Suppression of 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia and inflammation by the dehydroepiandrosterone analog 16alpha-fluoro-5-androsten-17-one and its reversal by NADPH liposomes.

Dehydroepiandrosterone and related steroids produce cancer-preventive and other potentially important therapeutic effects in laboratory animals. These steroids are potent uncompetitive inhibitors of mammalian glucose-6-phosphate dehydrogenase, the first enzyme in the pentose phosphate pathway. Inhibition of this pathway could have profound effects on the supply of 5-carbon sugars required for nucleic acid synthesis as well as on the availability of nicotinamide adenine dinucleotide phosphate (NADPH) and the cellular redox state. NADPH is a source of reducing equivalents for the production of oxygen free radicals, which act as intermediate messengers stimulating mitogenesis and up-regulating the inflammatory response. Using a mixture of NADPH and cationic liposomes to facilitate uptake of the normally impenetrable dinucleotide, we found that intradermal injections of NADPH-liposomes reversed the anti-inflammatory and anti-hyperplastic effects of the dehydroepiandrosterone analog, 16alpha-fluoro-5-androsten-17-one, in mouse skin treated with 12-O-tetradecanoylphorbol-13-acetate, whereas similar treatment had no apparent effect on the anti-hyperplastic and anti-inflammatory effect of corticosterone.

Inhibition of 7,12-dimethylbenz[a]anthracene-induced lung tumorigenesis in A/J mice by food restriction is reversed by adrenalectomy.

Prior work has demonstrated that food restriction of mice markedly suppresses 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion of skin papillomas and adrenalectomy prior to initiating food restriction completely reverses the tumor inhibitory effect of underfeeding. In the present experiment the effect of food restriction, with or without prior adrenalectomy, on 7,12-dimethylbenz[a]anthracene (DMBA)-induced lung tumor development in A/J mice was explored. Food restriction (27%), beginning 3 weeks after a single oral dose of 0.5 mg DMBA and continued for the duration of the experiment (14 weeks), significantly inhibited lung adenoma development, whereas adrenalectomy 2 weeks before initiating food restriction abolished the tumor inhibitory effect of underfeeding and also enhanced tumor development in the ad libitum fed mice. Plasma corticosterone levels were significantly elevated in food-restricted A/J mice, whereas plasma dehydroepiandrosterone (DHEA) levels showed no apparent change. These studies suggest that adrenal gland secretory products may play a general role in the tumor preventive effect of food restriction in laboratory mice.

Cancer prevention with dehydroepiandrosterone and non-androgenic structural analogs.

There is increasing evidence that the adrenocortical steroid, dehydroepiandrosterone (DHEA), is an important mammalian hormone. Administration of DHEA to laboratory mice and rats inhibits development of experimental tumors of the breast, lung, colon, liver, skin and lymphatic tissue. In the two-stage skin tumorigenesis model in mice, DHEA treatment inhibits tumor initiation, as well as tumor promoter-induced epidermal hyperplasia and promotion of papillomas. There is much evidence that DHEA produces its antiproliferative and tumor preventive effects by inhibiting glucose-6-phosphate dehydrogenase and the pentose phosphate pathway. This pathway is an important source of NADPH, a critical reductant for many biochemical reactions that generate oxygen free radicals, which may act as second messengers in stimulating hyperplasia. The therapeutic use of DHEA in humans may be limited by its sex hormonal side effects. DHEA is metabolized in vivo to both testosterone and estrone, producing both androgenic and estrogenic effects in laboratory animals. We have developed a synthetic steroid, 16 alpha-fluoro-5-androsten-17-one, which does not demonstrate the androgenic or estrogenic activity of DHEA, yet retains the antiproliferative and cancer preventive activity of the native steroid.

Antihyperglycemic effect of dehydroepiandrosterone analogue 16 alpha-fluoro-5-androsten-17-one in diabetic mice.

The adrenocortical steroid, dehydroepiandrosterone, has been shown previously to produce an antidiabetic effect in C57BL/KsJ db/db mice. Preliminary clinical data suggest that this steroid may enhance insulin sensitivity in humans. The therapeutic use of dehydroepiandrosterone may be limited by its androgenic action. In a previous study, high-dose dehydroepiandrosterone therapy to postmenopausal women produced marked elevations in plasma testosterone (9-fold) and dihydrotestosterone (20-fold) levels. We previously developed the synthetic steroid, 16 alpha-fluoro-5-androsten-17-one, which lacks the androgenic action of dehydroepiandrosterone yet has retained other biological activities of the native steroid. In this study, administration of 16 alpha-fluoro-5-androsten-17-one in the diet (0.2 and 0.3%) to male C57BL/KsJ db/db mice markedly reduced plasma glucose levels. In contrast, treatment with dehydroepiandrosterone was effective in reducing plasma glucose levels at the 0.2% dose but had no effect at the 0.3% dose, possibly as a result of the androgenic state induced at the higher dose. Dehydroepiandrosterone treatment also produced a 25-fold elevation in plasma testosterone levels and a significant increase in seminal vesicle weights, whereas treatment with 16 alpha-fluoro-5-androsten-17-one had no apparent effect on the weight of the seminal vesicle glands.

