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Expression and prognostic value of metalloproteinases in human gastric carcinomas
April 1995
• HELICOBACTER PYLORI BROTH CULTURE SUPERNATANTS ENHANCE OXIDANT-INDUCED CELL INJURY TO HUMAN GASTRIC EPITHELIAL CELLS. K.G. Vernon, A. Asseffa, M.H. Earlington, T.L. Cover, D.T. Smoot. Depts. of Medicine & Biochemistry, Howard Univ. College of Medicine, Washington, D.C.; Dept. of Medicine, Vanderbilt Univ. & Nashville VAMC, Nashville, TN. Risks for gastric cancer in relation to H. pylori (HP), diet and other environmental factors are receiving renewed attention. The exact role and interactions between HP and oxidants remains to be elucidated. The following experiments were performed to determine if exposure of gastric cells to HP increases oxidant associated cell injury. A human gastric epithelial cell line (HAE), which was established by transfecting normal human gastric epithelial cells with the SV4O Large T-antigen gone, was used for these experiments. Cells were grown on 96-well clusters, then exposed overnight to broth culture supernatants from isogenic HP strains with VacA (vacuolating cytotoxin) activity, without VacA activity or Brucella broth control. The next day cells were exposed to xanthine and xanthine oxidase (to generate superoxide radical) for one hour. Cell viability was measured by two methods, metabolism of MTS using a non-radioactive proliferation assay (Promega Corp, Madison, WI) and by dual fluorescent cell labefing using a Live/Dead cell assay (Molecular Probes, Eugene, OR). The %viability of cells exposed to superoxide, as measured by MTS metabolism, incubated with medium alone, control broth, VacA-neg broth and VaoA-pos broth was 100%, 97.7%, 70.5% and 63.5%, respectively. With the fluorescent Live/Dead cell assay, cell viability after exposure to supei'oxide was 94.4%, 65.9% and 57.4%, for cells exposed to control broth, VacA-neg broth and VaeA-P0S broth, respectively. These data show that HP products signitlcantly enhance the susceptibility of HAE gastric epithelial cells to 0xyradieai associated cell injury, which is likely the outcome of maeromolecular damage to membrane lipids and cytoplasmic proteins. The mechanism by which HP products enhances the susceptibility of HAE cells to superoxide is unknown; however, the VacA cytotoxin does not appear to be the primary bacterial product responsible. Further studies are needed to identify the bacterial products responsible and elucidate their mechanism(s) of action. Increased suceptibility to superoxide induced cell injury also increases the likelyhood of oxidant related DNA damage and carcinogenic transformation.
• PROGNOSTIC IMPACT OF THE TISSULAR PLASMINOGEN ACTIVATION SYSTEM IN PATIENTS WITH GASTRIC CANCER. H.W. Verspaqet, S. Ganesh, C.F.M. Sier, M.M. Heerding, G. Gdffioen, C.B.H.W. Lamers. Dept. of Gastroenterology and Hepatology, University Hospital, 2333 AA Leiden, The Netherlands. Gastric cancer has a poor prognosis and can only be cured in an early stage by surgery. Identification by pathophysiological markers of subgroups of patients with a good or poor prognosis might help to select patients for adjuvant therapies. Plasminogen activation (PA) parameters, important in tumor invasion and metastasis, have been shown to be of prognostic value in some human malignancies. We evaluated the relation of several plasminogen activation parameters in tissue of patients with gastdc cancer (n=50) with the overall survival of at least 2 years, and a compadson was made with standard clinicopathological parameters. Univadate Cox analysis showed that a low tissue-type PA (t-PA) activity in normal mucosa and in carcinomas as well as a high antigen level of inhibitor type-1 (PAl-l) and urokinase-type PA (u-PA) receptor in carcinomas are significantly associated with a poor overall survival. From 14 clinicopathological parameters, including age, WHO and TNM classification etc., only the number of eosinophils in the tumors was found to be associated with survival. Multivariate Cox analysis with all variables revealed that the PA parameters, apart from the u-PA receptor level in carcinoma, remained significantly (0.02
Gastrointestinal Oncology A549
