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Expression of alternatively spliced isoforms of tight junction protein ZO-1 in gastrointestinal tumors
phosphorylation in T cells exposed to hydrogen peroxide prior to activation, suggesting that increased c-Jun dephosphorylation was not involved. However, an inhibitor of tyrosinespecific phosphatases, orthovanadate, did restore activation-induced phosphorylation of cJun and cytokine production. A correlation ofc-Jun phosphorylation and cytokine production was also seen in T cells derived from cancer patients. Conclusion: Oxidative stress mimicked by hydrogen peroxide treatment inhibited T cell function. This inhibition is at least in part due to a reduction in c-Jun phosphorylation. This effect is mediated by tyrosine phospatase activity and possibly effects upstreamJNK, which requires phosphorylation of its threonin and tyrosine residues to phosphorylate c-Jun. A reduced susceptibility of c-Jun phosphorylation following stimulation was also observed in T cells derived from cancer patients. In accordance with our previous results this data suggests that the cellular immune response in cancer patients is influenced by the redox status of T cells and supports the concept of an antioxidant therapy or a treatment that reduces granulocyte activity.
$1005
Expression of Alternatively Spliced Isoforms of Tight Junction Protein ZO-1 in Gastrointestinal Tumors Shigeru Kanaoka, Ken-lchi Yoshida, Mutsuhiro Ikuma, Naoyuki Miura, Haruhiko Sugimura, Masayoshi Kajimura Background: In vitro model of epithelial neoplasia have shown that there is association between increased tumor promoter-induced tight junction (TJ) permeability, transepithellal flux of growth factors and development of epithelial tumors. Alternatively spliced isoforms of TJ protein ZO-1 (ZO-1 a plus and ZO-1 cx minus) have been reported to correlate with junctional plasticity; ZO-1 a minus is expressed in structurally dynamic junctions, whereas ZO-1 a plus is expressed in those which are less dynamic. ZO-2, a functional component of TJ, differentially expressed in normal pancreatic duct compared to pancreatic adenocarcinoma. We aimed to determine the expression of alternatively spliced isoforms of ZO-1 in gastrointestinal tumors. Methods: Tissues were obtained from 20 patients with gastric cancer, 17 patients with coloretal cancer and 7 patients with colorectal adenoma. Matched tumor and normal mucosal specimens were obtained from endoscopidforceps biopsy. RNA was extracted and used as a template for RT-PCR usmg specific primers straddled ZO-1 c~ domain. Results: The expression level of ZO- 1 a plus was higher than that of ZO-i a minus in every normal gastric mucosa and every normal colorectal mucosa. The median expression ratios of ZO-1 a minus to a plus (ZO-1 c~ minus/plus) were 0.61 +/- 0.17 and 0.60 +/0.11 respectively, while those in gasmc carcinoma, colorectal carcinoma and adenoma were 0.81 +/- 0.38 (p < 0.05), 0.91 +/- 0.31 (p < 0.005) and 1.1 +/- 0.25 (p < 0.05), respectively (p value of Wilcoxon signed-rank test are shown in parenthesis). The expression ratio of ZO-1 oc minus/plus in gastric and colorectal tumors was significantly higher than that in corresponding normal mucosa, especially in coIorectal tumors. ZO-2 differentially expressed between in pancreatic cancer and in normal pancreatic duct. Similarly, expression pattern of ZO-1 isoforms in gastrointestinal tumors is different from that of normal mucosa. Conclusion: These results suggest that the change of expression pattem of ZO-1 isoforms may influence junctional plasticity, which give growth advantage of gastrointestinal tumors through direct contact with luminal various growth factors.
