RESUMEN
The aromatic fatty acid phenylacetate (PA) and its analogs have come under intense investigation due to their ability to cause the growth arrest of a variety of neoplasia, including human breast cancer. We have determined that PA and its halide derivative 4-chlorophenylacetate (4-CPA) showed marked antiproliferative activity on 3 of 6 human breast cancer cell lines tested. Interestingly, the 3 cell lines that were growth inhibited by PA and 4-CPA were estrogen receptor (ER) positive (T47-D, MCF-7 and ZR-75-1) whereas those that were little affected by these compounds were ER-negative (MDA-MB-157, MDA-MB-231 and SK-Br-3). Dose response studies indicated that 4-CPA inhibited the growth of the sensitive (ER+) cell lines with a potency 3-4 times that of PA. These findings suggest that there is "cross-talk" between the PA and estrogen signaling pathways such that PA can directly inhibit estrogen-dependent events. This hypothesis was directly tested in vitro using ER+ MCF-7 cells that were stably transfected with a luciferase reporter construct driven by the full length (1745 bp) cyclin D1 promoter (MCF-7-D1). Our experiments with MCF-7-D1 cells indicated that PA and 4-CPA inhibited basal and estrogen-induced reporter gene activity by up to 90%, resulting in almost complete elimination of estrogen-dependent cyclin D1 gene activation. Using a reporter gene construct (ERE(V)-tk-Luc) containing a canonical estrogen response element that was transiently transfected into MCF-7 and MDA-MB-231 cells, we have also demonstrated inhibition of promoter activity by PA and 4-CPA that was directly mediated by blockage of activity through the ERE. Taken together, these findings indicate that PA analogs possess potent antiestrogen properties that may, at least partly, account for their antiproliferative effects on ER+ breast cancer cells. The data suggests a novel mechanism of action that might bypass some of the limitations of conventional antiestrogen therapy.
Asunto(s)
Neoplasias de la Mama/patología , Estrógenos/farmacología , Expresión Génica/efectos de los fármacos , Fenilacetatos/farmacología , Antimetabolitos Antineoplásicos/farmacología , Neoplasias de la Mama/genética , División Celular/efectos de los fármacos , Ciclina D1/genética , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Regiones Promotoras Genéticas/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Aromatic fatty acids, of which phenylacetate is a prototype, constitute a class of low toxicity drugs with demonstrated antitumor activity in experimental models and in humans. Using in vitro models, we show here a tight correlation between tumor growth arrest by phenylacetate and activation of peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily. In support are the following observations: (a) the efficacy of phenylacetate as a cytostatic agent was correlated with pre-treatment levels of PPARgamma, as documented using established tumor lines and forced expression models; (b) in responsive tumor cells, PPARgamma expression was up-regulated within 2-9 h of treatment preceding increases in p21waf1, a marker of cell cycle arrest; (c) inhibition of mitogen-activated protein kinase, a negative regulator of PPARgamma, enhanced drug activity; and (d) phenylacetate interacted directly with the ligand-binding site of PPARgamma and activated its transcriptional function. The ability to bind and activate PPARgamma was common to biologically active analogues of phenylacetate and corresponded to their potency as antitumor agents (phenylacetate < phenylbutyrate < p-chloro-phenylacetate < p-iodo-phenylbutyrate), whereas an inactive derivative, phenylacetylglutamine, had no effect on PPARgamma. These findings point to PPARgamma as a novel target in cancer therapy and provide the first identification of ligands that have selective antitumor activity in patients.
