RESUMO
Retinoids, vitamin A analogues that bind to retinoic acid receptor (RAR) or retinoid X receptor (RXR), play important roles in regulating cell proliferation, apoptosis, and differentiation. Recently, RXR-selective ligands, also referred to as rexinoids, have been investigated as potential chemopreventive agents for breast cancer. Our previous studies demonstrated that the rexinoid bexarotene significantly prevented ER-negative mammary tumourigenesis with less toxicity than naturally occurring retinoids in animal models. To determine whether bexarotene prevents cancer at the early stages during the multistage process of mammary carcinogenesis, we treated MMTV-erbB2 mice with bexarotene for 2 or 4 months. The development of preinvasive mammary lesions such as hyperplasias and carcinoma-in-situ was significantly inhibited. This inhibition was associated with reduced proliferation, but no induction of apoptosis. We also examined the regulation of a number of rexinoid-modulated genes including critical growth and cell cycle regulating genes using breast cell lines and mammary gland samples from mice treated with rexinoids. We showed that two of these genes (DHRS3 and DEC2) were modulated by bexarotene both in vitro and in vivo. Identification of these rexinoid-modulated genes will help us understand the mechanism by which rexinoid prevents cancer. Such rexinoid-regulated genes also represent potential biomarkers to assess the response of rexinoid treatment in clinical trials.
Assuntos
Anticarcinógenos/uso terapêutico , Genes erbB-2 , Neoplasias Mamárias Experimentais/prevenção & controle , Vírus do Tumor Mamário do Camundongo/genética , Lesões Pré-Cancerosas/prevenção & controle , Tetra-Hidronaftalenos/uso terapêutico , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Bexaroteno , Proliferação de Células/efeitos dos fármacos , Ciclina D , Ciclinas/genética , Feminino , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos , Camundongos TransgênicosRESUMO
The present study determined the influence of a retinoid X receptor agonist bexarotene on angiogenesis and metastasis in solid tumours. In the experimental lung metastasis xenograft models, treatment with bexarotene inhibited the development of the lung tumour nodule formation compared to control. In vivo angiogenesis assay utilising gelfoam sponges, bexarotene reduced angiogenesis in sponges containing vascular endothelial growth factor, epidermal growth factor and basic fibroblast growth factor to various extent. To determine the basis of these observations, human breast and non-small-cell lung cancer cells were subjected to migration and invasion assays in the presence of bexarotene. Our data showed that bexarotene decrease migration and invasiveness of tumour cells in a dose-dependent manner. Furthermore, bexarotene inhibited angiogenesis by directly inhibiting human umbilical vein endothelial cell growth and indirectly inhibiting tumour cell-mediated migration of human umbilical vein endothelial cells through Matrigel matrix. Analysis of tumour-conditioned medium indicated that bexarotene decreased the secretion of angiogenic factors and matrix metalloproteinases and increased the tissue inhibitor of matrix metalloproteinases. The ability of bexarotene to inhibit angiogenesis and metastasis was dependent on activation of its heterodimerisation partner peroxisome proliferator-activated receptor gamma. Collectively, our results suggest a role of bexarotene in treatment of angiogenesis and metastasis in solid tumours.
Assuntos
Anticarcinógenos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Metástase Neoplásica/prevenção & controle , Neovascularização Patológica/prevenção & controle , Tetra-Hidronaftalenos/farmacologia , Animais , Bexaroteno , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Endoteliais/fisiologia , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Receptores X de Retinoides/agonistas , Transplante Heterólogo , Cordão Umbilical/irrigação sanguíneaRESUMO
Clinical studies have demonstrated that retinoids, including retinol (Vitamin A) and its synthetic derivatives, can eradicate leukoplakia and suppress the formation of squamous cell carcinoma of the head and neck (SCCHN). Nonselective retinoids have been shown to abrogate transcriptional activation of transforming growth factor-alpha (TGF-alpha) and epidermal growth factor receptor (EGFR), which characterize SCCHN. LGD1069 (Targretin) is a potent RXR-selective retinoic acid agonist with a reduced toxicity profile compared with other nonselective retinoids. We examined the effect of LGD1069 (10 microm) on cellular proliferation and expression of putative intermediate biomarker genes including TGF-alpha, EGFR, and RAR-beta in seven SCCHN cell lines. A quantitative reverse transcription-PCR assay using a novel "primer dropping" method was used to determine expression levels of EGFR, TGF-alpha, and RAR-beta before and after treatment with LGD1069 (10 microM). SCCHN proliferation was reduced by a mean of 50% at 4 days in seven SCCHN cell lines after LGD1069 treatment (P < or = 0.05). EGFR expression levels were decreased by a mean of 58.4% (P = 0.007), TGF-alpha levels were decreased by a mean of 28.8% (P = 0.01), and RAR-beta levels were increased by a mean of 60% (P = 0.03). TGF-alpha stimulation of EGFR is associated with constitutive signal transducer and activator of transcription 3 (Stat3) activation in SCCHN. Abrogation of constitutive Stat3 activation was seen with LGD1069 treatment. These results suggest that an RXR-selective retinoic acid decreases SCCHN proliferation in part by interfering with TGF-alpha/EGFR autocrine signaling.
