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Possible relationship between the expression of membrane-associated phospholipase A2 and the proliferation of interstitial tissue in human pancreatic cancer
International Congress Series 1255 (2003) 375 – 379
Possible relationship between the expression of membrane-associated phospholipase A2 and the proliferation of interstitial tissue in human pancreatic cancer Hideo Kiyohara, Hiroshi Egami *, Takashi Kurizaki, Kazuya Murata, Hideki Ohmachi, Junji Akagi, Shigeki Ohshima, Shinichi Yamamoto, Yuji Shibata, Michio Ogawa Department of Surgery II, Kumamoto University Medical School, Honjo 1-1-1, Kumamoto 860-8556, Japan Received 27 February 2003; accepted 5 March 2003
Abstract The immunohistochemical localization of membrane-associated phospholipase A2 (M-PLA2) in normal human pancreases and 30 cases of pancreatic ductal carcinomas was investigated. In pancreatic ductal carcinomas, the immunoreactivity was observed in 25 cases (83%). Among the clinocopathological factors of pancreatic cancer, the incidence of expression of this enzyme is significantly higher in infiltrative type cancers. Furthermore, the expression of M-PLA2 was significantly increased in tumors, which had a larger amount of interstitial tissue. On the other hand, human M-PLA2 added exogenously to the fibroblast cell lines Swiss 3T3 and BALB/3T3 was found to augment their DNA synthesis. The stimulation of DNA synthesis was not affected by treatment with indomethacin. These results suggest that this enzyme could be involved directly in the proliferation of interstitial tissue through its own function. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Pancreatic cancer; Group II PLA2; Immunohistochemistry; Proliferation of interstitial tissue
* Corresponding author. Tel.: +81-96-373-5212; fax: +81-96-371-4378 E-mail address: [email protected] (H. Egami). 0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00204-8
376
H. Kiyohara et al. / International Congress Series 1255 (2003) 375–379
1. Introduction Membrane-associated phospholipase A2 (M-PLA2) is thought to be involved in regulating cellular phospholipids metabolism, release of prostaglandins (PGs) precursor and to regulate several other important metabolic pathways [1]. Serum immunoreactivity of M-PLA2 was reported to elevate in patients with surgical trauma, which suggests that it is an acute phase reactant [2,3]. Moreover, the serum immunoreactivity has been found to be elevated in patients with malignancies of digestive organs, especially pancreatic cancer [4]. However, the mechanism of the elevation of immunoreactivity in a serum and the function of the enzyme are still unclear. In this study, immunohistochemical expression of PLA2 was examined in normal human pancreases of fetuses and adults, and pancreatic ductal carcinoma on comparative bases. In addition, the effect of the enzyme on the interstitial tissue proliferation was examined in order to clarify its biological function.
2. Immunohistochemical distribution of M-PLA2 in the human pancreas Normal fetal and adult human pancreases, five chronic pancreatitises and 30 pancreatic ductal carcinomas were examined by the ABC method using monoclonal anti-human M-PLA2 antibodies. In pancreatic ductal carcinomas, the immunohistochemical findings were correlated with some clinicopathological parameters.
3. Normal pancreas In the adults, almost all acinar cells were immunoreactive. A few epithelial cells of small and large ducts were also immunoreactive, as were some of the islet cells. The immunoreactivity was increased in the cells of hyperplastic ducts [5]. It has been reported that PLA2 is produced in acinar cells and secreted as zymogen in the pancreas [6]. The immunoreactivity of group II PLA2 was observed in the apical zymogen granule portion of the acinar cells and sometimes in the ductal secretory material [7], but not in the epithelial cells of pancreatic ducts [6 –8]. Our findings clearly showed that pancreatic ductal cells have the ability to produce MPLA2.
4. Chronic pancreatitis In five chronic pancreatitises examined in this study, the immunoreactivity was retained in islet cells, ductal cells and several acini. However, staining was observed to be diminished in acinar cells of atrophic lobules. On the other hand, the immunoreactivity was increased in the cells of hyperplastic ducts found in tissue of chronic pancreatitis [5].