SIO03
Butyrate Impairs Intestinal Tumor Cell Induced Angiogenesis by Inhibiting HIFl a Nuclear Translocation Dimitrios Zgouras, Juergen Stein Angiogenesis is a complex process encompassing endothelial cell migration, proliferation, and tube formation. Inhibition of angiogenesis is considered to be one of the most promising strategies in the development of novel anti-neoplastic therapies. Butyrate is known to stimulate proliferation of normal crypt ceils, it inhibits growth and induce apoptosis in colon cancer cells. In this study we examined the effects of butyrate on colon cancer (Caco-2) cell induced angiogenesis. Methods: Condiotioned media were obtained by Caco-2 cells, incubated with serum free medium containing either the solvent (PBS) or butyrate (1 or 2 raM). Proliferation of HUVEC cells was assesed by a BrdU incorporation assay. VEGF production in the supernatant of Caco-2 cells was quantified by use of a colorimetric ELISA. Furthermore HIF-lu expression in total cell lysate and in nuclear extracts was detected by Western Blot. Intracelhilar distribution of HIF-lot was additionally observed by immunofluorescence. Nuclear HIF- lc~ DNA-binding activity was detected by art HIF-la transcription assay. Results: HUVEC cell proliferation was significantly inhibited up to 46% of controls when incubated with medium conditioned by butyrate treated Caco-2 cells. Simultaneously, VEGF, a proangiogenic vascular endothelial growth factor, was significantly reduced. Protein levels of HIF-la, which is a nuclear transcription factor known to be a key regulator in hypoxia induced angiogenesis, were upregulated by butyrate. This is in contrast to its importance as a VEGF regulating component. However Western blot analysis of nuclear extracts showed a downregulation of H1F-la protein. Nuclear HIF-la DNA-binding activity was also decreased by butyrate. Immunofluoresoence experiments indicate that HIF 1 nucleolar sequestration is repressed by butyrate, through inhibition of nuclear translocation. Conclusion: Taken together our data indicate that butyrate inhibits not only growth, but also angiogenic properties of colon cancer cells (Caco-2). This effects are likely to be induced by inhibition of HIF-ltx nuclear transloeation. We postulate that diminished HIF-la nuclear presence and activity in butyrate treated Caco-cells could be one reason for decreased VEGF expression and antiangiogenic effects.
S1006
Differential Ca2 + Channel Activity in Human Primary Neuroendocrine Tumor Cell Cultures from Fore- and Midgut Tumors Stefan Mergler, Adriana Drost, Wolf-Otto Bechstein, Peter Nenhans, Bertram Wiedenmann BACKGROUND: Depending on the primary location, gastroenteropancreatic neu'roendocdne tumor (GEP-NET) cells release hormones and biogenic amines in distinct patterns leading to typical hypersecretion syndromes. For example, the release ol serotonine is almost exclusively observed in midgut location whereas the release of hormones (e.g. gastdn, insulin) is primarily found in foregnt location. GEP-NET cells also express voltage-operated Ca 2+ channels (VOCCs) of the L-, N-, and P/Q-type. So far, it is unclear if a specific VOCC pattern of various Ca2+ channel subtypes correlates with these clinical observations. AIM: To investigate whether clinically distinct primary location of NETs are associated with specific patterns of VOCC expression. METHODS: Electrophysiological properties of human GEP-NET primary cell cultures (n = 9) and permanent GEP-NET cell cultures (n = 5) were analysed by the whole-cen patch-clamp technique and by contour plots. RESULTS: We could demonstrate specific Ca~§ channel properties in GP-NET cells [rum midgut tumors which were not found in GEP-NET cells from foregut location. While a sustained voltage-dependent Ca2 + channel inward current with low current density (about -2 pApF-~) was found in primary NET ceils from foregut tumors, a transient Ca2+ channel current with high current density (15.5 + 3.3 pApFZ; n = 4) was found in primary NET cells from midgut tumors. CONCLUSION: The differences in Ca2~ channel subtype pattern in GEP-NET cells from foregnt vs. midgnt may be functionally relevant for the hypersecretion syndrome predominantly found in foregut GEP-NETs. Thus, the use of specific Ca2. channel subtype inhibitors may help the development of a novel therapy for GEP-NET disease.
