Inhibition Of Nitidine Chloride On The Malignant Proliferation Of Hepato-Cellular Carcinoma Cell And Its Regulation On P125Expression

Objective:To investigate the inhibition of nitidine chloride on the malignant proliferation of hepatocellular carcinoma cell and its effect on the expression of p125which is the DNA polymerase δ catalytic subunit, to further explore the inhibition mechanism of nitidine chloride on hepatocellular carcinoma cell’s proliferation via p125signaling pathway.Methods:1. MTT assay and cell clony formation assay were used to detect the inhibition of nitidine chloride on the malignant proliferation of hepatocellular carcinoma cell such as SMMC-7721or BEL-7402.2. The expression of p125was detected by western blot and its encoding gene POLD1was tested by RT-PCR in hepatocellular carcinoma cell and normal liver cell.3. Then hepatocellular carcinoma cell was treated with different concentrations of nitidine chloride, and evaluated the expression of p125by western blot.4. The expression of p53and p21was detected by Real-time PCR, followed by ELISA to quantify the expression of P53and P21.5. SMMC-7721cells were transfected with plasmid pGPU6/GFP/neo-p53-shRNA, pGPU6/GFP/neo-p21-shRNA and negative control vector pGPU6/GFP/neo-NCshRNA respectively. Stable transfected cell lines7721-p53shRNA,7721-p21shRNA and7721-NCshRNA were screened by G418. And the changes of p53and p21expression were tested by RT-PCR after transfecting. Western blot was used to detect the changes of p125expression in7721-p53shRNA and7721-p21shRNA cell lines. Nitidine chloride was used to treat the stable transfected cell lines, and the changes of the effect on p125expression were detected by western blot.6. Interaction of P53and p125was detected by immunoprecipitation assay (IP).7. Real-time PCR was applied to detect the changes of the effect on p21expression after transfecting p53plasmid in SMMC-7721cells.8. The expression of CDK2, CyclinE, E2F, c-myc and Rb-1mRNA was detected by Real-time PCR after treating on nitidine chloride.Results:1. Nitidine chloride could inhibit the proliferation of SMMC-7721and BEL-7402cells. And it showed a concentration-dependent manner. The IC50values of48h were (1.08±0.13)mg/L and (1.35±0.21)mg/L respectively. Nitidine chloride inhibited the formation of cell colonies. The colony formulation rate of SMMC-7721was (83.58±6.452)%,(25.35±2.765)%, (11.52±0.921)%and0%after0,0.25,0.50and1.0mg/L nitidine chloride treating respectively. The colony formulation rate of BEL-7402was (87.13±7.312)%,(30.38±1.783)%,(13.22±1.421)%and (4.35±0.937)%after0,0.25,0.50and1.0mg/L nitidine chloride treating respectively.2. The expression of p125in hepatocellular carcinoma cell was much higher than that in normal liver cell (0.98±0.14vs0.33±0.21, P<0.01). And the expression of POLD1was higher, too (P<0.05).3. Compared with negative control group, nitidine chloride significantly decreased p125expression (P<0.05).4. Nitidine chloride increased expression of p53(P53) and p21(P21)(P<0.05).5. When cells were transfected with shRNA targeted on p53and p21, their expression of mRNA levels was down-regulated, but the level of p125expression was up-regulated at the same time. When hepatocellular carcinoma cell was transfected with shRNA of p53and p21, the effect of nitidine chloride down-regulating p125was weakened.6. IP assay showed that there was not detectable p125in P53antibody precipitated protein complex.7. P21was significantly lower in7721-p53shRNA group (P<0.05), and much higher in7721-p53group (P<0.05). But the effect of nitidine chloride on regulating P21was increased more in7721-p53shRNA group than in7721-NCshRNA group (P<0.01).8. The expression of CyclinE was down-regulated (P<0.01), but the expresion of E2F and Rb-1was up-regulated after nitidine chloride treating (P<0.01). Nitidine chloride had no effect on the expression of CDK2and c-myc (P>0.05). Conclusion:Nitidine chloride can significantly inhibit malignant proliferation of hepatocellular carcinoma cell, and reduce the level of abnormal expression of p125in hepatocellular carcinoma cell. P53and p21can inhibit the expression of p125. The regulation of p125on nitidine chloride correlates with up-regulation of p53and p21expression in SMMC-7721cells. Nitidine chloride can increase the expresion of p21by p53-dependent or independent pathway. Nitidine chloride may inhibit CyclinE through increasing p21, thereby inhibit the p125expression, and ultimately suppress hepatocellular carcinoma cell’s proliferation.