Abstract 1900: Mechanistic study of aspirin, folic acid and calcium (AFAC) combinations delivered using targeted polymeric nanoparticles in colon cancer prevention
Authors: Thakkar Arvind , Chenreddy Sushma , Wang Jeffrey , Prabhu Sunil 
Published: August 2015

Abstract
Colorectal cancer (CRC) is the third most common cause of cancer death in the US, accounting for ∼51,000 deaths each year. Although the prognosis of this disease is improving due to better treatment options, attention has focused on the chemoprevention of this disease rather than on diagnosis and treatment. The objective of the present study was to evaluate the mechanisms of action of orally administered, colon targeted chemopreventive regimens aspirin (ASP), folic acid (FA) and calcium (Ca) alone and in combination AFAC (ASP+FA+Ca) in in-vitro and in-vivo rat model. We used colon cancer cells, HCT-116 and HT-29 as the in-vitro model. For colon targeting, ASP and FA was encapsulated in polylactide-co-glycolide (PLGA 50:50) polymer nanoparticles. Our previous studies demonstrated that combination of ASP (1mM), FA (1.5mM) and Ca (1.5mM) showed synergistic effect on inhibition of colon cancer cell viability. Follow-up study on azoxymethane (AOM) induced colon cancer in rats showed that the low (40+3.2+65 mg/kg), medium (80+6.4+130 mg/kg) and high dose (133+11+216 mg/kg) of modified AFAC-PLGA nanoparticle chemopreventive regimens demonstrated 1.7 to 2.0 fold reduction in the number of aberrant crypt foci (ACF). In continuation, a mechanistic approach was evaluated whereby colon cancer cells HCT-116 and HT-29 were treated with unmodified AFAC for 6h to determine gene expression using RT-qPCR analysis, and treated for 24h for protein expression study using western blot. The results obtained from the in-vitro study were further validated on rat colon tissue treated AFAC-PLGA nanoparticles, using immunohistochemistry. The RT-qPCR analysis showed that AFAC promoted reduction in cell viability was associated with down-regulation of proliferation genes (PCNA, and MKI67), cell cycle genes (CCND1, RB1, CDK4, E2F1, and JUN), and up-regulation of anti-apoptotic p21 gene. The protein expression studies revealed that AFAC treatment inhibited phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2), and phospho-p38 MAPK protein levels. The AFAC treatment also inhibited protein expression of PCNA, MKI67 and p-RB1 compared to control and individual drug treatment. Immunohistochemistry study using rat colon tissues revealed that the low, medium and high dose of AFAC-PLGA nanoparticles showed significant reduction in cell proliferation at 10.6%, 26.5%, and 48.5% as evidenced by PCNA protein expression. The AFAC treatment significantly increased apoptosis as evaluated by Tunnel assay, as well as decreased p-ERK1/2 and p-RB1 index. Overall, our results illustrate the mechanisms of action which confirms anti-proliferative activity of AFAC-PLGA nanoparticles, thus confirming the importance of novel combination chemoprevention strategies against colon cancer.
Citation Format: Arvind Thakkar, Sushma Chenreddy, Jeffrey Wang, Sunil Prabhu. Mechanistic study of aspirin, folic acid and calcium (AFAC) combinations delivered using targeted polymeric nanoparticles in colon cancer prevention. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1900. doi:10.1158/1538-7445.AM2015-1900