Biological aromatization of delta4,6- and delta1,4,6-androgens and their 6-alkyl analogs, potent inhibitors of aromatase

Enzymic aromatization of Δ⁶- and Δ^1,6-derivatives of the natural substrate androstenedione with human placental aromatase was first studied using gas-chromatography-mass spectrometry. The two steroids were aromatized with apparent K_m and V_max values of 62 nM and 32 pmol/min/mg protein for the Δ⁶-steroid and 167 nM and 10 pmol/min/mg protein for the Δ^1,6-steroid, respectively. We next explored the aromatization of a series of 6-alkyl (methyl, ethyl, n-propyl, and n-pentyl)-substituted Δ⁶-androstenediones and their Δ^1,6-analogs, potent competitive inhibitors of aromatase, to gain insight into the relationships between the inhibitory activity of the 6-alkyl-C₁₉ steroids and their ability to serve as a substrate of aromatase. In a series of the Δ^1,6-androstenediones, all the 6-alkyl steroids were more efficient substrates than the parent Δ^1,6-steroid in which the aromatization rates of the alkyl steroids were about 2-fold that of the parent steroid, in contrast, all of the 6-alkyl-substituted Δ⁶-androstenediones were converted into the corresponding 6-alkyl-Δ⁶-estrogens with the rates of less than about a half that of the parent steroid. These results indicate that the 6-alkyl function decreases the aromatization rate of the Δ⁶-steroid but enhances that of the Δ^1,6-steroid. The relative apparent K_m values for the C₁₉ steroids obtained in this study are different from the relative K_i values obtained previously, indicating that a good inhibitor is not essentially a good substrate in the 6-alkyl-substituted Δ⁶- and Δ^1,6-androstenedione series.     

Aromatase activity and the effect of estradiol and testosterone on DNA synthesis in endometrial carcinoma cell lines

Human endometrial and breast carcinoma cell lines were examined for aromatase activity and the effects of sex steroids (estradiol and testosterone) on DNA synthesis. Aromatase activity was high (greater than 500 fmol/10⁷ cells/24 h) in the cell lines MCF-7 and OMC-2, moderate (100–499 fmol/10⁷ cells/24 h) in the cell lines HEC-59 and Ishikawa, and low (less than 100 fmol/10⁷ cells/24 h) in the HHUA cell line. A substantial stimulation of DNA synthesis by estradiol (10⁻⁹M) was observed in cell lines HEC-59, OMC-2, and MCF-7, with an increase in [³H]thymidine uptake of over 250%. The Ishikawa cell line was stimulated moderately (115–249%). No estradiol-induced increase in DNA synthesis was observed in HHUA. Responsiveness of DNA synthesis to testosterone was observed in cell lines that showed the greatest response to estradiol, namely HEC-59, OMC-2, and MCF-7. Otherwise, estrogen-responsiveness did not always correlate with a significant aromatase activity. These data suggest that some but not all endometrial carcinomas may possess an aromatase-dependent growth stimulating system.     

Steroid metabolism in the hormone dependent MCF-7 human breast carcinoma cell line and its two hormone resistant subpopulations MCF-7/LCC1 and MCF-7/LCC2

Androgen and estrogen metabolism was investigated in the hormone-dependent human breast cancer cell line MCF-7 and its two hormone-resistant sublines MCF-7/LCC1 and MCF-7/LCC2. Using the product isolation method, the activity of aromatase, 5-reductase, 3/β-hydroxysteroid oxidoreductase and 17β-hydroxysteroid oxidoreductase were investigated isolating the following steroids: estriol (E₃), estradiol (E₂), estrone (E₁), 3/β-androstanediol (A-diol), testosterone (T), dihydrotestosterone (DHT), androsterone (AND), androstenedion (4-AD) and androstanedione (A-dion). For all experiments, cells were preincubated with cortisol and subsequently incubated with [¹⁴C]T or [¹⁴C]4-AD as the substrate in medium without phenol red and with serum charcoal stripped of steroids. The results showed no aromatase activity in any of the cell lines under the experimental conditions used, and preincubation with cortisol had no effect on the enzyme activity. With [¹⁴C]T as the substrate, the metabolized level of DHT was very similar in the three cell lines, though MCF-7/LCC1 and MCF-7/LCC2 utilized the substrate to a much lesser extent. The amount of DHT and 4-AD produced were comparable in the two hormone-resistant cell lines, while the amount of 4-AD was significantly higher in MCF-7 cells. No differences in enzyme activity were found in the three cell lines when [¹⁴C]4-AD was used as the substrate. This study showed an altered androgen metabolism in the MCF-7/LCC1 and MCF-7/LCC2 sublines compared to the parent MCF-7. However, since treatment with DHT and T inhibited cell growth equally well in all three tumor cell lines, it is unlikely that the found differences in steroid metabolism was involved in the acquisition of the endocrine resistance of the two MCF-7 sublines.     