$1004
The TGF Beta-1 Signaling Pathway, Mitogenic and Fibrotie Pathways in Ileal Carcinoids Mark Kidd, Irvin M. Modlin, Kevin D. Lye, Toshinori Hinoue Background and Aims: Neuroendocrine tumors (NETs) of the small bowel are slowly growing carcinoids. Despite medical and therapeutic advances, they often present with pronounced fibrosis around tumor cells and in the peritoneal cavity. The prevalence of carcinoid heart disease, a potentially life-threatening complication, is increasing due to the prolonged life expectancy of carcinoid patients, secondary to improved treatment protocols for carcinoid tumors. Transforming growth factor-beta (TGFb) is a well known proliferative and fibrotic regulator and recent studies have indicated that TGFb leads to the induction of connective ttssue growth factor (CTGF), which acts in concert with TGFb to drive overproduction of collagen. CTGF itself may be an autocrine growth-regulator. We examined the expression and biological significance of TGFb signaling components in neuroendocrine carcinoid tumors of the small bowel. Methods: Alterations in TGFb signaling components was examined in two surgically resected ileal NET (carcinoid) specimens by Affymetrix GeneChip (U133A) analysis and compared to unaffected adjacent tissue, lmmunohistochemistry for TGFb, CTGF and cyclin D1, a marker for cell cycle progression, was undertaken in a carcinoid tissue microarray (TMA) (n = 81 carcinoids; including 33 small bowel, 17 appendiceal, 5 colon, 5 gastric carcinoids), staining intensity scored and correlated with disease status. CTGF, cyclin D1 and the cell cycle inhibitor p21, were investigated by reverse transcriptasepolyrnerase chain reaction in a tissue databank of 28 samples. Results: TGFb receptor II and BMP-2 were down-regulated in the two ileal carcinoids compared to control tissue. In contrast, Smad-3, a mediator of TGFb signaling and CTGF were up-regulated (p < 0.01) compared to normal tissue. Examination of the TMA demonstrated that 80% of carcinoid specimens were both TGFb/CTGF-positive, with a correlation between staining of r = 0.57, p < 0.0001. Small bowel tumors exhibited significantly higher staining scores for cyclin D1 (p < 0.05). More patients with earcinoids expressed CTGF message (85%) and p21 message (70%) compared to normal tissue (p < 0.03); none of the non-neoplastic tissue expressed cyclin D1. Conclusions: CTGF was upregulated in small bowel carcinoids which also exhibited elevations of Smad-3, cyclln D 1 and p21. These data suggest that modifications in the TGFb-1 pathway in ileal carcinoids result in alterations in a known regulator of fibrosis (CTGF) and in the cell cycle.
S1007
The Influence of Platelets on Promotion of Tumor Cells Invasion and it's inhibition by Antiplatelet Agents Keiichi Suzuki, Koichi Aiura, Masakazu Ueda, Masaki Kitajima The effect of platelets on facilitation of tumor ceils invasion has been noted, but the mechanism remains to be unclear. In the present study, we show that platelets promote the invasiveness of five human pancreatic carcinoma cell lines (SW. 1990, SU 86.86., Capan-2, BxPC-3 and AsPC-1) by using Chemoinvasion assay. Gelatin zymography was performed for the detection of MMP-9 secreted from tumor cells in presence of platelets. Moreover, the effects of antiplatelet agents (prostacyclin, eicosapentaenoic acid, and cilostazol) on tumor cells invasiveness and secretion level of MMP-9 from tumor cells were evaluated by using chemoinvasion assay and ELISA analysis. In chemoinvasion assay, the number of traversed tumor cells was significantly increased in condition incubated with platelets compared to without platelets in all cell lines. In gelatin zymography analysis, the band on 92 k-Da was detected in all pancreatic carcinoma cell lines, and the intensity was obviously greater in condition incubated with platelets than without platelets. In the experiment to evaluate the effects of antiplatelet agents on invasiveness, by using chemoinvasion assay, it was confirmed that invasiveuess of tumor ceils was significantly decreased by incubation with dlostazol, one of antiplatelet agents, depending on concentration in spite ot presence of platelets. The secrete level o[ MMP-9 from tumor cells was also significantly decreased in ELISA analysis. We postulate that platelet activated invasiveness of tumor cells because of secretion of MMP-9 from tumor cells was increased. Furthermore, antiplatelet drugs may inhibit invasiveness of tumor cells and this inhibition may lead to suppression of tumor cell invasion. These results suggested that antiplatelet agents were applicable to clinical treatment as antimetastasis of malignam tumor cells.
