Study On Antiglioma Effects Of β-diketone-cobalt Complexes And Its Mechanisms

Study on antiglioma effects ofβ-diketone- cobalt complexes and its mechanismsBackground:The incidence of glioma is the highest of nervous system tumors,and and the mortality also is the highest among the nervous system tumors. As with other malignancies, Surgery, radiotherapy,chemotherapy are the main methods. However,the malignant behavior is the most outstanding performance (ie,invasive growth), and micro-surgery has the difficulty to completely remove it,so the prognosis is poor. Therefore,postoperative radiotherapy and chemotherapy have important significance. Studies have shown that certain anti-glioma radiation radiotherapy in glioma treatment, so the benefit is limited. Chemotherapy is most active in current areas of glioma therapy. Currently temozolomide of status in glioma treatment has been recognized, however, a large number of studies have showed that the role of a temozolomide is still not satisfied,the development of new,high efficiency,low toxicity of the anti-glioma drugs has been imminent.With the multi-disciplinary collaboration,cooperation in-depth,the mixture of medicine and chemistry, more and more studies present using a variety of new compounds to solve medical problems. The research of the chemical effect between metal complexes with DNA emerged continuously,especially in the interaction between the small molecular with DNA,which induce many biological effects frequently and has Far-reaching significance. Cobalt is a high melting point,good stability of magnetic hard metal. Cobalt content in the normal human is 1.1 ~ 1.5mg,of which 14% is located in the bone,43% distributed in the muscle,and the remaining distributed in other soft tissues of the human body. Advances in the anti-tumor activity of cobalt compounds have been discussed by us and we found that lots of cobalt complexes,the anti-tumor activity of which mainly plays in the interaction with DNA, showed significant inhibition to tumor cells. Therefore,the research and development of new cobalt-containing complexes in the field of cancer therapy will show important practical significance. Jensen synthesizedβ- diketone firstly in 1959, as a chelating agent, which has a wide range of chelating ability. The chelation was studied,and the results show that it has good chelation with metal. Based on the above finding,we researchedβ– diketone– cobalt,a new metal complex,in the role of anti-glioma in-depth through the following experiments. And we expect to find new anti-glioma drugs with high efficiency and low toxicity.Object:In order to investigate the inhibition ofβ-diketone-cobalt complexes to brain glioma, and the mechanism of anticarcinoma,which can provide the powerful experimental foundation and theoretical basis for clinic,and expand the new field of new type anti-carcinoma drug.Research methods and results:1.In vitro investigation of the inhibition ofβ-diketone- cobalt complexes in gliomas.After preliminary screening of antitumor activity in vitro, higher antitumor activityβ- diketone- cobalt complexes are chosen as tested compound, by checking their roles in the cell growth inhibition using C6 rats glioma line by MTT method. The result shows thatβ-diketone-cobalt complexes significantly inhibits cell growth inhibition rate of C6 rats glioma line in a dose dependant manner (p<0.05), and the high concentration can obviously increase the inhibition effect, the max value showed 83.1% (100ug/ml). Furthermore, theβ-diketone- cobalt complexes showed more sensitivity in C6 rats glioma than fluorouracil.2 . Investigation ofβ-diketone-cobalt complexes toxicity.The 80 Kunming mice were divided into 8 groups and were subjected to different concentrations ofβ-diketone-cobalt for two weeks, to analyze the response to different doses, sensitivity, toxicity and mortality rate. The mice were dissected and analyzed histopathologically. We found that some mice died 6 hours after injection, and the mice were suffering from diarrhea and weight loss before death. And some mice grossly showed that the liver and intestinal tract appeared different degrees of damage. LD50 ofβ-diketone-cobalt was 3099.1mg/kg.3. Effect ofβ-diketone-cobalt complexes on DNA synthesis of the C6 rats glioma cellsThe C6 rats glioma cells were isolated at entered the logarithmic growth phase and cultured in 96-well culture plates. Tested drugs and 3H-TdR were added respectively,and then oven dried after reasonable culture. After adding scintillation liquid, cpm values and inhibition rate were determined.β-diketone-cobalt complexes showed obvious inhibition in the DNA synthesis of C6 rats glioma cells,a dose-dependent relationship and the maximum inhibition rate reaching 50%.4. Effect ofβ-diketone-cobalt complexes on cell cycle and apoptosis C6 rats glioma lines.The cell percentage of each phase and the influence ofβ-diketone-cobalt complexes on cell cycle and apoptosis in C6 rats glioma lines were checked by flow cytometry. The percentage of apoptotic cell in the experiment group is significantly higher than that in control group (p < 0.01), but the percentage of S stage cells showed no change along with time and dose concentration obviously.5. Effect ofβ-diketone-cobalt complexes on apoptosis of C6 rats glioma cell line.The C6 rats glioma cells were treated with the different concentration ofβ-diketone- cobalt complexes and stained with DAPI, the change of morphology and confluent were observed. We found that the cells confluent and cells bulk decreased, and some cells showed round or rupture. Furthermore, some fluorescence image found in the cellular nuclear of apoptotic cell in the experimental group, and granular DNA piece were observed in some cells of high concentration group, which showed nuclear rupture in the apoptotic cells.Effect ofβ-diketone-cobalt complexes on apoptotic factor expression of C6 rats glioma cell were detected with Anexin V and PI flow cyometry. In contrast to the control group, the higher apoptotic rate were found in the experimental group. Apoptotic rate increased with exposure of high dose and time prolonged, which showed obvious dose–time -effect relationship.6. Effect ofβ-diketone-cobalt complexes on apoptotic factor expression of C6 rats glioma cell.Western blot for bcl-2 and bax proteins were performed, after C6 rats glioma cells were treated withβ-diketone- cobalt complexes for 48 hours. We found that decreased bcl-2 and increased bax protein were detected with high dose.Conclusion:1.β-diketone- cobalt complexes shows obvious proliferate inhibition effect on the C6 rats glioma cells and obvious dose -effect relationship; 2.β-diketone- cobalt complexes has low toxicity and less damage in the kidney, only targets liver and intestinal tract;3.β-diketone-cobalt complexes inhibits the synthesis of DNA in the tumor cells arresting the cell replication and proliferation;4.β-diketone-cobalt complexes inhibits the tumor cell into the next cycle and proliferation by blocking S stage cells;5.β-diketone-cobalt complexes induces the tumor cell apoptosis;6. The anti-tumor mechanism ofβ-diketone-cobalt complexes has closely relations with the expression of pro-apoptotic factor Bax and reduction in apoptosis inhibitor factor bcl-2.The above findings demonstrate thatβ-diketone-cobalt complexes show strong anti- glioma cells role. We find that this role has closely relationship with inhibition of DNA synthesis, cellular block, inducing cellular apoptosis and apoptotic factor expression. However,the anti-brain glioma mechanism ofβ-diketone-cobalt complexes still needs more research in-depth level.