Asunto(s)
Neoplasias/prevención & control , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Animales , Antimetabolitos Antineoplásicos/metabolismo , Antimetabolitos Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Fenilacetatos/metabolismo , Fenilacetatos/farmacología , Fosforilación , Unión Proteica , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ensayo de Unión Radioligante , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/efectos de los fármacos , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
The aromatic fatty acid phenylbutyrate (PB) induces cytostasis, differentiation, and apoptosis in primary myeloid leukemic cells at clinically achievable concentrations. In the present study, we have investigated the structural and cellular basis for PB-induced cytostasis, using the ML-1 human myeloid leukemia cell line as a model system. PB induced a dose-dependent increase in cells in G1 with a corresponding decrease in cells in S-phase of the cell cycle. At comparable doses, PB induced expression of CD11b, indicating myeloid differentiation. At higher doses, the drug induced apoptosis. The antitumor activity was independent of the aromatic ring, as butyric acid (BA) was of equal or greater potency at producing these biological changes. In contrast, shortening of the fatty acid carbon chain length, as demonstrated with phenylacetate (PA), significantly diminished drug potency. Consistent with their effects on cell cycle, PB and BA, but not PA, induced the cyclin-dependent kinase inhibitor, p21(WAF1/CIP1), and led to the appearance of hypophosphorylated Rb, suggesting a role for p21(WAF1/CIP1) in PB-induced cytostasis. Therefore, it appears that the fatty acid moiety of PB, rather than its aromatic ring, is critical for its activity in myeloid leukemic cells. These data provide a potential mechanistic basis for the increased potency of PB over PA previously demonstrated in primary leukemic samples, and support the further clinical development of PB in the treatment of hematologic malignancies.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Fase G1/efectos de los fármacos , Leucemia Mieloide/tratamiento farmacológico , Leucemia Mieloide/patología , Fenilbutiratos/farmacología , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Fenilbutiratos/química , Fenilbutiratos/uso terapéutico , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Although Burkitt's lymphoma (BL) is a readily treated malignancy, recurrences, as well as disease arising in immunosuppressed patients, are notoriously resistant to conventional therapeutic approaches. The EBV is associated with a significant proportion of these lymphomas that evade immune surveillance through decreased expression of both viral and cellular antigens. Increasing the immunogenicity of BL cells may, therefore, represent a potentially beneficial therapeutic maneuver. Using in vitro models of EBV-transformed lymphoblastoid as well as BL cell lines, we demonstrate increased expression of genes coding for HLA class I and EBV latent proteins by the differentiation inducer phenylbutyrate (PB). The aromatic fatty acid also caused cytostasis associated with sustained declines in c-myc expression, a direct antitumor effect that was independent of the EBV status. We conclude, therefore, that differentiation therapy of BL with PB may lead to growth arrest with increased tumor immunogenicity in vivo. The findings may have clinical relevance because the in vitro activity has been observed with PB concentrations that are well tolerated and nonimmunosuppressive in humans, a desirable feature for the different patient populations afflicted with this disease.
Asunto(s)
Linfoma de Burkitt/patología , Linfoma de Burkitt/virología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Herpesvirus Humano 4/genética , Fenilbutiratos/farmacología , Linfoma de Burkitt/metabolismo , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , ADN Viral/biosíntesis , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Herpesvirus Humano 4/crecimiento & desarrollo , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Células Tumorales Cultivadas , Regulación hacia Arriba , Proteínas de la Matriz Viral/biosíntesis , Activación Viral/efectos de los fármacosAsunto(s)
Antineoplásicos/farmacología , Inhibidores de Histona Desacetilasas , Leucemia Promielocítica Aguda/tratamiento farmacológico , Fenilbutiratos/farmacología , Transcripción Genética/efectos de los fármacos , Tretinoina/farmacología , ADN de Neoplasias/metabolismo , Sinergismo Farmacológico , Histona Desacetilasas/genética , Humanos , Leucemia Promielocítica Aguda/enzimología , Leucemia Promielocítica Aguda/genética , Metilación/efectos de los fármacos , Neovascularización Patológica/prevención & controlRESUMEN