A-135
AGA Abstracts
$1005
Expression of Alternatively Spliced Isoforms of Tight Junction Protein ZO-1 in Gastrointestinal Tumors Shigeru Kanaoka, Ken-lchi Yoshida, Mutsuhiro Ikuma, Naoyuki Miura, Haruhiko Sugimura, Masayoshi Kajimura Background: In vitro model of epithelial neoplasia have shown that there is association between increased tumor promoter-induced tight junction (TJ) permeability, transepithellal flux of growth factors and development of epithelial tumors. Alternatively spliced isoforms of TJ protein ZO-1 (ZO-1 a plus and ZO-1 cx minus) have been reported to correlate with junctional plasticity; ZO-1 a minus is expressed in structurally dynamic junctions, whereas ZO-1 a plus is expressed in those which are less dynamic. ZO-2, a functional component of TJ, differentially expressed in normal pancreatic duct compared to pancreatic adenocarcinoma. We aimed to determine the expression of alternatively spliced isoforms of ZO-1 in gastrointestinal tumors. Methods: Tissues were obtained from 20 patients with gastric cancer, 17 patients with coloretal cancer and 7 patients with colorectal adenoma. Matched tumor and normal mucosal specimens were obtained from endoscopidforceps biopsy. RNA was extracted and used as a template for RT-PCR usmg specific primers straddled ZO-1 c~ domain. Results: The expression level of ZO- 1 a plus was higher than that of ZO-i a minus in every normal gastric mucosa and every normal colorectal mucosa. The median expression ratios of ZO-1 a minus to a plus (ZO-1 c~ minus/plus) were 0.61 +/- 0.17 and 0.60 +/0.11 respectively, while those in gasmc carcinoma, colorectal carcinoma and adenoma were 0.81 +/- 0.38 (p < 0.05), 0.91 +/- 0.31 (p < 0.005) and 1.1 +/- 0.25 (p < 0.05), respectively (p value of Wilcoxon signed-rank test are shown in parenthesis). The expression ratio of ZO-1 oc minus/plus in gastric and colorectal tumors was significantly higher than that in corresponding normal mucosa, especially in coIorectal tumors. ZO-2 differentially expressed between in pancreatic cancer and in normal pancreatic duct. Similarly, expression pattern of ZO-1 isoforms in gastrointestinal tumors is different from that of normal mucosa. Conclusion: These results suggest that the change of expression pattem of ZO-1 isoforms may influence junctional plasticity, which give growth advantage of gastrointestinal tumors through direct contact with luminal various growth factors.
