| Objective: Approximately 15% to 20% of breast patients are specifically human epidermal growth factor receptor 2(HER2)-positive,and this group of breast cancers is associated with poorer survival outcomes.Although the use of trastuzumab has significantly improved the prognosis of HER2-positive breast cancer patients,treatment failure due to drug resistance still occurs in more than 30% of patients.Therefore,it is important to develop novel drugs to overcome trastuzumab resistance.Oleanolic acid(OA)has significant advantages in the treatment of tumor and inflammatory diseases,but its clinical application is limited by target uncertainty and other issues.Our group and the Dalian Polytechnic University group synthesized a series of oleanolic acid derivatives.The basis of previous work suggests that the novel oleanolic acid derivatives have significant inhibitory effects on a variety of tumors,but the targets and mechanisms of drug action have not been fully elucidated.In this study,we intend to investigate whether the novel oleanolic acid derivative ZQL-3d can play a role in overcoming trastuzumab resistance in HER2-positive breast cancer,and further investigate the mechanism of ZQL-3d’s action in overcoming drug resistance.Methods: 1.To detect trastuzumab sensitivity and resistance in HER2-positive breast cancer cell lines.The antitumor activity of trastuzumab against HER2-positive breast cancer was examined in vitro and in vivo by Western blot,cell proliferation assay(Cell Counting Kit-8,CCK-8),flow cytometry,scratch healing assay,and construction of a nude mouse transplantation tumor model.2.To investigate the role of the novel oleanolic acid drug ZQL-3d in JIMT-1.The effect of ZQL-3d drug on PI3K/m TOR/Akt signaling pathway in JIMT-1 was assessed by CCK-8,scratch healing assay,reactive oxygen(ROS)probe staining assay,flow cytometry,Hoechst 33342 staining,Western blot technique,and fluorescence real-time quantitative PCR technique.3.To explore the effect of drug combination application to overcome the resistance of HER2-positive breast cancer cells to trastuzumab.We examined the anti-tumor effects of drug combination application on resistant strains of xenograft tumors in vitro and in vivo by CCK-8 technique,scratch healing assay,ROS probe staining assay,flow cytometry,fluorescence real-time quantitative PCR technique,Hoechst 33342 staining,Western blot technique and the construction of a trastuzumab-resistant transplant tumor model in nude mice.4.To explore the targets of drug combination application to overcome trastuzumab resistance in HER2-positive breast cancer.We analyzed the change indicators caused by drug combination application by transcriptomic,proteomic,and metabolomic methods,screened the action target of drug combination application stearoyl-Co A desaturase(SCD)by combined multi-omics analysis,simulated the molecular interaction between ZQL-3d drug and SCD protein by using molecular docking technique,used Western blot method and fluorescence real-time quantitative PCR method in We verified that drug co-treatment could regulate SCD in vitro,and detected the effect of drug coapplication on intracellular lipid droplet content using oil red O staining,and detected the alteration of protein and m RNA levels of lipid metabolism-related factors by drug coapplication in vitro using Western blot method and fluorescence real-time quantitative PCR method.5.To explore the molecular mechanism by which SCD protects cells from lipotoxicity-mediated apoptosis and thus inhibits the combination of drugs to overcome trastuzumab resistance.In vitro,SCD overexpression cell model was constructed by lentiviral transfection on the basis of JIMT-1.CCK-8,flow cytometry,Western blot technique,fluorescence real-time quantitative PCR technique,Hoechst 33342 staining,oil red O staining,and construction of trastuzumab-resistant transplant tumor model overexpressing SCD were used to detect the effect of drug combination treatment in vivo changes in antitumor effects on drug-resistant strains of xenograft tumors.Results:1.HER2-positive breast cancer trastuzumab-sensitive and drug-resistant cell lines were clarified.Western blot demonstrated significant overexpression of HER2 protein in both SK-BR-3 and JIMT-1.CCK-8 assay confirmed that the proliferative activity of trastuzumab-sensitive cell lines was significantly lower than that of drug-resistant lines as the concentration of trastuzumab gradually increased.Flow cytometry showed that trastuzumab blocked the cell cycle progression of SK-BR-3 and induced late apoptosis.Scratch healing assays demonstrated that the rate of scratch healing in SK-BR-3 cells slowed down with increasing trastuzumab concentration.In vivo models demonstrated a significant reduction in tumor volume and weight in the S-treat group treated with trastuzumab compared to the control group.In an ex vivo model of JIMT-1 cells,trastuzumab had no significant antitumor effect.2.The novel oleanolic acid drug ZQL-3d had antitumor effects in trastuzumab-resistant cell lines.CCK-8 assay demonstrated that the IC50 of JIMT-1cells treated with ZQL-3d was 1.896 μmol/L at 24 h and 1.194 μmol/L at 48 h.JIMT-1cell lines showed dose-dependent and time-dependent.The scratch healing assay confirmed that the scratch healing rate of JIMT-1 cells slowed down when the concentration of ZQL-3d was increased,and the ratio of green fluorescent cells to live cells in JIMT-1 cells was increased when the concentration of ZQL-3d was