Tumor Stroma Interactions Induce Chemoresistance in Pancreatic Ductal Carcinoma Cells Involving Increased Secretion and Paracrine Effects of Nitric Oxide and Interleukin-1β

Pancreatic ductal carcinoma is characterized by a profound chemoresistance. As we have shown previously, these tumor cells can develop chemoresistance by interleukin (IL)-1β in an autocrine and nuclear factor-κB-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-1β in T3M4 and PT45-P1 cells was increased by the fibroblasts, and IL-1β-receptor blockade abolished the resistance-inducing effect during cocultivation. This stimulated IL-1β secretion could be attributed to nitric oxide (NO) released by the fibroblasts as an IL-1β-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-1β 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-1β 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-1β by the tumor cells. These findings substantiate the implication of tumor–stromal interactions in the chemoresistance of pancreatic carcinoma.

[1]  H. Kalthoff,et al.  Usage of the NF‐κB inhibitor sulfasalazine as sensitizing agent in combined chemotherapy of pancreatic cancer , 2003, International journal of cancer.

[2]  M. Löhr,et al.  A comprehensive characterization of pancreatic ductal carcinoma cell lines: towards the establishment of an in vitro research platform , 2003, Virchows Archiv.

[3]  A. Doseff,et al.  Presentation of Nitric Oxide Regulates Monocyte Survival through Effects on Caspase-9 and Caspase-3 Activation* , 2003, The Journal of Biological Chemistry.

[4]  J. Abbruzzese,et al.  A novel model system for studying the double-edged roles of nitric oxide production in pancreatic cancer growth and metastasis , 2003, Oncogene.

[5]  V. Trajković,et al.  The role of interleukin-17 in inducible nitric oxide synthase-mediated nitric oxide production in endothelial cells , 2003, Cellular and Molecular Life Sciences CMLS.

[6]  P. Chan,et al.  Aspirin Inhibits p44/42 Mitogen-Activated Protein Kinase and Is Protective Against Hypoxia/Reoxygenation Neuronal Damage , 2003, Stroke.

[7]  N. Fusenig,et al.  Tumor-stroma interactions directing phenotype and progression of epithelial skin tumor cells. , 2002, Differentiation; research in biological diversity.

[8]  Ding-I Yang,et al.  NO‐Mediated Chemoresistance in C6 Glioma Cells , 2002, Annals of the New York Academy of Sciences.

[9]  A. Arlt,et al.  Autocrine Production of Interleukin 1β Confers Constitutive Nuclear Factor κB Activity and Chemoresistance in Pancreatic Carcinoma Cell Lines , 2002 .

[10]  U. Fölsch,et al.  Isolation, Long-term Culture, and Characterization of Rat Pancreatic Fibroblastoid/Stellate Cells , 2001, Pancreas.

[11]  R H Hruban,et al.  Invasion-specific genes in malignancy: serial analysis of gene expression comparisons of primary and passaged cancers. , 2001, Cancer research.

[12]  H. Kalthoff,et al.  Inhibition of NF-κB sensitizes human pancreatic carcinoma cells to apoptosis induced by etoposide (VP16) or doxorubicin , 2001, Oncogene.

[13]  F. Sigaux,et al.  Contribution of Nitric Oxide to the Apoptotic Process in Human B Cell Chronic Lymphocytic Leukaemia , 2001, Leukemia & lymphoma.

[14]  Marty W. Mayo,et al.  NF-κB Induces Expression of the Bcl-2 Homologue A1/Bfl-1 To Preferentially Suppress Chemotherapy-Induced Apoptosis , 1999, Molecular and Cellular Biology.

[15]  Claus Scheidereit,et al.  NF-κB Function in Growth Control: Regulation of Cyclin D1 Expression and G0/G1-to-S-Phase Transition , 1999, Molecular and Cellular Biology.

[16]  J. Cusack,et al.  Control of inducible chemoresistance: Enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-κB , 1999, Nature Medicine.

[17]  C. Y. Wang,et al.  NF-kappaB antiapoptosis: induction of TRAF1 and TRAF2 and c-IAP1 and c-IAP2 to suppress caspase-8 activation. , 1998, Science.

[18]  Z. Ao,et al.  IEX-1L, an Apoptosis Inhibitor Involved in NF-κB-Mediated Cell Survival , 1998 .

[19]  F. Sigaux,et al.  B-cell chronic lymphocytic leukemia cells express a functional inducible nitric oxide synthase displaying anti-apoptotic activity. , 1998, Blood.

[20]  James B. Mitchell,et al.  The multifaceted roles of nitric oxide in cancer. , 1998, Carcinogenesis.

[21]  I. Fidler,et al.  Therapy of cancer metastasis by activation of the inducible nitric oxide synthase , 1998, Cancer and Metastasis Reviews.

[22]  B. Dörken,et al.  Constitutive nuclear factor-kappaB-RelA activation is required for proliferation and survival of Hodgkin's disease tumor cells. , 1997, The Journal of clinical investigation.

[23]  A. E. Rogers,et al.  Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer. , 1997, The Journal of clinical investigation.

[24]  I. Fidler,et al.  Destruction of bystander cells by tumor cells transfected with inducible nitric oxide (NO) synthase gene. , 1997, Journal of the National Cancer Institute.

[25]  T. Billiar,et al.  New insights into the regulation of inducible nitric oxide synthesis. , 1994, The American journal of physiology.

[26]  C Caldas,et al.  Allelotype of pancreatic adenocarcinoma. , 1994, Cancer research.

[27]  S. Moncada,et al.  Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor , 1987, Nature.

[28]  Seaver,et al.  Nitric oxide as a secretory product of mammalian cells , 2004 .

[29]  R. Hruban,et al.  Exploring the host desmoplastic response to pancreatic carcinoma: gene expression of stromal and neoplastic cells at the site of primary invasion. , 2002, The American journal of pathology.

[30]  J. K. Lee,et al.  Inducible nitric oxide synthase (iNOS) immunoreactivity and its relationship to cell proliferation, apoptosis, angiogenesis, clinicopathologic characteristics, and patient survival in pancreatic cancer , 2001, International journal of pancreatology : official journal of the International Association of Pancreatology.

[31]  Douglas B. Evans,et al.  The Nuclear Factor-κB RelA Transcription Factor Is Constitutively Activated in Human Pancreatic Adenocarcinoma Cells , 1999 .

[32]  K. Lillemoe Palliative therapy for pancreatic cancer. , 1998, Surgical oncology clinics of North America.

[33]  S. Snyder,et al.  Nitric oxide: a neural messenger. , 1995, Annual review of cell and developmental biology.