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  • To assess the roles of

    2021-10-25

    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) Cobimetinib 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.
    Conflict of interest statement
    Acknowledgments This work was supported by JSPS KAKENHI Grant Number 24590493 and by Grants from the Faculty of Science and Engineering, Kinki University.
    Introduction Free fatty acids (FFAs) are essential dietary nutrients and mediate several biological effects via binding to FFA receptors (FFARs), which belong to a member of G-protein-coupled receptors (GPCRs). GPCR 120 (GPR120) and GPR40 are identified as GPCRs for unsaturated medium- and long-chain (C8-C22) FFAs [1], [2], [3], [4]. GPR120 are highly expressed in gastrointestinal tract, lung, adipocytes and macrophages, while high expression of GPR40 is found in pancreatic beta cells. GPR120 induces hormone secretion from pancreatic tissues and digestive tract and regulates anti-inflammatory responses [1], [5], [6], [7]. On the other hand, the insulin secretion by glucose is stimulated through GPR40 in pancreatic islet cells [4]. Therefore, GPR120 and GPR40 are considered as potent target molecules for the treatment of metabolic diseases, inflammation and cardiovascular disorders [8], [9]. 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.