EXPRESSION AND PROGNOSTIC VALUE OF METALLOPROTEINASES IN HUMAN GASTRIC CARCINOMAS. H.W. Verspaqet, C.F.M. Sier, S. Ganesh, M.M. Heerding, G. Griffioen, C.B.H.W. Lamers. Department of Gastroenterology and Hepatology, University Hospital, 2333 AA Leiden, The Netherlands. Proteinases are involved in the degradation and remodeling of extracallular matrix and basement membranes during tumor invasion and metastasis. The levels of matrix metalloproteinases MMP-2 and MMP-9 have been shown to be enhanced in some human carcinomas compared with adjacent normal tissue. In this study the levels of MMP-2 and MMP-9 were determined in 50 gastric carcinomas and corresponding normal gastric mucosa using quantitative gelatin-zymography with subsequent laser densitometry and expressed in arbitrary units related to a uniform standard. MMP levels were significantly enhanced in gastric carcinomas compared with normal gastric mucosa (MMP-2:2.6+0.2 versus 1.5:L,-0.1, P=0.0005; MMP-9:5.9-J:2..3 versus 3.7:L~0.2, P=0.0005). No relation was found between carcinoma MMP levels and clinicopathological classifications like Borrrnann's, Laur6n's and TNM. In contrast, evaluation of the quantities of MMPs within the carcinomas revealed that low-invasive tumors~ restricted to the submucosa, contained MMP levels comparable with normal tissues (MMP-2: 1.3:LK).3; MMP-9: 2.5+1.1), whereas high-invasive tumors had much higher quantities (MMP-2: 2.5~-0.3; MMP-9: 6.0~0.4). Univariate Cox survival analysis, using optimal cutoff values for both MMPs, showed that patients with a high level of MMP-2 (Hazard Ratio 2.6, P=0.O2) or MMP-9 (HR 2.0, P=0.04) in their carcinoma had a significantly worse survival. In contrast, MMP-levels in normal mucosa were not related with survival. The prognostic value of the MMP levels in the carcinomas remained significant (MMP-2: HR 2.5, P=0.O5; MMP-9: HR 2.1, P=0.05) in extensive multivariate analyses including all clinicopathological parameters, e.g. sex, age, tumor diameter, and tumor classifications according to Borrmann, Laur6n, and TNM. These data emphasize the role of matrix metalloproteinases in gastric carcinogenesis and their possible usefulness as independent prognostic parameters for overall survival of patients with gastric cancer.
HEPARIN-BINDING GROWTH FACTOR SECRETED FROM MKN-I IS A PLATELET-DERIVED GROWTH FACTOR (PDGF)-LIKE PROTEIN, A POTENT MITOGEN FOR HUMAN GASTRIC FIBROBLASTS. K. Wada, C. Sakamoto, K. Matsuda, T. Uchida, H. Noguchi, H. Mizuno, M. Kasuga. The Second Dept. of Internal Medicine, Kobe University School of Medicine, Kobe 650, JAPAN It has been suggested that growth factors secreted by stromal cells may play an important role in the paracrine regulation of human gastric cancer growth. On the other hand, it is not well clarified whether gastric epithelial cells regulate the stromal cell growth via paracrine mechanism. In the present study, therefore, to understand the epitheliaI-stromal interaction of gastric cancer tissue, we examined the mitogenic effects of conditioned media obtained from MKN- 1, a cell line established from a human gastric carcinoma, on either BALB/c 3T3 cells or human fibroblasts obtained from gastric tissues. Conditioned media obtained from MKN-1 stimulated a 3fold increase in DNA synthesis of BALB/c 3T3 cells. When the conditioned media were applied on heparin-affinity chromatography, the growth activity for BALB/c 3T3 cells was eluted at approximately 0.4 M NaCI. This fraction, however, had no bioactivity for chinese hamster ovary cells overexpressing human EGF receptors, suggesting that the bioactive material in the fraction is unlikely to be EGF-like growth factors. The bioactivity was heat stable and abolished under the reducing condition. In addition, antiPDGF antibodies, which neutralized 100% of the human recombinant PDGF, neutralized about 60-70% of the bioactivity eluted at 0.4 M NaCI, suggesting that the fraction may contain PDGF. This fraction also stimulated tyrosine phosphorylation of the PDGF a-receptors in BALB/c 3T3 cells. Moreover, PDGF A-chain was detected in the 0.4 M NaCI fraction by immunoblotting, indicating more direct evidence that the fraction actually contains PDGF. Interestingly, the fraction had no mitogenic activity for MKN-1 itself, but had mitogenic activity for human gastric fibroblasts. Gastric fibroblasts more remarkably responded to PDGF than other growth factors such as basic fibroblast growth factor and transforming growth factor-a as compared to BALB/c 3T3 cells. Furthermore, the fraction was partially additive with saturating concentrations of both PDGF-AA and -BB, regarding the mitogenic activity, suggesting the presence of another growth factor in the fraction. These results suggest that hepadn binding growth factors including PDGF containing A-chain are released from gastric cancer cells and may act as a paracrine growth l~_ctorfor production of fibrous stroma in gastric cancer tissue.
