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  • Recently we indicated that cell motility was

    2022-01-13

    Recently, we indicated that cell motility was regulated by the different induction of GPR120 and GPR40 in liver epithelial WB-F344 NMS-873 treated with chemical agents. The cell motility of WB-F344 cells was stimulated by phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) which is a potent tumor promoting agent, correlating with the induction of GPR120 [11]. In contrast, cell motility of WB-F344 cells was suppressed by GPR40 induced by ethionine which is a potent liver carcinogen [12]. These findings suggest that GPR120 acts as a positive regulator and GPR40 as a negative regulator of the cell motility of WB-F344 cells. In the present study, we investigated the effects of GPR120 and GPR40 on cellular functions of three pancreatic cancer cell lines. The expression patterns of GPR120 and GPR40 genes by semi-quantitative RT-PCR analysis are shown in Fig. 1A. HPD1NR cells expressed Gpr40 but did not express Gpr120. While Gpr120 was expressed in HPD2NR cells, Gpr40 expression was undetectable. On the other hand, GPR40 and GPR120 expressions were detected in PANC-1 cells. Based on these findings, to evaluate effects of GPR120 and GPR40 on cell motility of pancreatic cancer cells, cells were pretreated with GW9508 (10 μM) which is an agonist of GPR120 and GPR40 [16], [17], [18]. The cell motile activity of HPD1NR cells were suppressed by GW9508, while GW9508 stimulated the cell motile activity of HPD2NR cells (Fig. 1B). Since PANC-1 cells expressed GPR120 and GPR40 genes, cells were treated with GW1100, a GPR40 antagonist, before cell motility assay [16], [17], [18]. The cell motility of PANC-1 cells was inhibited by GW9508. In contrast, the cell motile activity of PANC-1 cells was markedly enhanced by pretreatment of GW9508 and GW1100 (Fig. 1C). Therefore, these results demonstrated that GPR120 enhanced and GPR40 inhibited the cell motility of pancreatic cancer cells, similar as observed with WB-F344 cells. To assess the roles of GPR120 and GPR40 in malignant properties of pancreatic cancer cells, we generated GPR120 and GPR40 knockdown (PANC-sh120 and PANC-sh40, respectively) cells from PANC-1 (Fig. 2A). While no difference of cell growth rate between control PANC-RFP cells and PANC-sh120 cells was observed, PANC-sh40 cells showed the low cell growth rate (Fig. 2B). The cell growth activity of PANC-sh40 cells was significantly stimulated by GW9508 at a concentration of 10 μM, but not PANC-RFP cells and PANC-sh120 cells (Fig. 2C). To measure effects of GPR120 and GPR40 knockdown on cell motile activity of PANC-1 cells, cells were pretreated with GW9508 (10 μM). The cell motility of PANC-sh120 cells was markedly inhibited by GW9508 in comparison with PANC-RFP cells. In contrast, GW9508 significantly enhanced the cell motile activity of PANC-sh40 cells (Fig. 3A). To confirm the cell motile activities regulated by GPR120 and GPR40, scrape assay was performed. PANC-sh120 cells indicated the low cell motility, compared with PANC-RFP cells. The cell movement of PANC-sh40 cells was markedly faster than that of PANC-RFP cells (Fig. 3B). In a recent study, GPR120 activation stimulated cell motile activity and angiogenesis process in colorectal carcinoma cells [10]. Taken together with our previous studies, GPR120 positively regulated the cell motile activity of pancreatic cancer cells as well as colon cancer cells, whereas GRR40 suppressed the cell motile activity. It is well known that activations of matrix metalloproteinase-2 (MMP-2) and MMP-9 are closely involved in the enhancement of invasive and metastatic potency of cancer cells [19], [20]. To assess the roles of GPR120 and GPR40 in tumor progression, we measured activity levels of MMP-2 and MMP-9 in GPR120 and GPR40 knockdown cells. In gelatin zymography, MMP-2 activity was reduced in PANC-sh120 cells and enhanced in PANC-sh40 cells, compared with PANC-RFP cells. No activation of MMP-9 was observed in all cells. Treatment of GW9508 did not affect MMP-2 and MMP-9 activations (Fig. 4A and B). Furthermore, we investigated whether GPR120 and GPR40 knockdown cells exhibit colony formation using colony assay, which is used to evaluate the tumorigenic activity of a target gene [21]. Cells were seeded into conditioned medium containing low-melting-point agarose. After 10 days, the large sized colonies were significantly formed in PANC-sh40 cells, compared with PANC-RFP cells. However, no colony formation was observed in PANC-sh120 cells (Fig. 4C and D). These results showed that GPR20 enhanced and GPR40 suppressed the tumor progression of pancreatic cancer cells as well as cell motile activity.