Nicandro Mendoza-Patiño

Decrease of cyclin D1 in the human lung adenocarcinoma cell line A-427 by 7-hydroxycoumarin

Coumarin in vivo has antitumor activity in various types of cancer. In vitro, coumarin and 7-hydroxycoumarin, its major biotransformation product in humans, inhibit the proliferation of several human tumor cell lines. The molecular mechanisms of these effects are unknown. To gain information about these mechanisms, we studied the effects of coumarin and 7-hydroxycoumarin in the human lung adenocarcinoma cell line A-427 on the inhibition of: (i) cell proliferation; (ii) cell cycle progression; and (iii) expression of cyclins D1, E and A. The inhibitory concentrations 50 (IC(50)) of both compounds were estimated by cytostatic assays of tetrazolium (MTT) reduction. The effects on cell cycle progression were assayed with propidium iodide and BrdU using DNA histograms and multiparametric flow cytometry. The percentages of cells expressing cyclins D1, E, and A were estimated by means of bivariate flow cytometry using propidium iodide, and FITC-conjugated monoclonal antibodies for each cyclin. The IC(50) (+/-S.E.M. n=3) of 7-hydroxycoumarin and coumarin at 72 h exposure, were 100+/-4.8 and 257+/-8.8 microg/ml, respectively. 7-Hydroxycoumarin at the concentration of 160 microg/ml (1 mM), inhibited the G(1)/S transition of the cell cycle, an action consistent with the cytostatic effect. No significant decreases of cyclins E and A were observed. In contrast, cyclin D1 significantly decreased, which appears to indicate an action of 7-hydroxycoumarin in early events of phase G(1). However, messenger RNA of cyclin D1, assayed by RT-PCR, did not change. This suggests a posttranscriptional effect. The effects of coumarin were not significant. Cyclin D1 is overexpressed in many types of cancer, and its inhibition has been proposed as a pharmacological and therapeutic target for novel antitumor agents. Knowledge of the decrease of cyclin D1 by 7-hydroxycoumarin may lead to its use in cancer therapy, as well as to the development of more active compounds.

4-Hydroxycoumarin disorganizes the actin cytoskeleton in B16–F10 melanoma cells but not in B82 fibroblasts, decreasing their adhesion to extracellular matrix proteins and motility

This study determined the in vitro effects of 4-hydroxycoumarin (4-HC) employing the melanoma cell line B16-F10 and the non-malignant fibroblastic cell line B82. 4-HC disorganized the actin cytoskeleton in B16-F10 cells, but not in B82 fibroblasts. Cytoskeletal disorganization correlated with reductions in cell adhesion to four extracellular matrix proteins and inhibition of random motility. 4-HC did not modify cell viability or actin expression, but decreased tyrosine phosphorylation of several proteins in melanoma cells. Because adhesion of tumor cells to extracellular matrix is required during the metastatic process, 4-HC might be useful as an adjuvant therapy for melanoma.

Anticoagulant and estrogenic effects of two new 17β-aminoestrogens, butolame [17β-(4-hydroxy-1-butylamino)-1, 3,5(10)-estratrien-3-ol] and pentolame [17β-(5-hydroxy-1-pentylamino)-1, 3,5(10)-estratrien-3-ol]

Abstract The syntheses and characterizations of two new 17β-aminoestrogens, butolame [17β-(4-hydroxy-1-butylamino)-1, 3,5(10)-estratrien-3-ol] and pentolame [17β-(5-hydroxy-1-pentylamino)-1,3,5(10)-estratrien-3-ol], are presented. Both compounds, when administered in single subcutaneous injections to male mice and rats, produce dose-dependent increases in blood clotting times that may last several days. The estrogenic effects assessed by the vaginal cornification test are of relatively short duration.

