Study On The Mechanism Of Novel Microtubule Inhibitor TH320 To Overcome Multidrug Resistance In A549/Taxol Cells

Background: According to the statistics,the mortality of lung cancer ranks first in the world,and the incidence of lung cancer ranks second in the world.Non-small-cell lung cancer(NSCLC)accounts for the highest proportion of lung cancer,and the 5-year survival rate is low.For the treatment of NSCLC,paclitaxel is one of the basic chemotherapeutic drugs,but multidrug resistance(MDR)is the main factor affecting the efficacy of paclitaxel.Studies have shown that in the process of tumor treatment,MDR is the main reason that affects chemotherapy,and the clinical production of MDR chemotherapeutic drugs can easily affect the therapeutic effect.Microtubules are important targets for the treatment of tumors.Compared with tubulin inhibitors at the paclitaxel site and vincristine site,tubulin inhibitors acting at the colchicine site can overcome P-gp regulated MDR.Objective: To investigate the effect and mechanism of novel tubulin inhibitor TH320 on multidrug resistance of A549/Taxol cells.Methods: MTT assay was used to detect the effect of TH320 on the growth inhibition of multiple tumor cells and the drug resistance ratio of A549/Taxol cells to many kinds of compounds.Rhodamine(Rh123)accumulation experiment using high-throughput live cell imaging system(HSC)to detect the effect of TH320 on the efflux function and expression of P-gp.The effect of TH320 on the morphology of A549 and A549/Taxol cells was observed by inverted fluorescence microscope.The effect of TH320 on intracellular microtubules was observed by immunofluorescence β-tubulin staining.The effects of TH320 on apoptosis(Annexin V/PI),cell cycle(PI),autophagy(MDC)and reactive oxygen species(DHE)of A549 and A549/Taxol cells were detected by flow cytometry.Hoechst33342 staining was used to observe the effect of TH320 on the nuclear changes of A549/Taxol cells.Senescence of A549/Taxol cells induced by TH320 was observed by SA-β-Gal staining.Western blot was used to detect the effects of TH320 on cycle-associated proteins(cyclin B1,p-H3,cdc2,p-cdc2),apoptosis proteins(Caspase-3,Caspase-8,Caspase-9,Bax,Bcl-2,XIAP)and Cytochrome C,senescence proteins(Rb,p-Rb,p21,p16),autophagy proteins(ATG5,LC3,SQSTM/p62)and P-gp protein in A549/Taxol cells.Results: 1.TH320 showed excellent cytotoxic activity in many tumor cells.Compared with CA-4,it was less toxic to normal cells and inhibited the proliferation of A549 and A549/Taxol cells in a dose-and time-dependent manner.2.A variety of compounds were used to detect the drug resistance of A549/Taxol cells,and the drug resistance of A549/Taxol cells was higher than that of paclitaxel and CA-4,which indicated that the drug resistance of Taxol was strong.The resistance index(RI)to TH320 was only 0.6,which indicated that TH320 could resist resistance.3.Immunofluorescence was used to detect the effects of TH320,CA-4 and Taxol on A549/Taxol tubulin.Confocal observation was made to observe that TH320 and CA-4 inhibited microtubule polymerization,while Taxol promoted microtubule polymerization.4.P-gp was highly expressed in A549/Taxol cells by Western blot,which indicated that A549/Taxol had drug resistance,and TH320 decreased the expression of P-gp in A549/Taxol cells in a time-dependent manner.Rhodamine accumulation assay showed that TH320 inhibited P-gp efflux.5.TH320 induced different morphological and nuclear changes in A549 and A549/Taxol.Cell shrinkage and roundness in A549.The nucleus of A549/Taxol became enlarged and appeared multinucleated and micronucleated,and the cell morphology became larger and flattened.6.TH320 induced apoptosis,cycle arrest,senescence,autophagy and reactive oxygen species in A549/Taxol in a time-dependent manner.7.Western blot showed that TH320 regulated the expression of apoptosis protein active caspase-3,active caspase-8,active caspase-9,Bax up and Bcl-2,XIAP down;cycle protein cyclin B1,p-H3,cdc2 down and p-cdc2 up;aging protein Rb,p21,p16 up and p-Rb down;autophagy protein ATG5,LC3Ⅱ/LC3Ⅰ up and SQSTM/p62 down.8.Flow cytometry showed that proteasome inhibitors MG132,Bortezomib,Carfilzomib inhibited TH320-induced apoptosis,cycle arrest,autophagy,reactive oxygen species and senescence.9.Western blot was used to detect the effects of proteasome inhibitors MG132,Bortezomib,Carfilzomib to reverse the regulation of TH320 on apoptotic,cycly,aging and autophagy protein.Conclusions: TH320,a novel tubulin inhibitor,has outstanding inhibitory ability against the growth of A549/Taxol cells.With tubulin as the target,by inhibiting microtubule polymerization,induce apoptosis,and can cause cycle arrest,senescence and autophagy,produce reactive oxygen species and play the role of drug resistance.Proteasome plays an important role in TH320 resistance.