AIMS AND BACKGROUND: Decitabine (5-aza-2'-deoxycytidine) is an S-phase-specific pyrimidine analog with hypomethylation properties. In laboratory models of prostate cancer (PC-3 and DU-145), decitabine induces cellular differentiation and enhanced expression of genes involved in tumor suppression, immunogenicity, and programmed cell death. METHODS: We conducted a phase II study of decitabine in 14 men with progressive, metastatic prostate cancer recurrent after total androgen blockade and flutamide withdrawal. Decitabine was administered at a dose of 75 mg/m2/dose i.v. as a 1 hour infusion every 8 hours for three doses. Cycles of therapy were repeated every 5 to 8 weeks to allow for resolution of toxicity. RESULTS: Two of 12 patients evaluable for response had stable disease with a time to progression of more than 10 weeks. This activity was seen in 2 of 3 African-American patients. Toxicity was similar to previously reported experience. No significant changes in urinary concentrations of the angiogenic factor bFGF, a potential biomarker of tumor activity, were identified over time in 7 unselected patients with progressive disease. CONCLUSIONS: We conclude that decitabine is a well tolerated regimen with modest clinical activity against hormone-independent prostate cancer. Further investigations in patients of African-American origin may be warranted.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Azacitidina/análogos & derivados , Biomarcadores de Tumor/sangre , Metilasas de Modificación del ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/uso terapéutico , Factor 2 de Crecimiento de Fibroblastos/sangre , Neoplasias de la Próstata/tratamiento farmacológico , Anciano , Azacitidina/uso terapéutico , Decitabina , Humanos , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Neoplasias de la Próstata/sangre , Resultado del TratamientoRESUMEN
The aromatic fatty acids phenylacetate (PA) and phenylbutyrate (PB) are novel antitumour agents currently under clinical evaluation. Their ability to induce tumour differentiation in laboratory models and their low clinical toxicity profile makes them promising candidates for combination with conventional therapies. In the present studies, we characterized the interactions between these aromatic fatty acids and radiation, using as a model cell lines derived from cancers of the prostate, breast, brain and colon. Analysis of the radiation response of the tumour lines using the linear-quadratic model, demonstrated that cellular exposure to pharmacological, non-toxic concentrations of either PA or PB resulted in time-dependent and contrasting changes in radiation response. While drug pretreatment for 24 h reduced radiation sensitivity (significant alterations in both alpha and beta parameters), pre treatment for 72 h significantly increased radiosensitivity (significant alterations in alpha and beta parameters). In replicating tumour cells, these changes were accompanied by a gradual G1-phase arrest. Cytostasis alone, however, could not explain radiosensitization, as similar alterations in radiation response were documented also in non-cycling cells. Modulation of tumour radiobiology by PA and PB was tightly correlated with early rise followed by decline in intracellular glutathione levels and the activity of antioxidant enzymes such as catalase, superoxide dismutase, glutathione reductase, glutathione peroxidase and glutathione S-transferase. Although in vitro findings identify the aromatic fatty acids PA and PB as a new class of non-toxic modulators of radiation response, the antagonistic effect of these compounds on radiation response needs further examination. Our data strongly suggest that for PA or PB to have a role in clinical radiotherapy, appropriate scheduling of combination therapies must take into account their time-dependent effects in order to achieve clinical radiosensitization.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Fenilacetatos/farmacología , Fenilbutiratos/farmacología , Tolerancia a Radiación/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Diferenciación Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Humanos , Células Tumorales CultivadasRESUMEN
Phenylacetate and analogs represent a new class of pleiotropic growth regulators that alter tumor cell biology by affecting gene expression at both the transcriptional and post transcriptional levels. Based on these findings, NaPA and NaPB entered clinical trials at the National Cancer Institute. Ongoing phase I studies with NaPA, involving adults with prostate and brain cancer, have confirmed that therapeutic levels can be achieved with no significant toxicities, and provide preliminary evidence for benefit to patients with advanced disease (Thibault et al., submitted).
