William L. Alworth

Binding of MCF-7 cell mitochondrial proteins and recombinant human estrogen receptors α and β to human mitochondrial DNA estrogen response elements

Our previous studies have shown that 17β estradiol (E2) enhances the transcript levels of mitochondrial DNA (mtDNA)‐encoded genes and mitochondrial respiratory chain (MRC) activity via estrogen receptors (ER). Others have reported the presence of putative estrogen responsive elements (ERE) in human mtDNA (mtEREs) and detection of ERs in mitochondria of rat uterine and ovary cells. Recently, we demonstrated the E2‐enhanced mitochondrial localization of ERα and ERβ, and E2‐induced mtDNA transcript levels in MCF‐7 cells. In this study, we applied electrophoresis mobility shift assays (EMSAs) and surface plasmon resonance (SPR) to determine if mitochondrial extracts, recombinant human ERα (rhERα), and rhERβ interact with mtEREs. Using EMSAs, we observed that ER‐containing mitochondrial extracts bound to mtEREs and the binding was enhanced by E2, whereas the binding of mitochondrial proteins from ERβ‐deficient cells was almost undetectable. Both rhERα and rhERβ bound to the mtEREs and their binding was altered by their respective antibodies. However, the ERα antibodies did not alter the binding of MCF‐7 cell mitochondrial extracts to mtEREs whereas the binding MCF‐7 and MDA‐MB‐231 cell mitochondrial extracts to mtEREs was reduced by ERβ antibody. These results suggest that the mtERE‐bound mitochondrial protein is ERβ. Using SPR, we observed the binding of both ERs to mtEREs and that the binding was increased by E2. These results indicate that the mitochondrial ERs can interact with mtEREs and suggest that they may be directly involved in E2 induction of mtDNA transcription.

4-Hydroxy-3-Methoxybenzoic Acid Methyl Ester: A Curcumin Derivative Targets Akt/NFκB Cell Survival Signaling Pathway: Potential for Prostate Cancer Management

Transcription factor NFkappaB and the serine/threonine kinase Akt play critical roles in mammalian cell survival signaling and have been shown to be activated in various malignancies including prostate cancer (PCA). We have developed an analogue of curcumin called 4-hydroxy-3-methoxybenzoic acid methyl ester (HMBME) that targets the Akt/NFkappaB signaling pathway. Here, we demonstrate the ability of this novel compound to inhibit the proliferation of human and mouse PCA cells. HMBME-induced apoptosis in these cells was tested by using multiple biochemical approaches, in addition to morphologic analysis. Overexpression of constitutively active Akt reversed the HMBME-induced growth inhibition and apoptosis, illustrating the direct role of Akt signaling in HMBME-mediated growth inhibition and apoptosis. Further, investigation of the molecular events associated with its action in LNCaP cells shows that: 1) HMBME reduces the level of activated form of Akt (phosphorylated Akt); and 2) inhibits the Akt kinase activity. Further, the transcriptional activity of NFkappaB, the DNA-binding activity of NFkappaB, and levels of p65 were all significantly reduced following treatment with HMBME. Overexpression of constitutively active Akt, but not the kinase dead mutant of Akt, activated the basal NFkappaB transcriptional activity. HMBME treatment had no influence on this constitutively active Akt-augmented NFkappaB transcriptional activity. These data indicate that HMBME-mediated inhibition of Akt kinase activity may have a potential in suppressing/decreasing the activity of major survival/antiapoptotic pathways. The potential use of HMBME as an agent that targets survival mechanisms in PCA cells is discussed.

Biosynthesis of N-acetyl dopamine by the American cockroach

Abstract Tyrosine is transaminated to para-hydroxyphenylpyruvic acid, decarboxylated to para-hydroxyphenethylamine (tyramine), and hydroxylated to dihydroxyphenylalanine (dopa) by crude homogenates of freshly ecdysed American cockroaches. Dopa is further decarboxylated to dihydroxyphenethylamine (dopamine) which is subsequently acetylated to N-acetyldopamine. Tyramine is acetylated to N-acetyltyramine, and these two N-acetyl derivatives are postulated to be the precursors of the sclerotization quinones. The addition of purified bursicon preparations to each of the reaction mixtures fails to accelerate or inhibit. This suggests that cockroach bursicon does not exert a direct effect on the synthesis of the two N-acetyl derivatives.

