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Low BAX protein expression correlates with disease-recurrence in preoperatively irradiated rectal cancer
Tg mice (p2-4 told especially at the proximal end of the colon. In response to AOM, the incidence of neoplastic leswns in the proximal colon of hGAS mice was also slgnificandy increased (Gastro, 2000), suggesting the possibihty that the anti-apoptotic effects of PG may be mediating the obseFeed increase in colon carcinogenesis in the proximal colon o{ tbese mice. Since PG is expressed in patients ~aJth hypergastrinemia and in patients with colorectal cancers, it ts possible that both endocrine and autocrine PG play an important role in colon carcinogenesis. Potent anti-apoptotic effects of PG on the colonic epithelial cells, as observed in the current studies, may {beretbre play, an important role in colon carcinogenesis in patients expressing PG
T986 Deoxycholic acid-induced signal transduction in HT-29 cells: Role of NF-KB and Interleukin-8 H)'an4on Kim, Dong Ki Lee, Sun Young Park, Soon Koo Balk, Sang Ok Kwon Background/Aims: Secondary bile acid, particularly &oxycholic acid (DCA), has been appeared to be endogeneous colon tumor promoter in vivo and to enhance ceil transformation in vitro. Recently, nuclear factor-kappa 8 (NF4r highly- expressed in colonic adenomatous polyp and cancer, has been related to chemoresistance through anti-apoptosis. Also Interleukin-8 (IL-8) expression diredly correlated with increasing metastatic potential of colon cancer m vitro and in vivo. In this study, we investigated whether DCA reduces NF-gB activation and IL-8 expression and w'hed:er taurousodeoxycholic acid (TUDC), a known chemopreventive agent, or nonselective COX-2 inhibitor, Sulindac, inhibits this signaling in HT-29 cells. Methods: After dose-&pen&nt DCA treatment, time courses of NF-KB binding activity tn the nuclear extracts of cells were &termnied by dectrophoretic mobility shift assay (EMSA) using as probes radiotabeled oligonucleoti&s representing gel retardation oligonudeotides of NF4r (Promega Corp., Wl). Also, we performed Western blotting of IKBa (Santa cruze, CA) to confirm NF-KB activation, Time and concentration courses of DCAinduced secretion of IL-8 were measured with ELISA (R&D System, MN) in supernatams of cultured media from cells. To evaluate the role of NF4r on the expression of IL-8, lL-8 levels were assessed after pretreatment with nsmg phosphorothioate-modified anti-sense oligonncleoti&s (ODN), complementary sequences of ATG start codon for p50 and p65 mRNA of NF-tCB. Moreover, DCA-induced secretions of IL-8 were measured after 2hr pretreatment of TUDC or Sulindac, Results: DCA dose-&pen&ntly induced prominent NFKB binding complexes (p50/50 and p65/50) from 30 rain to 8 hr. In addition, DCA caused dose-dependent &gradation of IKBa in Western blotting. The secretions of IL-8 were increased with DCA (50-200 taM) treatment in a time and dose-dependent manner (6hr24hr), and 200 taM of DCA caused approximately 3-fold increase in II--8 after 24hr. Proincubation of the cells with TUDC (0.1-10/~M) or Sulindac (100-400 ~M) for 2 hr caused significant dose-&pen&nt decreases in DG~ induced IL-8 secretion. However, transient n'ansfection using p50 or p65 AS-ODN by DOTAP method caused no influence on DCA induced IL-8 secretion. Conclusion; DCA may act as a colonic tumor promoter through anti-apoptototic effect of NF-ICBand IL-8 expression, and d:isDCA-induced NF-KB independent lL-8 expression is inhibited by TUDC or nonselective COX-2 inhibitor.
