Abstract A170: Analysis of marine sponge Cribrochalina vasculum compounds demonstrate selective antitumor properties by activation of intrinsic apoptotic signaling and impaired growth factor receptor signaling cascades.

Abstract

Abstract Introduction and Aim of Study: Marine derived compounds have been explored and considered as possible antitumor agents. Some natural products from marine organisms are currently in clinical trials, such as: Eribulin, an analogue of halichondrin B, isolated from the marine sponge Halichondria okadai, which is approved by FDA for the treatment of metastatic breast cancer; and Trabectedin, from Ecteinascidia turbinate, approved for treatment of soft tissue sarcomas and ovarian cancer also in Europe. In this study we analyzed extracts of the sponge Cribrochalina vasculum for the inhibition of tumor growth and hence hold potential to be used as novel antitumor agents. Material and Methods: Extracts were obtained from the Caribbean sponge Cribrochalina vasculum. The tumor specific cytotoxicity of the extracts was determined by MTT assay using non-small cell lung carcinoma (NSCLC) cells and normal lung fibroblasts WI-38. The active crude extract showing tumor specific toxicity was fractionated by HPLC to obtain pure compounds and their structures elucidated by physiochemical methods. Various concentrations of the pure compounds were rescreened on several non-small and small lung carcinoma cell lines (NSCLC and SCLC), on ovarian carcinoma cells as well as on normal lung fibroblasts WI-38. Induction of apoptosis was assessed as caspase-3, caspase-9 and PARP cleavage on western blot. To detect and quantify apoptotic morphology, cells treated were stained with DAPI and examined using a fluorescence microscope. Conformational change in proapoptotic protein Bak in response to treatment for 16 h was examined in NSCLC U-1810 using flow cytometric analyses. Cell cycle distribution was examined in NSCLC and diploid fibroblast by propidium iodide (PI) staining, using a flow cytometer. Results: Screening of crude extracts from C. vasculum identified certain extracts that showed tumor specific toxicity while having no effect on diploid fibroblasts. From these extracts purified compounds were identified which showed antitumor activity in both NSCLC, SCLC as well as in ovarian carcinoma cells. The purified compounds were found to cause a time dependent increase in activation of apoptotic signaling in NSCLC cells, but not in normal fibroblasts. Thus exposure of NSCLC cells to these compounds caused cleavage of caspase-9, caspase-3 and PARP, as well as induction of apoptotic cell morphology and Bak conformational change. Cell cycle analyses revealed that these compounds caused a pronounced block in the G2/M phase of the cell cycle of NSCLC cells whereas normal fibroblasts were not influenced. In vivo analyses of the compounds in mice tumor models is ongoing. Conclusion: We have identified, by screening extracts from the sponge C. vasculum, compounds that hold promise as antitumor agents in NSCLC and SCLC as well as in ovarian carcinoma. Further work aims to identify putative cellular targets of these compounds within tumor cells. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A170. Citation Format: Ana Zovko, Kristina Viktorsson, Petra Hååg, Micha Ilan, Dimitry Kovalerchick, Shmuel Carmeli, Andrea Alimonti, Rolf Lewensohn. Analysis of marine sponge Cribrochalina vasculum compounds demonstrate selective antitumor properties by activation of intrinsic apoptotic signaling and impaired growth factor receptor signaling cascades. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A170.