seocalcitol and Laryngeal-Neoplasms

The objective of this study is to evaluate the role of the cyclin-dependent kinase inhibitor p57 in EB1089-inhibited proliferation of human laryngeal squamous carcinoma cells (HEp-2). HEp-2 cells were treated with the vitamin D3 analogue EB1089 for 48 h and total RNA was extracted for reverse transcription-PCR amplification using primers for the p57 coding sequence. Proteins were detected by Western blot analysis. For interference using silencing RNA (siRNA), HEp-2 cells were transfected with siRNA specific for p57 (siRNA-p57) or a negative control sequence (siRNA-con) followed by treatment with 10 nmol/L EB1089. The effects of EB1089 on cell proliferation were evaluated by 5-bromo-2'-deoxyuridine incorporation and '3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide assay. Cell death and cell cycle dynamics were monitored using flow cytometry. EB1089 significantly inhibited HEp-2 cell proliferation and increased p57 mRNA and protein levels; this was blocked by siRNA-p57 but not by siRNA-con. The EB1089-induced suppression of HEp-2 cell proliferation recovered to near-normal levels with siRNA-p57 transfection. EB1089 inhibits the proliferation of HEp-2 cells and p57 plays an important role in this.

Topics: Animals; Antineoplastic Agents; Calcitriol; Carcinoma, Squamous Cell; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p57; Humans; Laryngeal Neoplasms; Mice; Mice, Nude; RNA, Small Interfering; Time Factors; Tumor Cells, Cultured

The biologically active 1,25(OH)2 vitamin D3 and its analogs have been shown to have antiproliferative and differentiating effects in a variety of malignant and non-malignant cells. For squamous carcinoma cell lines of the head and neck (SCCHN) we could show that this antiproliferative activity of 1,25(OH)2 vitamin D3 is due to induced expression of the cell-cycle inhibitory proteins p21 and p27, causing an arrest in the G0/G1 cell-cycle phase.. In this work we investigated the effects of three vitamin D3 analogs, EB1089, MC1288 and CB1093, on proliferation behavior and cell-cycle status in a laryngeal carcinoma cell line (JPPA) as well as in control human immortalized keratinocytes (HaCaT). To study the molecular mechanism the functional activity of the promoter region of p21, a potential target gene of vitamin D3 transcriptional regulation, was investigated. For this reason a 2.7-kb fragment of the p21 promoter was isolated by polymerase chain reaction from HaCaT, JPPA and SCC9 (tongue carcinoma) cells and directionally cloned into an enhanced green fluorescence protein (EGFP) reporter gene vector system. A construct was used to stably transfect HaCaT cells and to monitor the expression of the EGFP gene by confocal microscopy.. Analysis of proliferation and cell-cycle status revealed decreased growth rates and G0/G1I cell-cycle arrest in cells treated with 1,25(OH)2 vitamin D3 and its analogs The EGFP reporter gene-transfected cells showed distinct fluorescence under the influence of 1,25(OH)2 vitamin D3 and its analogs compared to control cells.. These results demonstrate that the cell-cycle inhibitor protein p21 is a direct target gene of biologically active 1,25(OH)2 vitamin D3, inducing G0/G1 cell-cycle arrest. The ability of vitamin D analogs to act via the same molecular mechanism as the natural hormone but with less hypercalcemic activity may have therapeutic implications for patients with SCCHN malignancy.

Topics: Antineoplastic Agents; Calcitriol; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cell Survival; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Reporter; Green Fluorescent Proteins; Humans; Laryngeal Neoplasms; Luminescent Proteins; Promoter Regions, Genetic; Proto-Oncogene Proteins p21(ras); Resting Phase, Cell Cycle; Transcription, Genetic; Transfection; Tumor Cells, Cultured