Investigation On The Role Of IFIT2 On Curcumin-induced Apoptosis In Leukemia

Objective: To explore the role IFIT2 play in the anticancer effect of curcumin and provide a theoretical basis for curcumin or combined with interferon in the cancer therapeutics.Method: MTT was used to analyze the growth viability;the percent of apoptotic cells and the cell cycle progeression was dectected by Annexin-V/PI staining assay and PI single staining assay,respectively.Change at molecular biological level was determined by RT-PCR and western-blot.In order to explore the molecular mechanisms underlying antitumor effects of curcumin,we analyzed change of gene expression in U937 after treated with curcumin(10 m M)for 24 h by gene expression chip.Results: 1.After treated with different concentration of curcumin,the concentration of curcumin inhibiting 50% cell proliferation(IC50)in leukemia cell lines(NB4,NB4-R1,K562,HL60 and U937)were: 8.63±2.34?mol/L for U937;27.67± 2.72?mol/L for K562;15.77±2.17 ?mol/L for NB4;16.67±1.63?mol/L for NB4-R1;27.86±2.45?mol/L for HL60 respectively.2.With Annexin-V/PI staining assay detection on concentrations respectively of 0,5,10?mol/L of curcumin on U937 cells after 24 hours,the percentage of apoptotic cells were 4.2±1.44%,21.9±3.64%,33.4±4.74%;With Annexin-V/PI staining assay detection on concentrations respectively of 0,5,10?mol/L of curcumin on K562 cells after 24 hours,the percentage of apoptotic cells were 4.2±2.2%,6.9±1.9%,8.8±2.1%.3.After treated with different concentration of curcumin for 24 h,the apoptosic proteins(cleaved caspase3 and PARP)in U937 were markedly increased and the apoptosis inhibitor protein Bcl-2 was decreased.However,the proteins in the K562 changed slightly.4.After treated with different concentration of curcumin for 24 h,in U937,an significantly increased number of cells were detected in G2/M phase and corresponding reduction in G0/G1 phase were observed.However,in K562 cells,the proportion of G2/M phase slightly increased and accompanied by a decrease in the S phase.5.We found that some of the interferon regulatory genes,especially IFIT2 gene expression was significantly upregulated in U937 after treated with curcumin by gene chip.6.After exposed to 10?mol/L curcumin,the percentage of apoptotic cells was found to be obviously increased in the IFIT2 overexpressed cells.7.The percentage of apoptotic cells was reduced significantly in IFIT2-sh RNA infected cells after treating with 10?mol/L curcumin.8.The apoptosis determined by Annexin-V/PI staining assay showed that the percentage of apoptotic cells in group treated with curcumin changed slightly,while the percentage of apoptotic cells of group treated with IFN? was between group treated with curcumin and group treated with IFN? combined with curcumin,and percentage of apoptotic cells in group treated with IFN? combined with curcumin increased significantly than other group.Conclusion: 1.Sensitivity of leukemia cell lines to curcumin is various,U937 is the most sensetive cell line to curcumin,the sensetive of NB4,NB4-R1 is moderate and K562,HL-60 is even lower.2.Curcumin induces apoptosis in leukemia cell lines.3.Curcumin induces G2/M phase arrest in leukemia cells.4.Our results suggested for the first time that IFIT2 enhance the effect of curcumin.5.Our results supported that there is a synergistic effect between curcumin and IFN?.