Identification of cembratriene-4,6-diol as antitumor-promoting agent from cigarette smoke condensate

Cigarette smoke condensate (CSC) was separated into several fractions and each was tested for an inhibitory effect on the early antigen (EA) of Epstein-Barr virus (EBV) which can be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Two diastereoisomers of 2,7,11-cembratriene-4,6-diol (alpha- and beta-CBT) were isolated from the neutral fractions of CSC and these showed potent inhibitory effects on the induction of EBV-EA by TPA. The doses of alpha- and beta-CBT required for 50% inhibition of EBV-EA induction by TPA were 7.7 and 6.7 micrograms/ml, respectively, which are comparable with those of retinoic acid, a potent inhibitor of induction of epidermal ornithine decarboxylase (ODC) activity and tumor promotion by TPA in mice. Application of alpha- and beta-CBT to mouse skin prior to treatment with TPA inhibited TPA-induced ODC activity. The degree of inhibition was dependent on the dose and application of 16.5 mumol/mouse of alpha- and beta-CBT resulted in a 50 and 40% reduction, respectively, of the maximum of the ODC activity induced as a result of treatment with TPA. In initiation-promotion experiments, alpha-CBT markedly inhibited the promoting effect of TPA on skin tumor formation in mice which were initiated with 7,12-dimethylbenz[a]anthracene, but beta-CBT was found to be less effective. Application of 3.3 mumol of alpha-CBT 40 min prior to treatment with TPA (1 microgram) resulted in a 53% reduction in the number of papillomas per mouse. Our present data suggest that EBV-EA inhibition assay using Raji cells is effective for the first screening of inhibitors of tumor promotion, and provide evidence that CSC contains antitumor-promoting agents in addition to carcinogenic and tumor-promoting agents already reported.