Fred B. Lih

Testosterone and dihydrotestosterone tissue levels in recurrent prostate cancer

Purpose: Prostate cancer eventually recurs during androgen deprivation therapy despite castrate levels of serum androgens. Expression of androgen receptor and androgen receptor–regulated proteins suggests androgen receptor activation in recurrent prostate cancer. Many groups have pursued mechanisms of ligand-independent androgen receptor activation but we found high levels of testicular androgens in recurrent prostate cancer tissue using RIA. Experimental Designs: Prostate specimens from 36 men were procured preserving blood flow to prevent ischemia and cyropreserved immediately. Recurrent prostate cancer specimens from 18 men whose cancer recurred locally during androgen deprivation therapy and androgen-stimulated benign prostate specimens from 18 men receiving no hormonal treatments were studied. Tissue levels of testosterone and dihydrotestosterone were measured in each specimen using liquid chromatography/electrospray tandem mass spectrometry. Testosterone and dihydrotestosterone levels were compared with clinical variables and treatment received. Results: Testosterone levels were similar in recurrent prostate cancer (3.75 pmol/g tissue) and androgen-stimulated benign prostate (2.75 pmol/g tissue, Wilcoxon two-sided, P = 0.30). Dihydrotestosterone levels decreased 91% in recurrent prostate cancer (1.25 pmol/g tissue) compared with androgen-stimulated benign prostate (13.7 pmol/g tissue; Wilcoxon two-sided, P < 0.0001) although dihydrotestosterone levels in most specimens of recurrent prostate cancer were sufficient for androgen receptor activation. Testosterone or dihydrotestosterone levels were not related to metastatic status, antiandrogen treatment, or survival (Wilcoxon rank sum, all P > 0.2). Conclusions: Recurrent prostate cancer may develop the capacity to biosynthesize testicular androgens from adrenal androgens or cholesterol. This surprising finding suggests intracrine production of dihydrotestosterone and should be exploited for novel treatment of recurrent prostate cancer.

Epoxyeicosanoids stimulate multiorgan metastasis and tumor dormancy escape in mice

Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.

Activation of the Androgen Receptor by Intratumoral Bioconversion of Androstanediol to Dihydrotestosterone in Prostate Cancer

The androgen receptor (AR) mediates the growth of benign and malignant prostate in response to dihydrotestosterone (DHT). In patients undergoing androgen deprivation therapy for prostate cancer, AR drives prostate cancer growth despite low circulating levels of testicular androgen and normal levels of adrenal androgen. In this report, we demonstrate the extent of AR transactivation in the presence of 5α-androstane-3α,17β-diol (androstanediol) in prostate-derived cell lines parallels the bioconversion of androstanediol to DHT. AR transactivation in the presence of androstanediol in prostate cancer cell lines correlated mainly with mRNA and protein levels of 17β-hydroxysteroid dehydrogenase 6 (17β-HSD6), one of several enzymes required for the interconversion of androstanediol to DHT and the inactive metabolite androsterone. Levels of retinol dehydrogenase 5, and dehydrogenase/reductase short-chain dehydrogenase/reductase family member 9, which also convert androstanediol to DHT, were lower than 17β-HSD6 in prostate-derived cell lines and higher in the castration-recurrent human prostate cancer xenograft. Measurements of tissue androstanediol using mass spectrometry demonstrated androstanediol metabolism to DHT and androsterone. Administration of androstanediol dipropionate to castration-recurrent CWR22R tumor-bearing athymic castrated male mice produced a 28-fold increase in intratumoral DHT levels. AR transactivation in prostate cancer cells in the presence of androstanediol resulted from the cell-specific conversion of androstanediol to DHT, and androstanediol increased LAPC-4 cell growth. The ability to convert androstanediol to DHT provides a mechanism for optimal utilization of androgen precursors and catabolites for DHT synthesis.