Differential effects of esculetin and daphnetin on in vitro cell proliferation and in vivo estrogenicity

Esculetin (6,7-dihydroxycoumarin) and daphnetin (7,8-dihydroxycoumarin) are secondary metabolites of plants used in folk medicine. These compounds have showed great antiproliferative activity in several tumor cell lines and have been proposed as potential anticancer agents. However, the estrogenic potential of these two compounds has to date not been reported. The present study compared esculetin and daphnetin on the inhibition of cell proliferation and cell cycle progression of the MCF-7 estrogen-responsive human carcinoma cell line. In vivo and in vitro estrogenic activity for both compounds was also evaluated. Esculetin inhibited cell proliferation after 72 h exposure (IC50=193 ± 6.6 μM), while daphnetin evidenced inhibiting effects starting at 24-h exposure (72 h, IC50=73 ± 4.1 μM). Both effects showed changes in cyclin D1 gene expression. In non-estrogenic conditions (E-screening assay), esculetin produced biphasic response on proliferation of the MCF-7 cells; at 10(-8)-10(-6)M, concentrations induced proliferative effects as EC50=4.07 × 10(-9)M (E(2)=2.91 × 10(-12)M); at higher concentrations (10(-5)-10(-4)M), cell proliferation was inhibited. Relative proliferative effect at E(2) was 52% (E(2)=100), relative proliferative potency was 0.072 (E(2)=100). Additionally, esculetin tested in vivo showed estrogenic effects at 50-100mg/kg doses; relative uterotrophic effect at E(2) was 37%, with relative uterotrophic potency registered at 0.003. In contrast, daphnetin did not induce estrogenic effects in vitro or with in vivo models. The low estrogenic activity of esculetin could prove useful in postmenopausal therapy but not as a safe antitumor agent in estrogen-dependent tumors. Daphnetin-based antiproliferative selectivity with MCF-7 cells showed that daphnetin is a promising antitumoral agent also acting on estrogen dependent tumors.

Synthesis and molecular structure of prolame, N-(3-hydroxy-1,3,5(10)-estratrien-17β-yl)-3-hydroxypropylamine; an amino-estrogen with prolonged anticoagulant and brief estrogenic effects

The synthesis and molecular structure of prolame, N-(3-hydroxy-1,3,5(10)-estratrien-17 beta-yl)-3-hydroxypropylamine, is described. It was characterized by ir, nmr, mass spectrometry and chemical analysis. The crystal structure of this compound was determined by single-crystal x-ray diffraction. Prolame belongs to space group P212121. Cell dimensions are: a = 8.356(2), b = 13.343(4) and c = 16.119(4) A. Z = 4; R = 4.1%.

Reduced paxillin expression contributes to the antimetastatic effect of 4-hydroxycoumarin on B16-F10 melanoma cells.

Background4-Hydroxycoumarin (4-HC) is a coumarin that lacks anticoagulant activity. 4-HC affects the cytoskeletal stability and decreases cell adhesion and motility of the melanoma cell line B16-F10. Together with integrins and other cytoskeletal proteins, paxillin participates in the regulation of cell adhesion and motility, acting as an adapter protein at focal adhesions. The present study determined the participation of paxillin in the reported effects of 4-HC and analyzed the role of paxillin in the formation of melanoma metastases.Results4-HC decreased protein and mRNA levels of α- and β-paxillin isoforms in B16-F10 cells. Paxillin downregulation correlated with an inadequate translocation of paxillin to focal adhesions and a reduced phosphotyr118-paxillin pool. Consequently, 4-HC altered paxillin-mediated signaling, decreasing the phosphorylation of FAK and the level of GTP-bound Rac-1. These results partially explain the mechanism of the previously reported effects of 4-HC. Additionally, we studied the effect of 4-HC on metastatic potential of B16-F10 cells through experimental metastasis assays. In vitro treatment of cells with 4-HC inhibited their capability to originate pulmonary metastases. 4-HC did not affect cell proliferation or survival, demonstrating that its antimetastatic effect is unrelated to changes on cell viability. We also studied the importance of paxillin in metastasis by transfecting melanoma cells with paxillin-siRNA. Transfection produced a modest reduction on metastatic potential, indicating that: i) paxillin plays a role as inducer of melanoma metastasis; and ii) paxillin downregulation is not sufficient to explain the antimetastatic effect of 4-HC. Therefore, we evaluated other changes in gene expression by differential display RT-PCR analysis. Treatment with 4-HC produced a downregulation of Adhesion Regulating Molecule-1 (ARM-1), which correlated with a decreased adhesion of melanoma cells to lung slides.ConclusionThis study shows that reduced paxillin expression is associated with the impaired cell adhesion and motility seen in 4-HC-treated cells and partially contributes to the antimetastatic effect of 4-HC. In contrast, the role of ARM-1 reduced expression in the effects of 4-HC is still to be clarified. The antimetastatic effect of 4-HC suggests that this compound, or others with similar mode of action, might be useful for the development of adjuvant therapies for melanoma.

