Caspase-8 Fluorometric Assay Kit It has been reported that

It has been reported that aberrant GPR120 expressions were detected in colorectal carcinomas, compared with normal tissues. In addition, GPR120 stimulated cell motile activity and angiogenic property in colon cancer cells [10]. Recently, we indicated that GPR40 suppressed cell motile and invasive activities of fibrosarcoma cells [11]. However, the biological effects of GPR120 and GPR40 on cellular functions of cancer cells are not fully understood. It is well known that 12-O-tetradecanoylphorbol-13-acetate (TPA) is a tumor promoting agent of skin carcinogenesis in rodents [12]. TPA stimulates a variety of cellular functions in cancer cells, including cell motile activity [13], [14]. In our recent study, when liver epithelial cells were treated with TPA, the cell motile activity was stimulated, correlating with GPR120 expression [15]. In the present study, to assess whether GPR120 and GPR40 are involved in regulation of cellular functions induced by TPA in melanoma cells, we generated GPR120 knockdown cells from melanoma cells and investigated effects of GPR120 knockdown on cell motile activity of melanoma cells treated with TPA.
Materials and methods
Results
Discussion TPA regulates cell motile activity through several intracellular signaling pathways. The cell motile activity was stimulated through activation of protein kinase C induced by TPA in some cancer cells [13], [14], [18]. In A375 cells, induction of COX-2, PGE2 and PGE2 receptors by TPA contributed to regulation of cell motile activity [19]. Our previous study showed that TPA treatment enhanced cell motile activity of liver epithelial cells, correlating with lysophosphatidic Caspase-8 Fluorometric Assay Kit (LPA) receptor-3 (LPA3) expression [20]. First, we assessed whether GPR120 and GPR40 regulate intrinsic cell motile activities of melanoma cells. Although A375 and G361 cells endogenously expressed GPR120 and GPR40 genes by semi-quantitative RT-PCR analysis, the GPR40 expression in G361 cells was relatively lower than that in A375 cells. In a previous study, the high expression level of GPR40 gene was detected in A375 cells, compared with several neuroblastoma and breast cancer cell lines [21]. The cell motile activity of A375 cells was increased by GW9508, while GW9508 inhibited the cell motile activity of G361 cells. Therefore, the diverse effects of GW9508 on the cell motile activities of A375 and G361 cells may be due to different levels of GPR40 expression. In addition, when cells were pretreated with GW9508 and GW1100, the cell motile activities of A375 and G361 cells were significantly lower than those of untreated cells. These findings demonstrate that GPR120 negatively regulates the cell motile activities of A375 and G361 cells. Recently, we reported that cell motile activity and GPR120 expression were elevated in liver epithelial cells treated with TPA [15]. In the present study, to evaluate whether GPR120 and GPR40 are involved in cell motile activities induced by TPA in melanoma cells, A375 and G361 cells were treated with TPA. The short-term TPA treatment increased the cell motile activity and GPR40 gene expression in A375 cells. The cell motile activity stimulated by TPA was markedly enhanced by GPR120 knockdown. In contrast, the cell motile activity of G361 cells was significantly inhibited by TPA, correlating with GPR120 and GPR40 gene expressions. Therefore, it is suggested that GPR120 induced by TPA may act as a dominant negative regulator of cell motile activity in G361 cells. On the other hand, whereas GPR120 and GPR40 expressions were elevated in the long-term TPA treated cells, the cell motile activity was significantly elevated. The cell motile activity stimulated by the long-term TPA treatment was suppressed by GW1100 and enhanced by GPR120 knockdown, similar as observed with the short-term TPA treated cells. These results suggest that opposite roles of GPR120 and GPR40 regulate cell motile activity stimulated by TPA in melanoma cells. However, it is unclear why TPA indicated the diverse effects on cell motile activities of A375 and G361 cells. It is possible that different induction levels of GPR120 and GPR40 by TPA may result in positive or negative regulation of cell motile activity in A375 and G361 cells.