Endothelial expression of human cytochrome P450 epoxygenases lowers blood pressure and attenuates hypertension-induced renal injury in mice

Renal cytochrome P450 (CYP)‐derived epoxyeicosatrienoic acids (EETs) regulate sodium transport and blood pressure. Although endothelial CYP‐derived EETs are potent vasodilators, their contribution to the regulation of blood pressure remains unclear. Consequently, we developed transgenic mice with endothelial expression of the human CYP2J2 and CYP2C8 epoxygenases to increase endothelial EET biosynthesis. Compared to wild‐type littermate controls, an attenuated afferent arteriole constrictor response to endothelin‐1 and enhanced dilator response to acetylcholine was observed in CYP2J2 and CYP2C8 transgenic mice. CYP2J2 and CYP2C8 transgenic mice demonstrated modestly, but not significantly, lower mean arterial pressure under basal conditions compared to wild‐type controls. However, mean arterial pressure was significantly lower in both CYP2J2 and CYP2C8 transgenic mice during coadministration of N‐nitro‐l‐arginine methyl ester and indomethacin. In a separate experiment, a high‐salt diet and subcutaneous angiotensin II was administered over 4 wk. The angiotensin/high‐salt‐induced increase in systolic blood pressure, proteinuria, and glomerular injury was significantly attenuated in CYP2J2 and CYP2C8 transgenic mice compared to wild‐type controls. Collectively, these data demonstrate that increased endothelial CYP epoxygenase expression attenuates afferent arteriolar constrictor reactivity and hypertension‐induced increases in blood pressure and renal injury in mice. We conclude that endothelial CYP epoxygenase function contributes to the regulation of blood pressure.—Lee, C. R., Imig, J. D., Edin, M. E., Foley, J., DeGraff, L. M., Bradbury, J. A., Graves, J. P., Lih, F. B., Clark, J., Myers, P., Perrow, A. L., Lepp, A. N., Kannon, M. A., Ronnekleiv, O. K., Alkayed, N.J., Falck, J. R., Tomer, K B., Zeldin, D. C. Endothelial expression of human cytochrome P450 epoxygenases lowers blood pressure and attenuates hypertension‐induced renal injury in mice. FASEB J. 24, 3770–3781 (2010). www.fasebj.org

Endothelial CYP epoxygenase overexpression and soluble epoxide hydrolase disruption attenuate acute vascular inflammatory responses in mice

Cytochrome P‐450 (CYP)‐derived epoxyei‐cosatrienoic acids (EETs) possess potent anti‐inflammatory effects in vitro. However, the effect of increased CYP‐mediated EET biosynthesis and decreased soluble epoxide hydrolase (sEH, Ephx2)‐mediated EET hydrolysis on vascular inflammation in vivo has not been rigorously investigated. Consequently, we characterized acute vascular inflammatory responses to endotoxin in transgenic mice with endothelial expression of the human CYP2J2 and CYP2C8 epoxygenases and mice with targeted disruption of Ephx2. Compared to wild‐type controls, CYP2J2 transgenic, CYP2C8 transgenic, and Ephx2−/− mice each exhibited a significant attenuation of endotoxin‐induced activation of nuclear factor (NF)‐κB signaling, cellular adhesion molecule, chemokine and cytokine expression, and neutrophil infiltration in lung in vivo. Furthermore, attenuation of endotoxin‐induced NF‐κB activation and cellular adhesion molecule and chemokine expression was observed in primary pulmonary endothelial cells isolated from CYP2J2 and CYP2C8 transgenic mice. This attenuationwas inhibited bya putative EET receptor antagonist and CYP epoxygenase inhibitor, directly implicating CYP epoxygenase‐derived EETs with the observed anti‐inflammatory phenotype. Collectively, these data demonstrate that potentiation of the CYP epoxygenase pathway by either increased endothelial EET biosynthesis or globally decreased EET hydrolysis attenuates NF‐κB‐dependent vascular inflammatory responses in vivo and may serve as a viable anti‐inflammatory therapeutic strategy.—Deng, Y., Edin, M. L., Theken, K N., Schuck, R N., Flake, G. P., Kannon, M. A., DeGraff, L. M., Lih, F. B., Foley, J., Bradbury, J. A., Graves, J. P., Tomer, K. B., Falck, J. R., Zeldin, D. C., Lee, C. R. Endothelial CYP epoxygenase overexpression and soluble epoxide hydrolase disruption attenuate acute vascular inflammatory responses in mice. FASEB J. 25, 703–713 (2011). www.fasebj.org

