Abstract A39: The marine natural product manzamine A targets vacuolar ATPases and autophagy in pancreatic cancer cells

Background and Aims: Vacuolar ATPases are proton transporters located in the membranes of intracellular compartments such as lysosomes, which play an important role in the autophagic pathway. Vacuolar ATPases play a crucial role in drug resistance and tumor metastasis, by changing the pH gradient between intracellular organelles and the cytoplasma or between extracellular and intracellular compartments. Thus, vacuolar ATPases may represent a novel target for anti-cancer strategies. Manzamine A, a marine sponge derived alkaloid, was recently shown to have activity against pancreatic cancer cells. To elucidate the mechanism of action of manzamine A, its effect on vacuolar ATPases and the autophagic pathway was studied. Methods: The HIP/HOP (haploinsufficency profiling/homozygous profiling) assay to chemogenomically profile manzamine A was conducted at Novartis. Results suggested that manzamine A is an uncoupler of vacuolar ATPases. To confirm this function, expression of vacuolar ATPases in pancreatic cancer cell lines was analyzed by immunocytochemistry in the presence or absence of manzamine A. To evaluate the effect of manzamine A on vacuolar ATPase proton pump activity/ lysosomal acidification, cells were stained with Lysosensor pH indicator followed by detection of acidic lysosomes by immunoflorescent staining and flow cytometry. To analyze the effect of manzamine A on autophagy, pancreatic cancer cells were treated with manzamine A in presence or absence of the autophagy inhibitor bafilomycin, followed by the detection of the autophagy markers, Light Chain 3 (LC 3-II) and Sequestosome 1 (p62/ SQSTM1) by Western blot analysis. Results: Treatment of four pancreatic cancer cell lines (AsPC-1, MIA-PaCa2, BxPc-3, Panc-1) with manzamine A had no effect on expression of vacuolar ATPases. However, it resulted in an increase of intra-lysosomal acidity, which suggests interference with proton pump activity of vacuolar ATPases. Manzamine A significantly increased the LC 3-II and p62/SQSTM1 levels in AsPC-1 cells. Treatment with manzamine A in combination with bafilomycin A1 did not change the levels of LC 3-II when compared to cells treated with bafilomycin A1 alone, indicating that manzamine A is a potential inhibitor of autophagy (inhibitor of autophagosome turnover). Conclusion: Results indicate that manzamine A interferes with proton pump activity of vacuolar ATPases in pancreatic cancer cells, induces an increase of intralysosomal acidity, and affects autophagosome turnover, making it a potential inhibitor of autophagy likely to act at the level of autophagosome-lysosome fusion. Citation Format: Georgios Kallifatidis, Dominic Hoepfner, Sven Schuierer, Nicole Hartmann, Esther Guzman, Amy Wright. The marine natural product manzamine A targets vacuolar ATPases and autophagy in pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A39.