Licochalcone B inhibits growth and induces apoptosis of human non-small-cell lung cancer cells by dual targeting of EGFR and MET

Abstract

Background

Epidermal growth factor receptor (EGFR) gene alterations are associated with sensitization to tyrosine kinase inhibitors such as gefitinib in lung cancer. Some patients suffering from non-small cell lung cancer (NSCLC) have difficulty in treating the cancer due to resistance acquired to gefitinib with MET amplification. Therefore EGFR and MET may be attractive targets for lung cancer therapy.

Purpose

This study aimed to investigate the anti-cancer activity of Licochalcone (LC)B extracted from Glycyrrhiza inflata, in gefitinib-sensitive or gefitinib-resistant NSCLC cells, and to define its mechanisms.

Study design

We investigated the mechanism of action of LCB by targeting EGFR and MET in human NSCLC cells.

Methods

We used the HCC827 and HCC827GR lines as gefitinib-sensitive and –resistant cells respectively, and determined the effects of LCB on both, by performing cell proliferation assay, flow cytometry analysis and Western blotting. Targets of LCB were identified by pull-down/kinase assay and molecular docking simulation.

Results

LCB inhibited both EGFR and MET kinase activity by directly binding to their ATP-binding pockets. The ability of this interaction was verified by computational docking and molecular dynamics simulations. LCB suppressed viability and colony formation of both HCC827 and HCC827GR cells while exhibiting no cytotoxicity to normal cells. The induction of G2/M cell-cycle arrest and apoptosis by LCB was confirmed by Annexin V/7-AAD double staining, ER stress and reactive oxygen species induction, mitochondrial membrane potential loss and caspase activation as well as related-proteins regulation. Inhibition of EGFR and MET by LCB decreased ERBB3 and AKT axis activation.

Conclusion

We provide insights into the LCB-mediated mechanisms involved in reducing cell proliferation and inducing apoptosis in NSCLC cells. This occurs through dual inhibition of EGFR and MET in NSCLC cells regardless of their sensitivity or resistance to gefitinib. LCB may be a promising novel therapeutic medicine for gefitinib-sensitive or resistant NSCLC treatment.

Introduction

LCB is an active flavonoid extracted from roots of Chinese medicinal herb Glycyrrhiza inflata (Wang et al., 2013). It is the most important component with a variety of pharmacological effects, anti-bacterial, anti-oxidant and anti-tumor activities (Wang et al., 2013). The anti-cancer effect of LCB has been documented in a number of cancer cell lines, including human bladder cancer T24 and EJ cells, oral cancer HN22 and HSC4 cells, breast cancer MCF-7 cells, skin melanoma A375 cells and skin cancer A431 cells (Kang et al., 2017, Oh et al., 2016, Yu et al., 2016, Yuan et al., 2014). Previous reports demonstrated that LCB affected cancer cell growth and inhibition of metastasis, cell cycle arrest, and apoptosis induction (Oh et al., 2016, Yu et al., 2016, Yuan et al., 2014). However, its mechanism in lung cancer cells has not yet been elucidated.

Lung cancer is the leading cause of cancer deaths in both men and women in the world, and 85% of lung cancer patients have NSCLC (Molina et al., 2008). Despite treatments with surgery, radiotherapy, chemotherapy, and combinations of these, for patients with advanced NSCLC the overall 5-year survival rate is only 10∼20% (Molina et al., 2008). Chemotherapy treatment prolongs survival of patients with advanced NSCLC, but is often accompanied by a variety of adverse side effects such as significant toxicity and increasing drug resistance (Nguyen et al., 2009).

Molecular targeting of receptor tyrosine kinases (RTK)s such as EGFR and hepatocyte growth factor (HGF)/MET has emerged as a promising therapeutic strategy in the treatment of lung cancer. The RTK family of cell surface receptors, ERBB1 (EGFR), ERBB2, ERBB3 and ERBB4 can mediate cell signaling (Sharma et al., 2007). When ligand activates the receptor, the receptor forms homo- or hetero-dimers and autophosphorylation of its tyrosine kinase domain results in a cascade of intracellular signal transduction events (Puri and Salgia, 2008). EGFR and MET have generated considerable attention as targets for lung cancer therapy due to their high expression levels in lung tumor cells.

Gefitinib, a tyrosine kinase inhibitor (TKI), is a small-molecule drug that specifically targets EGFR and has been used for treating NSCLC. Gefitinib serves as a competitive inhibitor that targets the ATP-binding cleft at the active site of EGFR kinase (Sharma et al., 2007). Most advanced NSCLC patients initially respond to gefitinib, but then develop acquired resistance to this drug as treatment continues (Nguyen et al., 2009). Resistance to gefitinib is relatively common in EGFR T790M mutations, MET amplification, or HGF overexpression (Comoglio et al., 2008, Pao and Chmielecki, 2010). Therefore, additional strategies are needed to combat this resistance, including new drug combinations or development of novel compounds.

The Engelman group established the gefitinib-resistant HCC827GR cell line following long-term exposure to gefitinib in EGFR mutant HCC827 cells which are sensitive to gefitinib. They found that amplification of MET induced gefitinib resistance through activation of ERBB3/PI3K/AKT signaling in lung cancer (Engelman et al., 2007). Interestingly, cell viability of HCC827GR cells was not affected by treatment with gefitinib or MET inhibitor alone, but treatment with both resulted in decreased cell growth and induced apoptosis. The key observation here was that dual inhibition of EGFR and MET may be an effective treatment for EGFR-TKI resistant cells.

The aim of the current study therefore was to investigate the role of EGFR and MET in inducing apoptosis following treatment with LCB, using human gefitinib-sensitive or -resistant NSCLC cells as a model. We investigated whether LCB treatment could suppress EGFR and MET activities and reduce the expression of downstream molecules of both the EGFR and MET signaling pathways. To investigate whether LCB induces apoptosis, we further examined NSCLC cell proliferation, colony formation, cell cycle distribution, reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP) depolarization and caspase activation. The results of the present study shed light on the molecular mechanisms of LCB in NSCLC and may provide new insights into efforts to cure cancer.