Inhibition of steroid sulphatase activity by steroidal methylthiophosphonates: Potential therapeutic agents in breast cancer

The hydrolysis of steroid sulphates, by steroid sulphatase, is an important source of oestrogenic steroids (oestrone, oestradiol and 5-androstene-3β,17β-diol) which are found in tumours. In the present study, we have examined the effect of dehydroepiandrosterone-3-O-methylthiophosphonate (DHA-3-MTP), pregnenolone-3-O-methylthiophosphonate (pregnenolone-3-MTP) and cholesterol-3-O-methylthiophosphonate (cholesterol-3-MTP) on the inhibition of oestrone sulphatase as well as DHA sulphatase activities in intact MCF-7 breast cancer cells and in placental microsomes. All three methylthiophosphonates significantly (P< 0.01) inhibited the hydrolysis of oestrone sulphate (E₁ S) in intact MCF-7 cells (31–85% inhibition at 1 μM and 53–97% inhibition at 10 μM). Significant inhibition of DHA sulphatase was also achieved. At a concentration of 50 μM, all three compounds inhibited the hydrolysis of dehydroepiandrosterone sulphate (DHAS) by > 95%. Using human placental microsomes, the K_m and V_max of E₁S were determined to be 8.1 μM and 43 nmol/h/mg protein. The corresponding K_i values for DHA-3-MTP, pregnenolone-3-MTP and cholesterol-3-MTP were found to be 4.5, 1.4 and 6.2 μM, respectively. Such inhibitors which are resistant to metabolism may have considerable potential as therapeutic agents and may have additional advantage over aromatase inhibitors in also reducing tumour concentrations of the oestrogenic steroid, 5-androstene-3β,17β-diol, by inhibiting the hydrolysis of DHAS.     

Tibolone and metabolites induce prolactin production in human endometrial stromal cells in vitro: evidence for cell-specific metabolism

In this study, we assessed the effects of tibolone and its metabolites on the production of a progesterone sensitive parameter, prolactin, in human endometrium stroma cells in vitro. In addition, the metabolism of the compounds by isolated stromal and epithelial cells was evaluated.

The reference compounds, progesterone, Org 2058, and DHT all induced prolactin production. Oestradiol also slightly induced prolactin production and enhanced the response to Org 2058. Tibolone and Δ⁴-tibolone were similar with regard to potency to induce prolactin levels in the culture supernatant. Their potency was lower than that of Org 2058, similar to that of progesterone and higher than that of DHT. The efficacies of tibolone, Δ⁴-tibolone and Org 2058 were similar (200-fold induction). The estrogenic tibolone metabolites 3- and 3β-OH tibolone also significantly stimulated prolactin production. Their potency, however, was low since significance was reached only at the highest concentrations tested.

The PR antagonist Org 31710 inhibited both tibolone- and Δ⁴-tibolone-induced prolactin production. The responses of tibolone and Δ⁴-tibolone were not affected by co-incubation with the androgen receptor antagonist OH-flutamide. The effect of tibolone, but not Δ⁴-tibolone, was antagonized approximately 50% in combination with the highest dose (1 μM) estrogen receptor antagonist, ICI 164384. The induction of prolactin by 3- and 3β-OH tibolone was antagonized most potently by Org 31710, but also by ICI 164384 and OH-flutamide.

Tibolone is metabolized differently in epithelial and stromal cells of the human endometrium. The epithelial cells mostly produce the progestagenic/androgenic Δ⁴-tibolone. The stromal cells produce predominantly the 3β-OH tibolone, and some Δ⁴-tibolone, but the net effect observed with regard to prolactin production is progestagenic. When the metabolites 3-OH, 3β-OH, and Δ⁴-tibolone were added to the cultures no conversions were observed. The HPLC analyses showed no evidence for the production of sulfated metabolites.

