Abstract C13: Ascorbate induces cytotoxicity in prostate cancer cells through an autophagy pathway

Abstract

Abstract Ascorbate (Vitamin C) has been shown to act as a pro-oxidant agent under pharmacological concentrations, and thus can cause cancer cell death in vitro and inhibit tumor growth in animal models, through formation of H2O2. The cell death mechanisms induced by ascorbate need to be further elaborated. The goal of our study was to determine whether ascorbate is cytotoxic to prostate cancer cells and if so, to determine the mechanism of ascorbate-induced cytotoxicity. We hypothesized that through generation of H2O2, pharmacological concentrations of ascorbate can deplete prostate cancer cell ATP and cause cell death through autophagy pathway. A panel of prostate cancer cell lines PC-3, LNCap, C4-2, LaPC4 and 22RV1 were incubated with millimolar range of ascorbate for two hours. Then ascorbate was washed out and cells were incubated in fresh media for 24 hrs before cell viability was assessed. Cell ATP levels were determined by HPLC. The results showed that 4 out of the 5 tested cell lines have IC50s < 5 mM, while the most resistant cell line LaPC4 has IC50 at 12 mM. Addition of catalase - a H2O2 scavenger to the culture media, completely protected the cells from the death caused by ascorbate, confirming the death is mediated by H2O2. PC-3 cell ATP levels significantly decreased after 2 hrs of incubation with 5 mM ascorbate, and did not recover over the following 24 hrs. To determine if ascorbate induced autophagy, western blot of PC-3 cells for LC3 was performed. The results showed an increase of LC3-II form at 2 hrs of ascorbate treatment, indicating the processing of LC3 protein to its lipidated form. At 24 hrs the LC3-II bands were decreased, which may due to degradation of LC3 proteins at the autophagosomes. We also generated cells (PC3-LC3G) that express a fusion protein of LC-3 and GFP. The GFP-LC3 protein punctation were observed at 4 hrs of ascorbate treatment, indicating a translocation of LC3 to the autophagic membranes. These results suggest that ascorbate induces cytotoxicity in prostate cancer cells through depletion of cell ATP and induction of autophagy. To further confirm this pathway, an autophagy inhibitor 3-MA is under testing, the degradation of GFP-LC3 is also under analysis to determine whether ascorbate increases the autophagic flux. Also, to confirm the role of ATP drop in ascorbate-induced autophagy, 3-aminibenzamide (3AB) and 4-hydroxyquinazoline (4HQ) are being used to prevent the drop in ATP level. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C13.