Function Receptor And Signal Pathway Of GABA Inhibitory On The Growth Of Cholangiocarcinoma QBC939Cells

Introduction Cholangiocarcinoma is the most frequently occurred biliary malignancy.It is the second most common primary hepatic malignancy after hepatocellular cancer. Itis difficult to diagnose owing to its anatomic location, growth patterns, and lack of definite diagnostic criteria. The prognosis of this malignancy is dismal owing to itssilent clinical character, difficulties in early diagnosis, and limited therapeutic options.The influence of neuropsychological on the disease has been valued and proved. Manyneurotransmitters play an important role in tumor development, metastasis and invasion,which have been a hotspot research for tumor prevention and treatment. GABA, as amajor inhibitory neurotransmitter, we showed that GABA inhibited the growth ofhuman cholangiocarcinoma QBC939cells in vitro in our previous study, but thefunction receptor and signal pathway of inhibition effect were not fully understood. Inthis study, we tried to explore the function receptor and signal pathway byimmunohistochemical, MTT, FCW, Western blot and animal experiments. Clinicalapplication and experimental basis were provided for the pharmacological action ofGABA receptor drugs.Objective To investigate which receptor and signal pathway truly plays a role in theinhibitory effect of GABA on human cholangiocarcinoma QBC939cells.Methods An initial immunohistochemistry study of the expressions of GABAAR(β3),GABABR(R1) and GABACR(ρ2) in cholangiocarcinoma and normal bile duct tissueswas followed by the culture and treatment of QBC939cells for48h with control,GABA, GABA+bicuculine (GABAAreceptor antagonist), GABA+phaclofen (GABABreceptor antagonist). MTT and Annexin V-FITC/PI binding assays were used todetermine the proliferation and apoptosis of the QBC939cells. The expression of thesignal transducer and activator of transcription3(STAT3) and phosphorylated STAT3[p-STAT3(Tyr705)] was evaluated by the western blot assay. The effect of GABA on thegrowth of QBC939xenograft tumors in athymic nu/nu mice was examined; includingthe body weight and tumor volume. The p-STAT3(Tyr705) expression in xenografttumors was detected by immunohistochemistry and western blot. Results A significant difference was only observed in GABABreceptor expressionbetween cholangiocarcinoma and normal bile tissues (61.9%vs.16.7%, x2=7.65,P<0.05), and the expression of GABABR(R1) protein had a correlation with histologicaldifferentiation, perneural invasion, local invasion and lymph node metastasis incholangiocarcinoma (P<0.05). MTT and FCM assays all showed that the effect ofGABA inhibitory on the proliferation (15.30%0.80%vs.2.66%0.74%, t=23.15，P<0.05) and induced apoptosis (23.15%0.21%vs.4.30%0.69%, t=52.40，P<0.05) ofQBC939cells could be antagonized by phaclofen, but not bicuculine. The expression ofSTAT3and p-STAT3(Tyr705) proteins were all observed in the QBC939cells. Ascompared with control group, GABA significantly down-regulated p-STAT3(Tyr705)protein expression (0.770.00vs.0.450.01, t=63.14, P<0.05), this action was alsoantagonized by phaclofen (0.450.01vs.0.760.01, t=56.25, P<0.05). Xenograft tumorvolume [(0.620.03vs.0.340.03) cm3, t=13.45，P<0.05] and the expression ofp-STAT3(Tyr705) protein were significantly decreased in GABA-treated group ascompared with control group.Conclusion GABA may inhibit the growth of cholangiocarcinoma cells QBC939through GABABreceptor, and down-regulation of the p-STAT3(Tyr705) expressionperhaps is one of its anti-tumor mechanisms.