Mechanistic Study Of Compounds On Enhancing The Anti-breast Cancer Effects And Attenuating The Cardiotoxicity Of Doxorubicin Based On A Small Molecule Probe

Objective: Doxorubicin(Dox)is one of the most effective chemotherapeutic agents for breast cancer.Dox-induced cardiotoxicity limits its further clinical applications.In this study,we aimed to screen compounds that can enhance the anti-breast cancer effects and attenuate Dox-induced cardiotoxicity,and the underlying mechanisms would also be investigated.Methods: 1 Effects of BAA combined with Dox on anti-breast cancer and Dox-induced cardiotoxicity MDA-MB-231 cells and H9c2 cells were treated with different concentrations of BAA alone or in combination with Dox for 24 h.The Cell viability was measured by MTT assay,and the cell cytotoxicity was detected by lactate dehydrogenase release assay.Respectively,Western Blot was used to explore the mechanisms of BAA which enhance the anti-breast cancer effects and attenuate the cardiotoxicity of Dox,inhibitors were added to further clarify the mechanisms of BAA.In vivo,zebrafish were distributed into a 12-well microplate,heart function of zebrafish were evaluated by assessing stroke volume and heart rate after co-treatment with Dox and various concentrations of BAA for 36 h.2 The potential screened compounds that synergize with Dox to enhance the anti-breast cancer effects and attenuate cardiotoxicity based on the chemical structure of BAA As the synthesis of BAA was difficult and costly,compounds with high structural similarity were screened based on the chemical structure of BAA.Firstly,MDA-MB-231 cells and H9c2 cells were treated with different concentrations of compounds alone or in combination with Dox for 24 h.Cell morphologies were photographed,the cell viability were evaluated by MTT method and the cell cytotoxicity were detected by lactate dehydrogenase release assay.Secondly,MDA-MB-231 and H9c2 were treated with different concentrations of compounds alone or in combination with Dox for 24 h,cells apoptosis was performed by DAPI staining.3 The mechanistic study of CAPE combined with Dox on anti-breast cancer and Dox-induced cardiotoxicity in vitro Because of high similarity between CAPE and BAA,we suspected that the mechanisms of CAPE may be similar to BAA.Therefore,Western Blot and inhibitors were used to verify the mechanisms of CAPE enhance the anti-breast cancer effects and attenuate the cardiotoxicity of Dox.Data Independent Acquisition(DIA)proteomics were performed to explore the mechanisms of CAPE enhance the anti-breast cancer effects and attenuate the cardiotoxicity of Dox.4 The mechanistic study of CAPE combined with Dox on anti-breast cancer and Dox-induced cardiotoxicity in vivo 4T1 cells were subcutaneously implanted into the left armpit of mice to establish tumor xenografts,the mice were randomly divided into four groups: Vehicle group,Dox group,CAPE group and Dox+CAPE group.All animals were killed at the end of the study,and their tumors and hearts were collected for further analysis.Serum creatine kinase activities and serum lactate dehydrogenase activities were detected by automatic biochemical analyzer.Results: 1 BAA showed synergistic effects with Dox enhance anti-breast cancer and attenuate Dox-induced cardiotoxicity,the underlying mechanisms were involved in ER stress.In MDA-MB-231 cells,different concentrations of BAA combined with Dox further decreased the cell viability.In contrast,BAA attenuated Dox-induced cell injury in H9c2 cells.Western blot analysis showed that BAA significantly upregulated Dox-induced ER stress in MDA-MB-231 cells,4-PBA compromised the synergistic anti-tumor effect of Dox and BAA in MDA-MB-231 cells.By contrast,BAA attenuated Dox-induced ER stress in H9c2 cells,4-PBA could significantly attenuate the protective effect of BAA.These findings indicated that the mechanisms of BAA which attenuate cardiac dysfunction and improve antitumor activity of Dox in breast cancer cells were involved in ER stress.In zebrafish model,it could be seen that the systolic and diastolic function of the ventricle were damaged after Dox treatment,which were partly reversed by BAA,indicated by stroke volume and heart rate,while co-treatment with BAA significantly