Assuntos
Anticarcinógenos/farmacologia , Receptores ErbB/fisiologia , Tetra-Hidronaftalenos/farmacologia , Fator de Crescimento Transformador alfa/fisiologia , Animais , Bexaroteno , Biomarcadores Tumorais/biossíntese , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/fisiologia , Receptores ErbB/biossíntese , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Camundongos , Camundongos Nus , Receptores do Ácido Retinoico/agonistas , Receptores X de Retinoides , Fator de Transcrição STAT3 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transativadores/fisiologia , Fatores de Transcrição/agonistas , Fator de Crescimento Transformador alfa/biossíntese , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targretin (LGD1069; a high-affinity ligand for the retinoid X receptors) is an efficacious chemotherapeutic and chemopreventive agent in the N-nitroso-N-methylurea-induced rat mammary carcinoma model. To evaluate the molecular action of LGD1069 in mammary carcinoma we have examined gene expression patterns in controls and nonresponding tumors compared with tumors undergoing regression (responding) by LGD1069. When compared with controls or nonresponding tumors, the expression of adipocyte-related genes such as adipocyte P2 (aP2), adipsin, peroxisome proliferator-activated receptor gamma (PPARgamma), and lipoprotein lipase was elevated in LGD1069-responding tumors. Further analysis showed that gene expression changes occurred rapidly, in as little as 6 h, after the first dose of LGD1069. Immunohistochemical analysis showed that aP2 protein was also highly expressed in responding tumors when compared with control or nonresponding tumors. More importantly, aP2 protein was localized in the tumor cells in addition to the adipocytes present in the tumors. Similar changes in gene expression and inhibition in growth were seen in tumor cells (cloned from N-nitroso-N-methylurea-induced carcinoma) exposed to LGD1069 in vitro. These data suggest that tumor regression by LGD1069 involves differentiation induction along the adipocyte lineage.
Assuntos
Adipócitos/efeitos dos fármacos , Anticarcinógenos/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Receptores do Ácido Retinoico/agonistas , Tetra-Hidronaftalenos/farmacologia , Fatores de Transcrição/agonistas , Complexo 2 de Proteínas Adaptadoras , Subunidades alfa do Complexo de Proteínas Adaptadoras , Proteínas Adaptadoras de Transporte Vesicular , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Bexaroteno , Carcinógenos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Clonais , Fator D do Complemento , Feminino , Expressão Gênica/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Ligantes , Lipase Lipoproteica/biossíntese , Lipase Lipoproteica/genética , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Metilnitrosoureia , Ratos , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
The conventional treatment of uterine leiomyomas, or fibroids, with gonadotropin-releasing hormone (GnRH) agonists is often associated with serious side effects, necessitating short-term, palliative use of this therapy. Therefore, we examined a retinoid X receptor (RXR)-selective ligand, LGD1069, as a possible treatment for leiomyoma. LGD1069 has demonstrated efficacy as a chemopreventive agent in the N-nitroso-N-methylurea (NMU)-induced rat mammary carcinoma model and is a therapeutic agent in several epithelial tumor models. Previous studies have shown that it has both antitumor effects and antiestrogenic activity in the rat uterus, suggesting the potential utility of this agent for treatment of hormonally dependent uterine fibroids. The expression of retinoid receptors in tumors and cell lines derived from leiomyomas arising in the Eker rat was confirmed by Northern analysis. After treatment for 4 months with LGD1069, the number of grossly observable tumors was substantially reduced although the total incidence of tumors, including microscopic lesions, remained unaffected, suggesting an effect of the compound on tumor growth kinetics rather than on tumor initiation. Analysis of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and determination of 5-bromo-2-deoxyuridine (BrdU) incorporation indicated that the reduction in grossly observable tumors that occurred in treated animals was mediated by a significant increase in the level of apoptosis rather than a decrease in cell proliferation. These results suggest that LGD1069 may be an effective therapeutic agent for uterine leiomyoma that may inhibit tumor growth and, consequently, alleviate the symptoms associated with this disease.