H. Kiyohara et al. / International Congress Series 1255 (2003) 375–379
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Table 1 The relationship between group II PLA2 expression and clinicopathological parameters of pancreatic ductal carcinoma Group II PLA2 expression +
++
Incidence, %
p Value
Interstitial tissue Medullary type Intermediate type Scirrhous type
2 3 0
0 7 2
0 7 9
0/2 (0.0) 14/17 (82.4) 11/11 (100)
< 0.01
The growth pattern Expanding type Intermediate type Infiltrative type
1 4 0
0 7 2
0 6 10
0/1 (0.0) 13/17 (76.5) 12/12 (100)
< 0.05
Total
5
9
16
25/30 (83.3)
There were no obvious relationship between group II PLA2 expression and tumor size, vascular involvement, lymphatic involvement, perineural invasion, lymph node metastasis, and clinical stage.
This suggests that, in chronic pancreatitis, the acinar cells with atrophic change loose the ability to produce M-PLA2.
5. Pancreatic cancer Of the 30 pancreatic carcinomas examined, 25 (83.3%) showed immunoreactivity with M-PLA2. Strong expressions (over 30% of cancer cells) were found in 16 cases (53.3%). There was no significant difference between the PLA2 expression and the histologic cancer type. Table 1 shows the relationship between the expression and clinicopathological parameters of pancreatic cancer. A significant relationship was found between the degree of PLA2 expression and amount of interstitial tissue in the tumor. The staining intensity tended to increase in relation to the amount of interstitial tissue ( p < 0.01) [5]. A similar relationship was observed between the staining intensity and the growth pattern of the tumor. More cancer cells in the infiltrating and intermediary cancer expressed PLA2 than in the expanding type ( p < 0.05) [5]. This result suggests that the production and the secretion of M-PLA2 are related to the proliferation of interstitial tissue.
6. The stimulating effect of M-PLA2 on DNA synthesis The stimulating effect of M-PLA2 and group-I PLA2 (P-PLA2) on DNA synthesis in the two murine fibroblast cell lines, Swiss 3T3 and BALB/3T3, was examined by [3H] thymidine incorporation into the cell. In addition, the involvement of arachidonate products, such as prostaglandins, was investigated by using indomethacin, an inhibitor of cyclooxygenase.
378
H. Kiyohara et al. / International Congress Series 1255 (2003) 375–379
Both P-PLA2 and M-PLA2 stimulated DNA synthesis in a dose-dependent manner in two murine fibroblast cell lines [9]. To investigate the involvement of arachidonate products such as prostaglandins in the observed mitogenic effects, BALB/3T3 and Swiss 3T3 cells were treated with indomethacin, an inhibitor of cyclooxygenase. In Swiss 3T3 cells, the treatment with indomethacin did not alter the mitogenic effect of either PLA2. In BALB/3T3, however, the mitogenic effect of P-PLA2 was suppressed by indomethacin in a dose-dependent manner, whereas the mitogenic effect of M-PLA2 was not affected [9]. The present results indicate that M-PLA2 directly stimulates DNA synthesis, without involving arachidonate products which are known to stimulate mitogenesis in several fibroblast cell lines [10,11].
7. Conclusion M-PLA2 is produced in large amounts in pancreatic ductal carcinoma cells. Furthermore, the expression of M-PLA2 increases in relation to the amount of interstitial tissue of pancreatic cancer. M-PLA2 stimulates DNA synthesis in a dose-dependent manner in two murine fibroblast cell lines, BALB/3T3 and Swiss 3T3, and the mitogenic effect of MPLA2 is not suppressed by indomethacin, an inhibitor of cyclooxygenase. These results suggest that M-PLA2 could be involved in the proliferation of interstitial tissue directly through its own function.