T984 Selective COX-2 Inhibitor in Liver Cancer Cells: Role and Mechanism Sandeep S. Sekbon, Tamara Sneddon, Wilham Bowen, George Michalopo~tlos, Satdarshan Pal S. Monga Hepatocellular cancer remains a disease of grim prognosis and limited therapeutic options. 13-Catenin stabilization is seen in a large percentage of hver tumors. Cox-2, a know target gene of the Wnt/~,-catenm pathway has been shown to be upregnlatad in tumors exhibiting such stabilization. We wanted to investigate d:e efl?ct of selective cox-2 inhibitor (Celeeoxib) on the Wnt/[3-cateinn pathway and on the growth of liver cancer cells. We utilized 2 mt beparoma ceil lines-JMI and JM2 that have been characterized previously. JMI cells (isdated from highly metastatic tumor) are smaller, rounded, rapidly growing ceils and lack ~-catenin. JM2 ceils are larger, bepatocyte-like and slow growing cells that express ~-catenin. Cox-2 inhibitoss were added to the celt cultures every 24 hours at doses ranging from 20-100 lag/ ml. Proliferation assay was pertbrmed by tby~idine uptake. Also, treated cells were utilized for protein extraction for western anal?,~is There was a clear decrease in cell confluence and number in response to Celecoxib within 5 days in both JM1 and JM2 cells~ There was also increased cell detachment and cell &afh in these cultures. After 10 days of drug traatmem, there was a massive change in the cell nun:ber, morphology" and viability in the JM1 ceils. JM2 cells displayed complete cell death and no viable cell was detectable after 10 days A two-fold decrease in thyanidine uptake was evident niJM1 cells after 48 hours in culture in preseoce nf Celecomb. JM2 cells showed a 2,5 fold decrease in thyraidine uptake at 48 hours and a further decrease by 50% at 96 hours, in response to treatmem. Western Blot analysis utilizing extracts from treated JM1 and JM2cells demonstrated a more than 2-5 fold decrease in total cyclin-D1 keels as compared to the untreated controls There was a significant decrease in total ~-catenin levds m the JM2 cells following Celecoxib treatment. We conclude that, selective cox-2 inhibitor Celecoxib demonstrates an in vitro anti-tumor activW in liver cancer cells by affecting proliferation and enhancing apoptosis. Celecoxib signiflcanfly decreases cyclin-Dl Ievels in these tumor cells, suggesting a potential role in blunting tumor progressiun. It exerts its anti-tumor role via 13-caterdn dependent and independent mechanisms. Finally cyclin-D1 in liver seems to be also regulated via 13catenin dependent and independent mechauisms as can be seen by effect of Celecoxib on cyclm-D1 levels in both bepatoma cell lines
T987 Low BAX Protein Expression correlates with Disease-Recurrence m Preoperatively Irradiated Rectal Cancer Oliver Nehls, Thomas Okech, Chi-Jen Hsieh, Franz Borchard, Hans-Helmnt Gmenagel, Vera Gaco, Michael Gregor, Bodo Klump Purpose: To deternbine the prognostic impact of BAX protein expression in correlation to clinico-pathological variables and patient outcome as well as in view of the p53/BAX pathway. Experimental design: We nivestigated retrospectively 92 patients with preoperativdy irradiated rectal cancer. After irradiation with a cumulative dose of 30 Gray, all patients nn&r~vent surgical resection at the same institution. Immunohistochemistry was performed on paraffin sections of pretreatment biopsy samples using a monoclonal antibody that recognizes the cytophsmatic amino epitope of BA,X protein. The p53 expression data, which we had been previously determined in this cohort, had also been considered. Results: BAX protein expression was classified as high and low in 63 (685%) and 29 (31,5%) tumors, respectively" Univariate analysis revealed no correlation between BAXprotein expression and cfinicopathological variables like age, gender, tumor location, TNM stage, pT category, pN category, or histologic grade. However, high BAX expression was significantly associated ~nth improved disease free-survival by univariate analysis (p = 0.048). Moreover, in multivariate analyses, high BAX expression was an independent prognostic marker tbr both local recurrence flee interval and disease tree-survival Concerning the p53~AX pathway, subgroup analysis yielded no association between p53 immunonegative/BAX high versus p53 immunopositive/ BAX low expressing tumors and overall, disease-free or local recurrence flee survival m either uinvariate (P = 0.88, 0.54 and O.16, respectively) or multivariate analysis, Conclusions: This study demonstrates that BAX protein expression might help to predict disease recun'enee in preoperatively" irradiated rectal cancer, whereas &termination of the p53/BAX pathway adds no further information.
