| Background and Objective:Temozolomide is an orally safe and effective anticancer drugs, which pass easilythrough the blood-brain barrier, today, as first-line anti-brain cancer chemotherapy drugs for clinical use. TMZ exert cytotoxic effects by alkylating DNA guanine and formation of O6-methylguanine (O6-MeG). In the cancer, there is widespread O6-alkyl guanine DNA-alkyl transferase (O6-methylguanine-DNA methyltransferase, MGMT), which could be transfer the alkylating group from the O6-guanine to own 145 cysteine residues irreversibly and repair the damage of DNA, thus weakening the cytotoxic effects of TMZ. Therefore, Malcolm Professor Stevens,The inventors of TMZ, designs new drugs 377/465,that are derivative of TMZ. which may be like TMZ,under the physiological conditions, opening by itselves and alkylating DNA, but the new drugs are not to identificate and repair by MGMT, and have a broad-spectrum anti-cancer activity. Such derivatives have been synthesized successfully, but the current study has yet to reveal them unclear,how to evade recognition of MGMT repair, how to form of DNA adducts.so, the purpose of this study intend to explore that,new drugs how to bing with DNA; DNA damage pathways; cell cycle and apoptosis.Methods:1.MTT method has be used to detecte the half inhibitory concentration in several cell lines.2. To determine the interaction of new drugs with DNA by UV spectroscopy.3. flow cytometry assay is used to detect the state of cell cycle in and HCT116 cell.4.Immunofluorescence analysis the phosphorylationof histone H2AX.5. To detect the apoptosis after exposured to new drugs Annexin-V-PI double staining.6. To detect the proteins expression of PARP, Caspase9, P53, p-P53, Chk2, p-Chk2 byimmunoblotting after exposured to new drugs.Results:1.MTT assay experiment shows that,the cytotoxicity reduction of new drugs may have not connection with MGMT and MRR, and the new drugs have the broad-spectrum toxicity toanti-cancer.2.TMZ and its derivatives can bind with DNA, but not at identical binding sites.3.the resulting cell cycle in HCT116:the cell cycle is blockaded. The TMZ derivatives 377 can arrest the cells in G2-M stage, pre-G1 is more and more obvious when the cells are exposured to new drugs for a long time. in an low concentration or a high concentration, the blocking of cells is not obvious after exposured to 465, and the pre-Gl peak is more obvious more than 377 in corresponding time after processing.4. The role of TMZ derivatives to the double-stranded DNA:the phosphorylation of H2AX is a standard to judge DNA double-strand breaks. In the HCT116 and U87-MG cells,after expored to 377 and 465, the phosphorylation of H2AX can be detected.377 and 465 can cause DNA double-strand breaks.5. The experiments of apoptosis assay:in the colon cancer cells HCT116 and glioma cells U87-MG,377 and 465 can cause apoptosis after 24 hours.6.To test the cycle arrest protein and apoptosis protein by Western blotting:after HCT116 and U87-MG cells processed by 377 and 465,the activity of p-P53, p-CHK2, PARP, Caspase-9 have a time-dependent manner; to 377, the activity of P-P53, p-CHK2 have a dose-dependent manner. After for 465 treatment, the activity of p-p53, p-CHK2, PARP, caspase-9 also have a time-dependent manner, but no significant dose-dependent.377 and 465 can modify the expression of proteins which can arrest cycle or induce apoptosis. |
PDF Full Text Request