First-line
therapy for
pancreatic cancer is
gemcitabine. Although
tumors may initially respond to the
gemcitabine treatment, soon
tumor resistance develops leading to treatment failure. Previously, we demonstrated in human MIA PaCa-2
pancreatic cancer cells that
N-acetyl-l-cysteine (NAC), a
glutathione (GSH) precursor, prevents NFκB activation via S-glutathionylation of p65-NFκB, thereby blunting expression of survival genes. In this study, we documented the molecular sites of S-glutathionylation of p65, and we investigated whether NAC can suppress NFκB signaling and augment a therapeutic response to
gemcitabine in vivo. Mass spectrometric analysis of S-glutathionylated p65-NFκB
protein in vitro showed post-translational modifications of cysteines 38, 105, 120, 160 and 216 following oxidative and nitrosative stress. Circular dichroism revealed that S-glutathionylation of p65-NFκB did not change secondary structure of the
protein, but increased
tryptophan fluorescence revealed altered tertiary structure.
Gemcitabine and NAC individually were not effective in decreasing MIA PaCa-2
tumor growth in vivo. However, combination treatment with NAC and
gemcitabine decreased
tumor growth by approximately 50%. NAC treatment also markedly enhanced
tumor apoptosis in
gemcitabine-treated mice. Compared to untreated
tumors,
gemcitabine treatment alone increased p65-NFκB nuclear translocation (3.7-fold) and
DNA binding (2.5-fold), and these effects were blunted by NAC. In addition, NAC plus
gemcitabine treatment decreased anti-apoptotic
XIAP protein expression compared to
gemcitabine alone. None of the treatments, however, affected extent of tumor hypoxia, as assessed by EF5 staining. Together, these results indicate that adjunct
therapy with NAC prevents NFκB activation and improves
gemcitabine chemotherapeutic efficacy.