EFFECTS OF CINOBUFAGIN ON APOPTOSIS IN U-2OS OSTEOSARCOMAS CELLS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

CAOFei ¹ , KANGXiaohong ² ,  WANGLifang ³

1. Department of Orthopaedics, First People's Hospital of Pingdingshan, Pingdingshan Henan, 467000, P. R. China;
2. Department of Clinical Oncology, First People's Hospital of Pingdingshan, Pingdingshan Henan, 467000, P. R. China;
3. Department of Medical Oncology, Longhua Hospital Affiliated to Shanghai University of Traditional Chinese Medicine;

Corresponding author：

WANGLifang, Email: wanglifang1976@yahoo.com.cn

Keywords：

Cinobufagin; U-2OS osteosarcomas cells; Cell apoptosis; Caspase-3

DOI：

10.7507/1002-1892.20140078

Video：

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Objective To investigate the effects of cinobufagin on the apoptosis in U-2OS osteosarcomas cells (U-2OS cells) and explore its potential mechanism. Methods The cytostatic effects of cinobufagin (10, 20, 50, 100, 200, and 400 nmol/L) on U-2OS cells were evaluated by MTT assay at 24, 48, and 72 hours after culture; simple U-2OS cells served as control group. The impact of cinobufagin (100 nmol/L) on the apoptosis in U-2OS cells was determined by flow cytometry at 48 hours after culture, which were treated with cinobufagin (experimental group) or with cinobufagin plus Z-VAD-FMK (control group), and simple U-2OS cells served as blank control group. The Caspase-3 activity was measured by Caspase-3 activity assay kit at 48 hours after culture, which were treated with cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group.The expression of apoptosis signal pathway related proteins in U-2OS cells treated with cinobufagin were detected by Western blot at 48 hours after culture, which were treated with cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group. Results The results of MTT assay showed that cinobufagin inhibited the proliferation of U-2OS cells in a dose- and time-dependent manners. At each time point, the growth rate of U-2OS cells was significantly reduced with the increasing cinobufagin concentration, and as time prolonged, the growth rate of U-2OS cells behaved the same way in the same group. There were significant differences among different time points and groups (P<0.05). The apoptotic rate of experimental group (46.87%±11.23%) was significantly higher than that of the control group (2.34%±0.98%) and blank control group (1.04%±0.25%) (P<0.05). The Caspase-3 activity in 20, 50, and 100 nmol/L groups were 1.14±0.32, 1.31±0.41, and 1.92±0.54, respectively, which were significantly higher than that in control group (P<0.05). Compared with 20 and 50 nmol/L groups, 100 nmol/L group significantly increased the Caspase-3 activity in U-2OS cells (P<0.05). Compared with the control group, the expressions of cleaved Caspase-3, cleaved Caspase-9, and Bax were obviously up-regulated; the Bcl-2 expression was down-regulated; and the ratio of Bax/Bcl-2 was increased in different cinobufagin-treated groups (P<0.05). The same tendency was seen in different cinobufagin-treated goups, showing significant differences among groups (P<0.05). Conclusion Cinobufagin can inhibite the proliferation of U-2OS cells, and induce cell apoptosis. The potential mechanism of cinobufagin-induced apoptosis may be related to the mitochondria-mediated pathway.

Citation： CAOFei, KANGXiaohong, WANGLifang. EFFECTS OF CINOBUFAGIN ON APOPTOSIS IN U-2OS OSTEOSARCOMAS CELLS. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(3): 349-353. doi: 10.7507/1002-1892.20140078 Copy

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7.	Zhang DM, Liu JS, Tang MK, et al. Bufotalin from Venenum Bufonis inhibits growth of multidrug resistant HepG2 cells through G2/M cell cycle arrest and apoptosis. Eur J Pharmacol, 2012, 692(1-3):19-28.
8.	He X, Hu H, Wu Y, et al. Urinary metabolites of cinobufagin in rats and their antiproliferative activities. Nat Prod Res, 2012, 26(6):489-499.
9.	Castets M, Coissieux MM, Delloye-Bourgeois C, et al. Inhibition of endothelial cell apoptosis by netrin-1 during angiogenesis. Dev Cell, 2009, 16(4):614-620.
10.	Alenzi FQ. BCL-6 prevents mammary epithelial apoptosis and promotes cell survival. J Pak Med Assoc, 2008, 58(9):494-497.
11.	Schultz DR, Hardngton WJ Jr. Apoptosis:programmed cell death at a molecular level. Semin Arthritis Rheum, 2003, 32(6):345-369.
12.	Baker JE, Su J, Hsu A, et al. Human thrombopoietin reduces myocardial infarct size, apoptosis, and stunning following ischaemia/reperfusion in rats. Cardiovasc Res, 2008, 77(1):44-53.
13.	Schwiekart M, Huang X, Lill JR, et al. Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell survival. Nature, 2010, 463(7277):103-107.
14.	Gareia S, Mera A, Gómez-Reino JJ, et al. Poly (ADP-ribose) polymerase suppression protects rheumatoid synovial fibroblasts from Fas-induced apoptosis. Rheumatology (Oxford), 2009, 48(5):483-489.
15.	Abu-Qare AW, Abou-Donia MB. Biomarkers of apoptosis:release of cytochrome c, activation of caspase-3, induction of 8-hydroxy-2'-deoxyguanosine, increased 3-nitrotyrosine, and alteration of P53 gene. J Toxicol Environ Health B Crit Rev, 2001, 4(3):313-332.
16.	Heath-Engel HM, Chang NC, Shore GC. The endoplasmic reticulum in apoptosis and autophagy:role of the Bcl-2 protein family. Onocogene, 2008, 27(50):6419-6433.
17.	Kang MH, Reynolds CP. Bcl-2 inhibitors:targeting mitochondrial apoptotic pathways in cancer therapy. Clin Cancer Res, 2009, 15(4):1126-1132.
18.	Fisher DE. Apoptosis in cancer therapy:crossing the threshold. Cell, 1994, 78(4):539-542.
19.	Yu CH, Kan SF, Pu HF, et al. Apoptotic signaling in bufalin- and cinobufagin-treated androgen-dependent and -independent human prostate cancer cells. Cancer Sci, 2008, 99(12):2467-2476.

