Induction of apoptosis by an oleanolic acid derivative in SMMC-7721 human hepatocellular carcinoma cells is associated with mitochondrial dysfunction

Fan,Xinfeng; Wang,Penglong; Sun,Yaogui; Jiang,Junbing; Du,Haiyuan; Wang,Zhirui; Duan,Zhibian; Lei,Haimin; Li,Hongquan

doi:10.3892/ol.2017.7653

Induction of apoptosis by an oleanolic acid derivative in SMMC-7721 human hepatocellular carcinoma cells is associated with mitochondrial dysfunction

Authors:
- Xinfeng Fan
- Penglong Wang
- Yaogui Sun
- Junbing Jiang
- Haiyuan Du
- Zhirui Wang
- Zhibian Duan
- Haimin Lei
- Hongquan Li
View Affiliations

Affiliations: College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu, Shanxi 030801, P.R. China, Department of Traditional Chinese Medicinal Chemistry, Beijing University of Chinese Medicine, Beijing 100000, P.R. China, Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA
Published online on: December 19, 2017 https://doi.org/10.3892/ol.2017.7653
Pages: 2821-2828
Copyright: © Fan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The aim of the present study was to investigate the effects of an oleanolic acid derivative, a novel antitumor drug, on the growth of SMMC‑7721 human hepatocellular carcinoma cells and the underlying mechanism. An MTT assay was performed to determine the cytotoxicity of the oleanolic acid derivative. Cell membrane integrity was assessed using fluorescence microscopy to assess the uptake of annexin V‑FITC/propidium iodide (PI). Western blotting was used to detect the apoptosis‑associated proteins B cell lymphoma‑2 (Bcl‑2), Bax, caspase‑9 and caspase‑3. A spectrophotometer was used to analyze the intracellular adenosine triphosphate (ATP) expression level. The loss of mitochondrial membrane potential was detected by performing the JC‑1 assay. ELISA was used to evaluate the content of cytochrome c (Cyt‑C). The oleanolic acid derivative reduced the cell viability of SMMC‑7721 cells in a dose‑ and time‑dependent manner. The half maximal inhibitory concentration values of the oleanolic acid derivative in SMMC‑7721 cells at 24, 48 and 72 h were 26.80, 11.85, and 6.66 µM, respectively. The antiapoptotic‑protein Bcl‑2 was downregulated, and the proapoptotic protein Bax was upregulated following treatment with the oleanolic acid derivative for 48 h. The oleanolic acid derivative induced the cleavage of caspase‑9 and caspase‑3 as well as promoted annexin V‑FITC/PI uptake in SMMC‑7721 cells. Furthermore, treatment of SMMC‑7721 cells with the oleanolic acid derivative induced a reduction of the intracellular ATP expression level, loss of ΔΨm and Cyt‑C release from the mitochondria. The oleanolic acid derivative induced apoptosis in SMMC‑7721 human cells. Mitochondrial dysfunction was involved in the anticancer effects of this derivative on SMMC-7721 human cells.

