The Research On The Growth Inhibition Of T47D Cell And Reversion Of P16 Hypermethylation By CDP

Background & Objective Pathogenesis of cancer involves a stepwise accumulation of genetic alterations that disrupt either the normal machinery of cell proliferation or its regulation cellular oncogenes or tumor suppressor genes. Cellular proliferation is mediated by progression through the cell cycle, where the most important checkpoints are located at G1/S. Upon activation by mitogens, upregulation of D-type cyclins results in activation of cyclin-dependent kinases(CDK) namely CDK4 and CDK6, inducing phos-phorylation of pRB. Hyperphosphorylated pRB leads to release of E2F, which activates transcription of S phase specific genes, and irreversible commitment to cell cycle progression. Negative regulatory elements to the cell cycle progression include the p16 that give rise to cell cycle arrest. The protein encoded by p\6 binds with CDK4 and 6 competing with cyclin D, arrest at G1 by inhibition of pRB phosphorylation. The inactivation of p16 in cell cycle may cause cell lost the ability to activate pRB and grow out of control. Numerous classical genetic changes are involved in the transformation of normal cells into a tumor. Some events include loss of tumor suppressor gene function through gene deletions and other types of mutations, as well asconstitutive activation or amplification of dominant oncogenes. More recently, it has become clear that epigenetic alterations, particularly tumor suppressor gene silencing via DNA methylation, also plays a critical role in the initiation and progression of cancer. p16 is one of the most relevant genetically inactivated tumor suppressor genes. Studies using DNA methyltransferase inhibitors like 5-azacytidine (5-aza-CR) and 5-aza-2'- deoxycytidine (decita-bine) in order to arrest DNA methylation, show that demethylation of 5' gene sequences can activate gene expression. This nucleoside analog is incorporated into DNA, but unlike cytosine, forms a covalent bond with DNA methyltransferase (DNMT1) that inactivates the enzyme, thus leading to demethylation of DNA. These two are the most commonly used demethylating drugs, but they may induce cytotoxicity and mutagenesis. Safe and effective drugs are needed. In the present study, we will investigate one element of Chinese drug, CDP, on reversion of p16 hypermethylation in human T47D breast cancer cell, in order to explore a new substitute for 5-aza-CR and decitabine for demethylation. With 5-aza-CR as a control, our experiments monitored the cell proliferation, cell cycle changed and pl6 methylation status, searching for the mechanism of CDP on reversing hypermethylation of tumor suppress gene and inhibiting growth of tumor, and provided primary experimental foundation for further therapy application of tumors.Methods T47D cells were cultured with different concentration of 5-aza-CR and CDP several days. The growth rate assessed by cell proliferation experiment (MTT colorimetric assay); The apoptic peak and cell cycle distribution was detected by flow cytometry (FCM); The level of methylationand unmethylation status of p16 was detected by methylation specific PCR(MSP).Results Afer incubation with the two drugs, the cell growth rate decreasedwith the concentration raised (P<0.05). when 5-aza-CR increased to 5u.mol/L and CDP to 250u.mol/L, most of the cells were dead. On the growing balanced phase(5th day), the growth inhibiting rate moved up as the concentration increased. 5-aza-CR: IC50=1.79 umol/L; CDP: IC50= 45.38 umol/L. The cell cycle was influenced under the well-chosen concentration of 5-aza-CR (2umol/L) and CDP (50umol/L): the cell number increased in G0/G1 phase from 65.1% to 71.3%, 84.3%, and decreased in S phase from 19.4% to 14.3%, 7.2%. The ratio of apoptic cell had no significantly influenced. DNA was abstracted before and after the drug application, Only methylating PCR product appeared before treatment with drugs, Conversely, Only demethylating PCR amplification product showed after treatment of any of the two drugs for 6 days.Conclusion MTT assay indicated T47D cell growth rate decrease in adose and time depend...