RESUMEN
Triple-negative breast cancer (TNBC) is unresponsive to antiestrogen and anti-HER2 therapies, requiring the use of cytotoxic drug combinations of anthracyclines, taxanes, cyclophosphamide, and platinum compounds. Multidrug therapies achieve pathological cure rates of only 2040%, a consequence of drug resistance and cumulative dose limitations necessitated by the reversible cardiotoxic effects of drug therapy. Safer and more effective treatments for TNBC are required to achieve durable therapeutic responses. This study describes the mechanistic analyses of the novel anthracycline, pivarubicin, and its in vivo efficacy against human primary TNBC. Pivarubicin directly activates PKCd, triggers rapid mitochondrial-dependent apoptosis, and circumvents resistance conferred by overexpression of P-glycoprotein, Bcl-2, Bcl-XL, and Bcr-Abl. As a consequence, pivarubicin is more cytotoxic than doxorubicin against MDA-MB-231, and SUM159 TNBC cell lines grown in both monolayer culture and tumorspheres. Comparative in vivo efficacy of pivarubicin and doxorubicin was performed in an orthotopic NSG mouse model implanted with MDA-MB-231 human TNBC cells and treated with the maximum tolerated doses (MTDs) of pivarubicin and doxorubicin. Tumor growth was monitored by digital caliper measurements and determination of endpoint tumor weight and volume. Endpoint cardiotoxicity was assessed histologically by identifying microvacuolization in ventricular cardiomyocytes. Primary tumors treated with multiple rounds of doxorubicin at MTD failed to inhibit tumor growth compared with vehicle-treated tumors. However, administration of a single MTD of pivarubicin produced significant inhibition of tumor growth and tumor regression relative to tumor volume prior to initiation of treatment. Histological analysis of hearts excised from drug- and vehicle-treated mice revealed that pivarubicin produced no evidence of myocardial damage at a therapeutic dose. These results support the development of pivarubicin as a safer and more effective replacement for doxorubicin against TNBC as well as other malignancies for which doxorubicin therapy is indicated.
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Antineoplásicos/farmacología , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Animales , Antraciclinas/farmacología , Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Cardiotoxicidad/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Femenino , Humanos , Dosis Máxima Tolerada , Ratones , Ratones SCID , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tryptophan hydroxylase (TPH) activity was detected in cultured epidermal melanocytes and dermal fibroblasts with respective Km of 5.08 and 2.83 mM and Vmax of 80.5 and 108.0 µmol/min. Low but detectable TPH activity was also seen in cultured epidermal keratinocytes. Serotonin and/or its metabolite and precursor to melatonin, N-acetylserotonin (NAS), were identified by LC/MS in human epidermis and serum. Endogenous epidermal levels were 113.18 ± 13.34 and 43.41 ± 12.45 ng/mg protein for serotonin (n = 8/8) and NAS (n = 10/13), respectively. Their production was independent of race, gender, and age. NAS was also detected in human serum (n = 13/13) at a concentration 2.44 ± 0.45 ng/mL, while corresponding serotonin levels were 295.33 ± 17.17 ng/mL (n = 13/13). While there were no differences in serum serotonin levels, serum NAS levels were slightly higher in females. Immunocytochemistry studies showed localization of serotonin to epidermal and follicular keratinocytes, eccrine glands, mast cells, and dermal fibrocytes. Endogenous production of serotonin in cultured melanocytes, keratinocytes, and dermal fibroblasts was modulated by UVB. In conclusion, serotonin and NAS are produced endogenously in the epidermal, dermal, and adnexal compartments of human skin and in cultured skin cells. NAS is also detectable in human serum. Both serotonin and NAS inhibited melanogenesis in human melanotic melanoma at concentrations of 10-4 -10-3 M. They also inhibited growth of melanocytes. Melanoma cells were resistant to NAS inhibition, while serotonin inhibited cell growth only at 10-3 M. In summary, we characterized a serotonin-NAS system in human skin that is a part of local neuroendocrine system regulating skin homeostasis.
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Epidermis/metabolismo , Fibroblastos/metabolismo , Queratinocitos/metabolismo , Melatonina/metabolismo , Serotonina/análogos & derivados , Envejecimiento de la Piel , Adulto , Anciano , Anciano de 80 o más Años , Línea Celular Tumoral , Femenino , Humanos , Masculino , Persona de Mediana Edad , Serotonina/metabolismoRESUMEN
OBJECTIVE: To determine whether recombinant AMH (rAMH) could prevent post-transplant follicular depletion by acting on the stemness markers Oct-4, Sox2, and NANOG. MATERIALS AND METHODS: This was an experimental study where 12 ovariectomized nude mice were xenotransplanted with vitrified/warmed ovarian cortex obtained from a pre-pubertal girl and Alzet pumps delivering rAMH, or placebo (control), were inserted intra-abdominally. Previously vitrified/warmed ovarian cortex fragments were transplanted after 7 days and then harvested after 14 days from pump placement. We performed real-time RT-PCR analyses, ELISA for AMH, FSH, and estradiol, histologic measurement of ovarian follicles, and immunohistochemistry for Ki67 and TUNEL. The main outcome measures were serum levels and tissue expression of the parameters under investigation and follicle count. RESULTS: Serum AMH, FSH, and estradiol reflected post-ovariectomy profiles and were mildly influenced by rAMH administration. Ovarian cortex expression of AMH, AMH-R2, VEGF, GDF9, Oct-4, and Sox2 was lower in rAMH mice than in controls, while NANOG was upregulated. There was a non-significant decrease in primordial follicles after vitrification-warming, and xenotransplantation further decreased this number. There were lower cell replication and depressed apoptosis in the rAMH group. CONCLUSIONS: Administration of recombinant AMH in the peri-transplant period did not protect the initial follicular depletion but decreased apoptosis and cellular activation and regulated stem cell markers' tissue expression. These results aid our understanding of the inhibitory effects of AMH on follicular development and show the benefit of administering exogenous AMH at the time of pre-pubertal ovarian cortex transplant to protect the follicles from pre-activation and premature depletion.
