Inpp4B Over-expression Enhances The Anti-tumor Efficacy Of PARP Inhibitor AG014699in MDA-MB-231Triple-negative Breast Cancer Cells

Purpose: The main aim of this work was to evaluate whether over-expression inositol poly-phosphate4-phosphatase type II (INPP4B), an newly found tumor suppressor gene, enhanced the antitumour effect of Poly-(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitor used for the treatment of Triple-negative Breast Cancer (TNBC).Method:TNBC cell line MDA-MB-231was transfected with the INPP4B gene using a lentiviral vector. To determine the effect of the combined therapy, MDA-MB-231transfected and non-transfected cells were exposed to PARP inhibitor AG014699at different concentrations. The levels of expression of INPP4B, AKT, phosphorylated AKT (p-Akt) were performed by western blot assay. The growth-inhibitory effect of over-expression INPP4B and/or PARP inhibitor was assessed by CCK-8assay. Apoptosis modulation and cell cycle distribution was determined by flow cytometric analysis.Result:1. MCF-7cells which have been proved to have high levels of INPP4B were used as positive controls. Result showed that INPP4B in MDA-MB-231cells presented a low expression or non-expression utilizing DNA agarose gel electrophoresis and western blotting method2. The Lenti-INPP4B was constructed to infect MDA-MB-231cells, and Lenti-GFP was used as negative control to reflect transfection efficiency indirectly. Results showed that transfection efficiency reached80%in the group of MOI=20. To confirm the expression of INPP4B at the mRNA and protein levels, real-time PCR and western blotting were performed, respectively. INPP4B mRNA expression in Lenti-INPP4B group was about110times higher than in the control/Lenti-GFP group at MOI of20. Consistently, similar results were obtained by western blotting analysis of INPP4B protein. This indicated that lentivirus made MDA-MB-231cells over-express INPP4B successfully.3. Western blotting analysis showed that levels of p-Akt were significantly decreased upon treatment with over-expression of INPP4B. Furthermore, our results also demonstrated that PARP inhibitor AGO14699increased p-Akt levels in a dose-dependent manner, and when INPP4B was over-expressed, the levels of p-Akt reduced.4. Effect of INPP4B over-expressionin in MDA-MB-231cellsGrowth curve of vector-infected MDA-MB-231cells was detected by CCK-8. A remarkable drop in growth rate was observed when comparing the Lenti-INPP4B group with the control/Lenti-GFP group. This difference increased gradually as time went on. Annexin V-FITC/PI flow cytometric analysis showed that the sum of early apoptosis rate and late apoptosis rate was2.85%and5.48%respectively in the control group and Lenti-INPP4B group, indicating that INPP4B over-expression did not increase apoptosis rate but rather induced growth arrest. As a result, the percentage of G1-stage cells in Lenti-INPP4B group was74.54%, as comparing with62.86%in the control group, indicating that INPP4B over-expression could block cells in Phase Gl.5. Anti-tumor effects of PARP inhibitor AG014699in MDA-MB-231cellsCCK-8results showed that the inhibition effect of PARP inhibitor AGO14699on MDA-MB-231cells was dependent on concentration. Under0.1,1,10,20and40μM concentration for24hours, the cell viability was94.67%,79.35%,73.07%,38.11%and29.45%, respectively. The inhibition effect of PARP inhibitor AGO14699on proliferation of MDA-MB-231cell was dependent on time. Cell viability rate was82.62%、72.51%and40.33%, respectively, after8,36and54hours. Annexin V-FITC/PI flow cytometric analysis showed that the sum of early apoptosis rate and late apoptosis rate was9.98%,53.10%and62.38%, respectively, in1,20and40μM groups. These cells showed a gradual increase in apoptosis rate along with drug concentration increased. PI staining cycle distribution showed that the percentage of G2/M stage cells were60.37%,69.82%and82.93%, respectively, in1,20and40μM groups, indicating PARP inhibitor AG014699could block cell cycle progression in G2/M phase.6. INPP4B overexpression combined with PARP inhibitor in MDA-MB-231cellsThe proliferation inhibition rate in combination treatment group was elevated about10%and35%compared with INPP4B over-expression/PARP inhibitor AG014699alone group after24hours. The combination treatment showed potent anti-proliferative effects in a time-dependent pattern. Cell inhibition rate was29.21%,61.37%and84.62%respectively after8,24and36hours. Annexin V-FITC/PI flow cytometric analysis showed that the sum of early apoptosis rate and late apoptosis rate was17.68%and17.71%respectively in the AGO14699group and the combination of treatment group. Our results showed that the combination treatment did not result in any further significant increase in apoptosis rate compared with PARP inhibitor AG014699alone. Then the cell cycle distribution was detected. PI staining cycle distribution showed that Phase G1ratio was72.54%and72.62%, respectively, in the Lenti-INPP4B group and the combination treatment group. The combination treatment would not affect the cycle distribution which might transfer to G2/M, but cells would still be blocked in G1phase。Conclusion:Our in vitro results indicated that experimental therapeutic strategy based in the combined therapy was greater than obtained using over-expression INPP4B or PARP inhibitor alone, may be of potential therapeutic value as a new strategy for patients with TNBC.