Abstract
Curcumin activates diverse anticancer activities that lead to inhibition of cancer cell and tumor growth, induction of apoptosis, and antiangiogenic responses. In this study, we observed that curcumin inhibits Panc28 and L3.6pL pancreatic cancer cell and tumor growth in nude mice bearing L3.6pL cells as xenografts. In addition, curcumin decreased expression of p50 and p65 proteins and NFkappaB-dependent transactivation and also decreased Sp1, Sp3, and Sp4 transcription factors that are overexpressed in pancreatic cancer cells. Because both Sp transcription factors and NFkappaB regulate several common genes such as cyclin D1, survivin, and vascular endothelial growth factor that contribute to the cancer phenotype, we also investigated interactions between Sp and NFkappaB transcription factors. Results of Sp1, Sp3, and Sp4 knockdown by RNA interference demonstrate that both p50 and p65 are Sp-regulated genes and that inhibition of constitutive or tumor necrosis factor-induced NFkappaB by curcumin is dependent on down-regulation of Sp1, Sp3, and Sp4 proteins by this compound. Curcumin also decreased mitochondrial membrane potential and induced reactive oxygen species in pancreatic cancer cells, and this pathway is required for down-regulation of Sp proteins in these cells, demonstrating that the mitochondriotoxic effects of curcumin are important for its anticancer activities.
Highlights
Specific gene polymorphisms [4, 5]
Curcumin decreased several nuclear factor B (NFB)-regulated genes in tumors and these include cyclin D1, c-myc, bcl-2, cyclooxygenase-2 (COX-2),3 and vascular endothelial growth factor (VEGF) [19]. Recent studies in this laboratory demonstrated that the anticancer activity of curcumin in bladder cancer cells and tumors was associated with repression of specificity protein (Sp) tran
Cells were transfected iSp and NFB-luc, and luciferase activity was estimated as described under “Experimental Procedures.” -Actin and Lamin served as a loading control and similar results were observed in duplicate experiments (A–C)
Summary
Cell Lines—The Panc cell line was a generous gift from Dr Paul Chiao and L3.6pL cells were kindly provided by Dr Isaiah Fidler Western Blots—Pancreatic cancer cells were seeded in DMEM/Ham’s F-12 medium containing 2.5% charcoalstripped FBS and after 24 h, cells were treated with either vehicle (DMSO) or the indicated compounds. The electrophoretic mobility shift assay reaction was carried out in the reporter lysis buffer supplemented with 0.1 mol/liter of KCl. Each reaction contained 2 g of nuclear protein, 1 g of poly(dI-dC) (Roche Molecular Biochemicals) with or without unlabeled competitor oligonucleotides, and 10 fmol of labeled probe; the mixture was incubated for 15 min on ice. Protein-DNA complexes were resolved by 5% native PAGE at 160 V at room temperature for 1.5 h and visualized after exposing it to ImageTek-H autoradiography X-Ray film. The Panc and L3.6pL pancreatic cancer cell lines were seeded (6 ϫ 104 per well) in 12-well plates in DMEM/Ham’s F-12 medium supplemented with 2.5% charcoal-stripped FBS without antibiotic and left to attach for 1 day. IC50 values were calculated using linear regression analysis and expressed in micromolar at 95% confidence intervals
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