Inhibition of phorbol ester-induced tumor promotion in mice by vitamin A analog and anti-inflammatory steroid.

The effects of a vitamin A analog, TMMP ethyl retinoate [or ethyl-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl-trans-2,4,6,8-nonatetraenoate] (abbreviated Ro 10-9359), and an anti-inflammatory steroid, fluocinolone acetonide (or 6 alpha, 9 alpha-difluoro-11 beta, 16 alpha, 17,21-tetrahydroxypregna-1,4-diene-3,20-dione cyclic 16,17-acetal) (abbreviated FA), given alone or together were studied in a two-stage carcinogensis system. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was used as the tumor promoter in a 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin system. Two stocks of female mice, CD-1 and Sencar, which differ in their degrees of sensitivity to skin carcinogenesis, were used. A dose-dependent inhibition of carcinogenic expression, as determined by a decreased number of papillomas per animal, was observed in each mouse stock with the use of both FA and Ro 10-9359 when given alone. When FA and Ro 10-9359 were given together, an enhanced effect on the lowering of tumor incidence was noted. FA effectively inhibited tumor formation in the sensitive mouse stock even when the steroid was given 1 day prior to TPA treatment under conditions of unusually high doses of initiator (DMBA) and/or promoter (TPA). These results suggest that both anti-inflammatory steroids and retinoids inhibit tumor promotion and can be effectively used as a combination regimen for increased chemopreventive response.

Potent tumor-initiating activity of the 3,4-dihydrodiol of 7,12-dimethylbenz(a)anthracene in mouse skin.

The abilities of the racemic trans-3,4-, 5,6-, and 8,9-dihydrodiols of 7,12-dimethylbenz(a)anthracene to initiate skin tumors in mice were determined by using a two-stage system of tumorigenesis. The 7,12-dimethylbenz(a)anthracene trans-3,4-dihydrodiol was found to be much more active as a tumor initiator than the parent hydrocarbon. The 7,12-dimethylbenz(a)anthracene trans-5,6- and 8,9-dihydrodiols were essentially inactive as skin tumor initiators. Our results suggest that the 3,4-dihydrodiol of 7,12-dimethylbenz(a)anthracene is a proximal carcinogen and that the "bay region" diol-epoxide may be the ultimate carcinogenic form of DMBA.

Effects of 12-O-tetradecanoylphorbol-13-acetate and mezerein on epidermal ornithine decarboxylase activity, isoproterenol-stimulated levels of cyclic adenosine 3':5'-monophosphate, and induction of mouse skin tumors in vivo.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the antileukemic agent mezerein are diterpene esters of plant origin with certain structural similarities. Both compounds, when applied topically to mouse skin, were equipotent on a molar basis in inducing hyperplasia, inflammation, and ornithine decarboxylase activity, as well as in reducing cyclic adenosine 3':5'-monophosphate accumulation in response to beta-adrenergic stimulation. In contrast, mezerein was much less effective as a tumor promoter; the phorbol ester at 8.5 nmol/application yielded 78-fold more tumors than did 8.5 nmol mezerein per application to similarly initiated SENCAR mice. The superiority of the phorbol ester was nearly as great in CD-1 mice.

Comparison of the skin tumor initiating activity of 3-methylcholanthrene and 3,11-dimethylcholanthrene in mice.

The abilities of 3-methylcholanthrene (3-MC) and 3,11-dimethylcholanthrene (3,11-DMC) to initiate skin tumors in Sencar mice were determined by using a 2-stage system of tumorigenesis. 3,11-DMC was found to have very weak skin tumor initiating activity when compared to the potent activity of 3-MC. The only difference between 3-MC and 3,11-DMC is the substitution of a methyl group in position 11 which is part of the 'K-region' or the 'peri' position. From these results, we suggest that an unhindered peri position adjacent to an angular benzene ring is necessary for carcinogenic activity of 3-MC.

Indomethacin enhancement of TPA tumor promotion in mice.