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Hormona Antimülleriana/genética , Xenoinjertos/metabolismo , Folículo Ovárico/trasplante , Ovario/trasplante , Animales , Hormona Antimülleriana/administración & dosificación , Hormona Antimülleriana/sangre , Apoptosis/genética , Estradiol/sangre , Femenino , Hormona Folículo Estimulante/sangre , Regulación del Desarrollo de la Expresión Génica , Xenoinjertos/efectos de los fármacos , Xenoinjertos/crecimiento & desarrollo , Humanos , Ratones , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/metabolismo , Ovariectomía , Ovario/efectos de los fármacos , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Factores de Transcripción SOXB1/genética , Trasplante Heterólogo , VitrificaciónRESUMEN
Catalytic oligonucleotides, known as DNAzymes, are a new class of nucleic acid-based gene therapy that have recently been used in preclinical animal studies to treat various cancers. In this study the systemic distribution, pharmacokinetics, and safety of intravenously administered anti-MMP (matrix metalloproteinase)-9 DNAzyme (AM9D) were determined in healthy FVB and in MMTV-polyoma virus middle T (PyMT) transgenic mice bearing mammary tumors. MMP-9 is known to be involved in tumor cell development, angiogenesis, invasion, and metastasis. Sulfur-35 ((35)S) labeled ([(35)S]-AM9D) administered intravenously, without the use of carrier molecules, to healthy and mammary tumor bearing MMTV-PyMT transgenic mice distributed to all major organs. The order of percentages of [(35)S]-AM9D accumulation in different organs of healthy and MMTV-PyMT mice were blood>liver>kidney>lung>spleen>heart and mammary tumor>blood≈liver>kidney>spleen>lung>heart, respectively. The amount of AM9D accumulated in mammary tumors 2 hours post injection was 0.6% and 0.2% higher than in either blood or liver, respectively, and its rate of initial clearance from mammary tissue was at least 50% slower than the other organs. Approximately 43% of the delivered dosage of [(35)S]-AM9D was cleared from the system via feces and urine over a period of 72 hours. No evidence of acute or chronic cytotoxicity, local or widespread, associated with AM9D treatment (up to 75 mg AM9D /kg of body weight) was observed in the organs examined. These data suggest that DNAzyme in general and AM9D in particular can be used systemically as a therapeutic agent to treat patients with breast cancer or other metastatic and surgically inaccessible tumors.
Asunto(s)
Antineoplásicos/administración & dosificación , ADN Catalítico/administración & dosificación , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Metaloproteinasa 9 de la Matriz/metabolismo , Administración Intravenosa , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , ADN Catalítico/farmacocinética , ADN Catalítico/toxicidad , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Neoplasias Mamarias Experimentales/metabolismo , Virus del Tumor Mamario del Ratón , Ratones , Ratones Transgénicos , Poliomavirus , Distribución TisularRESUMEN
Indolic and kynuric pathways of skin melatonin metabolism were monitored by liquid chromatography mass spectrometry in human keratinocytes, melanocytes, dermal fibroblasts, and melanoma cells. Production of 6-hydroxymelatonin [6(OH)M], N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and 5-methoxytryptamine (5-MT) was detected in a cell type-dependent fashion. The major metabolites, 6(OH)M and AFMK, were produced in all cells. Thus, in immortalized epidermal (HaCaT) keratinocytes, 6(OH)M was the major product with Vmax = 63.7 ng/10(6) cells and Km = 10.2 µM, with lower production of AFMK and 5-MT. Melanocytes, keratinocytes, and fibroblasts transformed melatonin primarily into 6(OH)M and AFMK. In melanoma cells, 6(OH)M and AFMK were produced endogenously, a process accelerated by exogenous melatonin in the case of AFMK. In addition, N-acetylserotonin was endogenously produced by normal and malignant melanocytes. Metabolites showed selective antiproliferative effects on human primary epidermal keratinocytes in vitro. In ex vivo human skin, both melatonin and AFMK-stimulated expression of involucrin and keratins-10 and keratins-14 in the epidermis, indicating their stimulatory role in building and maintaining the epidermal barrier. In summary, the metabolism of melatonin and its endogenous production is cell type-dependent and expressed in all three main cell populations of human skin. Furthermore, melatonin and its metabolite AFMK stimulate differentiation in human epidermis, indicating their key role in building the skin barrier.
