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Metastatic intraperitoneal spread is initiated by phospholipase A2
Medical Hypotheses Medical Hypotheses (1995) 44, 392-394 © Pearson Professional Ltd 1995
Metastatic Intraperitoneal Spread is Initiated by Phospholipase A2 M. SNOJ Department of Surgery, Institute of Oncology, Zaloska 2, 61105 Ljubljana, Slovenia Abstract - - A new hypothesis is introduced, i.e. that intraperitoneal tumor spread is initiated by activation of phospholipase A 2. This view may well have some new therapeutic implications.
Introduction Metastatic intraperitoneal tumor spread is common in different gynecological (1) and gastrointestinal (2) tumors and contributes to marked additional morbidity in these patients. By far the most common complication of intraperitoneal tumor spread is ascites and intestinal obstruction. Both of these could lead to a fatal outcome. The complexity of the metastatic process has obliged investigators to focus on one step at a time in order to reduce the number of variables to a reasonable level. Of the current theories, the one which is most widely accepted is the three-step theory which explains the metastatic process in sequence of attachment, local proteolysis and migration (3). However, the research into intraperitoneal metastatic spread has been mainly limited to the collecting of various clinical data and to offering some therapeutic options (1,2,4); so far, no specific theory has been introduced. Therefore, some new theoretical approaches, especially those with therapeutic implications, would now seem to be highly warranted. We have postulated that intra-abdominal adhesion formation is initiated by phospholipase A z (5). There is strong evidence that, during the process of attach-
ment of the intraperitoneal metastastatic cells, the destruction of phospholipid layer, due to increased activity of phospholipase A2, takes part.
Phospholipids in the abdominal cavity In patients, surface active materials could be detected in the pleural cavity (6), the stomach surface layer (7) and in the lung alveoli (8). Surface active materials were found in the peritoneal dialysate at a concentration of 11-25 mg/L (9). Thin-layer chromatography demonstrated that 81% of these were phosphatidylcholines, 5% lysophosphatidylcholines, 6.5% sphingomielines, 3.5% phosphatidylcholines and inositols, and 4% phosphatidylethanolamines. Since they are composed of both hydrophobic and hydrophilic parts, it was suggested that they adhere to the negatively charged peritoneum by its positively charged part, so that their hydrophobic parts would be oriented into the cavity and would thus come into contact with the hydrophobic parts from the opposing side (9). This would enable adequate lubrication and would prevent the neighbouring organs from coming into a constant and close position and thus forming adhesions. Therefore, it seems logical that no malignant cell could be attached unless this phospholipid layer is broken.
Date received27 June 1994 Date accepted28 September1994 392
METASTATIC INTRAPERITONEAL SPREAD IS INITIATED BY PHOSPHOLIPASE A2
Phospholipase A 2
The hydrolysis of phospholipid is catalysed by phospholipases. In rabbits (10), the phospholipase A 2 was isolated from inflammatory ascitic fluid, serum and polymorphonuclear leukocytes, and in mice from the peritoneal macrophages (11), so the phospholipid layer could be destroyed in the presence of inflammation. However, the activity of phospholipase A 2 w a s detected in some malignant cells such as the human hepatoma cell line HePG (12) and the Chinese hamster ovary gip-2 oncogene cells (13), and in undifferentiated U937 cells (14).
Intraperitoneal metastasing The process of intraperitoneal metastasing has been described in detail in rats inoculated by rat ascites hepatoma AH100B cells (15). After inoculation of the abdominal cavity with tumor cells, no peritoneal changes were noted macroscopically for 1-3 days. Scanning electron microscopy revealed adhesion of some tumor cells to the mesothelium by pseudopodium-like protoplasmatic processes and/ or microvilli. However, no changes were noted in the mesothelial cells. After the third day, scanning microscopy showed a single-layer, horizontal proliferation of tumor cells on the mesothelium. The tumor cells adhered to the mesothelial cells by microvilli or by pseudopodia. 5-45 days after inoculation, the formation of indurations composed of multiple layers of tumor cells was noted. Marked irregularity and atrophy were observed in the mesothelial cells surrounding the tumor cell mass (15).
