Rakicidin A: A Hypoxia-Selective Cytotoxin

Abstract

Regions of hypoxia are present in the majority of solid tumors mainly because of poor and defective vascularization.1,2) Evidence indicating that hypoxic cells exist in the tumors has been obtained from microscopic analysis of histological sections 3,4) and radiobiological studies. 5) Oxygen is required for the cytotoxic effects of many cancer chemotherapeutic drugs and radiation, and hypoxic tumor cells are resistant to the conventional chemotherapy and radiotherapy. 6)Moreover, hypoxia also selects for more aggressive and metastatic cancer phenotypes that are associated with poor prognosis. 7)To conquer the therapeutic resistance induced by hypoxia, several compounds have been developed as prodrugs that are preferentially bioactivated in the hypoxic cells. The most advanced bioreductive drug, tirapazamine, is in phase III clinical trials in combination with cisplatin. 8)To identify a novel hypoxia-selective cytotoxin, we screened 20000 cultured broths of microorganisms and found that rakicidin A showed significant hypoxia-selective cytotoxicity. Rakicidin A was approximately 17.5-fold more cytotoxic under hypoxic than under normoxic conditions. In this paper, we describe the hypoxia-selective cytotoxicity of rakicidin A. MATERIALS AND METHODSCell Culture CHO chinese hamster ovary cells were maintained in minimum essential medium alpha medium (a-MEM) supplemented with 10% fetal bovine serum (ICN Biomedicals, Costa Mesa, CA, U.S.A.) and kanamycin (50 mg/ml). Human colorectal adenocarcinoma cell lines HCT-8 and DLD-1, human glioblastoma cell lines U251 and SF268, human prostate cancer cell lines PC-3 and LNCaP, human pancreatic adenocarcinomas cell line PANC-1 were maintained in RPMI1640 with 10% fetal bovine serum and kanamycin (50 mg/ml). A stable transformant of CHO cells was established by the transfection of the HIF-1-dependent luciferase (5ϫHRE/pGL3/VEGF/E1b) and neomycin resistant genes as previously described.9) Stably transfected cells were maintained in a-MEM supplemented with 10% fetal bovine serum and kanamycin (50 mg/ml). Cells were cultured at 37°C in a humidified incubator containing 5% CO 2 (normoxia).Materials Materials used were as follows: [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT), CoCl 2 , actinomycin D, bleomycin, camptothecin, chlorambucil, doxorubicin, etoposide, melphalan, mitomycin C, streptonigrin, taxol and tubercidin from Sigma Aldrich, St. Louis, U.S.A.; cisplatin from BioVision, California, U.S.A.; neocarzinostatin from Astellas Pharma, Inc., Japan; glutathione from Cayman Chemical Company, Michigan, U.S.A.; a-tocopherol and N-acetyl-L-cysteine from LKT Laboratories, Inc., Minnesota, U.S.A. All reagents were initially dissolved in dimethyl sulfoxide (DMSO) (Sigma) and diluted with the medium for assay.Screening CHO cells were seeded at a density of 1ϫ10 4 /100 ml medium per well into a pair of 96-well microplates and cultured for 24 h to assure complete adherence of the cells to the plates. The cultured cells were treated with fermentation broths of microor...

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