Asunto(s)
Melatonina/metabolismo , Redes y Vías Metabólicas , Piel/metabolismo , 5-Metoxitriptamina/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Cromatografía Líquida de Alta Presión , Células Epidérmicas , Epidermis/efectos de los fármacos , Epidermis/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Queratina-10/metabolismo , Queratina-14/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Cinética , Kinuramina/análogos & derivados , Kinuramina/metabolismo , Kinuramina/farmacología , Melanocitos/citología , Melanocitos/metabolismo , Melanoma/metabolismo , Melanoma/patología , Melatonina/análogos & derivados , Melatonina/farmacología , Serotonina/análogos & derivados , Serotonina/metabolismo , Piel/citología , Espectrometría de Masa por Ionización de Electrospray , PorcinosRESUMEN
The discovery that 7-dehydrocholesterol (7DHC) is an excellent substrate for cytochrome P450scc (CYP11A1) opens up new possibilities in biochemistry. To elucidate its biological significance we tested ex vivo P450scc-dependent metabolism of 7DHC by tissues expressing high and low levels of P450scc activity, placenta and epidermal keratinocytes, respectively. Incubation of human placenta fragments with 7DHC led to its conversion to 7-dehydropregnenolone (7DHP), which was inhibited by dl-aminoglutethimide, and stimulated by forskolin. Final proof for P450scc involvement was provided in isolated placental mitochondria where production of 7DHP was almost completely inhibited by 22R-hydroxycholesterol. 7DHC was metabolized by placental mitochondria at a faster rate than exogenous cholesterol, under both limiting and saturating conditions of substrate transport, consistent with higher catalytic efficiency (k(cat)/K(m)) with 7DHC as substrate than with cholesterol. Ex vivo experiments showed five 5,7-dienal intermediates with MS spectra of dihydroxy and mono-hydroxy-7DHC and retention time corresponding to 20,22(OH)(2)7DHC and 22(OH)7DHC. The chemical structure of 20,22(OH)(2)7DHC was defined by NMR. 7DHP was further metabolized by either placental fragments or placental microsomes to 7-dehydroprogesterone as defined by UV, MS and NMR, and to an additional product with a 5,7-dienal structure and MS corresponding to hydroxy-7DHP. Furthermore, epidermal keratinocytes transformed either exogenous or endogenous 7DHC to 7DHP. 7DHP inhibited keratinocytes proliferation, while the product of its pholytic transformation, pregcalciferol, lost this capability. In conclusion, tissues expressing P450scc can metabolize 7DHC to biologically active 7DHP with 22(OH)7DHC and 20,22(OH)(2)7DHC serving as intermediates, and with further metabolism to 7-dehydroprogesterone and (OH)7DHP.
Asunto(s)
Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Deshidrocolesteroles/metabolismo , Células Epidérmicas , Queratinocitos/enzimología , Placenta/metabolismo , Adolescente , Adulto , Animales , Cromatografía Líquida de Alta Presión , Colforsina/farmacología , Deshidrocolesteroles/química , Femenino , Humanos , Queratinocitos/efectos de los fármacos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Redes y Vías Metabólicas/efectos de los fármacos , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Placenta/efectos de los fármacos , Embarazo , Pregnenolona/análogos & derivados , Pregnenolona/química , Pregnenolona/metabolismo , Sus scrofa , Factores de Tiempo , Adulto JovenRESUMEN
Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D(3), producing 20S-hydroxyvitamin D(3) [20(OH)D(3)] and 20S,23-dihydroxyvitamin D(3) [20,23(OH)(2)D(3)] as the major metabolites. These are biologically active, acting as partial vitamin D receptor (VDR) agonists. Minor products include 17-hydroxyvitamin D(3), 17,20-dihydroxyvitamin D(3), and 17,20,23-trihydroxyvitamin D(3). In the current study, we have further analyzed the reaction products from cytochrome P450scc (P450scc) action on vitamin D(3) and have identified two 22-hydroxy derivatives as products, 22-hydroxyvitamin D(3) [22(OH)D(3)] and 20S,22-dihydroxyvitamin D(3) [20,22(OH)(2)D(3)]. The structures of both of these derivatives were determined by NMR. P450scc could convert purified 22(OH)D(3) to 20,22(OH)(2)D(3). The 20,22(OH)(2)D(3) could also be produced from 20(OH)D(3) and was metabolized to a trihydroxyvitamin D(3) product. We compared the biological activities of these new derivatives with those of 20(OH)D(3), 20,23(OH)(2)D(3), and 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. 1,25(OH)(2)D(3), 20(OH)D(3), 22(OH)D(3), 20,23(OH)(2)D(3), and 20,22(OH)(2)D(3) significantly inhibited keratinocyte proliferation in a dose-dependent manner. The strongest inducers of involucrin expression (a marker of keratinocyte differentiation) were 20,23(OH)(2)D(3), 20,22(OH)(2)D(3), 20(OH)D(3), and 1,25(OH)(2)D(3), with 22(OH)D(3) having a heterogeneous effect. Little or no stimulation of CYP24 mRNA expression was observed for all the analogs tested except for 1,25(OH)(2)D(3). All the compounds stimulated VDR translocation from the cytoplasm to the nucleus with 22(OH)D(3) and 20,22(OH)(2)D(3) having less effect than 1,25(OH)(2)D(3) and 20(OH)D(3). Thus, we have identified 22(OH)D(3) and 20,22(OH)(2)D(3) as products of CYP11A1 action on vitamin D(3) and shown that, like 20(OH)D(3) and 20,23(OH)(2)D(3), they are active on keratinocytes via the VDR, however, showing a degree of phenotypic heterogeneity in comparison with other P450scc-derived hydroxy metabolites of vitamin D(3).
