Antitumor Effects And Mechanisms Of Olaparib Combination With Carboplatin And BKM120 On Human Triple Negative Breast Cancer Cells

Background and Objective: Triple-negative breast cancer(TNBC)is a heterogeneous disease group characterized by lack of expression of estrogen receptor(ER),progesterone receptor(PR),and human epidermal growth factor2(Her2).The Cancer Genome Atlas has revealed shared genomic alterations of triple negative BC(TNBC)including extensive copy number alterations,p53 mutations,PI3 K pathway activation,and deficiencies in DNA damage repair and homologous recombination(HR).TNBC is based on the use of traditional cytotoxic drugs with a plethora of side effects,which have not significantly benefited.So far,there is still no standard treatment for TNBC,and more effective targeted treatment for it is urgently needed to be introduced into the clinic.Poly(ADP-ribose)polymerase(PARP)inhibitors,phosphatidylinositol 3-kinase(PI3K)inhibitors,and carboplatin(CBP)have demonstrated efficacy and safety for its use as individual drugs for the treatment of TNBC.The phase I study of the oral PI3 K inhibitor BKM120 and the oral PARP inhibitor Olaparib did not show any drug drug interactions when combination used showing the effect both BRCAm and BRCAwt.However,the effect of a combination of the three type of drugs on TNBC has not been elucidated.The primary objectives of this study were to determine the effects of a combination of CBP,olaparib and NVP-BKM120(BKM120)and investigate their underlying mechanism on TNBC cells.Methods: 1.Drug toxicity of Olaparib?CBP?BKM120 were determined essentially with Cell Proliferation Assay(MTT),the combination use of the three drugs was tested,and the combination index(CI)value was calculated using the Calcu Syn software(Biosoft,Cambridge,UK)based on the Chou and Talalay method in CAL51 and MDA-MB-231 cell lines.2.The capacity of cells to survive and proliferate after Olaparib,CBP and BKM120 treatment on CAL51 and MDA-MB-231 of tri-negative breast cancer cell lines was evaluated by Colony forming assay.3.The effect of three drugs(Olaparib,CBP,BKM120)on the cell cycle of CAL51 and MDA-MB-231 cells were detected by Flow cytometric analysis,for determining the combination of the three drugs whether has an effect on the cell cycle so as to achieve the effect of cell growth inhibition.4.Western blotting was used to test the expression of H2 AX,cleaved PARP,PADPR,ATM,Chk1 and Chk2 proteins in the tri-negative breast cancer cell lines CAL51 and MDA-MB-231 cells treated with three drugs of Olaparib,CBP and BKM120 at different concentrations,respectively,in order to speculate its promoting effect on DNA damage.5.Using Western blotting to detect the the expression of H2 AX,PADPR,53BP1,p27,p21 and p-ATM in the CAL51 and MDA-MB-231 cells in single,double,or three drugs(Olaparib,CBP,BKM120)respectively,to predict the DNA damage of cells and to evaluate the functionality of NHEJ repair axis after treatment with the combination of the three drugs.6.The Immunofluorescence used to indicate the degree of DNA damage in the cells of CAL51 and mda-mb-231 with combination of Olaparib,CBP and BKM120,and the activation of HR and NHEJ pathways of DNA repair.Results: 1.Olaparib,CBP,and BKM120 exhibited dose-dependent inhibition of both MDA-MB-231 and CAL51 cells,however,Olaparib toxicity for both CAL51 and MDA-MB-231 cells was very modest(IC50 44.12 ?M and 95.07 ?M,respectively)while MCF-7 cells was no obvious inhibition of cell growth.2.After treated cells with all three drugs together at different concentrations,the combination indexes were below 1.0,indicating a synergistic inhibitory effect.And no similar synergies were seen in MCF-7 cell lines.3.In the coloby formation,individual drugs inhibited proliferation of TNBC cells to some extent,combination of two drugs improved inhibition of proliferation in MDA-MB-231 cells,although this was only observed for the combination of olaparib and CBP in CAL51 cells.the stronger inhibitory effect was observed when the three drugs were used in combination.4.Cell cycle progression by flow cytometry showed that the strongest effect on the cell cycle blockage was observed when the three drugs were combined;CAL51 cells showing a 20.6% increase of cells in G2/M,and MDA-MB-231 cells a 14.2% increase at the S phase.The cells in the G0/G1 phase of the two cell lines after three drugs was significantly reduced compared with the control group.However,when comparing the OLA+CBP group and the three drugs group,the proportion of G1 stage cells was significantly increased due to the addition of BKM120.5.The phosphorylated levels of H2 AX increased in both MDA-MB-231 and CAL51 cells after treatment with each of the three drugs,whereas the levels of poly(ADP-ribose)polymer(PADPR),which is synthesized by PARP,decreased in both cell lines,and,as expected,more dramatically after addition of BKM120.Phosphorylation of checkpoint kinases Chk1 and Chk2,whose activation by ATM results in cell cycle arrest and cell death(confirmed by PARP cleavage),increased also after treatment with olaparib,CBP and BKM120.DNA damage was more obvious with the increase of drug dosage.6.The expression of 53BP1,p53 and p-ATM was increased in both MDA-MB-231 and CAL51 cells treated with a combination of the three drugs,which proved that the combination of the three drugs led to the increase of the activation of the NHEJ repair pathway.Compared with OLA+CBP group,the expression of p27 and p21 after three-drug treatment was significantly increased,reflecting the addition of BKM120,which increased the inhibition of PI3 K pathway and activated G1 phase block of cell cycle.Conclusion: The combination of CBP,olaparib and BKM120 has synergistic inhibitory effects on both MDA-MB-231 and the CAL51 TNBC cell lines.The combined use of the three drugs,through the synergistic effect of DNA damage,inhibits the homologous recombinant repair(HR)pathway,increases its non-homologous terminal connection(NHEJ)pathway for DNA repair,thus leading to increased DNA damage and decreased repair ability,ultimately enhances the killing effect on TNBC cells.This suggests a strong rationale for the exploration of using Olaparib in combination with CBP and BKM120 for patients with TNBC.