Studies On The Anti-tumor Activity And The Mechanisms Of A Novel Colchicine Binding Site Inhibitor BZML In Multidrug Resistacnce Non-small-cell Lung Cancer Cells

Lung cancer is in the first place in the incidence and mortality of malignant cancer in China.According to the histological features,non-small-cell lung cancer(NSCLC)accounts for more than 80%of all lung cancers.Currently,chest film and low dose CT scan have been widely used in the early detection and diagnosis of NSCLC,but there are 70%～80%of NSCLC patients have lost the excellent opportunity of surgical treatment.In recently years,molecularly targeted therapy,radiotherapy and immunotherapy have made remarkable progress in NSCLC-treatment.But traditional chemotherapy has been the mainstay of treatment for decades.However,mulitidrug resistance(MDR)is a major limiting factor influencing the effect of chemotherapy.Microtubules,a component of the cytoskeleton,have been developed as attractive chemotherapeutic targets for anti-cancer drugs.It is widely reported that colchicine binding site inhibitors(CBSIs)as potent tubulin inhibitors,have a potent anti-tumor activity via induction of different modes of cell death.Importantly,some CBSIs have been reported to be able to effectively circumvent MDR.Therefore,a series of novel CBSIs have been designed,synthesized and screened by our group.Among these compounds,5-(3,4,5-trimethoxybenzoyl)-4-methyl-2-(p-tolyl)imidazole(BZML)exhibits a desirable anti-tumor activity against various human cancer cell lines including MDR cells.In this study,we intend to elucidate the anti-tumor activity and molecular mechanisms of BZML against MDR NSCLC in vitro and in vivo.It will provide a theoretical basis for BZML used as a promising anti-cancer agent to overcome MDR NSCLC.Our results show that BZML had broad spectrum anti-tumor activity against various human cancer cells,especially MDR cancer cells in vitro.Additionally,the proliferation of both A549 and A549/Taxol cells were inhibited by BZML in a time-and concentration-dependent manner.Meanwhile,BZML could also inhibit the growth of human tumour xenografts in A549 and A549/Taxol cells-bearing mice,but did not show obvious toxicity to nude mice.Importantly,BZML acted as a CBSI,but not a P-gp substrate.Additionally,it could irreversibly inhibit P-gp function via decreasing P-gp expression at the protein and mRNA levels.Therefore,BZML has a potent anti-tumour activity against MDR NSCLC.Further,BZML could cause cell cycle arrest in mitosis phase in both A549 and A549/Taxol cells.Importantly,BZML induced mitotic catastrophe(MC)that undergoes mitotic slippage due to cyclin B1 degradation in A549/Taxol cells.Meanwhile,BZML drove A549/Taxol cells to die by MC,which could circumvent MDR in A549/Taxol cells.MC is an apoptotic-like cell death mode,which is different in nature from cell apoptosis.Differently,BZML induced A549 cells to die by apoptosis through the mitochondrial apoptotic pathway.Although,P-gp is an important factor which results in MDR in A549/Taxol cells.The anti-apoptosis property of A549/Taxol cells is originated from the defect in activation of the mitochondrial apoptotic pathway.Meanwhile,BZML mediated-ROS generation could cause DNA damage and GSH depletion in both A549 and A549/Taxol cells.In addition to,accompanied with MC occurrence,irreverible cellular senescence is activated in BZML-treated A549/Taxol cells.Importantly,we found that autophagy acted as a non-protective type of autophagy during BZML-induced apoptosis in A549 cells,whereas it acted as a type of cytoprotective autophagy against BZML-induced MC in A549/Taxol cells.Therefore,these findings suggest that autophagy can block the occurrence and development of MC,but cannot contribute to resistance to mitochondrial apoptosis to induce MC in BZML-treated A549/Taxol cells.Meanwhile,the inhibition of autophagy can accelerate the death of BZML-induced MC cells.In conclusion,BZML is a CBSI and exhibits a potent anti-tumor activity against MDR NSCLC in vitro and in vivo.Our study will provide a theoretical basis for BZML used as a promising anti-cancer agent to overcome MDR NSCLC.Notably,it gives a new research direction and strategy for overcoming MDR.Further,we proved that MC is an apoptotic-like cell death mode,which is different in nature from cell apoptosis.