Suicide inhibitors of cytochrome P450 1A1 and P450 2B1

The inhibition of the P450 1A1 dependent de-ethylation of 7-ethoxyphenoxazone (7EPO) and the P450 2B1 dependent de-pentylation of 7-pentoxyphenoxazone (7PPO) by 1-ethynylnaphthalene (1EN), 2-ethynylnaphthalene (2EN), 1-ethynylanthracene (1EA), 2-ethynylanthracene (2EA), 9-ethynylanthracene (9EA), 2-ethynylphenathrene (2EPh), 3-ethynylphenanthrene (3EPh), 9-ethynylphenanthrene (9EPh), 1-ethynylpyrene (1EP) and 2-ethynylpyrene (2EP) was studied in hepatic microsomal preparations from rats. Although all of the polycyclic aromatic acetylenes studied inhibited the dealkylation of 7EPO or 7PPO, only some of the acetylenes produced a mechanism-based irreversible inactivation (suicide inhibition) of the P450 dependent dealkylation of 7EPO or 7PPO. Of the molecules tested, only 1EP, 1EN, 2EN, 2EPh and 3EPh were effective suicide inhibitors of the P450 1A1 dependent de-ethylation of 7EPO and only 1EN, 2EN, 1EA and 9EPh were effective suicide inhibitors of the P450 2B1 dependent de-pentylation of 7PPO. In addition to the size and shape of the polycyclic aromatic ring system, placement of the carbon--carbon triple bond on the ring system was critical for suicide inhibition. In contrast to 1EP, 2EP was not a mechanism-based inhibitor of P450 1A1; 9EPh, but not 2EPh or 3EPh, was a suicide inhibitor of P450 2B1. None of the aryl acetylenes tested produced heme destruction under assay conditions that produced the suicide inhibition of the P450 dependent 7EPO or 7PPO dealkylation activities. Because a precise orientation of the terminal acetylene is required to produce suicide inhibition without heme destruction, acetylenic suicide inhibitors can potentially be used to differentiate between P450 isozymes and to establish some distinguishing geometric features of the active site of these isozymes.

Combination of 2-methoxyestradiol (2-ME2) and eugenol for apoptosis induction synergistically in androgen independent prostate cancer cells

Lack of effective treatment options for the management of hormone refractory prostate cancer (PCA) reinforce the great need to develop novel compounds that act singly or in combination. 2-Methoxyestradiol (2-ME(2)) is an endogenous estrogenic metabolite that has been reported to work as an antiproliferative agent in various tumor models including prostate. Recently conducted clinical trial in hormone refractory prostate cancer (HRPC) patients concluded that 2-ME(2) was safe and well tolerated. However this study identified bioavailability of 2-ME(2) as a limiting factor. Here we report the ability of a combination of 2-ME(2) and eugenol (4-allyl-2-methoxyphenol) as an approach for enhancing anticancerous activities in prostate cancer cells. Combining 2-ME(2) with eugenol (i) inhibited growth of prostate cancer cells and induced apoptosis at lower concentrations than either single agent alone; (ii) analysis of the data using combination index (CI) showed CI values of 0.4 indicating strong synergistic interaction; (iii) increased population of cells G(2)/M phase by 4.5-fold (p=0.01); (iv) significantly reduced expression of antiapoptotic protein Bcl-2 and enhanced expression of proapoptotic protein Bax. Combination induced apoptosis was not affected in PC-3 cells that over-express or lack Bcl-2 but was associated with loss of mitochondrial membrane potential. Since 2-ME(2) was well tolerated in phase II trail in patients with HRPC; and eugenol is consumed by humans in the form of spices, the combination of 2-ME(2) with eugenol may offer a new clinically relevant treatment regimen. Combining these agents may allow ameliorating any adverse effects of either 2-ME(2) or eugenol alone by reducing their individual concentrations should these two agents be developed for human use.

Chemical Inactivation of the Cinnamate 4-Hydroxylase Allows for the Accumulation of Salicylic Acid in Elicited Cells

The cinnamate (CA) 4-hydroxylase (C4H) is a cytochrome P450 that catalyzes the second step of the main phenylpropanoid pathway, leading to the synthesis of lignin, pigments, and many defense molecules. Salicylic acid (SA) is an essential trigger of plant disease resistance. Some plant species can synthesize SA from CA by a mechanism not yet understood. A set of specific inhibitors of the C4H, including competitive, tight-binding, mechanism-based irreversible, and quasi-irreversible inhibitors have been developed with the main objective to redirect cinnamic acid to the synthesis of SA. Competitive inhibitors such as 2-hydroxy-1-naphthoic acid and the heme-coordinating compound 3-(4-pyridyl)-acrylic acid allowed strong inhibition of C4H activity in a tobacco (Nicotiana tabacum cv Bright Yellow [BY]) cell suspension culture. This inhibition was however rapidly relieved either because of substrate accumulation or because of inhibitor metabolism. Substrate analogs bearing a methylenedioxo function such as piperonylic acid (PIP) or a terminal acetylene such as 4-propynyloxybenzoic acid (4PB), 3-propynyloxybenzoic acid, and 4-propynyloxymethylbenzoic acid are potent mechanism-based inactivators of the C4H. PIP and 4PB, the best inactivators in vitro, were also efficient inhibitors of the enzyme in BY cells. Inhibition was not reversed 46 h after cell treatment. Cotreatment of BY cells with the fungal elicitor β-megaspermin and PIP or 4PB led to a dramatic increase in SA accumulation. PIP and 4PB do not trigger SA accumulation in nonelicited cells in which the SA biosynthetic pathway is not activated. Mechanism-based C4H inactivators, thus, are promising tools for the elucidation of the CA-derived SA biosynthetic pathway and for the potentiation of plant defense.

