Mechanisms Of Lysophosphatidic Acid-stimulated Cell Motility Of Pancreatic Cancer

Background:Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer related death in men and the fifth leading cause in women, with only3%-5%of five-year survival rate. Pancreatic cancer is a highly metastasis disease characterized by widespread peritoneal dissemination and ascites formation. Extensive invasion and metastasis is the principle reason for the poor prognosis. Thus, clarifying mechanisms of pancreatic cancer metastasis plays an important role in development of new therapies. In previous studies, lysophosphatidic acid (LPA) was a critical component of ascites and promoted the invasion and metastasis of pancreatic cancer, and LPA1/3may mediate the motility of human pancreatic cancer cells. The administration of Ki16198, an effective antagonist of LPA1and LPA3, inhibited LPA-induced cell migration and invasion in pancreatic cancer cells both in vitro and in vivo. Whereas LPA2mediated inhibition of migration of pancreatic cancer cells in response to LPA. These data suggest that LPA had a pivotal role in invasion and metastasis of pancreatic cancer. Two focal adhesion proteins focal adhesion kinase(FAK) and paxillin played a critical role in cell migration, cytoskeleton reorganization and focal adhesions dynamics. Tyrosine phosphorylation of FAK and paxillin located at the periphery and focal adhesions, indicating that phosphorylation of multiple tyrosines in FAK and paxillin was necessary for the temporospatial regulation of focal adhesion formation, actin cytoskeletal organization and cell motility. LPA induced tyrosine phosphorylation and translocation of FAK and paxillin to the focal adhesions at the periphery, a drastic increase of actin bundles, and focal adhesion assembly in colon cancer cells and ovarian cancer cells. These evidence has emerged linking LPA with FAK and paxillin activation and cell motility. However, the molecular mechanism of LPA-induced cell migration in pancreatic cancer remains poorly understood. In the present study, we investigated the possible involvement of LPA1/3specific inhibitor Kil6425in the LPA-induced responses in PANC-1.Part I Effects of LPA on activation of FAK and paxillin and cell motility in pancreatic cancer cellsObjective: This research was to explore the effect of LPA on tyrosine phosphorylation and cellular localization of FAK and paxillin, and the influence of LPA on cellular migration ability in pancreatic cancer cells. The aim was to investigate the role of FAK and paxillin in LPA stimulation of pancreatic cancer cell motility, preliminary explore the possible mechanism of LPA in pancreatic cancer invasion and metastasis, in order to further provide theoretical basis for the treatment of pancreatic cancer.Methods:The human pancreatic carcinoma cell line PANC-1was used in this research. LPA-stimulated cellular protein expression of total FAK, pY397FAK, total paxillin, pY31paxillin and pY118paxillin was analyzed by western blotting. The subcellular localization of FAK and paxillin was visualized by immunofluorescence. LPA-stimulated cell migration was measured by using a transwell migration chamber. All data were expressed as mean±tandard deviation(SD). Differences between groups were calculated by one-way ANOVA analysis. A P<0.05was considered statistically significant. All experiments were repeated at least three times. Calculations were performed by using the SPSS computer software version13.0(SPSS Inc., Chicago, IL, USA).Results:The results of western blotting showed that LPA treatment significantly enhanced Tyr397phosphorylation of FAK and Tyr31and Tyr118phosphorylation of paxillin in a dose-and time-dependent manner. A maximum response was observed at10?M LPA, which was used in all subsequent experiments. In addition, the optimal time interval was45min for tyrosine phosphorylation of both FAK and paxillin. Immunofluorescent staining demonstrated that in the absence of LPA, FAK and paxillin was predominantly distributed diffusely in the cytoplasm. These quiescent, serum-starved cells had very few focal adhesions and the amount of FAK and paxillin staining at the focal adhesions was greatly small. After LPA stimulation, FAK and paxillin were recruited and translocated to the focal adhesions. The intensity and number of focal adhesions containing FAK and