Trifluoperazine Promotes Glioma Cells Proliferation And Reduce Calcium-dependent Apoptosis

Objective: According to statistics,90% glioma patients also suffer from mental disorders and receive assisted antipsychotic medication treatment.However,in previous studies,the relationship between antipsychotic treatment and cancer progression is still uncertain.In our previous study,bioinformatics analysis results suggest that phenothiazine may promote the proliferation of glioma cells.Therefore,we designed this experiment to further explore the relationship between phenothiazine and glioma progression in vitro and vivo,so as to provide some reference for the linical treatment of glioma patients.Methods: The experiments were divided into three parts.Part 1 The bioinformatics analysis of the glioma cell lines expression profile drug screening in Connectivity-Map database 1.Detected the SWOZ2 and its drug resistant cell lines SWOZ2-BCNU expression profile gene chip whose chip model is Affymetrix Human Genome U133 Plus 2.0.2.Standardized the gene chip of the two cell lines,screened differentilally expressed genes and study the clustering analysis of differentially expressed genes fuction.3.Screened small molecular compounds according to the selected differentially expressed genes by the Connectivity-Map database.Part 2 The biological study of Trifluoperazine on glioma cell lines.1.MTT assay was performed to detect the activity of SWOZ2,SWOZ2-BCNU,U87,U251,C6,PC12,PC3,SHSY-5Y,SW480,and other tumor cells lines.2.Plate colony and Ed U fluorescence staining were performed to detect the effect ofTrifluoperazine on SWOZ2,SWOZ2-BCNU,U87 and U251 cell lines proliferation.3.Flow cytometry and TUNEL staining were performed to detect the apoptosis of SWOZ2,SWOZ2-BCNU,U87 and U251 cell lines in order to clear the effect of Trifluoperazine on the apoptosis of human glioma cell lines.Part 3 To observe the effect of Trifluoperazine on the subcutaneous xenograft tumor in nude mice.1.U251 cells mixed with matrigel was injected into the right shouler of balb/c nude mice,2×106 cells per nude mice.2.The transplanted tumor volume curves of nude mice were recorded for 2 weeks.Immunohistochemical and TUNEL were performed to detect the proliferation and apoptosis of the glioma cells in order to clear the effect of Trifluoperazine on human glioma cell lines in vivo.Results: Part 1.The bioinformatics analysis of the glioma cell lines expression profile drug screening in Connectivity-Map database 1.Totally,1181 unique genes and other transcripts were shown to be differentially expressed between the various comparisons perform,in which 643 up-regulated and 538 down-regulated genes.2.Then,these genes were analyzed with Gen Cli P 2.0 software to annotate gene functions.The top-ranking terms were all about apoptosis,programmed cell death.The results suggest that SWOZ2-BCNU was likely to achieved resistance through escaping of apoptosis.3.In order to find drugs that target these genes set,we uploaded the probe list of differentially expressed genes to Connectivity-Map database.The results showed that in the top 20 of the p-value ranking,there were ten drugs' enrichment scores were positive,while other 10 were negative.Among these drugs,we observed that trifluoperazine,thioridazine and fluphenazine enrichment scores were positive,and they all belong to the same class of phenothiazines.Part 2.The biological effects of Trifluoperazine on glioma cell lines.1.To determine the effect of TFP treatment on cell viability,different types of cell lines were exposed to TFP(0 ?M,2?M,4 ?M,6 ?M,8 ?M and 10 ?M)and cell viability was measured at 24 h post-TFP treatment.Interestingly,our data showed that low concentrations of TFP exposure resulted in a significant increase in the viability of gliomas related cell types U87,U251,SWOZ2,SWOZ2-BCNU and C6(p < 0.05),however,these effects were slight in neuroendocrine tumor cells,gastrointestinal tumor cells and prostatic cancer cells(p > 0.05).2.To examine the effect of TFP on gliomas cells proliferation,the clone formation ability of gliomas cells SWO-Z2 and SWO-Z2-BCNU were compared after treated with TFP(2?M)or control,respectively.TFP-treated SWO-Z2 and SWO-Z2-BCNU cells exhibited much more colonies compared with the control(P<0.01).After TFP treatment,Ed U assay was also carried out to determine the effect of TFP on gliomas cells proliferation.TFP treatment appeared to increase the percentage of Ed U-positive cells compared with control cells(P<0.01).3.After incubation with control or different concentrations of TFP,the effect of TFP on the apoptosis of SWO-Z2 and SWO-Z2-BCNU cells were examined using flow cytometry after Annexin V-FITC/PI staining and TUNEL staining.We found that TFP significantly decreased apoptosis in gliomas cells(P<0.05).Part 3.To observe the effect of Trifluoperazine on the subcutaneous xenograft tumor in nude mice.1.To investigate whether TFP promotes tumor cell proliferation in vivo,glioma cells U-251 were injected subcutaneously into the dorsal flank of nude mice.After the xenograft tumor formed,TFP(2?M)or control was injected to mice for every three days.Two weeks later,xenograft tumor volumes of the TFP-treated group was significantly bigger compared with the control group(P < 0.05).2.We used immunohistochemical assay to detect the expression levels of PCNA and Ki67 between these two groups.Immunohistochemical results showed that both the staining intensity and the number of hyperproliferative Ki-67+ and PCNA+ tumor cells were significantly increased compared with the control group(P < 0.05).And with the results of TUNEL staining.,we found that TFP significantly decreased apoptosis in xenograft gliomas cells(P<0.05).Conclusion: Our experimental results show that the tumor-promoting effect of antidepressant drugs,especially trifluoperazine,needs to be fully evaluated before used in depressive and neurobehavioural disorder glioma patients.