s-nitro-n-acetylpenicillamine and Adenocarcinoma

Pancreatic ductal carcinoma is characterized by a profound chemoresistance. As we have shown previously, these tumor cells can develop chemoresistance by interleukin (IL)-1beta in an autocrine and nuclear factor-kappaB-dependent fashion. Because pancreatic ductal carcinoma contains many mesenchymal stromal cells, we further investigated how tumor-stroma interactions contribute to chemoresistance by using a transwell coculture model, including murine pancreatic fibroblasts and the chemosensitive human pancreatic carcinoma cell lines T3M4 and PT45-P1. If cultured with fibroblast-conditioned medium or kept in coculture with fibroblasts, both cell lines became much less sensitive toward treatment with etoposide than cells cultured under standard conditions. Furthermore, the secretion of IL-1beta in T3M4 and PT45-P1 cells was increased by the fibroblasts, and IL-1beta-receptor blockade abolished the resistance-inducing effect during cocultivation. This stimulated IL-1beta secretion could be attributed to nitric oxide (NO) released by the fibroblasts as an IL-1beta-inducing factor. Although both tumor cells secreted only little NO, which was in line with undetectable inducible nitric oxide synthase (iNOS) expression, fibroblasts exhibited significant iNOS expression and NO secretion that could be further induced by the tumor cells. Incubation of T3M4 and PT45-P1 cells with the NO donor S-Nitroso-N-acetyl-D,L-penicillamine up-regulated IL-1beta secretion and conferred resistance toward etoposide-induced apoptosis. Conversely, the resistance-inducing effect of the fibroblasts was significantly abolished, when the specific iNOS inhibitor aminoguanidine was added during coculture. Immunohistochemistry on tissue sections from human pancreatic ductal carcinoma also revealed iNOS expression in stromal cells and IL-1beta expression in tumor cells, thus supporting the in vitro findings. These data clearly demonstrate that fibroblasts contribute to the development of chemoresistance in pancreatic carcinoma cells via increased secretion of NO, which in turn leads to an elevated release of IL-1beta by the tumor cells. These findings substantiate the implication of tumor-stromal interactions in the chemoresistance of pancreatic carcinoma.

Topics: Adenocarcinoma; Carcinoma, Pancreatic Ductal; Cell Communication; Cell Line, Tumor; Drug Resistance, Neoplasm; Fibroblasts; Humans; Interleukin-1; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pancreatic Neoplasms; Penicillamine; Stromal Cells

Nitrogen monoxide (NO) is a cytotoxic effector molecule produced by macrophages that results in Fe mobilization from tumour target cells which inhibits DNA synthesis and mitochondrial respiration. It is well known that NO has a high affinity for Fe, and we showed that NO-mediated Fe mobilization is markedly potentiated by glutathione (GSH) generated by the hexose monophosphate shunt [Watts, R.N. & Richardson, D.R. (2001) J. Biol. Chem. 276, 4724-4732]. We hypothesized that GSH completes the coordination shell of an NO[bond]Fe complex that is released from the cell. In this report we have extended our studies to further characterize the mechanism of NO-mediated Fe mobilization. Native PAGE 59Fe-autoradiography shows that NO decreased ferritin-59Fe levels in cells prelabelled with [59Fe]transferrin. In prelabelled cells, ferritin-59Fe levels increased 3.5-fold when cells were reincubated with control media between 30 and 240 min. In contrast, when cells were reincubated with NO, ferritin-59Fe levels decreased 10-fold compared with control cells after a 240-min reincubation. However, NO could not remove Fe from ferritin in cell lysates. Our data suggest that NO intercepts 59Fe on route to ferritin, and indirectly facilitates removal of 59Fe from the protein. Studies using the GSH-depleting agent, L-buthionine-(S,R)-sulphoximine, indicated that the reduction in ferritin-59Fe levels via NO was GSH-dependent. Competition experiments with NO and permeable chelators demonstrated that both bind a similar Fe pool. We suggest that NO requires cellular metabolism in order to effect Fe mobilization and this does not occur via passive diffusion down a concentration gradient. Based on our results, we propose a model of glucose-dependent NO-mediated Fe mobilization.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cell Membrane Permeability; Cell-Free System; Cytosol; Deferoxamine; Female; Ferritins; Fibroblasts; Glutathione; Humans; Iron; Iron Chelating Agents; Macrophage Activation; Mice; Neuroblastoma; Neuroectodermal Tumors, Primitive, Peripheral; Nitric Oxide; Nitric Oxide Donors; Nitrogen Oxides; Oxidation-Reduction; Penicillamine; S-Nitrosoglutathione; Spermine; Tumor Cells, Cultured