Effects On Proliferation And NF-κB By Pf In Melanoma Cell Line A375

ObjectiveTo investigate the effects of different concentrations of PF ( paeoniflorin ) on the cell proliferation inhibition rate and cell apoptosis rate, and the effects on the mRNA transcription level of p16INK4a,p14INK/ARF and p65, one subunit of nuclear factor-κB (NF-κB ) in human melanoma cells A375, with the purpose of providing foundation for clinical application and the further study.MethodsHuman melanoma cells A375 were cultured, divided into the blank control group without PF and five corresponding experimental group, which pretreated with PF contents 0.01 mg / ml, 0.1 mg / ml, 0.5 mg / ml, 1.0 mg / ml and 2.0 mg / ml. The Purity of PF≥92%. (1) Observe the changes in density and shape of A375 cells by inverted microscope; (2) Detect the effect of cell proliferation inhibition rate by MTT on human melanoma cells A375, which cultured for 24 hours, 48 hours and 72 hours with the drugs; (3) Detect the effect of apoptosis rate by Annexin V / PI staining kit and flow cytometry on A375, which cultured for 24 hours with the drugs; (4) The mRNA transcription level of p16INK4a,p14INK/ARF and p65, one subunit of NF-κB in human melanoma cells A375, were studied by SYBR Green-PCR.Results1,Inverted microscope results showed that PF could inhibit the growth of A375 cells, whose density was decreasing and whose shapes becoming shorter and thicker rather than the normal kind of slender bipolar dendritic-like cell bodies with the concentrations increasing. 2,MTT results showed that PF can inhibit A375 cells proliferation, and the effects of inhibition was concentration dependent, but no time dependence. 3,Annexin V / PI results showed that there was no significant difference between the control groups and experimental groups in the apoptosis rate of A375 (P> 0.05). 4,SYBR Green-PCR results showed:(1)PF could promote the mRNA transcription level of p16INK4a in A375, compared with the control groups, there were significant differences (P <0.05), and the promotion was concentration dependent, but no time dependence; (2)PF could inhibit the mRNA transcription level of p65, one subunit of NF-κB in A375, compared with the control groups, there were significant differences (P <0.05), and the inhibitation was concentration dependent, but no time dependence; (3)There was no significant difference in the mRNA level of p14INK/ARF between the control groups and experimental groups in A375 (P> 0.05).Conclusions1,PF could inhibit the proliferation of human melanoma cells A375, and the effects of inhibition was concentration dependent, but no time dependence. PF could not have significantly effects on its apoptosis,it may be associated with tolerance in melanoma. 2,There was a possibility that PF promoted the expression of p16INK4a, then inhibited the expression of p65, one subunit of NF-κB, and the effects of enhancement or inhibition was concentration dependent, but no time dependence. There was no significant difference in the transcription of p14INK/ARF by PF. It shows that PF may inhibit the proliferation of A375 by promoting p16INK4a, inhibiting the expression of NF-κB subunit p65.