Department of Medical Oncology, Tangshan Gongren Hospital, Tangshan 063000, China.
Department of Thoracic Suegery, Tangshan Gongren Hospital, Tangshan 063000, China.
Exp Mol Pathol. 2020 Dec;117:104564. doi: 10.1016/j.yexmp.2020.104564. Epub 2020 Oct 31.
The current study aimed to explore the mechanism of autophagy-regulating chemoresistance in esophageal cancer (EC) cells. Methods: 45 cases of esophageal cancer cell tissue and 25 cases of adjacent normal tissue excised in the surgical resection were collected from the tumor pathology department of our hospital from March to November 2017. The above cancer cells and paracancerous cells were cultured according to the cell culture procedures. The autophagy was induced by cisplatin in human esophageal cancer EC9706 cells line. The effect of autophagy on the survival of EC9706 cells was observed by autophagy inhibitor 3-MA. Cell viability was also measured by cell counting kit-8 (CCK-8). Apoptosis and cell cycle were detected by flow cytometry. Furthermore, monodansylcadaverine (MDC) was used to detect autophagy. Western blot was applied to determine the molecular changes during treatment. Diketopyrrolopyrrole (DPP) is able to inhibit cell proliferation, induce cell death and cell cycle arrest in the S phase. In addition, autophagy was activated through PI3K-III pathway. Results: 3-MA inhibitor plus 10% fetal bovine serum were added for culture, and the cell culture temperature and humidity were the best conditions. There were few autophagic vesicles in the stationary cells, where their brightness was weakened. There were more and brighter green fluorescent particles in the DPP group without a 3-MA inhibitor, indicating that autophagic parameters actually exist in this process. The apoptosis rate of DDP-induced cell death was not found to be the best, but was higher than that of the control group (P<0.05). The combination of DDP and 3-MA had a more obvious catalytic effect on apoptosis, and the apoptosis rate was much higher than that of single DDP (P<0.05), indicating that DDP was capable of inducing significant apoptosis after inhibiting autophagy. The combination of DDP and 3-MA had an obvious catalytic effect on apoptosis, and the apoptosis rate was higher than that of DDP alone (P < 0.05), suggesting that DDP could significantly improve the ability to induce apoptosis after inhibiting autophagy. The expression level of autophagy-related proteins was also detected by Western blotting. Our findings indicated that autophagy may be a self-protective mechanism of esophageal cancer cells induced by DDP, and its inhibition may be a new strategy for adjuvant chemotherapy in esophageal cancer.
本研究旨在探讨自噬调控食管癌（EC）细胞化疗耐药的机制。方法：收集我院肿瘤病理科 2017 年 3 月至 11 月手术切除的 45 例食管癌组织标本和 25 例癌旁正常组织标本。根据细胞培养程序培养上述癌细胞和癌旁细胞。用顺铂诱导人食管癌细胞 EC9706 系自噬。用自噬抑制剂 3-MA 观察自噬对 EC9706 细胞存活的影响。通过细胞计数试剂盒-8（CCK-8）测量细胞活力。通过流式细胞术检测细胞凋亡和细胞周期。此外，用单丹磺酰尸胺（MDC）检测自噬。采用 Western blot 检测治疗过程中的分子变化。二酮吡咯并吡咯（DPP）能够抑制细胞增殖，诱导细胞死亡和细胞周期停滞在 S 期。此外，自噬通过 PI3K-III 途径被激活。结果：添加 3-MA 抑制剂加 10%胎牛血清进行培养，细胞培养温度和湿度为最佳条件。静止细胞中的自噬小泡较少，其亮度减弱。DPP 组无 3-MA 抑制剂时，绿色荧光颗粒较多且较亮，表明该过程中确实存在自噬参数。未发现 DDP 诱导细胞死亡的凋亡率最佳，但高于对照组（P<0.05）。DDP 与 3-MA 联合应用对凋亡有更明显的催化作用，凋亡率明显高于单独 DDP（P<0.05），表明 DDP 抑制自噬后能显著诱导凋亡。DDP 与 3-MA 联合应用对凋亡有明显的催化作用，凋亡率高于单独 DDP（P<0.05），表明 DDP 抑制自噬后能显著提高诱导凋亡的能力。Western blot 检测自噬相关蛋白的表达水平。我们的研究结果表明，自噬可能是 DDP 诱导的食管癌细胞的一种自我保护机制，其抑制可能是食管癌辅助化疗的新策略。
