Study On The Roles And Action Mechanisms Of Novel Pyrazoline Coumarin Derivatives And Ferrocenyl Pyrazole Derivatives In Modulation Of Cell Apoptosis,Autophagy And Cell Cycle

It is reported that the morbidity and mortality of lung cancer is increasing recently all over the world,which greatly threatens human health.The chemotherapy drugs,the choice drugs for the treatment of lung cancer,have side effects and drug resistance.Therefore,it is urgent to develop novel anti-lung cancer drugs.Promoting cell apoptosis,modulating autophagy and indicing cell cycle arrest have been considered as effective strategies to the development of novel anti-cancer drugs.By utilization of new small molecules,chemical genetics can discover new key factors and new signal pathways involved in the modulation of apoptosis,autophagy and cell cycle,which is very helpful for future drug development.Small fluorescent molecules are potent tools to monitor biological events in living cells and whole organisms.However,very few fluorescent anti-cancer drugs have been developed.Many pyrazoline and cotumarin derivatives show antitumor property.In addition,pyrazoline and coumarin are also used as fluorescent chromophores.Here,we evaluated whether a series of novel fluorescent pyrazoline coumarin derivatives could inhibit cell proliferation of A549 lung cancer.Among these derivatives 4-(3-(7-(Diethylamimo)-2-oxo-2H-chromen-3-yl)-5-phenyli4,5-dihydro-1H-pyrazol-1-yl)benzoic acid(DPB)was the most effective small molecule.In the present study,we identified the target of screened DPB as HSP90 by taking advantage of fluorescence combined with LC-MS/MS and chemoinformatics technique.Immunoprecipitations(IPs)results showed that DPB blocked the binding of HSP90 with its monoclonal antibody in a dose-dependent way,suggesting that the compound might bind to HSP90 directly.Results of molecular docking and the detection of client proteins also verified HSP90 as the target of DPB.Further study indicated that DPB also inhibited the growth of H322 and H1299 lung cancer cells in dose-dependent manner.More importantly,DPB had no inhibitory effect on the growth of normal HUVECs.DPB at 20?M induced apoptosis rather than necrosis in A549 cells.Autophagy,an important cellular homeostatic mechanism,plays a vital role in tumor development and cancer therapy.Thus,it may help us develop more effective therapeutic strategy and improve clinical outcomes to investigate the influence of DPB on autophagy in cancer cells.DPB,HSP90 inhibitor,induced autophagy in mTOR-independent manner in A549 lung cancer cells.Autophagy inhibitor 3MAand 3BDO increased the toxic effect of DPB on A549 cells,which indicated that DPB combined with autopbay inhibitors might be more effective in cancer treatment.Although inhibitors of HSP90 are emerging as novel promising therapeutic agents for cancer therapy and more than 17 inhibitors of HSP90 have entered clinical trials,most of them have no exact curative effects.The reason for the lack of efficacy is currently not clear.Some compounds suppressing cell growth at high concentrations can promote cell growth at tower concentrations.That is,their dose-response curve is'bell-shaped'.The low dose stimulation and high dose inhibiton phenomenon,biphasic dose response,is called hormesis in the field of toxicology.Hormesis has currently become a central concept in biological and biomedical scieinces with significant implications for clinical medicine.Presently,there are only few researches on the hormesis of HSP90 inhibitors.This may be the main reason that HSP90 inhibitors often have poor efficacy.It was shown that low-dose DPB and AUY-922 could unexpectedly restrain apoptosis induced by serum starvation in HUVECs.This hormesis was accompanied by the increase of p-AKT1.So the dose and administration of HSP90 inhibitors in clinical trials may need reevaluation.Many ferrocenyl pyrazole derivatives display antitumor activity.In our previous study,we evaluated a series of novel ferrocenyl pyrazole derivatives.Although it was shown that these compounds inhibited the growth of A549 lung cancer cells,but their anti-cancer mechanisms are still unclear.The modification of the structure of ferrocenyl pyrazole derivatives and the identification of the linteraction mechanisms between small molecules with their targets are of great importance for developing new drugs.Molecular chirality is very important for biology,pharmacotherapy and new drug development.Herein,we analyzed the effects of a series of novel ferrocenyl pyrazole derivatives on the proliferation of lung cancer cells and explored the mechanisms.The results showed that almost all the ferrocenyl pyrazole derivatives suppressed the growth of A549 lung cancer cells.Among the compounds,(S)-and(R)-2a,2g,(R)-2e and(S)-2f were more effective and might perform their functions through cell cycle arrest.Although the inhibitory effects of most R and S enantiomers had no significant differences,(S)-2f displayed more effective inhibition than(R)-2f.Moreover,compounds(S)-and(R)-2a,2g and(S)-2f induced the cell cycle arrest in p53 dependent manner,whilethe cell cycle arrest induced by compound(R)-2e was p53 independent.