Assuntos
Antineoplásicos/farmacologia , Leiomioma/tratamento farmacológico , Receptores do Ácido Retinoico/metabolismo , Tetra-Hidronaftalenos/farmacologia , Fatores de Transcrição/metabolismo , Neoplasias Uterinas/tratamento farmacológico , Animais , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Bexaroteno , Divisão Celular/efeitos dos fármacos , Endométrio/efeitos dos fármacos , Feminino , Leiomioma/metabolismo , Leiomioma/patologia , Ligantes , Ratos , Receptores X de Retinoides , Tetra-Hidronaftalenos/metabolismo , Células Tumorais Cultivadas , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia , Vagina/efeitos dos fármacosRESUMO
Retinoids have been investigated as potential agents for the prevention and treatment of human cancers. These compounds play an important role in regulating cell growth, differentiation, and apoptosis. 9-cis-Retinoic acid (9cRA) is a naturally occurring ligand with a high affinity for both the retinoic acid receptors and the retinoid X receptors. We hypothesized that treatment with 9cRA would prevent mammary tumorigenesis in transgenic mice that spontaneously develop mammary tumors. To test this hypothesis, C3(1)-SV40 T antigen (Tag) mice, which develop mammary tumors by the age of 6 months, were treated daily p.o. with vehicle or two different dose levels of 9cRA (10 or 50 mg/kg) from 5 weeks to 6 months of age. Tumor size and number were measured twice each week, and histological samples of normal and malignant tissue were obtained from each mouse at time of sacrifice. Our results demonstrate that 9cRA suppresses mammary tumorigenesis in C3(1)-SV40 Tag-transgenic mice. Time to tumor development was significantly delayed in treated mice; median time to tumor formation for vehicle-treated mice was 140 days versus 167 days for mice treated with 50 mg/kg 9cRA (P = 0.05). In addition, the number of tumors per mouse was reduced by >50% in mice treated with 9cRA (3.43 for vehicle, 2.33 for 10 mg/kg 9cRA, and 1.13 for 50 mg/kg 9cRA, P < or = 0.002). Histological analysis of the mammary glands from vehicle and treated mice demonstrated that 9cRA treatment also did not affect normal mammary gland development. Immunohistochemical staining of normal and malignant breast tissue and Western blot analysis demonstrated that SV40 Tag expression was not affected by treatment with retinoids. Single doses of 10 and 50 mg/kg resulted in peak plasma concentrations of 3.4 and 6.71 microM, respectively. Daily doses of 9cRA for 28 days resulted in plasma concentrations of 0.86 and 1.68 microM, respectively, concentrations consistent with that seen in humans treated with 9cRA in clinical trials. These results demonstrate that 9cRA suppresses mammary carcinogenesis in transgenic mice without any major toxicity and suggest that retinoids are promising agents for the prevention of human breast cancer.
Assuntos
Anticarcinógenos/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Neoplasias Mamárias Experimentais/prevenção & controle , Tretinoína/farmacologia , Alitretinoína , Animais , Antígenos Transformantes de Poliomavirus/biossíntese , Antígenos Transformantes de Poliomavirus/genética , Transformação Celular Neoplásica/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/imunologia , Camundongos , Camundongos Transgênicos , Tretinoína/sangueRESUMO
Retinoids modulate the growth and differentiation of cancer cells presumably by activating gene transcription via the nuclear retinoic acid receptor (RAR) alpha, beta, and gamma and retinoid X receptor (RXR) alpha, beta, and gamma. We analyzed the effects of 38 RAR-selective and RXR-selective retinoids on the proliferation of 10 human head and neck squamous cell carcinoma (HNSCC) cell lines. All of these cell lines expressed constitutively all of the receptor subtypes except RARbeta, which was detected in only two of them. Most of the RAR-selective retinoids inhibited the growth of HNSCC cells to varying degrees, whereas the RXR-selective retinoids showed very weak or no inhibitory effects. Three RAR antagonists suppressed growth inhibition by RAR-selective agonists, as well as by RAR/RXR panagonists such as 9-cis-retinoic acid. Combinations of RXR-selective and RAR-selective retinoids exhibited additive growth-inhibitory effects. Furthermore, we found that CD437, the most potent growth-inhibitory retinoid induced apoptosis and up-regulated the expression of several apoptosis-related genes in HNSCC cells. These results indicate that: (a) retinoid receptors are involved in the growth-inhibitory effects of retinoids; (b) RXR-RAR heterodimers rather than RXR-RXR homodimer are the major mediators of growth inhibition by retinoids in HNSCC cells; and (c) induction of apoptosis can account for one mechanism by which retinoids such as CD437 inhibit the growth of HNSCC cells. Finally, these studies identified several synthetic retinoids, which are much more effective than the natural RAs and can be good candidates for chemoprevention and therapy of head and neck cancers.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Divisão Celular/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Receptores do Ácido Retinoico/antagonistas & inibidores , Retinoides/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Apoptose/genética , Northern Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Análise Mutacional de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Mutação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Ácido Retinoico/agonistas , Receptores do Ácido Retinoico/genética , Receptores X de Retinoides , Fator de Transcrição AP-1/antagonistas & inibidores , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: We have previously shown that a retinoid X receptor (RXR)-selective ligand (a rexinoid), called LGD1069, is highly efficacious in both the chemoprevention and the chemotherapy for N-nitrosomethylurea-induced rat mammary carcinomas. To evaluate a possible role for rexinoids in breast cancer therapy further, we have designed and characterized a novel carcinogen-induced model to mimic the clinical situation in which the tumors of patients stop responding to tamoxifen therapy and develop resistance to this drug. METHODS: Rats with experimentally induced mammary tumors were treated with tamoxifen to select a population with primary tumors that failed to respond completely to the drug. Once the failure of tamoxifen therapy had been established, LGD1069 was added to the treatment regimen, and the tumors in these animals were compared with tumors in a group of animals that remained on tamoxifen alone. RESULTS: LGD1069 in combination with tamoxifen for up to 20 weeks yielded an overall objective response rate of 94% (95% confidence interval [CI] = 86%-100%) (includes complete and partial responses) in primary tumors compared with a rate of 33% (95% CI = 11%-56%) in primary tumors treated with tamoxifen alone, a statistically significant difference (two-sided P<.0001). In addition, the LGD1069 and tamoxifen combination was associated with a statistically significant decrease in total tumor burden (two-sided P =.03). In a second study, tumors that failed to respond to tamoxifen therapy exhibited a 51% (95% CI = 34%-71%) objective response rate when treated with LGD1069 alone for 6 weeks after tamoxifen therapy was withdrawn. CONCLUSION: We have demonstrated that the RXR-selective ligand LGD1069 in combination with tamoxifen is a highly efficacious therapeutic agent for tumors that fail to respond completely to tamoxifen. This finding suggests that rexinoid therapy offers a novel approach to the treatment of breast tumors that may have developed resistance to antihormonal therapies such as tamoxifen.