Asunto(s)
Calcifediol/análogos & derivados , Colecalciferol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Hidroxicolecalciferoles/química , Hidroxicolecalciferoles/metabolismo , Piel/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Calcifediol/química , Calcifediol/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colecalciferol/química , Dihidroxicolecalciferoles/química , Dihidroxicolecalciferoles/metabolismo , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Piel/citología , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Vitamina D3 24-HidroxilasaRESUMEN
Treatment of CHO cells expressing human Y receptors (Y(1), Y(2) or Y4 subtype) with pertussis toxin results in a large decrease in functional receptors, with a preferential loss of heteropentameric assemblies of receptor dimers and G-protein trimers. This occurs in parallel to inactivation of the nucleotide site of Gi α subunits, with a half period of about 4 h. The loss could be mainly due to proteolysis at the level of recycling/perinuclear endosomes, and of receptor completion in the ER, since it is reduced by co-treatment with ammonium chloride, an inhibitor of particulate proteinases. Antagonists do not strongly decrease the heteropentameric fraction. These findings indicate that the upkeep of Y receptor dimers in epithelial cell lines depends on the association of receptor oligomers with functional Gi α subunits. This interaction could use the juxtamembrane helix 8 in the fourth intracellular domain, and could also be supported by the C-terminal helix of the third intracellular loop, as outlined in the companion review (Parker et al., Amino Acids, doi: 10.1007/s00726-010-0616-1 , 2010).
Asunto(s)
Células Epiteliales/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptores de Neuropéptido Y/metabolismo , Animales , Células CHO , Bovinos , Cricetinae , Cricetulus , Dimerización , Proteínas de Unión al GTP Heterotriméricas/antagonistas & inhibidores , Proteínas de Unión al GTP Heterotriméricas/genética , Humanos , Toxina del Pertussis/metabolismo , Unión Proteica , Ratas , Receptores de Neuropéptido Y/antagonistas & inhibidores , Receptores de Neuropéptido Y/química , Receptores de Neuropéptido Y/genética , PorcinosRESUMEN
We have synthesized 3ß,21-dihydroxypregna-5,7-dien-20-one (21(OH) 7DHP) and used UVB radiation to induce its photoconversion to analogues of vitamin D (pD), lumisterol (pL) and tachysterol (pT). The number and character of the products and the dynamics of the process were dependent on the UVB dose. The main products: pD and pT compounds were characterized by UV absorption, MS and NMR spectroscopy after RP-HPLC chromatography. In addition, formation of multiple oxidized derivatives of the primary products was detected and one of these derivatives was characterized as oxidized 21-hydroxyisotachysterol compound (21(OH)oxy-piT). These newly synthesized compounds inhibited growth of human melanoma cells in a dose dependent manner, with greater or equal potency to calcitriol. 3ß,21-Dihydroxy-9ß,10α-pregna-5,7-dien-20-one (21(OH)pL) and 21(OH)oxy-piT had higher potency against pigmented melanoma cells, while the EC(50) for compounds 21(OH)7DHP and (5Z,7E)-3ß,21-dihydroxy-9,10-secopregna-5,7,10(19)-trien-20-one (21(OH)pD) were similar in both pigmented and non-pigmented cells. Moreover, 21(OH)7DHP and its derivatives inhibited proliferation of human epidermal HaCaT keratinocytes, albeit at a lower activity compared to melanoma cells. Importantly, 21(OH)7DHP derivatives strongly inhibited the colony formation of human melanoma cells with 21(OH)pD being the most potent. The potential mechanism of action of newly synthesized compounds was similar to that mediated by 1,25(OH)(2)D(3) and involved ligand-induced translocation of vitamin D receptor into the nucleus. In summary, we have characterized for the first time products of UVB-induced conversion of 21(OH)7DHP and documented that these compounds have selective, inhibitory effects on melanoma cells.
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Antineoplásicos/farmacología , Melanoma/tratamiento farmacológico , Pregnadienodioles/farmacología , Secoesteroides/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Melanoma/patología , Conformación Molecular , Fotoquímica , Pregnadienodioles/síntesis química , Pregnadienodioles/química , Secoesteroides/síntesis química , Secoesteroides/química , Estereoisomerismo , Rayos UltravioletaRESUMEN
20S-hydroxyvitamin D3 (20S-(OH)D3), an in vitro product of vitamin D3 metabolism by the cytochrome P450scc, was recently isolated, identified and shown to possess antiproliferative activity without inducing hypercalcemia. The enzymatic production of 20S-(OH)D3 is tedious, expensive, and cannot meet the requirements for extensive chemical and biological studies. Here we report for the first time the chemical synthesis of 20S-(OH)D3 which exhibited biological properties characteristic of the P450scc-generated compound. Specifically, it was hydroxylated to 20,23-dihydroxyvitamin D3 and 17,20-dihydroxyvitamin D3 by P450scc and was converted to 1alpha,20-dihydroxyvitamin D3 by CYP27B1. It inhibited proliferation of human epidermal keratinocytes with lower potency than 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in normal epidermal human keratinocytes, but with equal potency in immortalized HaCaT keratinocytes. It also stimulated VDR gene expression with similar potency to 1,25(OH)2D3, and stimulated involucrin (a marker of differentiation) and CYP24 gene expression, showing a lower potency for the latter gene than 1,25(OH)2D3. Testing performed with hamster melanoma cells demonstrated a dose-dependent inhibition of cell proliferation and colony forming capabilities similar or more pronounced than those of 1,25(OH)2D3. Thus, we have developed a chemical method for the synthesis of 20S-(OH)D3, which will allow the preparation of a series of 20S-(OH)D3 analogs to study structure-activity relationships to further optimize this class of compound for therapeutic use.
