The Study On Molecular Mechanism Of Phenothiazine Antidepressant (Trifluoperazine) Induced Apoptosis In Non-small Cell Lung Cancer Cells

Chapter 1 Trifluoperazine induces apoptosis and autophagy in NSCLC cellsObjective:To study the effect of trifluoperazine on apoptosis and autophagy in NSCLC cells.Materials and Methods:1.The cytotoxicity of trifluoperazine on NSCLC cell lines(H1792 H1299 and Calu-1)was investigated via sulforhodamine B(SRB)assay.2.Western blot(WB)was used to test the effect of trifluoperazine on apoptosis in NSCLC cells.3.The number of autophagosomes in NSCLC cells treated with trifluoperazine was observed by Transimission Electronic Microscopy(TEM).4.The expression of LC3B-II was detected via WB in NSCLC cells treated with trifluoperazine at different concentrations and different time.5.The number of intracellular EGFP fluorescence spots in calu-1-EGFP-LC3 B cells treated with trifluoperazine at different concentrations and different time was observed by confocal laser scanning microscope.6.The expression changes of LC3B-II were detected via WB when NSCLC cells were treated by trifluoperazine combined with LY294002/ chloroquine/knockdown ATG5 separately,which was used to determine change of autophagy flow.7.H1299 cells transfected with ATG5-siRNA were treated with trifluoperazine for24 h,and cell apoptpsis was detected via WB and flow cytometry.Results:1.Trifluoperazine inhibits the survival of NSCLC cells.2.Trifluoperazine can induce the apoptosis of NSCLC cells in both dose-dependent and time-dependent manner.3.Trifluoperazine induces autophagy in NSCLC cell.3.1 TEM found that the number of intracellular autophagosomes increased significantly after treatment with trifluoperazine.3.2 WB showed that LC3B-II level increased in both time-dependent and dose-dependent manner after treatment with trifluoperazine.3.3 In Calu-1-EGFP-LC3 B cell,confocal laser scanning microscope showed that fluorescence spots also increased in both time-dependent and dose-dependent manner after treatment with trifluoperazine.3.4 WB showed LY294002 could increase the expression of LC3B-II induced by trifluoperazine,while knockdown of ATG5 or CQ reduced the expression.So,trifluoperazine could activate autophagy flow in NSCLC cells.4.Autophagy induced by trifluoperazine promoted apoptosis of NSCLC cells.Conclusion:Trifluoperazine induces the apoptosis and autophagy in NSCLC cells,and autophagy induced by trifluoperazine could promote cell apoptosis.Chapter 2 RNA-Seq and Bioinformatic analysis show trifluoperazine might induce endoplasmic reticulum stress in NSCLC cellsObjective:To detect differentially expressed genes(DEGs)and explore potential molecular mechanism of apoptosis and autophagy in NSCLC cells induced by trifluoperazine using RNA-Seq and bioinformatics analysis.Materials and Methods:1.Total RNA was extracted in H1299 cells exposing in trifluoperazine(20 μM)for 8h and used for RNA-Seq.2.Raw reads were quality controlled and aligned by Sickle and Hisat2 respectively.3.RSEM was used to quantify gene abundances,and DEGs were screened with DESeq2.4.GO and KEGG functional-enrichment analysis were performed with Goatools and Signaling Pathway Impact Analysis(SPIA)package.5.GSEA for DEGs was performed with MSig DB.Results:1.RNA extracted from H1299 cells met the requirements for RNA-Seq.2.The ratio of Q20 was high(more than 98%)in clean reads,and GC% accounted for 50% nearly;there was not obviously separation of AT and GC.Furthermore, more than 93.48% of sequences could be uniquely mapped to the reference genome in all samples,indicating the reference genome was also appropriate.3.1947 DEGs were identified,including 1059 up-regulated and 888down-regulated genes.Among proteins producted by top 10 genes with significant differential expression,five proteins were associated with endoplasmic reticulum(ER)stress and two proteins were involved in