Background To investigate the role of P16 (INK4a)-extracellular signal related kinase 1/2 (ERK1/2) signaling pathway in cisplatin (DDP) resistance induced by multidrug resistance protein 1 (MDR1), also known as P-glycoprotein (P-gp), in cervical adenocarcinoma. Methods A human DDP-resistant HeLa cell line (HeLa/DDP) was constructed using the combination of incremental and intermittent administration of DDP. Cell Counting Kit-8 (CCK-8) assay was used to measure the IC50 and resistance index (RI) of cells. The morphological changes and population doubling time were observed under an inverted microscope. Plate cloning formation assay was performed to evaluate the cell proliferation and tumorigenic ability. Cell invasion and migration were determined by transwell assays. Besides, the expression of P16, phosphorylated extracellular signal related kinase 1 and 2 (pERK1/2), total ERK1/2 and MDR1 were measured using western blot analysis. The ERK-specific inhibitor U0126 and agonist TPA was used to explore the role of ERK. Results The DDP-resistant cervical adenocarcinoma HeLa/DDP cell line was successfully established, which showed stronger cell growth, invasion, and migration. In the HeLa/DDP cells, pERK1/2 was downregulated, P-gp was upregulated and P16 was downregulated. Overexpression of P16 led to a significant decrease in the proliferation rate, migration ability, and invasion ability of the HeLa/DDP cells. Furthermore, overexpression of P16 increased and the decreased expression of pERK1/2 and P-gp in the HeLa/DDP cells, respectively. Treatment of HeLa/DDP cells transfected with P16 plasmid with ERK-specific inhibitor U0126 significantly decreased the expression of pERK1/2 and increased the expression of P-gp from 6 h to 48 h. Moreover, after 72 h, the expression of pERK1/2 was up-regulated and the expression of P-gp was inhibited. Conclusion Overexpression of P16 could partially reverse the MDR1-mediated DDP resistance in the cervical adenocarcinoma by the enhancement of phosphorylation of ERK signaling pathway, which provided a theoretical basis for the treatment of DDP resistance in cervical adenocarcinoma.

中文翻译：

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P16的过表达通过激活ERK1/2信号通路逆转了宫颈腺癌中MDR1介导的DDP耐药。

背景 研究 P16 (INK4a)-细胞外信号相关激酶 1/2 (ERK1/2) 信号通路在多药耐药蛋白 1 (MDR1) 诱导的顺铂 (DDP) 耐药中的作用，也称为 P-糖蛋白 (P- gp)，在宫颈腺癌中。方法采用增量给药和间歇给药相结合的方法构建人DDP抗性HeLa细胞系（HeLa/DDP）。细胞计数试剂盒8（CCK-8）测定用于测量细胞的IC50和抗性指数（RI）。倒置显微镜下观察形态变化和种群倍增时间。进行平板克隆形成测定以评估细胞增殖和致瘤能力。细胞侵袭和迁移由transwell 测定法确定。此外，P16 的表达，使用蛋白质印迹分析测量磷酸化的细胞外信号相关激酶 1 和 2 (pERK1/2)、总 ERK1/2 和 MDR1。ERK 特异性抑制剂 U0126 和激动剂 TPA 用于探索 ERK 的作用。结果成功建立了抗DDP宫颈腺癌HeLa/DDP细胞系，细胞生长、侵袭、迁移能力更强。在 HeLa/DDP 细胞中，pERK1/2 下调，P-gp 上调，P16 下调。P16的过表达导致HeLa/DDP细胞的增殖率、迁移能力和侵袭能力显着下降。此外，HeLa/DDP 细胞中 P16 的过表达增加，pERK1/2 和 P-gp 的表达减少。用 ERK 特异性抑制剂 U0126 处理用 P16 质粒转染的 HeLa/DDP 细胞显着降低了 pERK1/2 的表达，并使 P-gp 的表达从 6 小时增加到 48 小时。此外，72 h后，pERK1/2的表达上调，P-gp的表达受到抑制。结论 P16过表达可通过增强ERK信号通路磷酸化部分逆转MDR1介导的宫颈腺癌DDP耐药，为宫颈腺癌DDP耐药的治疗提供理论依据。