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Diferenciación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Fenilacetatos/farmacología , Fenilacetatos/uso terapéutico , Fenilbutiratos/farmacología , Adulto , Antimetabolitos Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Ensayos Clínicos Fase I como Asunto , Humanos , Masculino , Neoplasias/tratamiento farmacológico , Fenilbutiratos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Transcripción Genética/efectos de los fármacos , Células Tumorales CultivadasAsunto(s)
Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Colesterol/biosíntesis , Ácido Mevalónico/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Terpenos/farmacología , Adenocarcinoma , Neoplasias Encefálicas , Neoplasias de la Mama , Radioisótopos de Cobalto , Neoplasias del Colon , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/biosíntesis , Ciclohexenos , Relación Dosis-Respuesta en la Radiación , Femenino , Fibrosarcoma , Rayos gamma , Genes ras/efectos de los fármacos , Genes ras/efectos de la radiación , Glioblastoma , Humanos , Limoneno , Neoplasias Pulmonares , Masculino , Ácido Mevalónico/antagonistas & inhibidores , Neoplasias de la Próstata , Células Tumorales CultivadasRESUMEN
Sodium phenylacetate (PA) and sodium phenylbutyrate (PB) are aromatic fatty acids that can effect differentiation in a variety of cell lines at doses that may be clinically attainable. We have studied the impact of these two agents on lineage- and differentiation stage-specific antigen expression, proliferation, apoptosis, and clonogenic cell survival in primary cultures of bone marrow samples from patients with myeloid neoplasms at presentation and in remission and from normal volunteers. PB inhibited the proliferation of primary acute myeloid leukemia cells in suspension culture with an ID50 of 6.6 mM, similar to its ED50 in cell lines. At higher doses (>/=5 mM), PB also induced apoptosis. PB inhibited clonogenic leukemia cell growth with a median ID50 of less than 2 mM; however, colony-forming units-granulocyte/macrophage from patients with myelodysplasia and normal volunteers were inhibited with a similar ID50. In contrast to PB, its metabolite PA had no significant effect on either acute myeloid leukemia proliferation or apoptosis. Expression of the monocytic marker CD14 was increased in monocytic and myelomonocytic leukemias in response to PB, and to a lesser extent, PA. Surprisingly, both agents appeared to increase expression of the progenitor cell antigen CD34, as well as the DR locus of the human leukocyte antigen. These data indicate that PB, but not its metabolite PA, has significant cytostatic and differentiating activity against primary neoplastic myeloid cells at doses that may be achievable clinically.
Asunto(s)
Leucemia Mieloide/patología , Fenilacetatos/farmacología , Fenilbutiratos/farmacología , Enfermedad Aguda , Antígenos de Diferenciación/biosíntesis , Antígenos de Diferenciación/genética , Antígenos de Neoplasias/biosíntesis , Antígenos de Neoplasias/genética , Apoptosis/efectos de los fármacos , Médula Ósea/patología , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Linaje de la Célula , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Humanos , Síndromes Mielodisplásicos/patología , Células Tumorales Cultivadas/efectos de los fármacos , Ensayo de Tumor de Célula MadreRESUMEN
Phenylacetate (PA) and related aromatic fatty acids constitute a novel class of relatively nontoxic antineoplastic agents. These compounds induce tumor cytostasis and growth inhibition and differentiation of cancer cells, but little is known regarding the molecular events mediating these biological effects. Using human breast carcinoma MCF-7 cells as a model, we show here that PA-induced growth arrest is associated with enhanced expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 and dephosphorylation of the retinoblastoma protein (pRB). The induction of p21WAF1/CIP1 mRNA by PA was independent of the cellular p53 status. To directly assess the contribution of p21Waf1/Cip1 to PA-mediated cytostasis, we compared the effects of PA in parental MCF-7 cells and cells expressing reduced levels of p21Waf1/Cip1 protein (clones AS.3 and AS.4), accomplished through constitutive expression of antisense p21Waf1/Cip1 transcripts. In contrast to parental cells, AS.3 and AS.4 cells did not show reduced pRB phosphorylation following PA treatment, indicating that p21Waf1/Cip1 induction by PA is required for dephosphorylation (inactivation) of pRB, a known mediator of cell cycle control. A prominent role for p21Waf1/Cip1 in mediating PA-induced growth arrest was further supported by the demonstration that embryonal fibroblasts derived from a p21WAF1/CIP1 knockout mouse (p21-/- mouse embryonal fibroblasts) did not growth arrest following PA treatment, whereas PA effectively induced p21WAF1/CIP1 mRNA and growth inhibition of the wild-type mouse embryonal fibroblasts. Taken together, our findings strongly support a role for p21Waf1/Cip1 in the PA-mediated inhibition of cell growth.