Celastrol induces apoptosis of human osteosarcoma cells via the mitochondrial apoptotic pathway

Yu,Xiaolong; Zhou,Xin; Fu,Changlin; Wang,Qiang; Nie,Tao; Zou,Fan; Guo,Runsheng; Liu,Hucheng; Zhang,Bin; Dai,Min

doi:10.3892/or.2015.4124

Celastrol induces apoptosis of human osteosarcoma cells via the mitochondrial apoptotic pathway

Authors:
- Xiaolong Yu
- Xin Zhou
- Changlin Fu
- Qiang Wang
- Tao Nie
- Fan Zou
- Runsheng Guo
- Hucheng Liu
- Bin Zhang
- Min Dai
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Affiliations: Department of Orthopedics, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China
Published online on: July 13, 2015 https://doi.org/10.3892/or.2015.4124
Pages: 1129-1136
Copyright: © Yu et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

Celastrol is an active compound extracted from the root bark of Triptergium wilfordii Hook F., also known as ̔Thunder of God Vine̓. It is a well-known Chinese medicinal herb that was found to inhibit tumor cell growth and promote apoptosis in several tumor cell lines. However, research into its effects on osteosarcoma cell apoptosis is still extremely limited. The present study was undertaken to determine the effect of celastrol on viability and apoptosis of osteosarcoma cells and furthermore, to illuminate the molecular mechanism of celastrol-induced osteosarcoma cell apoptosis. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to evaluate the viability of the cells following treatment with celastrol. The effect of celastrol on the apoptotic rate of the cells was evaluated by flow cytometry using Annexin V-PE/7-AAD staining assay. Fluorescence microscopy was used to detect the morphological changes in the human osteosarcoma U-2OS cell lines. The expression of Bcl-2 family proteins, caspase-3, caspase-8, caspase-9, cytochrome c and PARP was measured by western blotting. We found that celastrol significantly inhibited the growth of osteosarcoma cells in a dose-dependent manner, particularly U-2OS cells. Furthermore, we observed that celastrol upregulated the expression of the pro-apoptotic proteins Bax and cytochrome c and altered the ratio of Bax/Bcl-2, and triggered the mitochondrial apoptotic pathway, resulting in caspase-3 and -9 activation and PARP cleavage. To conclude, the results indicate that celastrol inhibits the proliferation of human osteosarcoma cancer cells by inducing apoptosis via the mitochondrial-dependent pathway.

