apicidin and Uterine-Cervical-Neoplasms

It was recently reported that the reduced expression of hypoxia-inducible factor prolyl 4-hydroxylase PHD2 inhuman cancers correlates with increased angiogenesis. We used HeLa, CaSki, C33A, and SiHa cervical cancer cells to show the effect of apicidin on cellular levels of PHD2 enzyme. Using reverse transcription, real-time quantitative PCR, and western blot analysis, we established that apicidin upregulates PHD2 transcript and protein levels in HeLa, CaSki, and C33A, but not in SiHa cervical cancer cells. Bisulfite sequencing showed that the increase in PHD2 expression was accompanied by demethylation ofCpG islands located in the first exon of the PHD2 gene.As decreased PHD2 expression supports tumor progression, our findings may validate the usefulness of apicidin as an anticancer drug.

Topics: Antineoplastic Agents; Cell Line, Tumor; Female; Gene Expression; Humans; Hypoxia-Inducible Factor-Proline Dioxygenases; Peptides, Cyclic; Procollagen-Proline Dioxygenase; Up-Regulation; Uterine Cervical Neoplasms

Virtually all cervical cancer morbidities are associated with genital skin or mucosa cell infection with human papillomavirus (HPV). The HPV oncogenic proteins E6 and E7 are able to inactivate p53 and Rb proteins, which results in malignant transformation. Employing quantitative real-time PCR and Western blot analysis, we observed that apicidin histone deacetylase (HDAC) inhibitor significantly reduced HPV16-E6 and -E7 transcripts and protein levels in SiHa cervical cancer cells. Moreover, we found that apicidin lowered HPV16-E6 and -E7 transcript stability and significantly decreased these transcripts' half-life from approximately 5h to 2h and from 6h to 3h, respectively. Our results from experiments with protein biosynthesis inhibitor suggest the involvement of an RNase and/or mRNA stabilization protein in HPV16-E6 and -E7 transcript stabilization. Since the HPV type 16 is associated with most cervical cancer incidence and HDAC inhibitors are being tested in anti-cancer clinical trials, our observations may have clinical significance.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; Down-Regulation; Female; Gene Expression Regulation, Viral; Histone Deacetylase Inhibitors; Human papillomavirus 16; Humans; Oncogene Proteins, Viral; Papillomavirus E7 Proteins; Peptides, Cyclic; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Uterine Cervical Neoplasms

Dysregulation of DNA methyltransferase (DNMT)1 expression is associated with cellular transformation, and inhibition of DNMT1 exerts antitumorigenic effects. Here, we report that DNMT1 abnormally expressed in HeLa cells is downregulated by a histone deacetylase (HDAC) inhibitor apicidin, which is correlated with induction of repressive histone modifications on the promoter site. Apicidin selectively represses the expression of DNMT1 among DNMTs in HeLa cells, independent of cell cycle arrest at G0/G1. Furthermore, apicidin causes a significant reduction in the recruitment of RNA polymerase II into the promoter. Chromatin immunoprecipitation analysis shows that even though apicidin causes global hyperacetylation of histone H3 and H4, localized deacetylation of histone H3 and H4 occurs at the E2F binding site, which is accompanied by the recruitment of pRB and the replacement of P/CAF with HDAC1 into the sites. In addition, K4-trimethylated H3 on nucleosomes associated with the transcriptional start site is depleted following apicidin treatment, whereas repressive markers, K9- and K27-trimethylation of H3 are enriched on the site. The downregulation of DNMT1 expression seems to require de novo protein synthesis, because the apicidin effect is antagonized by cycloheximide treatment. Moreover, knock down of DNMT1 with siRNA induces the apoptosis of HeLa cells, indicating that downregulation of DNMT1 might be a good strategy for therapeutics of human cervix cancer. Collectively, our findings will provide a mechanistic rationale for the use of HDAC inhibitors in cancer therapeutics.

Topics: Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Down-Regulation; E2F Transcription Factors; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Histone Deacetylase Inhibitors; Histones; Humans; Peptides, Cyclic; Promoter Regions, Genetic; Protein Transport; Repressor Proteins; Retinoblastoma Protein; Uterine Cervical Neoplasms

Down-regulation of gelsolin expression is associated with cellular transformation and induction of gelsolin exerts antitumorigenic effects. In this study, we show that protein kinase C (PKC) signaling pathway is required for the induction of gelsolin by the histone deacetylase inhibitor apicidin in HeLa cells. Apicidin induces gelsolin mRNA independently of the de novo protein synthesis. Inhibitor study has revealed that the PKC signaling pathway is involved in the gelsolin expression. Furthermore, inhibition of PKCepsilon by either siRNA or dominant-negative mutant completely abrogates the expression of gelsolin by apicidin, indicating that PKCepsilon is the major isoform for this process. In parallel, apicidin induction of gelsolin is antagonized by the inhibition of Sp1 using dominant-negative Sp1 or specific Sp1 inhibitor mithramycin, and inhibition of PKC leads to suppression of Sp1 promoter activity. Our results provide mechanistic insights into molecular mechanisms of gelsolin induction by apicidin.

Topics: Binding Sites; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Gelsolin; Gene Expression Regulation, Neoplastic; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Mutation; Peptides, Cyclic; Protein Kinase C-epsilon; RNA, Messenger; RNA, Small Interfering; Uterine Cervical Neoplasms