Ammonium Tetrathiomolybdate Enhanced The Antitumor Efficacy Of Cisplatin In Esophageal Squamous Cell Carcinoma And Its Molecular Mechanisms

Background and ObjectiveEsophageal cancer(EC)is one of the most common digestive tract tumors,ranking as the eighth most common cancer and the sixth leading cause of death worldwide.According to histologic subtypes,EC is split into esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).In China,ESCC as a master histologic type displays high mortality,poor prognosis,low 5-year survival rate and considerable geographic variation.At present,the main treatment strategies for ESCC comprise of surgery,radiotherapy,chemotherapy,etc.For advanced and metastatic patients with ESCC,chemotherapy is still the most important option for prolonging patients' survival and improving patients' prognosis.However,acquirement of resistance and toxicity are the main causes for the failure of chemotherapy.Therefore,it is imperative to seek for a novel and promising strategy to prevent,diagnose and treat ESCC patients.Ammonium tetrathiomolybdate(TM)is one of the trace elements molybdenum related compounds to human body,and it can form a high affinity ternary complex with copper ions and blood albumin,which can chelate free copper ions in the blood and reduce copper content in the body.Mounting evidence has demonstrated that TM plays a pivotal role in suppressing angiogenesis,tumor growth and distant metastasis as well as promoting tumor necrosis,etc.Several studies have revealed that TM enhances the sensitivity of cisplatin on gynecologic tumors,such as breast carcinoma,cervical cancer and ovarian cancer,etc,and reduces resistance during chemotherapy by targeting copper transportation-related proteins,which will trigger strong antitumor efficacy.Cisplatin is currently the most widely used first-line standard chemotherapy drug for the treatment of multiple malignancies,including cervical cancer,ovarian cancer,head and neck cancer,lung cancer,gastrointestinal cancer,breast cancer,esophageal cancer,etc.However,due to its side effects and neurotoxicity,its dosage and therapeutic efficacy have been severely limited.Therefore,how to reduce its therapeutic dose and enhance its anti-tumor effect as well as elucidate the molecular mechanism of its drug resistance has become the problem demanding prompt solution.Accumulating evidence has shown that Stat3 signaling pathway is tightly implicated in the occurrence and development of a plethora of tumors,and may be an effective therapeutic target for a wide array of tumors.Subsequent investigations have revealed that Stat3 signaling pathway is closely associated with drug resistance in a myriad of tumors,and its activation is one of the most important signaling pathways that mediates tumor resistance.However,whether antitumor efficacy exerted by TM combined with cisplatin is tightly related to the activation of Stat3 signaling pathway,which will be the direction of our studies.Therefore,in the current study,the antitumor efficacy and possible molecular mechanisms mediated by TM were elucidated by cell proliferation assay,flow cytometry,migration and invasion related experiments,and transcriptome analysis in ESCC cells.Stepwise investigation confirmed the synergistic antitumor efficacy of TM combined with cisplatin and its underlying molecular mechanisms.The current studies will provide a theoretical basis for the treatment of ESCC patients using TM in future.Part I In vitro antitumor efficacy of ammonium tetrathiomolybdatein esophageal squamous cell carcinoma and its molecularmechanisms Method1.Effects of different doses of TM(0,5,10,20,30,50,80,100,150 and 200?M)on cell proliferation abilities of ESCC cells(EC1,Eca109,EC9706 and TE1)were evaluated at different time points(24,48,72 and 96h)using CCK-8 method.2.Flow cytometry was used to detect the effects of different concentrations TM on cell cycle and apoptosis at 48 h in ESCC cells,respectively.Hoechst staining was utilized to investigate the effects of different concentrations TM on cell apoptosis at 48 h in ESCC cells.3.Cell scratching test and Transwell chamber were employed to examine the effects of different concentrations TM on cell migration and invasion abilities in ESCC cells.4.Affymetrix gene chip expression profile was exploited to detect the effects of TM on transcription expression profiles in ESCC Eca109 cells.Western blot was used to detect the effects of different concentrations TM on the expression changes of cell cycle,apoptosis and invasion-related proteins at 48 h in a panel of ESCC cells,meanwhile,non-receptor tyrosine kinase c-Abl expression in gene chip result was detected in corresponding ESCC cells.5.Statistical assay: Statistical assay was performed using GraphPad Prism 6.0 software.Comparisons of three groups or above were analyzed using One-way ANOVA,and the comparisons of two groups were investigated using t test.A P value less than 0.05 was considered to be statistical significance.Result1.The results of CCK-8 revealed that different concentration TM displayed various suppressive effects on ESCC cell proliferation,and the suppressive effect became more significant with the progress of time.2.Cell cycle assay demonstrated that different concentration TM blocked cell cycle in S phase in a panel of ESCC cells.The results