GABA and GABA receptors act as an inhibitory neurotransmitter in the mature CNS, but their functions in non-neuronal cells or tumor cells are not well addressed. Previous investigation has reported that GABA is significantly decreased in NSCLC tissues . The previous observation maybe explained by our current findings demonstrating that GABA exerts inhibitory effects on human NSCLC cell. Our results further demonstrate that GABAB receptors play an important role in mediating the GABA-inhibitory effects on NSCLC cells since the effects were blocked by using the antagonist CGP35348 specific for GABAB receptor. Decreased GABA level might have resulted in impaired inhibitory effects on cancer cell proliferation as seen clinically . A compensatory mechanism may be required by enhancing the expression of GABAB receptors in order to redeem the impaired inhibitory effects.
The etiological factors of lung cancer are still not clear, but the tumor progression is associated with genetic changes and is reflected in phenotypic changes such as altered gene expression profiles. In this study, we found that the six genes of GABA receptors are expressed in most of NSCLC cells and tissues, including GABRA3, GABRB3, GABRE, GABRP, GABBR1 and GABBR2. These subunits might compose functional GABAA and GABAB receptor. These genetic changes suggest that GABA receptors have close relationship with NSCLC progression.
As for GABAB receptors, it is now well accepted that GABAB receptors assemble into heteromers composed of one GABBR1 and one GABBR2 subunit, which are both required for normal receptor function [6, 32]. Some recent reports have suggested that GABA inhibits neoplastic proliferation via GABAB receptor [23, 26]. Since GABAB receptor could strongly inhibit base level and isoproterenol-induced cAMP, p-CREB, cyclic adenosine monophosphate response element-luciferase activity and p-extracellular regulated kinase-1 (ERK1)/2 and effectively blocked DNA synthesis and cell migration. The inhibitory cancer cells arrest in G(0)/G(1) phase which is associated with down-regulation of intracellular cAMP level . In our study, GABA inhibited proliferation of NSCLC cells in a dose-dependent and a time-dependent manner. This inhibitory effect could be blocked in the presence of GABAB receptor inhibitor CGP35348. However, co-cultured with GABAA receptor inhibitor picrotoxin, there were no significant proliferative effects on cancer cells versus control. These results imply that GABA inhibition of NSCLC cell proliferation was associated with GABAB receptor which coincides with the report by Schuller and Al-Wadei [23, 26]. The high level expression of GABAB receptor gene in NSCLC tissues compared with the adjacent non-tumor lung tissues, implicated that GABA and GABAB receptor pathways could be a critical factor in regulation of NSCLC cells proliferation. This pathway might be a promising molecular target for the development of new therapeutic strategies for antineoplaston.
As for GABAA receptors, it has been demonstrated that GABAA receptors are usually composed of two α subunits, two β subunits, and one γ subunit, and sometime the γ subunit is replaced by other subunits, such as δ, ϵ, π and θ . Different GABAA receptor subunits have been detected in many cancer cell lines and tissues. Li et al.  detected overexpression of GABRQ in hepatocellular carcinoma cell line HepG2, and half of the tested hepatocellular carcinoma tissues. Takehara  identified the overexpression of GABA receptor pi subunit (GABRP) in PDAC cells. In gastric cancer, more than five GABAA receptor subunits were associated with stimulating KATO III cells . The similar results that GABAA receptors are related to cancer cell proliferation were reported in prostate cancer , breast carcinoma , even in normal human small airway epithelial cells . These findings imply that the inward Cl— ionic current transport and the activated mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) cascade via GABAA receptors positively promotes cell proliferation. Our study revealed that the GABRA3 receptor gene was overexpression in NSCLC tissues compare with paired non-cancerous tissues, this was consistent with the study reported by Liu et al. . In addition, beside overexpression of GABRA3 and GABRE genes, the other GABAA receptor genes expression in our study including GABRB3 and GABRP were also detected in lung cancers, although there was no significant difference between NSCLC tissues and paired non-cancerous tissues. These GABAA receptor subunits may form a functional pentameric chloride channel. We observe that the overexpression GABRA3 and GABRE genes were not associated with proliferative effects on cancer cells. This indecipherable phenomenon will lend us to take more investigations of their roles in lung cancers.
Furthermore, our clinical data analysis showed that both GABAA receptors (GABRA3) and GABAB receptor (GABBR2) genes were significantly expressed in the early pathological stage (stage I and II) of the lung cancer patients, and the expression was gone in stage III and IV. This suggests that high level of gene expression of these GABA receptors may be critical in inhibition of early stage of cancer cells and this regulatory effect got impaired in advanced stage. We speculate that therapeutic intervention approaches that enhance GABA receptors maybe beneficial in late stages of NSCLC patients.
We observe a correlation between the high expression of GABBR2 gene and the greater survival rate in patients with NSCLC. The overexpression of GABBR2 gene was mostly seen in female patients who had better outcome. This suggests that patients with higher level GABBR2 might have better outcome. This observation is consistent with previous in vitro data showing that high level GABBR2 gene expression is associated with inhibition of cancer cell proliferation [26, 39, 40].
In contrast, high level GABRA3 gene expression is correlated with cancer cell development [12, 35, 41], and thus these patients had worse outcome. We indeed showed that the higher gene expression of GABRA3 was mostly detected in those male patients who had a worse prognosis.
Our data was based on a relatively small sample size of 61 patients with NSCLC who were followed for 3–5 years. Our data suggests that the gene expression of certain GABA receptor subunits may be useful for prediction of NSCLC prognosis. We believe that a longer term follow-up study with larger sample size would be required to confirm our current findings.