Assuntos
Anticarcinógenos/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Tamoxifeno/uso terapêutico , Tetra-Hidronaftalenos/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bexaroteno , Carcinógenos , Progressão da Doença , Feminino , Neoplasias Mamárias Experimentais/induzido quimicamente , Metilnitrosoureia , Ratos , Ratos Sprague-Dawley , Falha de Tratamento , Resultado do TratamentoRESUMO
Estrogen receptor (ER)-positive human breast cancer cells are hormonally regulated and are inhibited by retinoids, whereas most ER-negative breast cancer cells are not. Here, we compared retinoid-induced transcriptional activation and growth inhibition in the ER-negative breast cancer cell line MDA-MB-231, stably transfected to express wild-type ER (S30), with that of the ER-positive MCF-7 line and the ER-negative parental line. Retinoids inhibited growth of the ER-expressing S30 clone but not of the parental MDA-MB-231 cells. Unlike a previously reported MDA-MD-231 subclone that was transfected to express a mutated ER (G400V), S30 did not express increased levels of retinoid receptor RNA or protein, nor was there increased binding activity to retinoid-responsive DNA elements. However, stable expression of ER increased retinoid activation of transcription of a retinoic acid (RA) response elements from the low level in MDA-MB-231 to approach the level of MCF-7. The restored growth inhibition and transcriptional regulation by RA were unaffected by treatment with ER agonists or antagonists. Transient expression of ER but not of other nuclear receptors in MDA-MB-231 cells also activated retinoid-induced transcription, showing that this response is specific to ER. Furthermore, the effect of exogenously expressed ER on retinoid response was much greater than that obtained by overexpression of RA receptor alpha and/or retinoid X receptor alpha. Finally, a panel of ER mutants showed that enhancement of retinoid-induced transcriptional activity was dependent on the integrity of the DNA binding domain.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Receptores de Estrogênio/metabolismo , Receptores do Ácido Retinoico/metabolismo , Ativação Transcricional/efeitos dos fármacos , Tretinoína/farmacologia , Divisão Celular/efeitos dos fármacos , Feminino , Humanos , Receptores de Estrogênio/genética , Receptores X de Retinoides , Fatores de Transcrição/metabolismo , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Recently, we reported that LGD1069, a high-affinity ligand for the retinoid X receptors (RXRs), was shown to have an efficacy equivalent to that of tamoxifen (TAM) as a chemopreventive agent in the N-nitroso-N-methylurea-induced rat mammary carcinoma model. Furthermore, LGD1069 was very well tolerated during 13 weeks of chronic therapy with no classic signs of "retinoid-associated" toxicities. Due to the high efficacy and benign profile of this RXR agonist as a suppressor of carcinogenesis, we examined its role as a therapeutic agent on established mammary carcinomas. In the rat mammary carcinoma model, N-nitroso-N-methylurea was used to induce tumors, and the tumors were allowed to grow to an established size prior to initiation of treatment. LGD1069-treated animals showed complete regression in 72% of treated tumors and had a reduced tumor load compared to control. In addition, the combination of LGD1069 and TAM showed increased efficacy over either agent alone. Histopathological analysis showed a reduction of LGD1069-treated tumor malignancy, an increase in differentiation, and a sharp decrease in cellular proliferation compared to vehicle-treated control tumors. These data demonstrate that the RXR-selective ligand LGD1069 is a highly efficacious therapeutic agent for mammary carcinoma and enhances the activity of TAM.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Receptores do Ácido Retinoico/agonistas , Tetra-Hidronaftalenos/uso terapêutico , Fatores de Transcrição/agonistas , Animais , Anticarcinógenos/uso terapêutico , Antineoplásicos Hormonais/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bexaroteno , Divisão Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/patologia , Metilnitrosoureia , Ratos , Ratos Sprague-Dawley , Receptores X de Retinoides , Tamoxifeno/administração & dosagem , Tetra-Hidronaftalenos/administração & dosagemRESUMO
