfunction and endoplasmic reticulum stress. Environ Toxicol. 32:17–27. 2015. View Article : Google Scholar : PubMed/NCBI
44	Mohan S, Abdelwahab SI, Kamalidehghan B, Syam S, May KS, Harmal NS, Shafifiyaz N, Hadi AH, Hashim NM, Rahmani M, et al: Involvement of NF-kappaB and Bcl2/Bax signaling pathways in the apoptosis of MCF7 cells induced by a xanthone compound Pyranocycloartobiloxanthone A. Phytomedicine. 19:1007–1015. 2012. View Article : Google Scholar : PubMed/NCBI
45	Ma YS, Hsu SC, Weng SW, Yu CC, Yang JS, Lai KC, Lin JP, Lin JG and Chung JG: Crude extract of Rheum palmatum L induced cell death in LS1034 human colon cancer cells acts through the caspase-dependent and -independent pathways. Environ Toxicol. 29:969–980. 2014. View Article : Google Scholar : PubMed/NCBI
46	Wu J, Tan Z, Chen J and Dong C: Cyclovirobuxine D inhibits cell proliferation and induces mitochondria-mediated apoptosis in human gastric cancer cells. Molecules. 20:20659–20668. 2015. View Article : Google Scholar : PubMed/NCBI
47	Chiang JH, Yang JS, Ma CY, Yang MD, Huang HY, Hsia TC, Kuo HM, Wu PP, Lee TH and Chung JG: Danthron, an anthraquinone derivative, induces DNA damage and caspase cascades-mediated apoptosis in SNU-1 human gastric cancer cells through mitochondrial permeability transition pores and Bax-triggered pathways. Chem Res Toxicol. 24:20–29. 2011. View Article : Google Scholar : PubMed/NCBI