In conclusion, the net effects on endometrial stromal cells are predominantly progestagenic. Tibolone is converted by epithelial cells into Δ⁴-tibolone which displays progestagenic and androgenic activities, whereas in stromal cells also the estrogenic metabolites 3- and 3β-OH tibolone are formed.     

Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ dpm ³H/ng, ³H/¹⁴C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.     

Diverse function of aromatase and the N-terminal sequence deleted form

The diverse function of human placental aromatase including estradiol 6-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6,7-³H₂,4-¹⁴C]estradiol, 7-ethoxycoumarin, and [N-methyl-³H₃]cocaine. 6-Hydroxy[7-³H,4-¹⁴C]estradiol was isolated as the metabolite of estradiol and the ³H-water release method based on the 6-³H label was established. The initial rate kinetics of the 6-hydroxylation gave K_m of 4.3 μM, V_max of 4.02 nmol min⁻¹mg⁻¹, and turnover rate of 0.27 min⁻¹. Testosterone competed dose-dependently with the 6-hydroxylation and showed the K_i of 0.15 μM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed K_m of 200 μM, V_max of 12.5 nmol min⁻¹mg⁻¹ and turnover rate of 1.06 min⁻¹. The N-demethylation of cocaine was analysed by the ³H-release method, giving K_m of 670 μM, V_max of 4.76 nmol min⁻¹mg⁻¹, and turnover rate of 0.49 min⁻¹. All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 ± 83 pmol min⁻¹mg⁻¹ (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min⁻¹.     

Estradiol inhibits the estrone sulfatase activity in normal and cancerous human breast tissues

It is well accepted that estradiol (E₂) plays an important role in the genesis and evolution of breast cancer. Quantitative evaluation indicates that in human breast tumor, estrone sulfate (E₁S) ‘via sulfatase’ is a much more likely precursor for E₂ than is androstenedione ‘via aromatase’. In previous studies, it was demonstrated that in isolated MCF-7 and T-47D breast cancer cell lines, estradiol can block estrone sulfatase activity. In the present study, the effect of E₂ was explored using total normal and cancerous breast tissues. This study was carried out with post-menopausal patients with breast cancer. None of the patients had a history of endocrine, metabolic or hepatic diseases or had received treatment in the previous 2 months. Each patient received local anaesthetic (lidocaine 1%) and two regions of the mammary tissue were selected: (A) the tumoral tissue and (B) the distant zone (glandular tissue) which was considered as normal. Samples were placed in liquid nitrogen and stored at –80 °C until enzyme activity analysis. Breast cancer histotypes were ductal and post-menopausal stages were T2. Homogenates of tumoral or normal breast tissues (45–75 mg) were incubated in 20 mM Tris–HCl, pH 7.2 with physiological concentrations of [³H]-E₁S (5 × 10⁻⁹ M) alone or in the presence of E₂ (5 × 10⁻⁵ to 5 × 10⁻⁷ M) during 30 min or 3 h. E₁S, E₁ and E₂ were characterized by thin layer chromatography and quantified using the corresponding standard. The sulfatase activity is significantly more intense with the breast cancer tissue than normal tissue, since the concentration of E₁ was 3.20 ± 0.15 and 0.42 ± 0.07 pmol/mg protein, respectively after 30 min incubation. The values were 27.8 ± 1.8 and 3.5 ± 0.21 pmol/mg protein, respectively after 3 h incubation. Estradiol at the concentration of 5 × 10⁻⁷ M inhibits this conversion by 33% and 31% in cancerous and normal breast tissues, respectively and by 53% and 88% at the concentration of 5 × 10⁻⁵ M after 30 min incubation. The values were 24% and 18% for 5 × 10⁻⁷ M and 49% and 42% for 5 × 10⁻⁵ M, respectively after 3 h incubation. It was observed that [³H]-E₁S is only converted to [³H]-E₁ and not to [³H]-E₂ in normal or cancerous breast tissues, which suggests a low or no 17β-hydroxysteroid dehydrogenase (17β-HSD) Type 1 reductive activity in these experimental conditions. In conclusion, estradiol is a strong anti-sulfatase agent in cancerous and normal breast tissues. This data can open attractive perspectives in clinical trials using this hormone.     

