6a,A Novel Synthetic Microtubule Destabilizing Agent,Inhibits Growth Of Vincristine-resistant Oral Epidermoid Carcinoma Cells

Background and objectiveNowadays,one of the most common problems in the treatment of malignant tumors is multidrug resistance(MDR),which the drug resistant tumors exhibit remarkable resistance to functionally and structurally unrelated classes of antitumor drugs.moreover,MDR in the clinical application severely restricts the therapeutic effect and long-term application of chemotherapy agents.Oral cancer is a serious malignant disease with high mortality worldwide.Vincristine(VCR),as one of the basic chemotherapy drugs for oral cancer,limits its clinical efficacy by the development of multidrug resistance phenomenon.Unlike taxane(e.g.paclitaxel)or vinca alkaloid binding sites(e.g.vincristine),the colchicine binding sites may provide a structural advantage to overcome the multidrug resistance regulated by P-gp.6a,2-(6-ethoxy-3-(3-ethoxyphenylamino)-l-methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-y loxy)acetamide,is a novel synthetic compound targeting on colchicine binding site with high affinity and exhibiting potent anti-tumor activity.In this study,we examined the anti-tumor efficacy of 6a against various sensitive and resistant tumor cell lines.Furthermore,the molecular mechanism of 6a against the vincristine-resistant oral epidermoid carcinoma cell line(KB/V cell)was elucidated.Methods1.The growth inhibitory activity of 6a was detected in vitro.To test the drug resistance ability of various resistant cells(Bel7402/5-FU,MCF-7/A and KB/N)and either anti-proliferation effect of 6a or colchicine on above cell lines were assessed using MTT assay.The anti-tumor effects of 6a on KB/V cells were confirmed by colony formation assay.2.The inhibitory effect of 6a in vivo was detected by nude mouse KB/V cells model.3.Target detection of 6a.The efflux function and expression of P-glycoprotein(P-gp)were evaluated by Rhodamine123 accumulation assay and western blotting assay,respectively.The microtubule organization was investigated by immunofluorescence staining(a-tubulin).4.Investigating the mechanism of 6a on KB/V cells.Flow cytometry was used to analyze cell cycle distribution(PI staining),apoptosis(Annexin V-FITC/PI staining)and mitochondrial membrane potential(JC-1 staining).The morphological change on apoptosis cells were measured by Hoechst 33342 nuclear staining.Western blotting assay was used to determine the effects of 6a on the PTEN mediated signal pathway.Results1.In vitro studies,results showed that 6a exhibited stronger anti-proliferative activity than colchicine in KB/V cells.Significantly,the weak cytotoxicity effect of 6a on BEL7402/5-FU and MCF-7/A resistant cell lines indicated that it might have high antitumor selectivity to KB/V cell.2.Studies in vivo revealed that 6a inhibited the KB/V xenografts by 75.8%at a dose of 30 mg/kg(ip)without significant toxicity.3.Further studies suggested that 6a inhibited cancer growth by inducing G2/M phase arrest and apoptosis via activating mitochondria mediated intrinsic apoptosis pathway.6a might be an activator of PTEN dephosphorylation in KB/V cells,with down-regulation of PI3K/Akt/NF-?B pathway.Interestingly,the inhibitory effect of 6a on the growth of KB/V cells was unrelated to both the efflux function and expression of P-gp.6a,targeting the colchicine binding site,significantly induced the inhibition of tubulin polymerization.4.The possible molecular regulation mechanisms of 6a on KB/V cells are described as follows:The effect of 6a on G2/Marrest might associate with the down-regulation of cdc2,p-cdc2 and cdc25B and up-regulation of cyclinB1.The mitochondria dependent apoptosis induction was partly due to reduce the mitochondrial membrane potential,Bcl-2/Bax ratio and increase the level of cytochrome C,cleaved-caspase9 and cleaved-PARP.In addition,6a could regulate the PTEN/Akt/NF-?B signal pathway by promoting dephosphorylation of PTEN and down-regulation of p-Akt,NF-?B.ConclusionThe results in vivo and in vitro reveal that 6a has outstanding inhibitory ability against the growth of KB/V cells.The underlying mechanism of 6a was related to regulating the PTEN/Akt/NF-?B signaling pathway,inducing cell apoptosis(mitochondrial-dependent)and G2/M phase arrest,and inhibiting tubulin polymerization.Significance6a might be developed as a potential therapeutic agent against MDR in human oral epithelial carcinoma.