| Purpose and Significance The successful application of cisplatin as anticancer drug in clinical, has attracted a great deal of attention in metal-based anticancer agents, opend a new era of research and development of metal anticancer drug. However in the clinical use of platinum based drugs, it was found that the drug has strong toxicity and the application scop is narrow have limied clinical utility. In order to overcome these limitations, the new metal anticancer drugs with more effective and less toxic have become a research hotspot, researcher pay attention to the ruthenium metal which belongs to group VIII, the physicochemical properties is similar to platinum, but toxic and side effect is lower, the ruthenium based drug is considered one of the most promising antitumor agent. Enthusiasm of ruthenium was stimulated by the clinical trials of NAMI-A and KP1019. The researchers conducted a gerat of structural modification on ruthenium metal, a large number of new ruthenium complexes have been obtained, some complexes have been confirmed that they show high cytotoxic against tumor cells. In recent years, the research on the anti-tumor activity of ruthenium complexes has attracted a great deal of attention, and many studies have showed that ruthenium complexes have a significant inhibitory effect on the activity of many kinds of tumor cells. This study was on the basis of the original work, we selected [Ru（bpy）2（taptp）]（ClO4）2（Ru1）for research, detect of the influence on SGC-7901 cells, and to reveal the related mechanism of ruthenium complex. The study is screen the tumor cell which is sensitive to ruthenium complex, detecting the cytotoxic against tumor cells and then research its possible mechanism. The results will be helpful for the development of novel ruthenium complexes as potential anticancer agents with high efficiency and low toxicityMethods Detecting of the inhibition rate of ruthenium complex on tumor cells This study selected gastric carcinoma SGC-7901 cells, cervical cancer Hela cells, breast cancer MDA-MB-231 cells for experimental, treated with different concentrations of [Ru（bpy）2（taptp）]（ClO4）2（Ru1） 24 h on tumor cells, respectively. Detection of inhibition rate of tumor cells by MTT test, then calculate the IC50 value. Determination of tumor cells number after treated with Ru1 by crystal violet staining assay, and choose the tumor cells which the most sensitive to complexe for subsequent experiments. The celluar uptake of ruthenium complex Based on the MTT method, The SGC-7901 cells were exposed to different concentrations of Ru1. By DAPI staining method, compare Ru1 itself fluorescence and DAPI fluorescence overlay, detect the distribution of Ru1 in SGC- 7901 cells. Detection of the influence of ruthenium complex on cell cycle of SGC-7901 cells In order to further study the molecular mechanism of Ru1 inhibit tumor cells whether is associated with SGC-7901 cell cycle, the cells were exposed to designated concentrations of tested Ru1 for 24, and the the cell cycle was analyzed by flow cytometry. Detection of the influence of ruthenium complex on SGC-7901 cells DNA In order to further investigate whether Ru1 can combine with DNA, after treatment of SGC- 7901 cells with Ru1 24 h, the comet assay was used to detect the DNA damage. Detection of the influence of ruthenium complex on apoptosis of SGC-7901 cells Based on the Ru1 contribute to cell cycle arrest at G0/G1 phase and induce DNA damage on SGC-7901 cells, after treatment of cells with Ru1, the apoptosis was analyzed by flow cytometry. Detection of the influence of ruthenium complex on cell cycle related proteins and apoptosis related proteins. The underlying pathway of Ru1-induce cell cycle arrest and the induction of apoptosis was also studied in detail by western blot analysis, including ATR, phospho-ATR, H2 AX, γH2AX, p53, phospho-p53, ERK1/2, phospho-ERK1/2, Bcl-2, Bax.Through expression of the checkpoints related proteins and apoptosis related proteins, to reveal the related mechanism of Ru1.Results 1. Detecting of the inhibition rate of ruthenium complex on tumor cells The MTT assays data clearly show that the Ru1 showed dose-dependent cytotoxic effect against SGC-7901 cells, Hela cells, MDA-MB-231 cells, and the complex had a higher cytotoxic effect on SGC-7901 cells with an IC50 value of 26.5μM. Crystal violet assay confirmed that Ru1 significantly decreased the viability of SGC-7901 cells in a concentration-dependent manner, consistent with the results obtained from the MTT assay. It was believed the Ru1 had a better cytotoxic effect on SGC-7901 cells, so SGC-7901 cells was selected for subsequent experimental. Besides, the results revealed that the Ru1 had a higher toxicity on SGC-7901 cells than that on amniotic fluid cells. 2. The celluar uptake of ruthenium complex These observations suggest that Ru1 can be successfully taken up by SGC-7901 cells in a concentration-dependent manner. Increasing concentrations of the complex led to increased fluorescent spot number and intensity. Fluorescence was present in the cytoplasm and accumulated in cell nuclei. 3. Detection of the influence of ruthenium complex on cell cycle of SGC-7901 cells After treatment SGC-7901 cells with Ru1, there was a clear enhancement of the percentage of cells in G0/G1 phase, indicating that the Ru1 contribute to cell cycle arrest at G0/G1 phase on SGC-7901 cells with increasing the amount of the synthesized compound more cells accumulated in G0/G1 phase were found. 4. Detection of the influence of ruthenium complex on SGC-7901 cells DNA The comet assay data suggest that after exposure of cells to Ru1 for 24 h, tail length, comet length, cell tail moment and olive tail moment were incread in a concentration-dependent manner, indicating that Ru1 can induced DNA damage. 5. Detection of the influence of ruthenium complex on apoptosis of SGC-7901 cells After exposure of cells to Ru1 for 24 h, the percentage of apoptotic cells were incread. Thus ruthenium complex leads to a dose-dependent induction of apoptosis in SGC-7901cells. 6. Detection of the influence of ruthenium complex on cell cycle related proteins and apoptosis related proteins Western blot analysis reveals that Ru1 activates ATR, H2 AX, Erk1/2 and p53, by upregulation of the levels of phospho-ATR, γH2AX, phospho-Erk1/2, phospho-p53. Additionally, blotting of mitochondrial Bcl-2 family members showed that anti-apoptotic Bcl-2 expression was downregulated and pro-apoptotic Bax expression was upregulatedConclusions 1. Ru1 showed dose-dependent cytotoxic effect against SGC-7901 cells, Hela cells, MDA-MB-231 cells, and the complex had a higher cytotoxic effect on SGC-7901 cells. 2. Ru1 can be successfully taken up by SGC-7901 cells, and distributed in the cytoplasm and accumulated in cell nuclei. 3. DNA is the targe of Ru1, that the complex can induced DNA damage in a concentration-dependent manner. 4. Ru1 can induce cell cycle arrest and induce apoptosis. 5. The mechanism of anti-tumor role of Ru1 is induce DNA damage and activate of cell cycle checkpoint, phosphorylating a series of cell cycle related proteins and apoptosis related proteins, like as phospho-ATR, phospho-p53, p53, phospho-ERK1/2, upregulate Bax and downregulate Bcl-2. |
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