The Inhibition In Platelet Aggregation And The Research In Mechanism Of Inducing Human Hepatocarcinoma Cells Apoptosis By Triflusal And Its Metal Complex

Background: Aspirin--- appears to have chemopreventive and chemotherapeutic effects on myocardial and cerebral infarction events, not only the common household drug to inhibit platelet activation, but also numerous clinical observations and laboratory studies having shown that regular use of aspirin is associated with a reduced risk for colorectal, oesophagus, lung, uterine cervix,breast, liver and skin cancers.However, aspirin is not currently prescribed for cancer prophylaxis owing to its adverse effects such as the risk of upper gastrointestinal bleeding. Triflusal, or2-acetyloxy-4-trifluoromethyl benzoic acid, is structurally related to acetylsalicylic acid,which presents similar effectiveness as an antiplatelet agent. It is extensively used on patients with ischemic stroke, cerebral infarction or transient ischemic attack and a better safety profile. Studies have found that many kinds of cancer such as gastric cancer, lung cancer, gynecological cancer patients accompanied by the phenomenon of thrombocytosis. Platelets protect tumor cells from a variety of immune cells killing in the blood, while promote tumor cells epithelial-mesenchymal transition and the metastasis process. Therefore, the research of drugs inhibition of platelet aggregation,applied to the associated with the thrombocytosis tumor prevention and treatment is of great significance.Copper is an essential metal with key roles in many biological processes. The critical functions associated with copper have resulted from its potentredox activity. This has been driven by research demonstrating critical changes to copper homeostasis in many disorders including cancer and neurodegeneration. More recently, mixtures of copper have been found to act as efficient proteasome inhibitors and apoptosis inducers, specifically in cancer cells for chemoprevention and chemotherapy. However, the relationship of triflusal with cancer is poorly studied.Supported by the strong evidence from aspirin, we speculate that triflusal may have an ever-expanding therapeutic repertoire on tumor’s progressing.Objective: 1. To detect the inhibition of platelet aggregation by triflusal and its metal ion complex, zhr-315. 2. To further explore and compare the effects of triflusal and zhr-315 on the proliferation of human hepatocarcinoma cancer cell Hep G2 and Bel7402.3. To observe the changes in morphology of Hep G2 and Bel7402 after drug’s inducing.4. To compare the differences of triflusal and zhr-315 effects on cell apoptosis. 5. To investigate the possible molecular mechanism of apoptosis, and to afford evidences for the efforts of triflusal and its metal complex on cancer.Methods: 1. Adapting the in vitro methods and preparing platelet-rich plasma to detect the inhibition of triflusal and zhr-315 on platelet aggregation. 2. MTT was performed to detect the inhibition of cell proliferation. 3. Apoptotic Cell Hoechst 33258 Detection Kit was used to compare the differences of drugs on cell apoptosis. 4. Western blot was performed to detect the expression levels of apoptosis corresponding proteins in Hep G2 and Bel7402 cell lines.Results: 1. Both triflusal and zhr-315 exerted inhibition of ADP-induced platelet aggregation of mouse, the IC50 is 1.764 m M and 0.513 m M for each other. 2. The results of MTT showed that triflusal inhibit the proliferation of Hep G2 and Bel7402 as well as zhr-315, for Hep G2,the IC50 is 0.933 ± 0.021 m M, 0.716 ± 0.141 Mm respectively; for Bel7402, the IC50 is 0.175±0.039 m M, 0.137±0.044 m M respectively.2. The results of MTT showed that triflusal inhibit the proliferation of Hep G2 and Bel7402 as well as zhr-315,but zhr-315 had the greater effects. 3. At the same concentrations, zhr-315 exerted greater ability to induce cell apoptosis compared with triflusal. 4. Triflusal and zhr-315 caused activations of caspase 3, caspase9 and PARPprotein, we detected the cleaved-caspase, that is both the drugs activated the mitochondrial intrinsic pathway. Moreover, the protein expression level is correspond with the concentration of drugs. 5. Both triflusal and zhr-315 increased the expression of Bax, decreased the expression of Bcl-xl and Bcl-2, and the protein expression level is correspond with the concentration of drugs.Conclusion: 1. Both triflusal and zhr-315 can inhibit the aggregation of platelet. 2. Both triflusal and zhr-315 caused inhibition of hepatoma Hep G2 and Bel7402 cell’s proliferation.3. zhr-315 induced cell apoptosis compared to triflusal with more effective.4. Triflusal and zhr-315 activated the intrinsic pathway to induced cell apoptosis. Taken together, triflusal and its metal complex may play a rule in cancer prevention, forecasted that triflusal and its metal complex can be considered an a supplementary treatment in cancer associated with thrombocytosis.