D in cells treated with Chrysin (Chry) and TSA at 40 M and 4 M concentrations for 24 h respectively. Chrysin was shown to decrease the HDAC-2, 3, 8 protein level.analogues such as oroxylin A and methoxy-chrysin suggesting that chrysin as a potent HDAC-8 inhibitor. To further confirm the inhibitory action of chrysin on HDAC-8 we carried out HDAC1/2 assay. We did not observe any change in the activity of HDAC1/2 upon addition of chrysin (Figure 3B). To measure the effect of chrysin and TSA on protein levels of HDACs a series of western blot analyses was performed using cell lysate extracted from treated A375 cells. The levels of HDAC-2, 3 and 8 proteins were significantly decreased by treatment of chrysin (40 M) or TSA (4 M) (Figure 3C). However their effect is less pronounced in HDAC-1 protein. In contrast , cells treated with chrysin and TSA did not show significant effect on HDAC-4 and 6 protein levels relative to control (C) untreated cells (Figure 3C 3D). Therefore chrysin functions as HDAC-2 8 inhibitor. Similarly apigenin, aflavonoid and a close analog of chrysin was found to inhibt the histone deacetyalse activity [33].Regulation of cell cycle componentsPrevious PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/25636517 studies have established that deacetylation of histones by HDAC enzymes cause inactivation of tumor suppressor genes leading to neoplastic transformation [34] Inhibition of HDAC enzymatic activity restores the expression of many tumor suppressor genes. Thus the amount of tumor suppressor proteins p53, p27 and p21WAF1 was estimated from the cell lysates of A375 neoplastic cells incubated with 0.1 DMSO, chrysin and TSA containing media. The p21WAF1 protein in A375 cells was increased 4folds in 40 M chrysin, while there was no significant change in the level of p27 protein. The expression of tumor suppressor protein p53 was drastically reduced in chrysin treated cells when compared to control untreated cellsPal-Bhadra et al. BMC Cancer 2012, 12:180 http://www.biomedcentral.com/1471-2407/12/Page 6 of(Figure 4A). Trichostatin A, a class I and class II HDAC inhibitor has shown similar effect on mRNA and protein levels of p21 and p53 [1]. Apigenin, a flavonoid caused p21 Necrostatin-1 supplier induction with the increase of p53 in 22Rv1 cells and the same compound caused p21 induction in PC-3 prostate cancer cells which lacks p53. Thus p21 induction is cell type dependent and is independent of p53 status [33]. P21 is a cdk inhibitor whose activation leads to cell cycle arrest or apoptosis [35]. Although the role of p21 in apoptosis is controversial, the HDAC inhibitor sodium butyrate has induced apoptosis in MCF-7 breast cancer cells. Thus acts as a modulator of apoptosis [36]. As cyclin-dependent kinases (Cdks) and cyclins are required for the complex formation with p21WAF1, the level of Cdk2, Cdk4 and Cyclin D1, that regulate G1-S phase transition was also estimated in chrysin (40 M) and TSA (4 M) treated cells (Figure 4A). Reduction in cyclin D1 and cdk2 protein level with no change in cdk4 was noticed. To confirm the importance or dependency of p21 induction on p53 and STAT-1 status A375 cells were treated with chrysin (40 M) and TSA (40 M) in K562 (p53 null) [37] and U3A ( STAT-1 null) cells for 24 h and conducted western and RT-PCR analysis. We observed pronounced increase (3? folds) of both mRNA and protein level of p21 in case of K562 cells and drastic reduction in case of U3A cells. This shows the functional dependency of p21 on STAT-1 protein than p53 protein. Similar results of STAT-1.