References [1] H. Van den Bosch, Intracellular phospholipase A, Biochim. Biophys. Acta 604 (1980) 191 – 246. [2] M. Ogawa, H. Arakawa, S. Yamashita, K. Sakamoto, S. Ikei, Postoperative elevations of serum interleukin 6 and group II phospholipase A2: group II phospholipase A2 in serum is an acute phase reactant, Res. Commun. Chem. Pathol. Pharmacol. 75 (1992) 109 – 115. [3] Y. Matsuda, M. Ogawa, K. Sakamoto, S. Yamashita, A. Kanda, M. Kohno, N. Yoshida, J. Nishijima, A. Murata, T. Mori, Development of a radioimmunoassay for human group II phospholipase A2 and demonstration of postoperative elevation, Enzyme 45 (1991) 200 – 208. [4] M. Ogawa, S. Yamashita, K. Sakamoto, S. Ikei, Elevation of serum group II phospholipase A2 in patients with cancers of digestive organs, Res. Commun. Chem. Pathol. Pharmacol. 74 (1991) 241 – 244. [5] H. Kiyohara, H. Egami, H. Kako, Y. Shibata, K. Murata, S. Oshima, K. Sei, S. Suko, R. Kurano, M. Ogawa, Immunohistochemical localization of group II phospholipase A2 in human pancreatic carcinoma, Int. J. Pancreatol. 13 (1993) 49 – 57. [6] P. Stommer, Immunocytochemical evidence of phospholipase A2 in pancreatic tumors—diagnostic values, Klin. Wochenschr. 67 (1989) 136 – 140. [7] T.J. Nevalainen, H.J. Aho, J.U. Eskola, K. Suonpaa, Immunohistochemical localization of phospholipase A2 in human pancreas in acute and chronic pancreatitis, Acta Pathol. Microbiol. Immunol. Scand. 91 (1983) 97 – 102. [8] H. Tatsumi, H. Tojo, T. Senda, T. Ono, H. Fujita, M. Okamoto, Immunocytochemical studies on the localization of pancreatic-type phospholipase A2 in rat stomach and pancreas, with special reference to the stomach cells, Histochemistry 94 (1990) 135 – 140. [9] T. Kurizaki, H. Egami, K. Murata, H. Kiyohara, N. Yoshida, M. Ogawa, Membrane-associated phospho-
H. Kiyohara et al. / International Congress Series 1255 (2003) 375–379
379
lipase A2 stimulates DNA synthesis in two murine fibroblasts, Res. Commun. Chem. Pathol. Pharmacol. 78 (1992) 39 – 45. [10] E. Rozengurt, M. Collins, M. Keehan, Mitogenic effect of prostaglandin E1 in Swiss 3T3 cells; role of cyclic AMP, J. Cell. Physiol. 116 (1983) 379 – 384. [11] R.D. Nolan, R.M. Danilowicz, T.E. Eling, Role of arachidonic acid metabolism in the mitogenic response of BALB/c 3T3 fibroblast to epidermal growth factor, Mol. Pharmacol. 33 (1988) 650 – 656.
Possible relationship between the expression of membrane-associated phospholipase A2 and the proliferation of interstitial tissue in human pancreatic cancer Hideo Kiyohara, Hiroshi Egami *, Takashi Kurizaki, Kazuya Murata, Hideki Ohmachi, Junji Akagi, Shigeki Ohshima, Shinichi Yamamoto, Yuji Shibata, Michio Ogawa Department of Surgery II, Kumamoto University Medical School, Honjo 1-1-1, Kumamoto 860-8556, Japan Received 27 February 2003; accepted 5 March 2003
Abstract The immunohistochemical localization of membrane-associated phospholipase A2 (M-PLA2) in normal human pancreases and 30 cases of pancreatic ductal carcinomas was investigated. In pancreatic ductal carcinomas, the immunoreactivity was observed in 25 cases (83%). Among the clinocopathological factors of pancreatic cancer, the incidence of expression of this enzyme is significantly higher in infiltrative type cancers. Furthermore, the expression of M-PLA2 was significantly increased in tumors, which had a larger amount of interstitial tissue. On the other hand, human M-PLA2 added exogenously to the fibroblast cell lines Swiss 3T3 and BALB/3T3 was found to augment their DNA synthesis. The stimulation of DNA synthesis was not affected by treatment with indomethacin. These results suggest that this enzyme could be involved directly in the proliferation of interstitial tissue through its own function. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Pancreatic cancer; Group II PLA2; Immunohistochemistry; Proliferation of interstitial tissue
* Corresponding author. Tel.: +81-96-373-5212; fax: +81-96-371-4378 E-mail address: [email protected] (H. Egami). 0531-5131/03 D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0531-5131(03)00204-8
376
H. Kiyohara et al. / International Congress Series 1255 (2003) 375–379
1. Introduction Membrane-associated phospholipase A2 (M-PLA2) is thought to be involved in regulating cellular phospholipids metabolism, release of prostaglandins (PGs) precursor and to regulate several other important metabolic pathways [1]. Serum immunoreactivity of M-PLA2 was reported to elevate in patients with surgical trauma, which suggests that it is an acute phase reactant [2,3]. Moreover, the serum immunoreactivity has been found to be elevated in patients with malignancies of digestive organs, especially pancreatic cancer [4]. However, the mechanism of the elevation of immunoreactivity in a serum and the function of the enzyme are still unclear. In this study, immunohistochemical expression of PLA2 was examined in normal human pancreases of fetuses and adults, and pancreatic ductal carcinoma on comparative bases. In addition, the effect of the enzyme on the interstitial tissue proliferation was examined in order to clarify its biological function.
