Galanin is an epigenetically silenced tumor suppressor gene in gastric cancer cells

Abstract

Galanin is a 30 amino-acid active neuropeptide that acts via three G-protein coupled galanin receptors, GALR1, GALR2 and GALR3. Recently, GALR1 was also suggested as a tumor suppressor gene that was frequently silenced in head and neck squamous cell carcinoma; moreover, galanin and GALR1 were reported to inhibit human oral cancer cell proliferation. However, the exact role of galanin in gastric cancer is unclear. Here, we describe the epigenetic silencing of galanin in human gastric cancer. Five gastric cancer cell lines (SNU-1, SNU-601, SNU-638, KATOIII, and AGS) showed a significant reduction in galanin expression that was restored by the demethylating agent 5-aza-2'-deoxycytidine. We confirmed the hypermethylation of CpG islands in the galanin promoter region by methylation-specific and bisulfate sequencing polymerase chain reaction (PCR). Interestingly, hypermethylated galanin did not affect galanin receptor expression. Exogenous galanin expression in silenced cells induced apoptosis and decreased phosphorylated Akt expression. Taken together, these data suggest that galanin hypermethylation impairs its tumor suppressor function in gastric cancer carcinogenesis.

Fig 1. Decreased galanin expression and restoration by demethylating agent.

(A) mRNA expression of Galanin and its receptors (GARL1-3) were examined by RT-PCR. (B) Recovery of Galanin mRNA expression was examined by RT-PCR following treatment with demethylating agent 5-aza-dC in SNU-601 and AGS cells.

Fig 2. Methylation analysis of galanin in gastric cancer cell lines.

(A) Galanin methylation status was analyzed by methylation-specific PCR (MSP) with methylation-specific primers (M) and ummethylation-specific primers (U). (B) Analysis of galanin hypermethylation by bisulfate-sequencing PCR (BSP). Schematic CpG island of galanin indicated 16 CpG dinucleotides (bars) in the genomic region that were examined using BSP primers (arrows). C and T represent methylated and unmethylated CpG islands, respectively. Representative chromatograms demonstrated methylated and unmethylated clones. PBLC is genomic DNA extracted from peripheral blood lymphocytes.

Fig 3. Induced apoptosis after galanin overexpression.

(A) After 24 hours of transfection with each vector, apoptosis was analyzed by annexin-V and PI staining. Representative histograms from triplicate experiments show cells stained with annexin-V and propidium iodide. (B) Percentage of early apoptotic cells stained with only annexin-V (AV+PI-) are compared in three cell lines after galanin overexpression. Significant differences are marked with * (p < 0.01), *** (p < 0.0001), respectively, with mean ± standard error (SE).

Fig 4. Apoptosis in galanin-overexpressing cell lines.

(A) Sub-G1 population was compared between galanin- (pEGFP-Galanin) and empty vector (pEGFP)-transfected cells. Significant differences are marked with *** (p < 0.0001). (B) Protein level and phosphorylation were examined by western blot analysis. Enhanced PARP cleavage was shown in galanin-transfected cells (arrow).