title: N-acetyl-L-cysteine sensitizes pancreatic cancers to gemcitabine by targeting the NFκB pathway.
abstract: 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.
authors.affiliation: Center for Cell Death, Injury & Regeneration, Medical University of South Carolina, Charleston 29425, SC, USA; Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, DD505 Drug Discovery Building, 70, President Street, Charleston 29425, SC, USA; Hollings Cancer Center, Medical University of South Carolina, Charleston 29425, SC, USA.
authors.forename: Suparna
authors.initials: S
authors.lastname: Qanungo
authors.affiliation: Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston 29425, SC, USA; Hollings Cancer Center, Medical University of South Carolina, Charleston 29425, SC, USA.
authors.forename: Joachim D
authors.initials: JD
authors.lastname: Uys
authors.affiliation: Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston 29425, SC, USA; Hollings Cancer Center, Medical University of South Carolina, Charleston 29425, SC, USA.
authors.forename: Yefim
authors.initials: Y
authors.lastname: Manevich
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland 44106, OH, USA; Louis Stokes Veterans Affairs Medical Research Center, Cleveland 44106, OH, USA.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Distler
authors.affiliation: Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, DD505 Drug Discovery Building, 70, President Street, Charleston 29425, SC, USA.
authors.forename: Brooke
authors.initials: B
authors.lastname: Shaner
authors.affiliation: Department of Public Health Sciences, Medical University of South Carolina, Charleston 29425, SC, USA; Hollings Cancer Center, Medical University of South Carolina, Charleston 29425, SC, USA.
authors.forename: Elizabeth G
authors.initials: EG
authors.lastname: Hill
authors.affiliation: Department of Pharmacology, Case Western Reserve University, Cleveland 44106, OH, USA; Louis Stokes Veterans Affairs Medical Research Center, Cleveland 44106, OH, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Mieyal
authors.affiliation: Center for Cell Death, Injury & Regeneration, Medical University of South Carolina, Charleston 29425, SC, USA; Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, DD505 Drug Discovery Building, 70, President Street, Charleston 29425, SC, USA; Department of Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston 29425, SC, USA; Hollings Cancer Center, Medical University of South Carolina, Charleston 29425, SC, USA.
authors.forename: John J
authors.initials: JJ
authors.lastname: Lemasters
authors.affiliation: Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, DD505 Drug Discovery Building, 70, President Street, Charleston 29425, SC, USA; Hollings Cancer Center, Medical University of South Carolina, Charleston 29425, SC, USA.
authors.forename: Danyelle M
authors.initials: DM
authors.lastname: Townsend
authors.affiliation: Center for Cell Death, Injury & Regeneration, Medical University of South Carolina, Charleston 29425, SC, USA; Department of Drug Discovery & Biomedical Sciences, Medical University of South Carolina, DD505 Drug Discovery Building, 70, President Street, Charleston 29425, SC, USA; Hollings Cancer Center, Medical University of South Carolina, Charleston 29425, SC, USA. Electronic address: nieminen@musc.edu.
authors.forename: Anna-Liisa
authors.initials: AL
authors.lastname: Nieminen
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D003545:Cysteine / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008297:Male; D051379:Mice; D008819:Mice, Nude; D016328:NF-kappa B / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism; D000093542:Gemcitabine
chemical_list: D016328:NF-kappa B; D051636:X-Linked Inhibitor of Apoptosis Protein; D003841:Deoxycytidine; D005978:Glutathione; D003545:Cysteine; D000111:Acetylcysteine; D000093542:Gemcitabine
keywords: Gemcitabine; Glutathione; N-acetyl-l-cysteine; Pancreatic cancer; S-glutathionylation