| Nasopharyngeal carcinoma(NPC)is one of the most common malignant tumor in China,so far its pathogenesis is not clear,what's more,the therapeutic effect and prognosis were poor.It has certain theoretical significance and application value for searching new treatment and targets.Some studies have found that the regulation of intracellular and extracellular ion channels are closely related to cell apoptosis.Research shows that the volume-sensitive outwardly rectifying chloride current of CNE-2Z cells recorded by the patch clamp has changed after the treatment of gambogic acid,but the relevance between this change and the anti-tumor mechanism of gambogic acid has not been reported.Previous studies have shown that the action of inhibitting the proliferation of various tumor cells of Gambogenic Acid(GNA)is related to the induction of tumor cell apoptosis,however,it is worth to study that whether it is related to the activation of endoplasmic reticulum pathway.Endoplasmic reticulum stress-mediated apoptosis is a pathway different from the apoptosis mediated by death receptors and mitochondrial injury.Therefore,this study is focused on using GNA to intervene human nasopharyngeal carcinoma cell line CNE-2Z to investigate whether the activation of VSOR Cl~-channels and endoplasmic reticulum pathway are related to the induction of apoptosis in CNE-2Z cells,expecting to provide some theoretical foundation for further development and utilization of GNA.Objective: The aim of the study is to investigate the role of volume sensitive outward rectifying chloride channels in GNA induced apoptosis in nasopharyngeal carcinoma CNE-2Z cells and explore the role and possible mechanisms involved in endoplasmic reticulum stress.Methods:1?The effect of GNA on the proliferation of CNE-2Z cells was detected by MTT method,and the morphology of CNE-2Z cells was observed by inverted microscope.DAPI staining and Annexin V-FITC/PI staining were used to observe the apoptosis of CNE-2Z cells induced by GNA.The apoptosis rate of CNE-2Z cells induced by GNA was detected by flow cytometry.2?The effect of GNA on the volume sensitivity of outwardly rectifying chloride ion current on the surface of CNE-2Z cell membrane was detected by using MQAE and patch clamp technique.3?The effect of GNA on the proliferation of CNE-2Z cells after the non-specific chloride channel blocker(DIDS)and the specific volume sensitive outward rectifying chloride channel blocker(DCPIB)preconditioning was detected by MTT method.DAPI staining and Annexin V-FITC/PI staining were used to observe the effects of DIDS and DCPIB on the apoptosis of CNE-2Z cells induced by GNA after DIDS and DCPIB preconditioning.DAPI staining was used to observe the effect of silencing Cl C-3 on apoptosis of CNE-2Z cells induced by GNA.The expression of Cl C-3 chloride channel protein after silencing Cl C-3 was detected by Western Blotting.The effect of GNA on the expression level of endoplasmic reticulum related protein(GRP78,ATF4,CHOP)in CNE-2Z cells after GNA,DIDS and DCPIB preconditioning was detected by Western Blotting.Results:1?MTT results show that the cell survival rate in experimental groups was decreased after treated by different concentration of GNA for 24 h or 48 h,compared with the blank group.DIDS group and DCPIB group had little effect on the survival rate of cells.Compared with GNA group,the survival rate in DIDS(20 ?mol/L)+ GNA(2?mol/L)group and DCPIB(20 ?mol/L)+ GNA(2 ?mol/L)group increased,the difference was statistically significant(P <0.01).2?Observed by inverted microscope,the cells were firmly adherent,the edge was clear,and there are no floating cells in the control group.In the low and medium dose GNA group,some cells turn round,the volume became smaller,and a few cells showed a semi adherent state.In the high concentration GNA group,In the high concentration GNA group,the cells were more exfoliated and floating in the culture medium.3?DAPI nuclear staining,it was observed that the nuclear were uniform pale blue in the control group by fluorescence microscopy,however,in the low and medium concentrations of GNA,the nuclei of the treated group were bright blue,and the fragments were dense and dense,a few nuclei showed fragmentation in the nucleus,these all showed obvious apoptotic characteristics.In the high concentration GNA administration group,the number of apoptotic cells increased obviously,and the brightness tended to be bright blue.A few cells were dyed bright blue in the DIDS group and DCPIB group and compared with the GNA group,the number of bright blue cells decreased in the DIDS(400 ?mol/L)+ GNA(2 ?mol/L)group and DCPIB(20 ?mol/L)+ GNA(2 ?mol/L)group.4?AV/PI staining,it was observed that a few cells were stained with dark green in the control group,DIDS group