Mechanism of synergistic inhibition of prostate cancer cells by Olaparib combined with Enzalutamide
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摘要:
目的：探讨奥拉帕利联合恩杂鲁胺协同抑制前列腺癌细胞生长的作用机制。方法：利用CCK-8在前列腺癌细胞系 C4-2中测定奥拉帕利及恩杂鲁胺的半抑制浓度以及两药联用后的联合指数。通过体外增殖、克隆形成实验、流式细胞术验证两药联用对前列腺癌细胞增殖及凋亡的影响。对联合及单独使用奥拉帕利及恩杂鲁胺处理的前列腺癌细胞进行二代高通量测序并探索两药协同作用的机制。使用免疫荧光观察联用两药后其双链损伤标志物的积累。利用qRT-PCR和Western blot检测协同作用通路关键基因的mRNA及蛋白表达水平。结果：联合指数表明奥拉帕利和恩杂鲁胺在前列腺癌细胞系C4-2中有强协同作用。联合使用两药相较于单药显著抑制细胞增殖及细胞克隆能力。流式细胞仪检测发现联用两药相较于单药显著促进细胞凋亡。免疫荧光发现联用两药后其双链损伤明显增加。二代高通量测序结果分析发现两药联用导致凋亡等通路被激活。mRNA及蛋白检测发现联用两药会导致促凋亡基因肿瘤坏死因子α诱导蛋白2(tumor necrosis factor alpha-induced pro- tein 2，TNFAIP2)、肿瘤坏死因子α诱导蛋白8样蛋白1(tumor necrosis factor alpha-induced protein 8-like protein 1，TNFAIP8L1)的高表达以及抗凋亡基因肿瘤坏死因子α诱导蛋白8(tumor necrosis factor alpha-induced protein 8，TNFAIP8)的低表达。结论：奥拉帕利联合恩杂鲁胺可以促进促凋亡基因TNFAIP2、TNFAIP8L1的表达，并抑制抗凋亡基因TNFAIP8的表达，从而协同促进细胞凋亡进而抑制前列腺癌细胞生长。
Abstract:
Objective：To investigate the mechanism of olaparib combined with enzalutamide in synergistically inhibiting the growth of prostate cancer cells. Methods：CCK -8 was used to determine the half -inhibitory concentrations of olaparib and enzalutamide in prostate cancer cell line C4-2，as well as the combined index of the two drugs. The effects of the combination of the two drugs on the proliferation and apoptosis of prostate cancer cells were verified by in vitro proliferation，clone formation experiments and flow cytometry. The accumulation of double - strand damagewas observed by immunofluorescence. Next - generation high - throughput sequencing of prostate cancer cells treated with olaparib and enzalutamide in combination and alone was used to explore the mechanism of the synergistic effect of the two drugs. The mRNA and protein expression levels of key genes in the synergistic pathway were detected by qRT - PCR and Western blot. Results：The combination index showed that the combination of olaparib and enzalutamide had strong synergistic effects in prostate cancer cell line C4-2. Compared with the single drug，the combined use of the two drugs significantly inhibited cell proliferation and cell cloning ability. Flow cytometry showed that the combination of the two drugs significantly promoted cell apoptosis compared with the single drug. Immunofluorescence showed that the double - strand damagewas significantly increased after the combination of the two drugs. Analysis of the results of next -generation high -throughput sequencing showed that the combination of the two drugs led to the activation of apoptosis and other pathways. Through the detection of mRNA and protein，it was found that the combination of the two drugs would lead to the high expression of the pro-apoptotic genes TNFAIP2 and TNFAIP8L1，and the low expression of the anti - apoptotic gene TNFAIP8. Conclusion：Olaparib combined with enzalutamide can promote the expression of pro-apoptotic genes TNFAIP2 and TNFAIP8L1 and inhibit the expression of anti-apoptotic gene TNFAIP8 to synergistically promote cell apoptosis and inhibit the growth of prostate cancer cells.
