Summary
Intact mycobacteria and mycobacterial cell wall extracts have been shown to inhibit the growth of human and murine bladder cancer. Their mechanism of action is, however, poorly understood. Mycobacterium phleimycobacterial cell complex (MCC) is a cell wall preparation that has mycobacterial DNA in the form of short oligonucleotides complexed on the cell wall surface. In this study, we have investigated the possibility that MCC has anti-cancer activity that is mediated by two different mechanisms – a direct effect on cancer cell proliferation and viability and an indirect effect mediated by the production of interleukin 12 (IL-12), a cytokine known to possess anti-cancer activity. We have found that, although MCC is a potent inducer of IL-12 and IL-6 synthesis in monocytes and macrophages either in vitro or in vivo, it is unable to induce the synthesis of either IL-12, IL-6 or granulocyte–macrophage colony-stimulating factor (GM-CSF) by the human transitional bladder cancer cell lines HT-1197 and HT-1376. MCC is not directly cytotoxic towards these cancer cells, but induces apoptosis as determined by nuclear DNA fragmentation and by the release of nuclear mitotic apparatus protein. Mycobacterium phlei DNA associated with MCC is responsible for the induction of apoptosis. Our results indicate that MCC directly effects bladder cancer cells by inhibiting cellular proliferation through the induction of apoptosis, and has the potential for an indirect anti-cancer activity by stimulating cancer-infiltrating monocytes/macrophages to synthesize IL-12.
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References
Alexandroff, A. G., Black, J., Esuvaranathan, K. & James, K. (1997). Antiproliferative effect of IL-6 on transitional cell carcinoma of the bladder. Insight into mechanisms of bacillus Calmette-Guérin in immunotherapy. Biochem Soc Trans 25: 270S–270S.
Bevers, R. F., de Boer, E. C., Kruth, L. H. & Schamhart, D. H. (1997). Effects of isoniazid on the proliferation and cytokine production of bladder cancer cells in vitro induced by bacille Calmette-Guérin. Br J Urol 80: 35–39.
Brunda, M. J., Luistro, L., Warrier, R. R., Wright, R. B., Hubbard, B. R., Murphy, M., Wolf, S. F. & Gately, M. K. (1993). Antitumour and antimetastatic activity of interleukin-12 against murine tumors. J Exp Med 178: 1223–1230.
Chen, L., Chen, D., Block, E., O’Donnell, M., Kufe, D. W. & Clinton, S. K. (1997). Eradication of murine bladder carcinoma by intratumoral injection of a bicistronic adenoviral vector carrying cDNAs for the IL-12 heterodimer and its inhibition by the IL-12 p40 subunit homodimer. J Immunol 159: 351–359.
Chin, J. L., Kadhim, S. A., Batislam, E., Karlik, S. J., Garcia, B. M., Curtis Nickel, J. & Morales, A. (1996). Mycobacterium cell wall: an alternative to intravesical bacillus Calmette Guerin (BCG) therapy in orthotopic murine bladder cancer. J Urol 156: 1189–1193.
De Reijke, T. M., Vos, P. C., de Boer, E. C., Bevers, R. F., de Munick, K., Kurth, K. H. & Schambart, D. H. (1993). Cytokine production by the human bladder carcinoma cell line T24 in the presence of bacillus Calmette-Guérin (BCG). Urol Res 21: 349–352.
Dive, C., Evans, C. A. & Whetton, A. D. (1992). Induction of apoptosis: new targets for cancer chemotherapy. Semin Cancer Biol 3: 417–427.
El-Demiry, M. I. M., Hargreave, T. B., Busuttil, A., James, K. & Chrisholm, G. D. (1986). Immunohistochemical identification of lymphocyte subsets and macrophages in normal human urothelium using monoclonal antibodies. Br J Urol 58: 436–442.
Esuvaranathan, K., Alexandroff, A. G., McIntyre, M., Jackson, A. M., Prescott, S., Chisholm, G. D. & James, K. (1995). Interleukin-6 production by bladder tumors is upregulated by BCG immunotherapy. J Urol 154: 572–575.
Filion, M. C. & Phillips, N. C. (1997). Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells. Biochim Biophys Acta 1329: 345–356.
Filion, M. C., Lépicier, P. & Phillips, N. C. (1997). Mycobacterium phleicell wall complex, a new antitumoral agent, induces IL-12 synthesis by monocytes/macrophages. Blood 90: 58b–58b.
Filion, M. C. et al (1998) (manuscript in preparation)
Hawkyard, S. J., Jackson, A. M., Prescott, S., James, K. & Chisholm, G. D. (1993). The effect of recombinant cytokines on bladder cancer cells in vitro. J Urol 150: 514–518.