Effects of 1-ethynylpyrene and related inhibitors of P450 isozymes upon benzo[a]pyrene metabolism by liver microsomes.

The effects of three aryl acetylenes, 1-ethynylpyrene (EP), 2-ethynylnaphthalene (EN) and 3-ethynylperylene (EPE), upon the metabolism of benzo[a]pyrene (BaP) by microsomes isolated from rat liver were investigated. These aryl acetylenes all inhibited the total metabolism of BaP. Formation of BaP 7,8-dihydrodiol and BaP tetrol products by microsomal preparations from rats that had been pretreated with 3-methylcholanthrene (3MC) were preferentially inhibited. The effects of EP upon the metabolism of BaP 7,8-dihydrodiol by microsomes from rat liver were also studied. This aryl acetylene strongly inhibited the formation of BaP tetrols from BaP 7,8-dihydrodiol by liver microsomes both from untreated rats and from rats pretreated with 3MC, but enhanced the conversion of the BaP dihydrodiol into other metabolites.

Mineralization of benzo[a]pyrene by Marasmiellus troyanus, a mushroom isolated from a toxic waste site

Mycelia from the mushroom Marasmiellus troyanus were grown in the presence of radiolabeled benzo[a]pyrene in liquid culture. After 15 days, 8.1% of the label from M. troyanus cultures was recovered in CO2 as compared to 1.1% for Phanerochaete chrysosporium and 0.2% for Aspergillus niger. M. troyanus efficiently transformed B[a]P into water soluble metabolites with 64% of the label recovered in the water soluble fraction as compared to 11.7% for P. chrysosporium and 4.1% for A. niger. Glucuronic acid and sulfate conjugates of B[a]P were identified from the aqueous fraction of cultures of M. troyanus, after 17 days.

Mechanism-based inhibition of mouse P4502b-10 by selected arylalkynes

Suicide inhibitors of cytochrome P450 families are excellent tools to predict which isoforms mediate the metabolism/activation of a variety of chemical agents. We compared the inhibitory effects of several arylalkynes on mouse cytochromes P450 with published data for the rat model. The inhibition of P4502b specific dealkylation of benzyloxyresorufin by 2-ethynylnaphthalene (2-EN), 5-phenyl-1-pentyne (PPY), 4-phenyl-1-butyne (PBY), and 9-ethynylphenanthrene (9-EPh) was measured in hepatic microsomes from male mice treated with 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP) to induce cytochrome P4502b. Pulmonary microsomes were prepared from untreated mice. 9-EPh, 2-EN, and PPY caused a time-, concentration-, and NADPH-dependent loss in P4502b activity in both tissues. PBY, however, demonstrated this type of inhibition only in liver microsomes. The IC50 was calculated for both liver and lung microsomes and compared with published Ki (concentration required for half-maximal inhibition) or KI (concentration required for half-maximal inactivation) values for the rat. PPY, PBY, and 9-EPh were equally effective inhibitors of mouse P4502b and rat P4502B1. 2-EN was a 5- to 10-fold less potent inhibitor of mouse P4502b, as compared with the rat, even though it was shown to bind to the active site of the mouse isoform as demonstrated by its metabolism to 2-naphthylacetic acid. These data suggest that the active site of the mouse P4502b enzyme is functionally similar to the rat P4502B isoform, with the exception of the disparity in its susceptibility to inactivation by 2-EN as measured by the Ki values.

4-Methylcatechol-induced oxidative stress induces intrinsic apoptotic pathway in metastatic melanoma cells

There has been a steady rise in fatalities associated with thick melanomas (>4mm). Although understanding of the biology of the disease has improved, effective treatment strategies for patients with advanced metastatic melanoma remain elusive. Therefore, more intensive testing of agents with therapeutic potential are needed to improve survival of patients with metastatic malignant melanoma. We have tested the ability of 4-methylcatechol, a metabolite of quercetin; a naturally occurring compound that is commonly found in a variety of fruits for its potential as an anti-melanoma agent. Our results show that 4-methylcatechol inhibits proliferation of melanoma cells in culture while not affecting the growth of normal human epidermal melanocytes. Further, the ability of metastatic melanoma cells to form colonies on soft agar was also inhibited. 4-Methylcatechol caused the accumulation of cells in G2/M phase of the cell cycle and induced apoptosis. There was an increase in reactive oxygen species following treatment with 4-methylcatechol that led to apoptosis through the intrinsic mitochondrial pathway. Treatment also inhibited cell survival mediated by Akt, a key player in melanoma cell survival. Taken together our results suggest that 4-methylcatechol exhibits cytotoxicity towards metastatic malignant melanoma cells while sparing normal melanocytes and should be tested further as a potential drug candidate for malignant melanoma.