Asunto(s)
Calcifediol/análogos & derivados , Colecalciferol/síntesis química , Colecalciferol/farmacología , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Animales , Calcifediol/síntesis química , Calcifediol/química , Calcifediol/metabolismo , Calcifediol/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colecalciferol/química , Colecalciferol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Cricetinae , Humanos , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Modelos Moleculares , Conformación MolecularRESUMEN
BACKGROUND: Cytochrome P450scc metabolizes vitamin D3 to 20-hydroxyvitamin D3 (20(OH)D3) and 20,23(OH)(2)D3, as well as 1-hydroxyvitamin D3 to 1alpha,20-dihydroxyvitamin D3 (1,20(OH)(2)D3). It also cleaves the side chain of 7-dehydrocholesterol producing 7-dehydropregnenolone (7DHP), which can be transformed to 20(OH)7DHP. UVB induces transformation of the steroidal 5,7-dienes to pregnacalciferol (pD) and a lumisterol-like compounds (pL). METHODS AND FINDINGS: To define the biological significance of these P450scc-initiated pathways, we tested the effects of their 5,7-diene precursors and secosteroidal products on leukemia cell differentiation and proliferation in comparison to 1alpha,25-dihydroxyvitamin D3 (1,25(OH)(2)D3). These secosteroids inhibited proliferation and induced erythroid differentiation of K562 human chronic myeloid and MEL mouse leukemia cells with 20(OH)D3 and 20,23(OH)(2)D3 being either equipotent or slightly less potent than 1,25(OH)(2)D3, while 1,20(OH)(2)D3, pD and pL compounds were slightly or moderately less potent. The compounds also inhibited proliferation and induced monocytic differentiation of HL-60 promyelocytic and U937 promonocytic human leukemia cells. Among them 1,25(OH)(2)D3 was the most potent, 20(OH)D3, 20,23(OH)(2)D3 and 1,20(OH)(2)D3 were less active, and pD and pL compounds were the least potent. Since it had been previously proven that secosteroids without the side chain (pD) have no effect on systemic calcium levels we performed additional testing in rats and found that 20(OH)D3 had no calcemic activity at concentration as high as 1 microg/kg, whereas, 1,20(OH)(2)D3 was slightly to moderately calcemic and 1,25(OH)(2)D3 had strong calcemic activity. CONCLUSIONS: We identified novel secosteroids that are excellent candidates for anti-leukemia therapy with 20(OH)D3 deserving special attention because of its relatively high potency and lack of calcemic activity.
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Antineoplásicos/farmacología , Calcifediol/análogos & derivados , Colecalciferol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Deshidrocolesteroles/metabolismo , Regulación Leucémica de la Expresión Génica , Hipercalcemia/metabolismo , Animales , Calcifediol/farmacología , Línea Celular Tumoral , Proliferación Celular , Deshidrocolesteroles/química , Células HL-60 , Humanos , Ratones , Pregnenolona/análogos & derivados , Pregnenolona/química , Células U937RESUMEN
Since melatonin production has been documented in extrapineal and extraneuronal tissues, we investigated the expression of molecular elements of the melatoninergic system in human RPE cells (ARPE-19). The expression of key enzymes for melatonin synthesis: tryptophan hydroxylases (TPH1 and TPH2); arylalkylamine N-acetyltransferase (AANAT) and hydroxyindole-O-methyltransferase (HIOMT) was detected in ARPE-19 cells using RT-PCR. TPH1 and AANAT proteins were detected in ARPE by Western blotting, while sequential metabolism of tryptophan, serotonin and N-acetylserotonin to melatonin was shown by RP-HPLC. We also demonstrated, by means of RT-PCR, that ARPE expressed mRNA encoding the melatonin receptors: MT2 (but not MT1), two isoforms of nuclear receptor (RORalpha1 and RORalpha4/RZR1), and quinone oxidoreductase (NQO2). By analogy with other peripheral tissues, for example the skin, the expression of these metabolic elements in RPE cells suggests that the RPE represents an additional source of melatonin in the eye, to regulate local homeostasis and prevent from oxidative damage in intra-, auto- and/or paracrine fashions.
