5 folds increase in H3ack12 and 2. 5 fold decrease in H3k9 methylation in ?2894 to ?1753 region was observed. The STAT 1 protein levels were increased in both the regions. Therefore histone tail modifications at the STAT response element are required for p21WAF1 induction and might serve as a switch for p21WAF1 induction by controlling his tone modifications. CC-5013 To further confirm the greater accumulation of STAT 1 protein occurred by chrysin expos ure, p21 protein was immunoprecipitated and is followed by western blot analyses using STAT 1, 3 and 5 antibodies. Both STAT 1, 3 proteins were increased at an equal level after TSA and chrysin treatment where as STAT 5a was found to be decreased. Probably the ratio of STAT1 and 3 might regulate the cell death event.

We have also conducted RT PCR experiment to study the change in the STAT 1 mRNA level in chrysin trea ted cells. We found an increase in STAT 1 mRNA level in chrysin treated cells. The acetylated histone pattern was increased 2. 5 to 3 folds by the incubation in chrysin and TSA containing media, while histone H3K9 methylation showed a pro found reduction in 40 uM chrysin and 4 uM TSA treated cells. Conversely no changes in the histone acetylation and methylation levels were detected in the p27 promoter by the chrysin incubation. STAT response element is important for chrysin mediated p21WAF1 promoter activity ChIP analyses reveal that STAT binding site of the p21WAF1 promoter is critical for p21 induction by chrysin. Thus A375 cells were transfected with 1 ug of p21 promoter and 500 ng of CMV B galac tosidase followed by incubation in 0.

1 % DMSO, 40 uM chrysin and 4 uM TSA for 24 h and assayed the lucifer ase activity. B galactosidase values obtained were used for normalization of luciferase activity. Here DMSO trea ted cells was used as control. TSA incubated cells have shown 3 folds of p21WAF1 promoter activity whereas chrysin treatment caused a marked increase in promoter activity. But the activity was reached to basal level in cells transfected with STAT mutated p21 construct followed by chrysin and TSA treatment. The mutation of the STAT site fail to activate the chry sin induced p21WAF1 activity, which suggests STAT re sponse element at ?692 to ?684 is critical for chrysin mediated transactivation of p21WAF1 promoter.

It indi cates that chrysin is capable of activating p21 transcrip tion through the promoter element in the region ?742 to ?488 bp containing STAT1 3 5 binding site. Effect of Carfilzomib chrysin on apoptosis STAT 1 and p21 are essential proteins that are involved in modulation and regulation of apoptotic process. Recent studies have also focussed on HDAC inhibitors and their repressive role on NF kB dependent genes i. e Bcl xL, Survivin to control cell prolifera tion. Thus we have treated A375 cells with chrysin and TSA for 72 h and lysates were sub jected to western blot analyses.