The Effect Of Sodium Cantharidinate And Vitamin B6 On Signaling Transduction Of MAPK And JAK-STAT In SMMC-7721 Cells

Objective: Hepatocellular carcinoma (HCC) is a common tumor of digestive tract with poor prognosis, environment and genetic factors contribute to carcinogenesis of this cancer, but the true mechanism remains unknown. Cell signaling transduction has a role on carcinogenesis, any subtle changes on transduction way may induce cell to different outcome as apoptosis, immortal and senescence. Ras/Raf/ERK and JAK-STAT signaling of tyrosine protein kinase pathway contributed to tumor process influences HCC development dramatically. As the traditional Chinese medieine, Cantharidin has antitmuor function and has been used to treat cancer, for example cervical cancer, nasopharyngeal cancer, skin cancer, myeloma, leukemia, ovarian cancer and liver cancer. In this study, compared with MEK signaling transduction way inhibitor PD98059 and U0126, we treated HCC cell line of SMMC-7721 cell with Sodium Cantharidinate and Vitamin B6(SCAVB6) of the effetive composition of the Cantharidin to study their effect on MAPK,JAK-STAT signaling pathway to HCC cell.Methods: 1 The effect of SCAVB6 on SMMC-7721 cells proliferation was measured with MTT methods. 2 The mRNA expressional levels of erk, stat3, stat5 and c-jun after treated with SCAVB6, SCAVB6 plus PD98059 or U0126 on SMMC-7721 cells were measured by RT-PCR.3 The protein expressional levels of ERK, STAT3, STAT5, P-ERK, P-STAT3, P-STAT5 and C-JUN after treated with SCA VB6, SCAVB6 plus PD98059 or U0126 on SMMC-7721 cells were measured by Western blotting.4 The protein expression of ERK, STAT3, STAT5, P-ERK, P-STAT3, P-STAT5 and C-JUN after treated with SCAVB6, SCAVB6 plus PD98059 or U0126 on SMMC-7721 cells was measured by laser scanning confocal microscopy.Results: 1 The result of MTT : The proliferation of SMMC-7721 cells was significantly inhibited in time and dose-dependent manner after treated with SCAVB6. Treated with SCAVB6 plus PD98059 or U0126, the inhibition ratios were higher. Compared with SCAVB6, there was obvious difference (P<0.05).2 The result of RT-PCR : After treated with SCAVB6, the expressional levels of erk, stat3, stat5, c-jun mRNA in SMMC-7721 cells were significantly lower than those in control group (P<0.05).After treated with SCAVB6 plus PD98059 or U0126, the expressional levels of erk, stat3, stat5, c-jun mRNA in SMMC-7721 cells were significantly lower than those in control group(P<0.05).Compared with SCAVB6, there were obvious difference of the mRNA expressional levels of erk, stat3 and stat5 in SMMC-7721 cells after treated with PD98059 or U0126 plus SCAVB6 (P<0.05).3 The result of Western blotting : After treated with SCAVB6, the protein expressional levels of ERK, STAT3, STAT5, P-ERK, P-STAT3, P-STAT5, C-JUN in SMMC-7721 cells were significantly lower than those in control group (P<0.05).Compared with control group, the protein expressional levels of ERK, STAT3, STAT5, P-ERK, P-STAT3, P-STAT5, C-JUN in SMMC-7721 cells were significantly decreased after treated with PD98059 or U0126 plus SCAVB6 (P<0.05).Compared with SCAVB6, there were obvious difference of the protein expressional levels of ERK, P-ERK, P-STAT3, P-STAT5, C-JUN in SMMC-7721 cells after treated with U0126 plus SCAVB6 (P<0.05). Compared with SCAVB6, there were obvious difference of the protein expressional levels of STAT3, P-STAT3, C-JUN in SMMC-7721 cells after treated with PD98059 plus SCAVB6 (P<0.05).4 The result of laser scanning confocal microscopy: After treated with SCAVB6, the fluorescence intensity of ERK, STAT3, STAT5, P-ERK, P-STAT3, P-STAT5, C-JUN was significantly weaker than that in control group.After treated with PD98059 or U0126 plus SCAVB6, the fluorescence intensity of all treated groups was weaker than that in control group. Conclusions: 1 The mRNA expressional levels of erk, stat3, stat5 and c-jun could be significantly down-regulated in SMMC-7721 cells and the MAPK, JAK-STAT signaling transduction pathway could be inhibited through blocking of the activiation of ERK, STAT3 and STAT5 protein and their downsteam signal C-JUN by Sodium cantharidinate and Vitamin B6(SCAVB6). The proliferation of SMMC-7721 cells could be inhibited by SCAVB6.2 The inhibition ratios of SMMC-7721 cells treated with PD98059 or U0126 could be enhanced by SCAVB6.And their effect could be increased .