2. Immunohistochemical distribution of M-PLA2 in the human pancreas Normal fetal and adult human pancreases, five chronic pancreatitises and 30 pancreatic ductal carcinomas were examined by the ABC method using monoclonal anti-human M-PLA2 antibodies. In pancreatic ductal carcinomas, the immunohistochemical findings were correlated with some clinicopathological parameters.
3. Normal pancreas In the adults, almost all acinar cells were immunoreactive. A few epithelial cells of small and large ducts were also immunoreactive, as were some of the islet cells. The immunoreactivity was increased in the cells of hyperplastic ducts [5]. It has been reported that PLA2 is produced in acinar cells and secreted as zymogen in the pancreas [6]. The immunoreactivity of group II PLA2 was observed in the apical zymogen granule portion of the acinar cells and sometimes in the ductal secretory material [7], but not in the epithelial cells of pancreatic ducts [6 –8]. Our findings clearly showed that pancreatic ductal cells have the ability to produce MPLA2.
4. Chronic pancreatitis In five chronic pancreatitises examined in this study, the immunoreactivity was retained in islet cells, ductal cells and several acini. However, staining was observed to be diminished in acinar cells of atrophic lobules. On the other hand, the immunoreactivity was increased in the cells of hyperplastic ducts found in tissue of chronic pancreatitis [5].
H. Kiyohara et al. / International Congress Series 1255 (2003) 375–379
377
Table 1 The relationship between group II PLA2 expression and clinicopathological parameters of pancreatic ductal carcinoma Group II PLA2 expression +
++
Incidence, %
p Value
Interstitial tissue Medullary type Intermediate type Scirrhous type
2 3 0
0 7 2
0 7 9
0/2 (0.0) 14/17 (82.4) 11/11 (100)
< 0.01
The growth pattern Expanding type Intermediate type Infiltrative type
1 4 0
0 7 2
0 6 10
0/1 (0.0) 13/17 (76.5) 12/12 (100)
< 0.05
Total
5
9
16
25/30 (83.3)
There were no obvious relationship between group II PLA2 expression and tumor size, vascular involvement, lymphatic involvement, perineural invasion, lymph node metastasis, and clinical stage.
This suggests that, in chronic pancreatitis, the acinar cells with atrophic change loose the ability to produce M-PLA2.
5. Pancreatic cancer Of the 30 pancreatic carcinomas examined, 25 (83.3%) showed immunoreactivity with M-PLA2. Strong expressions (over 30% of cancer cells) were found in 16 cases (53.3%). There was no significant difference between the PLA2 expression and the histologic cancer type. Table 1 shows the relationship between the expression and clinicopathological parameters of pancreatic cancer. A significant relationship was found between the degree of PLA2 expression and amount of interstitial tissue in the tumor. The staining intensity tended to increase in relation to the amount of interstitial tissue ( p < 0.01) [5]. A similar relationship was observed between the staining intensity and the growth pattern of the tumor. More cancer cells in the infiltrating and intermediary cancer expressed PLA2 than in the expanding type ( p < 0.05) [5]. This result suggests that the production and the secretion of M-PLA2 are related to the proliferation of interstitial tissue.
6. The stimulating effect of M-PLA2 on DNA synthesis The stimulating effect of M-PLA2 and group-I PLA2 (P-PLA2) on DNA synthesis in the two murine fibroblast cell lines, Swiss 3T3 and BALB/3T3, was examined by [3H] thymidine incorporation into the cell. In addition, the involvement of arachidonate products, such as prostaglandins, was investigated by using indomethacin, an inhibitor of cyclooxygenase.