autophagy.4.Both GO and KEGG analysis demonstrated DEGs were enriched significantly in ER stress-related GO terms and pathways.5.GSEA indicated ER stress-related gene set(HALLMARK_UNFOLDED_PROTEIN_RESPONSE)was enriched,and autophagy-related gene set(HALLMARK_MTORC1_SIGNALING)was also enriched.Conclusion:RNA-seq showed that trifluoperazine induced ER stress in NSCLC cell.Chapter 3 Trifluoperazine induces apoptosis and autophagy in NSCLC cells through endoplasmic reticulum stressObjective:To explore potential molecular mechanism of apoptosis and autophagy in NSCLC cells induced by trifluoperazine.Materials and Methods:1.qRT-PCR was used for relative quantitative analysis of gene expression and verifying the reliability of RNA-Seq.2.The expression of ER stress-related proteins in NSCLC cells treated with trifluoperazine was detected by WB.WB was also used to detect the expression change of apoptosis-related and autophagy-related proteins when co-treated with 4-PBA,an ER stress antagonist,and trifluoperazine in NSCLC cells.3.Protein–Protein Interaction network analysis of DDIT3 was performed via existing STRING database.4.The molecular mechanism of apoptosis induced by trifluoperazine in NSCLC cells was investigated using siRNA transfection,WB and flow cytometry.5.The molecular mechanism of autophagy induced by trifluoperazine in NSCLC cells was investigated using siRNA transfection and WB.6.The expression of apoptosis-related proteins in H1299 cell was detected using WB when co-treated with trifluoperazine and rapamycin.Results:1.qRT-PCR indicated the expression trend of 5 DEGs(DDIT3,ATF4,BIP,TRIB3 and WIPI1)were consistent with the results detected by RNA-Seq.The correlation between RNA-Seq and qRT-PCR data was very significant.2.Trifluoperazine could induce ER stress in NSCLC cells and regulate apoptosis and autophagy through ER stress.3.DDIT3 interacted with BCL2 and TRIB3.4.Trifluoperazine induced apoptosis in NSCLC cells via ATF4-DDIT3 signaling pathway.5.Trifluoperazine induced autophagy in NSCLC cells via ATF4-DDIT3-TRIB3- Akt-mTOR signaling pathway.6.Rapamycin promoted apoptosis induced by trifluoperazine in NSCLC cells.Conclusion:Trifluoperazine induced autophagy in NSCLC cells via ATF4-DDIT3-TRIB3-Akt-MTOR signaling pathway,and induced apoptosis via ATF4-DDIT3 signaling pathway also.Moreover,the chemotherapy drug rapamycin(an autophagy inducer also)could promote cell apoptosis induced by trifluoperazine.Chapter 4 Trifluoperazine inhibits NSCLC growth in vivoObjective:To study the effect of trifluoperazine on the growth of NSCLC in vivo.Materials and Methods:1.H1299 cells were injected subcutaneously in nude mice,and intraperitoneal injection was given when the tumor grew to about 80mm~3.PBS(control group),trifluoperazine(40mg/kg),rapamycin(1mg/kg)or trifluoperazine(40mg/kg)+rapamycin(1mg/kg)were injected every other day.The tumor growth was measured every 3 days.2.Three weeks later,mice were killed and tumors were made into paraffin sections.Immunohistochemical LC3 B,Ki-67 and BIP staining were performed in order to determine the effects in vivo of trifluoperazine on autophagy,proliferation and ER stress in NSCLC cells.3.TUNEL was used to explore the effect in vivo of trifluoperazine on apoptosis in NSCLC cells.Results:1.Trifluoperazine inhibits the growth of NSCLC in vivo,which can be further increased by rapamycin.2.Immunohistochemistry shows that trifluoperazine can induce autophagy and ER stress in NSCLC cells in vivo.The number of cells with Ki-67 positive in drug combination group is significantly less than it in the trifluoperazine group.3.TUNEL shows that rapamycin increases apoptosis of NSCLC cells induced by trifluoperazine in vivo.Conclusion:Trifluoperazine inhibits significantly the growth of NSCLC cells in vivo.Rapamycin can increase the apoptosis of NSCLC cells induced by trifluoperazine,and enhanced its anti-tumor effect.