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William L Alworth

Publications and source records attributed to William L Alworth.

9 recordsLinked to original sources
PubMed2004

Eugenol causes melanoma growth suppression through inhibition of E2F1 transcriptional activity.

Metastatic malignant melanoma is an extremely aggressive cancer, with no currently viable therapy. 4-Allyl-2-methoxyphenol (eugenol) was tested for its ability to inhibit proliferation of melanoma cells. Eugenol but not its isomer, isoeugenol (2-methoxy-4-propenylphenol), was found to be a potent inhibitor of melanoma cell proliferation. In a B16 xenograft study, eugenol treatment produced a significant tumor growth delay (p = 0.0057), an almost 40% decrease in tumor size, and a 19% increase in the median time to end point. More significantly, 50% of the animals in the control group died from metastatic growth, whereas none in the treatment group showed any signs of invasion or metastasis. Eugenol was well tolerated as determined by measurement of bodyweights. Examination of the mechanism of the antiproliferative action of eugenol in the human malignant melanoma cell line, WM1205Lu, showed that it arrests cells in the S phase of the cell cycle. Flow cytometry coupled with biochemical analyses demonstrated that eugenol induced apoptosis. cDNA array analysis showed that eugenol caused deregulation of the E2F family of transcription factors. Transient transfection assays and electrophoretic mobility shift assays showed that eugenol inhibits the transcriptional activity of E2F1. Overexpression of E2F1 restored about 75% of proliferation ability in cultures. These results indicate that deregulation of E2F1 may be a key factor in eugenol-mediated melanoma growth inhibition both in vitro and in vivo. Since the E2F transcription factors provide growth impetus for the continuous proliferation of melanoma cells, these results suggest that eugenol could be developed as an E2F-targeted agent for melanoma treatment.

Animals↗
PubMed2004

Binding of MCF-7 cell mitochondrial proteins and recombinant human estrogen receptors alpha and beta to human mitochondrial DNA estrogen response elements.

Our previous studies have shown that 17beta 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 ERalpha and ERbeta, 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 ERalpha (rhERalpha), and rhERbeta 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 ERbeta-deficient cells was almost undetectable. Both rhERalpha and rhERbeta bound to the mtEREs and their binding was altered by their respective antibodies. However, the ERalpha 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 ERbeta antibody. These results suggest that the mtERE-bound mitochondrial protein is ERbeta. 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.

Antibodies↗
PubMed2004

Novel reversible inactivation of cytochrome P450 2E1 T303A by tert-butyl acetylene: the role of threonine 303 in proton delivery to the active site of cytochrome P450 2E1.

This report investigates and characterizes the mechanism for the novel reversible inactivation of a T303A mutant of rabbit cytochrome P450 (P450) 2E1 by tert-butyl acetylene (tBA). P450 2E1 T303A was inactivated in a time-, concentration-, and NADPH-dependent manner through the formation of two tBA adducts to the P450 heme. Interestingly, losses in enzymatic activity and in the reduced CO spectrum of the tBA-inactivated T303A mutant could be restored to the samples after an overnight incubation at 4 degrees C. Removal of free tBA and NADPH from the tBA-inactivated T303A samples by spin column gel filtration demonstrated that the observed reversibility was time-dependent and was not significantly affected by the presence or absence of NADPH or tBA. Furthermore, the recovery of native heme was dependent on the native P450 enzyme structure. Electrospray ionization liquid chromatography-tandem mass spectrometry analysis under nondenaturing conditions of a preacidified tBA-inactivated T303A sample yielded two tBA adducts (m/z of 661 Da) with ion fragmentation patterns characteristic of a tBA adduct to the P450 heme. These adducts were absent in nonacidified samples subjected to the same conditions. In contrast, tandem mass spectrometry analysis of both non- and preacidified tBA-inactivated wild-type 2E1 samples yielded two tBA adducts (m/z of 661 Da) with ion fragmentation patterns similar to the preacidified T303A mutant adducts. These results lend insight into the reversible inactivation mechanism of the tBA-inactivated T303A mutant and suggest a role for the highly conserved threonine 303 residue in proton donation to the P450 2E1 active site and the stabilization of a reactive intermediate during substrate metabolism by P450.

Acetylene↗
PubMed2003

Differential expression of CYP102 in Bacillus megaterium by 17-beta-estradiol and 4-sec-butylphenol.

