Activity And Mechanism Of Action Research About Chiral Ruthenium Complexes ∧-OMe Targeted Anti-tumor Effect

Objective:Telomerase was an important marker to replication and proliferation of tumor cells, unlimited proliferation of tumor was related to telomerase activity or the change of telomere maintenance mechanism. This article studied the pathway of chiral ruthenium complexes (Ⅱ) to tumor cells from telomere and telomerase activity, cell cycle and cell apoptosis in three aspects, clarified the specific selection function of chiral isomers to tumor cells, and maked its anti-tumor targets clear to provide experimental and theoretical basis for the antitumor drugs development with inhibition of telomerase activity and efficient low toxicity.Methods:1. Filtrated cells and chiral ruthenium complexes (Ⅱ). Cultured cells, and then detected relevant cells and drugs by MTT method. Using the best chiral ruthenium complexes (Ⅱ) and tumor cells of drug susceptibility effect had filtrated to the next step.2. Telomere and telomerase activity detection. Detection how chiral ruthenium complexes (Ⅱ) influenced the expression of telomere related Trfl, Trf2 proteins by Western Blotting method; detection how chiral ruthenium complexes (Ⅱ) influenced telomerase activity by TRAP-PCR argentation.3. Cell cycle detection. Detection how chiral ruthenium complexes (Ⅱ) influenced tumor cell cycle arrest by PI single staining; Detection how chiral ruthenium complexes (Ⅱ) influenced to the expression of important regulatory factors p21mRNA cycle by RT-PCR.4. Cell apoptosis detection. Detection the morphology of cell apoptosis by hoechest33342 staining and Tunnel method; Annexin V-FITC/PI staining method was used for the quantitative determination of cell apoptosis; detection how chiral ruthenium complexes (Ⅱ) influenced the expression of cell apoptosis related Caspase-3、 Bcl-2、 Bax mRNA genes by RT-PCR.Results:1. MTT test:Chiral ruthenium complexes (Ⅱ) △/∧-[Ru (phen) 2 p-MOPIP] 2+(△/∧-ome) and raceme [Ru (phen) 2 p-MOPIP] 2+(dl-ome) had curative effect on detected tumor cells and chiral ruthenium complexes (Ⅱ) of ∧-OMe were most sensitive for MGC-803 gastric cancer cells. When ∧-OMe affected on MGC-803 for 48 hours, IC50 were 7.4μg/ml. And ∧-OMe induced MGC-803 cell deaths had obvious dosage and time dependence.2. Telomere and telomerase activity detection:Chiral ruthenium complexes (Ⅱ)∧-OMe can influence the expression of telomere related proteins and inhibit telomerase activity, and along with the increase of drug concentration, the telomerase activity became smaller. With the increase of drug concentration, the expression of Trf1 protein showed increasing trend, the expression of Trf2 protein showed a trend of decline when ∧-OMe processed MGC-803 cells after 48 hours. But the TRAP-PCR argentation detection showed that telomerase bands were the most and the darkest in no drug treatment group (A stripe); Negative control group (B stripe) had the least bands and the most shallow color; C, D, E bands were 5μg/ml, 10μg/ml,20 μ g/ml respectively, with higher drug concentration, the bands decreased, and the color became shallow.3. Cell cycle detection:Chiral ruthenium complexes (Ⅱ) ∧-OMe can block the cell cycle in G0/G1 phase. Joining complexes(Ⅱ) ∧-OMe 24 hours later, the G0/G1 phase had higher proportion along with the increase of drug concentration; Cells in S period decreased with the increase of drug concentration instead. The result of cycle related genes showed that the expression level of p21 mRNA raising along with the increase of drug concentration at the same time, but no obvious time dependence from the result comparison 24 and 48 hour later.4. Cell apoptosis detection:The chiral ruthenium complexes (Ⅱ) ∧-OMe can promote apoptosis of tumor cells with time and dose dependent. When chiral ruthenium complexes (Ⅱ)∧-OMe dealed with MGC-803 cells after 48 hours, the normal group cells grew well, normal cells decreased in drug treatment group, and cell morphology such as nuclear shrinkage, fragmentation, and apoptosis of apoptosis body can be seen. Along with the increase of drug concentration and the extension of time, cell apoptosis rate increased; Along with the increase of chiral ruthenium complexes (Ⅱ) ∧-OMe, the expression of Bax mRNA、 Caspase-3mRNA of MGC-803cells up regulation, the expression of Bcl-2 mRNA down regulation.Conclusions:Chiral ruthenium complexes (Ⅱ) ∧-OMe has obvious anti-tumor effecting to the gastric cancer cell MGC-803, mainly through the following ways:1. Chiral ruthenium complexes (Ⅱ) ∧-OMe can block the cell cycle to suppress the proliferation of MGC-803 cells. And the cycle arrest is in GO /Gl phase.2. Chiral ruthenium complexes (Ⅱ) ∧-OMe can regulate the expression of telomere related protein to inhibit telomerase activity and to block the extending of telomeres then block tumor cell proliferation.3. Chiral ruthenium complexes (Ⅱ) ∧-OMe can regulate the expression of apoptosis related gene, induce MGC-803 cells apoptosis so as to reach the purpose of anti-tumor.