Galanin-induced relaxation in gastric smooth muscle cells is mediated by cyclic AMP

Galanin has numerous effects on gastrointestinal motility in different species; however, its cellular basis of action in mediating these effects is unclear. Dispersed gastric smooth muscle cells have been shown to possess high-affinity galanin receptors that increase cAMP and cause relaxation. Recent studies show some smooth muscle relaxants such as VIP cause relaxation by both cAMP-dependent and -independent mechanisms. It is unknown if galanin's cellular basis of relaxation is similar or different from that of VIP. To investigate galanin's relaxant effect and compare it to VIP's effect, dispersed smooth muscle cells from guinea pig stomach were prepared by collagenase digestion. The mean length in resting cells was 110 ± 2 μm and, with carbachol treatment, contracted to 89 ± 2 μm. VIP and galanin alone had no effect on cell length, but each caused a dose-dependent inhibition of carbachol-induced contraction and both had an EC₅₀ of 3–7 nM. Galanin (1 μM) and VIP (1 μM) increased cellular cAMP from 118 ± 10 pmol/10⁶ cells in control to 212 ± 14 and 214 ± 12 pmol/10⁶ cells, respectively. The protein kinase A inhibitor, Rp-cAMPS, at 100 μM, completely inhibited the relaxant effect of an EC₅₀ concentration of galanin (3 nM), but only inhibited that by VIP by 80% (p < 0.05). Adding the nitric oxide inhibitor, -NNA ( ), at 100 μM did not alter the length of resting cells or inhibit carbachol-induced contraction. However, -NNA (100 μM) decreased VIP-induced relaxation by 45%, whereas it had no effect on galanin-induced relaxation. To determine the ability of each peptide to activate nitric oxide, the incorporation of [³H]arginine into [³H]citrulline was determined. Galanin (1 μM) did not cause nitric oxide generation whereas VIP (1 μM) increased nitric oxide generation above the control by 97 ± 14% (p < 0.01). These results demonstrated that with galanin, in contrast to VIP, nitric oxide is not involved in its ability to cause gastric smooth muscle cell relaxation. The relaxant action of galanin can be accounted for completely by its ability to activate protein kinase A and therefore resembles recent results with β-adrenergic agents.     

In vitro steroid metabolic studies in human testes II: Metabolism of cholesterol, pregnenolone, progesterone, androstenedione and testosterone by testes of an estrogen-treated man

The effect of long-term in vivo estrogen treatment on in vitro steroidogenesis by the testes of a young man was investigated. In vitro incubation of testicular tissue of this man with ³H-pregnenolone, ³H-progesterone, ³H-androstenedione and ³H-testosterone demonstrated suppression of 17-hydroxylase activity, with little or no effect of the treatment on Δ⁵-3β-hydroxysteroid oxidoreductase, 5a-reductase and aromatase. Increased 20-hydroxysteroid oxidoreductase activity was observed. Determination of intratesticular steroid concentrations led to similar conclusions.     

Estrogen synthesis in human colon cancer epithelial cells

Epidemiological and experimental data suggest an involvement of estrogen in the development and progression of colorectal cancer. In order to determine whether local synthesis of estrogen occurred in human colonic cancer cells, two colorectal cancer cell lines, HCT8 and HCT116, were evaluated for gene expression and enzyme activity of cytochrome P450 aromatase. In addition, the effect on aromatase expression of charcoal-stripped fetal calf serum, of quercetin and genistein and of tamoxifen and raloxifene was investigated in both cell lines. RT-PCR analysis revealed that colorectal adenocarcinoma cell lines contain aromatase as a major component. The conversion of [³H]-androstenedione to estrone and labeled water was dose-dependently inhibited by 4-hydroxyandrostenedione and obeyed Michaelis–Menten kinetic with apparent Km values of 20 nM and V_max values of approx. 200 and 500 fmol/mg protein/h for HCT8 and HCT116 cells, respectively. After 24 h incubation, genistein (1 μM) significantly increased aromatase activity in HCT8 cells, with no effect on HCT116 cells. In accord with previous observation in reproductive tissues, quercetin (1 μM) significantly inhibited the enzyme activity in both cell lines. Also tamoxifen (100 nM) acted as inhibitor, while raloxifene (10 nM) decreased the enzyme activity only in HCT116 cells. The aromatase gene expression modulation by these effective agents was consistent with their effects on enzyme activity. These findings demonstrate for the first time that colorectal adenocarcinoma cell lines express aromatase. Interestingly, the enzyme activity was inhibited by quercetin, one major dietary flavonoid, by tamoxifen, a hormonal therapeutic agent for breast cancer, and by raloxifene, used in the prevention of postmenopausal osteoporosis.     