SIO03
Butyrate Impairs Intestinal Tumor Cell Induced Angiogenesis by Inhibiting HIFl a Nuclear Translocation Dimitrios Zgouras, Juergen Stein Angiogenesis is a complex process encompassing endothelial cell migration, proliferation, and tube formation. Inhibition of angiogenesis is considered to be one of the most promising strategies in the development of novel anti-neoplastic therapies. Butyrate is known to stimulate proliferation of normal crypt ceils, it inhibits growth and induce apoptosis in colon cancer cells. In this study we examined the effects of butyrate on colon cancer (Caco-2) cell induced angiogenesis. Methods: Condiotioned media were obtained by Caco-2 cells, incubated with serum free medium containing either the solvent (PBS) or butyrate (1 or 2 raM). Proliferation of HUVEC cells was assesed by a BrdU incorporation assay. VEGF production in the supernatant of Caco-2 cells was quantified by use of a colorimetric ELISA. Furthermore HIF-lu expression in total cell lysate and in nuclear extracts was detected by Western Blot. Intracelhilar distribution of HIF-lot was additionally observed by immunofluorescence. Nuclear HIF- lc~ DNA-binding activity was detected by art HIF-la transcription assay. Results: HUVEC cell proliferation was significantly inhibited up to 46% of controls when incubated with medium conditioned by butyrate treated Caco-2 cells. Simultaneously, VEGF, a proangiogenic vascular endothelial growth factor, was significantly reduced. Protein levels of HIF-la, which is a nuclear transcription factor known to be a key regulator in hypoxia induced angiogenesis, were upregulated by butyrate. This is in contrast to its importance as a VEGF regulating component. However Western blot analysis of nuclear extracts showed a downregulation of H1F-la protein. Nuclear HIF-la DNA-binding activity was also decreased by butyrate. Immunofluoresoence experiments indicate that HIF 1 nucleolar sequestration is repressed by butyrate, through inhibition of nuclear translocation. Conclusion: Taken together our data indicate that butyrate inhibits not only growth, but also angiogenic properties of colon cancer cells (Caco-2). This effects are likely to be induced by inhibition of HIF-ltx nuclear transloeation. We postulate that diminished HIF-la nuclear presence and activity in butyrate treated Caco-cells could be one reason for decreased VEGF expression and antiangiogenic effects.
S1006
Differential Ca2 + Channel Activity in Human Primary Neuroendocrine Tumor Cell Cultures from Fore- and Midgut Tumors Stefan Mergler, Adriana Drost, Wolf-Otto Bechstein, Peter Nenhans, Bertram Wiedenmann BACKGROUND: Depending on the primary location, gastroenteropancreatic neu'roendocdne tumor (GEP-NET) cells release hormones and biogenic amines in distinct patterns leading to typical hypersecretion syndromes. For example, the release ol serotonine is almost exclusively observed in midgut location whereas the release of hormones (e.g. gastdn, insulin) is primarily found in foregnt location. GEP-NET cells also express voltage-operated Ca 2+ channels (VOCCs) of the L-, N-, and P/Q-type. So far, it is unclear if a specific VOCC pattern of various Ca2+ channel subtypes correlates with these clinical observations. AIM: To investigate whether clinically distinct primary location of NETs are associated with specific patterns of VOCC expression. METHODS: Electrophysiological properties of human GEP-NET primary cell cultures (n = 9) and permanent GEP-NET cell cultures (n = 5) were analysed by the whole-cen patch-clamp technique and by contour plots. RESULTS: We could demonstrate specific Ca~§ channel properties in GP-NET cells [rum midgut tumors which were not found in GEP-NET cells from foregut location. While a sustained voltage-dependent Ca2 + channel inward current with low current density (about -2 pApF-~) was found in primary NET ceils from foregut tumors, a transient Ca2+ channel current with high current density (15.5 + 3.3 pApFZ; n = 4) was found in primary NET cells from midgut tumors. CONCLUSION: The differences in Ca2~ channel subtype pattern in GEP-NET cells from foregnt vs. midgnt may be functionally relevant for the hypersecretion syndrome predominantly found in foregut GEP-NETs. Thus, the use of specific Ca2. channel subtype inhibitors may help the development of a novel therapy for GEP-NET disease.