Hypothesis In order to become attached to the mesothelial cells of the peritoneum or to the basal lamina in the peritoneal cavity, tumor cells should penetrate the phospholipid layer. Most likely, this could be achieved by hydrolysis with phospholipase A2.
Experimental data It has been shown that the areas of trauma of the peritoneum are the preferred sites for metastatic growth (16). This has been especially shown for the colonic anastomosis in an experimental animal model (17). Furthermore, it has been demonstrated by experiment with rats that metastasizing in the anastomotic region could be considerably reduced if omentectomy were performed (16). It is known that the omentum has a
393
high concentration of macrophages (17) which could be activated by inflammation in the anastomotic region close by. This could lead to increased activity of phospholipase A2, which in turn destroys the phospholipid layer. Moreover, we have shown that intraperitoneal adhesions after the anastomosis of the small intestine in the rat were reduced when PC was instilled intraperitoneally (18). It is known that the healing of anastomosis involves several steps. After the period of inflammation, the fibroblasts proliferate and deposit collagen. Thus, we could speculate that endogeneous phospholipid is hydrolysed by the activated phospholipase A 2 in the inflammatory phase of the healing of anastomosis, since its activity has been demonstrated in leukocytes and in the inflammatory ascitic fluid in rabbits (10). This speculation was further supported by our work on bacterial peritonitis (19). We showed that while the instillation of L-PC over three subsequent days reduced intra-abdominal adhesion formation, it also significantly increased mortality. If L-PC was replaced by DL-PC, which is hydrolysed by phospholipase A2 to 50% only (20), the mortality rate dropped to the control level, and the adhesion formation was markedly reduced. Since it is known that phospholipase A2 activity is present in leukocytes and in inflammatory ascitic fluid in the rabbit - and that the products of hydrolysis of L-PC are lysophosphatidylcholine, which is toxic (21,22), and arachidonic acid (23), which is the precursor of eicosanoids - the increased activity of phospholipase A2 might be responsible for the increased mortality. Therefore, we could speculate that phospholipase A2, activated in the process of inflammation due to the healing of anastomosis, would destroy the phospholipid layer, and hence the unprotected area of anastomosis could be exposed to attack by malignant cells.
Conclusions Phospholipase A2 plays a major role in the destruction of the peritoneal phospholipid layer. It is assumed that the peritoneal phospholipid layer does not allow malignant cells to attach to the peritoneum. This layer could be broken by hydrolysis with phospholipase A> Phospholipase A2 could be present, either in malignant cells or in the areas of traumatized peritoneum, as the inflammatory reaction occurs. Therefore, the activation of phospholipase A2 is the first step, and a prerequisite, if malignant cells are to become attached to the peritoneum. This new view could have some important therapeutic implications: phospholipase A2-resistant phos-
394