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1	Mirabello L, Troisi RJ and Savage SA: International osteosarcoma incidence patterns in children and adolescents, middle ages and elderly persons. Int J Cancer. 125:229–234. 2009. View Article : Google Scholar : PubMed/NCBI
2	Longhi A, Errani C, De Paolis M, Mercuri M and Bacci G: Primary bone osteosarcoma in the pediatric age: State of the art. Cancer Treat Rev. 32:423–436. 2006. View Article : Google Scholar : PubMed/NCBI
3	Chou AJ and Gorlick R: Chemotherapy resistance in osteosarcoma: Current challenges and future directions. Expert Rev Anticancer Ther. 6:1075–1085. 2006. View Article : Google Scholar : PubMed/NCBI
4	Schwartz CL, Gorlick R, Teot L, Krailo M, Chen Z, Goorin A, Grier HE, Bernstein ML and Meyers P; Children's Oncology Group: Multiple drug resistance in osteogenic sarcoma: INT0133 from the Children's Oncology Group. J Clin Oncol. 25:2057–2062. 2007. View Article : Google Scholar : PubMed/NCBI
5	Wafa H and Grimer RJ: Surgical options and outcomes in bone sarcoma. Expert Rev Anticancer Ther. 6:239–248. 2006. View Article : Google Scholar : PubMed/NCBI
6	Oertel S, Blattmann C, Rieken S, Jensen A, Combs SE, Huber PE, Bischof M, Kulozik A, Debus J and Schulz-Ertner D: Radiotherapy in the treatment of primary osteosarcoma - a single center experience. Tumori. 96:582–588. 2010.PubMed/NCBI
7	Mirabello L, Troisi RJ and Savage SA: Osteosarcoma incidence and survival rates from 1973 to 2004: Data from the Surveillance, Epidemiology, and End Results Program. Cancer. 115:1531–1543. 2009. View Article : Google Scholar : PubMed/NCBI
8	Picci P, Mercuri M, Ferrari S, Alberghini M, Briccoli A, Ferrari C, Pignotti E and Bacci G: Survival in high-grade osteosarcoma: Improvement over 21 years at a single institution. Ann Oncol. 21:1366–1373. 2010. View Article : Google Scholar
9	Arai K, Sakamoto R, Kubota D and Kondo T: Proteomic approach toward molecular backgrounds of drug resistance of osteosarcoma cells in spheroid culture system. Proteomics. 13:2351–2360. 2013. View Article : Google Scholar : PubMed/NCBI
10	Robert RS, Ottaviani G, Huh WW, Palla S and Jaffe N: Psychosocial and functional outcomes in long-term survivors of osteosarcoma: A comparison of limb-salvage surgery and amputation. Pediatr Blood Cancer. 54:990–999. 2010.PubMed/NCBI
11	Shangguan WJ, Li H and Zhang YH: Induction of G2/M phase cell cycle arrest and apoptosis by ginsenoside Rf in human osteosarcoma MG 63 cells through the mitochondrial pathway. Oncol Rep. 31:3051–313. 2014.
12	Li Z, Yu Y, Sun S, Qi B, Wang W and Yu A: Niclosamide inhibits the proliferation of human osteosarcoma cell lines by inducing apoptosis and cell cycle arrest. Oncol Rep. 33:1763–1768. 2015.PubMed/NCBI
13	Wei J, Zhu Y, Xu G, Yang F, Guan Z, Wang M and Fang Y: Oxymatrine extracted from Sophora flavescens inhibited cell growth and induced apoptosis in human osteosarcoma MG-63 cells in vitro. Cell Biochem Biophys. 70:1439–1444. 2014. View Article : Google Scholar : PubMed/NCBI
14	Ahmed S, Anuntiyo J, Malemud CJ and Haqqi TM: Biological basis for the use of botanicals in osteoarthritis and rheumatoid arthritis: A review. Evid Based Complement Alternat Med. 2:301–308. 2005. View Article : Google Scholar : PubMed/NCBI
15	Li PP, He W, Yuan PF, Song SS, Lu JT and Wei W: Celastrol induces mitochondria-mediated apoptosis in hepatocellular carcinoma Bel-7402 cells. Am J Chin Med. 43:137–148. 2015. View Article : Google Scholar : PubMed/NCBI
16	Mi C, Shi H, Ma J, Han LZ, Lee JJ and Jin X: Celastrol induces the apoptosis of breast cancer cells and inhibits their invasion via downregulation of MMP-9. Oncol Rep. 32:2527–2532. 2014.PubMed/NCBI
17	Ni H, Zhao W, Kong X, Li H and Ouyang J: NF-kappa B modulation is involved in celastrol induced human multiple myeloma cell apoptosis. PLoS One. 9:e958462014. View Article : Google Scholar : PubMed/NCBI
18	Zhao X, Gao S, Ren H, Huang H, Ji W and Hao J: Inhibition of autophagy strengthens celastrol-induced apoptosis in human pancreatic cancer in vitro and in vivo models. Curr Mol Med. 14:555–563. 2014. View Article : Google Scholar : PubMed/NCBI
19	Lee HW, Jang KS, Choi HJ, Jo A, Cheong JH and Chun KH: Celastrol inhibits gastric cancer growth by induction of apoptosis and autophagy. BMB Rep. 47:697–702. 2014. View Article : Google Scholar : PubMed/NCBI
20	Zhang J, Song J, Wu D, Wang J and Dong W: Hesperetin induces the apoptosis of hepatocellular carcinoma cells via mitochondrial pathway mediated by the increased intracellular reactive oxygen species, ATP and calcium. Med Oncol. 32:1012015. View Article : Google Scholar : PubMed/NCBI