The kinetics and energetics of formation of a molecular hydrogen complex involving a dinuclear ruthenium(II) centre

The reversible equilibrium conversion under H₂ of [RuCl(dppb) (μ-Cl)]₂ (1) to generate (η²-H₂) (dppb) (μ-Cl)₃RuCl(dppb) in CH₂Cl₂ (dppb = Ph₂P(CH₂)₄PPh₂) has been studied at 0–25 °C by UV-Vis and ³¹P{¹H} NMR spectroscopy, and by stoppe kinetics; the equilibrium constant and corresponding thermodynamic parameters, and the forward and reverse rate constants at 25 °C have been determined. A measured ΔH° value of 0 kJ mol⁻¹ allows for an estimation of an exothermicity of 60 kJ mol⁻¹ for binding an η²-H₂ at an Ru(II) centre; a ΔS° value of 60 J mol⁻¹ K⁻¹ indicates that in solution 1 contain s coordinated CH₂Cl₂. The kinetic and thermodynamic data are compared to those obtained from a previously studied hydrogenation of styrene catalyzed by 1. Preliminary findings on related systems containing Ph₂P(CH₂)₃PPh₂ and (C₆H₁₁)₂P(C₆H₁₁)₂ are also noted.     

Stimulation of platelet serotonin and Rb uptake by N-ethylmaleimide

The effects of N-ethylmaleimide (NEM) on mouse platelet serotonin (5-HT) and ⁸⁶Rb⁺ uptake were studied. The 5-HT transport system showed a biphasic response to increasing concentrations of NEM, with low concentrations (25–50 μM) stimulating and high concentrations (200–400 μM) inhibiting 5-HT transport. Fluoxetine, an inhibitor of the platelet 5-HT transporter, blocked NEM-induced stimulation of 5-HT transport. The kinetics of 5-HT uptake indicated that NEM (50 μM) markedly increased the maximal rate of 5-HT transport (V_max control = 28.4±1.4 pmol/10⁸ platelets/4 min vs V_max NEM = 64.5±9.5 pmol/10⁸ platelets/4 min but had no significant effect on the K_m value. Platelet Na⁺ K⁺ ATPase activity was determined by measuring ⁸⁶Rb⁺ uptake. Platelet ⁸⁶Rb⁺ uptake showed a biphasic response to NEM, with low concentrations (25–100 μM) significantly stimulating and high concentrations (400 μM) inhibiting uptake. These changes in platelet ⁸⁶Rb⁺ uptake paralleled the biphasic changes in 5-HT transport. In the presence of fluoxetine, 5-HT transport was markedly inhibited but no change in the ability of NEM to stimulate ⁸⁶Rb⁺ uptake was observed. These data suggest that low concentrations of NEM activate plasma membrane Na⁺ K⁺ ATPase which results in a marked stimulation of platelet 5-HT transport.     

Growth of the androgen-dependent tumor of the prostate: Role of androgens and of locally expressed growth modulatory factors

