过氧化氢通过调控自噬增强宫颈癌的放疗敏感性

doi:10.6040/j.issn.1671-7554.0.2022.0855

摘要/Abstract

摘要： 目的探讨过氧化氢(H₂O₂)联合透明质酸钠是否可以提高宫颈癌细胞的放疗敏感性,并探讨其潜在机制。方法将细胞分为对照组、透明质酸钠组、4 Gy组、透明质酸钠+4 Gy组,Western blotting实验检测自噬相关蛋白水平。将细胞分为对照组,透明质酸钠组,200 μmol/L H₂O₂组、500 μmol/L H₂O₂组、700 μmol/L H₂O₂组和1 000 μmol/L H₂O₂组,CCK8实验检测H₂O₂对细胞活力的影响;将细胞分为对照组和200 μmol/L H₂O₂组,分别进行0、2、4、6、8 Gy放疗,克隆形成实验评估H₂O₂的放疗增敏作用。将细胞分为对照组、200 μmol/L H₂O₂组、4 Gy组、200 μmol/L H₂O₂+4 Gy组,Western blotting实验和免疫荧光实验检测自噬相关蛋白水平。结果对照组与透明质酸钠组的自噬相关蛋白LC3 II、p62 表达差异无统计学意义(P>0.05);4 Gy组与透明质酸钠+4 Gy组的自噬相关蛋白LC3 II、p62 表达差异无统计学意义(P>0.05)。CCK8实验结果表明,放疗前后,对照组和透明质酸钠组的细胞增殖率差异无统计学意义(P>0.05);与对照组相比,200 μmol/L H₂O₂组促进细胞增殖(P<0.05),500 μmol/L H₂O₂组对细胞增殖的影响无统计学意义(P>0.05),700 μmol/L H₂O₂组和1 000 μmol/L H₂O₂组抑制细胞增殖(P<0.05)。克隆形成实验结果表明,H₂O₂增加了细胞对放疗的敏感性(P<0.05)。Western blotting实验和免疫荧光实验表明,与对照组相比,200 μmol/L H₂O₂组抑制了自噬(P<0.05);与4 Gy组相比,200 μmol/L H₂O₂+4 Gy组促进了自噬(P<0.05)。与对照组相比,200 μmol/L H₂O₂组p-AKT和p-mTOR蛋白表达增加(P<0.05);与4 Gy组相比,200 μmol/L H₂O₂+4 Gy组p-AKT和p-mTOR的表达减少(P<0.05)。结论 H₂O₂通过调控AKT/mTOR通路进而调控自噬来提高宫颈癌的放疗敏感性。

关键词: 自噬, 过氧化氢, 透明质酸钠, 放疗敏感性, AKT/mTOR通路, 宫颈癌

Abstract: Objective To investigate whether hydrogen peroxide, represented as H₂O₂, combined with sodium hyaluronate can improve the sensitivity of cervical cancer cells to radiotherapy and to explore the underlying mechanisms. Methods The cells were divided into control group, sodium hyaluronate group, 4 Gy group, sodium hyaluronate+4 Gy group, and the expressions of autophagy related proteins were detected with Western blotting assay. Then, the cells were divided into control group, sodium hyaluronate group, 200 μmol/L H₂O₂ group, 500 μmol/L H₂O₂ group, 700 μmol/L H₂O₂ group and 1 000 μmol/L H₂O₂ group, and CCK8 experiment was used to detect the effect of H₂O₂ on cell viability. The cells were divided into control group and 200 μmol/L H₂O₂ group, and were subjected to 0, 2, 4, 6, 8 Gy radiotherapy, respectively, and the clonogenic assay was performed to assess the radiosensitizing effect of H₂O₂. The cells were divided into control group, 200 μmol/L H₂O₂ group, 4 Gy group and 200 μmol/L H₂O₂+4 Gy group, and the expressions of autophagy-related proteins were measured by Western blotting assay and immunofluorescence assay. Results The expressions of autophagy-related proteins LC3 II and p62 between control group and sodium hyaluronate group was not statistically significant (P>0.05). The expressions of autophagy-related proteins LC3 II and p62 between 4 Gy group and sodium hyaluronate+4 Gy group was not statistically significant(P>0.05). The results of CCK8 experiment showed that the cell proliferation rate was not statistically significant between control group and sodium hyaluronate group before and after radiotherapy(P>0.05); compared with control group, 200 μmol/L H₂O₂ promoted cell proliferation(P<0.05), 500 μmol/L H₂O₂ had no statistically significant effect on cell proliferation(P>0.05), and 700 μmol/L H₂O₂ and 1 000 μmol/L H₂O₂ inhibited cell proliferation(P<0.05). The results of clonogenesis assay showed that H₂O₂ increased the sensitivity of cells to radiotherapy(P<0.05). Western blotting assay and immunofluorescence assay showed that 200 μmol/L H₂O₂ group inhibited autophagy compared with the control group(P<0.05), and the 200 μmol/L H₂O₂+4 Gy group promoted autophagy compared with the 4 Gy group(P<0.05). The expressions of p-AKT and p-mTOR proteins were increased in 200 μmol/L H₂O₂ group compared with control group(P<0.05); while the expressions of p-AKT and p-mTOR were decreased in the 200 μmol/L H₂O₂+4 Gy group compared with 4 Gy group(P<0.05). Conclusion H₂O₂ improves radiosensitivity by regulating AKT/mTOR pathway, and then regulating autophagy.

Key words: Autophagy, Hydrogen peroxide, Sodium hyaluronate, Radiosensitivity, AKT/mTOR pathway, Cervical cancer

中图分类号:

R574

任慧欣,郑茂金,韩文灿,王超群,周云,裴冬生. 过氧化氢通过调控自噬增强宫颈癌的放疗敏感性[J]. 山东大学学报 (医学版), 2023, 61(6): 22-28.

REN Huixin, ZHENG Maojin, HAN Wencan, WANG Chaoqun, ZHOU Yun, PEI Dongsheng. Hydrogen peroxide enhances radiotherapy sensitivity of cervical cancer by regulating autophagy[J]. Journal of Shandong University (Health Sciences), 2023, 61(6): 22-28.

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