J. Fernandez-Moure1,2, D. Kirui1, J. Van Eps1,2, N. Dhanani1,4, F. Cabrera1, M. Ferrari1,3, E. Tasciotti1 1Houston Methodist Research Institute,Nanomedicine,Houston, TX, USA 2Houston Methodist Hospital,Surgery,Houston, TX, USA 3University Of Texas Health Science Center At Houston,Houston, TX, USA 4Texas A & M Health Science Center College Of Medicine,Bryan, TX, USA
Introduction:
Gemcitabine (GEM) is the first-line treatment for pancreatic adenocarcinoma (PAC). Despite such broad use, intrinsic and acquired chemoresistance is common. Loss of the tumor suppressor SMAD4 is present in more than 50% of PAC and is known to play a role in chemoresistant mechanisms. Nitric oxide (NO) is the predominant species responsible for the cytotoxic action of macrophages against cancer cells yet localized delivery is difficult given the short half-life and volatility. Silica (Si) nanoparticle mediated local delivery has been shown to be an effective strategy for NO delivery and controlled release. We sought to study the effects of locally delivered NO on GEM mediated PAC cytotoxicity and the potential role of SMAD4 in this effect. We hypothesized that NO would enhance the cytotoxicity of GEM in a SMAD4 independent manner.
Methods:
NO-Silica nanoparticles (NO-Si) were synthesized via a co-condensation of tetraethoxysilane with aminoalkoxysilane under high pressure nitrous oxide for 3 or 4 days. Particle characterization included scanning electron microscopy, dynamic light scattering, and Fourier transform infrared spectroscopy. NO release was measured using a chemiluminescence nitric oxide analyzer. A SMAD4 negative PAC cell line (SMAD4-) was made using lentiviral knockdown of Panc1 PAC cells and confirmed by western blot. Panc1 and SMAD4- cells were then treated with gemcitabine (100nm to 30μm), 30 mg NOSi particles (NOSihi or NOSilo), or both for 72 hours. Cell viability was then quantified by MTS cell proliferation assay.
Results:
Half-life of NOSi NO release is 3.5±0.6 hours. NOSilo maximum concentration of NO release was 750ppb/mg and NOSihi was 2250ppb/mg. NOSilo alone reduced cell viability by 30% and 35% in SMAD4 and Panc1 respectively. When combined with GEM, NOSilo led to a significant reduction in cell viability of both cell lines at all concentrations used. The greatest effect was seen at 1μm where cytotoxicity was increased by 50% in Panc1 and 70% in SMAD4-. NOSihi led to >90% reductions in cell viability in Panc1 and no difference was seen when combined with GEM. NOSihi alone led to only 70% reduction in SMAD4- cells. When combined with GEM, cytotoxicity was improved to 90% at GEM concentrations as low as 5μm.
Conclusion:
We have demonstrated the in vitro dose dependent cytotoxic effects of Si nanoparticle NO controlled release on PAC. When combined with GEM there is a synergistic effect resulting in improved cytotoxicity seen in both Panc1 and SMAD4- PAC cells with a differential pattern of cell death seen at high concentrations of NO. These findings suggest not only that NOSi is useful chemosensitizing agent but that SMAD4 may play a role in its synergism with GEM. Creation of a novel class of therapeutics where local controlled release of NO is used as a method of chemosensitization may lead to a paradigm shift in not only treating PAC but all solid tumors where chemoresistance is common.