The crucial role played by androgens in the growth of prostatic carcinoma is now well established. However, the mechanisms of this proliferative action are still poorly understood. Experiments have been performed to clarify: (1) the metabolism of androgens in prostatic tumor cells; and (2) the role played by locally produced growth factors in the autocrine regulation of prostatic tumor cell proliferation and the possible regulation exerted by testosterone (T) on the activity of these factors. These studies have been performed by utilizing the human androgen-responsive prostatic cancer LNCaP cell line. (1) By incubating LNCaP cells with different ¹⁴C-labeled androgenic precursors, it has been shown that all the major key enzymes involved in the metabolism of androgens (5-reductase, 17β-hydroxysteroid-oxidoreductase, 3- and 3β-hydroxysteroid-oxidoreductases) are present and active in these cells. In particular, the 5-reductase, which converts T and Δ₄ to DHT and 5-A respectively, seems to be more active when Δ₄ is the substrate, suggesting a preference for this precursor. (2) The hypothesis that LNCaP cells might produce LHRH (or a LHRH-like peptide) has been verified by RT-PCR, performed in the presence of a pair of specific oligonucleotide primers. A cDNA band of the expected size (228 bp), which specifically hybridized with a ³²P-labeled LHRH oligonucleotide probe, has been obtained in LNCaP cells. To clarify the possible role played by this factor in the regulation of tumor growth, LNCaP cells, cultured in steroid-free conditions, have been treated with a LHRH antagonist; the treatment resulted in a significant increase of cell proliferation. Taken together, these data indicate that a LHRH (or LHRH-like) growth modulatory system is expressed in LNCaP cells and plays an inhibitory role in the regulation of tumor cell proliferation. This system seems to be regulated in a negative way by steroids. Growth factors endowed with stimulatory activity, such as EGF and TGF, have also been shown to be produced by LNCaP cells. The present studies show that the immunoprecipitation of the EGF receptor with a specific monoclonal antibody (Ab225) reveals a protein band of the expected size (170 kDa) which is phosphorylated even in basal conditions. Moreover, the treatment of LNCaP cells, cultured in serum-free conditions, either with a monoclonal antibody against the EGF receptor, or with immunoneutralizing antibodies against EGF and TGF, results in a significant decrease of cell proliferation. These observations clearly confirm the expression, in prostatic tumor cells, of an EGF/TGF loop which exerts a stimulatory action on cell proliferation. T seems to exert a positive regulation on this loop, at least in terms of EGF receptor concentration.     

Effect of diabetic sera on the conversion of eicosapentaenoic acid (EPA) to prostaglandin I3 by cultured bovine aortic endothelial cells

The effects of sera from diabetic patients and healthy donors on the synthesis of prostaglandin I₂ (PGI₂) and PGI₃in vitro were studied in confluent bovine aortic endothelial cells cultured with EPA. The products 6-keto-PGF₁ and Δ¹⁷-6-keto-PGF₁ were measured by GC/SIM as markers of PGI₂ and PG7₃ formation in growth medium after 60 min of incubation. PGI₂ and PG7₃ synthesis with 10% diabetic sera were less than with sera from healthy donors (p < 0.05). However, the total prostacyclin production (PGI₂ and PGI₃) in the cell cultures incubated with 10 μM EPA and 10% diabetic sera approximated that of the cultures incubated with the sera of healthy donors without EPA. These results suggest that the diabetic sera inhibits PGI₂ and PGI₃ synthesis in the cultured endothelial cells, and that EPA intake may reduce the complications of diabetes mellitus, such as microangiopathy and vaso-occlusive diseases, and enhances the production of PGI₃ which seems to exert a strong anti-aggregatory effect.     

Contribution of the heme propionate groups to the electron transfer and electrostatic properties of myoglobin

The role of the heme propionate groups in determining the electron transfer and electrostatic properties of myoglobin have been studied by thermodynamic, kinetic, and spectroscopic studies of horse heart myoglobin in which the heme propionate groups are esterified. Spectroelectrochemical analysis has established that the E_m,7 of dimethylester heme-substituted Mb (DME-Mb) (E_m,7 = 100.2(2) mV vs. NHE (Normal Hydrogen Electrode) (25 °C) is increased  40 mV relative to that of the native protein with ΔH° = −12.9(2) kcal/mol and ΔS° = −51.0(8) cal/mol/deg (pH 7.0, μ = 0.1 M (phosphate)). The second order rate constant for reduction of DME-metMb by Fe(EDTA)²⁻ is increased  > 400-fold relative to that for reduction of native metMb to a value of 1.34(2) × 10³ M⁻¹ s⁻¹ with ΔS^‡ = −13(1) cal/mol/deg and ΔH^‡ = 9.2(3) (pH 7.0, μ = 0.1 M (phosphate)). Analysis of the pH dependences of the reduction potential and rate constant for reduction by Fe(EDTA)²⁻ demonstrates that heme propionate esterification introduces significant changes into the electrostatic interactions in myoglobin. These changes are also manifested by differences in the pH dependences of the ¹H NMR spectra of native and DME-metMb that reveal shifts in pK_a values for specific His residues as the result of heme propionate esterification. In sum, the current results establish that heme propionate esterification not only affects the electron transfer properties of myoglobin but also influences the titration behavior of specific His residues.     

