A. D. Harrison1, R. Jaskula-Sztul1, R. Nair1, A. Dammalapati1, G. Winston-McPherson2, C. M. Schienebeck2, K. Kupcho3, M. Robers3, W. Tang2, H. Chen1 1University Of Wisconsin,Department Of Surgery,Madison, WI, USA 2University Of Wisconsin,Department Of Pharmacy,Madison, WI, USA 3Promega,Madison, WI, USA
Introduction: Carcinoid cancer consists of slowly growing neuroendocrine (NE) tumors within the gastrointestinal tract, which are often metastatic by the time of diagnosis making curative surgery infeasible. They cause excessive production of various bioactive hormones resulting in the carcinoid syndrome and a poor quality of life for these patients. The Notch pathway has proven to be tumor suppressive in NE cancer, resulting in a clinical need for therapeutic options to activate Notch. Herein, we describe the novel and potent histone deacetylase (HDAC) inhibitor, AB3, which induces Notch signaling and alters the malignant neuroendocrine phenotype of carcinoid cancer. We also fully characterized the activity and selectivity of AB3 against HDAC isoenzymes.
Methods: The cytotoxicity of AB3 on the GI carcinoid cell line (BON) was measured by determining the IC50 value via MTT assay (3-(4, 5-Dimethylthiazole-2-yl)-2, 5-dipenyltetrazolium bromide). The effect of AB3 on cell death was investigated by two independent assessments: 1) PI stain-based sub-G1 cell fraction analysis and 2) detection of protein expression of apoptotic markers via Western blot (cyclin D1, survivin, XIAP and cleaved PARP). Protein expression of NE tumor markers ASCL1 and CgA, and Notch1-3 isoforms were also detected by Western blot. Luciferase assay for CBF1 binding (Notch pathway mediator) was used to measure the functional activity of Notch. To characterize the activity and selectivity of AB3, HDAC-Glo TM assay and screening system was used.
Results: We demonstrated that the IC50 value for AB3 treatment in BON cells was 3 µM. The mechanism of growth inhibition was found to be apoptosis, evident by an increased sub-G1 cell fraction (G0 DNA content). In addition, protein expression of anti-apoptotic markers was decreased with concomitant cleavage of PARP. Western blot analysis revealed ASCL1 and CgA reduction. AB3 activated the Notch pathway as evident by an induction of Notch1- 3 isoforms. Furthermore, CBF1 binding analysis revealed that the Notch pathway is activated. We determined that AB3 exhibits selectivity for HDAC 1, 2, and 3, with less inhibitory potency against the HDAC 6, 8, and 10 isoforms.
Conclusion: We demonstrated that AB3 is a potent inhibitor of BON carcinoid cell proliferation at low µM concentrations. The high antitumor activity of AB3 strongly suggests that this HDAC inhibitor may hold great therapeutic potential for the treatment of carcinoid cancer.
