Abstract 2765A: Co-treatment of human pancreatic cancer MiaPaCa-2 Cells with PNC-27 and Gemzar

Abstract

Novel anti-cancer PNC-27 peptide, derived from the p53 binding domain of MDM2, induces necrosis of human pancreatic MiaPaCa-2 cancer cells but not normal cells in vitro, in a dose dependent fashion. Recent studies support a membranolytic mechanism of action for the peptide, initiated by binding of peptide monomers to the cells’ plasma membrane that subsequently aggregate to form transmembrane pores. Gemzar, on the other hand, is an established, non-selective chemotherapeutic used to treat pancreatic cancer. As a nucleoside analog, Gemzar inhibits DNA replication, resulting in cell cycle arrest, and ultimately apoptosis. Speculation regarding whether the two drugs, with different mechanisms, could produce a combined killing effect if administered simultaneously is investigated. Cell proliferation assays (MTT), cell cytotoxicity assays (LDH) and confocal microscopy were used to study the susceptibility of the MiaPaCa-2 cells to the co-treatment of PNC-27 and Gemzar. To determine the working concentration of PNC-27 that would result in pore formation but would not be cytotoxic to the cells, a dose response assay with serial dilutions was conducted. Results revealed that while MiaPaCa-2 cells treated with PNC-27 in DMEM have an LD50 = 150 ug/mL, the working concentration that would enable minimal pore formation without cell death was determined to be ∼10 ug/mL. Through confocal microscopy, it was then determined that propidium iodide (PI) (668.4 Da), a marker for dead cells and that is impermeable for intact cells, would nevertheless be able to enter the cell cytoplasm via PNC-27 pores. The results found PI staining in the cytoplasm but complete absence of nuclear staining. This observation not only indicates the sustained viability of the cells treated with 10 ug/mL PNC-27, but also supports the hypothesis that PI entered the cells through the PNC-27 induced pores. To explore a combined cytotoxic effect of Gemzar and PNC-27, MTT assays were carried out with Gemzar concentrations between 0 uM-100 uM for either 24 or 48 hours, in presence or absence of PNC-27. Our results demonstrate that co-treatment of MiaPaCa-2 cells with PNC-27 and Gemzar leads to a greater cytotoxic effect than with either Gemzar or PNC-27 alone. Additionally, the 48hr treatment resulted in a greater cytotoxic effect than did the 24hr condition. Our results indicate that the increased permeability of the cancer cells to Gemzar via the PNC-27-induced pores may allow for a more rapid mechanism of entry for Gemzar. Due to the selectivity of PNC-27, more Gemzar is able to target the cancer cells which have compromised membrane integrity caused by PNC-27. Therefore, co-treatment with Gemzar may allow for a dose reduction of Gemzar due to increased accessibility of the chemotherapeutic to its site of action. By reducing the dose of Gemzar required to achieve a significant cytotoxic effect, the severity of the side-effects of the drug experienced by patients may also be reduced. 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 2765A. doi:1538-7445.AM2012-2765A