Abstract A21: Cancer stem cell in multi-drug-resistant prostate cancer and green tea polyphenols restore drug sensitivity by reversal of epithelial mesenchymal transition

Abstract

Abstract Background: Prostate cancer (PC) is the most common cause of cancer death in men. In advanced PC, many patients suffer chemo-resistance therefore; establishment of a drug-resistant cell model to study the mechanism of action of drug resistance and increase in drug sensitivity is urgently needed. Recently, cancer stem cells (CSCs), and epithelial mesenchymal transition (EMT) have been suggested to play important roles in drug resistance in various cancer types. Green tea polyphenols, particularly the major component epigallocatechin-3-gallate (EGCG), have been shown to have chemopreventive properties in the malignant setting and recent studies have suggested that green tea catechins may enhance the chemo-sensitivity of drug-resistant cancer cells, such as breast cancer, although the mechanism of action is still unclear. Aims: (1) To establish a multi-drug-resistant prostate cancer cell line enriched for CSCs and (2) To investigate whether EGCG can restore drug sensitivity by limiting CSC expansion and/or by reversal of EMT. Experimental procedures: Chemo-resistant (CR) PC3M cells were developed by growth in increasing concentrations of a chemotherapeutic agent. Drug resistance was confirmed using increasing concentrations of doxorubicin (Dox), Taxotere (Tax), 5-fluorouracil (5FU) and cisplatin in fluorometric cell viability assays on resistant and wild-type (WT) cells. Immunofluorescent (IF) staining and flow cytometry were used to identify CSCs. Gene expression was assessed using quantitative real time-PCR. Cell morphology was observed using Nikon invert microscopy. Results: A chemo-resistant variant of WT-PC3M was derived by long-term culture in increasing concentrations of the chemotherapeutic, 5FU. As a result, the IC50 for WT PC3M to 5FU was 30μM as compared to >500μM for CR PC3M. In addition, the CR cells were also shown to be resistant to other chemotherapeutics including Dox and Tax, but not cisplatin. Enrichment for cells with CSC properties was determined in WT and CR cells. In comparison to WT PC3M, gene expression of both CD133 and ATP-binding cassette subfamily G2 (ABCG2), which have been reported as CSC markers in various cancer including prostate cancer, were significantly up-regulated by 10 and 2-folds, respectively in drug resistant cells (p<0.01). Protein expression of both CSC markers was confirmed to be elevated in CR PC3M by IF staining. Data from flow cytometry showed that there were 10–15% double-positive CD133/ABCG2 cells within CR cells whereas the percentage is <2% in WT PC3M cells. In addition, CD133 expression was confirmed to be down-regulated by EGCG. When EGCG was combined with 5FU, the IC50 of 5FU in CR PC3M decreased from 500μM to 100μM in CR PC3M, but it did not show a significant effect in WT PC3M, suggesting that EGCG had restored drug sensitivity in CR cells. It was noted that chemo-resistant PC3M had a more mesenchymal appearance when compared to WT PC3M. The expression of key EMT transcriptional regulator genes such as twist, snail, slug and zeb-1, were up-regulated whereas the epithelial marker gene E-cadherin was down-regulated in CR PC3M. After 48hours treatment with EGCG, all four EMT regulatory genes were significantly down-regulated by up to 3-folds whereas expression of E-cadherin was elevated. Conclusion: Green tea polyphenols restore drug sensitivity in chemo-resistant prostate cancer by reversal of EMT. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A21