唑来膦酸联合紫杉醇不同序贯用药方案对人鼻咽癌HNE1细胞的作用

Study of sequence-dependent in vitro effects of zoledronic acid and paclitaxel upon human nasopharyngeal carcinoma cell line HNE1

目的探讨唑来膦酸（ZOL）联合紫杉醇（TAX）不同序贯方案对人鼻咽癌低分化鳞癌HNE1细胞系增殖抑制和凋亡诱导作用，寻找两药联合合适序贯给药方式及相关机制。方法采用四甲基偶氮唑蓝法检测ZOL联合TAX不同序贯用药方案对HNE1细胞的增殖抑制，流式细胞术和原位末端标记法（TUNEL）检测各用药方案对HNE1细胞的凋亡影响，实时定量PCR和Western印迹法测定各用药方案对HNE1细胞Bcl-2、Bax、半胱氨酸天冬氨酸蛋白酶（Caspase）3和Caspase9mRNA和蛋白表达水平的影响。结果MTF法提示实验组均能抑制HNE1细胞增殖（P〈0．05）；其中以ZOL序贯TAX组作用最强（P〈0．05）。流式细胞术检测早期细胞凋亡，实验组的早期凋亡率则分别为13．89％±0．69％、11．73％±0．54％、23．97％±0．68％、10．45％±0．16％和8．59％±0．74％，与对照组（2．59％±0．28％）比较，差异均有统计学意义（均P〈0．05）；TUNEL检测细胞凋亡，实验组均能不同程度增加凋亡率（P〈0．05），ZOL序贯TAX组更为明显（P〈0．05）。实时定量PCR和Western印迹法检测表明用药组均下调抑凋亡蛋白Bcl-2和上调促凋亡蛋白Bax、Caspase9和Caspase3表达；ZOL序贯TAX组更为明显。结论ZOL能够增强TAX对HNE1增殖抑制和凋亡诱导能力，尤以ZOL序贯TAX作用最强；其机制可能通过影响Bcl-2家族，活化线粒体凋亡途径有关。

Objective To explore the in vitro effects of anti-proliferation and apoptosis-inducing with different sequence regimens of zoledronic acid plus paclitaxel in human nasopharyngeal carcinoma cell line HNE1 so as to explore the optimal sequence regimen of these two drugs and related mechanism. Methods The cytotoxic effects of different sequence schemes of zoledronic acid plus paclitaxel on HNE1 cells were detected by methyl-thiazol-tetrazolium （MTT） assay. Annexin V-FITC/PI double staining flow cytometry （FCM） and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL） assay were used to measure the effects of zoledronic acid plus paclitaxel upon apoptosis. The expressions of mRNA of Bcl-2, Bax, Caspase3 and Caspase9 gene were detected by real-time quantitative- polymerase chain reaction （PCR） and protein was detected by Western blot. Results All experiment groups enhanced the effect of anti-proliferation by MTT assay（ P 〈 0. 05 ） ; the treatment of zoledronic acid followed by paclitaxel was superior to the other two regimens （ P 〈 0.05 ）. As detected by FCM, the early apoptotic rate of control group was 2. 59% ± 0. 28% and the experiment groups were 13.89% ± 0. 69%, 11.73% ±0. 54%, 23. 97% ±0. 68%, 10. 45% ±0. 16% and 8. 59% ±0. 74% respectively（P 〈0. 05）. TUNEL assay detected the late apoptosis of HNE1 cells and the experiment groups enhanced the effect of apoptosis-inducing（P 〈 0. 05 ）. The treatment of zoledronic acid followed by paclitaxel was superior to the other regimens（ P 〈 0. 05）. Such an effect was due to the down-regulation of anti-apoptotic protein Bcl-2 and up-regulations of pro-apoptotic proteins Bax, Caspase3 and Caspase9 at the expression levels of mRNA and protein. There was a greater regulation in the group of zoledronic acid followed by paclitaxel. Conclusion Zoledronie acid can enhance the in vitro effects of anti-proliferation and apoptosis-indueing for paclitaxel on HNE1 cell. The treatment of zoledronic acid followed by paclitaxel may be the optimal regimen. Synergistic induction of apoptosis is via the effects of Bcl-2 family and through the mitochondrial pathway.