Asunto(s)
Melatonina/biosíntesis , Epitelio Pigmentado de la Retina/metabolismo , Acetilserotonina O-Metiltransferasa/genética , Acetilserotonina O-Metiltransferasa/metabolismo , N-Acetiltransferasa de Arilalquilamina/genética , N-Acetiltransferasa de Arilalquilamina/metabolismo , Línea Celular , Regulación de la Expresión Génica , Humanos , Receptores de Melatonina/genética , Receptores de Melatonina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serotonina/análogos & derivados , Serotonina/química , Serotonina/metabolismo , Triptófano/química , Triptófano/metabolismo , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismoRESUMEN
Since P450scc transforms 7-dehydrocholesterol (7DHC) to 7-dehydropregnenolone (7DHP) in vitro, we investigated sequential 7DHC metabolism by adrenal glands ex vivo. There was a rapid, time- and dose-dependent metabolism of 7DHC by adrenals from rats, pigs, rabbits and dogs with production of more polar 5,7-dienes as detected by RP-HPLC. Based on retention time (RT), UV spectra and mass spectrometry, we identified the major products common to all tested species as 7DHP, 22-hydroxy-7DHC and 20,22-dihydroxy-7DHC. The involvement of P450scc in adrenal metabolic transformation was confirmed by the inhibition of this process by DL-aminoglutethimide. The metabolism of 7DHC with subsequent production of 7DHP was stimulated by forscolin indicating involvement of cAMP dependent pathways. Additional minor products of 7DHC metabolism that were more polar than 7DHP were identified as 17-hydroxy-7DHP (in pig adrenals but not those of rats) and as pregna-4,7-diene-3,20-dione (7-dehydroprogesterone). Both products represented the major identifiable products of 7DHP metabolism in adrenal glands. Studies with purified enzymes show that StAR protein likely transports 7DHC to the inner mitochondrial membrane, that 7DHC can compete effectively with cholesterol for the substrate binding site on P450scc and that the catalytic efficiency of 3betaHSD for 7DHP (V(m)/K(m)) is 40% of that for pregnenolone. Skin mitochondria are capable of transforming 7DHC to 7DHP and the 7DHP is metabolized further by skin extracts. Finally, 7DHP, its photoderivative 20-oxopregnacalciferol, and pregnenolone exhibited biological activity in skin cells including inhibition of proliferation of epidermal keratinocytes and melanocytes, and melanoma cells. These findings define a novel steroidogenic pathway: 7DHC-->22(OH)7DHC-->20,22(OH)(2)7DHC-->7DHP, with potential further metabolism of 7DHP mediated by 3betaHSD or CYP17, depending on mammalian species. The 5-7 dienal intermediates of the pathway can be a source of biologically active vitamin D3 derivatives after delivery to or production in the skin, an organ intermittently exposed to solar radiation.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Alquenos/metabolismo , Deshidrocolesteroles/metabolismo , Piel/metabolismo , Alquenos/química , Animales , Proliferación Celular , Cromatografía Liquida , Deshidrocolesteroles/química , Inhibidores Enzimáticos , Enzimas/metabolismo , Femenino , Humanos , Queratinocitos/metabolismo , Masculino , Espectrometría de Masas , Melanocitos/metabolismo , Melanoma/metabolismo , Melanoma/patología , Redes y Vías Metabólicas , Pregnatrienos/química , Pregnatrienos/metabolismo , Secoesteroides/química , Secoesteroides/metabolismo , Piel/citología , Factores de Tiempo , Extractos de Tejidos/metabolismoRESUMEN
Pregna-5,7-dienes and their hydroxylated derivatives can be formed in vivo when there is a deficiency in 7-dehydrocholesterol (7-DHC) Delta-reductase function, e.g., Smith-Lemli-Opitz syndrome (SLOS). Ultraviolet B (UVB) radiation induces photoconversion of 7-DHC to vitamin D3, lumisterol3 and tachysterol3. Two epimers (20R and 20S) of pregna-5,7-diene-3beta,17alpha,20-triol (4R and 4S, respectively) were synthesized and their UVB photo-conversion products identified as corresponding 9,10-secosteroids with vitamin D-like and tachysterol-like structures, and 5,7-dienes with inverted configuration at C-9 and C-10 (lumisterol-like). The number and character of the products and the dynamics of the process were dependent on the UVB dose. At high UVB doses, the formation of multiple oxidized derivatives of the primary products, and the formation of 5,7,9(11)-triene, were observed. The production of vitamin D-like, tachysterol-like and lumisterol-like derivatives was also observed in human skin treated with 4R and 4S, and subjected to UV irradiation, as shown by RP-HPLC. Newly synthesized compounds inhibited melanoma growth in dose dependent manner, and some of them showed equal or higher potency than 1,25(OH)2D3. In summary, we have characterized for the first time the products of UV induced conversion of pregna-5,7-diene-3beta,17alpha,20-triols and documented that the newly synthesized compounds have antiproliferative properties against melanoma cells.
Asunto(s)
Melanoma/metabolismo , Melanoma/patología , Fotólisis/efectos de la radiación , Pregnadienos/química , Pregnadienos/farmacología , Pregnadienotrioles/química , Pregnadienotrioles/síntesis química , Pregnadienotrioles/farmacología , Acetilación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Pregnadienos/síntesis química , Pregnadienos/metabolismo , Pregnadienotrioles/metabolismo , Secoesteroides/análisis , Secoesteroides/química , Piel/metabolismo , Piel/efectos de la radiación , Estereoisomerismo , Rayos UltravioletaRESUMEN
Calcitriol (3beta,5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-1alpha,3beta,25-triol) is a powerful oncostatic form of vitamin D3 that is of limited clinical utility due to hypercalcemic (toxic) effects. Since the removal of the side chain reduces or eliminates the calcemic activity of vitamin D3, secosteroidal compounds lacking or with a shortened side chain are good candidates for anti-cancer drugs. In addition, 5,7-steroidal dienes without a side chain can be generated in vivo under pathological conditions. A series of androsta- and pregna-5,7-dienes was efficiently synthesized from their respective 3-acetylated 5-en precursors by bromination-dehydrobromination and deacetylation reactions. Ultraviolet B (UVB) irradiation was used to generate corresponding 9,10-secosteroids with vitamin D-like structures. Additional products with tachysterol-like (T-like) structures or 5,7-dienes with inverted configuration at C-9 and C-10 (lumisterol, L-like) were also detected. Different doses of UVB resulted in formation of various products. At low doses, previtamin D-, T- or L-like compounds were formed as the main products, while higher doses induced further isomerization, with formation of potentially oxidized derivatives. In summary, we describe dynamic UVB induced conversion of androsta- and pregna-5,7-dienes into vitamin D-like compounds and their rearranged analogues; additionally novel T-like and L-like structures were also produced and characterized. Further biological evaluation of newly synthesized compounds should help to select the best candidate(s) for potential treatment of hyperproliferative diseases including cancer.