T985 Colorectal cancer: trnphic factors, COX-2 and apoptotin proteins status in patients before and after radiotherapy Madej Gonciarz, Piotr PiemchalskL Wladyslaw Bielanskl, Monika Zuchowicz, Artur Hartwich, Stanislaw J. Konturek, jacek Staraewski, Wieslaw Pawlik Coloractal cancer (CRC) is one of the most common form of cancer and the second leading cause of death. The CRC has been attributed to genetic and/or dietary and envimnmemal lactors but PGE2 and trophic fact6rs such as progastrin, gastrin have also been implicated. Ibis study was &signed to determine: (1) the plasma levels of progastrin, amidated gastrni in CRC patients betore and 1 month after removal of the tunror, (2) the concentrations of progastrin, gastrin, and PGE2 in the minor tissue, (3) expression tbe gastrin mRNA and gastrirgCCKB - receptor mRNA, (4) expression of cyclooxygenase COX- 1 and COX-2 mRNA, (4) expression of Bax and IM2 mRNA in two groups of patients: 1- (N = 30) with, lI (N = 30) wnhout preceding radio therapy. We have found statistically signd'icant decrease in plasma p'rogastrin concentration in patients after tumor resection and significant difference of progastnn concentration between turnor tissue and its resection margin. Plasma gastrni levels in both groups (before and after removal of tumor) also significant by decreased after tumor resection. Concentrations of tissue PGE2 in tumor tended to decrease as compared to the resection margin in both groups PCR analysis showed an overexpeession of gastrin mRNA in 50% of tumor dss:m in group I and 90% in group II. Expression of CCKB - receptor mRNA in tumor tissue was shown in 70% and 100% of patients in group I and I1 ,respectively. There were no differences in COX-1 expression in both groups (40%) but COX-2 mRNA expression was ddlerent between groups and reacbed 60% in group I and 90% in group 1I Most striking diflerences between groups of patients were noted in apoptosis related proteins mRNA e~:pression. Group I : m 75% of a*mlyzed samples the abundance of bax mKNA was t0und to be higher in tumor tissue :hen in a resection margin whereas in group Ii only in 23%. For bcl-2 mRNA those value were 66% and 53% respectively. We conclude that 1. CRC tissue and its resection margin contain high concemrations of progastrin and gastrin that might sumulata minor growth via locally cwerexpressed CCKB - receptors; 2. Accordingly, the removal ot CRCs is t0llowed by reduction in plasma levels of both gastrm attd progastein; 3. COX-2 mRNA is overexpressed in tumor tissue and could contribute to reduction of apoptosis; 4. Radiotberapy causes the changes in equilibrium between box and bel-2 mDgA comparing to not-treated tissues indicating the induction of apoptotic process m irrad:ated tumor
AGA Abstracts
T988 Gastric Mucosa Cell Turnover from Patients with Early and Advanced Gastric Cancer Spiros D. Ladas, Konstantinos Triantatyllou, Panagiota Kitsanta, Dimitrios G. Karamanoha, Sotirios A. Raptis introduction: Early gastric cancer (EGC) is a different biologic entity dran advanced cancer (AGC), Epithelial cell turnover alterations play important role during oncogenesis. Aim: To investigate cell apoptosis and proliferation rates in early" and advanced gastric cancer and in the gastric mucosa adjacent to cancer. Methods: We examined tissue biopsies from 17 EGC, 15 AGC and 18 H, pylori positive dyspeptic patients (DPT), We also examined nondysplastic tissue specimens 5 cm apart from the margin of each tumor H, pylori status and cell proliferation were studied inmmnohistochemicafiy with an anti-H, pylori and MIB-1 