1. 孟琳, 李新志. 骨肉瘤的治疗进展. 医学综述, 2009, 15(16):2431-2434.
2. Marina N, Gebhardt M, Teot L, et al. Biology and therapeutic advances for pediatric osteosarcoma. Oncologist, 2004, 9(4):422-441.
3. Picci P, Mercuri M, Ferrari S, et al. Survival in high-grade osteosarcoma improvement over 21 years at a single institution. Ann Oncol, 2010, 21(6):1366-1373.
4. Sadikovic B, Yoshimoto M, Al-Romaih K, et al. In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma. PLoS One, 2008, 3(7):e2834.
5. Satonaka H, Kusuzaki K, Akeda K, et al. Acridine orange inhibits pulmonary metastasis of mouse osteosarcoma. Anticancer Res, 2011, 31(12):4163-4168.
6. Chen T, Hu W, He H, et al. A study on the mechanism of cinobufagin in the treatment of paw cancer pain by modulating local β-Endorphin expression in vivo. Evid Based Complement Alternat Med, 2013:ID 851256.
7. Zhang DM, Liu JS, Tang MK, et al. Bufotalin from Venenum Bufonis inhibits growth of multidrug resistant HepG2 cells through G2/M cell cycle arrest and apoptosis. Eur J Pharmacol, 2012, 692(1-3):19-28.
8. He X, Hu H, Wu Y, et al. Urinary metabolites of cinobufagin in rats and their antiproliferative activities. Nat Prod Res, 2012, 26(6):489-499.
9. Castets M, Coissieux MM, Delloye-Bourgeois C, et al. Inhibition of endothelial cell apoptosis by netrin-1 during angiogenesis. Dev Cell, 2009, 16(4):614-620.
10. Alenzi FQ. BCL-6 prevents mammary epithelial apoptosis and promotes cell survival. J Pak Med Assoc, 2008, 58(9):494-497.
11. Schultz DR, Hardngton WJ Jr. Apoptosis:programmed cell death at a molecular level. Semin Arthritis Rheum, 2003, 32(6):345-369.
12. Baker JE, Su J, Hsu A, et al. Human thrombopoietin reduces myocardial infarct size, apoptosis, and stunning following ischaemia/reperfusion in rats. Cardiovasc Res, 2008, 77(1):44-53.
13. Schwiekart M, Huang X, Lill JR, et al. Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell survival. Nature, 2010, 463(7277):103-107.
14. Gareia S, Mera A, Gómez-Reino JJ, et al. Poly (ADP-ribose) polymerase suppression protects rheumatoid synovial fibroblasts from Fas-induced apoptosis. Rheumatology (Oxford), 2009, 48(5):483-489.
15. Abu-Qare AW, Abou-Donia MB. Biomarkers of apoptosis:release of cytochrome c, activation of caspase-3, induction of 8-hydroxy-2'-deoxyguanosine, increased 3-nitrotyrosine, and alteration of P53 gene. J Toxicol Environ Health B Crit Rev, 2001, 4(3):313-332.
16. Heath-Engel HM, Chang NC, Shore GC. The endoplasmic reticulum in apoptosis and autophagy:role of the Bcl-2 protein family. Onocogene, 2008, 27(50):6419-6433.
17. Kang MH, Reynolds CP. Bcl-2 inhibitors:targeting mitochondrial apoptotic pathways in cancer therapy. Clin Cancer Res, 2009, 15(4):1126-1132.
18. Fisher DE. Apoptosis in cancer therapy:crossing the threshold. Cell, 1994, 78(4):539-542.
19. Yu CH, Kan SF, Pu HF, et al. Apoptotic signaling in bufalin- and cinobufagin-treated androgen-dependent and -independent human prostate cancer cells. Cancer Sci, 2008, 99(12):2467-2476.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECTS OF CINOBUFAGIN ON APOPTOSIS IN U-2OS OSTEOSARCOMAS CELLS

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