prevented stroke volume and heart rate in a dose-dependent manner.2 CAPE showed synergistic effects of Dox enhance the anti-breast cancer and attenuate the cardiotoxicity of Dox.Based on the chemical structure of BAA,we found that CAPE also showed similar effects on enhance anti-breast cancer and attenuate the cardiotoxicity of Dox,the activities of CAPE were about 10 times higher than that of BAA.In H9c2 cells,western blot analysis revealed that Dox alone significantly upregluted the expression of ER stress marker proteins,while CAPE co-treatment was obviously reversed Dox-induced ER stress,4-PBA could significantly attenuate the protective effect of CAPE.In MDA-MB-231 cells,the expression of ER stress marker proteins of CAPE co-treatment with Dox were not consistent with BAA.It could be seen that the mechanisms of CAPE combined with Dox to enhance anti-breast cancer effects and attenuate the cardiotoxicity of Dox were not involved in ER stress.3 The mechanisms of CAPE combined with Dox enhance the anti-breast cancer effects and attenuate the cardiotoxicity of Dox were related to the protein processing in endoplasmic reticulum pathway.Mechanistically,our data from the proteomics were found that 11 pathways were screened out which involved in anti-tumor and cardioprotection.We seen that the protein processing in the endoplasmic reticulum had the minimum Q value,the maximum gene ratio and the most number of genes were enriched in this pathway by enriched bubble map analysis.In MDA-MB-231 cells,20 genes were enriched in this pathway;In H9c2 cells,24 genes were enriched in this pathway.Then,this pathway was selected for KEGG visualization and enrichment analysis.It was found that 11 genes expressed in the completely opposite direction in upregulation or downregulation in MDA-MB-231 cells and H9c2 cells.By analyzing the proteins in the protein processing in the endoplasmic reticulum pathway,we found that in MDA-MB-231 cells,CAPE combined with Dox could further upregulate the expression levels of proteins ERp29,Bap31,p-e If2α and TRAF2 in MDA-MB-231 cells.In contrast,CAPE decreased the expression levels of protein processing in endoplasmic reticulum related proteins induced by Dox in H9c2 cells.Western blot analysis showed that CAPE could significantly upregulate the expression of Dox-induced apoptosis marker proteins in MDA-MB-231 cells.By contrast,CAPE decreased the expression levels of Dox-induced apoptosis marker proteins in H9c2 cells.Therefore,these results suggested that the mechanisms of CAPE combined with Dox to enhance the anti-breast cancer and attenuate the cardiotoxicity of Dox may be involved in the pathway of protein processing in endoplasmic reticulum.4 CAPE showed synergistic effects with Dox on enhancing anti-breast cancer and attenuates cardiotoxicity in mice Animal experiments showed that the tumor burden was markedly suppressed by CAPE in combination of BAA.HE staining and serum assays revealed that CAPE alone had no obvious toxicity to the heart of mice.Obviously,our datas showed cardiac damage after treating with Dox,mainly performed by the disorder of cell arrangement and the inflammatory of cell infiltration,and the activity of serum creatine kinase and lactate dehydrogenase were increased.While combined with CAPE,the above-mentioned cardiac damage caused by Dox could be significantly reversed.Conclusion: 1 BAA combined with Dox could significantly enhance the anti-tumor activity in MDA-MB-231 cells and reduce the toxicity in H9c2 cells,at least in part,by mediating ER stress activation.2 BAA protected against Dox-induced cardiotoxicity in zebrafish.3 CAPE was confirmed as a potential compound with double functions similar to that of BAA using chemical structure based screening and indicated experimental validation.4 CAPE showed synergistic effects with Dox on enhancing anti-breast cancer and attenuating cardiotoxicity in vitro.5 Proteomics analysis demonstrated that the protein processing in endoplasmic reticulum was involved in enhanced the anti-breast cancer effects and attenuated Dox-induced cardiotoxicity.6 CAPE combined with Dox could further suppressed tumor growth compared with Dox alone,and CAPE exerted a protective effect on Dox-induced cardiotoxicity in vivo.