摘要
目的探讨环氧化酶-2（COX-2）选择性抑制剂塞来昔布对人骨肉瘤MG-63细胞增殖和凋亡的影响及作用机制。方法塞来昔布（50μmol／L或100μmol／L）和（或）顺铂（10μg／ml）作用骨肉瘤MG-63细胞48h后，MTT法测定细胞增殖的抑制率，电子显微镜和流式细胞仪检测细胞凋亡，RT-PCR法检测基因水平COX-2表达变化，Western blot检测COX-2及凋亡相关蛋白表达变化。PI3K抑制剂Wortmannin作用MG-63细胞48h，检测蛋白表达变化。结果塞来昔布导致MG-63细胞阻滞在G1期，通过激活半胱氨酸天冬氨酸蛋白酶（caspase）-9的内源性凋亡途径诱导细胞凋亡；顺铂单药作用后骨肉瘤MG-63细胞凋亡率为5．93％，而联合应用塞来昔布50μmol／L或100μmol／L后，凋亡率分别为6．66％和37．15％，与顺铂联合具有明显的协同作用；COX-2蛋白表达未降低。塞来昔布联合顺铂明显降低PI3K／Akt、survivin、Bcl-2的表达，检测到caspase-9、caspase-3的激活和PARP裂解片段。Wortmannin作用MG-63细胞48h，检测到pAkt（Thr308）、Bcl-2、survivin表达下调。结论塞来昔布可通过非COX-2途径诱导骨肉瘤MG-63细胞凋亡，与PI3K／Akt、survivin、Bcl-2蛋白相关，并且PI3K／Akt途径在survivin、Bcl-2表达调控中发挥重要作用。这可能是塞来昔布药物干预的中心环节。
Objective To identify the anti-proliferation of celecoxib, a selective eyelooxygenase-2 （COX-2） inhibitor, and the combination of celecoxib and cisplatin in MG-63 cells, and to explore the potential molecular mechanisms involved. Methods MG-63 cells were treated with the combination of eeleeoxib （50 μmol/L or 100 μmol/L） and/or cisplatin （10μg/ml） for 48 h in serum-supplemented medium. Cell viability was measured by MTT assay; apoptosis was determined by electronmicroscope and flow eytometry （FCM）; gene transcription and protein expression were detected by RT-PCR and/or Western blot analysis. Results MG-63 cells were significantly inhibited by celecoxib at G1 phase. The eisplatin-indueed apoptosis was 5.93%, and potentiated to 6.66% and 37.15% while combining with celeeoxib （50 μmol/L and 100 μmol/L） respectively. There was significant synergetic effect between celecoxib and cisplatin. The protein expression of COX-2 did not occur in ceils treated with celecoxib. PI3K/Akt, survivin, Bcl-2 were significantly downregulated in cells treated with the combination of celecoxib and cisplatin. Moreover, the decreased expressions of pro-caspase-9, pro-easpase-3 and cleaved PARP-1 were detected by Western blot analysis. And pAkt （Thr308）, survivin and Bel-2 levels down-regulated in cells treating with Wortmannin for 48 h, a specific PI3K inhibitor. Conclusion Celecoxib exerts its anti-tumor activities through COX-2 independent mechanisms, which may be PI3K/Akt-dependent, and smwivin and Bcl-2-related. PI3K may be at the center of the celeeoxib effects, which play an essential role in the regulation of survivin and Bcl-2.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2009年第7期684-689,共6页
Chinese Journal of Orthopaedics
