蛋白酶体抑制剂联合紫杉醇对卵巢上皮性癌细胞药物敏感性的影响

Proteasome inhibitors sensitize ovarian cancer cells to paclitaxel induced apoptosis

目的研究蛋白酶体抑制剂波替单抗与紫杉醇单独作用及联合作用于卵巢上皮性癌（卵巢癌）细胞株SKOV3的效果，并对其诱导细胞凋亡的分子机制进行探讨。方法波替单抗与紫杉醇单独或联合（50nmol／L波替单抗、90nmol／L紫衫醇或50nmol／L波替单抗＋90nmol／L紫衫醇）作用于SKOV3细胞后，四甲基偶氮唑蓝比色法测定细胞增殖活性并计算细胞存活率，流式细胞仪检测细胞凋亡率，蛋白印迹法检测磷酸化蛋白激酶B（AKT）和磷酸化糖原合成酶激酶3B（GSK-3p）蛋白的表达水平。结果波替单抗与紫衫醇联合作用于SKOV3细胞，12、24、36、48及72h各时间点的细胞存活率分别为（65．2±5．8）％、（58．3±14．4）％、（35．3±5．0）％、（19．2±1．5）％和（11．4±2．5）％，与单用紫杉醇比较，细胞增殖抑制效果增强，各时间点分别比较，差异均有统计学意义（P〈0．05）。单用紫杉醇、单用波替单抗和两者联用的细胞凋亡率分别为（14．7±0．5）％、（15．1±0．8）％和（20．5±0．7）％，波替单抗与紫杉醇联用的细胞凋亡率明显增高，分别与单用紫杉醇或单用波替单抗比较，差异均有统计学意义（P〈0．05）。不同药物处理细胞后，磷酸化AKT和磷酸化GSK-3B蛋白的表达水平均有不同程度降低，以波替单抗与紫杉醇联用者降低最为明显[分别为（3．2±0．8）％、（19．3±0．4）％]，分别与单用紫杉醇、单用波替单抗、正常未处理细胞比较，差异均有统计学意义（P〈0．05）。结论蛋白酶体抑制剂波替单抗与紫杉醇联用能增强卵巢癌细胞对紫杉醇的药物敏感性；AKT／GSK-313信号通路可能在波替单抗与紫杉醇联用诱导细胞凋亡中发挥了重要作用。

Objective To explore the sensitivity of ovarian cancer cell line SKOV3 to paclitaxel, proteasome inhibitors, bortezomib, and their combination. Methods The methyl thiazolyl tetrazolium （MTT） assay was applied to examine the cell viability after treatment. The annexin V-propidium iodide apoptosis detection kit was used to determine the apoptosis rate of different groups. Western blot assay was used to evaluate the expression levels of phosphorylated protein kinase B （AKT） and glycogen synthase kinase-3 beta （GSK-3[3）. Results In MTT assay, the cell viability ratios of the combination group at serial time points from 12, 24, 36, 48 and 72 hours were （65.2 ±5.8）%, （58.3± 14.4）%, （35.3 ±5.0）%, （ 19. 2 ± 1.5） %, and （ 11.4 ± 2. 5 ） %, which were significantly lower than those of the paclitaxel group （P 〈 0. 05 ）. After drug treatments, apoptosis rates of paclitaxel group, bortezomib group and the combination group were （14. 7 ±0. 5）%, （ 15. 1 ±0. 8）% and （20. 5±0. 7）% respectively. The rate of the combination group was significantly higher than that of non-treated group and pacIitaxel group （P 〈 0. 05）. Western blot assay showed the changes in expression levels of phosphorylated AKT and GSK-3[3, which were decreased significantly after paclitaxel and bortezomib combination treatment [ （ 3.2 ± 0. 8 ） %,（ 19.3 ± 0.4 ） % ; P 〈 0. 05 ]. Conclusions The lethal effect of paclitaxel on tumor cells could be increased significantly by its combination with proteasome inhibitors, bortezomib. The AKT/GSK-313 signaling pathway plays an important role in the molecular mechanism of the combination treatment.