renal cancer-selective Englerin A induces multiple mechanisms of cell death and autophagy (vol 32, 57, 2013)

Williams et al. Journal of Experimental & Clinical Cancer Research 2013, 32:57 http://www.jeccr.com/content/32/1/57 RESEARCH Open Access Renal cancer-selective Englerin A induces multiple mechanisms of cell death and autophagy Richard T Williams 1 , Alice L Yu 1,2,3 , Mitchell B Diccianni 1 , Emmanuel A Theodorakis 4 and Ayse Batova 4* Abstract Renal cell carcinoma (RCC), the most common malignancy of the kidney, is refractory to standard therapy and has an incidence that continues to rise. Screening of plant extracts in search of new agents to treat RCC resulted in the discovery of englerin A (EA), a natural product exhibiting potent selective cytotoxicity against renal cancer cells. Despite the establishment of synthetic routes to the synthesis of EA, very little is known about its mechanism of action. The results of the current study demonstrate for the first time that EA induces apoptosis in A498 renal cancer cells in addition to necrosis. The induction of apoptosis by EA required at least 24 h and was caspase independent. In addition, EA induced increased levels of autophagic vesicles in A498 cells which could be inhibited by nonessential amino acids (NEAA), known inhibitors of autophagy. Interestingly, inhibition of autophagy by NEAA did not diminish cell death suggesting that autophagy is not a cell death mechanism and likely represents a cell survival mechanism which ultimately fails. Apart from cell death, our results demonstrated that cells treated with EA accumulated in the G 2 phase of the cell cycle indicating a block in G 2 /M transition. Moreover, our results determined that EA inhibited the activation of both AKT and ERK, kinases which are activated in cancer and implicated in unrestricted cell proliferation and induction of autophagy. The phosphorylation status of the cellular energy sensor, AMPK, appeared unaffected by EA. The high renal cancer selectivity of EA combined with its ability to induce multiple mechanisms of cell death while inhibiting pathways driving cell proliferation, suggest that EA is a highly unique agent with great potential as a therapeutic lead for the treatment of RCC. Keywords: Englerin A, Apoptosis, Autophagy, Necrosis, Renal cell carcinoma Introduction Renal cell carcinoma (RCC) is the most common type of malignant kidney tumor with an incidence that con- tinues to rise. Between 1992 and 2005, the incidence of RCC rose by 1.8% and 2.1% among white men and white women, respectively [1]. Although surgery can be cura- tive for tumors confined to the kidney, about 25% of pa- tients have metastatic disease at diagnosis, and another 20-40% develop metastases following surgery [2,3]. The two-year survival rate for patients with metastatic dis- ease is under 20% due to the poor response of these tu- mors to current treatments. Clear cell RCC (cc-RCC) which comprises 83% of RCC is one of the most radio- * Correspondence: abatova@ucsd.edu Department of Chemistry and Biochemistry, University of California, 9500 Gilman Drive, La Jolla, CA 92093, USA Full list of author information is available at the end of the article and chemo-resistant cancers and no curative treatment is available once metastases develop [4]. Investigations of the molecular biology of RCC have established that inactivating alterations in the Von Hippel Lindau (VHL) tumor suppressor gene are present in the majority (91%) of sporadic cc-RCC underscoring the central role of VHL in the regulation of growth and differentiation of renal epithelium [5-7]. The VHL gene product is involved in oxygen and energy sensing by regulating the activity of the hypoxia inducible factors (HIFs) [8]. Inactivation of VHL results in HIF stabilization and the activation of transcription of over 60 hypoxia- responsive genes involved in oncogenesis and tumor pro- gression including vascular endothelial growth factor (VEGF), the platelet-derived growth factor (PDGF), transforming growth factor alpha (TGF-α), epidermal growth factor (EGF), and glucose transporter-1 (GLUT-1) © 2013 Williams et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.