• HELICOBACTER PYLORI BROTH CULTURE SUPERNATANTS ENHANCE OXIDANT-INDUCED CELL INJURY TO HUMAN GASTRIC EPITHELIAL CELLS. K.G. Vernon, A. Asseffa, M.H. Earlington, T.L. Cover, D.T. Smoot. Depts. of Medicine & Biochemistry, Howard Univ. College of Medicine, Washington, D.C.; Dept. of Medicine, Vanderbilt Univ. & Nashville VAMC, Nashville, TN. Risks for gastric cancer in relation to H. pylori (HP), diet and other environmental factors are receiving renewed attention. The exact role and interactions between HP and oxidants remains to be elucidated. The following experiments were performed to determine if exposure of gastric cells to HP increases oxidant associated cell injury. A human gastric epithelial cell line (HAE), which was established by transfecting normal human gastric epithelial cells with the SV4O Large T-antigen gone, was used for these experiments. Cells were grown on 96-well clusters, then exposed overnight to broth culture supernatants from isogenic HP strains with VacA (vacuolating cytotoxin) activity, without VacA activity or Brucella broth control. The next day cells were exposed to xanthine and xanthine oxidase (to generate superoxide radical) for one hour. Cell viability was measured by two methods, metabolism of MTS using a non-radioactive proliferation assay (Promega Corp, Madison, WI) and by dual fluorescent cell labefing using a Live/Dead cell assay (Molecular Probes, Eugene, OR). The %viability of cells exposed to superoxide, as measured by MTS metabolism, incubated with medium alone, control broth, VacA-neg broth and VaoA-pos broth was 100%, 97.7%, 70.5% and 63.5%, respectively. With the fluorescent Live/Dead cell assay, cell viability after exposure to supei'oxide was 94.4%, 65.9% and 57.4%, for cells exposed to control broth, VacA-neg broth and VaeA-P0S broth, respectively. These data show that HP products signitlcantly enhance the susceptibility of HAE gastric epithelial cells to 0xyradieai associated cell injury, which is likely the outcome of maeromolecular damage to membrane lipids and cytoplasmic proteins. The mechanism by which HP products enhances the susceptibility of HAE cells to superoxide is unknown; however, the VacA cytotoxin does not appear to be the primary bacterial product responsible. Further studies are needed to identify the bacterial products responsible and elucidate their mechanism(s) of action. Increased suceptibility to superoxide induced cell injury also increases the likelyhood of oxidant related DNA damage and carcinogenic transformation.
• PROGNOSTIC IMPACT OF THE TISSULAR PLASMINOGEN ACTIVATION SYSTEM IN PATIENTS WITH GASTRIC CANCER. H.W. Verspaqet, S. Ganesh, C.F.M. Sier, M.M. Heerding, G. Gdffioen, C.B.H.W. Lamers. Dept. of Gastroenterology and Hepatology, University Hospital, 2333 AA Leiden, The Netherlands. Gastric cancer has a poor prognosis and can only be cured in an early stage by surgery. Identification by pathophysiological markers of subgroups of patients with a good or poor prognosis might help to select patients for adjuvant therapies. Plasminogen activation (PA) parameters, important in tumor invasion and metastasis, have been shown to be of prognostic value in some human malignancies. We evaluated the relation of several plasminogen activation parameters in tissue of patients with gastdc cancer (n=50) with the overall survival of at least 2 years, and a compadson was made with standard clinicopathological parameters. Univadate Cox analysis showed that a low tissue-type PA (t-PA) activity in normal mucosa and in carcinomas as well as a high antigen level of inhibitor type-1 (PAl-l) and urokinase-type PA (u-PA) receptor in carcinomas are significantly associated with a poor overall survival. From 14 clinicopathological parameters, including age, WHO and TNM classification etc., only the number of eosinophils in the tumors was found to be associated with survival. Multivariate Cox analysis with all variables revealed that the PA parameters, apart from the u-PA receptor level in carcinoma, remained significantly (0.02
Gastrointestinal Oncology A549
