Effect of ursolic acid on proliferation and apoptosis of human osteosarcoma cell line U2-OS_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HUANG Shifu ¹ , CAI Feng ² , CHENG Zhaoxian ² , ZHOU Rongping ² ,  HAO Liang ²

1. Department of Orthopaedics, the People’s Hospital of Jishui County, Jishui Jiangxi, 331600, P.R.China;
2. Department of Orthopaedics, the Second Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 33006, P.R.China;

Corresponding?author：

HAO Liang, Email: haoliang201@163.com

Keywords：

Ursolic acid; osteosarcoma; U2-OS cells; cell proliferation; cell apoptosis

DOI：

10.7507/1002-1892.201704089

Video：

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Objective To investigate the effect of ursolic acid on the proliferation and apoptosis of human osteosarcoma cell line U2-OS and analyze its mechanism. Methods Human osteosarcoma cell line U2-OS was divided into 4 groups, which was cultured with ursolic acid of 0, 10, 20, and 40 μmol/L, respectively. At 0, 24, 48, and 72 hours after being cultured, the cell proliferation ability was detected by cell counting kit 8 (CCK-8). At 48 hours, the effects of ursolic acid on cell cycle and apoptosis of U2-OS cells were measured by flow cytometry. Besides, the expressions of cyclin D1 and Caspase-3 were detected by real-time fluorescent quantitative PCR and Western blot. Results CCK-8 tests showed that the absorbance (A) value of each group was not significant at 0 and 24 hours (P>0.05); but the differences between groups were significant at 48 and 72 hours (P<0.05). Flow cytometry results showed that, with the ursolic acid concentration increasing, the G₁ phase of U2-OS cells increased, the S phase and G₂/M phase decreased, and cell apoptosis rate increased gradually. There were significant differences between groups (P<0.05). Compared with the 0 μmol/L group, the relative expressions of cyclin D1 mRNA and protein in 10, 20, and 40 μmol/L groups significantly decreased (P<0.05); whereas, there was no significant difference in relative expression of Caspase-3 mRNA between groups (P>0.05). However, with the ursolic acid concentration increasing, the relative expressions of pro-Caspase-3 protein decreased and the relative expressions of activated Caspase-3 increased; there were significant differences between groups (P<0.05). Conclusion Ursolic acid can effectively inhibit the proliferation of osteosarcoma cell line U2-OS, induce the down-regulation of cyclin D1 expression leading to G₀/G₁ phase arrest, increase the activation of Caspase-3 and promote cell apoptosis.

Citation： HUANG Shifu, CAI Feng, CHENG Zhaoxian, ZHOU Rongping, HAO Liang. Effect of ursolic acid on proliferation and apoptosis of human osteosarcoma cell line U2-OS. Chinese Journal of Reparative and Reconstructive Surgery, 2017, 31(11): 1371-1376. doi: 10.7507/1002-1892.201704089 Copy

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7.	Shan JZ, Xuan YY, Zheng S, et al. Ursolic acid inhibits proliferation and induces apoptosis of HT-29 colon cancer cells by inhibiting the EGFR/MAPK pathway. J Zhejiang Univ Sci B, 2009, 10(9): 668-674.
8.	Shanmugam MK, Rajendran P, Li F, et al. Ursolic acid inhibits multiple cell survival pathways leading to suppression of growth of prostate cancer xenograft in nude mice. J Mol Med (Berl), 2011, 89(7): 713-727.
9.	Shao JW, Dai YC, Wang JC, et al. In vitro and in vivo anticancer activity evaluation of ursolic acid derivatives. Eur J Med Chem, 2011, 46(7): 2652-2661.
10.	Prasad S, Yadav VR, Sung B, et al. Ursolic acid inhibits growth and metastasis of human colorectal cancer in an orthotopic nude mouse model by targeting multiple cell signaling pathways: chemosensitization with capecitabine. Clin Cancer Res, 2012, 18(18): 4942-4953.
11.	Gao N, Cheng S, Budhraja A, et al. Ursolic acid induces apoptosis in human leukaemia cells and exhibits anti-leukaemic activity in nude mice through the PKB pathway. Br J Pharmacol, 2012, 165(6): 1813-1826.
12.	Kowalczyk MC, Walaszek Z, Kowalczyk P, et al. Differential effects of several phytochemicals and their derivatives on murine keratinocytes in vitro and in vivo: implications for skin cancer prevention. Carcinogenesis, 2009, 30(6): 1008-1015.
13.	Zhu Z, Qian Z, Yan Z, et al. A phase I pharmacokinetic study of ursolic acid nanoliposomes in healthy volunteers and patients with advanced solid tumors. Int J Nanomedicine, 2013, 8(1): 129-136.
14.	Kim SH, Ryu HG, Lee J, et al. Ursolic acid exerts anti-cancer activity by suppressing vaccinia-related kinase 1-mediated damage repair in lung cancer cells. Sci Rep, 2015, 5: 14570.
15.	Yang L, Tang Q, Wu J, et al. Erratum to: Inter-regulation of IGFBP1 and FOXO3a unveils novel mechanism in ursolic acid-inhibited growth of hepatocellular carcinoma cells. J Exp Clin Cancer Res, 2016, 35: 81.
16.	盧夢玲, 閆超, 賴多, 等. Cyclin D1 與細胞周期調控. 生物技術通報, 2011, 25(10): 55-59.
17.	毛德文, 陳月橋, 王麗, 等. Caspase-8 及 Caspase-3 與細胞凋亡. 遼寧中醫藥大學學報, 2008, 10(10): 148-150.