Cancer chemoprevention with the adrenocortical steroid dehydroepiandrosterone and structural analogs.

Dehydroepiandrosterone (DHEA) is an adrenocortical steroid that produces broad-spectrum cancer chemopreventive action in mice and rats. In the mouse two-stage skin tumorigenesis model, DHEA treatment inhibits tumor initiation, as well as tumor promoter-induced epidermal hyperplasia and promotion of papillomas. There is considerable evidence that DHEA exerts its anti-proliferative and tumor-preventive action through the inhibition of glucose-6-phosphate dehydrogenase and the pentose phosphate pathway, which generate NADPH (required for mixed-function oxidase activation of chemical carcinogens, as well as for deoxyribonucleotide synthesis) and ribose 5-phosphate (also required for deoxyribonucleotide synthesis). Long-term DHEA treatment of mice also reduces weight gain (apparently by enhancing thermogenesis), and appears to produce many of the beneficial effects of food restriction, which have been shown to inhibit the development of many age-associated diseases, including cancer. Using the mouse two-stage skin tumorigenesis model, we found that adrenalectomy completely reverses the anti-hyperplastic and antitumor-promoting effects of food restriction. It is not unlikely that food restriction stimulates enhanced levels of adrenocortical steroids, such as the anti-inflammatory glucocorticoids and DHEA, which in turn mediate the tumor-inhibitory effect of underfeeding.

Reversal of food restriction-induced inhibition of mouse skin tumor promotion by adrenalectomy.

Restricting the food intake of laboratory mice and rats markedly reduces the incidence of spontaneous and experimentally induced cancers. Using the two-stage skin tumorigenesis model in CD-1 mice, we report now that food restriction suppresses 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of epidermal [3H]thymidine incorporation as well as TPA promotion of skin papillomas, whereas adrenalectomy completely reverses the inhibition in [3H]thymidine incorporation and tumor development. These results suggest that the adrenal gland may play an important role in mediating the tumor inhibitory effect of food restriction.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-promoted skin tumor formation in mice by 16 alpha-fluoro-5-androsten-17-one and its reversal by deoxyribonucleosides.

The work of ourselves and others has demonstrated that dehydroepiandrosterone (DHEA) dispalys a broad spectrum of cancer preventive action in laboratory rodents, with little toxicity. In the two-stage skin tumorigenesis model in mice, topical application of the synthetic DHEA analog 16 alpha-fluoro-5-androsten-17-one, a more potent preventive agent than DHEA without the sex-hormonal side-effects of the parent steroid, markedly inhibited promotion of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated tumor development by 12-O-tetradecanoylphorbol-13-acetate (TPA). DHEA is a powerful inhibitor of glucose-6-phosphate dehydrogenase (G6PDH), suggesting that its inhibiting effect in carcinogenesis may be due to a lack of NADPH and ribose-5-phosphate production for deoxyribonucleotide synthesis and subsequent DNA replication. Further evidence of a reduced NADPH and ribose-5-phosphate pool on the lowering of intracellular deoxyribonucleotide levels has been demonstrated in this paper by completely reversing the 16 alpha-fluoro-5-androsten-17-one-induced inhibition of tumor promotion by the addition of the four deoxyribonucleosides-deoxyadenosine, deoxycytidine, deoxyguanosine and thymidine--to the drinking water during the promotion period of tumorigenesis.

Inhibition of 7,12-dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted skin papilloma formation in mice by dehydroepiandrosterone and two synthetic analogs.

Previous work has demonstrated that the adrenal steroid, dehydroepiandrosterone (3-beta-hydroxy-5-androsten-17-one, DHEA), has broad spectrum tumor chemopreventive activity in laboratory mice and rats, inhibiting the development of spontaneous breast cancer and chemically induced tumors of the lung, colon, skin, thyroid and liver. DHEA treatment produces specific side-effects, including estrogenic and androgenic action and an increase in liver weight, which could limit its use as a cancer chemopreventive drug. It is now shown that oral administration of the synthetic steroid 16 alpha-fluoro-5-androsten-17-one, which lacks the side-effects of DHEA treatment, to CD-1 mice inhibits 7,12-dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted skin papilloma formation at both the initiation and promotion stage. The synthetic steroid is more potent as an inhibitor of papilloma formation than comparably administered DHEA.

