Activation condition of NF-κB pathway in THP-1 cells and the efficacy of pathernolide on cell's proliferation and apoptosis

Aim:To explore the activation condition of NF-κB pathway in acute monocytic leukemia cell lines THP-1,and the efficacy of pathenolide(PN),the inhibitor of NF-κB,on proliferation and apoptosis of THP-1 cells.Methods:THP-1 cell was cultured in medium DMEM at 37 ℃ in the presence of 5% CO2.①Cells in exponential growth stage were harvested.Total RNA was extracted from cells with Trizol reagent,and semi-quantitative RT-PCR was used to examine the P65,IκBα mRNA.②The total cellular protein and the nuclear protein were extracted using Nuclear and Cytoplasmic Extraction Reagents Kit.Western blotting was used to detect their protein expression in cytoplasm extracts or nuclear extracts,respectively.③Cells in 96-well plates were allocated into 10 groups(the blank control,the negative control and the tested wells(1,2,4,6,8,10,15,20 μmol/L PN),cell proliferation was determined using Cell Counting Kit-8;percentages of proliferation inhibition and 50%inhibiting concentration(IC50) were calculated at 12 h,24 h check-times.④Cells were allocated into 3 groups(the blank control,the negative control,the group treated with 6 μmol/L PN),and cultured for 6 days,then cell growth curves were made.⑤Cells were allocated into 5 groups and treated with 0,2,4,6,and 8 μmol/L PN respectively,Flow cytometry was used to determine the distribution of cell cycle.Annexin V-FITC staining was employed to analyze cell apoptosis.Results:①RT-PCR showed that P65 and IκBα mRNA expressed in THP-1 cells.Western blotting showed that both P65 and IκBα protein expressed in cytoplasm,and some p65 protein expressed in nuclear.②Growth inhibition was showed in THP-1 cells treated with PN in a concentration-dependant manner and the highest inhibiting rate was(69.56±2.52)%;the IC50 of PN on THP-1 for 12 h,24 h were 8~10 μmol/L,6~8 μmol/L,respectively.③The growth curve of cells treated with 6 μmol/L of PN was lower than that of controls.④The constituent ratio of cells in G0/G1 stage increased with the increasing of PN concentrations from 2 μmol/L to 6 μmol/L,which was up to(63.07±3.91)% in 6 μmol/L group.On the other hand,the constituent ratio of cells in S stage decreased which the increasing of PN concentrations,which was(23.55±4.34)% in 8 μmol/L group.⑤The early apoptosis rates of the blank control,2 μmol/L PN-group,4 μmol/L PN-group,6 μmol/L PN-group and 8 μmol/L PN-group were(3.84±1.50)%,(4.67±1.97)%,(12.67±2.13)%,(17.72±2.78)% and(23.62±3.36)% respectively,and the difference was significante(F=36.280,P0.001).Conclusion:Constitutively activated NF-κB signaling pathway exists in the THP-1 cells.PN,a NF-κB inhibitor,effectively inhibits cell proliferation and induces apoptosis.