and DCPIB group by fluorescence microscopy.In the low dose GNA group,the surface of the cells was bright green,indicating that the cells appeared the tendency of early apoptosis.In the medium and high concentration GNA group,the cytoplasm showed a different intensity of red,indicating that the cells appeared late apoptosis or necrosis.The number of red cells decreased,which indicated that the apoptosis rate decreased in the DIDS(400 ?mol/L)+ GNA(2?mol/L)group and DCPIB(20 ?mol/L)+ GNA(2 ?mol/L)group.5?Flow cytometry showed that compared with the control group,the apoptosis rate of CNE-2Z cells increased with the rising of GNA concentration after treatment with different concentrations of GNA for 24 h,the apoptosis rate of DIDS group and DCPIB group was not obvious.The apoptosis rate decreased significantly in the DIDS(400 ?mol/L)+ GNA(2 ?mol/L)group and DCPIB(20 ?mol/L)+ GNA(2?mol/L)group which compared with the GNA group.6?Patch clamp test results showed that the control group cells produced a small and stable background current.In GNA group,the cell produced a significantly outward current and the current density increased gradually with the time of GNA action,and finally reached the peak value and entered the stationary phase.The current had noapparent voltage dependent inactivation and temporal dependence inactivation.Compared with the GNA group,the current was inhibited significantly in the DIDS(400 ?mol/L)+ GNA(2 ?mol/L)group and DCPIB(20 ?mol/L)+ GNA(2 ?mol/L)group,the difference was statistically significant(P<0.05).The results suggested that the current activated by GNA was VSOR Cl~-current,and could be blocked by nonspecific chloride channel blockers and specific volume sensitive outward rectifier chloride channel blockers.7?The MQAE test showed that compared with the normal control group,MQAE fluorescence intensity increased significantly in GNA group,the difference was statistically significant(P<0.01).What's more,compared with the GNA group,MQAE fluorescence intensity was significantly reduced in the DIDS(400 ?mol/L)+GNA(2 ?mol/L)group and DCPIB(20 ?mol/L)+ GNA(2 ?mol/L)group,the difference was statistically significant(P<0.01).The results further suggested that GNA may activate VSOR Cl~-current,which resulting in the outflow of chloride ion.8?The transfection rate was detected by fluorescence microscopy and flow cytometry,the results suggested that Cl C-3 si RNA was successfully transferred into CNE-2Z cells.Western Blotting method was used to detect the silence of Cl C-3,compared with the normal control group,the expression of Cl C-3 chloride channel protein in Cl C-3 and si RNA groups was markedly inhibited,the difference was statistically significant(P<0.01).The results indicated that Cl C-3 chloride channel protein was successfully knocked out,which provided the basis for the next experiment.9?DAPI nuclear staining,it was observed that normal control group and Cl C-3 group si RNA cell nuclei showed uniform pale blue by fluorescence microscope,while the GNA group of nuclei was a bright blue and the fragments were dense and dense,we could also found fragmentation in the nucleus,these all showed obvious characteristics of apoptosis.Compared with the GNA group,the number of bright blue cells decreased in the Cl C-3 si RNA + GNA(2 ?mol/L)group.The results suggested that Cl C-3 si RNA could reduce the damage of GNA to CNE-2Z cells,and further suggested that the apoptosis of CNE-2Z cells induced by GNA may be related to VSOR Cl~-channels.10?The results of Western Blotting showed that compared with the normal control group,the expression of GRP78 protein was regulated down,the expression of ATF4,CHOP protein was up-regulated in the GNA group.The results suggested that GNA induced CNE-2Z cell apoptosis may be related to endoplasmic reticulum stress signaling pathway.Compared with the control group,GRP78,CHOP,ATF4 protein expression was no difference in DIDS group and DCPIB group.Compared with GNA group,the expression of GRP78 protein was significantly up-regulated and CHOP,ATF4 protein expression was significantly down regulated in the DIDS(400 ?mol/L)+ GNA(2 ?mol/L)group and DCPIB(20 ?mol/L)+ GNA(2 ?mol/L)group,the difference was statistically significant(P<0.01).The results suggested that DIDS,DCPIB can lowered the expression level of endoplasmic reticulum stress related protein ATF4,CHOP and raised the expression of GRP78 protein,which induced by GNA.This suggested that the occurrence of ERS may be related to the activation of VSOR Cl~-channels.Conclusion: GNA could activate the volume sensitive chloride channel,which leaded to the occurrence of ERS,thus induced the apoptosis of CNE-2Z cells and inhibited the proliferation of CNE-2Z cells. |
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