Histone deacetylase inhibitors inducing human cervical cancer cell apoptosis by decreasin~ DNA-methyltransferase 3B

Background Histone deacetylase （HDAC） inhibitors are a group of small chemical molecules that inhibit histone deacetylase. At cell level, HDAC inhibitors have multiple biological effects such as cell cycle arrest, apoptosis, cell differentiation and auotophagy. At molecular level, HDAC inhibitors cause histone and nonhistone acetylation and induce gene expression. HDAC inhibitors are widely used in cancer therapy because of its function of inducing apoptosis. However, the mechanisms of apoptosis effect are not fully understood. TSA is a classical HDAC inhibitor and widely used in epigenetic and anti-cancer research. In this study, we selected Trichostatin A （TSA） to investigate the mechanisms of HDAC inhibitors apoptotic effect on cancer cells. Methods Cervical cancer cell lines such as Hela, Caski and normal human keratinocyte line HaCaT were treated with various concentrations of TSA. Crystal violent assay and 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay were performed to determine cell number. PARP cleavage and FITC-AnexinV were performed to determine apoptosis. DNA-methyltransferase （DNMT）I, DNMT3A and DNMT3B were determined by regular PCR, qPCR and Western Blotting. Small interfering RNA （SiRNAi） was used to knock down DNMT3B. Results HDAC inhibitors only induce cervical cancer cell apoptosis. At 1 Iumol/L of TSA, 86% of Hela cell and 76% of Caski went apoptosis. For normal cells, HDAC inhibitors have no cytotoxic effect at therapeutic dosage, （90.0＋8.4）% of normal cell survive after treated with 1 IJmol/L of TSA. We compared 1 pmol/L group with untreated control with t-test. There was no significance between 1 pmol/L group and untreated control for normal cell （P 〉0.05）. HDAC inhibitors decreased DNMT3B in cancer cell but not in normal cell. Manually knock-down of DNMT3B induced Hela and Caski cell apoptosis. More than 99% of Hela and Caski cell went apoptosis after deprived of DNMT3B. Conclusions DNMT3B was essential to cervical cancer cell survival. Down-regulated DNMT3B by HDAC inhibitors may play an important role in the toxicity of HDAC inhibitors on cervical cancer cells.

Effects of phosphatidylinositol 3-kinase inhibitor on humancervical carcinoma cells in vitro

Phosphatidylinositol 3-kinase(PI3K)is a crucial cell survival pathway implicated in tumorigenesis because of its role in stimulating cell proliferation and suppressing apoptosis.This study was to investigate the regulation of proliferation and apoptosis by LY294002,an inhibitor of PI3K in cervical cancer cells and the expression of FLICE-like inhibitory protein(c-FLIP)in vitro.Human cervical cancer HeLa cells were used in this experiment and cultured.The cultured cells were treated with LY294002 at different concentrations(10,25,50 and 100µmol/L)for 6,12,24,and 48 h before harvesting for evaluation.Cell viability was measured by 3-(4,5)-dimethylthiazol(-2-y1)-3,5-di-phenyltetrazoliumbromide(MTT)assay.Apoptosis was analyzed byflow cytometry.The expression of c-FLIP was detected by Western blot.Cell viability was inhibited by LY294002 significantly(P<0.05).Flow cytometry analysis revealed that cell apoptosis was significantly increased in the presence of LY294002 as compared with the control group.Although the expression of c-FLIP was increased in a short time,the expression of c-FLIP was markedly suppressed after the treatment of LY294002 for 48 h.These results suggested that the PI3K/Akt signal pathway might be involved in the regulation of cell apoptosis in cervical cancer cells.Moreover,the regulation of c-FLIP expression through PI3K/Akt signal pathway in cervical cancer cells was observed in vitro.

Immune response to plasmid DNA encoding HPV16-L1 protein

Objective To test the immunogenicity of recombinant plasmid DNA containing human papillomavirus type 16 L1 (HPV16 L1) coding sequence of mice Methods The HPV16 L1 encoding sequence was generated by polymerase chain reaction (PCR), and inserted into TA cloning vector PCR Ⅱ, then cloned in the eukaryotic expression vector pcDNA3 1 with CMV promoter The recombinant plasmid DNA pcDNA L1 was transferred into Cos 7 cells and used to immunize BALB/c mice via muscular injection The expression of HPV16 L1 in transferred cells was identified by immunospot and immunocytochemistry, which tested specific anti HPV16 L1 antibody in the serum of immunized mice Results Using the immunospot technique, we found L1 protein expression in pcDNA L1 transferred cells The immunocytochemistry studies demonstrated that the L1 protein was located in nuclei In immunized mice, specific anti HPV16 L1 antibodies could be detected by immunospot and immunocytochemistry 28 days after the first immunization and last at least 41 days Conclusions We constructed HPV16 L1 eukaryotic expressing plasmid whose DNA could induce immuno humoral response in mice This observation will be helpful in designing HPV16 prophylactic vaccine