Roles Of NFκB In G1/S Cell Cycle Checkpoint And Apoptosis Induced By HCPT In Human Breast Carcinoma Cells

The nuclear factor NF k B family of eukaryotic transcription factors plays an important role in the regulation of immune response, embryo and cell lineage development, cell apoptosis, differentiation, cell-cycle progression, inflammation, cell migration ,oncogenesis, and resistance to chemotherapy or radiation.Increasing evidence indicates that activation of NF k B in cancer cells plays an important role in coordinating the control apoptotic cell death and cell cycle arrest, which either to promotes or inhibits apoptosis and cell cycle arrest. Up to now, more than 150 target genes had been modulated by NF k B .Many studies showed NF k B is overexpression in a lot of tumors, including gastric cancer, prostate cancer, multiple myeloma, ovarian cancer, colon cancer, breast cancer, liver cancer etc. blocking NF k B activation could enhance the effects of cytotoxic chemotherapy and inprove cell killing.However, the roles of NF k B is depended on the different cell types and apoptotoic stimuli. The same drug could cause different role of NF k B. We also do not know the relationship of NF k B and P53. so we need more study on the role of NF k B in the tumor.CPT11, TPT and HCPT are topoisomerase I targeting agents, which have been usded in the clinical. HCPT was invented by Shanghai drug institute in 1978.HCPT is a novel CPTanalogue that differs from CPT by the presence of an additional methylene group in the E-ring. DNA Topo I is a nuclear enzyme that is very important for solving topological problems arising during DNA replication .HCPT may cause cell apoptosis and cell cycle arrest by inhibiting the Topo I activity, which leads to an accumulation of DNA strand breaks (cleavable complexes) and, ultimately, cell death.Some studies indicated HCPT could cause a lot of tumor cell apoptosis and cell cycle arrest. But we do not know if there are other signal transduction pathway involved in cell apoptosis and cell cycle arrest except for Topo 1 inhibition.Contrary to some studies, our study indicated that NF k B play an pro-apoptosis role in Bcap37 cells .both Taxol and Vincristine could active NF k B and we found that the apoptosis and cell cycle arrest induced by Taxol or Vincristine suppressed after klocking the signal pathway of NF k B.However, the nuclear target is very little known. To shed light on the mechanism of action of HCPT at the cellular level, we seek to investigate the the relationship of NF k b and apoptosis induced by HCPT in Bcap37 cells. This study includes six sections. Section one Materials and MethodsReagents made according to < Molecular Clone: a laboarty manual > (3nd). Methods include MTT; Western Blotting; DNA gel shift; DIG-EMSA and Plasmid transfection.Section two The Apoptosis and Cell Cycle Arrest of Bcap37 Cells Induced by HCPT Objective: To investigate if HCPT cause cell apoptosis and cell cycle arrest in Bcap37 cells. Methods: FCM, MTT, DIG-EMSA, Western Blot, DNA cleavage agarose gel eletrophoresis and Immunocytochemistry.Results: HCPT could induce Bcap37 cells apoptotic DNA ladder with G1/S arrest and down-regulated IkB-a level with NF k B activation. Conclusion: HCPT induce cell apoptosis and cell cycle arrest by activation of NF k b.Section three The Mechanisms of Apoptosis and Cell Cycle Arrest Induced by HCPTin Bcap37 CellsObjective: To investigate the mechanisms of apoptosis and cell cycle arrest induced byHCPT in Bcap37 cellsMethods: DIG-EMSA, Western Blot and DNA cleavage agarose gel eletrophoresis.Results: HCPT could up-regulate the expression of P53, P21, Bax and inhibit the expressionof CDK4/ClnDl, Bcl-2 ,but no influence on G2/M cell cycle checkpoint. DEVD-CHO couldblock HCPT-induced apoptosis and activation of NF k B.Conclusion: P53, P2I, CDK4/ClnDl, Bax/Bcl-2, Caspase3 are involved in apoptosis and cellcycle arrest induced by HCPT.Section four The Effect of Mutant IkB-a on the Apoptosis and Cell Cycle ArrestInduced by HCPT in Bcap37 CellsObjecti...