Novel dehydroepiandrosterone analogues with enhanced biological activity and reduced side effects in mice and rats.

Treatment of laboratory mice and rats with the adrenal steroid, dehydroepiandrosterone (DHEA), produces antiobesity and broad spectrum tumor chemopreventive activity. Certain side effects are associated with DHEA administration which could limit its usefulness as a drug. DHEA can be metabolized into both testosterone and estrone and also increases liver weight and liver catalase activity. We have developed two synthetic steroids, 16 alpha-fluoro-5-androstan-17-one and 16 alpha-fluoro-5 alpha-androstan-17-one, which, unlike DHEA, do not stimulate uterine weight in sexually immature female rats or seminal vesicle weight in castrated male rats, nor stimulate liver weight and liver catalase activity in mice. 16 alpha-Fluoro-5-androsten-17-one is also about three times as potent as DHEA as an antiobesity agent and is more active when administered p.o. in inhibiting [3H]-7,12-dimethylbenz(a)-anthracene binding to skin DNA and tetradecanoylphorbol-13-acetate stimulation of epidermal [3H]thymidine incorporation in the mouse, two effects believed to be important in the tumor preventive action of DHEA. 16 alpha-Fluoro-5 alpha-androstan-17-one is as active as 16 alpha-fluoro-5-androsten-17-one in inhibiting [3H]-7,12-dimethylbenz(a)anthracene binding to skin DNA and tetradecanoylphorbol-13-acetate stimulation in epidermal [3H]thymidine incorporation but, on the contrary, is not more active than DHEA as an antiobesity drug. Compounds such as 16 alpha-fluoro-5-androsten-17-one and 16 alpha-fluoro-5 alpha-androstan-17-one, which lack specific side-effects of DHEA treatment and demonstrate greater potency, may have therapeutic application as drugs for humans.

Dehydroepiandrosterone and two structural analogs inhibit 12-O-tetradecanoylphorbol-13-acetate stimulation of prostaglandin E2 content in mouse skin.

Dehydroepiandrosterone, a naturally occurring adrenal steroid, is a highly effective tumor chemopreventive agent in laboratory mice and rats, inhibiting spontaneous breast cancer and chemically induced tumors of the lung, colon, skin, liver and thyroid. Dehydroepiandrosterone blocks three processes that have been implicated in experimental tumorigenesis: (i) carcinogen activation through the mixed-function oxidases, (ii) 12-O-tetradecanoylphorbol-13-acetate stimulation of superoxide anion production in neutrophils, and (iii) 12-O-tetradecanoylphorbol-13-acetate stimulation of [3H]thymidine incorporation in mouse epidermis. All of these effects of dehydroepiandrosterone very likely result from glucose-6-phosphate dehydrogenase inhibition and a lowering of the NADPH cellular pool. It is now reported that oral administration of dehydroepiandrosterone (0.2% in the diet) for two weeks inhibits the stimulation in prostaglandin E2 content in mouse epidermis produced by topical application of 12-O-tetradecanoylphorbol-13-acetate. Two synthetic steroids, 16 alpha-fluoro-5-androsten-17-one and 16 alpha-fluoro-5 alpha-androstan-17-one, which are more potent inhibitors of the above three processes in tumorigenesis and are also more effective than dehydroepiandrosterone in inhibiting skin papilloma development in the mouse, are more active in suppressing prostaglandin E2 induction by 12-O-tetradecanoyl-phorbol-13-acetate. These two structural analogs, which also lack specific side-effects associated with dehydroepiandrosterone treatment, may find application as cancer chemopreventive drugs in humans.

Food restriction inhibits [3H] 7,12-dimethylbenz(a)anthracene binding to mouse skin DNA and tetradecanoylphorbol-13-acetate stimulation of epidermal [3H] thymidine incorporation.

It has been known for many years that reducing the food intake of laboratory mice and rats inhibits the development of a broad spectrum of chemically induced and spontaneous tumors, but the mechanism of this effect is poorly understood. Food restriction of A/J mice for two weeks is now shown to inhibit the binding of topically applied [3H]7,12-dimethylbenz(a)anthracene (DMBA) to skin DNA by 50% and to abolish the stimulation of [3H]-thymidine incorporation in the epidermis produced by topical application of the tumor promoter tetradecanoylphorbol-13-acetate (TPA). Similar effects on the actions of DMBA and TPA are observed following topical application of the adrenal steroid, dehydroepiandrosterone (DHEA), a potent glucose-6-phosphate dehydrogenase (G6PDH) inhibitor, while food restriction for two weeks depresses epidermal G6PDH activity by 60%. It is suggested that both the inhibition of [3H]DMBA binding to skin DNA and the TPA stimulation in epidermal [3H]thymidine incorporation result from a reduction in the NADPH cellular pool as a result of G6PDH inhibition.

