Abstract 2409: Pharmacologic ascorbate inhibits pancreatic cancer cell EMT and sensitizes cancer cells to gemcitabine

Abstract

Abstract Pharmacologic concentrations of ascorbate have been shown to act as a pro-oxidant anti-cancer agent. Our previous work has showed that pharmacologic ascorbate worked synergistically with gemcitabine in 8 pancreatic cancer cells lines, and in gemcitabine responsive and non-responsive mouse tumor models. This study aimed to investigate the mechanisms for this synergistic effect. In PANC-1 pancreatic cancer cells treated with 2.5 mM ascorbate, real time RT-PCR showed increased mRNA expression levels of E-cadherin, and decreased levels of Vimentin, indicating a reversal/inhibition of epithelial-mesenchymal transition (EMT). The inhibition of EMT was attribute to up-regulation of microRNAs mir429 and mir 200 family, which binds to to 3′ URTs of Zeb-1 and Zeb-2 mRNAs, thus inhibited Zeb-1/2 mRNA translation and accelerated their deadenylation and subsequent exonucleolytic digestion. Consistant with the mir429 and 200 increase, real-time RT-PCR showed decreased in Zeb-1. Since Zeb-1 is a repressor of E-cadherin, the inhibition in Zeb-1 resulted in the increased level of E-cadherin. Moreover, mRNAs for multiple MMPs were also found inhibited by ascorbate treatment, while collagen type IV and type II were largely increased. Relatively, mir21 was found decreased by ascorbate treatment. Mir21 is an oncomir that promotes cancer invasion. It inhibits TIMP-3 and up-regulate MMPs, and thus its inhibition is associated with repression of MMPs. At cellular level, immulofluorescence clearly showed an increase in E-cadherin and decrease in Vimentin expression, which confirmed the RT-PCR results in indicating EMT inhibition. As EMT is highly related to drug resistance, we co-treated PANC-1 cell with ascorbate and gemcitabine. PANC-1 cells exhibited a highly “EMTed” phenotype and were resistant to gemcitabine single-drug treatment. The combination treatment of ascorbate and gemcitabine dramatically increased cell death compared to gemcitabine alone. Using Chou-Talalay's constant ratio design, gemcitabine IC50 was decreased 3.5-fold, and gemcitabine IC90 was decreased 11.5-fold. In PANC-1 orthotopic mouse tumor model (n=3 for each group), live animal imaging showed predominant tumor growth and metastasis in saline-treated controls, but tumor regression in ascorbate (4 g/kg/day, i.p.) treated mice. At day 54, 2 of the 3 ascorbate-treated mice were tumor-free. Experiments are underway with larger groups of mice and with ascorbate/gemcitabine combination treatment to examine tumor growth and metastasis. Collectively, these data demonstrated that ascorbate at pharmacological doses inhibited pancreatic cancer growth. Inhibition in EMT by ascorbate resulted in a strong synergistic effect between ascorbate and gemcitabine. These results suggest a rational combination of high dose parenteral ascorbate and gemcitabine in treating pancreatic cancer, one of the most lethal cancers worldwide. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2409. doi:1538-7445.AM2012-2409