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Quinasas CDC2-CDC28 , Ciclinas/metabolismo , Inhibidores Enzimáticos/metabolismo , Fenilacetatos/farmacología , Animales , Elementos sin Sentido (Genética) , Neoplasias de la Mama , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , División Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/genética , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Ratones , Ratones Noqueados , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Proteína de Retinoblastoma/metabolismo , Transducción de Señal/fisiología , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Phenylacetate and phenylbutyrate, two novel inducers of tumor cytostasis and differentiation, are currently in clinical trials for the treatment of cancer in adults. The purpose of our study was to evaluate the plasma protein-binding characteristics of phenylacetate and phenylbutyrate in the plasma of normal volunteers and that of patients with cancer. Drug plasma protein-binding analysis was examined using three separate devices: a micropartition system and two equilibrium dialysis systems, all of which exhibited similar results. Phenylacetate and phenylbutyrate concentrations were determined by high-performance liquid chromatography. Both drugs exhibited concentration-dependent binding. Our results showed sodium phenylacetate to have a higher free fraction than sodium phenylbutyrate at corresponding concentrations (> 0.442 +/- 0.008 and > 0.188 +/- 0.001, respectively). Plasma pH did not greatly affect protein binding of either drug. As albumin concentration decreased, an increase in free fraction of both drugs was observed, however alpha 1-acid glyco-protein showed no change in free fraction as its concentration increased. Patients with cancer with lower levels of albumin showed an increase in free fraction with both phenylacetate and phenylbutyrate. When phenylacetate and phenylbutyrate were added together in plasma, the free fraction of phenylacetate increased, whereas the phenylbutyrate free fraction slightly decreased. We conclude that phenylacetate and phenylbutyrate have high free fractions that change with varying albumin levels and when both phenylacetate and phenylbutyrate are present together in plasma.
Asunto(s)
Antimetabolitos Antineoplásicos/sangre , Fenilacetatos/sangre , Fenilbutiratos/sangre , Adulto , Antimetabolitos Antineoplásicos/uso terapéutico , Humanos , Concentración de Iones de Hidrógeno , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Fenilacetatos/uso terapéutico , Fenilbutiratos/uso terapéutico , Unión Proteica , Albúmina Sérica/metabolismoRESUMEN
Phenylbutyrate has been shown recently to induce fetal hemoglobin (HbF) production in patients with sickle cell anemia and beta thalassemia. We have now examined related aromatic fatty acids in order to define the range of active structures and identify plausible mechanisms of action. Structure-function analysis revealed that for effective stimulation of HbF in erythroid precursors: (1) the ideal length for the aliphatic side chain is four carbons; (2) oxygen or sulfur substitutions in the carboxylic chain are allowed, as evidenced by the equal or increased activity of phenoxypropionate, benzylthioglycolate, and benzyloxyacetate compared with phenylbutyrate; and (3) blocking the carboxylate group by conversion to the amide form greatly reduces potency. Molecular analysis indicated that the prototype agent, phenylbutyrate, increases HbF production through transcriptional activation of the gamma-globin gene. The latter contains a butyrate responsive promoter known to up-regulate transcription in the presence of short-chain fatty acids of three to five carbons. To determine whether stimulation of an element in this promoter by phenylbutyrate and its analogues might contribute to their mechanism of action, we used a transient expression system involving K562 erythroleukemia cells transfected with a luciferase reporter gene driven by the minimum gamma-globin promoter. Transcriptional activation in this experimental system correlated well with the capacity of an aromatic fatty acid to increase HbF production in erythroid precursors (r = 0.94). Our studies identify potent analogues of phenylbutyrate for the treatment of beta-chain hemoglobinopathies, and suggest that stimulation of a butyrate responsive promoter may be responsible for their activity.
