The Effects And Mechanisms Of Over Expression Of STGC3 Gene On Drug Resistance In CNE2/DDP Cells

[Study Objective]Chemotherapy is one of the important methods for the treatment of advanced nasopharyngeal carcinoma（Nasopharyngeal carcinoma,NPC）.It can effectively improve the quality of life and improve the 5-year survival rate.However,with the extension of treatment time,tumor multidrug resistance appears,resulting in poor effect of chemotherapy.Therefore,it is of great significance to study the molecular mechanism of NPC chemotherapy resistance.The previous research work of our group showed that STGC3 was low-expressed in NPC cells and overexpressed STGC3 gene,which induced apoptosis of CNE2 cells by inhibiting PI3K signal pathway.The abnormal activation of PI3K signal pathway and the escape of apoptosis are all the mechanisms of multidrug resistance in tumors.This study is based on previous studies to explore the effect of STGC3 on multidrug resistance（MDR）of CNE2/DDP cells and its possible molecular mechanism.[Methods]1.MTT assay to detect the difference in sensitivity of CNE2 and cisplatin-resistant cells CNE2/DDP to different chemotherapeutic drugs（cisplatin,adriamycin,vincristine）and to assess the multidrug resistance of CNE2/DDP cells.2.To detect the drug efflux ability of CNE2/DDP cells using rhodamine 123 cell accumulation assay.To detect the expression of PI3K/Akt pathway-related protein and multidrug resistance protein P-gp in CNE2 and CNE2/DDP by Western Blot,and to investigate the molecular mechanism of multidrug resistance in CNE2/DDP.3.To detect the difference of STGC3 expression in CNE2 and CNE2/DDP.To transfect CNE2/DDP cells with plasmids overexpressing STGC3 using liposome transient transfection technique.After successful transfection,the effect of STGC3 on the sensitivity of CNE2/DDP cells to different drugs（cisplatin,adriamycin,vincristine）was examined by CCK8 assay to assess the ability of STGC3 to regulate the multidrug resistance phenotype of CNE2/DDP cells.4.The effect of STGC3 on the drug efflux ability of CNE2/DDP cells was examined by rhodamine 123 cell accumulation assay.The expression of P-gp,PI3K,Akt,and p-Akt in CNE2/DDP cells after overexpression of STGC3 was examined by Western blot.The PI3K/Akt pathway agonist IGF-1 was applied for reversion assay.The inhibitory effect of STGC3 on the pathway was further confirmed.5.Apoptosis was detected by flow cytometry.The effect of STGC3on Bcl-2 protein expression was detected by Western Blot.6.All cells were cultured in drug-free medium for more than 1 week before the experiment.[Results]1.The IC₅₀ values and resistance indices of different drugs（cisplatin,adriamycin and vincristine）of CNE2 and CNE2/DDP were detected by MTT method,and the results showed that the susceptibility of drug-resistant strains to cisplatin（resistance index 6.919）,adriamycin（resistance index 18.329）and vincristine（resistance index 28.129）was significantly lower than that of the parental group,indicating that CNE2/DDP is consistent with multidrug resistance characteristics.2.Rhodamine 123 cell accumulation assay showed that the fluorescence intensity of CNE2/DDP was significantly lower than that of parental cells（P<0.001）.Western Blot confirmed that the expression of P-gp protein was significantly higher in CNE2/DDP cells than in CNE2cells（P<0.01）,and the expression of PI3K（catalytic subunit p110α）in CNE2/DDP cells and p-AKT expression was increased in CNE2/DDP cells（P<0.01）,and total AKT protein expression was not significantly changed.This indicates that the multidrug resistance of CNE2/DDP cells is related to the activation of PI3K/Akt pathway and upregulation of P-gp protein expression level.3.Western Blot results showed that the expression of STGC3 was lower in CNE2/DDP cell lines than in CNE2 cell lines.After successful construction of CNE2/DDP cell line with high STGC3 expression（CNE2/DDP-RFP-STGC3）and empty vector CNE2/DDP cell line（CNE2/DDP-RFP-EV）,CCK8 results showed that the survival rate of CNE2/DDP cells in the overexpression STGC3 group was significantly lower after 48 h of different chemotherapeutic drugs（P<0.05）,indicating that STGC3 could enhance the chemosensitivity of CNE2/DDP cells.4.Rhodamine 123 cell accumulation assay showed that the intracellular fluorescence intensity of CNE2/DDP-RFP-STGC3 cells was significantly enhanced by rhodamine 123 treatment（P<0.05）,indicating that the drug efflux ability of overexpressed STGC3 cells was diminished and STGC3 could enhance the chemosensitivity of human nasopharyngeal carcinoma CNE2/DDP cells by reducing drug efflux.Western Blot detected the differences in PI3K/Akt pathway protein and P-gp protein expression in each group of cells after overexpression of STGC3.The results showed that in CNE2/DDP cells overexpressing STGC3,the expression of pathway-related proteins PI3K（catalytic subunit p110α）and p-Akt were down-regulated（P<0.05）,and the total Akt protein content was not significantly changed（P=0.374）;the expression of multidrug resistance protein P-gp was also significantly down-regulated（P<0.05）.The application of the pathway agonist IGF-1was validated in a reply experiment,and consistent results were obtained.It indicates that STGC3 attenuates drug efflux from CNE2/DDP cells and correlates with inhibition of PI3K/Akt pathway activation and downregulation of P-gp protein expression.5.Flow and Western Blot results together showed that STGC3induced apoptosis in CNE2/DDP cells and down-regulated the expression of anti-apoptotic protein Bcl-2.This result suggested that STGC3 could mediate apoptosis of CNE2/DDP by down-regulating the expression of Bcl-2,thus enhancing the cytotoxic effect of antitumor drugs on CNE2/DDP.[Conclusions]1.Cisplatin-induced resistant cell line CNE2/DDP has multidrugresistance,which is related to the activation of PI3K/Akt pathway and the up-regulation of P-gp protein expression.2.The over expression of STGC3 gene can participate in theregulation of multidrug resistance in CNE2/DDP cells by inhibiting the activation of PI3K/Akt pathway,reducing the expression of P-gp protein and reducing the efflux of intracellular chemotherapeutic drugs.3.Over expression of STGC3 can down-regulate Bcl-2,to induce apoptosis of CNE2/DDP cells and enhance their chemosensitivity.