Epoxyeicosanoids promote organ and tissue regeneration

Epoxyeicosatrienoic acids (EETs), lipid mediators produced by cytochrome P450 epoxygenases, regulate inflammation, angiogenesis, and vascular tone. Despite pleiotropic effects on cells, the role of these epoxyeicosanoids in normal organ and tissue regeneration remains unknown. EETs are produced predominantly in the endothelium. Normal organ and tissue regeneration require an active paracrine role of the microvascular endothelium, which in turn depends on angiogenic growth factors. Thus, we hypothesize that endothelial cells stimulate organ and tissue regeneration via production of bioactive EETs. To determine whether endothelial-derived EETs affect physiologic tissue growth in vivo, we used genetic and pharmacological tools to manipulate endogenous EET levels. We show that endothelial-derived EETs play a critical role in accelerating tissue growth in vivo, including liver regeneration, kidney compensatory growth, lung compensatory growth, wound healing, corneal neovascularization, and retinal vascularization. Administration of synthetic EETs recapitulated these results, whereas lowering EET levels, either genetically or pharmacologically, delayed tissue regeneration, demonstrating that pharmacological modulation of EETs can affect normal organ and tissue growth. We also show that soluble epoxide hydrolase inhibitors, which elevate endogenous EET levels, promote liver and lung regeneration. Thus, our observations indicate a central role for EETs in organ and tissue regeneration and their contribution to tissue homeostasis.

Evaluation of cytochrome P450-derived eicosanoids in humans with stable atherosclerotic cardiovascular disease

OBJECTIVE Preclinical and genetic epidemiologic studies suggest that modulating cytochrome P450 (CYP)-mediated arachidonic acid metabolism may have therapeutic utility in the management of coronary artery disease (CAD). However, predictors of inter-individual variation in CYP-derived eicosanoid metabolites in CAD patients have not been evaluated to date. Therefore, the primary objective was to identify clinical factors that influence CYP epoxygenase, soluble epoxide hydrolase (sEH), and CYP ω-hydroxylase metabolism in patients with established CAD. METHODS Plasma levels of epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE) were quantified by HPLC-MS/MS in a population of patients with stable, angiographically confirmed CAD (N=82) and healthy volunteers from the local community (N=36). Predictors of CYP epoxygenase, sEH, and CYP ω-hydroxylase metabolic function were evaluated by regression. RESULTS Obesity was significantly associated with low plasma EET levels and 14,15-EET:14,15-DHET ratios. Age, diabetes, and cigarette smoking also were significantly associated with CYP epoxygenase and sEH metabolic activity, while only renin-angiotensin system inhibitor use was associated with CYP ω-hydroxylase metabolic activity. Compared to healthy volunteers, both obese and non-obese CAD patients had significantly higher plasma EETs (P<0.01) and epoxide:diol ratios (P<0.01), whereas no difference in 20-HETE levels was observed (P=NS). CONCLUSIONS Collectively, these findings suggest that CYP-mediated eicosanoid metabolism is dysregulated in certain subsets of CAD patients, and demonstrate that biomarkers of CYP epoxygenase and sEH, but not CYP ω-hydroxylase, metabolism are altered in stable CAD patients relative to healthy individuals. Future studies are necessary to determine the therapeutic utility of modulating these pathways in patients with CAD.

Dietary n-3 Polyunsaturated Fatty Acids Enhance Hormone Ablation Therapy in Androgen-Dependent Prostate Cancer

Hormone ablation therapy typically causes regression of prostate cancer and represents an important means of treating this disease, particularly after metastasis. However, hormone therapy inevitably loses its effectiveness as tumors become androgen-independent, and this conversion often leads to death because of subsequent poor responses to other forms of treatment. Because environmental factors, such as diet, have been strongly linked to prostate cancer, we examined the affects of dietary polyunsaturated fatty acids (PUFAs; at 1.5 wt%) on growth of androgen-dependent (CWR22) and androgen-independent (CWR22R) human prostatic cancer xenografts, the acute response of CWR22 tumors to ablation therapy, and their progression to androgen independence. Significant diet-induced changes in tumor n-3 or n-6 PUFA content had no affect on CWR22 or CWR22R tumors growing with or without androgen support, respectively. However, dietary changes that increased tumor eicosapentaenoic acid and linoleic acid content enhanced responses to ablation therapy, measured by cancer cell apoptosis and mitosis. In addition, relapse to androgen-independent growth (measured by renewed increases in tumor volume and serum prostate-specific antigen after ablation) positively correlated with tumor arachidonic acid content. There was no correlation between expression of 15-lipoxygenase isozymes or their products and tumor growth or responses to ablation. In conclusion, dietary n-3 PUFA may enhance the response of prostate cancer to ablation therapy and retard progression to androgen-independent growth by altering tumor PUFA content.