BACKGROUND: Retinoids, which are derivatives of vitamin A, induce differentiation of acute promyelocytic leukemia (APL) cells in vitro and in patients. However, APL cells develop resistance to retinoic acid treatment. Arsenic trioxide (As2O3) can induce clinical remission in patients with APL, including those who have relapsed after retinoic acid treatment, by inducing apoptosis (programmed cell death) of the leukemia cells. In this study, we investigated the molecular mechanisms by which As2O3 induces apoptosis in retinoic acid-sensitive NB4 APL cells, in retinoic acid-resistant derivatives of these cells, and in fresh leukemia cells from patients. METHODS: Apoptosis was assessed by means of DNA fragmentation analyses, TUNEL assays (i.e., deoxyuridine triphosphate labeling of DNA nicks with terminal deoxynucleotidyl transferase), and flow cytometry. Expression of the PML/RAR alpha fusion protein in leukemia cells was assessed by means of western blotting, ligand binding, and immunohistochemistry. Northern blotting and ribonuclease protection assays were used to evaluate changes in gene expression in response to retinoic acid and As2O3 treatment. RESULTS AND CONCLUSIONS: As2O3 induces apoptosis without differentiation in retinoic acid-sensitive and retinoic acid-resistant APL cells at concentrations that are achievable in patients. As2O3 induces loss of the PML/RAR alpha fusion protein in NB4 cells, in retinoic-acid resistant cells derived from them, in fresh APL cells from patients, and in non-APL cells transfected to express this protein. As2O3 and retinoic acid induce different patterns of gene regulation, and they inhibit the phenotypes induced by each other. Understanding the molecular basis of these differences in the effects of As2O3 and retinoic acid may guide the clinical use of arsenic compounds and provide insights into the management of leukemias that do not respond to retinoic acid.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Arsenicais/farmacologia , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/fisiopatologia , Óxidos/farmacologia , Receptores do Ácido Retinoico/efeitos dos fármacos , Tretinoína/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Trióxido de Arsênio , Arsenicais/uso terapêutico , Northern Blotting , Western Blotting , Regulação para Baixo , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Humanos , Óxidos/uso terapêutico , Receptor alfa de Ácido Retinoico , Transglutaminases/metabolismo , Tretinoína/uso terapêutico , Células Tumorais CultivadasRESUMO
Retinoic acid (RA) induces differentiation, followed by apoptosis in acute promyelocytic leukemia (APL) cells, both in vitro and in patients. One problem in understanding these mechanisms is to distinguish molecular events leading to differentiation from those leading to apoptosis. We have identified a leukemic cell line, PLB-985, where RA directly induces apoptosis with no morphologic, genetic, or cell-surface marker evidence of differentiation. These cells differentiate following dimethyl sulfoxide (DMSO), but not RA, treatment. Two-color flow cytometry showed no alteration of the cell cycle after RA treatment, and cell-surface marker analysis of CD11a, CD11b, and CD13 showed no modulation typical of differentiating cells. RNA expression of myeloblastin and transglutaminase, genes regulated by RA-induced differentiation in NB4 cells, was unchanged by RA treatment. Instead, RA induced apoptosis, as shown by typical apoptotic morphological features, genomic DNA laddering, and positive labeling in the TUNEL assay. We found that induction of apoptosis in this model requires a different pattern of retinoid receptor binding and transcriptional activation than is seen in APL cells. As previously described, treatment with retinoid receptor-selective ligands showed that stimulation of RAR alone is sufficient to induce differentiation and apoptosis in NB4 cells, and that stimulation of RXR has no effect on the parameters analyzed. In PLB-985 cells, on the other hand, apoptosis was induced only upon costimulation of both RAR and RXR. Stimulation of either receptor alone had no effect on the cells. Consistent with these findings, bcl-2 RNA and protein levels were downregulated after stimulation of both RAR and RXR, but not with an RAR-specific ligand alone, as in NB4 cells. The expression of several other bcl-2 family members (bcl-X, ich-1, bax, bag, and bak ) and retinoid receptors (RARalpha, RXRalpha, and RXRbeta) was not affected by treatment with RAR- and/or RXR-activating retinoids; RARbeta RNA was undetectable before and after retinoid treatment. Thus, our cell model provides a useful tool in determining the genetic events mediating apoptosis as a response to RA, unobscured by events implicated in differentiation.
