Chidamide Reverses Fluzoparib-resistance In Triple-negative Breast Cancer Cells By Down-regulating RAD51

Background:Triple negative breast cancer(TNBC)is difficult in the treatment of breast cancer.The emergence of poly-ADP-ribose polymerases inhibitor(PARPi)for DNA damage repair has opened a new chapter of targeted therapy for TNBC,but the drug resistance of tumor cells to PARPi due to the adaptability of tumor has become a new challenge for anti-tumor therapy.The reversal of drug resistance to PARPi has also become a hot and difficult subject in anti-tumor research.The most common cause of PARPi resistance is overexpression of DNA damage repair proteins.Histone deacetylase inhibitor(HDACi)can regulate DNA damage repair proteins related to angiogenesis.Therefore,it is theoretically feasible to reverse tumor cell resistance to PARPi by regulating DNA damage repair protein through HDACi.Objective:To study the mechanism of reversal of TNBC resistance to fluzoparib(PARPi)by chidamide(HDACi).Methods:1.The TNBC cell lines HCC1937-FR and MDA-MB-468-FR resistant to fluzoparib were constructed by concentration gradient increasing induction method.2.Key drug-resistant genes were identified by gene sequencing of drug-resistant cell lines.3.Fluzoparib-resistant cell lines were treated with different concentrations of chidamide and the appropriate concentration was selected according to the inhibition rate of the drug.4.Fluzoparib-resistant cell lines were treated by fluzoparib combined with optimal concentration of chidamide.MTT assay was used to determine the changes in the activity of fluzoparib-resistant cells in the combined of chidamide and fluzoparibtreated group and the fluzoparib-single group,and the IC50 value and combination index were calculated.5.The effects of fluzoparib and chidamide on the migration of fluzoparib-resistant cell lines HCC1937-FR and MDA-MB-468-FR were detected by scratch test.6.The effects of fluzoparib and chidamide on the invasion ability of fluzoparibresistant cell lines HCC1937-FR and MDA-MB-468-FR were examined by Transwell assay.7.The effect of fluzoparib and chidamide on the cycle and apoptosis and of fluzoparib-resistant cell lines HCC1937-FR and MDA-MB-468-FR were detected through flow cytometry.8.Western-blot was utilized to detect the changes of protein expression of fluzoparib and chidamide in fluzoparib-resistant cell lines HCC1937-FR and MDAMB-468-FR.9.Xenograft model was utilized to explore the effect of fluzoparib and chidamide on Balb/c nude female mice.Results:1.HCC1937-FR and MDA-MB-468-FR had acquired resistance to fluzopalil,and the resistance index was 10 and 5.33,respectively,compared with parental cells.2.Bioinformatics analysis identified the key drug resistance genes as RAD51,MRE11,POLA1,RAD54 L,RFC4 and MCM10.3.The inhibition rates of HCC1937-FR and MDA-MB-468-FR were smaller at3μg/m L and 6μg/m L of chidamide,and the subsequent experiments were performed at this concentration.4.Compared with single drug fluzoparib,the IC50 of HCC1937-FR and MDAMB-468-FR against fluzoparib decreased from 60μg/ m L to 9.6μg/ m L and 80μg/ m L to20μg/ml,respectively,in combination with chidamide(P<0.05).5.Wound-healing assay results revealed that fluzoparib and chidamide could significantly inhibit the migration of fluzoparib-resistant cancer cells(P<0.05).6.Transwell assay results indicated that fluzoparib and chidamide could significantly inhibit the invasion of fluzoparib-resistant cancer cells(P<0.05).7.Flow cytometry results showed that fluzoparib and chidamide could significantly promote the apoptosis of fluzoparib-resistant cancer cells and arrest the cell cycle at G2/M(P<0.05).8.Western blot analysis showed that chidamide combined with fluzoparib could significantly inhibit the expression of RAD51 and MRE11 in fluzoparib-resistant cancer cells(P<0.05).9.Animal model assay results shows that fluzoparib and chidamide could inhibit the growth of breast cancer xenograft in nude mice(P<0.05).Conclusions:This study found that TNBC cells with drug resistance to fluzoparib combined with chidamide could restore the sensitivity of cells to fluzoparib.Fluzoparib combined with chidamide could inhibit the proliferation,invasion and metastasis of drug-resistant cells and promote apoptosis of drug-resistant cells,and also showed good anti-tumor effect in vivo experiments.Fluzoparib combined with chidamide reverses fluzoparib resistance in TNBC cells by down-regulating the expression of RAD51 and MRE11.