Prostaglandin E2 protects murine lungs from bleomycin-induced pulmonary fibrosis and lung dysfunction

Prostaglandin E(2) (PGE(2)) is a lipid mediator that is produced via the metabolism of arachidonic acid by cyclooxygenase enzymes. In the lung, PGE(2) acts as an anti-inflammatory factor and plays an important role in tissue repair processes. Although several studies have examined the role of PGE(2) in the pathogenesis of pulmonary fibrosis in rodents, results have generally been conflicting, and few studies have examined the therapeutic effects of PGE(2) on the accompanying lung dysfunction. In this study, an established model of pulmonary fibrosis was used in which 10-12-wk-old male C57BL/6 mice were administered a single dose (1.0 mg/kg) of bleomycin via oropharyngeal aspiration. To test the role of prostaglandins in this model, mice were dosed, via surgically implanted minipumps, with either vehicle, PGE(2) (1.32 μg/h), or the prostacyclin analog iloprost (0.33 μg/h) beginning 7 days before or 14 days after bleomycin administration. Endpoints assessed at 7 days after bleomycin administration included proinflammatory cytokine levels and measurement of cellular infiltration into the lung. Endpoints assessed at 21 days after bleomycin administration included lung function assessment via invasive (FlexiVent) analysis, cellular infiltration, lung collagen content, and semiquantitative histological analysis of the degree of lung fibrosis (Ashcroft method). Seven days after bleomycin administration, lymphocyte numbers and chemokine C-C motif ligand 2 expression were significantly lower in PGE(2)- and iloprost-treated animals compared with vehicle-treated controls (P < 0.05). When administered 7 days before bleomycin challenge, PGE(2) also protected against the decline in lung static compliance, lung fibrosis, and collagen production that is associated with 3 wk of bleomycin exposure. However, PGE(2) had no therapeutic effect on these parameters when administered 14 days after bleomycin challenge. In summary, PGE(2) prevented the decline in lung static compliance and protected against lung fibrosis when it was administered before bleomycin challenge but had no therapeutic effect when administered after bleomycin challenge.

The novel structure of the cockroach allergen Bla g 1 has implications for allergenicity and exposure assessment.

BACKGROUND Sensitization to cockroach allergens is a major risk factor for asthma. The cockroach allergen Bla g 1 has multiple repeats of approximately 100 amino acids, but the fold of the protein and its biological function are unknown. OBJECTIVE We sought to determine the structure of Bla g 1, investigate the implications for allergic disease, and standardize cockroach exposure assays. METHODS nBla g 1 and recombinant constructs were compared by using ELISA with specific murine IgG and human IgE. The structure of Bla g 1 was determined by x-ray crystallography. Mass spectrometry and nuclear magnetic resonance spectroscopy were used to examine the ligand-binding properties of the allergen. RESULTS The structure of an rBla g 1 construct with comparable IgE and IgG reactivity to the natural allergen was solved by x-ray crystallography. The Bla g 1 repeat forms a novel fold with 6 helices. Two repeats encapsulate a large and nearly spherical hydrophobic cavity, defining the basic structural unit. Lipids in the cavity varied depending on the allergen origin. Palmitic, oleic, and stearic acids were associated with nBla g 1 from cockroach frass. One unit of Bla g 1 was equivalent to 104 ng of allergen. CONCLUSIONS Bla g 1 has a novel fold with a capacity to bind various lipids, which suggests a digestive function associated with nonspecific transport of lipid molecules in cockroaches. Defining the basic structural unit of Bla g 1 facilitates the standardization of assays in absolute units for the assessment of environmental allergen exposure.