Abstract B44: Panobinostat, a novel histone deacetylase inhibitor, enhances cisplatin and etoposide-mediated apoptosis in Burkitt's lymphoma cells.
Authors: dos Santos Ferreira Ana Carolina , Fernandes Renan Amphilophio , da Cunha Vasconcelos Flavia , Klumb Claudete Esteves , Kwee Jolie Kiemlian 
Published: June 2012

Introduction: Histone Deacetylase Inhibitors (HDACis) have been actively explored as a new generation of anticancer drugs, generally known as epigenetic therapeutics and emerging as promising agents for the treatment of T-cell lymphomas. Panobinostat (Novartis Pharmaceuticals, Basel, Switzerland) is a novel cinnamic hydroxamic acid analogue HDACi that inhibits proliferation and induces apoptosis in tumor cell lines. Recent findings indicate that HDACi repress angiogenesis, a process essential for tumor metabolism and progression, through hypoxiainducible factors (HIF) which are the master regulator of cellular adaptation to hypoxia. In addition, HDACis apoptotic-effect was detected to occur through reactivation of Bim expression, a proapoptotic BH3-only family protein, which has attracted increasing attention as a target for tumor therapy. However, its effects on Burkitt's lymphoma (BL) cells are not entirely elucidated. Here we evaluate the efficacy of combined treatment of HDACi, Panobinostat, with Cisplatin (CDDP) or Etoposide (VP-16) in well characterized BL cell lines in order to analyze the molecular basis affected by the single and combined agents.

Methods: Burkitt's Lymphoma cell lines Raji and Daudi were maintained in RPMI culture medium and incubated with Panobinostat 0.1nM combined with CDDP 1.0 and 2.5μM or VP-16 0.1 and 0.3 μM during 24h for Raji and 48h for Daudi cells. Apoptosis and mitochondrial membrane potential (MMP) were accessed by flow cytometry using Annexin-V/PI and DiOC6 respectively. Drug interaction was calculated by Fischel et al., 2005 method for determining combination index (R). Pro-Caspase-3, Pro-Caspase-9 and PARP cleavage was analyzed by western blotting using β-actin as loading control. Real-Time RT PCR was used to evaluate Bim and HIF-1α gene expressions.

Results: Apoptosis was enhanced by the combined treatments (Panobinostat/CDDP and Panobinostat/VP-16), when compared with single treatment. The combination of Panobinostat and VP-16 induced apoptosis in 59,9% of Raji cells compared to 20,4% for Panobinostat alone and 33.3% for VP-16 0.3 μM alone resulting in synergistic R value of 0.77. Moreover, in Daudi cell line we observed an additive effects (R=1.1) of both combinations. The apoptosis effects were followed by MMP decreased levels in a dose-dependent manner and by cleavage of pro-caspase-3, pro-caspase-9 and PARP in both BL cell lines. Additionally, apoptotic profile was correlated with Bim and HIF-1α mRNA upregulation.

Conclusions: These pre-clinical data suggests the potential therapeutic role of Panobinostat for the treatment of BL. In order to identify and validate possible targets and/or biomarkers for these combined treatments further studies are in progress.

Financial support: Novartis FAF2301422, Associação Vencer FAF2301402, INCT para Controle do Câncer, CNPq 573806/2008-0, FAPERJ E26/170.026/2008 and Ministério da Saúde (M.S.).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B44.