by the Avidin-Biotin Complex method. Apoptusis was measured by TUNEL method, "[ire rate of the positive stained cells was count using image analysis technique (SABA). Results: H. pylori was detected in 16/17 and 11/15 early and advanced gastric cancers, respectively. Median apoptosis index was significantly higher in EGC (10) and AGC (10) than in DPT (3) (p<0.001). Median proliferation index was not significantly different among EGC (35) AGC (25) and DPT (29) (p = 0.3). No significant differences were observed of either apoptosis or proliferation indexes between EGC and AGC. Median apoptosis index was significantly" lower in non-dysplastic tissue adjacent to EGC (25) and AGC (18) than in DPT (31) (p=O.05). Median proliferation index was significantly lower in EGC (8) and AGC (12) than in DPT (29) (p<0.fl01). Conclusions: Cell turnover in not different between early and
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T986 Deoxycholic acid-induced signal transduction in HT-29 cells: Role of NF-KB and Interleukin-8 H)'an4on Kim, Dong Ki Lee, Sun Young Park, Soon Koo Balk, Sang Ok Kwon Background/Aims: Secondary bile acid, particularly &oxycholic acid (DCA), has been appeared to be endogeneous colon tumor promoter in vivo and to enhance ceil transformation in vitro. Recently, nuclear factor-kappa 8 (NF4r highly- expressed in colonic adenomatous polyp and cancer, has been related to chemoresistance through anti-apoptosis. Also Interleukin-8 (IL-8) expression diredly correlated with increasing metastatic potential of colon cancer m vitro and in vivo. In this study, we investigated whether DCA reduces NF-gB activation and IL-8 expression and w'hed:er taurousodeoxycholic acid (TUDC), a known chemopreventive agent, or nonselective COX-2 inhibitor, Sulindac, inhibits this signaling in HT-29 cells. Methods: After dose-&pen&nt DCA treatment, time courses of NF-KB binding activity tn the nuclear extracts of cells were &termnied by dectrophoretic mobility shift assay (EMSA) using as probes radiotabeled oligonucleoti&s representing gel retardation oligonudeotides of NF4r (Promega Corp., Wl). Also, we performed Western blotting of IKBa (Santa cruze, CA) to confirm NF-KB activation, Time and concentration courses of DCAinduced secretion of IL-8 were measured with ELISA (R&D System, MN) in supernatams of cultured media from cells. To evaluate the role of NF4r on the expression of IL-8, lL-8 levels were assessed after pretreatment with nsmg phosphorothioate-modified anti-sense oligonncleoti&s (ODN), complementary sequences of ATG start codon for p50 and p65 mRNA of NF-tCB. Moreover, DCA-induced secretions of IL-8 were measured after 2hr pretreatment of TUDC or Sulindac, Results: DCA dose-&pen&ntly induced prominent NFKB binding complexes (p50/50 and p65/50) from 30 rain to 8 hr. In addition, DCA caused dose-dependent &gradation of IKBa in Western blotting. The secretions of IL-8 were increased with DCA (50-200 taM) treatment in a time and dose-dependent manner (6hr24hr), and 200 taM of DCA caused approximately 3-fold increase in II--8 after 24hr. Proincubation of the cells with TUDC (0.1-10/~M) or Sulindac (100-400 ~M) for 2 hr caused significant dose-&pen&nt decreases in DG~ induced IL-8 secretion. However, transient n'ansfection using p50 or p65 AS-ODN by DOTAP method caused no influence on DCA induced IL-8 secretion. Conclusion; DCA may act as a colonic tumor promoter through anti-apoptototic effect of NF-ICBand IL-8 expression, and d:isDCA-induced NF-KB independent lL-8 expression is inhibited by TUDC or nonselective COX-2 inhibitor.