increased by ROS probe staining.Hoechst 33342 staining and flow cytometry demonstrated that the ZQL-3d drug significantly induced apoptosis in JIMT-1 cells.The ZQL-3d drug could inhibit PI3K/m TOR/AKT signaling pathway as detected by q RT-PCR.3.Drug combination can overcome trastuzumab resistance in HER2-positive breast cancer.CCK-8 assay suggested that the cytostatic rate of the combination group could reach 32.64% when ZQL-3d was 0.8 μmol/L,which was more than 4 times higher than that of the single drug group.Scratch healing assay confirmed that the rate of scratch healing was significantly slower in the drug combination group,and ROS probe staining showed that the ratio of green fluorescent cells to live cells was significantly higher in the drug combination group,and the level of intracellular reactive oxygen species was increased.Flow cytometry demonstrated that the drug combination was able to block the JIMT-1 cell cycle in the G2/M phase,the expression levels of CDK family proteins and cyclin family proteins were increased and the expression levels of CIP/KIP family and other proteins were decreased in the drug combination group.Hoechst 33342 staining and flow cytometry demonstrated that the drug combination significantly induced apoptosis in JIMT-1 cells,with changes in the expression levels of the caspase family and Bcl-2 family proteins,etc.In the drug combination group,p-PERK,p-e IF2α,and other endoplasmic reticulum stress-related proteins were significantly increased in the drug combination treatment group.The in vivo model demonstrated that the tumor volume and weight of the drug combination group were significantly reduced.4.The drug combination group exerted a role in overcoming trastuzumab resistance by targeting SCD in HER2-positive breast cancer.A total of 330 transcripts,195 proteins,and 37 metabolites were screened by transcriptomics,proteomics,and metabolomics to have differential expression in drug combination treatment.The multi-omics data were integrated to identify 10 cross-over differential indicators,and the most closely interacting and statistically different indicator,SCD,was selected and validated in vitro using Western blot and q RT-PCR techniques.The interaction between ZQL-3d drug structure and multiple sites of SCD protein such as L35,M36,R31,etc.was observed by molecular docking technique through multiple chemical bonds.Oil red O staining demonstrated a significant reduction of intracellular lipid droplets in JIMT-1cells in the drug combination group.The protein and m RNA levels of key enzymes for lipid ab initio synthesis and transporter enzymes were significantly reduced and the m RNA expression levels of fatty acid β oxidation-related enzymes were increased in the drug co-application group as verified by Western blot and q RT-PCR techniques.5.SCD protected cells from lipotoxicity-mediated apoptosis thereby inhibiting the overcoming effect of trastuzumab resistance by the combination.An in vitro SCD overexpression model ov-SCD cell model was constructed and the CCK-8 assay suggested that the cytostatic rate of ZQL-3d alone was 9.92% when the cytostatic rate of the drug combination group was only 15.64%.Flow cytometry results suggested that the percentage of S phase in the drug combination treatment group of ov-SCD cells was5.27% and the percentage of G2/M phase was 40.6%,while the percentage of S phase in the drug combination treatment group of NC group cells was 23.1% and the percentage of G2/M phase was 30.4%.Western blot results demonstrated that the difference in cyclin expression between groups of NC cells was more significant than that of ov-SCD cells.Hoechst 33342 staining and flow cytometry showed that the percentage of apoptotic cells in all phases was less in the drug combination group than in the NC group,and the Western blot showed that p-PERK,p-e IF2α,ATF-4,and CHOP were significantly higher in the drug combination group.The inter-group differences were more significant in NC cells than in ov-SCD cells.Oil red O staining demonstrated a significant reduction of lipid droplets in NC cells compared to ov-SCD cells.The protein expression levels of SCD,Sterol regulatory element binding protein 1(SREBP1),and Fatty Acid Synthase(FASN)were significantly reduced in the drug combination treatment group as verified by Western blot and q RT-PCR techniques,and the inter-group differences were more significant in NC cells than in ov-SCD cells.The in vivo model demonstrated a significant reduction in tumor volume and weight in the drug combination treatment group.the difference between NC groups was more significant than that between ov-SCD groups.Conclusions: 1.The novel oleanolic acid drug ZQL-3d can exert good antitumor effects in HER2-positive breast cancer trastuzumab-resistant cells.2.The combination of drug ZQL-3d with trastuzumab can overcome the resistance of trastuzumab in HER2-positive breast cancer.3.In HER2-positive breast cancer,drug ZQL-3d in combination with trastuzumab exerted the effect of overcoming trastuzumab resistance by targeting binding to SCD.4.The combination of drug ZQL-3d with trastuzumab induces reprogramming of lipid metabolism in HER2-positive breast cancer trastuzumab-resistant cells by regulating lipid de novo synthesis.5.By targeting the protective function of SCD against lipotoxicity-mediated apoptosis,ZQL-3d in combination with trastuzumab was able to overcome trastuzumab resistance. |
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