View Figures

View References

1	El-Serag HB: Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology. 142:1264–1273.e1. 2012. View Article : Google Scholar
2	Bruix J and Sherman M: American Association for the Study of Liver Diseases: Management of hepatocellular carcinoma: An update. Hepatology. 53:1020–1022. 2011. View Article : Google Scholar
3	Ng IO, Liu CL, Fan ST and Ng M: Expression of P-glycoprotein in hepatocellular carcinoma. A determinant of chemotherapy response. Am J Clin Pathol. 113:355–363. 2000. View Article : Google Scholar
4	Vitale A, Volk ML, Pastorelli D, Lonardi S, Farinati F, Burra P, Angeli P and Cillo U: Use of sorafenib in patients with hepatocellular carcinoma before liver transplantation: A cost-benefit analysis while awaiting data on sorafenib safety. Hepatology. 51:165–173. 2010. View Article : Google Scholar
5	Liu L, Fu J, Li T, Cui R, Ling J, Yu X, Ji H and Zhang Y: NG, a novel PABA/NO-based oleanolic acid derivative, induces human hepatoma cell apoptosis via a ROS/MAPK-dependent mitochondrial pathway. Eur J Pharmacol. 691:61–68. 2012. View Article : Google Scholar
6	Wei J, Liu M, Liu H, Wang H, Wang F, Zhang Y, Han L and Lin X: Oleanolic acid arrests cell cycle and induces apoptosis via ROS-mediated mitochondrial depolarization and lysosomal membrane permeabilization in human pancreatic cancer cells. J Appl Toxicol. 33:756–765. 2013. View Article : Google Scholar
7	Zheng QY, Li PP, Jin FS, Yao C, Zhang GH, Zang T and Ai X: Ursolic acid induces ER stress response to activate ASK1-JNK signaling and induce apoptosis in human bladder cancer T24 cells. Cell Signal. 25:206–213. 2013. View Article : Google Scholar
8	Arur S, Uche UE, Rezaul K, Fong M, Scranton V, Cowan AE, Mohler W and Han DK: Annexin I is an endogenous ligand that mediates apoptotic cell engulfment. Dev Cell. 4:587–598. 2003. View Article : Google Scholar
9	Kuida K: Caspase-9. Int J Biochem Cell Biol. 32:121–124. 2000. View Article : Google Scholar
10	Bossy-Wetzel E, Newmeyer DD and Green DR: Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 17:37–49. 1998. View Article : Google Scholar
11	Mourdjeva M, Kyurkchiev D, Mandinova A, Altankova I, Kehayov I and Kyurkchiev S: Dynamics of membrane translocation of phosphatidylserine during apoptosis detected by a monoclonal antibody. Apoptosis. 10:209–217. 2005. View Article : Google Scholar
12	Joza N, Susin SA, Daugas E, Stanford WL, Cho SK, Li CY, Sasaki T, Elia AJ, Cheng HY, Ravagnan L, et al: Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature. 410:549–554. 2001. View Article : Google Scholar
13	Houston MA, Augenlicht LH and Heerdt BG: Stable differences in intrinsic mitochondrial membrane potential of tumor cell subpopulations reflect phenotypic heterogeneity. Int J Cell Biol. 2011:9785832011. View Article : Google Scholar
14	Hao Z, Duncan GS, Chang CC, Elia A, Fang M, Wakeham A, Okada H, Calzascia T, Jang Y, You-Ten A, et al: Specific ablation of the apoptotic functions of cytochrome C reveals a differential requirement for cytochrome C and Apaf-1 in apoptosis. Cell. 121:579–591. 2005. View Article : Google Scholar
15	Samuel S, Tumilasci VF, Oliere S, Nguyên TL, Shamy A, Bell J and Hiscott J: VSV oncolysis in combination with the BCL-2 inhibitor obatoclax overcomes apoptosis resistance in chronic lymphocytic leukemia. Mol Ther. 18:2094–2103. 2010. View Article : Google Scholar
16	Barbu EM, Shirazi F, McGrath DM, Albert N, Sidman RL, Pasqualini R, Arap W and Kontoyiannis DP: An antimicrobial peptidomimetic induces Mucorales cell death through mitochondria-mediated apoptosis. PLoS One. 8:e769812013. View Article : Google Scholar
17	Jang JH, Cho YC, Kim KH, Lee KS, Lee J, Kim DE, Park JS, Jang BC, Kim S, Kwon TK and Park JW: BAI, a novel Cdk inhibitor, enhances farnesyltransferase inhibitor LB42708-mediated apoptosis in renal carcinoma cells through the downregulation of Bcl-2 and c-FLIP (L). Int J Oncol. 45:1680–1690. 2014. View Article : Google Scholar
18	Wang X, Beitler JJ, Wang H, Lee MJ, Huang W, Koenig L, Nannapaneni S, Amin AR, Bonner M, Shin HJ, et al: Honokiol enhances paclitaxel efficacy in multi-drug resistant human cancer model through the induction of apoptosis. PLoS One. 9:e863692014. View Article : Google Scholar
19	Vacca RA, Valenti D, Bobba A, Merafina RS, Passarella S and Marra E: Cytochrome c is released in a reactive oxygen species-dependent manner and is degraded via caspase-like proteases in tobacco Bright-Yellow 2 cells en route to heat shock-induced cell death. Plant Physiol. 141:208–219. 2006. View Article : Google Scholar
20	Bobba A, Atlante A, Giannattasio S, Sgaramella G, Calissano P and Marra E: Early release and subsequent caspase-mediated degradation of cytochrome c in apoptotic cerebellar granule cells. FEBS Lett. 457:126–130. 1999. View Article : Google Scholar
21	Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES and Wang X: Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell. 91:479–489. 1997. View Article : Google Scholar
22	Zou H, Li Y, Liu X and Wang X: An APAF1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem. 274:11549–11556. 1999. View Article : Google Scholar
23	Yoshida T, Okuyama H, Nakayama M, Nishimura K, Nonomura N and Inoue M: Rapid decrease of ATP followed by necrosis-like cell death in bladder cancer cells after exposure to high-dose chemotherapeutics used in intravesical therapy. Cancer Res. 74:13412014. View Article : Google Scholar
24	Galluzzi L and Kroemer G: Necroptosis: A specialized pathway of programmed necrosis. Cell. 135:1161–1163. 2008. View Article : Google Scholar : PubMed/NCBI