pholipids, such as D-phospholipids, could be used for intraperitoneal instillation. Furthermore, phospholipase A2 inhibitors for oral use could be designed in order to inhibit peritoneal metastasizing. It is suggested that increased phospholipase A2 activity is the first step in intra-abdominal metastasing. References 1. Ferrandina G, Scambia G, Benedetti-Panici P et al. Type II estrogen-binding sites in human ovarian cancer: correlation with estrogen, progesterone and epidermal growth factor receptor. Gynecol Oncol 1993; 49: 67-72. 2. Sugerbaker P H, Zhu B W, Sese G B, Shmokler B. Peritoneal carcinomatosis from appendiceal cancer: results in 69 patients treated by cytoreductive surgery and intraperitoneal chemotherapy. Dis Colon Rectum 1993; 36: 323-329. 3. Liotta L A. Tumor invasion and metastases - role of extracellular matrix: Rhoads Memorial Award Lecture. Cancer Research 1986; 46: 1-7. 4. Melioli G, Sertoli M R, Bruzzone M e t al. A phase I study of recombinant interleukin-2 intraperitoneal infusion in patients with neoplastic ascites; toxic effects and immunologic results. Am J Clin Oncol 1991; 14: 231-237. 5. Snoj M. Intra-abdominal adhesion formation is initiated by phospholipase A 2. Med Hypotheses 1993; 41: 525-528. 6. Hills B A, Butler B D, Barow R E. Boundary lubrication imparted by pleural surfactants and their identification. J Appl Physiol 1982; 53(2): 463-469. 7. Wassef M K, Lin Y N, Horowitz M I. Molecular species of phosphatidylcholine from rat gastric mucosa. Biochim Biophys Acta 1979; 573: 222-226. 8. Hills B A. What is true role of surfactant in the lung? Thorax 1981; 36: 1-4. 9. Grahame G R, Torchia M G, Dankewich K A, Ferguson I A. Surface active material in peritoneal effluent of CAPD patients. Bull Periton Dial 1985; 5: 109-111. 10. Wright G W, Ooi C E, Weiss J, Elsbach P. Purification of a cellular (granulocyte) and extracellular (serum) phospholipase A 2 that participates in the destruction of E. coli in rabbit inflammatory exudate. J Biol Chem 1990; 265: 6675~5681.
MEDICAL HYPOTHESES 11. Wijkander J, Sundler R. A phospholipase A 2 hydrolyzing arachidonoyl-phospholipids in mouse peritoneal macrophages. FEBS Lett 1989; 244: 51-56. 12. Koumanov K S, Momchilova-Pankova A B, Wang S R, Infante R. Membrane phospholipid composition, fluidity and phospholipase A 2activity of human hepatoma cell line HepG2. Int J Biochem 1990; 22: 1435-1455. 13. Lowndes J M, Gupta S K, Osawa S, Johnson G L. GTPasedeficient G~a2oncogene gip2 inhibits adenylcyclase and attenuates receptor-stimulated phospholipase A2 activity. J Biol Chem 1991; 266: 14193-14197. 14. Galbraith W, Paschetto K A, Stevens T M, Kerr J S. Phospolipase A 2activity in undifferentiated U937 cells. Agents Actions 1989; 27: 422--424. 15. Koga S, Kudo H, Kiyasu Y e t al. A scanning electron microscopic study on the peritoneal implantations of ascites hepatoma AH100B cells in rats. Gann 1980; 71: 8-13. 16. Lawrance R J, Loizidou M, Cooper A J, Alexander P, Taylor I. Importance of the omentum in the development of intraabdominal metastases. Br J Surg 1991; 78:117-119. 17. Murphy P, Alexander P, Kirkham M, Fleming J, Taylor I. Pattern of spread of blood-borne tumors. Br J Surg 1986; 73: 829-834. 18. Snoj M, Ar'Rajab A, Ahren B, Bengmark S. Effect of phosphatidylcholine on postoperative adhesions after small bowel anastomosis in the rat. Br J Surg 1992; 79: 427-429. 19. Snoj M, Ar'Rajab A, Ahren B, Larsson K, Bengmark S. Phospholipase-resistant phosphatidylcholine reduces intraabdominal adhesions induced by bacterial peritonitis. Res Exp Med 1993; 193: 117-122. 20. Van Deenen L L M, De Haas G H. The substrate specificity of phospholipase A. Biochim Biophys Acta 1963; 70: 538-553. 21. Chi L M, Wu W G, Sung K L P, Chien S. Biophysical correlates of lysophosphatidylcholine- and ethanol-mediated shape transformation and hemolysis of human erythrocytes. Membrane viscoelasticity and NMR measurement. Biochim Biophys Acta 1990; 1027: 163-171. 22. Chang J, Musser J H, McGregor H. Phospholipase A2: function and pharmacologic regulation. Biochem Phannacol 1987; 36: 2429-2436. 23. Garcia M C, Fernandez-GaUardo S, Gijon M A, Garcia C, Nieto M L, Sanchez-Crespo M. Biosynthesis of platelet-activating factor (PAF) in human polymorphonuclear leucocytes. Biochem J 1990; 268: 91-98.
