RESUMEN
The liver X> or = receptor alpha (LXRalpha) is a nuclear receptor with a key role in bile acid biosynthesis and cholesterol metabolism. The present study investigated the expression and function of LXRalpha in the normal and malignant human breast. LXRalpha mRNA transcripts were detected by RT-PCR in nine breast carcinoma cell lines. The nucleotide sequence of the cloned PCR product was identical to the corresponding human LXRalpha cDNA sequence. Expression of LXRalpha protein was confirmed by immunoblot analysis of breast cancer cell lysates. LXRalpha mRNA was expressed in 14/15 (93%) of normal human breast mammoplasty specimens and in 11/15 (73%) of primary breast carcinomas. Oxysterol and nonsteroidal LXRalpha agonists at low micromolar concentrations inhibited proliferation of breast carcinoma cell lines in culture. The importance of LXRalpha signaling in cholesterol homeostasis and the observed expression of LXRalpha in normal breast tissue suggest that this nuclear oxysterol receptor has an important physiological function in the breast. LXRalpha gene expression is regulated by dietary fatty acids implicated in breast carcinogenesis and detection of LXRalpha expression in breast cancer cell lines and breast tumors in the present study indicates that LXRalpha may also be important in breast carcinogenesis. Inhibition of breast cancer cell proliferation suggests that pharmacological LXRalpha agonists may have potential preventive and/or therapeutic antitumor activity in breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma/genética , Carcinoma/patología , Transformación Celular Neoplásica , Perfilación de la Expresión Génica , Receptores Citoplasmáticos y Nucleares/biosíntesis , Mama/patología , Colesterol/metabolismo , Proteínas de Unión al ADN , Femenino , Humanos , Immunoblotting , Receptores X del Hígado , Receptores Nucleares Huérfanos , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Trichostatin A is a potent and specific histone deacetylase inhibitor with promising antitumor activity in preclinical models. Plasma pharmacokinetics of trichostatin A were studied following single-dose intraperitoneal administration of 80 mg/kg (high dose) or 0.5 mg/kg (low dose) to female BALB/c mice. Plasma trichostatin A concentrations were quantified by high performance liquid chromatography (HPLC)-UV assay (high dose) or by HPLC-multiple reaction monitoring assay (low dose). Trichostatin A was rapidly absorbed from the peritoneum and detectable in plasma within 2 min. Cmax of 40 microg/ml and 8 ng/ml occurred within 5 min, followed by rapid exponential decay in plasma trichostatin A concentration with t1/2 of 6.3 min and 9.6 min (high and low doses, respectively). Phase I metabolites at the high dose were identified by simultaneous UV and positive ion electrospray mass spectrometry. Trichostatin A underwent extensive metabolism: primary metabolic pathways were N-demethylation, reduction of the hydroxamic acid to the corresponding trichostatin A amide, and oxidative deamination to trichostatic acid. N-Monomethyl trichostatin A amide was the major plasma metabolite. No didemethylated compounds were identified. Trichostatic acid underwent further biotransformation: reduction and beta-oxidation of the carboxylic acid, with or without N-demethylation, resulted in formation of dihydro trichostatic acid and dinor dihydro trichostatic acids. HPLC fractions corresponding to trichostatin A and N-demethylated trichostatin A exhibited histone deacetylase-inhibitory activity; no other fractions were biologically active. We conclude that trichostatin A is rapidly and extensively metabolized in vivo following intraperitoneal administration to mice, and N-demethylation does not compromise histone deacetylase-inhibitory activity.
Asunto(s)
Inhibidores de Histona Desacetilasas , Ácidos Hidroxámicos/administración & dosificación , Ácidos Hidroxámicos/sangre , Animales , Cromatografía Líquida de Alta Presión/métodos , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/sangre , Inhibidores Enzimáticos/farmacocinética , Femenino , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/farmacocinética , Inyecciones Intraperitoneales , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas WistarRESUMEN
Germ-line mutations of the CDKN2A tumour suppressor gene have been reported in association with familial melanoma, sporadic melanoma with multiple primary lesions and also pancreatic cancer. We studied the hypothesis that patients with melanoma and additional unrelated cancers may harbour mutations in the CDKN2A gene. Twenty seven patients with histologically confirmed melanoma who also had additional cancers such as breast, colorectal, lymphoma and other neoplasms were studied. We also examined 17 additional patients, 13 of whom had a first-degree relative with melanoma and four who had two or more primary melanomas. Some patients belonged to more than one of these categories. No mutations of the CDKN2A tumour suppressor gene were detected among patients with melanoma and additional cancers. The previously described Met53Ile CDKN2A mutation located in exon 2 was detected in a female patient with melanoma metastatic to the regional lymph nodes, multiple primary cutaneous lesions, atypical naevi and a first-degree relative with melanoma. The studied cohort is too small for firm conclusions. However, it would appear that melanoma and additional, apparently unrelated, cancers developing in the same individual are likely to be related to a combination of low-risk susceptibility genes and environmental factors.
Asunto(s)
ADN de Neoplasias/análisis , Genes p16 , Melanoma/genética , Neoplasias Primarias Múltiples/genética , Neoplasias Cutáneas/genética , Adulto , Anciano , Anciano de 80 o más Años , Cromosomas Humanos Par 9 , Análisis Mutacional de ADN , Femenino , Genes Supresores de Tumor , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Detection of melanoma cells in the peripheral blood has been facilitated by the reverse transcriptase-polymerase chain reaction (RT-PCR), but their presence is of uncertain importance in the evolution of the disease. We studied the detection of melanoma cells using RT-PCR in the peripheral blood of 21 patients, four with regional lymph node metastases (American Joint Committee on Cancer [AJCC] stage III) and 17 with disseminated disease (AJCC stage IV). RNA was extracted from 10 ml of heparinized blood following density gradient centrifugation and converted into cDNA for PCR analysis. Assay sensitivity of 10 cells in 10(7) mononuclear cells and granulocytes obtained from 10 ml of peripheral blood was achieved using the G361 and C32 melanoma cell lines. Tyrosinase mRNA was not detected in control samples from healthy volunteers or patients with non-malignant disease. Six patients (one stage III, five stage IV) tested positive for tyrosinase mRNA (28.6%); with one exception, all patients were receiving chemotherapy at the time of sampling. Of the six positive results, three were from patients who initially tested negative but were subsequently positive after a 3-4 week interval. The low detection rates of melanoma cells in the peripheral blood of patients with widely disseminated disease is consistent with recent reports and correlates poorly with the clinical stage of melanoma. This may be partly explained by the clinically observed intermittent and random evolution of melanoma metastases.