Assuntos
Apoptose/efeitos dos fármacos , Ceratolíticos/farmacologia , Leucemia/patologia , Receptores do Ácido Retinoico/fisiologia , Fatores de Transcrição/fisiologia , Tretinoína/farmacologia , Diferenciação Celular , Humanos , Leucemia/fisiopatologia , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Retinoids are promising agents for cancer chemoprevention and therapy. Nuclear retinoic acid receptors (RARs; RARalpha, -beta, and -gamma) and retinoid X receptors (RXRs; RXRalpha, -beta, and -gamma) are thought to mediate most of retinoids' effects on cell growth and differentiation. Because the majority of human non-small cell lung carcinoma (NSCLC) cell lines are resistant to all-trans-retinoic acid, we searched for more potent retinoids. Therefore, we examined the effects of 37 natural and synthetic retinoids that exhibit specific binding to and transactivation of individual RARs or RXRs on the proliferation of eight human NSCLC cell lines. All of these cells expressed mRNAs of the three RXRs; however, they expressed varying levels of RARalpha and RARgamma, and only three of the eight cell lines expressed RARbeta mRNA. Cellular retinoic acid-binding proteins (CRABPs) I and II were detected in one and three of the eight cell lines, respectively. Only 8 of the 37 retinoids exhibited growth-inhibitory activity (IC50, < 10 microM) against at least two of the eight NSCLC cell lines. The active retinoids included one (TD550) of five RARalpha-selective, one (Ch55) of three RARbeta-selective, three (CD437, CD2325, and SR11364) of six RARgamma-selective, and one (CD271) of four RARbeta/gamma-selective retinoids. The potency of these retinoids was low (IC50, > 1 microM), except for CD437, which was very potent (IC50, 0.1-0.5 microM). The six RXR-selective retinoids were mostly inactive even at 10 microM. However, combinations of RAR-selective and RXR-selective retinoids exhibited additive effects. There appeared to be no simple correlation among the histological type of the NSCLC (adeno- or squamous), the levels of nuclear receptors or CRABPs, and the response of the cells to the growth-inhibitory effects of retinoids. Nevertheless, in contrast with former studies with natural retinoids, these results suggest that several synthetic retinoids do exhibit inhibitory activity against NSCLC cells, and some of them may be useful clinically.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Proteínas de Neoplasias/metabolismo , Receptores do Ácido Retinoico/metabolismo , Retinoides/farmacologia , Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Divisão Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Receptor alfa de Ácido Retinoico , Receptores X de Retinoides , Retinoides/química , Fator de Transcrição AP-1/antagonistas & inibidores , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Receptor gama de Ácido RetinoicoRESUMO
The unique t(15;17) of acute promyelocytic leukemia (APL) fuses the PML gene with the retinoic acid receptor alpha (RAR alpha) gene. Although retinoic acid (RA) inhibits cell growth and induces differentiation in human APL cells, resistance to RA develops both in vitro and in patients. We have developed RA-resistant subclones of the human APL cell line, NB4, whose nuclear extracts display altered RA binding. In the RA-resistant subclone, R4, we find an absence of ligand binding of PML-RAR alpha associated with a point mutation changing a leucine to proline in the ligand-binding domain of the fusion PML-RAR alpha protein. In contrast to mutations in RAR alpha found in retinoid-resistant HL60 cells, in this NB4 subclone, the coexpressed RAR alpha remains wild-type. In vitro expression of a cloned PML-RAR alpha with the observed mutation in R4 confirms that this amino acid change causes the loss of ligand binding, but the mutant PML-RAR alpha protein retains the ability to heterodimerize with RXR alpha and thus to bind to retinoid response elements (RAREs). This leads to a dominant negative block of transcription from RAREs that is dose-dependent and not relieved by RA. An unrearranged RAR alpha engineered with this mutation also lost ligand binding and inhibited transcription in a dominant negative manner. We then found that the mutant PML-RAR alpha selectively alters regulation of gene expression in the R4 cell line. R4 cells have lost retinoid-regulation of RXR alpha and RAR beta and the RA-induced loss of PML-RAR alpha protein seen in NB4 cells, but retain retinoid-induction of CD18 and CD38. Thus, the R4 cell line provides data supporting the presence of an RAR alpha-mediated pathway that is independent from gene expression induced or repressed by PML-RAR alpha. The high level of retinoid resistance in vitro and in vivo of cells from some relapsed APL patients suggests similar molecular changes may occur clinically.
Assuntos
