异丹叶大黄素下调XIAP基因的化疗增敏作用研究

Sensitizing Effect of Chemotherapy of Isorhapontigenin through Down-Regulating Expression of X-Linked Inhibitor of Apoptosis

目的观察异丹叶大黄素(ISO)对膀胱癌细胞增殖及X染色体连锁凋亡抑制蛋白(XIAP)的影响,并观察异丹叶大黄素对化疗药物多西他赛诱导膀胱癌细胞凋亡及其细胞毒性的增敏作用。方法以异丹叶大黄素作用于人源性膀胱癌T24T细胞,以ATPase法检测异丹叶大黄素对膀胱癌细胞的细胞毒性,并以软琼脂集落形成实验,检测异丹叶大黄素对T24T增殖能力的影响。以逆转录-聚合酶链反应和蛋白质印迹法检测异丹叶大黄素对膀胱癌细胞X染色体连锁凋亡抑制蛋白基因表达的影响。以ATPase法、倒置显微镜下以及流式细胞仪分别观察检测异丹叶大黄素对多西他赛诱导的膀胱癌细胞凋亡和细胞毒性的影响。结果异丹叶大黄素可显著抑制膀胱癌细胞的增殖和集落形成,其IC50约为(54.5±3.6)μmol·L-1。经蛋白印迹法和逆转录-聚合酶链反应法证实,异丹叶大黄素在作用于人源性膀胱癌T24T细胞后,其X染色体连锁凋亡抑制蛋白基因在蛋白和mRNA水平均显著性降低。经多西他赛处理T24T细胞后,IC50为(8.78±1.32)nmol·L-1;而联用异丹叶大黄素后,IC50仅为(1.02±0.38)nmol·L-1,两者之间有显著性差异(P<0.01)。经流式细胞仪检测,T24T细胞接受2.5 nmol·L-1多西他赛组凋亡率(21.07±2.79)%显著低于联用异丹叶大黄素治疗组的凋亡率(49.59±5.67)%(P<0.01)。结论异丹叶大黄素可抑制膀胱癌细胞增殖,并可通过下调X染色体连锁凋亡抑制蛋白基因表达,显著增强多西他赛诱导的膀胱癌细胞凋亡,并增强细胞毒性。

OBJECTIVE To observe the inhibitor effects of isorhapontigenin （ISO） on the proliferation of bladder cancer cells and expression of X-linked inhibitor of apoptosis （XIAP） ,and the sensitizing effect of chemotherapy of docetaxel to bladder cancer cells by ISO. METHODS The ATPase assay and anchorage-independent growth assay were used to detect the cytotoxicity and proliferative capacity of ISO on the T24T bladder cancer cell line, respectively. The reverse transcription polymerase chain reaetion （RT-PCR） and Western-blotting methods were used to detect XIAP gene expression after pretreated with ISO. Pretreatment with different concentration of docetaxel and ISO on T24T bladder cancer cell, ATPase methods were performed to measure in vitro cell viability. And the morpho-logical changes and apoptosis rates were detected by phase contrast microscope and flow cytometry assay, respectively. RESULTS ISO could exhibit significant inhibitory effects on human bladder cancer cell growth through ATPase assay and anchorage-independent growth assay. Further proved by Western-blotting and RT-PCR methods, the protein and mRNA level of XIAP gene in T24T cells were both significantly decreased. After pretreatment with ISO, the IC50 of T24T cells for doeetaxel was （ 1.02 ± 0. 38） nmol · L^-1 , signifi-cantly lower than that docetaxel without ISO, which was （8.78 ± 1.32） nmol · L^-1 for 24 h（P 〈0. 01 ）. Compared with the control group, the typical apoptosis morphological changes were observed in the T24T cells pretreatment with ISO combined with docetaxel. And the index of apoptnsis was （21.07 ± 2.79） % at 2. 5 nmol · L^-1 docetaxel, markedly lower than those of T24T cells pretreatment with 2. 5 nmol · L^-1 doeetaxel and ISO, which was （49.59 ± 5.67） % （P 〈 0. 01 ）. CONCLUSION ISO could exhibit significant inhibitory effects on human bladder cancer cell proliferation. And the XIAP gene could be down-regulated significantly by ISO, which could increase docetaxel-induced apoptosis and cytotoxic activity significantly.