(T1530-01-06) Targeted Combination Therapy for Pancreatic Cancer Management

Purpose: Pancreatic cancer is the 3^rd leading cause of cancer-related death in the USA, with a 5-year survival rate of 12%. Lack of tumor specificity, dose-related toxicity, and the tumor’s innate resistance to chemotherapeutic agents are significant barriers to improving treatment outcomes for pancreatic cancer. Thus, to resolve this shortcoming, researchers focus on developing tumor-targeted delivery of combination therapy to treat cancer effectively. Incorporating multiple anticancer drugs affecting different key pathways can reduce drug resistance and tumor growth at relatively low concentrations. Thus, the primary goal of this study was to evaluate a new approach for treating pancreatic cancer with a combination of individually nano-capsulated entinostat, a novel HDAC inhibitor, and oxaliplatin used at much lower concentrations.

Methods: Mouse pancreatic cancer (KPC) and human pancreatic cancer (PANC-1) cells were treated with various concentrations of oxaliplatin and entinostat individually as well as in combination. The cell viability was determined via MTT assay. The combination interactions between free oxaliplatin and entinostat were evaluated by synergy scoring models using Combenefit and Compusyn software. Additionally, a colony-forming assay was performed to verify further the synergistic interaction between oxaliplatin and entinostat using KPC and PANC-1 cells. Furthermore, western blotting techniques were performed to evaluate the synergistic mechanism to determine acetylated histone H3 and H4, and cleave caspase expression. Both drugs were individually encapsulated into poly(lactic-co-glycolic) acid (PLGA) nanoparticles using the emulsion solvent evaporation method. The nanoparticles were characterized for the surface charge, particle size, drug loading, and in vitro drug release profile. Furthermore, in vitro cytotoxicity of oxaliplatin and entinostat nanoparticles was compared with free drugs in KPC and PANC-1 cells.

Results: KPC and PANC-1 cells treated with different drug combinations showed synergistic potential at various combination ranges. The colony formation assay results also revealed synergism interaction of oxaliplatin and entinostat as the total number of colonies in KPC cells reduced to 74%, 76 %, and 29 % in entinostat (1µM), oxaliplatin (1µM), and combination (1µM entinostat + 1µM oxaliplatin) treatment, respectively, as compared to control. Similarly, colony numbers were reduced to 91%, 77%, and 52% for PANC-1 cells treated with entinostat, oxaliplatin, and combination, respectively, compared to the control. Western blot analysis revealed higher expression of acetylated H3, H4, and cleaved caspase 3 in combination treatment than in the individual treatments in both KPC and PANC-1 cells. Both oxaliplatin (size: 250 nm, charge: -12 mV, drug loading: 4 %w/w) and entinostat nanoparticles (size: 260 nm, charge: -14 mV, drug loading: 12 %w/w) were spherical and showed sustained in vitro drug release profiles. The nanoparticle-loaded oxaliplatin and entinostat showed significantly higher in vitro cytotoxicity than their solution counterpart.

Conclusion: The combination of oxaliplatin and entinostat showed great potential for pancreatic cancer management. The potential of this combination can further be elevated via tumor-targeted delivery using PLGA nanoparticles. In the future, the pharmacokinetic and anticancer efficacy of the synergistic combination of nanoformulations will be evaluated in an orthotopic mouse model of pancreatic cancer.

Acknowledgements: This work was supported by the National Institute of General Medical Sciences (NIGMS) COBRE award 2P20 GM109024.

Figure 1. Synergy analysis by the Loewe model for oxaliplatin and entinostat combination using Combenefit® software. A: KPC cells, B: PANC-1 cells.

Figure 2. (A) Oxaliplatin and entinostat combination reduced colony formation in KPC cells. Data represents mean ± SD (n = 3) ‘*, #, †’ significantly (p < 0.005) lower than control (Ctrl), entinostat (Ent), and oxaliplatin (Oxa), respectively. (B) Representative images.

Figure 3. Entinostat (0 and 10 µM) and oxaliplatin (0 and 5 µM) combination inhibits HDAC activity in PANC-1 and KPC cells. Western blotting analysis of acetylated histones and total histones. Ctrl: control, Ent: entinostat, Oxa: oxaliplatin.