Metastatic Intraperitoneal Spread is Initiated by Phospholipase A2 M. SNOJ Department of Surgery, Institute of Oncology, Zaloska 2, 61105 Ljubljana, Slovenia Abstract - - A new hypothesis is introduced, i.e. that intraperitoneal tumor spread is initiated by activation of phospholipase A 2. This view may well have some new therapeutic implications.
Introduction Metastatic intraperitoneal tumor spread is common in different gynecological (1) and gastrointestinal (2) tumors and contributes to marked additional morbidity in these patients. By far the most common complication of intraperitoneal tumor spread is ascites and intestinal obstruction. Both of these could lead to a fatal outcome. The complexity of the metastatic process has obliged investigators to focus on one step at a time in order to reduce the number of variables to a reasonable level. Of the current theories, the one which is most widely accepted is the three-step theory which explains the metastatic process in sequence of attachment, local proteolysis and migration (3). However, the research into intraperitoneal metastatic spread has been mainly limited to the collecting of various clinical data and to offering some therapeutic options (1,2,4); so far, no specific theory has been introduced. Therefore, some new theoretical approaches, especially those with therapeutic implications, would now seem to be highly warranted. We have postulated that intra-abdominal adhesion formation is initiated by phospholipase A z (5). There is strong evidence that, during the process of attach-
ment of the intraperitoneal metastastatic cells, the destruction of phospholipid layer, due to increased activity of phospholipase A2, takes part.
Phospholipids in the abdominal cavity In patients, surface active materials could be detected in the pleural cavity (6), the stomach surface layer (7) and in the lung alveoli (8). Surface active materials were found in the peritoneal dialysate at a concentration of 11-25 mg/L (9). Thin-layer chromatography demonstrated that 81% of these were phosphatidylcholines, 5% lysophosphatidylcholines, 6.5% sphingomielines, 3.5% phosphatidylcholines and inositols, and 4% phosphatidylethanolamines. Since they are composed of both hydrophobic and hydrophilic parts, it was suggested that they adhere to the negatively charged peritoneum by its positively charged part, so that their hydrophobic parts would be oriented into the cavity and would thus come into contact with the hydrophobic parts from the opposing side (9). This would enable adequate lubrication and would prevent the neighbouring organs from coming into a constant and close position and thus forming adhesions. Therefore, it seems logical that no malignant cell could be attached unless this phospholipid layer is broken.
Date received27 June 1994 Date accepted28 September1994 392
METASTATIC INTRAPERITONEAL SPREAD IS INITIATED BY PHOSPHOLIPASE A2
Phospholipase A 2
The hydrolysis of phospholipid is catalysed by phospholipases. In rabbits (10), the phospholipase A 2 was isolated from inflammatory ascitic fluid, serum and polymorphonuclear leukocytes, and in mice from the peritoneal macrophages (11), so the phospholipid layer could be destroyed in the presence of inflammation. However, the activity of phospholipase A 2 w a s detected in some malignant cells such as the human hepatoma cell line HePG (12) and the Chinese hamster ovary gip-2 oncogene cells (13), and in undifferentiated U937 cells (14).
Intraperitoneal metastasing The process of intraperitoneal metastasing has been described in detail in rats inoculated by rat ascites hepatoma AH100B cells (15). After inoculation of the abdominal cavity with tumor cells, no peritoneal changes were noted macroscopically for 1-3 days. Scanning electron microscopy revealed adhesion of some tumor cells to the mesothelium by pseudopodium-like protoplasmatic processes and/ or microvilli. However, no changes were noted in the mesothelial cells. After the third day, scanning microscopy showed a single-layer, horizontal proliferation of tumor cells on the mesothelium. The tumor cells adhered to the mesothelial cells by microvilli or by pseudopodia. 5-45 days after inoculation, the formation of indurations composed of multiple layers of tumor cells was noted. Marked irregularity and atrophy were observed in the mesothelial cells surrounding the tumor cell mass (15).