21	Pieme C, Santosh G, Tekwu E, Askun T, Aydeniz H, Ngogang J, Bhushan S and Saxena A: Fruits and barks extracts of Zanthozyllum heitzii a spice from Cameroon induce mitochondrial dependent apoptosis and G0/G1 phase arrest in human leukemia HL-60 cells. Biol Res. 47:542014. View Article : Google Scholar
22	Zhang K, Wang X, Wang C, Zheng H, Li T, Xiao S, Wang M, Fei C, Zhang L and Xue F: Investigation of quinocetone-induced mitochondrial damage and apoptosis in HepG2 cells and compared with its metabolites. Environ Toxicol Pharmacol. 39:555–567. 2015. View Article : Google Scholar : PubMed/NCBI
23	Green DR and Reed JC: Mitochondria and apoptosis. Science. 281:1309–1312. 1998. View Article : Google Scholar : PubMed/NCBI
24	Landriscina M, Laudiero G, Maddalena F, Amoroso MR, Piscazzi A, Cozzolino F, Monti M, Garbi C, Fersini A, Pucci P, et al: Mitochondrial chaperone Trap1 and the calcium binding protein Sorcin interact and protect cells against apoptosis induced by antiblastic agents. Cancer Res. 70:6577–6586. 2010. View Article : Google Scholar : PubMed/NCBI
25	Gross A, McDonnell JM and Korsmeyer SJ: BCL-2 family members and the mitochondria in apoptosis. Genes Dev. 13:1899–1911. 1999. View Article : Google Scholar : PubMed/NCBI
26	Manfredi G, Kwong JQ, Oca-Cossio JA, Woischnik M, Gajewski CD, Martushova K, D'Aurelio M, Friedlich AL and Moraes CT: BCL-2 improves oxidative phosphorylation and modulates adenine nucleotide translocation in mitochondria of cells harboring mutant mtDNA. J Biol Chem. 278:5639–5645. 2003. View Article : Google Scholar
27	Chang HY and Yang X: Proteases for cell suicide: Functions and regulation of caspases. Microbiol Mol Biol Rev. 64:821–846. 2000. View Article : Google Scholar : PubMed/NCBI
28	Cao X, Bennett RL and May WS: c-Myc and caspase-2 are involved in activating Bax during cytotoxic drug-induced apoptosis. J Biol Chem. 283:14490–14496. 2008. View Article : Google Scholar : PubMed/NCBI
29	Stennicke HR and Salvesen GS: Properties of the caspases. Biochim Biophys Acta. 1387:17–31. 1998. View Article : Google Scholar : PubMed/NCBI
30	Hui KK, Kanungo AK, Elia AJ and Henderson JT: Caspase-3 deficiency reveals a physiologic role for Smac/DIABLO in regulating programmed cell death. Cell Death Differ. 18:1780–1790. 2011. View Article : Google Scholar : PubMed/NCBI
31	Volkmann N, Marassi FM, Newmeyer DD and Hanein D: The rheostat in the membrane: BCL-2 family proteins and apoptosis. Cell Death Differ. 21:206–215. 2014. View Article : Google Scholar :
32	Renault TT and Chipuk JE: Death upon a kiss: Mitochondrial outer membrane composition and organelle communication govern sensitivity to BAK/BAX-dependent apoptosis. Chem Biol. 21:114–123. 2014. View Article : Google Scholar :
33	Westphal D, Kluck RM and Dewson G: Building blocks of the apoptotic pore: How Bax and Bak are activated and oligomerize during apoptosis. Cell Death Differ. 21:196–205. 2014. View Article : Google Scholar :
34	Chen PM, Cheng YW, Wu TC, Chen CY and Lee H: MnSOD overexpression confers cisplatin resistance in lung adenocarcinoma via the NF-κB/Snail/Bcl-2 pathway. Free Radic Biol Med. 79:127–137. 2015. View Article : Google Scholar
35	Donskow-Łysoniewska K, Brodaczewska K and Doligalska M: Heligmosomoides polygyrus antigens inhibit the intrinsic pathway of apoptosis by overexpression of survivin and Bcl-2 protein in CD4 T cells. Prion. 7:319–327. 2013. View Article : Google Scholar
36	Hengartner MO: The biochemistry of apoptosis. Nature. 407:770–776. 2000. View Article : Google Scholar : PubMed/NCBI
37	Gillies LA and Kuwana T: Apoptosis regulation at the mitochondrial outer membrane. J Cell Biochem. 115:632–640. 2014. View Article : Google Scholar : PubMed/NCBI
38	Jourdain A and Martinou JC: Mitochondrial outer-membrane permeabilization and remodelling in apoptosis. Int J Biochem Cell Biol. 41:1884–1889. 2009. View Article : Google Scholar : PubMed/NCBI
39	Wood WG, Igbavboa U, Muller WE and Eckert GP: Statins, Bcl-2, and apoptosis: Cell death or cell protection? Mol Neurobiol. 48:308–314. 2013. View Article : Google Scholar : PubMed/NCBI
40	Fulda S and Debatin KM: Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene. 25:4798–4811. 2006. View Article : Google Scholar : PubMed/NCBI
41	Villa-Morales M and Fernández-Piqueras J: Targeting the Fas/FasL signaling pathway in cancer therapy. Expert Opin Ther Targets. 16:85–101. 2012. View Article : Google Scholar : PubMed/NCBI
42	Gordon N and Kleinerman ES: Aerosol therapy for the treatment of osteosarcoma lung metastases: Targeting the Fas/FasL pathway and rationale for the use of gemcitabine. J Aerosol Med Pulm Drug Deliv. 23:189–196. 2010. View Article : Google Scholar : PubMed/NCBI