br Materials and methods br Results

Materials and methods
Results
Discussion The distribution of FFA1 and FFA4 expressed in normal tissues is distinguishable. FFA1 is expressed in the restricted organ, such as pancreatic beta transferases [18]. In contrast, FFA4 is highly expressed in the lung, gastrointestinal tract, adipocytes and macrophages [19], [20]. Therefore, it is suggested that FFA1 and FFA4 are closely involved in the regulation of metabolism and immune response [1], [6]. On the other hand, the patterns of FFA1 and FFA4 expressions are various in tumor cells. FFA1 was highly expressed in insulinoma cells, but not in glucagonoma cells [21]. Our recent studies indicated that FFAR1 and FFAR4 expressions were detected in pancreatic cancer, melanoma and osteosarcoma cells [9], [22], [23]. In colon cancer cells, FFA4 was expressed in HCT116 and SW480 cells, whereas no expression of FFA1 was observed [8]. In contrast, the present study showed that FFAR1 and FFAR4 genes were intrinsic expressed in DLD1, CaCo-2 and LoVo cells. The levels of FFAR1 and FFAR4 expressions fluctuated during anticancer drug treatment in cancer cells [10], [11]. FFAR1 and FFAR4 expressions were significantly decreased in lung cancer cells treated with CDDP [11]. CDDP treatment elevated FFAR1 expression, while no change of FFAR4 expression was observed [10]. In this study, we measured FFAR1 and FFAR4 expression levels in three colon cancer cells treated with 5-FU for 3 days. FFAR1 expressions were reduced and FFAR4 expressions were increased in 2 out of three colon cancer cells treated with 5-FU. Moreover, the long-term 5-FU treatment suppressed FFAR1 expression and elevated FFAR4 expression in DLD1 cells, similar as observed with the long-term CDDP treated DLD1 cells. FFA1 and FFA4 exhibited the different effects on cellular functions, depending on types of cancer cells [9], [10], [11], [22]. To evaluate the roles of FFA1 and FFA4 in cell motile activity of the long-term anticancer drug treated DLD1 cells, cells were pretreated with GW9508 and GW1100. In the absence of GW9508, the cell motile activity of DLD-5FU cells was higher than that of DLD1 cells. However, the high cell motile activity of DLD-5FU cells was suppressed by GW9508. While DLD-CDDP cells indicated the low cell motile activity in the absence of GW9508, the cell motile activity of DLD-CDDP cells was reduced by GW9508. Furthermore, pretreatment with GW1100 significantly reduced the cell motile activities of DLD-5FU and DLD-CDDP cells as well as DLD1 cells. Since GW9508 acts as the FFA1/FFA4 agonist and GW1100 as the FFA1 antagonist [14], [15], [16], these results suggest that FFA1 enhances and FFA4 inhibits the cell motile activity of the long-term anticancer drug treated cells. To confirm the roles of FFA1 and FFA4 in the cell motile activity, FFA4 knockdown cells were generated from DLD-5FU cells. The cell motile activity of DLD-5FU cells was markedly elevated by FFA4 knockdown. In addition, we confirmed that the cell motile activity of DLD-5FU-G cells was stimulated by the FFA4 antagonist AH 7614 [17]. The magnitude of the increased cell motility caused by AH 7614 was much greater than that of inhibition by GW9508. Therefore, these findings suggest that the inhibitory effects of FFA4 on cell motility and invasive activities may be stronger than the stimulatory effects of FFA1 in DLD1 cells. In a previous study, activation of FFA4 signaling promoted the cell motile activity and angiogenesis in colon cancer HCT116 and SW480 cells. These cells did not express FFAR1 gene [8]. To investigate the effects of FFA1 and FFA4 on the enhancement of cell motile and invasive activities, highly migratory hmDLD1 cells were established from DLD1 cells. The expression level of FFAR1 gene was markedly higher in hmDLD1 cells than in DLD1 cells, but not FFAR4 expression. The elevated cell motile and invasive activities of hmDLD1 cells were suppressed by GW1100, suggesting that FFA1 plays an important role in the enhancement of cell motile and invasive activities of DLD1 cells. In contrast, FFAR4 expression was increased in highly migratory osteosarcoma cells which endogenously expressed FFAR1 and FFAR4 genes, correlating with the cell motile activity [23]. It is unknown why FFA1 and FFA4 showed the different actions on the enhancement of cell motile activity in two highly migratory cells.