378
H. Kiyohara et al. / International Congress Series 1255 (2003) 375–379
Both P-PLA2 and M-PLA2 stimulated DNA synthesis in a dose-dependent manner in two murine fibroblast cell lines [9]. To investigate the involvement of arachidonate products such as prostaglandins in the observed mitogenic effects, BALB/3T3 and Swiss 3T3 cells were treated with indomethacin, an inhibitor of cyclooxygenase. In Swiss 3T3 cells, the treatment with indomethacin did not alter the mitogenic effect of either PLA2. In BALB/3T3, however, the mitogenic effect of P-PLA2 was suppressed by indomethacin in a dose-dependent manner, whereas the mitogenic effect of M-PLA2 was not affected [9]. The present results indicate that M-PLA2 directly stimulates DNA synthesis, without involving arachidonate products which are known to stimulate mitogenesis in several fibroblast cell lines [10,11].
7. Conclusion M-PLA2 is produced in large amounts in pancreatic ductal carcinoma cells. Furthermore, the expression of M-PLA2 increases in relation to the amount of interstitial tissue of pancreatic cancer. M-PLA2 stimulates DNA synthesis in a dose-dependent manner in two murine fibroblast cell lines, BALB/3T3 and Swiss 3T3, and the mitogenic effect of MPLA2 is not suppressed by indomethacin, an inhibitor of cyclooxygenase. These results suggest that M-PLA2 could be involved in the proliferation of interstitial tissue directly through its own function.
References [1] H. Van den Bosch, Intracellular phospholipase A, Biochim. Biophys. Acta 604 (1980) 191 – 246. [2] M. Ogawa, H. Arakawa, S. Yamashita, K. Sakamoto, S. Ikei, Postoperative elevations of serum interleukin 6 and group II phospholipase A2: group II phospholipase A2 in serum is an acute phase reactant, Res. Commun. Chem. Pathol. Pharmacol. 75 (1992) 109 – 115. [3] Y. Matsuda, M. Ogawa, K. Sakamoto, S. Yamashita, A. Kanda, M. Kohno, N. Yoshida, J. Nishijima, A. Murata, T. Mori, Development of a radioimmunoassay for human group II phospholipase A2 and demonstration of postoperative elevation, Enzyme 45 (1991) 200 – 208. [4] M. Ogawa, S. Yamashita, K. Sakamoto, S. Ikei, Elevation of serum group II phospholipase A2 in patients with cancers of digestive organs, Res. Commun. Chem. Pathol. Pharmacol. 74 (1991) 241 – 244. [5] H. Kiyohara, H. Egami, H. Kako, Y. Shibata, K. Murata, S. Oshima, K. Sei, S. Suko, R. Kurano, M. Ogawa, Immunohistochemical localization of group II phospholipase A2 in human pancreatic carcinoma, Int. J. Pancreatol. 13 (1993) 49 – 57. [6] P. Stommer, Immunocytochemical evidence of phospholipase A2 in pancreatic tumors—diagnostic values, Klin. Wochenschr. 67 (1989) 136 – 140. [7] T.J. Nevalainen, H.J. Aho, J.U. Eskola, K. Suonpaa, Immunohistochemical localization of phospholipase A2 in human pancreas in acute and chronic pancreatitis, Acta Pathol. Microbiol. Immunol. Scand. 91 (1983) 97 – 102. [8] H. Tatsumi, H. Tojo, T. Senda, T. Ono, H. Fujita, M. Okamoto, Immunocytochemical studies on the localization of pancreatic-type phospholipase A2 in rat stomach and pancreas, with special reference to the stomach cells, Histochemistry 94 (1990) 135 – 140. [9] T. Kurizaki, H. Egami, K. Murata, H. Kiyohara, N. Yoshida, M. Ogawa, Membrane-associated phospho-
H. Kiyohara et al. / International Congress Series 1255 (2003) 375–379
379
lipase A2 stimulates DNA synthesis in two murine fibroblasts, Res. Commun. Chem. Pathol. Pharmacol. 78 (1992) 39 – 45. [10] E. Rozengurt, M. Collins, M. Keehan, Mitogenic effect of prostaglandin E1 in Swiss 3T3 cells; role of cyclic AMP, J. Cell. Physiol. 116 (1983) 379 – 384. [11] R.D. Nolan, R.M. Danilowicz, T.E. Eling, Role of arachidonic acid metabolism in the mitogenic response of BALB/c 3T3 fibroblast to epidermal growth factor, Mol. Pharmacol. 33 (1988) 650 – 656.