Abdalla M. El-Mowafy

Endothelium-independent effect of estrogen on Ca2+-activated K+ channels in human coronary artery smooth muscle cells

OBJECTIVE Postmenopausal estrogen replacement therapy lowers the incidence of cardiovascular disease, suggesting that estrogens support cardiovascular function. Estrogens dilate coronary arteries; however, little is known about the molecular basis of how estrogen affects the human coronary circulation. The cellular/molecular effects of estrogen action on human coronary smooth muscle were investigated in the present study. METHODS Patch-clamp and fluorescent microscopy studies were performed on human coronary myocytes in the absence of endothelium. RESULTS Estrogen increased whole-cell currents over a range of membrane potentials, and further studies indicated that the large-conductance (186.5 +/- 3 pS), calcium- and voltage-activated potassium (BK(Ca)) channel was the target of estrogen action. Channel activity was stimulated approximately 15-fold by nanomolar concentrations of 17 beta-estradiol, and this stimulation was reversed >90% by inhibiting cGMP-dependent protein kinase activity with 300 nM KT5823. 17 beta-Estradiol increased the level of cGMP and nitric oxide in human myocytes, and the stimulatory effect of estrogen on channel activity and NO production was reversed by inhibiting NO synthase with 10 microM N(G)-monomethyl-L-arginine. CONCLUSIONS Our cellular and molecular studies identify the BK(Ca) channel as a target of estrogen action in human coronary artery smooth muscle. This response to estrogen involves cGMP-dependent phosphorylation of the BK(Ca) channel or a closely associated regulatory molecule, and further evidence suggests involvement of the NO/cGMP signaling system in coronary smooth muscle. These findings are the first to provide direct evidence for a molecular mechanism that can account for endothelium-independent effects of estrogen on human arteries, and may also help explain why estrogens reduce myocardial ischemia and stimulate coronary blood flow in patients with diseased coronary arteries.

Recognition of resveratrol by the human estrogen receptor-alpha: a molecular modeling approach to understand its biological actions.

Objectives: Resveratrol (RSVL) is an edible phytoestrogen with multifaceted health benefits that may originate from binding to the estrogen receptors. Despite its structural similarity to the estrogen receptor-α (ERα) agonist diethylstilbestrol (DES), RSVL showed distinct biological profiles in estrogen-responsive biological systems. The molecular basis of such biological profiles has been undefined. Methods: We considered possible orientations for RSVL in ERα binding pocket. These conformations have been analyzed based on: (i) alignment with the key pharmacophoric elements of DES; (ii) computational energy of interaction, and (iii) pattern of accommodation at the ERα binding pocket. The characteristics of the most favored RSVL orientation have been compared with those of DES. Results: Both RSVL and DES interacted with the catalytic amino acid triad of the ERα pocket (His524, Arg394 and Glu 353). However, unlike the Erα agonists DES and estradiol (E2), RSVL formed three additional hydrogen bonds with Gly521 and Leu525, two paramount ligand recognition residues, and with Met343 at the ERα binding cavity. Lastly, RSVL displayed a more favorable energy of interaction with the ERα binding cavity. Conclusions: The present study suggests, for the first time, that RSVL is well recognized by the human ERα but in a manner distinct from the pure agonists DES and E2. These variations may well entail the unique biological responses of RSVL in ER-responsive systems.

Synthesis, spectroscopic characterization, stability assessment and DNA-binding of new 2,6-piperidinedione derivatives

This work reports on structural characterization of new antineoplaston (ANP) representatives, namely 3-(benzoylamino)-2,6-piperidinedione (BPD), 3-(4-methoxybenzoylamino)-2,6-piperidinedione (MPD) and 3-(p-nitrobenzoylamino)-2,6-piperidinedione (NPD). These compounds were prepared by reacting N-(4-substituted benzoyl)-glutamines with N-hydroxysuccinimide to afford the corresponding esters, which were heated to produce the corresponding 2,6-piperidinedione (PD) compounds. Non-destructive analytical procedures such as 1H NMR and NIR analyses confirmed the postulated chemical structures of these PD compounds. HPLC chromatograms at an ambient temperature or from solutions preheated at 30, 40 or 60 degrees C displayed only a single peak for each compound. Combination of heat with pH modification had virtually no effect on the obtained peaks, thus attesting to the stability and purity of these compounds. MS analysis displayed molecular mass ions indicative of BPD, MPD and NPD at m/z 233.4, 263.2 and 278.3, respectively. The fragmentation patterns using MS/MS analyses conformed to the structural and molecular formulae of the prepared compounds. Furthermore, preliminary biological assessments showed the capacity of these compounds to bind to the DNA. NPD, but not BMP or MPD, had a superior affinity to the DNA than the prototype ANP-A10.

Progesterone inhibition of MDM2 p90 protein in MCF-7 human breast cancer cell line is dependent on p53 levels

The mdm2 gene encodes several protein isoforms with different molecular weights (p90, p80, p76 and p57). MDM2 p90 (usually considered to be the major MDM2 protein) binds to and inactivates P53. We have recently shown that growth inhibition of MCF-7 human breast cancer cells by progesterone is associated with P53 down-regulation. In this work, we analyzed the expression pattern of MDM2 proteins in three human breast cancer cell lines by western blotting with anti-MDM2 antibodies. We found a prominent expression of MDM2 p57 protein in cell lines which have non-functional P53 protein (T47D and MDA-MB-231) as compared to the p90, p80 isoforms, whereas p90 was the major protein isoform in MCF-7 cells that contain functional P53 protein. When MCF-7 cells were treated with 100 nM of progesterone, MDM2 p90 was inhibited but the highly expressed MDM2 p57 isoform was not. The inhibition of MDM2 p90 protein by progesterone was abrogated in MCF-7 cells transfected with a P53 expressing vector. To our knowledge, this is the first report linking progesterone-induced growth inhibition with down-regulation of the MDM2 protein. We present evidence that reestablishing of P53 expression by transient transfection of P53 cDNA in these cells enhances the expression level of MDM2 p90 isoform. The data indicate that expression of MDM2 p90 is regulated through a P53-dependent pathway in response to progesterone.