1. Lu J, Song G, Tang Q, et al. IRX1 hypomethylation promotes osteosarcoma metastasis via induction of CXCL14/NF-κB signaling. J Clin Invest, 2015, 125(5): 1839-1856.
2. Siegel HJ, Pressey JG. Current concepts on the surgical and medical management of osteosarcoma. Expert Rev Anticancer Ther, 2008, 8(8): 1257-1269.
3. 劉瑞, 花寶金. 中醫藥參與腫瘤綜合治療模式現狀與分析. 中國腫瘤, 2014, 23(4): 311-315.
4. 孟艷秋, 陳瑜, 王趲, 等. 熊果酸的研究進展. 中國新藥雜志, 2007, 16(01): 25-28.
5. 崔兵兵, 程麗芳, 侯立靜, 等. 熊果酸抑制人肝癌 SMMC-7721 細胞生長及凋亡的實驗研究. 中國實驗方劑學雜志, 2013, 19(18): 181-184.
6. 司福亭, 李靖靖, 曾超, 等. 熊果酸的抗腫瘤活性及作用機理研究進展. 化學與生物工程, 2010, 27(1): 9-12.
7. Shan JZ, Xuan YY, Zheng S, et al. Ursolic acid inhibits proliferation and induces apoptosis of HT-29 colon cancer cells by inhibiting the EGFR/MAPK pathway. J Zhejiang Univ Sci B, 2009, 10(9): 668-674.
8. Shanmugam MK, Rajendran P, Li F, et al. Ursolic acid inhibits multiple cell survival pathways leading to suppression of growth of prostate cancer xenograft in nude mice. J Mol Med (Berl), 2011, 89(7): 713-727.
9. Shao JW, Dai YC, Wang JC, et al. In vitro and in vivo anticancer activity evaluation of ursolic acid derivatives. Eur J Med Chem, 2011, 46(7): 2652-2661.
10. Prasad S, Yadav VR, Sung B, et al. Ursolic acid inhibits growth and metastasis of human colorectal cancer in an orthotopic nude mouse model by targeting multiple cell signaling pathways: chemosensitization with capecitabine. Clin Cancer Res, 2012, 18(18): 4942-4953.
11. Gao N, Cheng S, Budhraja A, et al. Ursolic acid induces apoptosis in human leukaemia cells and exhibits anti-leukaemic activity in nude mice through the PKB pathway. Br J Pharmacol, 2012, 165(6): 1813-1826.
12. Kowalczyk MC, Walaszek Z, Kowalczyk P, et al. Differential effects of several phytochemicals and their derivatives on murine keratinocytes in vitro and in vivo: implications for skin cancer prevention. Carcinogenesis, 2009, 30(6): 1008-1015.
13. Zhu Z, Qian Z, Yan Z, et al. A phase I pharmacokinetic study of ursolic acid nanoliposomes in healthy volunteers and patients with advanced solid tumors. Int J Nanomedicine, 2013, 8(1): 129-136.
14. Kim SH, Ryu HG, Lee J, et al. Ursolic acid exerts anti-cancer activity by suppressing vaccinia-related kinase 1-mediated damage repair in lung cancer cells. Sci Rep, 2015, 5: 14570.
15. Yang L, Tang Q, Wu J, et al. Erratum to: Inter-regulation of IGFBP1 and FOXO3a unveils novel mechanism in ursolic acid-inhibited growth of hepatocellular carcinoma cells. J Exp Clin Cancer Res, 2016, 35: 81.
16. 盧夢玲, 閆超, 賴多, 等. Cyclin D1 與細胞周期調控. 生物技術通報, 2011, 25(10): 55-59.
17. 毛德文, 陳月橋, 王麗, 等. Caspase-8 及 Caspase-3 與細胞凋亡. 遼寧中醫藥大學學報, 2008, 10(10): 148-150.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of ursolic acid on proliferation and apoptosis of human osteosarcoma cell line U2-OS

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