Study Of The Synergistic Anti-tumor Activity And Mechanism Of Ruthenium Complex And Doxorubicin

Objective:To explore the synergistic anti-tumor effect of ruthenium complexes and clinical anti-tumor drugs cisplatin,oxaliplatin and doxorubicin on high-incidence tumors.Study the in vitro synergistic effect and mechanism of the drug combination,and its anti-tumor proliferation effect and toxicity in transplanted tumors.Provide reference for more researches on the combined effects of ruthenium complexes and its clinical applications.Method:1.The synergistic effect in vitro was mainly used the MTT experiment to screen the drug combination,and the combination effect was calculated by the synergy analysis software Compu Syn to obtain the combination index CI(Combination Index).When the CI < 1,it can be considered the combination has potential synergy.We used the x CELLigence Real-Time Cell Analyzer System for verification.2.Calcein-AM staining was used to detect the live/dead cells on 2D cells and 3D cell spheres level.Hoechst staining and Annexin V staining were used to detect cell apoptosis;DCFH-DA staining method was used to detect the generation of reactive oxygen species(ROS)in 2D cells and 3D cell spheroids;Western-Blot was used to detect synergy related pathway proteins expression.3.Constructed an in vivo xenograft tumor model in nude mice to verify the synergistic anti-tumor effect of the drug combination.The body weight of the mice in the Dox group decreased significantly after the third administration,while the combined administration group had no significant difference compared with the control group.Mainly include:recording the tumor proliferation curve,tumor weight and weight change of mice;H&E staining,Sirius red staining and Prussian blue staining were used to detect the toxicity of the main organs of mice;TUNEL staining of tumor was used to detect the cell apoptosis;Immunohistochemically stained was used to detect the expression of synergy-related pathway proteins.Result:1.Through MTT experiment and Compu Syn software analysis results,we found that the ruthenium/doxorubicin combination had a significant synergistic effect on breast cancer MCF-7 cells.The results obtained by the cell real-time analysis system were consistent with the MTT results.2.The combination significantly inhibited tumor cells at the 2D cells and 3D cell spheroids level,and synergistically induced the generation of ROS in tumor cells.Further studies have found that the combination could significantly promoted MCF-7 cell apoptosis.Western-Blot results showed that the combination significantly affected the expression of PI3K/AKT pathway-related proteins,including down-regulating the expression of PI3 K,AKT,NF-κB and XIAP proteins and up-regulating the expression of PTEN and P53 proteins.3.The combination had a significant inhibition on MCF-7transplanted tumors in vivo,and could significantly decreased the toxic and side effects of doxorubicin.The results of H&E staining and TUNEL staining of tumor tissues showed that the combination of ruthenium/doxorubicin significantly promoted tumor cell apoptosis compared with single drug.The results were consistent with the results of in vitro experiments.H&E staining results showed that doxorubicin had obvious damage to the heart of mice,and Sirius red staining results showed it induced cardiac tissue fibrosis,which was alleviated after reducing the dose and combining with ruthenium complexes.The results of Prussian blue staining of heart tissue showed that doxorubicin caused iron accumulation in heart tissue.The results of immunohistochemically stained of cardiac tissue showed that doxorubicin could regulate iron accumulation in the ferroptosis pathway and regulate the expression of lipid peroxidation-related proteins,including the up-regulation of Tf,DMT1 and HO-1 expression and the down-regulation of Nrf2,SLC7A11 and GPX4.Conclusion:The ruthenium/doxorubicin combination has shown synergistic antitumor effects on breast cancer MCF-7 cells in both in vitro and in vivo.The combination promotes tumor cell apoptosis by increasing the production of ROS in tumor cells and regulating the expression of PI3K/AKT pathway related proteins.More importantly,while the combination in vivo studies synergistically promotes tumor growth inhibition,it can significantly reduce and alleviate the toxic side effects caused by doxorubicin,especially the cardiac injury.