T984 Selective COX-2 Inhibitor in Liver Cancer Cells: Role and Mechanism Sandeep S. Sekbon, Tamara Sneddon, Wilham Bowen, George Michalopo~tlos, Satdarshan Pal S. Monga Hepatocellular cancer remains a disease of grim prognosis and limited therapeutic options. 13-Catenin stabilization is seen in a large percentage of hver tumors. Cox-2, a know target gene of the Wnt/~,-catenm pathway has been shown to be upregnlatad in tumors exhibiting such stabilization. We wanted to investigate d:e efl?ct of selective cox-2 inhibitor (Celeeoxib) on the Wnt/[3-cateinn pathway and on the growth of liver cancer cells. We utilized 2 mt beparoma ceil lines-JMI and JM2 that have been characterized previously. JMI cells (isdated from highly metastatic tumor) are smaller, rounded, rapidly growing ceils and lack ~-catenin. JM2 ceils are larger, bepatocyte-like and slow growing cells that express ~-catenin. Cox-2 inhibitoss were added to the celt cultures every 24 hours at doses ranging from 20-100 lag/ ml. Proliferation assay was pertbrmed by tby~idine uptake. Also, treated cells were utilized for protein extraction for western anal?,~is There was a clear decrease in cell confluence and number in response to Celecoxib within 5 days in both JM1 and JM2 cells~ There was also increased cell detachment and cell &afh in these cultures. After 10 days of drug traatmem, there was a massive change in the cell nun:ber, morphology" and viability in the JM1 ceils. JM2 cells displayed complete cell death and no viable cell was detectable after 10 days A two-fold decrease in thyanidine uptake was evident niJM1 cells after 48 hours in culture in preseoce nf Celecomb. JM2 cells showed a 2,5 fold decrease in thyraidine uptake at 48 hours and a further decrease by 50% at 96 hours, in response to treatmem. Western Blot analysis utilizing extracts from treated JM1 and JM2cells demonstrated a more than 2-5 fold decrease in total cyclin-D1 keels as compared to the untreated controls There was a significant decrease in total ~-catenin levds m the JM2 cells following Celecoxib treatment. We conclude that, selective cox-2 inhibitor Celecoxib demonstrates an in vitro anti-tumor activW in liver cancer cells by affecting proliferation and enhancing apoptosis. Celecoxib signiflcanfly decreases cyclin-Dl Ievels in these tumor cells, suggesting a potential role in blunting tumor progressiun. It exerts its anti-tumor role via 13-caterdn dependent and independent mechanisms. Finally cyclin-D1 in liver seems to be also regulated via 13catenin dependent and independent mechauisms as can be seen by effect of Celecoxib on cyclm-D1 levels in both bepatoma cell lines
T987 Low BAX Protein Expression correlates with Disease-Recurrence m Preoperatively Irradiated Rectal Cancer Oliver Nehls, Thomas Okech, Chi-Jen Hsieh, Franz Borchard, Hans-Helmnt Gmenagel, Vera Gaco, Michael Gregor, Bodo Klump Purpose: To deternbine the prognostic impact of BAX protein expression in correlation to clinico-pathological variables and patient outcome as well as in view of the p53/BAX pathway. Experimental design: We nivestigated retrospectively 92 patients with preoperativdy irradiated rectal cancer. After irradiation with a cumulative dose of 30 Gray, all patients nn&r~vent surgical resection at the same institution. Immunohistochemistry was performed on paraffin sections of pretreatment biopsy samples using a monoclonal antibody that recognizes the cytophsmatic amino epitope of BA,X protein. The p53 expression data, which we had been previously determined in this cohort, had also been considered. Results: BAX protein expression was classified as high and low in 63 (685%) and 29 (31,5%) tumors, respectively" Univariate analysis revealed no correlation between BAXprotein expression and cfinicopathological variables like age, gender, tumor location, TNM stage, pT category, pN category, or histologic grade. However, high BAX expression was significantly associated ~nth improved disease free-survival by univariate analysis (p = 0.048). Moreover, in multivariate analyses, high BAX expression was an independent prognostic marker tbr both local recurrence flee interval and disease tree-survival Concerning the p53~AX pathway, subgroup analysis yielded no association between p53 immunonegative/BAX high versus p53 immunopositive/ BAX low expressing tumors and overall, disease-free or local recurrence flee survival m either uinvariate (P = 0.88, 0.54 and O.16, respectively) or multivariate analysis, Conclusions: This study demonstrates that BAX protein expression might help to predict disease recun'enee in preoperatively" irradiated rectal cancer, whereas &termination of the p53/BAX pathway adds no further information.