Asunto(s)
Ácidos Grasos/farmacología , Globinas/genética , Fenilbutiratos/farmacología , Células Cultivadas , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Ácidos Grasos/química , Hemoglobina Fetal/biosíntesis , Hemoglobinopatías/sangre , Hemoglobinopatías/tratamiento farmacológico , Hemoglobinopatías/genética , Humanos , Leucemia Eritroblástica Aguda , Fenilacetatos/farmacología , Fenilbutiratos/química , Regiones Promotoras Genéticas/efectos de los fármacos , Relación Estructura-Actividad , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The prostatic-specific antigen (PSA) is the tumor marker most widely relied upon for the monitoring of patients with prostate cancer. Recently, declines in the serum concentrations of PSA have been advocated as a surrogate marker of tumor response in clinical trials of investigational antitumor agents. We examined the hypothesis that this postulate may not apply to the evaluation of drugs such as phenylacetate, a differentiating agent endowed with mechanisms of action different from those of classic cytotoxic chemotherapy. Using human prostatic carcinoma LNCaP cells as a model, we show that phenylacetate induces PSA production despite inhibition of tumor cell proliferation. Incubation of LNCaP cultures with cytostatic doses of phenylacetate (3-10 mM) resulted in a three- to fourfold increase in PSA secretion per cell. This appears to result from upregulation of PSA gene expression, as indicated by elevated PSA mRNA steady-state levels in treated cells. The increase in PSA production per cell was confirmed in rats bearing subcutaneous LNCaP tumor implants that were treated systemically with phenylacetate. Further comparative studies indicate that upregulation of PSA is common to various differentiation inducers, including all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, and butyrate but is not induced by other antitumor agents of clinical interest such as suramin. We conclude that declines in PSA may be treatment specific and that the exclusive use of this criterion as a marker of disease response may mislead the proper evaluation of differentiating agents in prostate cancer patients.
Asunto(s)
Carcinoma/metabolismo , Fenilacetatos/farmacología , Antígeno Prostático Específico/biosíntesis , Neoplasias de la Próstata/metabolismo , Animales , Carcinoma/patología , Diferenciación Celular , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Antígeno Prostático Específico/genética , Neoplasias de la Próstata/patología , ARN Mensajero/metabolismo , Ratas , Células Tumorales CultivadasRESUMEN
The aromatic fatty acid phenylacetate and its analogs induce tumor cytostasis and differentiation in experimental models. Although the underlying mechanisms of action are not clear, effects on lipid metabolism are evident. We have now examined whether these compounds, structurally similar to the peroxisome proliferator clofibrate, affect the human peroxisome proliferator-activated receptor (hPPAR), a homolog of the rodent PPAR alpha, a transcriptional factor regulating lipid metabolism and cell growth. Gene transfer experiments showed activation of hPPAR, evident by the increased expression of the reporter gene chloramphenicol acetyltransferase linked to PPAR-response element from either the rat acyl-CoA oxidase or rabbit CYP4A6 genes. The relative potency of tested drugs in the co-transfection assay was: 4-iodophenylbutyrate > 4-chlorophenylbutyrate > clofibrate > phenylbutyrate > naphthylacetate > 2,4-D > 4-chlorophenylacetate > phenylacetate >> indoleacetate. Phenylacetylglutamine, in which the carboxylic acid is blocked, was inactive. The ability of the aromatic fatty acids to activate PPAR was confirmed in vivo, as CYP4A mRNA levels increased in hepatocytes of treated rats. Further studies using human prostate carcinoma, melanoma, and glioblastoma cell lines showed a tight correlation between drug-induced cytostasis, increased expression of the endogenous hPPAR, and receptor activation documented in the gene-transfer model. These results identify phenylacetate and its analogs as a new class of aromatic fatty acids capable of activating hPPAR, and suggest that this nuclear receptor may mediate tumor cytostasis induced by these drugs.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Hígado/efectos de los fármacos , Fenilacetatos/farmacología , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Factores de Transcripción/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Ácidos Grasos/química , Humanos , Conejos , Ratas , Transfección , Células Tumorales CultivadasRESUMEN
