The Mechanism Of ClC-3 In Starvation-induced Autophagy In Nasopharyngeal Carcinoma Cell CNE-2Z

Objectives:Autophagy is an important process of intracellular misfolded protein degradation in eukaryotes,which are particularly active in cancer cells.In human nasopharyngeal carcinoma cell line CNE-2Z,the cells suffer a long-term starvation and the autophagy activity is enhanced due to the infinite proliferation of cancer cells.Our previous studies have shown that the expression and functional activity of ClC-3 channel in CNE-2Z are increased.Based on these studies,we used EBSS to induce autophagy and explore whether ClC-3 channel is involved in autophagy induced by starvation.So it's significant to provide experimental basis for clinical treatment of nasopharyngeal cancer in the future.Methods:In this study,nasopharyngeal carcinoma cells CNE-2Z were used as the research object,and the following experiments were conducted.1)Whole-cell patch clamp was used to record the activated chlorine current induced by EBSS in CNE-2Z cells.2)Expression of autophagosomes and acidic lysosomes was observed by MDC staining.3)Western blot analysis of the expression of ClC-3,autophagy substrate protein P62 and autophagy marker protein LC3 in CNE-2Z cells.4)The changes of chloride current,P62 and LC3 in cells were observed after Knockdown of ClC-3.5)The intracellular ROS level in CNE-2Z cells was detected by flow cytometry.Results:EBSS activated a chloride current in CNE-2Z.Under EBSS-induced starvation,a current was induced in 107.48±21.32 min,which reached a peak within 174.16±18.88 min.The results showed an outward rectification with current density of 48.09±6.41 pA/pF at+80 mV and-35.36±4.53 pA/pF at-80 mV.EBSS-induced current had a trend of slow and smooth growth,showing a outward rectification,without time-dependent or voltage-dependent inactivation.The I-V curve demonstrated that the current reversed at-9.98±2.64mV,closed to the E^-_Cl with value of-13.5mV.Chloride channel blockers NPPB and DIDS as well as ClC-3 siRNA suppressed the chloride current induced by EBSS.2.EBSS promoted the increasing of autophagosomes and acid lysosomes in CNE-2Z cells.Chlorine channel blockers NPPB and DIDS and ClC-3 siRNA could both further increase the number of autophagosomes and acid lysosomes,3.EBSS promoted the increasing expression of ClC-3 and the degradation of autophagy substrate protein P62,and the transformation of autophagy marker protein LC3-I to LC3-II in CNE-2Z cells,and prevent the autophagy flux processing.4.The chloride channel blockers NPPB,DIDS and ClC-3 siRNA could inhibit the degradation of autophagy substrate protein P62 and the degradation of autophagy marker protein LC3-II,and prevent the autophagy flux processing..5.The antioxidant L-NAC reduced the ROS levels in CNE-2Z in NPC cells,inhibited the expression of ClC-3 and degradation of autophagy substrate protein P62 as well as the transformation of autophagy marker protein LC3-I into LC3-II,indicating that the expression of ClC-3 is inhibited by decreased ROS levels.EBSS induced the increased level of ROS in CNE-2Z cells,while the down-regulation of ClC-3 had little effect on the ROS level.Conclusion:ClC-3 involved in EBSS-induced autophagy in nasopharyngeal carcinoma cells CNE-2Z.After the increase level of ROS,the expression of ClC-3 chloride channel was up-regulated to mediate EBSS-induced autophagy.