T985 Colorectal cancer: trnphic factors, COX-2 and apoptotin proteins status in patients before and after radiotherapy Madej Gonciarz, Piotr PiemchalskL Wladyslaw Bielanskl, Monika Zuchowicz, Artur Hartwich, Stanislaw J. Konturek, jacek Staraewski, Wieslaw Pawlik Coloractal cancer (CRC) is one of the most common form of cancer and the second leading cause of death. The CRC has been attributed to genetic and/or dietary and envimnmemal lactors but PGE2 and trophic fact6rs such as progastrin, gastrin have also been implicated. Ibis study was &signed to determine: (1) the plasma levels of progastrin, amidated gastrni in CRC patients betore and 1 month after removal of the tunror, (2) the concentrations of progastrin, gastrin, and PGE2 in the minor tissue, (3) expression tbe gastrin mRNA and gastrirgCCKB - receptor mRNA, (4) expression of cyclooxygenase COX- 1 and COX-2 mRNA, (4) expression of Bax and IM2 mRNA in two groups of patients: 1- (N = 30) with, lI (N = 30) wnhout preceding radio therapy. We have found statistically signd'icant decrease in plasma p'rogastrin concentration in patients after tumor resection and significant difference of progastnn concentration between turnor tissue and its resection margin. Plasma gastrni levels in both groups (before and after removal of tumor) also significant by decreased after tumor resection. Concentrations of tissue PGE2 in tumor tended to decrease as compared to the resection margin in both groups PCR analysis showed an overexpeession of gastrin mRNA in 50% of tumor dss:m in group I and 90% in group II. Expression of CCKB - receptor mRNA in tumor tissue was shown in 70% and 100% of patients in group I and I1 ,respectively. There were no differences in COX-1 expression in both groups (40%) but COX-2 mRNA expression was ddlerent between groups and reacbed 60% in group I and 90% in group 1I Most striking diflerences between groups of patients were noted in apoptosis related proteins mRNA e~:pression. Group I : m 75% of a*mlyzed samples the abundance of bax mKNA was t0und to be higher in tumor tissue :hen in a resection margin whereas in group Ii only in 23%. For bcl-2 mRNA those value were 66% and 53% respectively. We conclude that 1. CRC tissue and its resection margin contain high concemrations of progastrin and gastrin that might sumulata minor growth via locally cwerexpressed CCKB - receptors; 2. Accordingly, the removal ot CRCs is t0llowed by reduction in plasma levels of both gastrm attd progastein; 3. COX-2 mRNA is overexpressed in tumor tissue and could contribute to reduction of apoptosis; 4. Radiotberapy causes the changes in equilibrium between box and bel-2 mDgA comparing to not-treated tissues indicating the induction of apoptotic process m irrad:ated tumor
AGA Abstracts
T988 Gastric Mucosa Cell Turnover from Patients with Early and Advanced Gastric Cancer Spiros D. Ladas, Konstantinos Triantatyllou, Panagiota Kitsanta, Dimitrios G. Karamanoha, Sotirios A. Raptis introduction: Early gastric cancer (EGC) is a different biologic entity dran advanced cancer (AGC), Epithelial cell turnover alterations play important role during oncogenesis. Aim: To investigate cell apoptosis and proliferation rates in early" and advanced gastric cancer and in the gastric mucosa adjacent to cancer. Methods: We examined tissue biopsies from 17 EGC, 15 AGC and 18 H, pylori positive dyspeptic patients (DPT), We also examined nondysplastic tissue specimens 5 cm apart from the margin of each tumor H, pylori status and cell proliferation were studied inmmnohistochemicafiy with an anti-H, pylori and MIB-1 by the Avidin-Biotin Complex method. Apoptusis was measured by TUNEL method, "[ire rate of the positive stained cells was count using image analysis technique (SABA). Results: H. pylori was detected in 16/17 and 11/15 early and advanced gastric cancers, respectively. Median apoptosis index was significantly higher in EGC (10) and AGC (10) than in DPT (3) (p<0.001). Median proliferation index was not significantly different among EGC (35) AGC (25) and DPT (29) (p = 0.3). No significant differences were observed of either apoptosis or proliferation indexes between EGC and AGC. Median apoptosis index was significantly" lower in non-dysplastic tissue adjacent to EGC (25) and AGC (18) than in DPT (31) (p=O.05). Median proliferation index was significantly lower in EGC (8) and AGC (12) than in DPT (29) (p<0.fl01). Conclusions: Cell turnover in not different between early and
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