大蒜素通过 ROS 介导的细胞死亡途径和抑制低氧诱导因子克服肺癌细胞缺氧介导的顺铂耐药性。

Allicin Overcomes Hypoxia Mediated Cisplatin Resistance in Lung Cancer Cells through ROS Mediated Cell Death Pathway and by Suppressing Hypoxia Inducible Factors.

机构信息

Department of Genetics, University of Delhi South Campus, New Delhi, India.

Department of Chemical Engineering, Imperial College London, London, United Kingdom.

出版信息

Cell Physiol Biochem. 2020 Aug 19;54(4):748-766. doi: 10.33594/000000253.

DOI:10.33594/000000253

PMID:32809300

Abstract

BACKGROUND/AIMS: The hypoxic microenvironment in NSCLC has been widely accepted as a contributor to both therapeutic resistance and tumor progression. In this study, we have explored Allicin, a key organosulfur compound present in garlic for its previously unreported effectiveness in the heterogeneous hypoxic tumor microenvironment of NSCLC.

METHODS

The effect of Allicin on the viability of NSCLC cells was determined by MTT assay. To determine the migration rate of treated cells compared to the control, scratch and transwell migration assays were performed. Flowcytometry was done to explore cell cycle distribution, apoptosis and ROS production in cells. Fluorescence microscopy was used to examine autophagy and DNA damage in cells. Dot blot was done to check genome wide methylation. RNA expression was detected by RT-PCR and protein expression by western blotting.

RESULTS

Allicin significantly decreases cell viability, proliferation and migration of NSCLC cells in both normoxia and hypoxia. It elicits both apoptosis and autophagy pathway in A549 cells by ROS accumulation and facilitating S/G2-M phase arrest in both normoxia as well as hypoxia. We suggest that ROS/MAPK and ROS/JNK signaling pathway together govern the cytotoxic effect of allicin in NSCLC cells. Notably, allicin suppresses the expression of HIF-1α and HIF-2α in hypoxic cells, pointing towards a mechanism of its effectiveness in hypoxia. A long term passive demethylation was observed, with decreased mC and no change in TET expression, thereby ruling out active demethylation by allicin. Furthermore, allicin synergistically enhances growth inhibitory activity of low dose cisplatin to effectively overcome hypoxia induced cisplatin resistance in A549 cells.

CONCLUSION

Altogether, our results elucidate a potential use of allicin in sensitizing hypoxic and chemoresistant NSCLC to cisplatin-based chemotherapy and provide new, affordable therapeutic strategy with reduced side effects.

摘要

背景/目的：非小细胞肺癌（NSCLC）的缺氧微环境被广泛认为是导致治疗抵抗和肿瘤进展的因素之一。在这项研究中，我们探索了大蒜中的一种关键有机硫化合物——大蒜素，其在非小细胞肺癌异质缺氧肿瘤微环境中的作用以前并未被报道过。

方法

通过 MTT 法测定大蒜素对 NSCLC 细胞活力的影响。为了确定与对照组相比，处理细胞的迁移率，进行划痕和 Transwell 迁移实验。通过流式细胞术研究细胞周期分布、凋亡和 ROS 产生。荧光显微镜用于检查细胞自噬和 DNA 损伤。点印迹用于检查全基因组甲基化。通过 RT-PCR 检测 RNA 表达，通过 Western blot 检测蛋白表达。

结果

大蒜素在常氧和缺氧条件下均显著降低 NSCLC 细胞的活力、增殖和迁移。它通过 ROS 积累和促进常氧和缺氧条件下的 S/G2-M 期阻滞，在 A549 细胞中引发凋亡和自噬途径。我们认为 ROS/MAPK 和 ROS/JNK 信号通路共同调控大蒜素对 NSCLC 细胞的细胞毒性作用。值得注意的是，大蒜素抑制缺氧细胞中 HIF-1α 和 HIF-2α 的表达，表明其在缺氧环境中有效的机制。观察到长期的被动去甲基化，mC 减少而 TET 表达不变，因此排除了大蒜素的主动去甲基化作用。此外，大蒜素与低剂量顺铂协同增强生长抑制活性，有效克服 A549 细胞中缺氧诱导的顺铂耐药性。