Study On Pt(Ⅳ) Prodrugs Based On Molecular Subtypes Of Breast Cancer And Inflammatory Microenvironment

Breast cancer（BC）is a big killer threatening women’s health.In 2011,the consensus of St.Gallen’s experts for the first time was to analyze the molecular“essence”from morphological“phenomenon”,that is,based on the expression of estrogen receptor（ER）,progesterone receptor（PR）and human epidermal growth factor receptor 2（HER2）molecules etc.,the BC was classified into different subtypes:Luminal A/B,HER2 positive,and triple negative breast cancer（TNBC）.This breakthrough is of great significance for the design of clinically precise treatment regimens.In addition,tumor metastasis and recurrence affected by inflammatory microenvironment have also become the key to clinical BC treatment.Cisplatin,as a first-line anticancer drug,is widely used in clinic,with the advantages of strong drug efficacy and broad anticancer spectrum.However,due to its poor selectivity and solubility,the side effects and inherent/obtained drug resistance problems seriously affect the patient’s treatment.In recent years,with the continuous deepening of platinum drug research,Pt（Ⅳ）stands out in numerous studies due to its unique stability and structural fabricability.Pt（Ⅳ）follows an action mode of prodrug,with kinetic inertness and less toxicity.Meanwhile,the introduction of axial ligands enhanced drug efficacy,changed original uptake mode,increased accumulation,improved bioavailability,overcame resistance of Pt（Ⅱ）drug etc.After entering cells,the ligands and parent nucleus of Pt（Ⅱ）drugs are released,which improves the traditional administration defects and effectively exerts the synergistic anticancer effect.Therefore,a series of molecules with unique pharmacological activity on BC subtypes and tumor microenvironment were selected as ligands and introduced into the axial positions of Pt（Ⅳ）,aiming to obtain some targeting precision drugs.These protocols not only effectively overcome the defects of BC clinical treatment,but also reduce the toxic and side effects caused by chemotherapy.Objective:In view of the molecular subtypes and tumor microenvironment characteristics of BC and the clinical treatment defects of platinum drugs,a series of targeted Pt（Ⅳ）prodrugs were designed and synthesized,displaying high effective synergistism in BC cells and low systemic toxicity.Methods:Herein,the derivatives melatonin,etodolac,carprofen,sulindac and ketoprofen,with（or modified）carboxyl and amino active groups,were selected and condensed with oxoplatin to obtain Pt（Ⅳ）prodrug candidates,respectively.The obtained compounds were physicochemically characterized by ¹H/¹³C NMR,HRMS,HPLC etc.MTT,ICP-MS,flow cytometry,western blot,Hoechst staining,comet assay,immunofluorescence,healing and invasion assay etc.were employed to explore the action mechanism of prodrugs.A tumor-bearing nude mouse model was established and H&E stained sections were produced to evaluate the anti-tumor activity and in vivo toxicity of prodrugs.Results:1.ER-positive BC is the most common disease in clinical,and ER exerts a biological function through combining with estrogen.Melatonin,as an endogenous product in human body,is closely related to BC occurrence and has a significant regulatory effect on estrogen-related enzymes and receptors.Following this,four melatonin-Pt（Ⅳ）molecules,Melatplatins,were firstly designed and synthesized.Among them,compound 3 showed an outstanding selectivity for ER⁺MCF-7 cells and potent cytotoxicity（nearly 100 times higher than cisplatin）,which is consistent with the increased receptor affinity and inhibitory to ER expression of 3.High drug-accumulation and synergistic effect of Pt-core and ligand MT released,induced S-phase arrest and DNA damage,and up-regulatedγH2AX and P53.In vivo,compound 3 caused severe necrosis of tumor tissue and alleviated systemic toxicity cpmpared to cisplatin.In addition,MT introduction to oxoplatin effectively activated immune system,promoted lymphocyte proliferation in spleen,and realized the possibility of immune-chemotherapy.2.To break through the clinical treatment defects of TNBC,the Pt（Ⅳ）prodrug Ketoplatin was firstly designed and synthesized,which showed a great selectivity and strong cytotoxicity for MDA-MB-231（TNBC）cells.Ketoplatin largely accumulated and then released Pt-core and ketoprofen in cells.On the one hand,Ketoplatin induced severe DNA damage,S-phase block,and promoted cells apoptosis.On the other hand,it significantly inhibited the expression of COX-2,vimentin and N-cadherin to reverse EMT process,and effectively inhibited the metastatic and invasive ability of tumor cells.In addition,Ketoplatin exerts an efficient and low-toxic anticancer effect in vivo.3.The inflammatory microenvironment is a basis for protecting and maintaining tumor development,recurrence and metastasis.Cyclooxygenase-2（COX-2）,as a pro-inflammatory factor,is closely related to it and is over-expressed in most cancer tissues.Following this,the bio-active mechanism of three NSAID-Pt（Ⅳ）molecules developed by our group were studied.Among them,Etoplatin showed an excellent inhibitory effect on MCF-7 cells,which improved the inflammation of the tumor microenvironment via inhibiting COX-2.And Etoplatin reduced vimentin and MMP-2 expression,and up-regulated E-cadherin to reverse EMT mechanism to inhibit the invasion and metastasis of tumor cells,and displayed a high effective and low-toxic anti-cancer effect in tumor-bearing mice.Conclusions:This paper designed and synthesized a series of Pt（Ⅳ）molecules for different subtypes and inflammatory microenvironment of BC.They changed the original action mode of cisplatin,played an excellent cytotoxicity in target cells,corrected the expression of oncogenes,induced severe DNA damage,cell cycle arrest and apoptosis,regulated tumor microenvironment,inhibited the metastasis and invasion of cancer cells,overcame tumor drug-resistance,activated body immunity,and played an high efficient and low-toxic antitumor effect in tumor-bearing mice.Thses result will provide an important theoretical basis for the later clinical application of Pt（Ⅳ）prodrug molecules.