Antígenos CD , Antineoplásicos/farmacologia , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 17/genética , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Promielocítica Aguda/patologia , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Translocação Genética , Tretinoína/farmacologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Antígenos CD18/biossíntese , Antígenos CD18/genética , Células COS , Diferenciação Celular/efeitos dos fármacos , Cromossomos Humanos Par 15/ultraestrutura , Cromossomos Humanos Par 17/ultraestrutura , Regulação Leucêmica da Expressão Gênica , Genes Dominantes , Humanos , Leucemia Promielocítica Aguda/genética , Glicoproteínas de Membrana , N-Glicosil Hidrolases/biossíntese , N-Glicosil Hidrolases/genética , Proteínas de Neoplasias/biossíntese , Receptores do Ácido Retinoico/biossíntese , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptores X de Retinoides , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
We have identified a novel retinoid, ALRT1550, that potently and selectively activates retinoic acid receptors (RARs). ALRT1550 binds RARs with Kd values of approximately equal to 1-4 nM, and retinoid X receptors with low affinities (Kd approximately equal to 270-556 nM). We studied the effects of ALRT1550 on cellular proliferation in squamous carcinoma cells. ALRT1550 inhibited in vitro proliferation of UMSCC-22B cells in a concentration-dependent manner with an IC50 value of 0.22 +/- 0.1 (SE) nM. 9-cis-Retinoic acid (ALRT1057), a pan agonist retinoid that activates RARs and retinoid X receptors, inhibited proliferation with an IC50 value of 81 +/- 29 nM. In vivo, as tumor xenografts in nude mice, UMSCC-22B formed well-differentiated squamous carcinomas, and oral administration (daily, 5 days/week) of ALRT1550, begun 3 days after implanting tumor cells, inhibited tumor growth by up to 89% in a dose-dependent manner over the range of 3-75 micrograms/kg. ALRT1550 (30 micrograms/kg) also inhibited growth of established tumors by 72 +/- 3% when tumors were allowed to grow to approximately equal to 100 mm3 before dosing began. In comparison, 9-cis retinoic acid at 30 mg/kg inhibited growth of established tumors by 73 +/- 5%. Interestingly, retinoids did not appear to alter tumor morphologies in UMSCC-22B tumors. Notably, ALRT1550 produced a therapeutic index of approximately equal to 17 in this model, indicating a separation between doses that inhibited tumor growth and that induced symptoms of hypervitaminosis A. In summary, ALRT1550 potently inhibits cellular proliferation in vitro and in vivo in this squamous cell carcinoma tumor model. These data support additional study of ALRT1550 for its potential for improving anticancer therapy in human clinical trials.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Bucais/tratamento farmacológico , Receptores do Ácido Retinoico/agonistas , Retinoides/uso terapêutico , Animais , Carcinoma de Células Escamosas/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Camundongos Nus , Neoplasias Bucais/metabolismo , Receptores do Ácido Retinoico/metabolismo , Transplante HeterólogoRESUMO
Recently, 9-cis retinoic acid, a high affinity ligand for retinoic acid receptors and retinoid X-receptors (RXRs), was shown to have efficacy superior to all-trans retinoic acid as a chemopreventive agent in the N-nitroso-N-methylurea-induced rat mammary carcinoma model. To further explore the specific contribution RXR activation may play in suppression of carcinogenesis, the efficacy of LGD1069 (Targretin), an RXR-selective ligand, in the N-nitroso-N-methylurea-induced rat mammary tumor model was studied. LGD1069-treated animals showed a 90% reduction in tumor burden and tumor incidence compared with vehicle-treated rats with an efficacy similar to that achieved with tamoxifen. LGD1069 was very well tolerated during 13 weeks of chronic therapy with no classic signs of "retinoid-associated" toxicities. These data demonstrate that LGD1069, an RXR-selective ligand, can act as a highly effective and benign chemopreventive agent for mammary carcinoma.
Assuntos
Anticarcinógenos/uso terapêutico , Neoplasias Mamárias Experimentais/prevenção & controle , Receptores do Ácido Retinoico/agonistas , Tetra-Hidronaftalenos/uso terapêutico , Fatores de Transcrição/agonistas , Animais , Bexaroteno , Carcinógenos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Neoplasias Mamárias Experimentais/sangue , Neoplasias Mamárias Experimentais/induzido quimicamente , Metilnitrosoureia , Tamanho do Órgão/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores X de Retinoides , Útero/efeitos dos fármacosRESUMO
Retinoic acid receptor (RAR) active retinoids have proven therapeutically useful for treating certain cancers and dermatological diseases. Herein, we describe the discovery of two new RAR active trienoic acid retinoids, (2E,4E,6E)-7-(3,5-di-tert-butylphenyl)-3-methylocta-2, 4,6-trienoic acid (10a, ALRT1550) and (2E,4E,6Z)-7-(3,5-di-tert-butylphenyl)-3-methylocta-2, 4,6-trienoic acid (10b, LG100567). ALRT1550 is a RAR selective retinoid which exhibits exceptional potency in both competitive binding and cotransfection assays. Moreover, it is the most potent antiproliferative retinoid described to date and thus has implications for the treatment of certain cancers. LG100567 is a potent panagonist which activates both RARs and retinoid X receptors.
