Role Of Volume-regulated Chloride Channel In The Proliferation Of Human Gastric Carcinoma Cell Line

AimsVolume-regulated chloride channel is expressed ubiquitously in mammalian cells. When cell volume is disturbed, such as growth and apoptosis, cells defend themselves by regulatory volume decrease ( RVD ) and regulatory volume increase (RVI) to maintain a constant volume. For example, during the Gi/S transition, cells prepare for entry into S phase and are committed to several tasks, including uptake of amino acids, metabolic substrates and material for the synthesis of DNA. Tumor cells usually have higher rates of metabolism, mitosis and migration, compared with normal cells, suggesting the volume-regulated chloride channel plays a more important role in cancerDespite its well-characterized electrophysiological properties, the molecular structure of volume-regulated chloride channel is not yetresolved. Three molecules such as P-gp , plcin and CLC-3 have been put forward in relation with Icl.vol CLC-3, one of the candidates, is the most controversial one, for there are different results in different cell types tested.The experiment purpose is to study the role of volume-regulated chloride channel in the proliferation of human gastric carcinoma cell line and the relationships between CLC-3 and volume-regulated chloride channel.Methods1. The culture of gastric carcinoma cell line.2. We adopted cell counting and MTTassay to observe the effects of TAM and NPPB on cell proliferation.3. Flow cytometry was used to determine the influence of TAM and NPPB on the DNAcontent of different stages of cell cycle.4. The currents of volume-regulated chloride channel activated by hypotonicity were recorded by whole-cell patch clamp.5. All cells were cultured for enough days to get the total RNA. The cDNA was synthesized from the total RNA, and PCR was used. Using these methods, CIC-3 mRNA was detected.6. The DNA sequence was assayed to determine whether the sequence of CLC-3 was what we wanted.7. Applying antisense oligonucleotide to block the expression of CLC-3 mRNA.8. Flow cytometry was used to determine the influence of blockade of CLC-3 on cell cycle.Results1. NPPB (100 umol/L) and TAM (10 umol/L) inhibited cell growth of SGC7901 reversibly.2. NPPB (80, 120, 160, 200nmol/L)and TAM(5, 10, 15, 20 umol/L) inhibited the proliferation of SGC7901 in a dose-dependent manner.3. Both NPPB (100 umol/L) and TAM (10 umol/L) can increase DNA level of G1 phase and decease that of S phase significantly. The blockers inhibit cell cycle progression at G1 phase.4. The currents which are in basal activity in isotonic solutions can be activated by hypotonic solutions and dramatically inhibited by NPPB (100 umol/L) and TAM (10 umol/L).5. CLC-3 mRNA was expressed in SGC7901.6. The results of DNA sequence assay were compared with the sequence in Genbank, and it is found that both of the sequence are some parts of CLC-3.7. Antisense oligonucleotide (4 ug/mL) can decrease the content of CLC-3 mRNA and make no influence on cell cycle.ConclusionsThe study shows that volume-regulated chloride channel plays an important role in gastric carcinoma cell line proliferation; CLC-3 mRNA was expressed in SGC7901, blocking expression of CLC-3 don't dramatically influence cell cycle. So CLC-3 may not be the molecular candidate of the chloride channel. The research lays the ground base and new target for gastric carcinoma clinical treatment,and provides more information about its molecular candidate.