Abstract A43: The Pi3K inhibitor GSK2126458 is highly toxic in breast ductal carcinoma
Authors: Alia Albawardi, Saeeda Almarzooqi, Mohamed Al Bashir, Abdul-Kader Souid
Journal: Clinical Cancer Research (2012)

Abstract Introduction: Breast carcinoma has been shown to harbor mutations that up-regulate phosphatidylinositol 3 kinase (PI3K) signaling pathway. Thus, targeting this pathway is expected to impair tumor survival. The aim of this work was to investigate the cytotoxicity of GSK2126458 (a highly potent and selective inhibitor of PI3K) in breast ductal carcinoma from a patient. Methods: A breast ductal carcinoma was collected immediately following resection. Tumor sections (0.5×0.5×0.1 cm) were immersed in ice-cold RPMI medium gassed with 95% O2:5% CO2. The samples were incubated at 37oC in RPMI (continuously gassed as above) with and without 50 nM GSK2126458 (Selleck Chemicals, cat. #S2658) for 90 min (final volume = 2.0 mL). At the end of the incubation period, 37 μM Ac-DEVD-AMC (a caspase-3 substrate) was added with and without the pan-caspase inhibitor zVAD-fmk (32.1 μM, added 10 min before the substrate). The reactions were allowed to continue at 37oC for 20 min before tissue processing for histology, immunohistochemistry for cleaved caspase -3 (Cell Signaling Technology, kit #8120), cellular respiration and caspase activation. Mitochondrial O2 consumption was determined as function of time from the phosphorescence decay rate (1/T) of Pd(II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. For caspase activity, the Ac-DEVD-AMC cleavage reaction was quenched with tissue disruption and passages through a 27-G needle. The supernatants were collected by centrifugation (∼16,300g for 90 min) through Microcentrifuge Filter (nominal molecular weight limit = 10,000 Dalton, Sigma©), separated on HPLC, and analyzed for the free fluorogenic AMC moiety. Results: This 73-year-old female had invasive ductal carcinoma (grade 3), measuring 4.5 cm. The tumor expressed estrogen and Her2-neu receptors. Ki-67 score was 70%. Compared to untreated tumor, the treated tumor revealed cellular disintegration with loss of cohesion, cytoplasmic vacuolization and numerous apoptotic bodies. Positive cleaved caspase-3 staining was noted in 10% of untreated tumor and 20% in treated tumor. The initial rate of mitochondrial O2 consumption (kc, in μM O2 min−1 mg−1) was 0.17. At 2 h, the value of kc for untreated tumor was 0.019 and for treated tumor 0.033. The AMC peak area (arbitrary units, reflecting intracellular caspase activation) in untreated tumor was 460,886, in tumor treated with GSK2126458 was 7,234,911 (15.7 fold increment), and in tumor treated with GSK2126458 plus zVAD-fmk was 1,523,682 (79% inhibition), confirming the cleavage of Ac-DEVD-AMC was mainly mediated by caspases. Conclusions: GSK2126458 induced remarkable necrosis in invasive ductal carcinoma. This preliminarily observation supports the role of PI3K activation in breast carcinogenesis and possible therapeutic utility of PI3K inhibitors in the treatment of invasive ductal carcinoma. The described in vitro methodology provides a rapid assessment of the cytotoxicity of PI3K/PTEN/Akt/mTOR inhibitors.