Laura L. Pashko

Dehydroepiandrosterone and Structural Analogs: A New Class of Cancer Chemopreventive Agents

Publisher Summary Over the past several years, several therapeutic effects of dehydroepiandrosterone (DHEA) in laboratory mice and rats have been demonstrated. These include cancer preventive, possible antiautoimmune, and antiatherogenic effects as well as antiobesity and antidiabetic activities. However, certain simplicity in the mechanism of the action of the DHEA class of steroids is now beginning to emerge. The well-documented capacity of DHEA to inhibit mammalian glucose-6-phosphate dehydrogenase (G6PDH)—the rate limiting enzyme in the pentose phosphate pathway, a major source of cytosolic NADPH—now appears central to the mechanism of the cancer preventive action of this steroid. Several laboratories have undertaken determinations of DHEAS plasma levels in women with breast cancer and in matched controls. The predominance of evidence in these case-control studies suggests that the women with breast cancer have subnormal DHEAS plasma concentrations. However, as in all case-control studies, it is uncertain whether the presence of the measured abnormality proceeds or is a consequence of the disease.

Inhibition of Tumor Development by Dehydroepiandrosterone and Related Steroids

The naturally occurring adrenal steroid, dehydroepiandrosterone (DHEA), is a potent non-competitive inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH). Oral administration of DHEA to mice inhibits spontaneous breast cancer and chemically induced tumors of the lung and colon. Topical application of DHEA to mouse skin inhibits 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and tetra-decanoylphorbol-13-acetate (TPA)-promoted papillomas and DMBA-induced carcinomas at both the initiation and promotion phase. Evidence is presented that critical steps in the initiation process (mixed-function oxidase activation of a carcinogen) and promotion process (enhanced rates of cell proliferation and superoxide formation) all require NADPH and may be inhibited by DHEA and structural analogs as a result of a lowering of the NADPH cellular pool. Results obtained by others with fibroblasts and lymphocytes from individuals with the Mediterranean variant of G6PDH deficiency also indicate that a reduction in the NADPH cellular pool confers resistance to benzo(a)pyrene. Preliminary data suggest that food restriction may depress G6PDH levels and this may contribute to the tumor preventive effect of underfeeding.

Dehydroepiandrosterone, glucose-6-phosphate dehydrogenase, and longevity

Dehydroepiandrosterone (DHEA) is an abundantly produced adrenal steroid whose biological role has never been clarified. DHEA is a potent uncompetitive inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH) and as a consequence lowers NADPH levels and reduces NADPH-dependent oxygen-free radical production. Overproduction of oxygen-free radicals, or oxidative stress, upregulates inflammation and cellular proliferation and is believed to play a critical role in the development of cancer, atherosclerosis, and Alzheimers disease, as well as the basic aging process. Both in vitro and in vivo experimental studies strongly indicate that DHEA and related steroids inhibit inflammation and associated epithelial hyperplasia, carcinogenesis, and atherosclerosis, at least in part, through the inhibition of G6PDH and oxygen-free radical formation. Recent epidemiological findings in Sardinian males bearing the Mediterranean variant of G6PDH deficiency are consistent with the hypothesis that reduced G6PDH activity has a beneficial effect on age-related disease development and longevity. Clinical trials with DHEA are encumbered by the high oral doses required as well as the conversion of DHEA into active androgens. The use of less androgenic congeners as well as non-oral formulations may facilitate testing of this class of compounds.

Dehydroepiandrosterone: An anti‐obesity and anti‐carcinogenic agent

Long-term treatment of female C3H-Avy/A (obese) and C3H-A/A (non-obese) mice with dehydroepiandrosterone, an adrenal steroid found in subnormal levels in women predisposed to develop breast cancer, reduces weight gain without suppressing appetite and significantly inhibits the development of spontaneous breast cancer. This steroid also antagonizes the capacity of the tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate, to stimulate 3H-thymidine incorporation in mouse epidermis and in a cultured rat kidney epithelial cell line.

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.

A novel dehydroepiandrosterone analog improves functional recovery in a rat traumatic brain injury model.

The purpose of this study was to investigate the efficacy of a novel steroid, fluasterone (DHEF, a dehydroepiandrosterone (DHEA) analog), at improving functional recovery in a rat model of traumatic brain injury (TBI). The lateral cortical impact model was utilized in two studies of efficacy and therapeutic window. DHEF was given (25 mg/kg, intraperitoneally) at the initial time point and once a day for 2 more days. Study A included four groups: sham injury, vehicle treated (n = 22); injured, vehicle treated (n = 30); injured, pretreated (5-10 min prior to injury, n = 24); and injured, posttreated (initial dose 30 min postinjury, n = 15). Study B (therapeutic window) included five groups: sham injury, vehicle treated (n = 17); injured, vehicle treated (n = 26); and three posttreatment groups: initial dose at 30 min (n = 18), 2 h (n = 23), or 12 h (n = 16) postinjury. Three criteria were used to grade functional recovery. In study A, DHEF improved beam walk performance both with pretreatment (79%) and 30-min posttreatment group (54%; p < 0.01, Dunnett vs. injured vehicle). In study B, the 12-h posttreatment group showed a 97% improvement in beam walk performance (p < 0.01, Dunnett). The 30-min and 12-h posttreatment groups showed a decreased incidence of falls from the beam, which reached statistical significance (p < 0.05, Dunnett). Tests of memory (Morris water maze) and neurological reflexes both revealed significant improvements in all DHEF treatment groups. In cultured rat mesangial cells, DHEF (and DHEA) potently inhibited interleukin-1beta-induced cyclooxygenase-2 (COX2) mRNA and prostaglandin (PGE2) production. In contrast, DHEF treatment did not alter injury-induced COX2 mRNA levels in the cortex or hippocampus. However, DHEF (and DHEA) relaxed ex vivo bovine middle cerebral artery preparations by about 30%, with an IC(50) approximately 40 microM. This was a direct effect on the vascular smooth muscle, independent of the endothelial cell layer. Fluasterone (DHEF) treatments improved functional recovery in a rat TBI model. Possible mechanisms of action for this novel DHEA analog are discussed. These findings suggest an exciting potential use for this agent in the clinical treatment of traumatic brain injury.

