A Study On The Effect And Mechanism Of Gamma-aminobutyric Acid (Inhibitory Neurotransmitter) On The Proliferation, Invasion And Metastasis In Human Cholangiocarcinoma Cell Line QBC939

As the development of modern neurobiology and suggestion of new medical model--- Biological- psychological- social Medicine Model, nervous system has been paid more and more attention. more other functions of neurotransmitters were been discovered, Recent years, many researches discovered that there are many neurotransmitter receptors in the surface of tumor cell. Moreover, many neurotransmitters can affect the growth, migration, invasion and metastasis. Gamma-aminobutyric acid(GABA) is the most important inhibitory neurotransmitter in the central nervous system. it can not only regulate the central nerve, but also to ettect the malignant tumor. At the same time, neuroendocrine system regulate the pathophysiology of cholangiocyte, many hormone, neuropeptide and neurotransmitters can affect the bionomics of cholangiocaicinoma. But the effect of GABA on the cholangiocarcinoma is unknown. So we design this experiment to explore the effect of GABA on the cholangiocarcinoma cell QBC939 in vitro, and understand the mechanism. the aim is to reveal the pharmacologic actions of GABA and to offer novel threads and plans to prevent and cure cholangiocarcinoma. ObjectionTo explore the effect of GABA(inhibitory Neurotransmitter) on the proliferation, invasion and metastasis of cholangiocarcinoma cell line----QBC939 in vitro, and to reveal the mechanism of this effect MethodsQBC939 cells were cultured in vitro and seeded into culture plates. At the state of culturing, observing the morphology of cells in culture flask by invert microscope every day. Following exposure to GABA at different concentrations in different times, then QBC939 cells were harvested and investigated.Cell growth inhibition was evaluated by MTT assay, The rate of apoptosis and cell cycle changes of QBC939 cell line induced by GABA were measured by means of flow Cytometry, The telomerase activity of QBC939 cells was examined by modified PCR-ELISA assay. Radioimmunoassay was used to measure the intracellular cyclic adenosine monophosphate (cAMP) content.Transwell cell culture chamber assay in vitro was used to detect the ability of invasion and metastasis of cholangiocarcinoma QBC939. The expression of matrix metalloproteinase (MMP) mRNA and enzymatic activity of QBC939 cells were detected by reverse transcription polymerase chain reaction (RT-PCR) and gelatin zymography, respectively. Results(1)MMT assay shows that the different concentrations of GABA inhibits the growth of QBC939 cells in a dose-dependent manner ,the rate of inhibition is from 2.6% to 26.82%(48h,P<0.05),especially obvious time is after 48 hours(2) GABA promotes the cell apoptosis, The apoptosis rate of QBC939 cells was increased from 4.8% to 28.03% which had significant difference (P < 0.05). It is no effect for distribution of cell cycle.(3)compared with control group, Telomerase activity was inhibited by GABA(0.82±0.048 vs 0.69±0.027, P<0.05)at the beginning of 10μmol/L.(4) The content of intracellular cAMP was increased with the increase of GABA concentration in a dose-dependent manner(100μmol/L, 0.59±0.049 nmol/L vs 0.71±0.029, P<0.05).(5) GABA inhibited tumor cells'ability of trans-matrigel (100μmol/L GABA, 42.67±4.04 vs 60±9.54, P<0.05)(6) PT-PCR shows that GABA inhibited MMP-2 and MMP-9 mRNA expression(100μmol/L处, MMP-2 mRNA: 0.632±0.065 vs 1.107±0.271,MMP-9 mRNA: 0.605±0.104 vs 0.891±0.167, P<0.05),Additionally, GABA attenuated the degrading activity of MMP-2 and MMP-9 (1000μmol/L处,Vmmp-2: 46.1±0.025 vs 73.2±0.14,Vmmp-9:11.2±0.035 vs 32.6±0.071, P<0.05)as demonstrated by gelatin zymography in a dose-dependent manner. Conclusion(1) GABA can inhibit the proliferation of QBC939 cells by promoting apoptosis and inhibitting telomerase activity, which may be mediated by the information transmission of post-receptor.(2)GABA can inhibit the invasion and metastasis of cholangiocarcinoma QBC939,the possible mechanism may be involved in the down-regulation of the secretion and activity of MMP-2, MMP-9.