| BackgroundBreast cancer is the most commonly diagnosed cancer in women,which is a serious threat to women in the whole world.Breast cancer can be classified into different subgroups depending on the expression of ER,PR or HER2.Since TNBC is lack of ER,PR or HER2 expression,the TNBC patients can’t benefit from endocrine or anti-HER2 therapy.Chemotherapy is a relatively effective therapeutic option for TNBC,and among these chemotherapy agents,doxorubicin is one of the most commonly used for TNBC.However,during clinical doxorubicin chemotherapy,a common and difficult problem is that patients can develop chemoresistance,which will induce chemotherapy failure or even tumor recurrence and metastasis.Therefore,doxorubicin resistance is an urgent problem that needs to be solved in the current clinical chemotherapy of TNBC patients.To address these issues,combined chemotherapy has become widely used in chemotherapeutic regimens.Traditional Chinese medicine has been applied to combined chemotherapy,and it can improve chemotherapeutic efficacy in clinical treatment.The essential oil of Rhizoma Curcumae is a traditional Chinese medicine,recorded in Pharmacopoeia of China,which has been widely applied to the therapy of tumor and has also achieved good results in clinical application.Currently,it has been reported that the essential oil of Rhizoma Curcumae can enhance chemotherapeutic efficacy in breast cancer.However,few reports have systematically illuminated the mechanism by which it improves chemotherapeutic efficacy,and it is still needed to be further clarified.Because the essential oil of Rhizoma Curcumae contains lots of active ingredients,it has brought difficulties to clearly clarify its mechanism and specific targets,which limits its use in clinical precision treatment and further research.Therefore,based on the theory of molecular Chinese pharmacology and modern pharmacological research methods,this project intends to clarify the main active components of the essential oil of Rhizoma Curcumae that enhance the sensitivity of doxorubicin in TNBC through a combined drug evaluation system.And we use this component as an entry point to clarify the effective mechanism by which it reverses chemotherapy resistance.These works would provide new methods and new ideas for improving the effect of breast cancer chemotherapy.AimsThe purpose of this study is to explore the sensitization effect of the essential oil of Rhizoma Curcumae and its main active ingredient,curcumol,on triple-negative breast cancer doxorubicin chemotherapy.And we intended to systematically elucidate the underlying mechanism through in vitro cytology experiments,in vivo animal experiments,and bioinformatics analysis,and to provide new ideas for improving the chemotherapy effect of TNBC treatment.Methods1.Combined drug evaluation was performed to identify the main active ingredients of the essential oil of Rhizoma Curcumae on chemosensitization of TNBC cells.2.Through in vitro experiments such as CCK8,cell apoptosis analysis,DNA damage comet assay,and in vivo experiment such as nude mice xenograft,the enhancement effects of curcumol on chemotherapy of doxorubicin in TNBC were evaluated.3.mi RNA-seq was used to analyze the main mi RNA molecules involved in doxorubicin chemosensity regulation of curcumol,and then we changed the expression of mi R-181b-2-3p through cell transfection experiments to clarify its effect on the efficacy of doxorubicin chemotherapy in TNBC cells.4.The mi R-181b-2-3p overexpressed cell was conducted and then collected for m RNA-seq;with the help of and bioinformatics analysis,the target genes regulated by mi R-181b-2-3p were identified.5.Reporter gene experiment was performed to validate the interaction between mi R-181b-2-3p and the target gene ABCC3.6.Transfected MDA-MB-231/ADR cell with mi R-181b-2-3p mimics/inhibitor,and western-blot,flow cytometry were used to analyzed whether ABCC3 expression,and intracellular doxorubicin drug efflux were affected by mi R-181b-2-3p.7.Verify the regulation effect of curcumol on mi R-181b-2-3p-ABCC3 axis by q RT-PCR,western-blot,flow cytometry and cell transfection.8.PDX animal models were constructed to evaluate the potential clinical efficacy of doxorubicin combined with curcumol;q RT-PCR and western-blot were used to measure the regulation effects of curcumol on mi R-181b-2-3p and ABCC3 expression in vivo.9.Transcription factor microarray was applied to detect activation of transcription factors in MDA-MB-231/ADR cells after curcumol treatment,and the transcriptional regulation of curcumol on mi R-181b-2-3p expression were preliminary detected by Ch IP experiments.Results1.Among the essential oil of Rhizoma Curcumae and its five main components,curcumol showed strongest synergistic effects with doxorubicin on the TNBC cells chemotherapy since it had the minimum CI value.2.Compared with doxorubicin alone administration group,curcumol-combined administration could significantly enhance the effect of doxorubicin on inhibiting TNBC cells proliferation,promoting cell DNA damage and inducing cell apoptosis;curcumol also enhanced the antitumor effect of doxorubicin in the breast fat pad-injection nude mouse model.3.mi RNA-seq showed that mi R-181b-2-3p was up-regulated by curcumol compared with the control group,and mi R-181b-2-3p was at a lower expression in doxorubicin-resistant cells;when mi R-181b-2-3p was up-regulated,the chemosensitivity of TNBC cells to doxorubicin increased;while mi R-181b-2-3p was down-regulated,the chemosensitivity of TNBC cells to doxorubicin decreased.4.Through m RNA-seq and bioinformatics analysis,transmembrane drug transporters were found to be regulated by mi R-181b-2-3p,then ABCC3 was founded to be significantly down-regulated.5.Reporter gene experiment confirmed that mi R-181b-2-3p could inhibit the expression of ABCC3 by directly targeted 3’UTR of ABCC3 gene,compared with the empty plasmid control group and the reporter gene plasmid group with mutant ABCC33’-UTR sequence.6.Western-blot and cell flow cytometry results showed that mi R-181b-2-3p reduced intracellular doxorubicin efflux by down-regulate the expression of ABCC3 protein on the cell membrane.7.Curcumol promoted mi R-181b-2-3p expression and inhibited ABCC3 expression in MDA-MB-231/ADR cells;while when mi R-181b-2-3p was interfered by transfection,curcumol could no more regulate ABCC3 expression.8.The combined treatment of curcumol could significantly enhance the antitumor effect of doxorubicin in PDX models,and curcumol could regulate the expression of mi R-181b-2-3p and ABCC3 in vivo.9.Curcumol activated the transcription factor NFAT1,and NFAT1 could target the promoter area of mi R-181b-2-3p to enhance its transcription;while the use of NFAT1 inhibitor blocked the up-regulation effect of curcumol on mi R-181b-2-3p expression.Conclusion1.In this study,through in vitro and in vivo experiments,we systematically verify that curcumol is the key active ingredient of the essential oil of Rhizoma Curcumae and it could enhance doxorubicin sensitivity in TNBC breast cancer.2.mi R-181b-2-3p is low-expressed in doxorubicin-resistant TNBC cells,and up-regulation of mi R-181b-2-3p could enhance doxorubicin sensitivity,which indicates that mi R-181b-2-3p plays an important role in doxorubicin-resistant of TNBC.3.Through in vitro and in vivo experiments,we verify curcumol could reverse doxorubicin-resistance of TNBC by regulating mi R-181b-2-3p-ABCC3 axis.4.Further research showed that,in TNBC cells,curcumol promotes mi R-181b-2-3p expression by activating the transcriptional activity of NFAT1. |
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