No abstract

The HIV transactivator protein, Tat, enhances HIV transcription by recruiting P-TEFb from the inactive 7SK snRNP complex and directing it to proviral elongation complexes. To test the hypothesis that T-cell receptor (TCR) signaling induces critical post-translational modifications leading to enhanced interactions between P-TEFb and Tat, we employed affinity purification–tandem mass spectrometry to analyze P-TEFb. TCR or phorbal ester (PMA) signaling strongly induced phosphorylation of the CDK9 kinase at Ser175. Molecular modeling studies based on the Tat/P-TEFb X-ray structure suggested that pSer175 strengthens the intermolecular interactions between CDK9 and Tat. Mutations in Ser175 confirm that this residue could mediate critical interactions with Tat and with the bromodomain protein BRD4. The S175A mutation reduced CDK9 interactions with Tat by an average of 1.7-fold, but also completely blocked CDK9 association with BRD4. The phosphomimetic S175D mutation modestly enhanced Tat association with CDK9 while causing a 2-fold disruption in BRD4 association with CDK9. Since BRD4 is unable to compete for binding to CDK9 carrying S175A, expression of CDK9 carrying the S175A mutation in latently infected cells resulted in a robust Tat-dependent reactivation of the provirus. Similarly, the stable knockdown of BRD4 led to a strong enhancement of proviral expression. Immunoprecipitation experiments show that CDK9 phosphorylated at Ser175 is excluded from the 7SK RNP complex. Immunofluorescence and flow cytometry studies carried out using a phospho-Ser175-specific antibody demonstrated that Ser175 phosphorylation occurs during TCR activation of primary resting memory CD4+ T cells together with upregulation of the Cyclin T1 regulatory subunit of P-TEFb, and Thr186 phosphorylation of CDK9. We conclude that the phosphorylation of CDK9 at Ser175 plays a critical role in altering the competitive binding of Tat and BRD4 to P-TEFb and provides an informative molecular marker for the identification of the transcriptionally active form of P-TEFb.

IKKα has been implicated as a key regulator of oncogenesis and driver of the metastatic process; therefore is regarded as a promising therapeutic target in anticancer drug development. In spite of the progress made in the development of IKK inhibitors, no potent IKKα inhibitor(s) have been identified. Our multistep approach of molecular modeling and direct binding has led to the identification of plant flavone apigenin as a specific IKKα inhibitor. Here we report apigenin, in micro molar range, inhibits IKKα kinase activity, demonstrates anti-proliferative and anti-invasive activities in functional cell based assays and exhibits anticancer efficacy in experimental tumor model. We found that apigenin directly binds with IKKα, attenuates IKKα kinase activity and suppresses NF-ĸB/p65 activation in human prostate cancer PC-3 and 22Rv1 cells much more effectively than IKK inhibitor, PS1145. We also showed that apigenin caused cell cycle arrest similar to knockdown of IKKα in prostate cancer cells. Studies in xenograft mouse model indicate that apigenin feeding suppresses tumor growth, lowers proliferation and enhances apoptosis. These effects correlated with inhibition of p-IKKα, NF-ĸB/p65, proliferating cell nuclear antigen and increase in cleaved caspase 3 expression in a dose-dependent manner. Overall, our results suggest that inhibition of cell proliferation, invasiveness and decrease in tumor growth by apigenin are mediated by its ability to suppress IKKα and downstream targets affecting NF-ĸB signaling pathways.

Methylation of DNA and histone proteins are mutually involved in the epigenetic regulation of gene expression mediated by DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs). DNMTs methylate cytosine residues within gene promoters, whereas HMTs catalyze the transfer of methyl groups to lysine and arginine residues of histone proteins, thus causing chromatin condensation and transcriptional repression, which play an important role in the pathogenesis of cancer. The potential reversibility of epigenetic alterations has encouraged the development of dual pharmacologic inhibitors of DNA and histone methylation as anticancer therapeutics. Dietary flavones can affect epigenetic modifications that accumulate over time and have shown anticancer properties, which are undefined. Through DNA binding and in silico protein-ligand docking studies with plant flavones viz. Apigenin, Chrysin and Luteolin, the effect of flavones on DNA and histone methylation was assessed. Spectroscopic analysis of flavones with calf-thymus DNA revealed intercalation as the dominant binding mode, with specific binding to a GC-rich sequence in the DNA duplex. A virtual screening approach using a model of the catalytic site of DNMT and EZH2 demonstrated that plant flavones are tethered at both ends inside the catalytic pocket of DNMT and EZH2 by means of hydrogen bonding. Epigenetic studies performed with flavones exhibited a decrease in DNMT enzyme activity and a reversal of the hypermethylation of cytosine bases in the DNA and prevented cytosine methylation in the GC-rich promoter sequence incubated with the M.SssI enzyme. Furthermore, a marked decrease in HMT activity and a decrease in EZH2 protein expression and trimethylation of H3K27 were noted in histones isolated from cancer cells treated with plant flavones. Our results suggest that dietary flavones can alter DNMT and HMT activities and the methylation of DNA and histone proteins that regulate epigenetic modifications, thus providing a significant anticancer effect by altering epigenetic processes involved in the development of cancer.