Effects Of CEMIP On Glutamine Transport And Its Mechanism On Anoikis Resistance In Prostate Cancer

Objective: To select appropriate human prostate cancer cell lines for establishing an anoikis-resistant cell modle,and to explore the effect of CEMIP on the anoikis,anchorage-independent growth,migration and invasion of prostate cancer cells.Methods: Human prostate cancer cell lines PC-3,DU145,C4-2,and LNCa P were cultured in suspension with ultra-low attachment cell culture plates(ULAP).The apoptosis rate was assessed by flow cytometry.The Transwell assay was used to detect the migration and invasion of PC-3,DU145,C4-2,and LNCa P cells.The anoikis-resistant cell model was established by both PC-3 and DU145,and the expression of CEMIP was silenced by sh RNA.The viability and proliferation ability of parental cells,anoikis-resistant cells and silencing CEMIP cells in ULAP were detected by CCK-8.Flow Cytometry was used to examine the effect of silencing CEMIP on the antiapoptotic capacity of anoikis-resistant cells.Soft agar cloning assay was cayyied out to detect the effect of silencing CEMIP on anchorage-independent growth.Western blotting was used to measure the expression of CEMIP,PARP and caspase-3 proteins in parental cells,anoikis-resistant cells,and silenced CEMIP cells.The effect of silencing CEMIP on the migration and invasion of anoikis-resistant cells was detected by Transwell chamber assay.Results: Flow cytometry showed that PC-3 and DU145 had a certain degree of tolerance to anoikis among PC-3,DU145,C4-2 and LNCa P cell lines.The apoptosis rate was fairly high in C4-2 and LNCa P cell lines under suspension culture conditions.Transwell chamber method showed that PC-3 and DU145 had a stronger ability of migration as well as invasion than C4-2 and LNCa P.The CCK-8 assay showed that the cell proliferation rate of anoikis-resistant cells was significantly higher than that of the corresponding parental cells or silenced CEMIP under suspension culture condition.Flow cytometry showed that the apoptosis rate of anoikis-resistant cells was significantly lower than that of the corresponding parental cells or silencing CEMIP cells under suspension culture condition.Soft agar cloning experiments showed that silencing of CEMIP significantly inhibited the formation of anoikis-resistant colonies.The results of western blotting showed that the expression of CEMIP was significantly higher in the anoikis-resistant cells than the corresponding parental cells and sh RNA could significantly silence the expression of CEMIP.High expression of CEMIP can inhibit the expression of cleaved PARP and cleaved caspase-3 protein.Transwell chamber method demonstrated that silencing CEMIP can inhibit migration and invasion in anoikis-resistant cells.Conclusion: PC-3 and DU145 cells were suitable for establishment of anoikis-resistant cell model.CEMIP could inhibit anoikis and promote anchorage-independent growth,migration and invasion.Objective: To investigate the effect of CEMIP on the oxidative stress and glutamine transport in anoikis-resistant prostate cancer cells under suspension conditions.Methord: The sh RNA was used to silence the expression of CEMIP in anoikis-resistant cells.The cells were suspended in ultra-low attachment culture plates for a certain period of time for subsequent experiments: Cells were stained with the fluorescent probe DCFH-DA,fluorescence microscope and flow cytometry was used to detect the level of reactive oxygen species in the cells.Mito SOXTM red reagent was used to detect the superoxide level in cells by fluorescence microscopy.The level of cellular DNA damage was detected by single cell gel electrophoresis.JC-1 Fluorescence probes were used to detect the mitochondrial membrane potential.GSH and GSSG detection kits were used to detect intracellular GSH/GSSG ratio levels.The effect of pro-oxidants and antioxidants on the cell survival rate was examined by CCK-8 method.We used glutamine detection reagent to determine the concentration of glutamine in the medium at different time points.The anoikis-resistant cells and silencing CEMIP cells were cultured with or without glutamine,and the effect of glutamine on cell viability was tested by CCK-8 assay.Results: After silencing CEMIP expression in anoikis-resistant cells,the intracellular reactive oxygen species,mitochondrial superoxide levels,DNA damage increased significantly,while mitochondrial membrane potential and GSH/GSSG ratio decreased under suspension culture conditions.Pro-oxidants further reduced cell survival after silencing CEMIP,whereas antioxidants reversed the decline in cell survival.After silencing CEMIP expression,anoikis-tolerant cells reduced glutamine consumption in the medium.Suspension culture of anoikis-resistant cells under glutamine deprivation resulted in a decrease in cell viability and a decrease in the ratio of GSH / GSSG.Conclusion: CEMIP might reduce the level of reactive oxygen species and promote the cell survival by promoting glutamine uptake.Objective: To investigate the molecular mechanism of CEMIP affecting glutamine uptake in human prostate cancer cells.Methods: quantitative reverse transcription-polymerase chain reaction was used to detect the change of m RNA expression of glutamine transporter(SLC1A5,SLC7A5,SLC38A3,SLC38A5)in anoiksi-resistant prostate cancer cells after silencing CEMIP by sh RNA.The protein expression level of SLC1A5 was further confirmed by western blotting.The human prostate cancer tissue microarray was used for immunohistochemical staining to study the expressive discrepancy of SLC1A5 protein between normal tissues and different grades of prostate cancer tissues.The differences in expression levels of SLC1A5 in prostate cancer compared with adjacent tissues were studied by Oncomine database.The Human Protein Atlas database was used to discover the relationship between expression of SLC1A5 and the prognosis of prostate cancer patients.The expression differences of ERK,p-ERK and c-MYC proteins were detected by western blotting.After silencing CEMIP expression,SLC1A5 overexpression functional response experiment was performed.The expression of SLC1A5 protein in cells was detected by western blotting.The GSH and GSSG levels were detected by GSH and GSSG detection kits.Results: Quantitative reverse transcription-polymerase chain reaction and western blotting showed that the m RNA and protein expression of SLC1A5 were significantly reduced after silencing CEMIP.The human prostate cancer tissue microarray results showed that the expression of SLC1A5 in prostate cancer tissues was significantly higher than that in paracancerous tissues.The expression of p-ERK and c-MYC protein in the anoikis-resistant prostate cancer cells after silencing CEMIP decreased.Overexpression of SLC1A5 followed by silencing CEMIP showed that the reduction of GSH / GSSG ratio could be reversed.Conclusions: Our finding revealed that CEMIP affected glutamine transport through regulating ERK/c-MYC/SLC1A5 pathways,thereby promoting anoikis resistance in prostate cancer cells.