Assuntos
Antineoplásicos/farmacologia , Receptores do Ácido Retinoico/agonistas , Retinoides/farmacologia , Divisão Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Estrutura Molecular , Receptores do Ácido Retinoico/metabolismo , Receptores X de Retinoides , Retinoides/química , Timidina/metabolismo , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Neoplasias do Colo do ÚteroRESUMO
Cisplatin (DDP) is commonly used to treat head and neck tumors. Therapy frequently fails due to development of DDP resistance or toxicities associated with DDP therapy. In this study, effects of ALRT1057 [9-cis retinoic acid (9-cis RA)] on DDP cytotoxicity were studied in a human oral squamous carcinoma xenograft model. Mice bearing xenografts were dosed p.o. daily 5 days/week with 30 mg/kg 9-cis RA and/or i.p. twice weekly with 0.3-0.9 mg/kg DDP. Maximum tolerated doses of 9-cis RA and DDP were approximately 60 and >/=2.9 mg/kg, respectively, under their dosing schedules and routes of administration. Control tumors grew rapidly with mean doubling times of 4 +/- 1 days and reached mean volumes of 1982 +/- 199 (SE) mm3 after 24 days. DDP at doses of 0.3, 0.45, and 0.9 mg/kg inhibited tumor growth by 28, 47, and 86%, respectively, 24 days after tumor cell implantation. Thirty mg/kg 9-cis RA inhibited tumor growth by 25%. In combination, 0.3 mg/kg DDP + 30 mg/kg 9-cis RA inhibited tumor growth by 68%; 0.45 mg/kg DDP + 30 mg/kg 9-cis RA inhibited growth by 78%. These decreases were greater than those that would have been produced by either agent summed separately. Of importance, at doses of 9-cis RA that enhanced DDP cytotoxicity, no change in dose tolerance was observed as compared to tolerances observed for either agent alone, indicating that 9-cis RA increased sensitivity to DDP without altering systemic toxicity. In addition, 9-cis RA profoundly altered squamous cell carcinoma phenotypes by suppressing squamous cell differentiation, resulting in tumors with increased numbers of basal cells. In contrast, DDP selectively depleted proliferating basal cells from carcinomas. In combination, morphological changes produced by 9-cis RA alone predominated, suggesting a possible basis for enhanced DDP sensitivity in tumors exposed to both agents. These data demonstrate that 9-cis RA enhances tumor sensitivity to DDP, and suggest that this combination should be tested in Phase I-II clinical trials for its potential for improving anticancer therapy of squamous cell cancers.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Cisplatino/administração & dosagem , Neoplasias Bucais/tratamento farmacológico , Tretinoína/administração & dosagem , Alitretinoína , Animais , Bromodesoxiuridina/metabolismo , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Bucais/patologia , Transplante de Neoplasias , Receptores do Ácido Retinoico/efeitos dos fármacos , Receptores X de Retinoides , Fatores de Transcrição/efeitos dos fármacos , Transplante HeterólogoRESUMO
9-cis retinoic acid (9-cis RA) is a retinoid receptor pan-agonist that binds with high affinity to both retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Using a variety of in vivo and in vitro cancer models, we present experimental data that 9-cis RA has activity as a potential chemotherapeutic agent. Treatment of the human promyelocytic leukemia cell line HL-60 with 9-cis RA decreases cell proliferation, increases cell differentiation, and increases apoptosis. Induction of apoptosis correlates with an increase in tissue transglutaminase (type II) activity. In vivo, 9-cis RA induces complete tumor regression of an early passage human lip squamous cell carcinoma xenograft. Finally, 9-cis RA inhibits the anchorage-independent growth of the human breast cancer cell lines MCF-7 and LY2 (an antiestrogen-resistant MCF-7 variant). Transient co-transfection assays indicate that 9-cis RA inhibits estrogen receptor transcription of an ERE-tk-LUC reporter through RAR or RXR receptors. These data suggest that retinoid receptors can antagonize estrogen-dependent transcription and provides one possible mechanism for the inhibition of cell growth by 9-cis RA in breast cancer cell lines. In summary, these findings present evidence that 9-cis RA has a wide range of activities in human cancer models.
Assuntos
Neoplasias/tratamento farmacológico , Tretinoína/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Carcinoma de Células Escamosas/tratamento farmacológico , Divisão Celular/efeitos dos fármacos , Células HL-60/efeitos dos fármacos , Humanos , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
Retinoids are promising agents for therapy of squamous cancers. In vitro, retinoids decrease expression of differentiation markers in head and neck squamous carcinoma cells. Little information is available on effects of retinoids on head and neck squamous carcinoma cell xenograft growth in vivo. To address this issue, head and neck squamous carcinoma cells (line 1483) were established as xenografts in nude mice. Control tumors grew rapidly with doubling times of 4-6 days to mean volumes of 1696 mm3 after 24 days. Histological analyses indicated the formation of well-differentiated squamous carcinoma cells exhibiting pronounced stratification (basal and suprabasal cells) and keratinization (keratin pearls) with abundant stroma. Cytokeratin 19 expression was restricted to the basal cell layers, and cytokeratin 4 expression was abundant in suprabasal cells. Mice were treated daily with 30 mg/kg 9-cis retinoic acid, 20 mg/kg all-trans-retinoic acid, or 60 mg/kg 13-cis retinoic acid by p.o. gavage on a schedule of 5 days/week over 4 weeks. Low micromolar (1.48-3.67 microM) and nanomolar (200-490 nM) concentrations of 9-cis retinoic acid and all-trans-retinoic acid were measured in plasmas and xenografts, respectively, 30 min after dosing. Retinoid treatment produced a marked suppression of the squamous cell differentiation of tumor cells manifest by decreased keratinization, loss of stratification, and accumulation of basal cells. This was accompanied by large decreases in the number of CK4-positive cells and concomitant increases of CK19-positive cells. REtinoic acid receptor-beta expression was also increased by 2.9-9.7-fold after chronic retinoid treatment. 9-cis retinoic acid and all-trans-retinoic acid decreased tumor volumes by 23 +/- 5 (SE) and 19 +/- 3%, respectively (P < or = 0.05); 13-cis retinoic acid was inactive. These retinoids did not decrease the rate of exponential tumor growth but increased the latent period until exponential growth began. These studies demonstrate that retinoids do not universally decrease tumor growth but profoundly suppress squamous cell differentiation in vivo in this xenograft model.