In Vivo Effects of Insulin and Free Fatty Acids on Matrix Metalloproteinases in Rat Aorta

OBJECTIVE—Obesity is associated with insulin resistance, hyperinsulinemia, elevated plasma free fatty acid (FFA), and increased risk for atherosclerotic vascular disease (ASVD). A part of this increased risk may be due to enhanced activation of matrix metalloproteinases (MMPs). Here, we have examined the effects of physiologically elevated levels of insulin and FFA on three MMPs and their inhibitors (tissue inhibitors of MMP [TIMPs]) in aortic tissue of male rats during euglycemic-hyperinsulinemic clamping. RESEARCH DESIGN AND METHODS—Four-hour euglycemic-hyperinsulinemic clamps with infusion of saline/glycerol, lipid/heparin, or insulin with or without lipid/heparin were performed in alert unrestrained male rats. RESULTS—Hyperinsulinemia increased MMP-2 (∼6-fold), MMP-9 (∼13-fold), membrane type 1-MMP (MT1-MMP; ∼8-fold) (all Western blots), and gelatinolytic activity (zymography) of MMP-2 (2-fold), while not affecting TIMP-1 and TIMP-2. Insulin increased IRS-1–associated PI 3-kinase (PI3K), extracellular signal–regulated kinases 1/2 (ERK1/2), and c-jun NH2-terminal kinase (JNK) (by Western blots with phospho-specific antibodies). FFA augmented the insulin-mediated increases in MMP-2 (from ∼6- to ∼11-fold), MMP-9 (from ∼3- to ∼23-fold), MT1-MMP (from ∼8- to ∼20-fold), MMP-2 gelatinolytic activity (from 2- to 3-fold), and JNK and p38 mitogen-activated protein kinase (MAPK) activities but decreased insulin-mediated activation of PI3K and ERK1/2. Raising FFA without raising insulin affected neither MMPs nor TIMPs. CONCLUSIONS—FFA augmented insulin stimulation of the MMP/TIMP balance of three proatherogenic MMPs and increased activities of two MAPKs (JNK and p38 MAPK), both of which are known to stimulate the production of proinflammatory cytokines. This may, over time, increase degradation of extracellular matrix and together with inflammatory changes promote development of ASVD.

Antihyperglycemic Effect of Dehydroepiandrosterone Analogue 16α-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, 16α-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 16α-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 16α-fluoro-5-androsten-17-one had no apparent effect on the weight of the seminal vesicle glands.

Suppression of 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia and inflammation by the dehydroepiandrosterone analog 16α-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.

Analysis: Potential Therapeutic Use of Dehydroepiandrosterone and Structural Analogs

221 ALTHOUGH DEHYDROEPIANDROSTERONE (DHEA) was first isolated from human urine by Butenandt and Tscherining in 1934, the biological significance of this steroid remains a mystery.1 In the first years of life, the human adrenal produces little DHEA until around age 8 (adrenarche), at which time the adrenal undergoes morphologic and functional changes giving rise to a prominent zona reticularis, the site of biosynthesis of DHEA and DHEA sulfate.2 The plasma levels of DHEA and DHEA sulfate increase quite rapidly from adrenarche, reaching their highest levels in the second decade, and thereafter decline to about one quarter of their peak level in old age, apparently as a result of a decline in the number of functional reticular cells.2,3 Cortisol plasma levels, on the contrary, progressively increase with age.4 Although not an androgen per se, DHEA is metabolized into testosterone and is weakly androgenic.5,6 However, over the past years, many investigators have demonstrated that DHEA treatment of laboratory animals produces various biologic effects, unrelated to androgenicity, that are potentially of great therapeutic importance. In numerous studies, DHEA treatment has produced cancer preventive,7 antiatherosclerotic,8 antiinflammatory,9,10 immunomodulating,11,12 as well as antiobesity13 and antidiabetic14 effects. The paper of Byrne and Bradlow15 is important since it demonstrates that DHEA treatment not only reduces plasma glucose levels in a rat model of type 2 diabetes but also attenuates the associated hypertriglyceridemia, which is an independent risk factor for coronary heart disease in type 2 diabetes.16 Interestingly, triglyceride lowering appears to be more sensitive to DHEA treatment than the reduction in plasma glucose levels. The authors took care to administer DHEAPC by osmotic infusion pump, instead of in the food, and thus avoided the reduced food consumption and weight gain seen in earlier studies with the Zucker rat.17 Coleman et al. found that dietary administration of DHEA to C57 BL/KsJ-db/db mice markedly reduced plasma glucose levels without affecting food consumption or weight,18 a finding that we subsequently confirmed.19 There is no evidence that DHEA is a ligand for a receptor belonging to the superfamily of steroid/thyroid hormone receptors, which act as ligand-activated transcription factors, and the mechanism by which DHEA exerts its biologic actions is largely unknown. The DHEA steroids, however, are potent uncompetitive inhibitors of mammalian glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme in the pentose phosphate pathway.20,21 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 NADPH and the cellular redox state. In-