$1004
The TGF Beta-1 Signaling Pathway, Mitogenic and Fibrotie Pathways in Ileal Carcinoids Mark Kidd, Irvin M. Modlin, Kevin D. Lye, Toshinori Hinoue Background and Aims: Neuroendocrine tumors (NETs) of the small bowel are slowly growing carcinoids. Despite medical and therapeutic advances, they often present with pronounced fibrosis around tumor cells and in the peritoneal cavity. The prevalence of carcinoid heart disease, a potentially life-threatening complication, is increasing due to the prolonged life expectancy of carcinoid patients, secondary to improved treatment protocols for carcinoid tumors. Transforming growth factor-beta (TGFb) is a well known proliferative and fibrotic regulator and recent studies have indicated that TGFb leads to the induction of connective ttssue growth factor (CTGF), which acts in concert with TGFb to drive overproduction of collagen. CTGF itself may be an autocrine growth-regulator. We examined the expression and biological significance of TGFb signaling components in neuroendocrine carcinoid tumors of the small bowel. Methods: Alterations in TGFb signaling components was examined in two surgically resected ileal NET (carcinoid) specimens by Affymetrix GeneChip (U133A) analysis and compared to unaffected adjacent tissue, lmmunohistochemistry for TGFb, CTGF and cyclin D1, a marker for cell cycle progression, was undertaken in a carcinoid tissue microarray (TMA) (n = 81 carcinoids; including 33 small bowel, 17 appendiceal, 5 colon, 5 gastric carcinoids), staining intensity scored and correlated with disease status. CTGF, cyclin D1 and the cell cycle inhibitor p21, were investigated by reverse transcriptasepolyrnerase chain reaction in a tissue databank of 28 samples. Results: TGFb receptor II and BMP-2 were down-regulated in the two ileal carcinoids compared to control tissue. In contrast, Smad-3, a mediator of TGFb signaling and CTGF were up-regulated (p < 0.01) compared to normal tissue. Examination of the TMA demonstrated that 80% of carcinoid specimens were both TGFb/CTGF-positive, with a correlation between staining of r = 0.57, p < 0.0001. Small bowel tumors exhibited significantly higher staining scores for cyclin D1 (p < 0.05). More patients with earcinoids expressed CTGF message (85%) and p21 message (70%) compared to normal tissue (p < 0.03); none of the non-neoplastic tissue expressed cyclin D1. Conclusions: CTGF was upregulated in small bowel carcinoids which also exhibited elevations of Smad-3, cyclln D 1 and p21. These data suggest that modifications in the TGFb-1 pathway in ileal carcinoids result in alterations in a known regulator of fibrosis (CTGF) and in the cell cycle.
S1007
The Influence of Platelets on Promotion of Tumor Cells Invasion and it's inhibition by Antiplatelet Agents Keiichi Suzuki, Koichi Aiura, Masakazu Ueda, Masaki Kitajima The effect of platelets on facilitation of tumor ceils invasion has been noted, but the mechanism remains to be unclear. In the present study, we show that platelets promote the invasiveness of five human pancreatic carcinoma cell lines (SW. 1990, SU 86.86., Capan-2, BxPC-3 and AsPC-1) by using Chemoinvasion assay. Gelatin zymography was performed for the detection of MMP-9 secreted from tumor cells in presence of platelets. Moreover, the effects of antiplatelet agents (prostacyclin, eicosapentaenoic acid, and cilostazol) on tumor cells invasiveness and secretion level of MMP-9 from tumor cells were evaluated by using chemoinvasion assay and ELISA analysis. In chemoinvasion assay, the number of traversed tumor cells was significantly increased in condition incubated with platelets compared to without platelets in all cell lines. In gelatin zymography analysis, the band on 92 k-Da was detected in all pancreatic carcinoma cell lines, and the intensity was obviously greater in condition incubated with platelets than without platelets. In the experiment to evaluate the effects of antiplatelet agents on invasiveness, by using chemoinvasion assay, it was confirmed that invasiveuess of tumor ceils was significantly decreased by incubation with dlostazol, one of antiplatelet agents, depending on concentration in spite ot presence of platelets. The secrete level o[ MMP-9 from tumor cells was also significantly decreased in ELISA analysis. We postulate that platelet activated invasiveness of tumor cells because of secretion of MMP-9 from tumor cells was increased. Furthermore, antiplatelet drugs may inhibit invasiveness of tumor cells and this inhibition may lead to suppression of tumor cell invasion. These results suggested that antiplatelet agents were applicable to clinical treatment as antimetastasis of malignam tumor cells.
A-135
AGA Abstracts