Abstract

Objective

Isoflurane is an extensively used inhalational anesthesia, and its carcinogenic or anti-cancerous effect has been identified recently. However, the specific role of isoflurane in cervical cancer remains unclear.

Aim

This study aimed to investigate the function of isoflurane in cervical cancer as well as the underlying mechanism.

Methods

After isoflurane treatment, HeLa cell viability, percentage of apoptotic cells, expression of active caspase-3/9 were examined by CCK-8 assay, Annexin V-FITC/PI double staining, and Western blot analysis, respectively. ROS generation, ratio of NAD⁺/NADH, and ATP level after isoflurane stimulation were determined using commercial assay kits. Afterwards, activation of AMPK and autophagy was assessed through Western blot analysis and immunofluorescence. Whether AMPK mediated the isoflurane-induced apoptosis and autophagy was explored by adding an AMPK inhibitor (Compound C). The in vivo function of isoflurane was finally investigated on a HeLa cell xenograft model.

Results

Isoflurane inhibited cell viability and induced apoptosis evidenced by upregulation of active caspase-3/9 in HeLa cells. Oxidative stress was triggered by isoflurane, as isoflurane elevated ROS level, and lowered ratio of NAD⁺/NADH and ATP level. Further results showed isoflurane activated the AMPK/mTOR pathway and induced autophagy. In addition, inhibition of AMPK led to ameliorated effects of isoflurane on apoptosis and autophagy. In vivo experiments proved isoflurane could repress tumorigenesis, activate AMPK, and induce autophagy in Xenograft mouse.

Conclusions

Isoflurane activated AMPK to inhibit proliferation and promote apoptosis and autophagy both in vitro and in vivo.

中文翻译：

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异氟醚激活AMP活化蛋白激酶抑制增殖，促进宫颈癌在体外和体内的凋亡和自噬

摘要

客观的

异氟醚是一种广泛使用的吸入麻醉剂，最近已确定其致癌或抗癌作用。然而，异氟醚在宫颈癌中的具体作用仍不清楚。

目标

本研究旨在探讨异氟醚在宫颈癌中的作用及其作用机制。

方法

异氟醚处理后，分别通过CCK-8法、Annexin V-FITC/PI双染法和Western印迹法检测HeLa细胞活力、凋亡细胞百分比、活性caspase-3/9的表达。使用商业化测定试剂盒测定异氟醚刺激后的 ROS 生成、NAD ^{+ /NADH 比率和 ATP 水平。}之后，通过蛋白质印迹分析和免疫荧光评估 AMPK 的激活和自噬。通过添加 AMPK 抑制剂（化合物 C）探索 AMPK 是否介导异氟醚诱导的细胞凋亡和自噬。最后在 HeLa 细胞异种移植模型上研究了异氟醚的体内功能。

结果

异氟醚抑制细胞活力并诱导细胞凋亡，这可以通过 HeLa 细胞中活性 caspase-3/9 的上调来证明。氧化应激是由异氟醚引发的，因为异氟醚升高了 ROS 水平，降低了 NAD ⁺ /NADH 和 ATP 水平的比率。进一步的结果显示异氟醚激活 AMPK/mTOR 通路并诱导自噬。此外，抑制 AMPK 可改善异氟醚对细胞凋亡和自噬的影响。体内实验证明异氟醚可以抑制异种移植小鼠的肿瘤发生、激活 AMPK 并诱导自噬。

结论

异氟醚激活 AMPK在体外和体内抑制增殖并促进细胞凋亡和自噬。