Abstract
Protein A (PA) of Staphylococcus aureus has been demonstrated to possess anti-tumor activity against a wide variety of tumors. In the current study we endeavored to obtain a mechanistic insight into PA-mediated Ehrlich's ascites carcinoma (EAC) killing. Our results indicate that PA stimulates generation of nitric oxide (NO) from murine peritoneal macrophages. Nitric oxide in turn induces cytotoxic damage to the tumor cells. Analysis of the morphological features and cell cycle phase distribution pattern of nuclear DNA revealed an induction of apoptosis (appearance of sub-G0/G1 population) in EAC after PA treatment. We have further elaborated the alterations in the expressions of the proto-oncoproteins p53 and Bax, together with a change in the ratio of Bcl-2/Bax in the treated tumor cells, which favor apoptosis. PA-induced apoptosis and changes in the expression of oncoproteins in the tumor cells was prevented by the suppression of NO release by the addition of L-NAME, the competitive NOS inhibitor, suggesting a possible mechanism by which PA exerts its anti-tumor activities involving nitric oxide through the alteration in the expressions of pro-apoptotic proteins.
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References
Das T, Sa G, Ray PK Mechanisms of protein A superantigeninduced signal transduction for proliferation of mouse B cell. Immunol Lett 1999; 70: 43–51.
Das T, Sa G, Sinha P, Ray PK Induction of cell proliferation and apoptosis: Dependence on the dose of the inducer. Biochem Biophys Res Commun 1999; 260: 105–110.
Das T, Sa G, Subbulakshmi V, et al Protein A-activated rat splenic lymphocyte proliferation involves tyrosine kinasephospholipase C-protein kinase C pathway. Immunopharmacol Immunotoxicol 2000; 22: 75–90.
Ghosh AK, Sinha P, Das T, Sa G, Ray PK S. aureus superantigen protein A expands CD4(+)/CD8(+)/CD19(+)/CD34(+) cells in mice: A potential immunorestorer. Biochem Biophys Res Commun 1999; 256: 142–146.
Ghosh AK, Jana S, Das T, Sa G, Mandal N, Ray PK Protection by protein A of apoptotic cell death caused by anti-AIDS drug zidovudine. Biochem Biophys Res Commun 1999; 264: 601–604.
Ray PK, Raychoudhury S, Allen P Mechanism of regression of mammary adenocarcinomas in rats following plasma adsorption over protein A containing Staphylococcus aureus. Cancer Res 1982; 42: 4970–4974.
Shukla Y, Verma AS, Mehrotra NK, Ray PK Antitumor activity of protein A in a mouse skin model of two-stage carcinogenesis. Cancer Lett 1996; 103: 41–47.
Verma AS, Dwivedi PD, Mishra A, Ray PK Ehrlich's ascites fluid adsorbed over proteinAcontaining Staphylococcus aureus Cowan I produces inhibition of tumor growth. Immunopharmacol Immunotoxicol 1999; 21: 89–108.
Prasad AK, Singh KP, Saxena AK, Mathur N, Ray PK Increased macrophage activity in protein A treated tumor regressed animals. Immunopharmacol Immunotoxicol 1987; 9: 541–561.
Singh KP, Saxena AK, Prasad AK, Dwivedi PD, Zaidi SIA, Ray PK Effect of protein A on mast cell numbers and macrophage phagocytic activity. Immunopharmacol Immunotoxicol 1987; 9: 281–297.
Mishra A, Dwivedi PD, Verma AS, Ray PK Mechanism of enhanced phagocytic response in protein A treated rat macrophages. Immunol Lett 1992; 34: 289–295.
Jackson PG, Evans SR Intraperitoneal macrophages and tumor immunity: A review. J Surg Oncol 2000; 75: 146–154.
Cui S, Reichner JS, Mateo RB, Albina JE Activated murine macrophages induce apoptosis in tumor cells through nitric oxide-dependent or-independent mechanisms. Cancer Res 1994; 54: 2462–2467.
Bhaumik S, Khar A Cytokine-induced production of NO by macrophages induces apoptosis and immunological rejection of AK-5 histiocytic tumor. Apoptosis 1998; 3: 361–368.
Stuehr DJ, Nathan CF Nitric oxide: A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 1989; 169: 1543–1555.
Farias-Eisener R, Sherman MP, Aeberhard E, Chaudhuri G Nitric oxide is an important mediator for tumoricidal activity in vivo. Proc Natl Acad Sci USA 1994; 91: 9407–9411.
Bredt DS Endogenous nitric oxide synthesis: Biological functions and pathophysiology. Free Radic Res 1999; 31: 577–596.
Stuehr DJ, Marletta MA Induction of nitrite/nitrate synthesis in murine macrophages in BCG infection, lymphokines or interferon-γ. J Immunol 1987; 139: 518–525.
