中华骨科杂志
中華骨科雜誌
중화골과잡지
CHINESE JOURNAL OF ORTHOPAEDICS
2009年
7期
684-689
,共6页
陶惠民%刘兵%师钟丽%冯洁
陶惠民%劉兵%師鐘麗%馮潔
도혜민%류병%사종려%풍길
骨肉瘤%顺铂%细胞凋亡%半胱氨酸天冬氨酸蛋白酶
骨肉瘤%順鉑%細胞凋亡%半胱氨痠天鼕氨痠蛋白酶
골육류%순박%세포조망%반광안산천동안산단백매
Osteosarcoma%Cisplatin%Apoptosis%Caspases
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(50 μmol/L或100 μmol/L)和(或)顺铂(10 μg/ml)作用骨肉瘤MG-63细胞48 h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.P13K抑制剂Wortmannin作用MG-63细胞48 h,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100 μmol/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P13K/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48 h,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P13K/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.
目的 探討環氧化酶-2(COX-2)選擇性抑製劑塞來昔佈對人骨肉瘤MG-63細胞增殖和凋亡的影響及作用機製.方法 塞來昔佈(50 μmol/L或100 μmol/L)和(或)順鉑(10 μg/ml)作用骨肉瘤MG-63細胞48 h後,MTT法測定細胞增殖的抑製率,電子顯微鏡和流式細胞儀檢測細胞凋亡,RT-PCR法檢測基因水平COX-2錶達變化,Western blot檢測COX-2及凋亡相關蛋白錶達變化.P13K抑製劑Wortmannin作用MG-63細胞48 h,檢測蛋白錶達變化.結果 塞來昔佈導緻MG-63細胞阻滯在G1期,通過激活半胱氨痠天鼕氨痠蛋白酶(caspase)-9的內源性凋亡途徑誘導細胞凋亡;順鉑單藥作用後骨肉瘤MG-63細胞凋亡率為5.93%,而聯閤應用塞來昔佈50μmol/L或100 μmol/L後,凋亡率分彆為6.66%和37.15%,與順鉑聯閤具有明顯的協同作用;COX-2蛋白錶達未降低.塞來昔佈聯閤順鉑明顯降低P13K/Akt、survivin、Bcl-2的錶達,檢測到caspase-9、caspase-3的激活和PARP裂解片段.Wortmannin作用MG-63細胞48 h,檢測到pAkt(Thr308)、Bcl-2、survivin錶達下調.結論 塞來昔佈可通過非COX-2途徑誘導骨肉瘤MG-63細胞凋亡,與P13K/Akt、survivin、Bcl-2蛋白相關,併且PI3K/Akt途徑在survivin、Bcl-2錶達調控中髮揮重要作用.這可能是塞來昔佈藥物榦預的中心環節.
목적 탐토배양화매-2(COX-2)선택성억제제새래석포대인골육류MG-63세포증식화조망적영향급작용궤제.방법 새래석포(50 μmol/L혹100 μmol/L)화(혹)순박(10 μg/ml)작용골육류MG-63세포48 h후,MTT법측정세포증식적억제솔,전자현미경화류식세포의검측세포조망,RT-PCR법검측기인수평COX-2표체변화,Western blot검측COX-2급조망상관단백표체변화.P13K억제제Wortmannin작용MG-63세포48 h,검측단백표체변화.결과 새래석포도치MG-63세포조체재G1기,통과격활반광안산천동안산단백매(caspase)-9적내원성조망도경유도세포조망;순박단약작용후골육류MG-63세포조망솔위5.93%,이연합응용새래석포50μmol/L혹100 μmol/L후,조망솔분별위6.66%화37.15%,여순박연합구유명현적협동작용;COX-2단백표체미강저.새래석포연합순박명현강저P13K/Akt、survivin、Bcl-2적표체,검측도caspase-9、caspase-3적격활화PARP렬해편단.Wortmannin작용MG-63세포48 h,검측도pAkt(Thr308)、Bcl-2、survivin표체하조.결론 새래석포가통과비COX-2도경유도골육류MG-63세포조망,여P13K/Akt、survivin、Bcl-2단백상관,병차PI3K/Akt도경재survivin、Bcl-2표체조공중발휘중요작용.저가능시새래석포약물간예적중심배절.
Objective To identify the anti-proliferation of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, and the combination of celecoxib and cisplatin in MG-63 cells, and to explore the poten-tial molecular mechanisms involved. Methods MG-63 cells were treated with the combination of celecoxib (50 μmol/L or 100 μmol/L) and/or cisplatin (10 μg/ml) for 48 h in serum-supplemented medium. Cell viabil-ity was measured by MTT assay; apoptosis was determined by electronmicroscope and flow cytometry (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 cisplatin-induced apoptosis was 5.93%, and potentiated to 6.66% and 37.15% while combining with celecoxib (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 cells treated with celecoxib. PI3K/Akt, survivin, Bcl-2 were significantly down-regulated in cells treated with the combination of celecoxib and cisplatin. Moreover, the decreased expres-sions of pro-caspase-9, pro-caspase-3 and cleaved PARP-1 were detected by Western blot analysis. And pAkt (Thr308), survivin and Bcl-2 levels down-regulated in cells treating with Wortmannin for 48 h, a spe-cific PI3K inhibitor. Conclusion Celecoxib exerts its anti-tumor activities through COX-2 independent mechanisms, which may be PI3K/Akt-dependent, and survivin and Bcl-2-related. PI3K may be at the center of the celecoxib effects, which play an essential role in the regulation of survivin and Bcl-2.
