The Effect Of Matrine, PD98059, U0126 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. New anticancer drug is hypercompetitive filed for HCC research so far to advanced breakthrough. 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. In this study, we treat HCC cell line of SMMC-7721 cell with Chinese herbal martrine, MEK signaling transduction way inhibitor PD98059 and U016 to study their effect on signaling way changes so as to HCC cell outcome.Methods: 1 SMMC-7721 cells proliferation was measured with MTT methods.2 The mRNA expressional levels of erk, stat3, stat5 and c-jun in 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 in 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 in SMMC-7721 cells was measured by laser scanning confocal microscopy.Results: 1 The effect of matrine, PD98059 and U0126 on SMMC-7721 cells proliferationMatrine, PD98059 and U0126 significantly inhibited SMMC-7721 cells proliferation in time and dose-dependent manner. Treated with matrine plus PD98059 or U0126, the inhibition ratios were higher. Compared with matrine, there was no obvious difference (P>0.05). But compared with PD98059 or U0126, the difference was significant (P<0.05).2 The mRNA expressional levels of erk, stat3, stat5 and c-jun in SMMC-7721 cellsAfter treated with matrine, 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 control group, there were no significant difference of the mRNA expressional levels of erk, stat3 and stat5 in SMMC-7721 cells after treated with PD98059 or U0126 for 48h. But the expression of c-jun was significantly lower than that in control cells (P<0.05).After treated with PD98059 or U0126 plus matrine for 48h, the mRNA expressional levels of erk, stat3, stat5, c-jun in SMMC-7721 cells were significantly lower than those in control group (P<0.05). Compared with PD98059 or U0126, the mRNA expressional levels of erk, stat3, stat5, c-jun in SMMC-7721 cell were significantly decreased after treated with PD98059 or U0126 plus matrine (P<0.05). Compared with matrine, there were no significant difference of the mRNA expressional levels of erk, stat3 and stat5 in SMMC-7721 cells after treated with PD98059 or U0126 plus matrine (P>0.05).3 The protein expressional levels of ERK, STAT3, STAT5, P-ERK, P-STAT3, P-STAT5 and C-JUN in SMMC-7721 cells measured by western blottingAfter treated with matrine, 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).After treated with PD98059 or U0126 for 48h, the protein expressional levels of P-ERK, P-STAT3, P-STAT5, C-JUN in SMMC-7721 cells were significantly lower than those in control group (P<0.05). But the protein expressional levels of ERK, STAT3 and STAT5 show no difference.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 matrine (P<0.05). Compared with PD98059 or U0126, the protein expressional levels of ERK, STAT3, STAT5 in SMMC-7721 cells were significantly decreased after treated with PD98059 or U0126 plus matrine (P<0.05). Compared with matrine, there were no significant difference of the protein expressional levels of ERK, STAT3, STAT5, P-ERK, P-STAT3, P-STAT5, C-JUN in SMMC-7721 cells after treated with PD98059 or U0126 plus matrine (P>0.05).4 The protein expressional levels of ERK, STAT3, STAT5, P-ERK, P-STAT3, P-STAT5 and C-JUN in SMMC-7721 cells measured by laser scanning confocal microscopyAfter treated with matrine, 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, the fluorescence intensity of P-ERK, P-STAT3, P-STAT5, C-JUN was significantly weaker than that in control group, but the fluorescence intensity of ERK, STAT3, STAT5 was no significant change. After treated with PD98059 or U0126 plus matrine, the fluorescence intensity of all treated groups was weaker than that in control group.Conclusions: 1 PD98059 and U0126 could significantly inhibit the proliferation of SMMC-7721 cells, because they could inhibit the activiation of ERK, STAT3 and STAT5 protein and decrease the expression of C-JUN.2 Matrine could significantly down-regulate the mRNA expressional levels of erk, stat3, stat5 and c-jun in SMMC-7721 cells and inhibit MAPK and JAK-STAT signaling transduction pathway through blocking of the activiation of ERK, STAT3 and STAT5 protein and their downsteam signal C-JUN, thus it could inhibit the proliferation of SMMC-7721 cells.3 Matrine could enhance the inhibition ratios of SMMC-7721 cells treated with PD98059 or U0126 and increase their effect.