Hypothesis In order to become attached to the mesothelial cells of the peritoneum or to the basal lamina in the peritoneal cavity, tumor cells should penetrate the phospholipid layer. Most likely, this could be achieved by hydrolysis with phospholipase A2.
Experimental data It has been shown that the areas of trauma of the peritoneum are the preferred sites for metastatic growth (16). This has been especially shown for the colonic anastomosis in an experimental animal model (17). Furthermore, it has been demonstrated by experiment with rats that metastasizing in the anastomotic region could be considerably reduced if omentectomy were performed (16). It is known that the omentum has a
393
high concentration of macrophages (17) which could be activated by inflammation in the anastomotic region close by. This could lead to increased activity of phospholipase A2, which in turn destroys the phospholipid layer. Moreover, we have shown that intraperitoneal adhesions after the anastomosis of the small intestine in the rat were reduced when PC was instilled intraperitoneally (18). It is known that the healing of anastomosis involves several steps. After the period of inflammation, the fibroblasts proliferate and deposit collagen. Thus, we could speculate that endogeneous phospholipid is hydrolysed by the activated phospholipase A 2 in the inflammatory phase of the healing of anastomosis, since its activity has been demonstrated in leukocytes and in the inflammatory ascitic fluid in rabbits (10). This speculation was further supported by our work on bacterial peritonitis (19). We showed that while the instillation of L-PC over three subsequent days reduced intra-abdominal adhesion formation, it also significantly increased mortality. If L-PC was replaced by DL-PC, which is hydrolysed by phospholipase A2 to 50% only (20), the mortality rate dropped to the control level, and the adhesion formation was markedly reduced. Since it is known that phospholipase A2 activity is present in leukocytes and in inflammatory ascitic fluid in the rabbit - and that the products of hydrolysis of L-PC are lysophosphatidylcholine, which is toxic (21,22), and arachidonic acid (23), which is the precursor of eicosanoids - the increased activity of phospholipase A2 might be responsible for the increased mortality. Therefore, we could speculate that phospholipase A2, activated in the process of inflammation due to the healing of anastomosis, would destroy the phospholipid layer, and hence the unprotected area of anastomosis could be exposed to attack by malignant cells.
Conclusions Phospholipase A2 plays a major role in the destruction of the peritoneal phospholipid layer. It is assumed that the peritoneal phospholipid layer does not allow malignant cells to attach to the peritoneum. This layer could be broken by hydrolysis with phospholipase A> Phospholipase A2 could be present, either in malignant cells or in the areas of traumatized peritoneum, as the inflammatory reaction occurs. Therefore, the activation of phospholipase A2 is the first step, and a prerequisite, if malignant cells are to become attached to the peritoneum. This new view could have some important therapeutic implications: phospholipase A2-resistant phos-
394
pholipids, such as D-phospholipids, could be used for intraperitoneal instillation. Furthermore, phospholipase A2 inhibitors for oral use could be designed in order to inhibit peritoneal metastasizing. It is suggested that increased phospholipase A2 activity is the first step in intra-abdominal metastasing. References 1. Ferrandina G, Scambia G, Benedetti-Panici P et al. Type II estrogen-binding sites in human ovarian cancer: correlation with estrogen, progesterone and epidermal growth factor receptor. Gynecol Oncol 1993; 49: 67-72. 2. Sugerbaker P H, Zhu B W, Sese G B, Shmokler B. Peritoneal carcinomatosis from appendiceal cancer: results in 69 patients treated by cytoreductive surgery and intraperitoneal chemotherapy. Dis Colon Rectum 1993; 36: 323-329. 3. Liotta L A. Tumor invasion and metastases - role of extracellular matrix: Rhoads Memorial Award Lecture. Cancer Research 1986; 46: 1-7. 4. Melioli G, Sertoli M R, Bruzzone M e t al. A phase I study of recombinant interleukin-2 intraperitoneal infusion in patients with neoplastic ascites; toxic effects and immunologic results. Am J Clin Oncol 1991; 14: 231-237. 5. Snoj M. Intra-abdominal adhesion formation is initiated by phospholipase A 2. Med Hypotheses 1993; 41: 525-528. 6. Hills B A, Butler B D, Barow R E. Boundary lubrication imparted by pleural surfactants and their identification. J Appl Physiol 1982; 53(2): 463-469. 7. Wassef M K, Lin Y N, Horowitz M I. Molecular species of phosphatidylcholine from rat gastric mucosa. Biochim Biophys Acta 1979; 573: 222-226. 8. Hills B A. What is true role of surfactant in the lung? Thorax 1981; 36: 1-4. 9. Grahame G R, Torchia M G, Dankewich K A, Ferguson I A. Surface active material in peritoneal effluent of CAPD patients. Bull Periton Dial 1985; 5: 109-111. 10. Wright G W, Ooi C E, Weiss J, Elsbach P. Purification of a cellular (granulocyte) and extracellular (serum) phospholipase A 2 that participates in the destruction of E. coli in rabbit inflammatory exudate. J Biol Chem 1990; 265: 6675~5681.
