HMQ-T-B10 induces human liver cell apoptosis by competitively targeting EphrinB2 and regulating its pathway.

Bingling Dai,Langchong He,Jie Zhang,Weina Ma,Yanmin Zhang,Xianpeng Shi,Nan Ma,Tianfeng Yang

doi:10.1111/jcmm.13729

Abstract

Hepatocellular carcinoma (HCC) is a highly prevalent cancer worldwide and it is necessary to discover and develop novel preventive strategies and therapeutic approaches for HCC. Herein, we report that EphrinB2 expression is correlated with liver cancer progression. Moreover, by using phosphorylated proteomics array, we reveal a pro‐apoptosis protein whose phosphorylation and activation levels are up‐regulated upon EphrinB2 knockdown. These results suggest that EphrinB2 may act as an anti‐apoptotic protein in liver cancer cells. We also explored the therapeutic potential of HMQ‐T‐B10 (B10), which was designed and synthesized in our laboratory, for HCC and its underlying mechanisms in vitro and in vivo. Our data demonstrate that B10 could bind EphrinB2 and show inhibitory activity on human liver cancer cells. Moreover, induction of human liver cancer cell apoptosis by B10 could be augmented upon EphrinB2 knockdown. B10 inhibited HCC cell growth and induced HCC cell apoptosis by repressing the EphrinB2 and VEGFR2 signalling pathway. Growth of xenograft tumours derived from Hep3B in nude mice was also significantly inhibited by B10. Collectively, these findings highlight the potential molecular mechanisms of B10 and its potential as an effective antitumour agent for HCC.

Highlights

Hepatocellular carcinoma (HCC) is a highly prevalent cancer worldwide and it is necessary to discover and develop novel preventive strategies and therapeutic approaches for HCC
We found that B10 inhibited HCC cell proliferation, induced HCC cell apoptosis, and suppressed xenograft growth in nude mice by targeting EphrinB2 and its pathway
We found that B10 could inhibit the VEGFR2 kinase activity in vitro by evaluating the phosphorylation of a peptide substrate by VEGFR2 kinase in a microtitre plate format using LANCE

Summary

| MATERIALS AND METHODS

Sorafenib was ordered from Dalian Meilun Biotech Co., Ltd (Dalian, China) and a novel agent, B10 (purity >98%), was designed and synthesized by Jie Zhang’s group in the Research and Engineering Center for Natural Medicine Xi’an Jiaotong University.[22,23] All compounds were dissolved in DMSO for use in biochemical assays. Human SMMC-7721, Hep3B, HepG2, Bel-7402 and 97 h liver cancer cell lines were purchased from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences (Shanghai, China). VEGFR2/HEK293 cell lines which over-expressed EphrinB2 and VEGFR2 were constructed at the Research and Engineering Center for Natural Medicine, Xi’an Jiaotong University. SMMC-7721, Hep3B, Bel-7402, HepG2 and 97 hours cells were seeded into 96-well plates and various concentrations of B10 and sorafenib were added; the plates were incubated for 48 hours. The expression profile of 12 signalling pathway phosphor-related proteins was detected and analysed using a human CSP100 Antibody Array kit (Full Moon Biosystems, Sunnyvale, CA, USA). The antibody microarray was blocked for 45 minutes, and dried and incubated with the biotin-labelled cell lysates at room temperature for 2 hours.

| RESULTS

| DISCUSSION

Findings

CONFLICT OF INTEREST