Ioachim-Velogianni, E., Stavropoulos, N. E., Kitsiou, E., Stefanaki, S. & Agnantis, N. J. (1994). HLA-DR antigen expression subsets in transitional cell carcinoma of the urinary bladder. An immunohistological study in frozen sections. J Pathol 174: 183–189.
Izquierdo, M. A., Degen, D., Sypek, J. P. & Von Hoff, D. D. (1996). Antiproliferative effects of IL-12 on human tumor colony-forming units taken directly from patients. Anticancer Drugs 7: 275–280.
Jackson, A. M., Alexandroff, A. B., Fleming, D., Prescott, S., Chisholm, G. D., Carter, G. D. & James, K. (1994). Bacillus Calmette-Guérin (BCG) organisms directly alter the growth of bladder tumour cells. Int J Oncol 5: 697–703.
Kadhim, S. A., Chin, J. L., Batislam, E., Karlick, S., Garcia, B. M., Curtis Nickel, J. & Morales, A. (1993). Modification of intravesical Bacillus Calmette Guerin (BCG) therapy in orthotopic murine bladder tumor: use of mycobacterial cell wall extract (MCWE). J Urol 149: A225–225.
Kawasaki, T., Tomita, Y., Bilim, V., Takeda, M., Takahashi, K. & Kumanishi, T. (1996). Abrogation of apoptosis induced by DNA-damaging agents in human bladder-cancer cell lines with p21/WAF1/CIP1 and/or p53gene alterations. Int J Cancer 68: 501–505.
Kudoh, S., Liu, X. X., Mori, K. & Suzuki, T. (1997). BCG-activated lymphocytes induce the death of bladder cancer cell via apoptosis in vitro. Br J Urol 80S2: 40–40.
Lagranderie, M. R. R., Balazuc, A-M, Deriaud, E., Leclerc, C. D., Gheorghiu, M. (1996). Comparison of immune responses of mice immunized with five different Mycobacterium bovisBCG vaccine strains. Infect Immunol 64: 1–9.
Lamm, D. L. (1995). BCG in perspective: advances in the treatment of superficial bladder cancer. Eur Urol 27S1: 2–2.
Lamm, D. L., van der Meijden, A. D. & Morales, A. (1992). Incidence and treatment of complications of bacillus Calmette-Guérin intravesical therapy in supperficial bladder cancer. J Urol 147: 596–600.
Lamm, D. L., Riggs, D. R., Traynelis, C. L. & Nseyo, U. N. (1995). Failure of intravesical chemotherapy prophylaxis in superficial transitional cell carcinoma of the bladder. J Urol 153: 1444–1450.
Mallick, B. B., Kishore, S., Das, S. K. & Garg, A. (1985). Non-specific immunostimulation against viruses. Comp Immunol Microbiol Infect Dis 8: 55–63.
Miller, T., Beausang, L. A., Meneghini, M. & Lidgard, G. (1993). Death-induced changes to the nuclear matrix: the use of anti-nuclear matrix antibodies to study agents of apoptosis. Biotechniques 15: 1042–1047.
Mizutani, Y., Okada, Y., Yoshida, O., Fukumoto, M. & Bonavida, B. (1997). Doxorubicin sensitizes human bladder carcinoma cells to Fas-mediated cytotoxicity. Cancer 79: 1180–1189.
Morales, A. (1984). Long term results of BCG therapy for bladder cancer. J Urol 132: 457–459.
Morales, A. & Chin, J. L. (1997). Mycobacterial cell wall (MCW) as an alternative to BCG in the treatment of carcinoma-in-situ (CIS) of bladder: an efficacy study. J Urol 157: A214–A214.
Mosman, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55–63.
Nastala, C. L., Edington, H. D., McKinney, T. G., Tahara, H., Nalesnik, M. A., Brunda, M. J., Gately, M. K., Wolf, S. F., Schreiber, R. D., Stewart, W. T., Storkus, J. & Lotze, M. T. (1994). Recombinant interleukin-12 (IL-12) administration induces tumor regression in association with interferon-γ production. J Immunol 153: 1697–1706.
Newell, M. K., Haughn, L. J., Maroun, C. R. & Julius, M. H. (1990). Death of mature T cells by separate ligation of CD4 and the T-cell receptor for antigen. Nature 347: 286–288.
Noguchi, Y., Richards, E. C., Chen, Y. T. & Old, L. J. (1995). Influence of interleukin-12 on p53 peptide vaccination against established Meth A sarcoma. Proc Natl Acad Sci USA 92: 2219–2223.
Okamoto, M., Hattori, K. & Oyasu, R. (1997). Interleukin-6 functions as an autocrine growth factor in human bladder carcinoma cell lines in vitro. Int J Cancer 72: 149–154.