from Western blot revealed that TM significantly downregulated the expressions of CDK4,Cyclin D1 and Cyclin A proteins in ESCC cells.3.Cell apoptosis assay demonstrated that different concentration TM markedly increased apoptotic cell numbers in ESCC cells,which displayed in a concentration-dependent manner.Hoechst staining showed that TM induced cell apoptosis in ESCC cells.In addition,TM promoted the expressions of apoptosis-related proteins cleaved-caspase-3,p-PARP and Bax,but suppressed the expression of Bcl-2 protein.4.Different concentration TM obviously inhibited cell migration in ESCC cells at 48 h and 72 h,and further Transwell chamber experiment demonstrated that various concentration TM evidently suppressed cell invasion ability in ESCC cells.Besides,TM significantly reduced the expressions of MMP-2 and MMP-9 proteins in ESCC cells.5.Affymetrix gene chip assay revealed that TM activated or suppressed multiple different signaling pathways in ESCC Eca109 cells,in which TM downregulated c-Abl mRNA level in Eca109 cells,and further Western blot assay demonstrated that different concentration TM markedly downregulated c-Abl expression in a panel of ESCC cells.Part II Ammonium tetrathiomolybdate enhances cisplatin sensitivityon esophageal squamous cell carcinoma cells via suppressing Stat3signaling pathwayMethod1.Effects of different doses of cisplatin(0,0.5,1,2,5,8,10,20 and 50?g/ml)on cell proliferation ability of ESCC cells(EC1,Eca109,EC9706 and TE1)were evaluated at different time points(24,48,72 and 96h)using CCK-8 method;meanwhile,the effects of TM combined various concentration cisplatin(0,1,2,5 and 10?g/ml)on cell proliferation were measured by CCK-8 kit in ESCC cells.2.Flow cytometry and Transwell chamber were exploited to detect the effects of TM combined with cisplatin on cell apoptosis and invasion ability at 48 h in ESCC cells,respectively.3.Western blot was employed to investigate the expressions effects of Stat3 and p-Stat3 proteins at 48 h in TM combined with cisplatin group and TM/cisplatin/IL-6 group in ESCC cells.Furthermore,CCK-8 kit was employed to detect the changes of cell proliferation in TM/cisplatin/IL-6 group.4.In vivo animal experiment: EC9706 cells were inoculated subcutaneously into nude mice,and experiments were split into 4 groups,including saline group,TM alone group;cisplatin alone group and TM combined with cisplatin group.Tumor volume was measured twice every week,and tumor growth curve was made using GraphPad Prism 6.0 software.After the end of measurement,tumor tissues were peeled off and paraffin embedded tissue section was made,and then immunohistochemistry was used to detect the expressions of angiogenesis related proteins VEGF,CD31 and ERG as well as total Stat3,p-Stat3 and Ki-67 in different groups.5.Statistical assay: Statistical assay was performed using GraphPad Prism 6.0 software.Comparisons of three groups or above were analyzed using One-way ANOVA,and the comparisons of two groups were investigated using t test.A P value less than 0.05 was considered to be statistical significance.Result1.CCK-8 assay revealed that different concentration cisplatin significantly inhibited cell proliferation in ESCC cells,and its suppressive efficacy was markedly enhanced with time extension.In addition,TM increased the inhibitive effect mediated by various concentration cisplatin.2.Flow cytometry assay demonstrated that compared with untreated group or treatment alone group,TM combined with cisplatin evidently induced cell apoptosis in ESCC cells,and further investigation from Transwell chamber showed that compared with untreated group or treatment alone group,TM in combination with cisplatin obviously suppressed cell invasion ability in ESCC cells.3.Western blot assay indicated that TM combined with cisplatin significantly reduced p-Stat3 levels,but didn't change the levels of total Stat3 proteins.IL-6 reversed the downregulation of p-Stat3 protein triggered by TM combined with cisplatin in ESCC cells.Stepwise investigation from CCK-8 kit demonstrated that IL-6 mediated the Stat3 activation dramatically reversed the suppressive effect of cell proliferation evoked by TM combined with cisplatin.4.Animal experiments verified that compared with saline group,TM alone group and cisplatin alone group,TM combined with cisplatin significantly inhibited tumor growth in EC9706 xenografted nude mice.Further investigation regarding immunohistochemistry demonstrated that TM combined with cisplatin markedly reduced angiogenesis-related proteins VEGF,CD31 and ERG protein levels and suppressed p-Stat3 and Ki-67 levels in EC9706 xenografted nude mice tissues.Conclusion1.TM triggers proliferation suppression,S phase blockage,cell apoptosis and the decreases of migration and invasion abilities in ESCC cells,indicating that TM may be a novel and promising antitumor agent for the therapy of ESCC patients.2.TM enhances antitumor efficacy of cisplatin in vitro and in vivo,which imply that TM will be a promising adjuvant drug for cisplatin in therapy of ESCC patients.3.TM combined with cisplatin significantly downregulates the levels of angiogenesis-related proteins VEGF,CD31 and ERG as well as reduces the levels of p-Stat3 and Ki-67 proteins,and thus may be a novel and important molecular target for evaluation of cisplatin resistance in clinic.