CAJ | 학술논문

目的研究蛋白酶体抑制剂--硼替佐米联合顺铂作用于卵巢上皮性癌(卵巢癌)顺铂耐药细胞的效果,并探讨其诱导细胞凋亡的分子学机制.方法实验分组:硼替佐米(50 nmol/L)+顺铂(40μmol/L)、硼替佐米(50 nmol/L)、顺铂(40 μmol/L)、对照组(未加药物),以未加药物和细胞,仅加培养基为空白对照(空白组).采用四甲基偶氮唑蓝比色法测定各组卵巢癌顺铂耐药细胞株C13细胞培养不同时间(12、24、36、48、60及72 h)后的增殖活性(以细胞存活率表示),膜联蛋白-碘化丙啶双染色法流式细胞仪检测各组细胞的凋亡率;并采用蛋白印迹法检测各组细胞内凋亡抑制蛋白短亚型(cFLIPs)蛋白的表达水平,半胱氨酸天冬氨酸蛋白酶8(caspase-8)活性检测试剂盒检测各组细胞内caspase-8的活性.结果硼替佐米+顺铂组细胞显示较好的抑制效果,作用12、24、36、48、60及72 h时其细胞存活率分别为(56.0±8.4)%、(44.7±7.3)%、(33.7±11.2)%、(27.6±8.0)%、(27.6±7.6)%和(28.1±2.4)%,均明显低于顺铂组各时间点(P<0.05).作用24 h时,顺铂、硼替佐米、硼替佐米+顺铂组细胞凋亡率分别为(16.7±l.7)%、(23.4±2.1)%和(26.9±1.6)%,硼替佐米+顺铂组明显高于顺铂或硼替佐米组(P<0.05).各药物处理组细胞内cFLIPs蛋白表达水平均不同程度下降,以硼替佐米+顺铂组[(43.2±2.3)%]降低最为明显,分别与顺铂、硼替佐米组[分别为(75.7±3.0)%、(67.9±2.1)%]比较,差异均有统计学意义(P<0.05).顺铂、硼替佐米和硼替佐米+顺铂组细胞内caspase-8活性分别为2.3±1.0、4.2±0.9和5.6±1.6,两两比较,差异均有统计学意义(P<0.05).结论蛋白酶体抑制剂--硼替佐米联合顺铂能增强卵巢癌顺铂耐药细胞的化疗敏感性;由药物作用后cFLIPs表达的降低和caspase-8活性的增强推测,cFLIPs/capsspe-8信号传导通路在硼替佐米联合顺铂诱导细胞凋亡中发挥了重要作用.
목적 연구단백매체억제제--붕체좌미연합순박작용우란소상피성암(란소암)순박내약세포적효과,병탐토기유도세포조망적분자학궤제.방법 실험분조:붕체좌미(50 nmol/L)+순박(40μmol/L)、붕체좌미(50 nmol/L)、순박(40 μmol/L)、대조조(미가약물),이미가약물화세포,부가배양기위공백대조(공백조).채용사갑기우담서람비색법측정각조란소암순박내약세포주C13세포배양불동시간(12、24、36、48、60급72 h)후적증식활성(이세포존활솔표시),막련단백-전화병정쌍염색법류식세포의검측각조세포적조망솔;병채용단백인적법검측각조세포내조망억제단백단아형(cFLIPs)단백적표체수평,반광안산천동안산단백매8(caspase-8)활성검측시제합검측각조세포내caspase-8적활성.결과 붕체좌미+순박조세포현시교호적억제효과,작용12、24、36、48、60급72 h시기세포존활솔분별위(56.0±8.4)%、(44.7±7.3)%、(33.7±11.2)%、(27.6±8.0)%、(27.6±7.6)%화(28.1±2.4)%,균명현저우순박조각시간점(P<0.05).작용24 h시,순박、붕체좌미、붕체좌미+순박조세포조망솔분별위(16.7±l.7)%、(23.4±2.1)%화(26.9±1.6)%,붕체좌미+순박조명현고우순박혹붕체좌미조(P<0.05).각약물처리조세포내cFLIPs단백표체수평균불동정도하강,이붕체좌미+순박조[(43.2±2.3)%]강저최위명현,분별여순박、붕체좌미조[분별위(75.7±3.0)%、(67.9±2.1)%]비교,차이균유통계학의의(P<0.05).순박、붕체좌미화붕체좌미+순박조세포내caspase-8활성분별위2.3±1.0、4.2±0.9화5.6±1.6,량량비교,차이균유통계학의의(P<0.05).결론 단백매체억제제--붕체좌미연합순박능증강란소암순박내약세포적화료민감성;유약물작용후cFLIPs표체적강저화caspase-8활성적증강추측,cFLIPs/capsspe-8신호전도통로재붕체좌미연합순박유도세포조망중발휘료중요작용.
Objective To explore the sensitivity and the molecular mechanism of cisplatinresistance ovarian cancer cell line C13 to proteasome inhibitors and the combination with cisplatin. Methods After different treatments, methyl thiazolyl tetrazolium (MTT) assay was applied to examine the cell viability, annexin-V/propidium iodide(PI) apoptosis detection kit was used to determine the apoptosis rate of different groups, western blot assay was introduced to evaluate the expression levels of Fas-associated death domain-like interleukin-1 beta converting enzyme inhibitory protein (cFLIPs), and the activity of caspase-8 was examined. Results MTT assay shown that the cell viability ratios of combination group at serial time points from 12, 24, 36, 48, 60, 72 hours were ( 56.0 ± 8.4 ) %, (44.7 ± 7.3 ) %, ( 33.7 ±11.2) %, (27.6 ± 8.0) %, (27. 6 ± 7.6) % and (28.1 ± 2.4) %, which were much lower than those of cisplatin group (P <0.05). After treated for 24 hours, apoptosis rates of cisplatin group, bortezomib group and combination group were ( 16.7 ± 1.7) %, (23.4 ± 2.1 ) % and (26.9 ± 1.6) %, respectively. The rate of combination group was much higher than that of non-treated group and that of cisplatin group or bortezomib group ( P < 0.05 ). Western blot assay showed the changes of expression levels of cFLIPs, which were downregulated seriously after cisplatin, bortezomib or combination treatment [ (43.2 ± 2.3 )% vs( 75.7 ± 3.0)%vs (67.9 ± 2.1 ) %, P < 0.05 ]. The caspase-8 activity of combination group was (5.6 ± 1.6) folds than that of non-treated group, which was higher than those of other two groups [ ( 2.3 ± 1.0) and (4.2 ± 0.9 ) folds,P < 0.05 ]. Conclusions The tumor cell lethal effect of cisplatin could be increase significantly by the combination application of proteasome inhibitors, bortezomib. And the cFLIPs/caspase-8 signaling pathway may be play an important role in the molecular mechanism of the combination treatment.