Abstract 2533: Synergistic interactions between topotecan and resveratrol in melanoma
Authors: Parmakhtiar Basmina , Trapp Valerie , Mossavat Maliheh , Eng Michael , Harris Mitchell 
Published: June 2012

Abstract
Reactive oxygen species (ROS) and the hypoxic microenvironment of skin may foster melanoma initiation and progression by promoting HIF mediated blockade of p53 tumor suppressor activity. Both ROS and hypoxia stabilize HIF-1α, thereby promoting angiogenesis and suppressing apoptosis. HIF functions in opposition to p53, which suppresses angiogenesis and promotes apoptosis. We previously reported that topotecan (TPT) inhibits HIF1α stabilization, leading to down regulation of vascular endothelial growth factor (VEGF). We have also found that resveratrol (RSV) depresses HIF levels and opposes angiogenesis. The aim of this study was to investigate the effects of TPT in combination with RSV on melanoma cell lines and primary melanoma tumors. We found that HIF-1α was stabilized in melanoma even under normoxic conditions, rising to significantly higher levels under hypoxic conditions (3- to 9-fold). We evaluated these agents against melanoma under normoxia and hypoxia (1% O2) in vitro using the XTT assay. Under normoxic/hypoxic conditions IC50 values for TPT on melanoma lines A375, YUZAZ6, and M14 were 20/21.7 nM, 94/32 nM, and 250/50nM, respectively and 246/56.54 nM for the MELV2 primary tumor. IC50 values for RSV were 25.3/33.4 uM, 22/17.8 uM, 75/65 uM, and 217/22.8 uM, respectively. These agents were synergistic (Calcusyn analysis, combination index (CI) &lt;1) against all three melanoma cell lines, with CI values of 0.5/0.709 (A375), 0.53/0.658 (YUZAZ6), 0.703/.850 (M14). Analysis of HIF and p53 by ELISA after drug treatment showed profound effects. A375 HIF levels dropped 6.7-fold after RSV, 2.2-fold after TPT, and 14.9-fold after their combination. Similar results were noted for M14 and YUZAZ6 cells. RSV and TPT treatment of hypoxic cells normalized HIF levels to those seen under normoxic conditions. However, RSV upregulated hypoxic A375 p53 by 2.8-fold, with a more modest increase of 1.3 fold in YUZAZ6 cells. M14 cells, which have mutant p53 and baseline p53 levels 5-fold higher than those in A375, showed a 24% decrease in p53. Upregulation of p53 was associated with increased apoptosis (Annexin V binding). Co-immunoprecipitation experiments with monoclonal p53 antibody showed that HIF-1α directly binds to p53, with decreased binding after 50uM resveratrol treatment. Furthermore, VEGF expression was suppressed and TSP-1, an anti-angiogenic protein promoted by p53, was increased after TPT and RSV. These data indicate that HIF binds to and blocks p53 function in melanoma cell lines. We postulate that HIF:p53 interactions in melanocytes are increased after ROS and hypoxia in skin and blockade of p53 tumor suppressor function may contribute to development and progression of malignant melanoma. TPT and RSV modulate this interaction in a favorable manner, blocking HIF protein accumulation and downstream transcriptional activity while restoring functional p53. Clinical evaluation of these agents is warranted.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2533. doi:10.1158/1538-7445.AM2011-2533