The Role Of CLC-3 Chloride Channel In Apoptosis Induced By Dihydroartemisinin In The Poorly Differentiated Human Nasopharyngeal Carcinoma CNE-2Z Cells

Objectives:Nasopharyngeal carcinoma(NPC)is a special head and neck tumor,mainly distributed in Southeast Asia.Radiotherapy for Nasopharyngeal carcinoma is preferred,but metastatic and radiation-tolerant Nasopharyngeal carcinoma also cooperate with chemotherapy.Recent studies have found that the traditional antimalarial drug artemisinin and its derivatives have multiple anti-tumor effects.Dihydroartemisinin(DHA),a semi-synthetic derivative of artemisinin,is not only traditionally anti-malarial,but also has an advantage of specific anti-tumor activity,but its mechanism is not clear.Previous results of our laboratory show that normal nasopharyngeal epithelial NP69-SV40 T cells had lower CLC-3 activity and less protein expression compared with nasopharyngeal carcinoma CNE-2Z cells.On this basis,the scientific problems of this subject are: 1)Whether CLC-3 chloride channel is a potential target for DHA selective anti-nasopharyngeal carcinoma cell line CNE-2Z;2)The mechanism of DHA inducing apoptosis in CNE-2Z cells through CLC-3 chloride channels.This will provide an experimental basis for making clear whether CLC-3 chloride channel is a potential target for DHA against Nasopharyngeal carcinoma and expand the clinical application of DHA.Methods:1)Experimental subjects: Nasopharyngeal carcinoma CNE-2Z cells and normal nasopharyngeal epithelial NP69-SV40 T cells;2)MTT assay was used to detect cell proliferation inhibition rate;3)Annexin V-PI staining assay was used to detect cell apoptosis rate;4)Dynamic image analysis assay was used to detect cell volume;5)Western blot was used to detect the protein expression of Cleaved-Caspase-3 and CLC-3 in cells;6)Patch clamp assay(whole cell recording)detect chloride current of cells;7)MQAE assay(chlorine ion probe)detected intracellular chloride ion fluorescence through fluorescent enzyme labeling;8)Fluo-3(calcium ion probe)detected intracellular calcium fluorescence through Cell Blow Meter;9)The data are expressed by mean ± standard error,and the data are analyzed by SPSS12.5 software.Results:1)DHA inhibits the proliferation of CNE-2Z cells.Treated with for 24 and 48 hours.the inhibition rate of cell proliferation is also increased with the increase of concentration,showing a time and concentration dependent manner.After calculation,the IC50 of DHA was 36.4 μmol/L.Compared with normal NP69-SV40 T cells,DHA had lower toxicity and IC50 was 189.7 μmol/L.Therefore,we choose DHA40 μmol/L as the follow-up experimental concentration.2)DHA induces apoptosis in CNE-2Z cells.The apoptosis rate of CNE-2Z cells at 24 and 48 hours was(14.14±1.75)% and(35.41±0.98)% respectively.And the apoptosis rate was only(1.32±1.05)% and(6.24±1.84)%(6.24±1.84))in normal NP69-SV40 T cells.Chloride channel blockers(DIDS,NPPB)and CLC-3 si RNA can inhibit the apoptosis of CNE-2Z cells induced by DHA.3)The expression of Cleaved-caspase 3 protein was increased induced by DHA in CNE-2Z cells,but there was no significant change in the expression of Cleaved-caspase 3 protein in normal NP69-SV40 T cells.4)The increased expression of CLC-3 protein was increased by DHA in CNE-2Z cells.CLC-3 protein began to increase at 1.5 hours,reached a peak at 6 hours and decreased in 12-24 hours(but higher than normal control).The expression of CLC-3 protein in normal NP69-SV40 T cells did not change significantly.5)DHA selectively activated the chloride current in CNE-2Z cells.At about 10.55 ±3.88 min,DHA activated the chloride current of cells,and the current reached the plateau stage after continuous action of 27.82±4.06 min.Under±80 voltage clamp,the maximum activation current is(40.09 + 2.54)p A/p F and(-26.52± 1.50)p A/p F respectively.For normal NP69-SV40 T cells,DHA could not activate chloride currents after 40 min treatment.Chloride channel blockers(DIDS、NPPB)and CLC-3 si RNA can inhibit DHA activated CNE-2Z chloride currents.6)DHA selectively induces chloride decrease in CNE-2Z cells.After DHA treatment of CNE-2Z cells 15 min,the intracellular Cl fluorescence decreased(29.67±6.22)%.The intracellular chloride ion outflow was stable,and the intracellular Cl fluorescence decreased(35.65±3.78)% after treatment with 30 min.7)DHA induced the apoptosis volume of CNE-2Z cells to shrink-AVD.After DHA perfusion of CNE-2Z cells 5min,cell volume could be observed.After continuous 120 min treatment,cell volume decreased(15 ± 4.58)%.CLC-3 si RNA and chloride channel blocker DIDS can inhibit DHA induced AVD.8)DHA induced the increase of calcium ion in CNE-2Z cells.After 6 hours of DHA action of CNE-2Z cells,the relative value of intracellular calcium fluorescence increased(77 ±18.52)%.After 24 hours treatment,the relative value of intracellular calcium fluorescence increased(207±36.78)%.CLC-3 si RNA inhibited DHA induced increase of calcium fluorescence in CNE-2Z cells,and the relative value of calcium fluorescence decreased from(307.76±40.53)% to(120.17±20.96)%.