Stamler JS Redox signaling: Nitrosylation and related target interactions of nitric oxide. Cell 1994; 78: 931–936.
Kim YM, Bergonia HA, Muller C, Pitt BR, Watkins WD, Lancaster JR Jr. Loss and degradation of enzyme-bound hemeinduced by cellular nitric oxide synthesis. J Biol Chem 1995; 270: 5710–5713.
Moncada S, Palmer RMJ, Higgs EA Nitric oxide: Physiology, pathophysiology and pharmacology. Pharmacol Rev 1991; 43: 19–142.
Sandau K, Pfeilschifter J, Brune B Nitric oxide and superoxide induced p53 and Bax accumulation during mesengial cell apoptosis. Kidney Int 1997; 52: 378–386.
Levine AJ p53, the cellular gatekeeper for growth and division. Cell 1997; 88: 323–331.
Basu A, Haldar S The relationship between Bcl2, Bax and p53: Consequences for cell cycle progression and cell death. Mol Hum Reprod 1998; 4: 1099–1109.
Miyashita T, Reed JC Tumor suppressor p53 is a direct transcriptional activator of human bax gene. Cell 1995; 80: 293–299.
Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR Analysis of nitrate, nitrite and 15N nitrate in biological fluid. Anal Bio Chem 1982; 126: 131–138.
Aggarwal BB, Mehta K Determination and regulation of nitric oxide production from macrophages by lipopolysaccharides, cytokines and retinoids. Methods Enzymol 1996; 269: 166–171.
Binder C, Schulz M, Hiddemann W, Oellerich M Induction of inducible nitric oxide synthase is an essential part of tumor necrosis factor-alpha-induced apoptosis in MCF-7 and other epithelial tumor cells. Lab Invest 1999; 79: 1703–1712.
Pollet S, Bottex-Gauthier C, Picot F, Potier P, Vidal D, Favier A Effect of a synthetic lipid immunomodulator on the regulation of the transcription factor NF-kappaB. Immunopharmacol Immunotoxicol 2000; 22: 653–669.
Stefano GB, Salzet M, Magazine HI, Bilfinger TV Antagonism of LPS and IFN-gamma induction of iNOS in human saphenous vein endothelium by morphine and anandamide by nitric oxide inhibition of adenylate cyclase. J Cardiovasc Pharmacol 1998; 31: 813–820.
Kroncke KD, Fehsel K, Kolb-Bachofen V Nitric oxide: Cytotoxicity versus cytoprotection—How, why, when and where? Nitric oxide. Biology and Chemistry 1997; 1: 107–120.
Brune B, von Knethen A, Sandau KB Nitric oxide: An effector of apoptosis. Cell Death Differ 1999; 6: 969–975.
Laurent M, Lepoivre M, Tenu JP Kinetic modelling of the nitric oxide gradient generated in vitro by adherent cells expressing inducible nitric oxide synthase. Biochem J 1996; 314: 109–113.
Messmer UK, Ankarcrona M, Nicotera P, Brune B p53 expression in nitric oxide-induced apoptosis. FEBS Lett 1994; 255: 23–26.
Adams JM, Cory S The Bcl-2 protein family: Arbiters of cell survival. Science 1998; 281: 1322–1326.
Reed JC Bcl-2 and the regulation of programmed cell death. J Cell Biol 1994; 124: 1–6.
Genaro AM, Hortelano S, Alvarez A, Martinez AC, Bosca L Spleenic B lymphocyte programmed cell death is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels. J Clin Invest 1995; 95: 1884–1890.
Albina JE, Martin B-A, Henry WL Jr, Louis CA, Reichner JS B cell lymphoma-2 transfected P815 cells resist reactive nitrogen intermediate-mediated macrophage dependent cytotoxicity. J Immunol 1996; 157: 279–283.
Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD The release of cytohrome C from mitochondria: A primary site for Bcl-2 regulation of apoptosis. Science 1997; 275: 1132–1136.
Rossé T, Olivier R, Monney L, et al Bcl-2 prolongs cell survival after Bax-induced release of cytochrome C. Nature 1998; 391: 496–499.
Jäätellä M Escaping cell death: Survival proteins in cancer. Exp Cell Res 1999; 248: 30–43.
Miyashita T, Krajewshi S, Krajewshi M, et al Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 1994; 9: 1799–1805.
Halder S, Negrini M, Monna M, Sabbioni S, Croce C Down regulation of bcl-2 by p53 in breast cancer cells. Cancer Res 1994; 54: 2095–2097.
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Chattopadhyay, S., Das, T., Sa, G. et al. Protein A-activated macrophages induce apoptosis in Ehrlich's ascites carcinoma through a nitric oxide-dependent pathway. Apoptosis 7, 49–57 (2002). https://doi.org/10.1023/A:1013512912160
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DOI: https://doi.org/10.1023/A:1013512912160