| Carcinoma of the pancreas is currently one of the leading causes of cancer death in the digest system and the fifth cause of death from malignant disease in Western society. In the United States incidence of pancreatic carcinoma has trebled in the last 50 years . Pancreatic carcinoma is associated with an especially poor prognosis . Neither radiotherapy nor chemotherapy improve 5 year survival rates, which do not exceed 5%. Despite umprovements in therapeutic strategies including surgical techniques as well as local and systemic adjuvant therapies, pancreatic cancer remains one of the leading cause of cancer death in industrialized countries. Therefore new therapeutic modalities are essential for treating pancreatic carcinoma. There has been increasing evidence that the steroid hormones such as vitamin D are naturally occurring agents controlling cellular differentiation and proliferation both in normal and malignant cells .The interest in vitamin D in relation to cancer emerged from the finding that VDRs are expressed in many types of cancer cells, including cells derived from tumors of breast, prostate, colon, cervix etc. In addition, it was found that EB1089 was capable of affecting growth and differentiation these tumor cells, it was also able to stimulate differentiation of tumors cells. Despite being 50 -200 times more potent than 1a,25( OH)2vitaminD3 with respect to regulation of cell growth and differentiation in almost every cell type studied, EB1089 displays a calcaemic activity in vivo which is approximately 50% weaker than that of 1a,25(OH)2vitaminD3[10].The molecular mechanisms of proliferation, invasion and metasta-sis are associated with the characters of tumor. It is well established that the cyclins play a positive role in promoting cell cycle transitions via their ability to associate with and activate their cognate cyclin - dependent kinases (Cdks). P21 is a potent, tight -binding inhibitor of Cdks and can inhibit the phosphorylation of Rb by the cyclins. We measure the level of expression of p21 in my experiment to value and analysis the growth inhibition and induction of differentiation of the cell line BxPC-3.The purpose of this experiment is tc investigate the EBI089 's effects of growth inhabination on the cell line of carcinoma of pancreas BxPC-3.MethodsCell cultureThe human pancreatic cancer cell line BxPC - 3 was received from the American Tissue Type Culture Collection ( ATCC) . Cell line was cultivated in RPMI medium ( Gibco, UK) supplemented with 10% heat - inactivated fetal bovine serum (FBS, Gibco, UK) and routinely tested of mycoplasma contamination.CompoundEB1089 was provided by Leo Pharmaceutical Products ( Balle-rup, Denmark ). The compound was dissolved in ethanol and stored in the dark at 20 t , Dilutions was made up in complete culture medium and the final DMSO concentration did not exceed 0.1% . The maximalconcentrations used 10 7M for EB1089.Growth inhibition assaysCells were plated in 96 - well plates at a density of 2 104 cells per well. After 24 -48 hour normal culture medium was exchanged for medium containing 2.5% serum and the inhibitory compounds. Control cells received 0. 1% DMSO and fresh medium was added every 2 -Sdays. At the end of each experiment cells were fixed in 10% DMSO and stained with MTT. In the final, the optical density was measured at 495 nm.Propidium Iodide staining and cell cycle analysisAfter treatment with EB1089 cells were harvested by trypsiniza-tion, washed twice with sample buffer (PBS) and fixed in 70% etha-nol at a density of 1 10 cells ml" . After > 18 h the cells were washed with sample buffer and resuspended in propidium Iodide (PI) staining solution containing 50 ug ml-1 PI and 20 ug ml-1 RNAase. fluorescence was measured cm a Becton - Dickinson FACScan and DNA histograms were analysed using CELLQUEST software.Western blottingCells were harvested and lysed in ice - cold lysis buffer (10 mM PBS. PH 7.4, 1% NP -40, 0. 5% sodium deoxycholate, 0. 1% SDS) supplemented with 1 mM sodium orth... |
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