Abstract B64: A novel chemoprevention strategy using nanotechnology-based delivery of combinations of piroxicam, curcumin, and sulforaphane (PCS) on human pancreatic cancer cell lines
Authors: Prabhu Sunil , Sutaria Dhruvitkumar , Grandhi Balgangadhar Karthik 
Published: June 2012

Abstract
According to the American Cancer Society (ACS), an estimated 37,660 deaths will occur in 2011 due to complications from pancreatic cancer. The onset of pancreatic cancer is often devoid of symptoms and is mostly diagnosed at a stage where survival rate is less than 5%. Thus, chemoprevention could play an important role in the fight against this silent yet deadly disease. The objective of our study was to use combinations of chemopreventive agents namely piroxicam (a non-steroidal anti-inflammatory drug), curcumin (a natural spice with significant cancer chemopreventive properties) and sulforaphane (a potent broccoli extract) in very low doses to inhibit cell proliferation and promote apoptosis on two human pancreatic cancer cell lines, MIA PaCA-2 and Panc-1. More importantly, we compared the effects of the chemopreventive combinations in both, unmodified (free drug) and modified forms, encapsulated within solid-lipid nanoparticles (SLNs). SLNs were prepared using a process of hot-melt oil in water (o/w) emulsion technique using stearic acid as the lipid backbone to which the chemopreventive agents were added and then recovered as nanoparticles (∼200 nm size) after a process of freeze-drying. MTS cell proliferation, apoptosis and cell colony forming assays were conducted on cultured cells. For the MTS assay, upon 75 % confluence, 2.5 × 103 cells for MIA PaCa-2 and 4 × 103 cells for Panc-1 were transferred into each well of 96-well plates. The PCS chemopreventive agents alone or in combination were added to the cells and incubated for 72 h. Absorbance was recorded at 490 nm using an ELISA plate reader. For the apoptosis assay, 3 × 105 cells were cultured in 6-well plates for both MIA PaCa-2 and Panc-1 cell-lines. PCS, individual or combinations, were added and the plates were incubated for 72 h. Cells were analyzed using flow cytometry, measuring the fluorescence emission at 530 nm (FL1) and &gt;575 nm (FL3). For the cell colony formation assays, MIA PaCa-2 and Panc-1 cells were seeded at a density of 1 ml containing 3 × 104 cells, incubated for a period of 24 h. After treatment with PCS alone and in combinations, cells were trypsinized then incubated for a period of 10 days. The number of colonies with &gt;50 cells were counted under a dissecting microscope. IC50 values obtained for the unmodified and modified forms of piroxicam were (663.1μM; 52.85μM), curcumin (16.05μM; 4.93μM) respectively and free sulforaphane at 11.82 μM/L for MIA PaCa-2 cells. For Panc-1 cells, the IC50 values for unmodified and modified piroxicam were (389.3μM; 66.96μM), curcumin (24.01μM; 7.5μM) respectively and free sulforaphane at 17.81μM/L. For MIA PaCa-2 cell line, unmodified combinations of piroxicam (250μM) with curcumin (10μM) and sulforaphane (5μM) showed significant reduction in cell viability of &gt; 60% using MTS assay. In comparison, lower concentrations of piroxicam SLN (25μM) and curcumin SLN (2.5μM) with free sulforaphane (5μM) mixtures showed decrease in cell viability by &gt;47.4%. Flow cytometry analysis demonstrated apoptosis of 71% (unmodified) and 38.4% (modified) for the combinations, at the same concentration ranges as above. Panc-1 cell line treated with similar concentrations of unmodified and modified SLN combinations showed a &gt;82% and &gt;45.79% decrease in cell viability and 91% and 54.6% apoptotic cells, respectively. Colony formation assays in both cell lines showed a significant decrease (p value &lt;0.0001) in cell counts. From this study, we believe the action of nanotechnology-based chemopreventive regimens on human pancreatic cell lines provides strong evidence of its potential as a viable tool to prevent the onset of pancreatic cancer.
Citation Information: Cancer Prev Res 2011;4(10 Suppl):B64.