一种新型1,2,3-三唑-靛玉红杂合物通过减轻肝细胞癌中的HGF/c-MET轴来抑制肿瘤生长和肺转移。

A new 1,2,3-triazole-indirubin hybrid suppresses tumor growth and pulmonary metastasis by mitigating the HGF/c-MET axis in hepatocellular carcinoma.

作者信息

Gowda Shalini V, Kim Na Young, Harsha Kachigere B, Gowda Darshini, Suresh Rajaghatta N, Deivasigamani Amudha, Mohan Chakrabhavi Dhananjaya, Hui Kam Man, Sethi Gautam, Ahn Kwang Seok, Rangappa Kanchugarakoppal S

机构信息

Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570006, Karnataka, India.

Department of Science in Korean Medicine, Kyung Hee University, 24 Kyungheedae-ro, Dongdaemun-gu, Seoul 02447, Republic of Korea.

出版信息

J Adv Res. 2025 Jul;73:341-356. doi: 10.1016/j.jare.2024.08.033. Epub 2024 Aug 30.

DOI:10.1016/j.jare.2024.08.033

PMID:39216686

Abstract

INTRODUCTION

Hepatocellular carcinoma (HCC) is a fatal cancer that is often diagnosed at the advanced stages which limits the available therapeutic options. The interaction of HGF with c-MET (a receptor tyrosine kinase) results in the activation of c-MET which subsequently triggers the PI3K/Akt/mTOR axis. Overexpression of c-MET in HCC tissues has been demonstrated to contribute to tumor progression and metastasis.

OBJECTIVES

We aimed to synthesize triazole-indirubin conjugates, examine their growth suppressor efficacy in cell-based assays, and investigate the antitumor as well as antimetastatic activity of lead cytotoxic agent in the orthotopic mice model.

METHODS

A series of triazole-indirubin hybrids were synthesized and cytotoxicity, apoptogenic, and antimigratory effect of the lead compound (CRI9) was evaluated using MTT assay, cell cycle analysis, annexin-V/PI assay, TUNEL assay, and wound healing assay. The effect of CRI9 on the operation of the HGF/c-MET/PI3K/Akt/mTOR axis was examined using western blotting and transfection experiments. Acute toxicity, antitumor, and antimetastatic activity of CRI9 were examined in NCr nude mice. The expression of c-MET/PI3K/Akt/mTOR, CD31, and Ki-67 was examined using immunohistochemistry and western blotting.

RESULTS

Among the new compounds, CRI9 consistently displayed potent cytotoxicity against HGF-induced HCC cells. CRI9 induced apoptosis as evidenced by increased sub G1 cells, annexin-V/PI cells, TUNEL cells, and cleavage of procaspase-3 and PARP. CRI9 inhibited HGF-induced phosphorylation of c-MET and subsequently suppressed the PI3K/Akt/mTOR axis. Also, depletion of c-MET or inhibition of c-MET by CRI9 resulted in suppression of the PI3K/Akt/mTOR axis. CRI9 showed no toxic effects in NCr nude mice and displayed a potent antitumor and antimetastatic effect in the orthotopic HCC mice model. CRI9 also reduced the levels of phospho-c-MET, CD31, and Ki-67 and suppressed the activation of the PI3K/Akt/mTOR axis in tumor tissues.

CONCLUSION

CRI9 has been identified as a new inhibitor of the c-MET/PI3K/Akt/mTOR axis in HCC preclinical models.

摘要

引言

肝细胞癌（HCC）是一种致命癌症，常于晚期被诊断出来，这限制了可用的治疗选择。肝细胞生长因子（HGF）与c-MET（一种受体酪氨酸激酶）相互作用会导致c-MET激活，随后触发PI3K/Akt/mTOR信号轴。已证实HCC组织中c-MET的过表达会促进肿瘤进展和转移。

目的

我们旨在合成三唑-靛玉红缀合物，在基于细胞的试验中检测它们的生长抑制功效，并在原位小鼠模型中研究先导细胞毒性剂的抗肿瘤及抗转移活性。

方法

合成了一系列三唑-靛玉红杂合物，并使用MTT试验、细胞周期分析、膜联蛋白V/碘化丙啶（PI）试验、末端脱氧核苷酸转移酶介导的缺口末端标记（TUNEL）试验和伤口愈合试验评估了先导化合物（CRI9）的细胞毒性、凋亡诱导和抗迁移作用。使用蛋白质印迹法和转染实验检测了CRI9对HGF/c-MET/PI3K/Akt/mTOR信号轴作用的影响。在NCr裸鼠中检测了CRI9的急性毒性、抗肿瘤和抗转移活性。使用免疫组织化学和蛋白质印迹法检测了c-MET/PI3K/Akt/mTOR、CD31和Ki-67的表达。

结果

在新化合物中，CRI9始终对HGF诱导的HCC细胞显示出强大的细胞毒性。CRI9诱导凋亡，表现为亚G1期细胞、膜联蛋白V/PI细胞、TUNEL细胞增多以及半胱天冬酶-3原和聚（ADP-核糖）聚合酶（PARP）的裂解。CRI9抑制HGF诱导的c-MET磷酸化，随后抑制PI3K/Akt/mTOR信号轴。此外，CRI9使c-MET耗竭或抑制c-MET会导致PI3K/Akt/mTOR信号轴受到抑制。CRI9在NCr裸鼠中未显示出毒性作用，并且在原位HCC小鼠模型中表现出强大的抗肿瘤和抗转移作用。CRI9还降低了肿瘤组织中磷酸化c-MET、CD31和Ki-67的水平，并抑制了PI3K/Akt/mTOR信号轴的激活。