The refractoriness of prostate cancer to androgen suppression is the landmark of clinically aggressive disease. In this study, the androgen-dependent LNCaP prostate cancer cells were transfected with the mutated c-Ha-ras gene from the T24 human bladder cancer. The derivative clone overexpressing T24-ras (LNCaP(T24-ras)) proliferated in androgen-depleted medium and showed increased growth. Protein isoprenylation and p21ras farnesylation in LNCaP(T24-ras) cells were tested in the presence of phenylacetate to document a possible relationship with the drug-induced inhibition of cell proliferation. Phenylacetate is a differentiation inducer that down-regulates in vitro the expression of the myc oncogene and activates the human peroxisome proliferator-activated nuclear receptor involved in cell growth regulation. The drug inhibited protein isoprenylation and p21ras farnesylation in LNCaP(T24-ras) cells; IC50 values were 3.1 and 3.3 mM, respectively, compared with controls. The drug reduced the cellular levels of endogenous farnesyl-PP (mean IC50 = 3.5 mM) and inhibited activation of the p21ras downstream target, p42(MAPK)/ERK2. LNCaP(T24-ras) was more sensitive than the parental line to both growth inhibition (mean IC50 = 3.01 and 7.1 mM, respectively) and apoptosis by phenylacetate. Exogenous farnesyl- and geranylgeranyl-PP indeed reduced the effects of the drug on proliferation and apoptosis in LNCaP(T24-ras) cells. In conclusion, the inhibition of protein isoprenylation and p21ras farnesylation by phenylacetate resulted in increased chemosensitivity of the androgen-independent LNCaP(T24-ras) cells compared with LNCaP, and this effect might contribute to the pharmacological activity of the drug.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Genes ras , Fenilacetatos/farmacología , Neoplasias de la Próstata/patología , Prenilación de Proteína/efectos de los fármacos , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Humanos , Masculino , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , TransfecciónRESUMEN
Cytotoxic chemotherapies often give rise to multidrug resistance, which remains a major problem in cancer management. In pursuit of alternative treatments for chemoresistant tumor cells, we tested the response of multidrug-resistant (MDR) tumor cell lines to the aromatic fatty acids phenylacetate (PA) and phenylbutyrate (PB), two differentiation inducers currently in clinical trials. Both compounds induced cytostasis and maturation of multidrug-resistant breast, ovarian, and colon carcinoma cells with no significant effect on cell viability. In contrast to their poor response to doxorubicin, the MDR cells were generally more sensitive to growth arrest by PA and PB than their parental counterparts. The aromatic fatty acids, like the differentiation-inducing aliphatic fatty acid butyrate, up-regulated mdr-1 gene expression. However, while butyrate increased multidrug resistance, PA and PB potentiated the cytotoxic activity of doxorubicin against MDR cells. The latter was associated with time-dependent declines in glutathione levels and in the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione S-transferase, the antioxidant enzymes implicated in cell resistance to free radical-based therapies. Taken together, our in vitro data indicate that PA and PB, differentiation inducers of the aromatic fatty acid class, may provide an alternative approach to the treatment of MDR tumors.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Fenilacetatos/farmacología , Fenilbutiratos/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Catalasa/metabolismo , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos , Glutatión/metabolismo , Humanos , Células Tumorales Cultivadas , Verapamilo/farmacologíaRESUMEN
PURPOSE: Benign prostatic hyperplasia (BPH) is the most common proliferative disease affecting men, and the obstructive uropathy it causes results in serious morbidity and financial cost. Phenylacetate (PA) is a small molecule that is a product of phenylalanine metabolism and is normally present in the mammalian circulation at very low levels. It has long been safely used in humans to treat the hyperammonemia resulting from urea synthesis disorders and liver failure. It has recently been investigated as an anticancer agent because it decreased growth and increased differentiation of a variety of human neoplasms, including prostate cancer in which a phase I trial has recently been completed. MATERIALS AND METHODS: Because of PA's growth-inhibitory effects on a variety of cell lines and the idea that BPH is due to a reawakening of embryonic-like inductive activity in prostatic stromal cells, which then induce development of epithelial nodules, we examined the effect of PA on serum-free organ cultures of developing rat prostates. RESULTS: We found that PA markedly decreased rat prostatic growth and ductal morphogenesis at concentrations that have previously been well tolerated in patients. In ventral prostates grown for 7 days in organ culture, histodifferentiation was inhibited as measured by a marked decrease in ductal lumen formation and ductal branching morphogenesis. This inhibition of differentiation was confirmed by using cytokeratin antibodies specific for basal and luminal cells. Synthesis of DNA was also significantly decreased per organ with PA. The growth inhibitory effects of PA were reversible, and the mechanism did not appear to be due to glutamine or glycine deprivation, or androgen receptor inhibition. CONCLUSIONS: In common with earlier studies, we found that PA inhibits prostatic growth; however, in our organ culture system, differentiation was also largely inhibited. These studies indicate that there may be a role for PA in treating BPH or in elucidating the mechanism by which it occurs since BPH apparently involves the neoformation of ductal-acinar tissue in aged men via mechanisms fundamentally similar if not identical to those in fetal prostatic development.