Inhibition of proteinuria development in aging Sprague-Dawley rats and C57BL/6 mice by long-term treatment with dehydroepiandrosterone.

Dehydroepiandrosterone (DHEA) is an adrenal steroid that previously has been shown to produce antiobesity, antidiabetic, cancer preventive, and antiautoimmune effects in laboratory rodents. DHEA, when administered in the diet to male Sprague-Dawley rats beginning at 2 months of age, inhibited the development of proteinuria at 19 months. The nontreated rats excreted 6.5 times as much urinary protein as the group treated with DHEA. Part of the effect of DHEA is apparently a result of reduced food intake in the treated rats (14% reduction), but this alone could not account for its action as a pair-fed group excreted significantly more urinary protein than the DHEA treated rats (2.3 times as much). A similar inhibition of proteinuria in 17-month-old male C57BL/6 mice was produced by DHEA treatment initiated at 10 months of age (5.8 times as much urinary protein excreted by non-DHEA treated mice). DHEA treatment reduced food intake by 11% in the C57BL/6 mice. This reduction in food intake had no apparent effect on proteinuria since a pair-fed group was found to excrete 6.5 times the amount of urinary protein as the DHEA-treated mice.

Inhibition of growth of HeLa and WI-38 cells by dehydroepiandrosterone and its reversal by ribo- and deoxyribonucleosides.

Dehydroepiandrosterone (DHEA), an adrenal steroid of no known biological function, is a potent inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH). DHEA inhibited the growth of two stains of HeLa and WI-38 cells in culture. One of the HeLa strains, TCRC-2, was about 10x as sensitive to growth inhibition as the two other cell lines. The G6PDH activity in cell extracts of HeLa TCRC-2 was also much more sensitive to DHEA inhibition than the G6PDH activities of the other cell lines. The addition of a combination of four deoxyribonucleosides and four ribonucleosides to the culture medium overcame the DHEA-induced growth inhibition in the HeLa TCRC-2 line.

Inhibition of 7,12-dimethylbenz(a)anthracene-induced skin papillomas and carcinomas by dehydroepiandrosterone and 3-beta-methylandrost-5-en-17-one in mice.

Topical application of the adrenal steroid, dehydroepiandrosterone, or the synthetic steroid, 3-beta-methylandrost-5-en-17-one, which unlike dehydroepiandrosterone is not demonstrably uterotrophic, inhibits 7,12-dimethylbenz(a)anthracene-induced skin papillomas and carcinomas in the CD-1 mouse.

Dehydroepiandrosterone (DHEA) and 3 beta-methylandrost-5-en-17-one: inhibitors of 7,12-dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin papilloma formation in mice.

Long-term oral administration of the adrenal steroid, dehydroepiandrosterone (DHEA), has previously been shown to inhibit the development of spontaneous breast cancer and chemically induced lung and colon tumors in various mouse strains. In the two-stage skin papilloma system in the mouse, topical application of DHEA inhibits both 7,12-dimethylbenz[a]anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate promotion of these tumors. The synthetic steroid, 3 beta-methylandrost-5-en-17-one, which, unlike DHEA, is not demonstrably estrogenic in the rat, also inhibits papilloma development.

Effect of food restriction, dehydroepiandrosterone, or obesity on the binding of 3H-7,12-dimethylbenz(a)anthracene to mouse skin DNA.

This study was undertaken to determine whether a reduction in body weight in laboratory mice by regimens that appear to delay the rate of aging (i.e., food restriction and chronic dehydroepiandrosterone (DHEA) treatment), or a production of obesity by the presence of the ob (obese) gene or by gold thioglucose-induced hyperphagia alter the rate of binding of 3H-7,12-dimethylbenz(a)anthracene (3H-DMBA) to mouse skin deoxyribonucleic acid (DNA). We have found that treating A/J mice with food containing .6% DHEA for 10 weeks or reducing the food intake of non-DHEA treated mice to 60% of ad libitum fed animals significantly reduces the amount of 3H-DMBA bound to mouse skin DNA 12 hours after a topical application of the carcinogen. Conversely, A/J mice made obese by a gold thioglucose-induced hyperphagia and C57BL/6J mice with the ob mutation bind significantly increased amounts of 3H-DMBA to skin DNA when compared to their nonobese counterparts.