The anticoagulant effect of prolame, N-(3-hydroxy-1,3,5(10) estratrien-17β-yl)-3-hydroxypropylamine, a novel amino-estrogen

The anticoagulant and estrogenic effects of prolame, N-(3-hydroxy-1,3,5(10)-estratrien-17 beta-yl)-3-hydroxypropylamine, are described. A single subcutaneous injection of prolame in male mice, ovariectomized mice, adult and infant male rats, produced dose-dependent increases of blood clotting time, which could be observed with the larger doses even after 4 days. In ovariectomized mice, prolame produced vaginal cornifications of shorter duration than those produced by estradiol-17 beta. The evidence suggests that, in contrast with currently used estrogens, prolame would not generate cardiovascular accidents if used for the treatment of prostatic carcinoma; it could also be exceptionally effective for the prevention of thrombosis.

Airway smooth muscle relaxation induced by 5-HT2A receptors: Role of Na+/K+-ATPase pump and Ca2+-activated K+ channels

AIMS Although 5-hydroxytryptamine (5-HT) contracts airway smooth muscle in many mammalian species, in guinea pig and human airways 5-HT causes a contraction followed by relaxation. This study explored potential mechanisms involved in the relaxation induced by 5-HT. MAIN METHODS Using organ baths, patch clamp, and intracellular Ca(2+) measurement techniques, the effect of 5-HT on guinea pig airway smooth muscle was studied. KEY FINDINGS A wide range of 5-HT concentrations caused a biphasic response of tracheal rings. Response to 32 microM 5-HT was notably reduced by either tropisetron or methiothepin, and almost abolished by their combination. Incubation with 10 nM ketanserin significantly prevented the relaxing phase. Likewise, incubation with 100 nM charybdotoxin or 320 nM iberiotoxin and at less extent with 10 microM ouabain caused a significant reduction of the relaxing phase induced by 5-HT. Propranolol, L-NAME and 5-HT(1A), 5-HT(1B)/5-HT(1D) and 5-HT(2B) receptors antagonist did not modify this relaxation. Tracheas from sensitized animals displayed reduced relaxation as compared with controls. In tracheas precontracted with histamine, a concentration response curve to 5-HT (32, 100 and 320 microM) induced relaxation and this effect was abolished by charybdotoxin, iberiotoxin or ketanserin. In single myocytes, 5-HT in the presence of 3 mM 4-AP notably increased the K(+) currents (I(K(Ca))), and they were completely abolished by charybdotoxin, iberiotoxin or ketanserin. SIGNIFICANCE During the relaxation induced by 5-HT two major mechanisms seem to be involved: stimulation of the Na(+)/K(+)-ATPase pump, and increasing activity of the high-conductance Ca(2+)-activated K(+) channels, probably via 5-HT(2A) receptors.

Protective effect of SnCl2 on K2Cr2O7-induced toxicity in LLC-PK1 cells.

The exposure to hexavalent chromium is often known to cause acute renal failure. It has been found that nonenzymatic antioxidants and the induction of heme oxygenase 1 have protective effects against nephrotoxicity induced by potassium dichromate in vivo. In this work, the effect of stannous chloride, an inducer of heme oxygenase 1, on potassium dichromate-induced toxicity in proximal tubular epithelial cells was studied. Hexavalent chromium levels, peroxynitrite content, reduced thiol content, heme oxygenase activity, reactive oxygen species production, and stannous chloride scavenging capacity were measured. It was found that stannous chloride protects proximal tubular epithelial cells from potassium dichromate-induced cell death. The decrease in extracellular and intracellular hexavalent chromium concentration, the induction of heme oxygenase 1, and the ability to scavenge reactive oxygen species and peroxynitrite are involved in the mechanism by which stannous chloride protects proximal tubular epithelial cells from potassium dichromate-induced toxicity.