Papapetropoulou, M., Tsintzou, A. & Vantarakis, A. (1997). Environmental mycobacteria in bottled table waters in Greece. Can J Microbiol 43: 499–502.
Phillips, N. C., Gagné, L., Ivanoff, N. & Riveau, G. (1996). Influence of phospholipid composition on antibody responses to liposome encapsulated protein and peptide antigens. Vaccine 14: 898–904.
Pryor, K., Stricker, P., Russell, P., Golovsky, D. & Penny, R. (1995a). Antiproliferative effects of bacillus Calmette-Guérin and interferon α2b on human bladder cancer cells in vitro. Cancer Immunol Immunother 41: 309–316.
Pryor, K., Goddard, J., Goldstein, D., Stricker, P., Russell, P., Govolsky, D. & Penny, R. (1995b). Bacillus Calmette-Guérin (BCG) enhances monocyte- and lymphocyte-mediated bladder tumour cell killing. Br J Cancer 71: 801–807.
Rahman, M. (1994). In vitro effects of high energy shock wave alone and combined with anticancer drugs on human bladder cancer cells. Urol Int 53: 12–17.
Rasheed, S., Gardner, M. B., Rongey, R. W., Nelson-Rees, A. & Arnstein, P. (1977). Human bladder carcinoma: characterization of two new tumor cell lines and search for tumor viruses. J Natl Cancer Inst 58: 881–887.
Sasaki, A., Kudoh, S., Mori, K., Takahashi, N. & Suzuki, T. (1997). Are BCG effects against urinary bladder carcinoma cell line T24 correlated with apoptosis in vitro?. Urol Int 59: 142–148.
Shemtov, M. M., Cheng, DL-W, Kong, L., Shu, W-P, Sassaroli, M., Droller, M. J. & Liu, BC-S (1995). LAK cell mediated apoptosis of human bladder cancer cells involves a pH-dependent endonuclease system in the cancer cell: possible mechanism of BGC therapy. J Urol 154: 269–274.
Stern, A. S., Magram, J. & Presky, D. H. (1996). Interleukin-12 an integral cytokine in the immune response. Life Sci 58: 639–654.
Stine, K. C., Warren, B. A. & Becton, D. L. (1996). Apoptosis induced by interleukin-12 measured by DNA electrophoresis and in situ end labelling in leukemia. Ann NY Acad Sci 795: 420–421.
Tewari, A. K., Sharma, N. N., Rao, J. R., Mishra, A. K. & Das, S. K. (1996). Effect of Mycobacterium phleion the development of immunity to Babesia bigemina. Vet Parasitol 62: 223–230.
Voest, E. E., Kenyon, M. N., O’Reilly, M. S., Truitt, G., D’Amato, R. J. & Folkman, J. (1995). Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst 87: 581–586.
Wyllie, S. H. (1980). Cell death: the significance of apoptosis. Int Rev Cytol 68: 251–306.
Zitvogel, L. & Lotze, N. H. (1995). Role of interleukin-12 (IL-12) as an anti-tumor agent: experimental biology and clinical application. Res Immunol 146: 628–638.
Zlotta, A. R., Drowart, A., Huygen, K., De Bruyn, J., Shekarsarai, H., Decock, M., Pirson, M., Jurion, F., Palfliet, K., Denis, O., Mascart, F., Simon, J., Schulman, C. C. & Van Vooren, J. P. (1997a). Humoral response against heat shock proteins and other mycobacterial antigens after intravesical treatment with bacille Calmette-Guérin (BCG) in patients with superficial bladder cancer. Clin Exp Immunol 109: 157–165.
Zlotta, A. R., Drowart, A., Van Vorren, J. P., de Cook, M., Pirson, M., Palfliet, K., Jurion, F., Vanonckelen, A., Simon, J., Schulman, C. C. & Huygen, K. (1997b). Evaluation and clinical significance of the T cell proliferative and cytokine response directed against the fibronectin binding antigen 85 complex of bacillus Calmette-Guérin during intravesical treatment of superficial bladder cancer. J Urol 157: 492–498.
Zou, J. P., Yamamoto, N., Fujii, T., Takenaka, H., Kobayashi, M., Herrmann, S. H., Wolf, S. F., Fujiwara, H. & Hamaoka, T. (1995). Systemic administration of rIL-12 induces complete tumor regression and protective immunity: response is correlated with a striking reversal of suppressed interferon-γ production by anti-tumor T cells. Int Immunol 7: 1135–1145.
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Filion, M., Lépicier, P., Morales, A. et al. Mycobacterium phlei cell wall complex directly induces apoptosis in human bladder cancer cells. Br J Cancer 79, 229–235 (1999). https://doi.org/10.1038/sj.bjc.6690038
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DOI: https://doi.org/10.1038/sj.bjc.6690038
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