| Objective:Tumor drug resistance is one of the main reasons for chemotherapy failure.Therefore,the development of new anti-tumor drugs or the combination of multiple anti-tumor drugs may be an effective means to deal with tumor drug resistance.Metal ruthenium complexes have the characteristics of variable structure,rich physicochemical properties and high-efficiency and low-toxicitythat it have become a hot spot in the research and development of metal anticancer drugs in recent years.In this paper,the synergistic killing effect of ruthenium complex Δ-Ru1 and clinical antitumor drugs paclitaxel and cisplatin on cancer cells was detected.The killing effect and mechanism of different drug combinations on drug-resistant tumor cells were studied,and their therapeutic effects and toxic reactions were explored in a nude mouse tumor-bearing model.To provide reference for the combined effect of ruthenium complexes with clinical antitumor drugs and their clinical applications.Method:1.Multiple cancer cells were screened for drug combination by MTT experiment to test the synergistic effect in vitro,and the synergy analysis software(Compu Syn)was used to calculate the combination index CI(Combination Index)value.When CI < 1,it means the combination is in synergistic effect under certain conditions.The synergy of the drug combination was then further verified using the Synergy Finder method,and the Synergy score was calculated according to the ZIP calculation method.ZIP Synergy score >0 indicates synergy(red area).2.After drug treatment,the morphological changes of cells were observed by taking pictures with a microscope.Lactate Dehydrogenase(LDH)release kit for detect cell membrane integrity;ELISA kit for detect the release of inflammatory factor Interleukin-1β(IL-1β);DCFH-DA fluorescent probe detection method is used to detect the effect of drug combination on the production of reactive oxygen species(ROS)in cells;Western-Blot detection of related protein expression.3.The paclitaxel-resistant cervical cancer xenograft model in BABL/c immunodeficient mice was constructed,and the in vivo therapeutic effect of the drug combination on cervical cancer-resistant cells was detected by recording the tumor size and body weight changes of the xenografted tumors in mice;H&E staining was used to detect The effects of drugs on the main organs of mice,the pulmonary toxicity caused by paclitaxel was detected by Sirius red staining;the neurotoxicity of paclitaxel was detected by mining experiments and benzylamine blue staining.Result:1.Through Compu Syn software and ZIP calculation method analysis results,it is found that the combination of ruthenium and paclitaxel has a significant synergistic killing effect on paclitaxel-resistant cervical cancer He La/Taxol cells.2.Δ-Ru1 & Taxol combination can induce pyroptosis of cells.After drug treatment,the cells swelled and formed many bubble-like protrusions,and eventually the entire cell swelled and ruptured,and the integrity of the cell membrane was lost.The combination of ruthenium and paclitaxel can synergistically induce the production of ROS in tumor cells,accompanied by the formation of inflammasomes and the release of inflammatory factors.Western-Blot results found that the Δ-Ru1 & Taxol combination affected the expression of Caspase-1/GSDMD pathwayrelated proteins,including the up-regulation of NLRP3,ASC,NF-κB,Caspase-1,and GSDMD protein expression.3.The combination of Δ-Ru1 & Taxol significantly inhibited the growth of xenograft tumors in mice,synergistically increased the sensitivity of He La/Taxol cells to paclitaxel,and significantly reduced the toxic and side effects of paclitaxel.After the fourth administration,compared with the control group,the weight of the mice in the Taxol group was significantly reduced,while there was no significant difference between the Δ-Ru1 & Taxol combined administration group and the Δ-Ru1 group.The results of H&E staining showed that Taxol had an obvious damage effect on the lung tissue of mice.Sirius red staining also showed that Taxol induced fibrosis in the lung tissue,while the Δ-Ru1 &Taxol combination group reduced the amount of Taxol through the combination of Taxol and Δ-Ru1.After the dose,the phenomenon of pulmonary fibrosis decreased.The results of benzylamine blue staining and mine field experiments showed that Taxol caused neurotoxicity,and the phenomenon was obviously alleviated after reducing the dose in combination with Δ-Ru1.Conclusion:In both in vitro and in vivo studies,the synergistic antitumor effect of Δ-Ru1 & Taxol on the paclitaxel-resistant cervical cancer cells He La/Taxol was found.The combination of Δ-Ru1 and Taxol synergistically induces pyroptosis of He La/Taxol cells by inducing the generation of ROS in tumor cells and regulating the expression of Caspase-1/GSDMD pathway-related proteins.In addition,the combination of Δ-Ru1 & Taxol not only synergistically inhibited tumor growth in mice,but also significantly increased the sensitivity of He La/Taxol cells to Taxol and alleviated the pulmonary and neurotoxicity caused by Taxol. |
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