鬼臼毒素靶向抑制 c-MET 促进半胱天冬酶依赖性凋亡并抑制吉非替尼耐药非小细胞肺癌细胞的生长。

Targeted inhibition of c-MET by podophyllotoxin promotes caspase-dependent apoptosis and suppresses cell growth in gefitinib-resistant non-small cell lung cancer cells.

机构信息

Department of Pharmacy, College of Pharmacy, Mokpo National University, Jeonnam 58554, Republic of Korea.

College of Korean Medicine, Dongshin University, Naju-si, Jeollanam-do 58245, Republic of Korea.

出版信息

Phytomedicine. 2021 Jan;80:153355. doi: 10.1016/j.phymed.2020.153355. Epub 2020 Sep 25.

DOI:10.1016/j.phymed.2020.153355

PMID:33039730

Abstract

BACKGROUND

Lung cancer has the highest incidence and cancer-related mortality of all cancers worldwide. Its treatment is focused on molecular targeted therapy. c-MET plays an important role in the development and metastasis of various human cancers and has been identified as an attractive potential anti-cancer target. Podophyllotoxin (PPT), an aryltetralin lignan isolated from the rhizomes of Podophyllum species, has several pharmacological activities that include anti-viral and anti-cancer effects. However, the mechanism of the anti-cancer effects of PPT on gefitinib-sensitive (HCC827) or -resistant (MET-amplified HCC827GR) non-small cell lung cancer (NSCLC) cells remains unexplored.

PURPOSE

In the present study, we investigated the underlying mechanisms of PPT-induced apoptosis in NSCLC cells and found that the inhibition of c-MET kinase activity contributed to PPT-induced cell death.

METHODS

The regulation of c-MET by PPT was examined by pull-down assay, ATP-competitive binding assay, kinase activity assay, molecular docking simulation, and Western blot analysis. The cell growth inhibitory effects of PPT on NSCLC cells were assessed using the MTT assay, soft agar assay, and flow cytometry analysis.

RESULTS

PPT could directly interact with c-MET and inhibit kinase activity, which further induced the apoptosis of HCC827GR cells. In contrast, PPT did not significantly affect EGFR kinase activity. PPT significantly inhibited the cell viability of HCC827GR cells, whereas the PPT-treated HCC827 cells showed a cell viability of more than 80%. PPT dose-dependently induced G2/M cell cycle arrest, as shown by the downregulation of cyclin B1 and cdc2, and upregulation of p27 expression in HCC827GR cells. Furthermore, PPT treatment induced Bad expression and downregulation of Mcl-1, survivin, and Bcl-xl expression, subsequently activating multi-caspases. PPT thereby induced caspase-dependent apoptosis in HCC827GR cells.

CONCLUSION

These results suggest the potential of PPT as a c-MET inhibitor to overcome tyrosine kinase inhibitor resistance in lung cancer.

摘要

背景

肺癌是全球所有癌症中发病率和癌症相关死亡率最高的癌症。其治疗方法主要集中在分子靶向治疗。c-MET 在各种人类癌症的发生和转移中起着重要作用，已被确定为有吸引力的潜在抗癌靶点。鬼臼毒素（PPT）是一种从鬼臼属植物根茎中分离出来的芳基四氢萘木脂素，具有多种药理活性，包括抗病毒和抗癌作用。然而，PPT 对 gefitinib 敏感（HCC827）或耐药（MET 扩增 HCC827GR）非小细胞肺癌（NSCLC）细胞的抗癌作用机制尚不清楚。

目的

本研究旨在探讨 PPT 诱导 NSCLC 细胞凋亡的潜在机制，发现抑制 c-MET 激酶活性有助于 PPT 诱导细胞死亡。

方法

通过下拉实验、ATP 竞争结合实验、激酶活性实验、分子对接模拟和 Western blot 分析检测 PPT 对 c-MET 的调节。采用 MTT 实验、软琼脂实验和流式细胞术分析检测 PPT 对 NSCLC 细胞生长抑制作用。

结果

PPT 可直接与 c-MET 相互作用并抑制其激酶活性，进而诱导 HCC827GR 细胞凋亡。相比之下，PPT 对 EGFR 激酶活性无明显影响。PPT 显著抑制 HCC827GR 细胞的活力，而 PPT 处理的 HCC827 细胞的活力超过 80%。PPT 呈剂量依赖性地下调 cyclin B1 和 cdc2 的表达，上调 p27 的表达，诱导 HCC827GR 细胞 G2/M 细胞周期阻滞。此外，PPT 处理诱导 Bad 表达，并下调 Mcl-1、survivin 和 Bcl-xl 的表达，随后激活多聚半胱天冬酶。PPT 从而诱导 HCC827GR 细胞 caspase 依赖性凋亡。