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Fenilacetatos/farmacología , Próstata/efectos de los fármacos , Animales , Animales Recién Nacidos , Diferenciación Celular/efectos de los fármacos , Inmunohistoquímica , Masculino , Técnicas de Cultivo de Órganos , Próstata/citología , Hiperplasia Prostática/tratamiento farmacológico , Ratas , Ratas Sprague-DawleyRESUMEN
Lovastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (the major regulatory enzyme of the mevalonate pathway of cholesterol synthesis), displays antitumor activity in experimental models. We therefore conducted a Phase I trial to characterize the tolerability of lovastatin administered at progressively higher doses to cancer patients. From January 1992 to July 1994, 88 patients with solid tumors (median age, 57 +/- 14 years) were treated p.o. with 7-day courses of lovastatin given monthly at doses ranging from 2 to 45 mg/kg/day. The inhibitory effects of lovastatin were monitored through serum concentrations of cholesterol and ubiquinone, two end products of the mevalonate pathway. Concentrations of lovastatin and its active metabolites were also determined, by bioassay, in the serum of selected patients. Cyclical treatment with lovastatin markedly inhibited the mevalonate pathway, evidenced by reductions in both cholesterol and ubiquinone concentrations, by up to 43 and 49% of pretreatment values, respectively. The effect was transient, however, and its magnitude appeared to be dose independent. Drug concentrations reached up to 3.9 micrometer and were in the range associated with antiproliferative activity in vitro. Myopathy was the dose-limiting toxicity. Other toxicities included nausea, diarrhea, and fatigue. Treatment with ubiquinone was associated with reversal of lovastatin-induced myopathy, and its prophylactic administration prevented the development of this toxicity in a cohort of 56 patients. One minor response was documented in a patient with recurrent high-grade glioma. Lovastatin given p.o. at a dose of 25 mg/kg daily for 7 consecutive days is well tolerated. The occurrence of myopathy, the dose-limiting toxicity, can be prevented by ubiquinone supplementation. To improve on the transient inhibitory activity of this dosing regimen on the mevalonate pathway, alternative schedules based on uninterrupted administration of lovastatin should also be studied.
Asunto(s)
Antineoplásicos/efectos adversos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/efectos adversos , Lovastatina/efectos adversos , Neoplasias/tratamiento farmacológico , Adulto , Anciano , Femenino , Humanos , Lovastatina/farmacología , Masculino , Persona de Mediana Edad , Neoplasias/sangre , Ubiquinona/sangreRESUMEN
Malignant gliomas, the most common form of primary brain tumors, are highly dependent on the mevalonate (MVA) pathway for the synthesis of lipid moieties critical to cell replication. Human glioblastoma cells were found to be uniquely vulnerable to growth arrest by lovastatin, a competitive inhibitor of the enzyme regulating MVA synthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase. The sodium salt of phenylacetic acid (NaPA), an inhibitor of MVA-pyrophosphate decarboxylase, the enzyme that controls MVA use, acted synergistically with lovastatin to suppress malignant growth. When used at pharmacologically attainable concentrations, the two compounds induced profound cytostasis and loss of malignant properties such as invasiveness and expression of the transforming growth factor-beta 2 gene, coding for a potent immunosuppressive cytokine. Supplementation with exogenous ubiquinone, an end product of the MVA pathway, failed to rescue the cells, suggesting that decreased synthesis of intermediary products are responsible for the antitumor effects observed. In addition to blocking the MVA pathway, lovastatin alone and in combination with NaPA increased the expression of the peroxisome proliferator-activated receptor, a transcription factor implicated in the control of lipid metabolism, cell growth, and differentiation. Our results indicate that targeting lipid metabolism with lovastatin, used alone or in combination with the aromatic fatty acid NaPA, may offer a novel approach to the treatment of malignant gliomas.