paxillin were higher compared with the untreated cells. The results of transwell migration experiments showed that, the amount of PANC-1cells migrating to the lower chamber was very small, while when LPA was loaded into the lower chamber of the transwell apparatus, PANC-1cells greatly migrated toward the lower chamber.Conclusions:Our results demonstrated that the treatment of PANC-1cells with LPA caused tyrosine phosphorylation and translocation of FAK and paxillin, leading to increased cell migration. This suggests that LPA can activation of FAK and paxillin, and affect the cell migration. It indicated the importance of FAK and paxillin in regulation of cell motility of pancreatic cancer cells in response to LPA. This study may provide the basis for the mechanisms of invasion and metastasis and new therapies to control the metastasis of pancreatic cancer.Part2Effect of LPA1/3receptors on LPA stimulated activation of FAK and paxillin and cell motility in pancreatic cancer cellsObjective:This research was to explore the effect of LPA1/3on LPA-stimulated tyrosine phosphorylation and cellular localization of FAK and paxillin, and the influence of LPA on cellular migration ability in pancreatic cancer cells. Selective LPA1/3antagonist Ki16425was used in this study. The aim was to explore the relationship between LPA and pancreatic cancer invasion and metastasis, to further provide theoretical basis for the treatment of pancreatic cancer.Methods:The human pancreatic carcinoma cell line PANC-1was used in this research. Ki16425was used for selective LPA1/3receptor antagonist. Reverse transcriptase polymerase chain reaction(RT-PCR) analysis was used to examine the mRNA expression of LPA receptors in PANC-1. Cellular protein expression of FAK and paxillin was analyzed by western blotting. The subcellular localization of FAK and paxillin was visualized by immunofluorescence. Cell migration was measured by using a transwell migration chamber. All data were expressed as mean±standard deviation(SD). Differences between groups were calculated by one-way ANOVA analysis. A P<0.05was considered statistically significant. All experiments were repeated at least three times. Calculations were performed by using the SPSS computer software version13.0(SPSS Inc., Chicago, IL, USA).Results:The result of RT-PCR showed that, three LPA receptors(LPAl, LPA2, and LPA3) were significantly expressed in PANC-1cells. The results of western blotting showed that, pretreatment with Ki16425(10?M) for30min prior to LPA treatment(10?M,45min) attenuated LPA-induced tyrosine phosphorylation of FAK and paxillin. Furthermore, the basal level of FAK tyrosine phosphorylation was also attenuated by Kil6425. Immunofluorescent staining demonstrated that in the absence of LPA, FAK and paxillin were predominantly distributed diffusely in the cytoplasm. These quiescent, serum-starved cells had very few focal adhesions and the amount of FAK and paxillin staining at the focal adhesions was greatly small. After LPA stimulation, FAK and paxillin were recruited and translocated to the focal adhesions. The intensity and number of focal adhesions containing FAK and paxillin were higher compared with the untreated cells. Ki16425significantly decreased LPA-induced formation of focal adhesions, inhibited the membrane localization of FAK and paxillin and reduced the number and the size of the membrane protrusions in PANC-1. Finally, we examined whether Ki16425can affect LPA-mediated the motility of the pancreatic cancer cell line. When LPA was loaded into the lower chamber of the transwell apparatus, PANC-1cells greatly migrated toward the lower chamber, suggesting that LPA stimulated directional migration(chemotaxis) of pancreatic cancer cells. When preincubating with Ki16425(10?M) for30min, the migration response to LPA was specifically inhibited.Conclusions:Our results demonstrated that the treatment of PANC-1cells with LPA caused tyrosine phosphorylation and activation of FAK and paxillin, leading to increased cell migration. Furthermore, we reported that LPA modulated these events through LPA1/3as Ki16425decreased LPA-induced effects. It indicated the importance of FAK and paxillin in regulation of cell motility of pancreatic cancer cells in response to LPA. This study may provide the basis for new therapies to control the metastasis of pancreatic cancer.