doi:10.1016/S0360-3016(03)00737-5 BIOLOGY CONTRIBUTION CYTOTOXICITY OF PERILLYL ALCOHOL AGAINST CANCER CELLS IS POTENTIATED BY HYPERTHERMIA KI-JUNG AHN, M.D.,* CHUNG K. LEE, M.D.,* EUN KYUNG CHOI, M.D., PH.D., † ROBERT GRIFFIN,PH.D.,* CHANG W. SONG,PH.D.,* AND HEON JOO PARK, M.D., PH.D.* ‡ *Radiobiology Laboratory, Department of Therapeutic Radiology, University of Minnesota Medical School, Minneapolis, MN; † Department of Therapeutic Radiology, College of Medicine, University of Ulsan, Seoul, Korea; ‡ Department of Microbiology, College of Medicine, Inha University, Inchon, Korea Purpose: Perillyl alcohol (POH) (4-isopropenyl-cyclohexenecarbinol) is a member of the monoterpenes, which are present in various fruits and vegetables. POH has been demonstrated to be cytotoxic against a variety of experimental cancer cells in vitro and in vivo. Phase I clinical trials have indicated that POH may be useful for human tumor treatment. The purpose of our study was to reveal whether the anticancer effect of POH could be enhanced by hyperthermia. Methods and Materials: SCK mammary carcinoma cells of A/J mice were used. The effects of POH or hyperthermia alone were studied by incubating the cells during exponential growth phase in culture with 0.25–1.0 mM of POH at 37°C for varying lengths of time or heating cells at 41– 43°C for varying lengths of time. The combined effect of POH and hyperthermia was investigated by heating the cells with 1 mM of POH at 41– 43°C for varying lengths of time. The effects of the treatments were evaluated using the clonogenic cell survival assay and three types of apoptosis assays. Results: An incubation of SCK cells with 1 mM of POH at 37°C for 60 min or hyperthermia at 43°C for 1 h decreased clonogenic cell survival to 40% and 60%, respectively. When the cells were heated at 43°C for 1 h in the presence of 1 mM of POH, clonogenic cell survival decreased to 0.2%, indicating that hyperthermia potentiated the effect of POH to cause clonogenic cell death. Hyperthermia also markedly increased the degree of POH-induced apoptosis. Conclusion: Hyperthermia synergistically potentiates the cytotoxicity of naturally occurring POH against cancer cells. © 2003 Elsevier Inc. Perillyl alcohol, Hyperthermia, Apoptosis, SCK tumor cells. INTRODUCTION Monoterpenes are naturally occurring compounds present in the extracts of fruits and vegetables and have been demon- strated to have a diverse array of pharmacologic effects, including antitumor properties (1–5). Perillyl alcohol (POH) (4-isopropenyl-cyclohexenecarbinol) is a hydroxylated monocyclic monoterpene found in cherries, lavender, mint, sage, cranberries, perilla, lemongrass, wild bergamot, gin- gergrass, savin, caraway, and celery seeds (4) and is the most potent anticancer agent among a variety of monoter- penes (3–5). POH has been demonstrated to inhibit the activation of carcinogens, suppress cell proliferation, and induce differentiation of cells, indicating that POH may be a potent chemopreventive drug (2–5). In addition, POH has been reported to induce apoptosis in a variety of cancer cells in vitro (6 –11) and in vivo (8, 12–14) and cause complete regression of chemically induced and transplanted animal tumors with little normal-tissue toxicity (8, 12–14). The initial results of Phase I clinical trials indicated that POH is relatively nontoxic, whereas it has therapeutic properties against some established human tumors (15, 16). The chemopreventive effect of monoterpenes has been attributed to detoxification through activation of carcino- gen-metabolizing enzymes and also to an inhibition of in- teraction of carcinogens with DNA (3–5). Among several molecular and metabolic changes that have been proposed as the cause of the chemotherapeutic effect of monoter- penes, activation of TGF- signaling seems to be the major mechanism (12, 14, 17, 18). Jirtle et al. (17) reported that the regression of rat mammary tumors caused by limonene, a monoterpene, was associated with a marked increase in Reprint requests to: Heon Joo Park, M.D., Ph.D., Radiation Biology Section, Department of Therapeutic Radiology-Radiation Oncology, University of Minnesota, Box 494, 420 Delaware St. SE, Minneapolis, MN 55455. Tel: (612) 626-6558; Fax: (612) 626-6245; E-mail: chaex001@tc.umn.edu This work was supported by grant CA-44114 from the National Cancer Institute, KOSEF 1999-2-208-003-3, and KISTEP Life Sci 2000. Acknowledgments—We thank Dr. S. Ahn and Mr. B. Williams for their expert technical assistance. Received Jan 29, 2003, and in revised form May 23, 2003. Accepted for publication May 30, 2003. Int. J. Radiation Oncology Biol. Phys., Vol. 57, No. 3, pp. 813– 819, 2003 Copyright © 2003 Elsevier Inc. Printed in the USA. All rights reserved 0360-3016/03/$–see front matter 813