Specific growth inhibition by alteration of metabolic pathway using Chinese herbal medicine in tyrosine kinase inhibitor resistant non-small cell lung cancer

OBJECTIVE Lung cancer is the leading cause of cancer death worldwide.Epidermal growth factor receptor(EGFR)mutation(s)is/are common in non-small cell lung cancer(NSCLC)in Asian population,resulting in lung tumor formation.L858 Rsubstitution mutation on exon 21 and in-flame deletion mutation on exon 19 are the two most common forms of EGFR mutation.Molecular targeted therapy using tyrosine kinase inhibitor(TKI)targeting EGFR shows promising initial response,however drug resistance is common.Therefore,it is needed to identify new inhibitors to tackle TKI-resistance.In this study,we aim to investigate the effect of multiple single purified compounds derived from Chinese herbal medicines(CMHs)on a panel of NSCLC cell lines with different EGFR mutational statuses and TKI sensitivity.We also examine the biological functional effect and drug action mechanism of these cell lines after drug treatment.METHODS We have reviewed the literature and selected ten single purified compounds derived from CMHs which exhibited the highest potential of cancer suppression effect in NSCLC.We have recruited three EGFR-dependent NSCLC cell lines for drug screening using cytotoxicity assay.A549 is used as EGFR wild-type control.Two TKI-resistant NSCLC cell lines were used,H1975 harbors double mutation(EGFRL858R+T790M)and H1650 harbors EGFRexon 19 deletion.H2228is a NSCLC cell line which harbours EML4-ALK fusion gene and was used as EGFR-independent cell line control.MTT assay was used to determine the drug efficacy and IC50 value.Then functional assays including cell cycle arrest analysis and apoptosis assay was used to determine the biological effect after drug treatment.RESULTS MTT assay revealed that six out of ten candidate agents showed significant cancer-inhibiting effects in H1650 and H1975cells.Three compounds exhibited IC50 value at micro-molar levels while another three compounds exhibited IC50 at as low as nano-molar levels.One compound exhibited specificity on EGFR-dependent NSCLC cell lines,which showed 10-fold more selective than the EGFR-independent H2228 cells.Cell cycle analysis and immunoblotting assay showed that one effective compound,designated was MUST-1,altered the metabolic pathway of glucose metabolism and lipid metabolism,and induced cell cycle arrest at G1 phase in NSCLC with EGFR mutation.However,the anti-proliferative effects were distinct in NSCLC cell lines with different EGFR mutation patterns.CONCLUSION MUST-1 significantly induced cell cycle arrest in four NSCLC in EGFR mutant cell lines but the inhibiting effect was not significant in EGFR wildtype cell line.Immunobloting assay revealed that overall intracellular lipid content,glycolytic enzymes PKM2 and cell cycle regulatory gene expression were altered after 72 hcompound treatment in the responsive cells.Further investigation is required to elucidate the underlying reason of drug selectivity and the role of glucose and lipid metabolism in regulating drug sensitivity.