Asunto(s)
Androstadienos/química , Pregnadienos/química , Androstadienos/síntesis química , Androstadienos/efectos de la radiación , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Modelos Moleculares , Conformación Molecular , Fotólisis , Pregnadienos/síntesis química , Pregnadienos/efectos de la radiación , Rayos UltravioletaRESUMEN
Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D3 to produce 20-hydroxyvitamin D3 and other poorly characterized hydroxylated products. The present study aimed to identify all the products of vitamin D3 metabolism by P450scc, as well as the pathways leading to their formation. Besides 20-hydroxyvitamin D3, other major metabolites of vitamin D3 were a dihydroxyvitamin D3 and a trihydroxyvitamin D3 product. The dihydroxyvitamin D3 was clearly identified as 20,23-dihydroxyvitamin D3 by NMR, in contrast to previous reports that postulated hydroxyl groups in positions 20 and 22. NMR of the trihydroxy product identified it as 17alpha,20,23-trihydroxyvitamin D3. This product could be directly produced by P450scc acting on 20,23-dihydroxyvitamin D3, confirming that hydroxyl groups are present at positions 20 and 23. Three minor products of D3 metabolism by P450scc were identified by MS and by examining their subsequent metabolism by P450scc. These products were 23-hydroxyvitamin D3, 17alpha-hydroxyvitamin D3 and 17alpha,20-dihydroxyvitamin D3 and arise from the three P450scc-catalysed hydroxylations occurring in a different order. We conclude that the major pathway of vitamin D3 metabolism by P450scc is: vitamin D3 --> 20-hydroxyvitamin D3 --> 20,23-dihydroxyvitamin D3 --> 17alpha,20,23-trihydroxyvitamin D3. The major products dissociate from the P450scc active site and accumulate at a concentration well above the P450scc concentration. Our new identification of the major dihydroxyvitamin D3 product as 20,23-dihydroxyvitamin D3, rather than 20,22-dihydroxyvitamin D3, explains why there is no cleavage of the vitamin D3 side chain, unlike the metabolism of cholesterol by P450scc.
Asunto(s)
Colecalciferol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Animales , Bovinos , Colecalciferol/química , Humanos , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Vitamina D/análogos & derivados , Vitamina D/química , Vitamina D/metabolismoRESUMEN
It has been shown that mammalian cytochrome P450scc can metabolize vitamin D3 to 20-hydroxyvitamin D3 (20(OH)D3) and 20,22(OH)2D3. To define the biological significance of this pathway, we tested the effects of 20(OH)D3 on the differentiation program of keratinocytes and on the expression of enzymes engaged in vitamin D3 metabolism. Immortalized HaCaT and adult human epidermal keratinocytes were used as a model and the effects of 20(OH)D3 were compared with those of 25(OH)D3 and 1,25(OH)2D3. 20(OH)D3 inhibited proliferation and caused G2/M arrest. 20(OH)D3 stimulated involucrin and inhibited cytokeratin 14 expression. The potency of 20(OH)D3 was comparable to that of 1,25(OH)2D3. 20(OH)D3 decreased the expression of cytochrome P450 enzyme (CYP)27A1 and CYP27B1, however, having only slight effect on CYP24. The effect of 20(OH)D3 was dependent on the vitamin D receptor (VDR). As shown by electrophoretic mobility shift assay, 20(OH)D3 stimulated the binding of nuclear proteins to the VDRE. Transfection of cells with VDR-specific siRNA decreased 20(OH)D3-stimulated transcriptional activity of the VDRE promoter and the expression of involucrin and CYP24 mRNA. Therefore, the above studies identify 20(OH)D3 as a biologically active secosteroid that induces keratinocyte differentiation. These data imply that the previously unreported pathway of vitamin D3 metabolism by P450scc may have wider biological implications depending, for example, on the extent of adrenal gland or cutaneous metabolism.