Skin tumor promotion in mice by 12-O-tetradecanoylphorbol-13-acetate (TPA) is characterized by hyperplasia and inflammation. Based on the inhibitory effect of the steroidal anti-inflammatory drugs, non-steroidal agents, such as indomethacin, were also expected to show some degree of inhibition; however, repeated tumor experiments demonstrate that indomethacin enhances TPA promotion in a dose-response manner. A time-of-application effect was evident such that indomethacin given 2 h prior to TPA resulted in the greatest enhancement. Flurbiprofen was also observed to enhance promotion slightly.

Studies on the mechanism of skin tumor promotion: evidence for several stages in promotion.

The effects of nonpromoting and weakly promoting diterpenes on skin tumor promotion by 12-O-tetradecanoylphorbol 13-acetate (TPA) were investigated. When phorbol and phorbol 12,13-diacetate (both nonpromoting) were given simultaneously with TPA after 7,12-dimethylbenz[a]-anthracene (DMBA) initiation in female mice, they had no effect on TPA promotion. However, the nonpromoter 4-O-methyl-TPA and the weak promoter mezerein were found to inhibit TPA promotion in a dose-dependent manner when given simultaneously with TPA. Because mezerein was found to be an effective inhibitor of TPA promotion when given simultaneously and because it induces many biological responses similar to those to TPA, the capacity of mezerein to act as an incomplete promoter in a two-stage promotion protocol was also investigated. Twice-weekly applications of 1,2, or 5 mug of TPA for 2 weeks after DMBA initiation produced 0, 0, and 0.5 papilloma per mouse, respectively, at 20 weeks. When the twice-weekly applications of TPA for 2 weeks were followed by twice-weekly treatments with 2 mug of mezerein for 18 weeks, the number of papillomas per mouse was 2.2, 3.5, and 9.0, respectively. Twice-weekly applications of 2 mug of TPA for 2 weeks followed by twice-weekly treatments with 1, 2, or 4 mug of mezerein for 18 weeks produced 2.1, 3.5, and 6.8 papillomas per mouse, respectively, in DMBA-treated mice. Twice-weekly doses as high as 40 mug of 4-O-methyl-TPA were not effective in producing tumors when given after a limited treatment with TPA; however, 4-O-methyl-TPA had weak activity as a first-stage promoter. The results suggest that although mezerein by itself is a weak promoter and mimics TPA in many biochemical and morphological effects it is a potent second-stage promoter in a two-stage promotion regimen.

Prostaglandin modulation of phorbol ester skin tumor promotion.

TPA promotion of skin tumors in mice can be modified by application of various prostaglandins or their precursors. The effects depend on the particular prostaglandin used: PGF2alpha enhances promotion, whereas PGE1 consistently inhibits promotion. Time of application of the prostaglandin with respect to TPA determines whether PGE2 enhances or inhibits. Dose-dependent inhibition was observed for arachidonic acid. The prostaglandins alone were unable to elicit tumors in initiated mice.

Lack of tumor-promoting ability of certain environmental chemicals in a two-stage mouse skin tumorigenesis assay.

The food antioxidants butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) were tested as tumor promoters on CD1 female mice initiated with 7, 12-dimethylbenz(a)anthracene (DMBA). At a dose of 1 mg twice weekly they did not promote skin tumors. Nor did they produce tumors when tested as a complete carcinogen without DMBA initiation. The polychlorinated biphenyl Aroclor 1254 (PCB) and the polybrominated biphenyl Firemaster-6 (PBB) were also tested for their ability to promote skin tumors; at a 100 microgram dose twice weekly they were inactive. The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at a dose of 0.1 microgram twice weekly did not promote skin tumors in DMBA-initiated mice. TCDD, PCB, and PBB did not promote spontaneous tumors. None of the compounds at the dosages tested significantly increased the intrafollicular epidermis, nor did they appear to be chronically toxic to the test animals. These results indicate that dosage may be an important factor in promotion, since several of the tested compounds are known to be promoters in pulmonary and hepatic systems.