Molecular mechanism of γ-aminobutyric acid inhibitory on the growth of cholangiocarcinoma QBC939 cell line / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the molecular mechanism of γ-aminobutyric acid (GABA) inhibitory on the growth of cholangiocarcinoma cell line (QBC939).</p><p><b>METHODS</b>QBC939 cells were cultured in different groups and treated with GABA, GABA + bicuculine (A receptor antagonist), GABA + phaclofen (B receptor antagonist) for 48 hours. MTT assay was used to determine the proliferation of QBC939 cells. Annexin V-FITC/PI binding assay was used to detect apoptosis in the QBC939 cells. Western blot was applied to check the expression of signal transducer and activator of transcription 3 (STAT3) and phosphorylation-STAT3 (p-STAT3) proteins in different groups of QBC939 cells. Animal models of cholangiocarcinoma bearing nude mice were established by subcutaneous injection of QBC939 cells and randomized into 2 groups: control and GABA-treated groups. The effect of GABA was evaluated after 5 weeks, including the body weight and tumor volume. The expression of p-STAT3 was detected by immunohistochemistry and Western blot in xenograft tumors.</p><p><b>RESULTS</b>MTT and FCM assays both showed that the effect of GABA inhibitory on the proliferation (15.30% ± 0.80% vs. 2.66% ± 0.74%, t = 23.15, P = 0.00) and induced apoptosis (23.15% ± 0.21% vs. 4.30% ± 0.69%, t = 52.40, P = 0.00) of QBC939 cells could be antagonized by phaclofen, but not bicuculine. The expression of STAT3 and p-STAT3 proteins were all observed in the QBC939 cells and GABA significantly down-regulated p-STAT3 protein expression (0.77 ± 0.00 vs. 0.45 ± 0.01, t = 63.14, P = 0.00), this action was also antagonized by phaclofen (0.45 ± 0.01 vs. 0.76 ± 0.01, t = 56.25, P = 0.00). Xenograft tumor volume ((0.62 ± 0.03) cm³ vs. (0.34 ± 0.03) cm³, t = 13.45, P = 0.00) and the expression of p-STAT3 protein were significantly decreased in GABA-treated group as compared with control group.</p><p><b>CONCLUSIONS</b>GABA may inhibit the growth of cholangiocarcinoma cells QBC939 through GABA(B) receptor, and down-regulation of the p-STAT3 expression perhaps is one of its anti-tumor mechanisms.</p>

Expression of WW domain containing oxidoreductase gene in cholangiocarcinoma and its effect on the biological behavior of cancer cell line RBE / 中华外科杂志

<p><b>OBJECTIVE</b>To study the effects of anti-oncogene WWOX on cell growth of cholangiocarcinoma.</p><p><b>METHODS</b>The expression of WWOX protein was detected with immunohistochemical method-SP in 54 patients with cholangiocarcinoma from July 2005 to May 2010 and 12 samples of normal bile duct tissues. The recombinant WWOX eukaryotic expression plasmid was introduced into RBE cells by liposome-mediated transfection and positive cell clones were selected and amplified. The mRNA and protein expressions in RBE cells stably transfected with WWOX were investigated by quantitative RT-PCR and Western Blot before and after transfection. Cell proliferation was tested by MTT, cell apoptosis was assessed by FCM, the alteration of mitochondria membrane potential (ΔΨm) was detected by JC-1 staining method, cell invasion was determined by Transwell chamber assay. The expression change of bcl-2, bax, FasL, caspase-3 mRNA and protein was detected by quantitative RT-PCR and Western Blot.</p><p><b>RESULTS</b>The expression of WWOX protein was significantly lower in cholangiocarcinoma than that in normal bile duct tissues and loss of WWOX protein expression was found in 40.7% of cholangiocarcinoma specimens (P < 0.05). RBE cells with stable transfection of WWOX were established. Quantitative RT-PCR showed that the expression of WWOX mRNA was significantly enhanced and Western Blot demonstrated that WWOX protein expression was markedly increased. MTT showed that WWOX gene transfection significantly decreased the proliferation of RBE cells (P < 0.05). FCM analysis showed that the apoptosis rate after transfection was significantly promoted [(1.1 ± 0.6)% vs. (1.7 ± 0.5)% vs. (35.2 ± 4.4)%, P < 0.01], JC-1 staining method indicated that the experimental group was loss of ΔΨm [(12.6 ± 1.9)% vs. (13.6 ± 1.8)% vs. (48.7 ± 2.9)%, P < 0.01], transwell chamber assay showed that the number of transfected cells that passed the transwell membrane was significantly less than those of control groups (77 ± 6 vs. 72 ± 8 vs. 48 ± 6, P < 0.01). Quantitative RT-PCR and Western blotting showed that the expression of bcl-2 mRNA and protein was markedly decreased and the expression of bax, caspase-3 were significantly increased. There was no significant change in the expression of FasL.</p><p><b>CONCLUSION</b>WWOX exerts its antitumor effect against proliferation through inducing cell apoptosis in cholangiocarcinoma.</p>