Asunto(s)
Calcifediol/análogos & derivados , Diferenciación Celular/efectos de los fármacos , Colecalciferol/metabolismo , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , 25-Hidroxivitamina D3 1-alfa-Hidroxilasa/metabolismo , Glándulas Suprarrenales/metabolismo , Calcifediol/metabolismo , Calcifediol/farmacología , Calcitriol/metabolismo , Diferenciación Celular/fisiología , Línea Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colestanotriol 26-Monooxigenasa/metabolismo , Humanos , Hidroxilación , Queratina-14/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Calcitriol/metabolismo , Piel/metabolismo , Esteroide Hidroxilasas/metabolismo , Vitamina D3 24-HidroxilasaRESUMEN
N-Benzyladriamycin-14-valerate (AD 198) is one of several novel anthracycline protein kinase C (PKC)-activating agents developed in our laboratories that demonstrates cytotoxic superiority over doxorubicin (Adriamycin; DOX) through its circumvention of multiple mechanisms of drug resistance. This characteristic is attributed at least partly to the principal cellular action of AD 198: PKC activation through binding to the C1b (diacylglycerol binding) regulatory domain. A significant dose-limiting effect of DOX is chronic, dose-dependent, and often irreversible cardiotoxicity ascribed to the generation of reactive oxygen species (ROS) from the semiquinone ring structure of DOX. Despite the incorporation of the same ring structure in AD 198, we hypothesized that AD 198 might also be cardioprotective through its ability to activate PKC-epsilon, a key component of protective ischemic preconditioning in cardiomyocytes. Chronic administration of fractional LD(50) doses of DOX and AD 198 to mice results in histological evidence of dose-dependent ventricular damage by DOX but is largely absent from AD 198-treated mice. The absence of significant cardiotoxicity with AD 198 occurs despite the equal ability of DOX and AD 198 to generate ROS in primary mouse cardiomyocytes. Excised rodent hearts perfused with AD 198 prior to hypoxia induced by vascular occlusion are protected from functional impairment to an extent comparable to preconditioning ischemia. AD 198-mediated cardioprotection correlates with increased PKC-epsilon activation and is inhibited in hearts from PKC-epsilon knockout mice. These results suggest that, despite ROS production, the net cardiac effect of AD 198 is protection through activation of PKC-epsilon.
Asunto(s)
Corazón/efectos de los fármacos , Proteína Quinasa C-epsilon/fisiología , Animales , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacología , Activación Enzimática , Femenino , Precondicionamiento Isquémico Miocárdico , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Wistar , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
Phospholipid scramblase 3 (PLS3) is a member of the phospholipid scramblase family present in mitochondria. PLS3 plays an important role in regulation of mitochondrial morphology, respiratory function, and apoptotic responses. PLS3 is phosphorylated by PKC-delta at Thr21 and is the mitochondrial target of PKC-delta-induced apoptosis. Cells with overexpression of PLS3, but not the phosphoinhibitory mutant PLS3(T21A), are more susceptible to apoptosis induced by AD198, an extranuclear targeted anthracycline that activates PKC-delta. Here we report that the phosphomimetic mutant of PLS3(T21D) by itself can induce apoptosis in HeLa cells. Using proteoliposomes with addition of pyrene-labeled phosphatidylcholine (PC) at the outer leaflet, we measured the lipid flip-flop activity of PLS3 and its phosphorylation mutant. PLS3(T21D) is more potent than wild-type PLS3 or PLS3(T21A) to transfer pyrene-PC from the outer leaflet to the inner leaflet of liposomes. Based on our previous finding that PLS3 enhances tBid-induced mitochondrial damages, we tested the hypothesis that PLS3 enhances cardiolipin translocation to mitochondrial surface and facilitates tBid targeting. Fluorescein-labeled tBid(G94E) was used as a probe to quantify cardiolipin on the surface of mitochondria. Mitochondria from cells treated with AD198 or cells expressing PLS3(T21D) had a higher level of tBid-binding capacity than control cells or cells expressing wild-type PLS3. These findings indicate that phosphorylation of PLS3 by PKC-delta induces PLS3 activation to facilitate mitochondrial targeting of tBid and apoptosis.
Asunto(s)
Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Mitocondrias/enzimología , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteína Quinasa C-delta/metabolismo , Apoptosis/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacología , Activación Enzimática/efectos de los fármacos , Células HeLa , Humanos , Mitocondrias/efectos de los fármacos , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteína Quinasa C-delta/genética , Transporte de Proteínas/efectos de los fármacos , TransgenesRESUMEN
Bcr-Abl activity in chronic myelogenous leukemia (CML) results in dysregulated cell proliferation and resistance against multiple cytotoxic agents due to the constitutive activation of proliferative signaling pathways. Currently, the most effective treatment of CML is the inhibition of Bcr-Abl activity by imatinib mesylate (Gleevec). Imatinib efficacy is limited by development of resistance through either expression of Bcr-Abl variants that bind imatinib less avidly, increased expression of Bcr-Abl, or expression of multidrug transport proteins. N-Benzyladriamycin-14-valerate (AD 198) is a novel antitumor PKC activating agent that triggers rapid apoptosis through PKC-delta activation and mitochondrial depolarization in a manner that is unaffected by Bcl-2 expression. We demonstrate that Bcr-Abl expression does not confer resistance to AD 198. Further, AD 198 rapidly induces Erk1/2 and STAT5 phosphorylation prior to cytochrome c release from mitochondria, indicating that proliferative pathways are active even as drug-treated cells undergo apoptosis. At sub-cytotoxic doses, AD 198 and its cellular metabolite, N-benzyladriamycin (AD 288) sensitize CML cells to imatinib through a supra-additive reduction in the level of Bcr-Abl protein expression. These results suggest that AD 198 is an effective treatment for CML both in combination with imatinib and alone against imatinib-resistant CML cells.