MEDICAL HYPOTHESES 11. Wijkander J, Sundler R. A phospholipase A 2 hydrolyzing arachidonoyl-phospholipids in mouse peritoneal macrophages. FEBS Lett 1989; 244: 51-56. 12. Koumanov K S, Momchilova-Pankova A B, Wang S R, Infante R. Membrane phospholipid composition, fluidity and phospholipase A 2activity of human hepatoma cell line HepG2. Int J Biochem 1990; 22: 1435-1455. 13. Lowndes J M, Gupta S K, Osawa S, Johnson G L. GTPasedeficient G~a2oncogene gip2 inhibits adenylcyclase and attenuates receptor-stimulated phospholipase A2 activity. J Biol Chem 1991; 266: 14193-14197. 14. Galbraith W, Paschetto K A, Stevens T M, Kerr J S. Phospolipase A 2activity in undifferentiated U937 cells. Agents Actions 1989; 27: 422--424. 15. Koga S, Kudo H, Kiyasu Y e t al. A scanning electron microscopic study on the peritoneal implantations of ascites hepatoma AH100B cells in rats. Gann 1980; 71: 8-13. 16. Lawrance R J, Loizidou M, Cooper A J, Alexander P, Taylor I. Importance of the omentum in the development of intraabdominal metastases. Br J Surg 1991; 78:117-119. 17. Murphy P, Alexander P, Kirkham M, Fleming J, Taylor I. Pattern of spread of blood-borne tumors. Br J Surg 1986; 73: 829-834. 18. Snoj M, Ar'Rajab A, Ahren B, Bengmark S. Effect of phosphatidylcholine on postoperative adhesions after small bowel anastomosis in the rat. Br J Surg 1992; 79: 427-429. 19. Snoj M, Ar'Rajab A, Ahren B, Larsson K, Bengmark S. Phospholipase-resistant phosphatidylcholine reduces intraabdominal adhesions induced by bacterial peritonitis. Res Exp Med 1993; 193: 117-122. 20. Van Deenen L L M, De Haas G H. The substrate specificity of phospholipase A. Biochim Biophys Acta 1963; 70: 538-553. 21. Chi L M, Wu W G, Sung K L P, Chien S. Biophysical correlates of lysophosphatidylcholine- and ethanol-mediated shape transformation and hemolysis of human erythrocytes. Membrane viscoelasticity and NMR measurement. Biochim Biophys Acta 1990; 1027: 163-171. 22. Chang J, Musser J H, McGregor H. Phospholipase A2: function and pharmacologic regulation. Biochem Phannacol 1987; 36: 2429-2436. 23. Garcia M C, Fernandez-GaUardo S, Gijon M A, Garcia C, Nieto M L, Sanchez-Crespo M. Biosynthesis of platelet-activating factor (PAF) in human polymorphonuclear leucocytes. Biochem J 1990; 268: 91-98.