Involvement of nitric oxide in anti-tumor effects of OK-432, a streptococcal anti-tumor immunotherapeutic agent Tetsuya Oshikawa a , Masato Okamoto a, * , Tomoyuki Tano a , Sharif Uddin Ahmed a , Akiko Sasai a , Shin Kan a , Yoichiro Moriya b , Yoshiki Ryoma b , Motoo Saito b , Mitsunobu Sato a a Second Department of Oral and Maxillofacial Surgery, Tokushima University School of Dentistry, Tokushima, 7708504, Japan b Product Research Laboratory, Chugai Pharmaceutical Co., Ltd., Tokyo, 1718545, Japan Received 17 August 2005; received in revised form 7 October 2005; accepted 14 November 2005 Abstract We examined the role of nitric oxide (NO) induced by OK-432, a streptococcal immunotherapeutic agent, in anti-tumor effects of the OK-432 by in vitro and in vivo experiments using an NO synthase inhibitor, N-monomethyl-l-arginine acetate (NMA). The in vitro treatment of mouse splenocytes with OK-432 increased the expression of inducible NO synthase (iNOS) gene and NO production in a dose-dependent manner. Although it is well known that OK-432 induces cytokines such as interferon (IFN)-g and tumor necrosis factor (TNF)-a, both of which are known to be potent NO inducers, we observed only a partial reduction of OK- 432-induced NO production with the addition of anti-IFN-g and/or anti-TNF-a neutralizing antibodies. The cytotoxicity of the splenocytes increased by in vitro OK-432 stimulation was almost completely inhibited by the treatment with NMA. OK-432 administration resulted in a marked prolongation of survival and a significant inhibition of tumor growth in syngeneic tumor- bearing mice, whereas NMA significantly inhibited the anti-tumor effects of OK-432. Although the increased cytotoxicity of adherent splenocytes derived from OK-432-treated tumor-bearing mice was almost completely inhibited by NMA, only partial inhibition by NMA was observed in the cytotoxicity of the nonadherent splenocytes. These findings strongly suggest that the iNOS/ NO induced by OK-432 is intimately involved in the anti-tumor effects of OK-432. D 2005 Elsevier B.V. All rights reserved. Keywords: OK-432; Nitric oxide; Cytotoxic activity; Anti-tumor immunity 1. Introduction OK-432, which is a penicillin-killed and lyophilized preparation of a low-virulence strain (Su) of Strepto- coccus pyogenes (group A) that was developed by Okamoto et al. in 1967 [1], is being successfully used as an immunotherapeutic agent in many types of ma- lignancies [2,3]. We have also reported that OK-432- based immunotherapy exerts a marked therapeutic effect in patients with oral squamous cell carcinomas [4,5]. It has been reported that OK-432 elicits anti- tumor effects by stimulating immunocompetent cells such as macrophages, T cells and natural killer (NK) cells [6,7], and that OK-432 induces interleukin (IL)-12 and polarizes the T-cell response to a helper T-cell 1 (Th1)-dominant state in mice [8]. Recently, we and other investigators demonstrated that OK-432 induces 1567-5769/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2005.11.010 * Corresponding author. Tel.: +81 88 633 7354; fax: +81 88 633 7462. E-mail address: mokamoto@dent.tokushima-u.ac.jp (M. Okamoto). International Immunopharmacology 6 (2006) 764 – 773 www.elsevier.com/locate/intimp