Cell-cycle arrest and apoptosis induction in human breast carcinoma MCF-7 cells by carboxymethylated β-glucan from the mushroom sclerotia of Pleurotus tuber-regium

The mechanism for the anti-tumor activity of a water-soluble carboxymethylated β-glucan (CMPTR), partially synthesized from an insoluble native glucan isolated from the sclerotia of Pleurotus tuber-regium, was studied using human breast carcinoma MCF-7 breast cancer cells in vitro. CMPTR-induced anti-proliferative activity dose-dependently, with an IC₅₀ of 204 μg/ml. CMPTR inhibited the cell proliferation of MCF-7 by arresting the G₁ phase of its cell cycle after 48 h of incubation as shown by flow cytometry. Such G₁ phase arrest was associated with the down-regulation of cyclin D1 and cyclin E expressions in the breast cancer cells. In addition, the CMPTR-treated MCF-7 cancer cells were associated with decreased expression of anti-apoptotic Bcl-2 protein and increased expression of Bax/Bcl-2 ratio. This study shows that CMPTR can inhibit the proliferation of MCF-7 by cell-cycle arrest and apoptosis induction. The potential development of this mushroom polysaccharide as a water-soluble anti-tumor agent requires further investigation.     

Heterobimetallic chloro bridgedcomplexes of (η:η−C10H16)-ruthenium(IV) with palladium(II), platinum(II), rhodium(III), iridium(III), copper(I) and rhodium(I)

Metathesis of [(η³:η³−C₁₀H₁₆)Ru(Cl) (μ−Cl)]₂ (1) with [R₃P) (Cl)M(μ-Cl)]₂ (M = Pd, Pt), [Me₂NCH₂C₆H₄Pd(μ-Cl)]₂ and [(OC)₂Rh(μ-Cl)]₂ affords the heterobimetallic chloro bridged complexes (η³:η³-C₁₀H₁₆) (Cl)Ru(μ-Cl)₂M(PR₃)(Cl) (M = Pd, Pt), (η³:η³-C₁₀H₁₆) (Cl)Ru(μ-Cl)₂PdC₆H₄CH₂NMe₂ and (η³:η³-C₁₀H₁₆) (Cl)Ru(μ-Cl)₂Rh(CO)₂, respectively. Complex 1 reacts with [Cp^*M(Cl) (μ-Cl)]₂ (M = Rh, Ir), [p-cymene Ru(Cl) (μ-Cl]₂ and [(Cy₃P)Cu(μ-Cl)]₂ to give an equilibrium of the heterobimetallic complexes and of educts. The structures of (η³:η³-C₁₀H₁₆)Ru(μ-Cl)₂Pd(PR₃) (Cl) (R = Et, Bu) and of one diastereoisomer of (η³:η³-C₁₀H₁₆)Ru(μ-Cl)₂IrCp^*(Cl) were determined by X-ray diffraction.     

Synthesis and pharmacology of the isomeric methylheptyl-Δ-tetrahydrocannabinols

The synthesis of the 3-heptyl, and the eleven isomeric 3-methylheptyl-Δ⁸-tetrahydrocannabinols (3–7, R and S methyl epimers, and 8) has been carried out. The synthetic approach entailed the synthesis of substituted resorcinols, which were subjected to acid catalyzed condensation with trans-para-menthadienol to provide the Δ⁸-THC analogue. The 1′-, 2′- and 3′-methylheptyl analogues (3–5) are considerably more potent than Δ⁸-THC. The 4′-, 5′- and 6′-methylheptyl isomers (6–8) are approximately equal in potency to Δ⁸-THC.     

Homeostasis of the protonmotive force in phosphorylating mitochondria

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (Δμ_H⁺) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and Δμ_H⁺ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of Δμ_H⁺, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on Δμ_H⁺; for example, the excess of oligomycin produced a 90% inhibition of the respiration while Δμ_H⁺ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing Δμ_H⁺ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of Δμ_H⁺, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and Δμ_H⁺.