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Recently we reported that the occurrence of lung adenoma caused by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was completely prevented by pretreatment of female A/J mice with 8-methoxypsoralen, a potent inhibitor of cytochrome P450 (P450 or CYP) 2A [Takeuchi et al. (2003) Cancer Res., 63, 7581-7583]. Thus, the aim of this study was to confirm that 8-methoxypsoralen exhibits chemopreventive effects by inhibiting CYP2A in the mouse lung. The involvement of CYP2A in the metabolic activation of NNK in the lung was first evidenced by the fact that the mutagenic activation of NNK by mouse lung microsomes was inhibited by 8-methoxypsoralen, coumarin and antibodies to rat CYP2A1. Supporting this, the mutagenic activation of NNK was efficiently catalyzed by mouse CYP2A4 and CYP2A5 co-expressed with NADPH-P450 reductase in a genetically engineered Salmonella typhimurium YG7108. The expression of mRNA for CYP2A5, but not for CYP2A4 or CYP2A12, in the mouse lung was proven by reverse transcriptase-polymerase chain reaction, probably indicating that CYP2A5 present in the mouse lung was involved in the metabolic activation of NNK. In accordance with these in vitro data, treatment of gpt delta transgenic mice with 8-methoxypsoralen prior to NNK completely inhibited the mutation of the gpt delta gene. The in vivo chemopreventive effects of 8-methoxypsoralen towards NNK-induced adenoma was seen only when the agent was given to female A/J mice prior to, but not posterior to, NNK, lending support to the idea that NNK is activated by CYP2A5 in the mouse lung as an initial step to cause adenoma. The inhibition by 8-methoxypsoralen of NNK-induced adenoma was seen in a dose-dependent manner: the dose to show apparent 50% suppression was calculated to be 1.0 mg/kg. To our surprise, CYP2A protein(s) was expressed in the lesion of NNK-induced lung adenomas, probably suggesting that 8-methoxypsoralen could inhibit the possible occurrence of further mutation of the adenoma cells induced by NNK. Based on these lines of evidence, we propose that 8-methoxypsoralen inhibits the CYP2A5-mediated metabolic activation of NNK in the mouse lung, leading to the prevention of NNK-induced adenoma.

We report that HSP105, identified by serological identification of antigens by recombinant expression cloning (SEREX), is overexpressed in a variety of human cancers, including colorectal, pancreatic, thyroid, esophageal, and breast carcinoma, but is not expressed in normal tissues except for the testis. The amino acid sequences and expression patterns of HSP105 are very similar in humans and mice. In this study, we set up a preclinical study to investigate the usefulness of a DNA vaccine producing mouse HSP105 whole protein for cancer immunotherapy in vivo using BALB/c and C57BL/6 mice, Colon26, a syngeneic endogenously HSP105-expressing colorectal cancer cell line, and B16.F10, a melanoma cell line. The DNA vaccine was used to stimulate HSP105-specific T-cell responses. C olorectal cancer (CRC) and melanoma are common and serious malignancies, for which surgery remains the main treatment, although the success of the treatment depends on the stage of the disease. Although adjuvant systemic chemotherapy or chemoradiation can confer a limited but significant survival advantage, novel and more effective therapies are needed. Identification of tumor associated antigens (TAA) expressed by CRC or melanomas remains one of the goals for designing novel immunological treatments for these tumors. Ideal targets for immunotherapy are gene products that are silenced in normal tissues except immune privilege tissue such as testis tissue, and that are overexpressed in cancer cells.More than 2000 candidate TAA have been identified by using the serological identification of antigens by recombinant expression cloning (SEREX) method. We have also reported TAA identified by using this method.(1-4) We earlier found that HSP105 (often called HSP110), as identified by SEREX was overexpressed specifically in a variety of human cancers, including colorectal, pancreatic, thyroid, esophageal, and breast carcinoma, but was not expressed in normal tissues except for testis tissue.(1,5) We recently found that HSP105 was also overexpressed in melanoma (unpublished data). If HSP105 can induce strong antitumor immunity, it may be a potential candidate as a target antigen for cancer immunotherapy. In the present study, we set up a preclinical study to investigate the usefulness of a HSP105-DNA vaccine, using BALB/c and C57BL/6 mice, the syngeneic endogenously HSP105-expressing CRC cell line Colon26, and the melanoma cell line B16.F10. Using these models, we analyzed both the antitumor effects and side-effects, including autoimmunity of the HSP105 DNA vaccination.The pioneering studies of Srivastava and colleagues led to the proposal that several HSP, including HSP70, HSP90 and gp96, bind antigenic peptides and deliver these peptides (through receptor-mediated endocytosis of the HSP) into the antigen-processing pathway of the antigen presenting cell (APC) for presentation on major histocompatibility complex (MHC) class I molecules. This HSP-involved pathway has been demonstrated to evoke potent antiviral and antitumor immune responses.(6) Howeve...

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