Previously we have reported the induction of CYP102 in Bacillus megaterium by 17beta-estradiol (E2) and 4-sec-butylphenol (4-sBP). Electrophoretic mobility shift assay analyses demonstrated that E2 and 4-sBP both cause a dose-dependent disassociation of the Bm3R1 repressor protein from its binding site on the operator sequence of the CYP102 gene. Equimolar combinations of E2 and 4-sBP demonstrated additive induction of CYP102 compared to equivalent samples of E2 and 4-sBP added alone. Two gene constructs were used in this investigation. One construct designated BMC143 contained the entire regulatory region of CYP102. The other gene construct, designated BMA45, had the "Barbie box" sequence deleted. While the induction of CYP102 by 4-sBP was much higher in the BMC 143 construct, E2 induced CYP102 in both constructs to the same extent. This difference in induction of CYP102 by these two inducers indicates that they act at different sites, either on the Bm3R1 repressor protein or on positive regulatory sites, or that they act, in part, through different mechanisms.

Bacillus megaterium↗
PubMed2002

Mechanism-based inactivation of cytochromes P450 2E1 and 2E1 T303A by tert-butyl acetylenes: characterization of reactive intermediate adducts to the heme and apoprotein.

The kinetics for the inactivation of cytochrome P450 2E1 and the mutant P450 2E1 T303A by tert-butyl acetylene (tBA) and tert-butyl 1-methyl-2-propynyl ether (tBMP) were investigated. The two acetylenes inactivated the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity of purified rabbit P450s 2E1 and 2E1 T303A in a reconstituted system in a time-, concentration-, and NADPH-dependent manner. The K(I) values for the inactivation of P450s 2E1 and 2E1 T303A by tBA were 1.0 and 2.0 mM, the k(inact) values were 0.20 and 0.38 min(-)(1), and the t(1/2) values were 3.5 and 1.8 min, respectively. The K(I) values for the tBMP-inactivated P450s were 0.1 and 1.0 mM, the k(inact) values were 0.12 and 0.07 min(-)(1), and the t(1/)(2) values were 5.9 and 10.2 min, respectively. Losses in enzyme activity occurred with concurrent losses in the P450 CO spectrum and P450 heme, which were accompanied by the appearance of two different tBA- or tBMP-modified heme products in each inactivated sample. LC-MS analysis of the adducts showed masses of 661 or 705 Da, consistent with the mass of an iron-depleted heme plus the masses of a tBA or tBMP reactive intermediate and one oxygen atom, respectively. Only the tBA-inactivated P450 2E1 revealed a tBA-adducted apoprotein with an increase in mass of 99 Da, corresponding to the mass of tBA plus one oxygen atom. Surprisingly, the inactivation, CO spectral and heme loss, and heme adduct formation of the tBA-inactivated T303A mutant were completely reversible after dialysis. In addition, metabolism of para-nitrophenol was not compromised by the tBA-inactivated T303A mutant. Therefore, our studies on the inactivation of P450s 2E1 and 2E1 T303A by tBA and tBMP suggest the existence of three distinct mechanisms for inactivation, among which includes a novel, reversible heme alkylation that has not been previously described with P450 enzymes.

Acetylene↗
PubMed2002

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 beta-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.

Benzoates↗
PubMed2002

Effect of 17-alpha-ethynylestradiol on activities of cytochrome P450 2B (P450 2B) enzymes: characterization of inactivation of P450s 2B1 and 2B6 and identification of metabolites.

17-alpha-Ethynylestradiol (17EE) inactivated purified, reconstituted rat hepatic cytochrome P450 (P450) 2B1 and human P450 2B6 in a mechanism-based manner. Little or no inactivation was observed when P450s 2B2 or 2B4 were incubated with 17EE. The inactivation of P450s 2B1 and 2B6 was entirely dependent on both NADPH and 17EE and followed pseudo-first order kinetics. The maximal rate constants for the inactivation of P450s 2B1 and 2B6 at 30 degrees C were 0.2 and 0.03 min(-1), respectively. For P450s 2B1 and 2B6 the apparent K(I) was 11 and 0.8 microM, respectively. Incubation of P450 2B1 with 17EE and NADPH for 20 min resulted in a 75% loss in enzymatic activity and a concurrent 20 to 25% loss of the enzyme's ability to form a reduced CO complex. With P450 2B6, an 83% loss in enzymatic activity and a 5 to 10% loss in the CO reduced spectrum were observed. The extrapolated partition ratios for 17EE with P450 2B1 and 2B6 were 21 and 13, respectively. Simultaneous incubation of an alternate substrate together with 17EE protected both enzymes from inactivation. A 1.3:1 stoichiometry of labeling for binding of the radiolabeled 17EE to P450 2B1 and 2B6 was seen. These results indicate that 17EE inactivates P450s 2B1 and 2B6 in a mechanism-based manner, primarily by the binding of a reactive intermediate of 17EE to the apoprotein. Analysis of the 17EE metabolites showed that 2B enzymes that become inactivated differ primarily by their ability to generate two metabolites that were not produced by P450s 2B2 or 2B4.

Animals↗
PubMedDate not supplied

4-Hydroxy-3-methoxybenzoic acid methyl ester: a curcumin derivative targets Akt/NF kappa B 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.

Animals↗