紫杉醇放射增敏作用的分子机制

Molecular mechanism of radiosensitizing effect of paclitaxel

背景与目的:紫杉醇作为一种放射增敏剂,能稳定微管系统,把细胞阻断在G2/M期从而改变肿瘤细胞的放射反应性。但其分子机制目前还未完全阐明,本文旨在研究紫杉醇的放射增敏作用及其分子机制。方法:克隆形成实验分析不同浓度紫杉醇联合不同剂量照射对KB细胞增殖抑制率的影响,多靶单击拟合模型方程测定各组细胞放射增敏比并评价增敏效果;流式细胞仪检测KB细胞经紫杉醇及射线共同作用后细胞周期分布变化;采用基因芯片技术筛选与紫杉醇放射增敏作用相关的差异表达基因;荧光定量PCR验证芯片提示细胞分裂相关基因PRC1、cyclinB2的变化。结果:紫杉醇与射线协同作用能明显抑制KB细胞增殖,20nmol/L药物协同射线作用时SERD0及SERDq分别为2.40±1.87、12.23±2.81。药物加照射处理后G1期细胞由48.32±2.40%下降为15.73±7.00%(P<0.01),G2/M期细胞由13.66±2.16%上升到52.51±5.02%(P<0.01)。基因芯片结果显示的差异变化基因中共有176条与紫杉醇放射增敏作用相关,10条在调控细胞分裂过程中起作用,其中上调表达2条,下调表达8条。荧光定量PCR证实与细胞分裂相关的PRC1和CyclinB2均下调表达,与芯片结果一致。结论:紫杉醇对KB细胞有明显的放射增敏作用,其机制可能是使细胞有丝分裂相关基因PRC1和CyclinB2表达特异性下调,导致双极纺锤体形成受阻,细胞无法完成正常的分裂,从而使细胞增殖受到抑制并最终死亡。

Background ＆ Objective： Paclitaxel is a radiosensitizer which may stabilize microtubules, block the GSM phase of the cell cycle and thus modulate the radioresponsiveness of tumor cells. However, its potential molecular mechanisms of radiosensitization have not been well understood yet. This study was to investigate the radiosensitizing effect of paclitaxel on human oral epithelium carcinoma （KB） cell line and to explore the molecular mechanism of radiosensitization. Methods：The survival of KB cells following the treatment with paclitaxel and/or radiation was determined by colony-forming assay. The radiosensitizing effect was evaluated by calculating the sensitizing enhancement ratio （SER） with multi-target single hit model. The cell cycle distribution was analyzed by flow cytometry. Differentially expressed genes related to paclitaxel radiosensitization were screened using human Oligo microarray. Expressions of protein regulating cytokinesis 1 （PRC1） and cyclin B2 genes were confirmed by real-time quantitative PCR. Results： The proliferation of KB cells was significantly inhibited by paclitaxel combined with ionizing radiation. The SERD0 and SERDq were （2.40±1.87） and （12.23±2.81） respectively, when the concentration of paclitaxel was 20 nmol/L After the treatment with paclitaxel in combination with irradiation, the percentage of G1 phase cells decreased from （48.32±2.40）% to （15.73±7.00）% （P〈0.01）, and the percentage of GSM phase cells increased from （13.66±2.16）% to （52.51±5.02）% （P〈0.01）. In total 176 differentially expressed genes were identified to be related to paclitaxel radiosensitization. Ten genes were found to regulate cell division, two of which were upregulated and eight were down-regulated after the treatment. Moreover, the expression of PRC1 and cyclin B2 was decreased. Conclusion： The radiosensitizing effect of paclitaxel on KB cells may be due to the down-regulated expression of PRC1 and cyclin B2, resulting in inhibition of mitotic spindle formation and cell necrosis.