Chloride Channels Involved In The Pathogenesis Of Glioma

Chloride channels are universally expressed all eukaryotic cells,which have been suggested to be related to the transport of charge, i.e.,to the electric current flowing through the channel, and on the otherhand to the transport of matter. It is now appreciated that thesechannels play important functional roles in diverse processes, such asvolume regulation, cell proliferation, invasiveness and metastasis,apoptosis and angiogenesis.Objective: In this study, we set up to investigate the relationshipbetween the chloride channels and invasiveness and proliferation ofglioma.Methods: ⑴ The expression of ClC-3 was observed byimmunohistochemical staining in 24 cases of human glioma and 4cases of brain metastic cancer specimens (with 5 cases of normal braintissue as control);meanwhile, the expression of ClC-3 mRNA wasobserved by RT-PCR in all the ClC-3 positive-staining cancerspecimens. ⑵HPLC was used to detect the relationship betweenchloride channels and amino acid efflux in RVD process in humanU251MG cells. ⑶We applied MTT assay to observe the effects ofchloride channel blockers, such as NPPB,DIDS, NFA and tamoxifenon the human U251MG cell proliferation. ⑷Trypan blue exclusiontest was applied to observe the effects of chloride channel blockers,such as NPPB,DIDS, NFA and tamoxifen on the human U251MGcell survival.. Acridine orange staining was used to investigated theeffects of NPPB on U251MG cell.⑸Flow cytometry and Ki-67immunohistochemistrical staining were applied to observe the effectsof chloride channel blocker NPPB on the cell cycle of synchronizedU251MG cell. ⑹RT-PCR was applied to observe the effects ofchloride channel blocker NPPB on the expression of cell cycle orapoptosis -related genes of synchronized U251MG cell.Results:⑴ClC-3 protein was mainly expressed in the membraneand/or cytoplasm of neoplastic cells and microvascular endothelialcells. ClC-3 protein was found negative in 4 cases of normal braintissues and positive in 19 cases of human glioma and 4 cases of brainmetastic cancer specimens. Moreover, the expression of ClC-3 proteinwas found highly positive in 3 cases of high malignantoligodendrogliomas whereas slightly positive in 7 cases of lowmalignant ones. ⑵The expression of ClC-3 mRNA was detected in 16cases of human glioma and 4 cases of brain metastasis cancerspecimens among the tissues with the positive expression of ClC-3protein. The level of protein and RNA of ClC-3 in high malignantoligodendrogliomas was higher than in low malignant ones. ⑶Theswelling-induced release of glutamate, aspartate, glycine, and lysineunder hypotonic condition was significantly increased. Interestingly, itwas decreased markedly by putative Cl– channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Treatment of cells with100μM NPPB significantly reduced the swelling-induced release ofglutamate, aspartate, glycine, and lysine with 47%, 17%, 24%, and63%, respectively.⑷Compared with the control group, the OD valueof the experimental groups decreased significantly;moreover, thechange of OD value depended on the change of drug concentration.⑸Trypan blue exclusion test showed that there was no significantdeference in the vitality of cells in control and experimental groups,except for the groups treated with NPPB and DIDS at theconcentration of 200μM. Furthermore, the rate of cell deathsignificantly increased when treated with NPPB and DIDS at theconcentration of 200μM. ⑹G0/G1 arrest and G1/S checkpoint failureoccurred at 12, 24, 48h and at 24h treated with NPPB, respectively. ⑺The rate of positive staining for Ki-67 was 94.5%±1.13% in thecontrol groups cultured for 24h. However, the rate of positive stainingfor Ki-67 apparently decreased to 74.4%±0.57% in experimentalgroups treated with NPPB. ⑻After the treatment with NPPB for 12and 24h, the expression of P57 and CDK6 increased significantly atmRNA level whereas cyclin D1 apparently decreased. Meanwhile, thechange of other observed genes expression was not found.Conclusions:⑴ The characteristics of ClC-3 expression in human glioma andbrain metastic cancer suggested that ClC-3 was probably correlatedwith malignant transforming, cell volume regulaion, proliferation andinvasiveness of human brain tumor. The characteristics of ClC-3expression in human oligodendrogliomas suggested that ClC-3 wasprobably correlated with the classification of its pathologicalmalignance.⑵ It indicate that these amino acids, glutamate, aspartate,glycine, and lysine, are implicated in the regulatory volume decreaseand the swelling-induced efflux of amino acids are dependent of theactivity of Cl– channel.⑶ Cl-channel blockers such as NPPB,DIDS,NFA andtamoxifen had inhibitory effects on the proliferation of glioma cellswith drug dependent characteristic feature. It suggested that Cl-channel was involved in the cell proliferation. Compared with controlgroups, the rate of positive staining for Ki-67 apparently decreased inexperimental groups treated with NPPB. It suggested that Cl-channelblocker lead the G0/G1 arrest and thus inhibited cell proliferation. G0/G1arrest and G1/S checkpoint failure occurred at 12, 24, 48h and at 24htreated with NPPB, respectively. It suggested that Cl-channel blockerinhibit cell proliferation by the control of cell cycle.⑷ After the treatment with NPPB for 12 and 24h, p57 andCDK6, increased significantly at mRNA level whereas cyclin D1apparently decreased. It suggested that Cl-channel blocker, NPPBregulated the expression of p57 and cyclinD1 , lead the occurrence ofG0/G1 arrest and G1/S checkpoint failure, and thus behave theinhibitory effect of cell proliferation. Meanwhile, the expression ofBax and bcl-2 was suggested there may be no direct correlationbetween inhibitory effect of cell proliferation and apoptosis.