CL-amidine In Combination With Docetaxel Enhances Chemosensitivity Of Tamoxifen-resistant Breast Cancer Mechanism Studies

Objective:Tamoxifen?TAM?is an important endocrine therapy for the treatment of estrogen receptor positive?ER+?breast or ovarian cancer.However,many patients eventually develop TAM resistance regardless of the state of the estrogen receptor expression.TAM resistance is a vital issue with unclear pathogenesis for the formation of drug resistance,which has now become a critical factor in the recurrence of breast cancer.Doc,also known as docetaxel,is a synthetic taxane antitumor drug,which has been applied to the treatment of patients with advanced breast cancer for its high anti-tumor activity.However,no studies have been reported on the efficacy of Doc in patients with TAM resistance.The peptidylarginine deiminases?PADs?are Ca²⁺-dependent modification enzymes,which play critical roles in inflammation,apoptosis and aging progression.CL-amidine?CLA?,as a broad inhibitor of PADs family,can block the activity of the enzymes to achieve the purpose of regulating the proliferation,differentiation and invasion of tumor cells.In recent years,it has been reported that the application of molecular targeted small-molecule anticancer drugs in combination with clinical chemotherapeutics can enhance the efficiency of chemotherapeutics to kill cancer cells,even with the reduced drug concentration.This study investigated the sensitivity and mechanism of the small molecular of CLA in sensitizing TAM-resistant breast cancer cells to clinical chemotherapy drug Doc.Methods:TAM-resistant breast cancer?TamR?and 293T cells were cultured in DMEM high glucose medium.The detections of the IC50 of CLA and Doc on TamR cells,followed by the proliferation in the control,Doc?0.1?M?,CLA?25?M?,Doc+CLA groups with or without MHY1485 treatment,were tested by CCK-8assays.The cells were harvested for western blot,immunofluorescence,flow cytometry and quantitative real-time PCR.TamR cells?1×10⁷?were injected into the right upper flank of the female BALB/c nude mice?6-week-old?.Before injection,the estrogen sustained-release capsules were implanted in the neck of these nude mice,which were were randomly divided into control,Doc?10mg/kg?,CLA?50mg/kg?and Doc+CLA groups to detect the size of the transplanted tumors.The status of mTOR pathway,proliferation and the expression of apoptosis-related protein markers were detected by Western blot.Results:As the inhibition of cell proliferation,CLA significantly enhanced the sensitivity of TamR cells to Doc.The combination of CLA and Doc evidently increased the apoptosis rate of TamR cells and arrested the cells in G2/M phase.Furthermore,AKT/mTOR signaling can be inhibited by the combination of CLA and Doc in vitro,after which been decreased by the application of mTOR activator MHY1485 accompanied by increased cell proliferation compared to the non-use of it.The Western blot assay indicated that the increased sensitivity of Doc to TamR cells treated with CLA may ultimately lead to the activation of p53 target genes and the occurrence of apoptosis and autophagy.Similarly,treatment with CLA and Doc could significantly inhibit the growth of transplanted tumors in nude mice.Conclusions:In our present study,we showed it for the first time that CLA significantly enhances the sensitivity of TAM-resistant breast cancer to Doc,which may help the occurrence of apoptosis,cycle arrest and autophagy in vitro and tumorigenensis in vivo.We therefore propose that that CLA can inhibit the PAD2activitation and co-activate p53 target genes with Doc to induce cell cycle arrest,apoptosis and autophagy.