EXPRESSION AND PROGNOSTIC VALUE OF METALLOPROTEINASES IN HUMAN GASTRIC CARCINOMAS. H.W. Verspaqet, C.F.M. Sier, S. Ganesh, M.M. Heerding, G. Griffioen, C.B.H.W. Lamers. Department of Gastroenterology and Hepatology, University Hospital, 2333 AA Leiden, The Netherlands. Proteinases are involved in the degradation and remodeling of extracallular matrix and basement membranes during tumor invasion and metastasis. The levels of matrix metalloproteinases MMP-2 and MMP-9 have been shown to be enhanced in some human carcinomas compared with adjacent normal tissue. In this study the levels of MMP-2 and MMP-9 were determined in 50 gastric carcinomas and corresponding normal gastric mucosa using quantitative gelatin-zymography with subsequent laser densitometry and expressed in arbitrary units related to a uniform standard. MMP levels were significantly enhanced in gastric carcinomas compared with normal gastric mucosa (MMP-2:2.6+0.2 versus 1.5:L,-0.1, P=0.0005; MMP-9:5.9-J:2..3 versus 3.7:L~0.2, P=0.0005). No relation was found between carcinoma MMP levels and clinicopathological classifications like Borrrnann's, Laur6n's and TNM. In contrast, evaluation of the quantities of MMPs within the carcinomas revealed that low-invasive tumors~ restricted to the submucosa, contained MMP levels comparable with normal tissues (MMP-2: 1.3:LK).3; MMP-9: 2.5+1.1), whereas high-invasive tumors had much higher quantities (MMP-2: 2.5~-0.3; MMP-9: 6.0~0.4). Univariate Cox survival analysis, using optimal cutoff values for both MMPs, showed that patients with a high level of MMP-2 (Hazard Ratio 2.6, P=0.O2) or MMP-9 (HR 2.0, P=0.04) in their carcinoma had a significantly worse survival. In contrast, MMP-levels in normal mucosa were not related with survival. The prognostic value of the MMP levels in the carcinomas remained significant (MMP-2: HR 2.5, P=0.O5; MMP-9: HR 2.1, P=0.05) in extensive multivariate analyses including all clinicopathological parameters, e.g. sex, age, tumor diameter, and tumor classifications according to Borrmann, Laur6n, and TNM. These data emphasize the role of matrix metalloproteinases in gastric carcinogenesis and their possible usefulness as independent prognostic parameters for overall survival of patients with gastric cancer.
HEPARIN-BINDING GROWTH FACTOR SECRETED FROM MKN-I IS A PLATELET-DERIVED GROWTH FACTOR (PDGF)-LIKE PROTEIN, A POTENT MITOGEN FOR HUMAN GASTRIC FIBROBLASTS. K. Wada, C. Sakamoto, K. Matsuda, T. Uchida, H. Noguchi, H. Mizuno, M. Kasuga. The Second Dept. of Internal Medicine, Kobe University School of Medicine, Kobe 650, JAPAN It has been suggested that growth factors secreted by stromal cells may play an important role in the paracrine regulation of human gastric cancer growth. On the other hand, it is not well clarified whether gastric epithelial cells regulate the stromal cell growth via paracrine mechanism. In the present study, therefore, to understand the epitheliaI-stromal interaction of gastric cancer tissue, we examined the mitogenic effects of conditioned media obtained from MKN- 1, a cell line established from a human gastric carcinoma, on either BALB/c 3T3 cells or human fibroblasts obtained from gastric tissues. Conditioned media obtained from MKN-1 stimulated a 3fold increase in DNA synthesis of BALB/c 3T3 cells. When the conditioned media were applied on heparin-affinity chromatography, the growth activity for BALB/c 3T3 cells was eluted at approximately 0.4 M NaCI. This fraction, however, had no bioactivity for chinese hamster ovary cells overexpressing human EGF receptors, suggesting that the bioactive material in the fraction is unlikely to be EGF-like growth factors. The bioactivity was heat stable and abolished under the reducing condition. In addition, antiPDGF antibodies, which neutralized 100% of the human recombinant PDGF, neutralized about 60-70% of the bioactivity eluted at 0.4 M NaCI, suggesting that the fraction may contain PDGF. This fraction also stimulated tyrosine phosphorylation of the PDGF a-receptors in BALB/c 3T3 cells. Moreover, PDGF A-chain was detected in the 0.4 M NaCI fraction by immunoblotting, indicating more direct evidence that the fraction actually contains PDGF. Interestingly, the fraction had no mitogenic activity for MKN-1 itself, but had mitogenic activity for human gastric fibroblasts. Gastric fibroblasts more remarkably responded to PDGF than other growth factors such as basic fibroblast growth factor and transforming growth factor-a as compared to BALB/c 3T3 cells. Furthermore, the fraction was partially additive with saturating concentrations of both PDGF-AA and -BB, regarding the mitogenic activity, suggesting the presence of another growth factor in the fraction. These results suggest that hepadn binding growth factors including PDGF containing A-chain are released from gastric cancer cells and may act as a paracrine growth l~_ctorfor production of fibrous stroma in gastric cancer tissue.