Effects And Mechanisms Of Pantoprazole On Elevating Sensitization Of Drug-resistant Oral Epidermoid Carcinoma Cells To Vincristine

Background and objectiveOral cancer is still a serious and growing health burden worldwide.Common treatments for oral cancer typically involve surgery and/or chemotherapy as well as radiotherapy.Despite recent advances in diagnosis and treatment,oral cancer still has lower 5-year survival rate.Vincristine(VCR),a classical microtubule-destabilizing agent,is one of essential medicines for oral cancer therapy.However,the treatment of VCR is limited by the development of multidrug resistance(MDR),the same as other chemotherapeutics in oral cancer.Proton pump inhibitors(PPIs),which are essentially H+-K+-ATPases inhibitors,are currently used in the treatment of acid related diseases.Recently,encouraging reports on the application of PPIs in cancer therapy have sprung up.Emerging evidences indicate that PPIs in combination with chemotherapeutic drugs might increase the therapeutic efficacy with unclear mechanisms.PPIs may really represent new chemosensitizers due to better safety and anti-tumor effect,which contributes to smaller dose of chemotherapeutic drugs and decreased toxicity in combination.Moreover,it was reported in clinical trials that PPIs combined with antiemetics could prevent gastrointestinal side reaction caused by chemotherapy.In the present study,we examined the synergistic anti-tumor efficacy and possible molecular mechanisms of combination treatment of pantoprazole(PPZ,a kind of PPI)and VCR on the proliferation,apoptosis,cell cycle,invasion and metastasis of KB/V cells in vitro and in vivo.Methods1.Anti-proliferative activity of PPZ and VCR on oral epidermoid carcinoma KB cells and vincristine-resistant KB/V cells.We first investigated the dose-dependent cytotoxic effects of PPZ as single treatment on KB and KB/V cells by MTT assay in order to select the suitable dosage of PPZ in the combination treatment.Then the cytotoxicity of VCR combined with 24h PPZ pretreatment on KB and KB/V cells were also determined by MTT and colony formation assays.Moreover,the cytotoxicity of VCR and PPZ on human normal cells was also investigated by MTT assay.2.The effect of combination treatment of PPZ and VCR on the apoptosis of KB/V cells was determined by Hoechst 33342 nuclear staining and Annexin V-FITC/PI double-staining assays.Furthermore,the expression of the apoptosis related proteins were detected by Western blotting assay.3.The effect of combination treatment of PPZ and VCR on cell cycle distribution was detected using PI staining by flow cytometry.The expression of the cell cycle regulation related proteins were detected by Western blotting assay.4.The effect of PPZ and VCR on migration and invasion of KB/V cells was examined using wound scratch assay and Transwell assay.The expressions of MMPs were detected by Western blotting assay.5.The mechanisms of PPZ enhancing the sensitivity of KB/V cells to VCR.Effect of PPZ and VCR on the efflux function and expression of P-gp in KB/V cells was evaluated by Rhodamine123(Rh123)accumulation assay and Western blotting assay respectively.The extracellular pH(pHe)of KB/V cells in unbuffered medium after PPZ or V-ATPase inhibitor Baf-Al treatment with different concentrations at various time points was detected by pH meter in order to investigate whether PPZ inhibit the acid secretion in KB/V cells.To further determine the relationship between the enhancement of PPZ on the cytotoxicity of VCR and V-ATPase,V-ATPase inhibitor Baf-A1 was added to observe the cytotoxicity of PPZ and VCR by MTT assay.Effect of PPZ and VCR on EGFR/MAPK and PI3K/Akt/mTOR signaling pathways in KB/V cells was evaluated by Western blotting assay.6.The inhibitory effect of PPZ and VCR on KB/V cells in vivo was detected in nude mouse KB/V xenografts model.The effects of VCR with or without PPZ pretreatment were compared by detecting the tumor weight.The toxicities of VCR with or without PPZ pretreatment were compared by measuring body weight and histopathological examination of mice gastric mucosal surface.The effect of PPZ and VCR on the apoptosis of KB/V cells in vivo was evaluated by detecting the expression of the apoptosis related proteins in KB/V xenografts.Results1.PPZ potently enhanced the susceptibility of KB cells or KB/V cells to VCR and the synergy was much more prominent in KB/V cells without obvious additional toxicity in human normal HUVECs,GES-1 cells and HL-7702 cells.2.Combination treatment of PPZ and VCR evoked mitochondrial dysfunction-related apoptosis in KB/V cells through the upregulation of cleaved-PARP,cleaved-Caspase 3 and Bax as well as the downregulation of Bcl-2 and Bcl-xL.3.Co-administration of PPZ and VCR induced G2/M phase arrest in KB/V cells through the upregulation of p21 and the downregulation of Cyclin B1,cdc2 and p-cdc2.4.PPZ combined with VCR suppressed the migration and invasion of KB/V cells via decreasing the protein levels of MMP-2 and MMP-9.5.Combination treatment of PPZ and VCR inhibited the function and expression of drug efflux-protein P-gp in KB/V cells.6.PPZ treatment did not lead to extracellular alkalization and had no effect on the expression of V-ATPase in KB/V cells.Furthermore,the intervention of Baf-A1 not only enhanced anti-proliferation effect of PPZ itself but also the combination treatment of PPZ and VCR significantly on KB/V cells.In all,PPZ treatment might not lead to an inability to eliminate protons in the extracellular compartment of KB/V cells by the inhibition of V-ATPase.7.Combination treatment of PPZ and VCR inhibited EGFR/MAPK and PI3K/Akt/mTOR signaling pathways in KB/V cells.8.Administration of PPZ and VCR induced significant synergistic inhibition of KB/V tumor growth in a mice xenograft model.Moreover,pretreatment with PPZ significantly attenuated VCR-induced toxicity.ConclusionIn conclusion,combination treatment of PPZ and VCR synergistically inhibited the proliferation of KB/V cells in vitro and in vivo.Furthermore,administration of PPZ and VCR not only induce apoptosis and G2/M phase arrest in KB/V cells but also suppress the migration and invasion of KB/V cells.The mechanism underlying synergistic anti-tumor effect of PPZ and VCR was related to the inhibition of the function and expression of P-gp and the downregulation of EGFR/MAPK and PI3K/Akt/mTOR signaling pathways in KB/V cells,but was independent of the effect of PPZ on acid inhition and the expression of V-ATPase in KB/V cells.SignificancePPZ may be used as a novel adjuvant for cancer chemotherapy.The combination of PPZ and VCR will provide a promising strategy for treatment on oral cancer,especially on VCR-resistant therapy.