Antitumor Effects of Miconazole on Human Colon Carcinoma Xenografts in Nude Mice through Induction of Apoptosis and G0/G1 Cell Cycle Arrest Chih-Hsiung Wu,* Jiiang-Huei Jeng,† Ying-Jan Wang,‡ Chia-Jen Tseng,§ Yu-Chih Liang, Chien-Ho Chen,§ Horng-Mo Lee,§ Jen-Kun Lin, ¶ Chien-Huang Lin,§ Shyr-Yi Lin,** Chung-Pei Li,§ and Yuan-Soon Ho§ ,1 *School of Medicine, §Institute of Biomedical Technology, Graduate Institute of Medical Sciences, and **Department of Internal Medicine, School of Medicine, Taipei Medical University, Taipei, Taiwan; †School of Dentistry and ¶ Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, Taiwan; and ‡Department of Environmental and Occupational Health, National Cheng Kung University Medical College, Tainan, Taiwan Received July 9, 2001; accepted December 14, 2001 Antitumor Effects of Miconazole on Human Colon Carcinoma Xenografts in Nude Mice through Induction of Apoptosis and G0/G1 Cell Cycle Arrest. Wu, C.-H., Jeng, J.-H., Wang, Y.-J., Tseng, C.-J., Liang, Y.-C., Chen, C.-H., Lee, H.-M., Lin, J.-K., Lin, C.-H., Lin, S.-Y., Li, C.-P., and Ho, Y.-S. (2002). Toxicol. Appl. Pharmacol. 180, 22–35. Miconazole (MIC), a promising oral antifungal agent, has been used worldwide in the treatment of superficial mycosis. In this study, we demonstrated that MIC dose dependently arrested var- ious human cancer cells at the G0/G1 phase of the cell cycle. The protein levels of p53, p21/Cip1, and p27/Kip1 were significantly elevated by MIC treatment in COLO 205 cells. Electrophoretic mobility gel shift assays showed that the nuclear extracts of the MIC-treated COLO 205 cells exerted a significant binding between wild-type p53 and its consensus-binding site present in the p21/ Cip1 promoter. These results suggested that the p53-associated signaling pathway is involved in the regulation of MIC-induced cancer cell growth arrest. By immunoblot analysis, we demon- strated that cyclin D3 and cyclin-dependent kinase-4 (CDK4) protein levels were inhibited by MIC treatment in the cancercells. Significant therapeutic effect was further demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with MIC (50 mg/kg ip). The protein expression of p53 was significantly increased in MIC-treated tumor tissues by immunohistochemical staining and Western blotting analysis. DNA fragmentation and TUNEL assay were performed and demonstrated that apoptosis occurred in tumor tissues treated with MIC. Our study provides the novel mechanisms of antitumor effects of MIC and such results may have significant applications for cancer chemotherapy. © 2002 Elsevier Science (USA) Key Words: miconazole; G0; G1 arrest; apoptosis; p53; nude mice. The discovery of the antifungal activity of azole compounds represented an important therapeutic advance. Miconazole (MIC) 2 , ketoconazole (KT), itraconazole, and fluconazole are currently commercially available (Bodey, 1992). Among their disadvantages are limited absorption in the absence of gastric acid and their potential for drug– drug interactions; many cli- nicians believe that topical MIC is a relatively effective agent for the treatment of most mycotic infections (Diehl, 1996). Because of its limited activity and toxicity, MIC has now been replaced by newer agents (such as terbinafine) (Leenutaphong et al., 1999; McClellan et al., 1999). MIC, KT, bifonazole, clotrimazole, econazole, isoconazole, and tioconazole are known inhibitors of cytochrome P450-dependent steroidogenic enzymes (Ayub and Levell, 1989). Another study indicated that KT and MIC inhibit cholesteryl ester formation in mac- rophages by blocking the intracellular transport of endocytosed cholesterol from lysosomes to the endoplasmic reticulum (Aikawa et al., 1999). These antifungal imidazoles, MIC and KT, are known to inhibit synthesis of essential cell membrane components. Furthermore, MIC can exert direct physicochem- ical cell membrane damage at relatively high levels, but KT cannot (Beggs, 1984). Although the mechanisms of antifungal activity of MIC and KT demonstrated were similar, several studies that identified some additional therapeutic effects of KT have been investi- gated. KT has been used in the treatment of hormone-depen- dent prostate cancer (Blagosklonny et al., 2000; Bok and Small, 1999; Heyns et al., 1985; Mahler and Denis, 1992; Trachtenberg and Pont, 1984; Trachtenberg, 1984). Moreover, KT has been found to exert a cytotoxic effect in various cancer cell lines (Rochlitz et al., 1988) and to potentiate the antitumor effect of interleukin-1 on murine RIF tumors (Braun- schweiger et al., 1990). Recent studies demonstrated that KT 1 To whom correspondence should be addressed at Graduate Institute of Biomedical Technology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Fax: 011– 886-2–2739-3422. E-mail: hoyuansn@ tmu.edu.tw. 2 Abbreviations used: CDK, cyclin-dependent kinase; CKIs, CDK inhibi- tors; EMSA, electrophoretic mobility gel shift assays; FACS, fluorescence- activated cell sorter; FCS, fetal calf serum; IP, immunoprecipitation; KT, ketoconazole; MIC, miconazole; PARP, poly(ADP ribose) polymerase. Toxicology and Applied Pharmacology 180, 22–35 (2002) doi:10.1006/taap.2002.9352, available online at http://www.idealibrary.com on 22 0041-008X/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.