RESUMO
Mouse mammary organ culture (MMOC) is used to evaluate the efficacy of chemopreventive agents against the development of carcinogen-induced preneoplastic lesions and is highly correlative to in vivo carcinogenesis models. Here, we developed a new ex vivo MMOC model, by introducing human breast cancer cells into the mouse mammary gland. This novel model, termed human breast cancer in MMOC (BCa-MMOC), mimics in vivo orthotopic breast cancer mouse models. To develop this model, estradiol- and progesterone-sensitized female mice were injected with letrozole-sensitive and -resistant T47D breast cancer cells in the mammary glands and then euthanized. The glands were cultured in vitro with hormone-supplemented media. On day 25, the glands were fixed and processed by histopathology and immunohistochemistry to evaluate for the presence of T47D cells, growth pattern, cancer markers and estradiol responsiveness. Histopathological analyses demonstrated an identical pattern of growth between the breast cancer cells injected ex vivo and in vivo Interestingly, clusters of cancer cells in the mammary gland stroma appeared similar to those observed in human breast tumors. The injected T47D cells survived and proliferated for 15â days maintaining expression of estrogen receptor alpha (ER), progesterone receptor (PR), epidermal growth factor receptor (EGFR), and aromatase. The aromatase-overexpressing T47D grown in the BCa-MMOC sufficiently metabolized estrogen, resulting in enhanced cell proliferation, induction of estrogen target genes (i.e. ER and PR-B), and showed typical changes to estrogenic milieu. In summary, here we show a novel, inexpensive ex vivo model, to potentially study the effects of therapeutic agents on cancer cells grown in an orthotopic micromilieu.This article has an associated First Person interview with the first author of the paper.
Assuntos
Neoplasias da Mama/etiologia , Neoplasias da Mama/metabolismo , Modelos Animais de Doenças , Animais , Elementos de Resposta Antioxidante/genética , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Células Cultivadas , Análise Custo-Benefício , Suscetibilidade a Doenças , Feminino , Humanos , Neoplasias Mamárias Experimentais , Camundongos , Técnicas de Cultura de Órgãos , Regiões Promotoras Genéticas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The present experiments were performed to determine the roles of estrogen receptors α and ß (ERα and ERß) in normal and neoplastic development in the mouse mammary gland. In wild-type mice, in vivo administration of estradiol (E) + progesterone (P) stimulated mammary ductal growth and alveolar differentiation. Mammary glands from mice in which the ERß gene has been deleted (ßERKO mice) demonstrated normal ductal growth and differentiation in response to E + P. By contrast, mammary glands from mice in which the ERα gene has been deleted (αERKO mice) demonstrated only rudimentary ductal structures that did not differentiate in response to E + P. EGF demonstrates estrogen-like activity in the mammary glands of αERKO mice: treatment of αERKO mice with EGF + P (without E) supported normal mammary gland development, induced expression of progesterone receptor (PR), and increased levels of G-protein-coupled receptor (GPR30) protein. Mammary gland development in ßERKO mice treated with EGF + P was comparable to that of wild-type mice receiving EGF + P; EGF had no statistically significant effects on the induction of PR or expression of GPR30 in mammary glands harvested from either wild-type mice or ßERKO mice. In vitro exposure of mammary glands to 7,12-dimethylbenz[a]anthracene (DMBA) induced preneoplastic mammary alveolar lesions (MAL) in glands from wild-type mice and ßERKO mice, but failed to induce MAL in mammary glands from αERKO mice. Microarray analysis of DMBA-treated mammary glands identified 28 functional pathways whose expression was significantly different in αERKO mice versus both ßERKO and wild-type mice; key functions that were differentially expressed in αERKO mice included cell division, cell proliferation, and apoptosis. The data demonstrate distinct roles for ERα and ERß in normal and neoplastic development in the mouse mammary gland, and suggest that EGF can mimic the ERα-mediated effects of E in this organ.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/metabolismo , Glândulas Mamárias Humanas/efeitos dos fármacos , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Primers do DNA/genética , Estradiol/administração & dosagem , Receptor alfa de Estrogênio/genética , Receptor beta de Estrogênio/genética , Feminino , Humanos , Imuno-Histoquímica , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/metabolismo , Camundongos , Camundongos Knockout , Análise em Microsséries , Progesterona/administração & dosagem , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Cancer related deaths in breast cancer patients are due to metastasis of the disease. Murine 4T1 cells (Murine mammary cancer cell line developed from 6-thioguanine resistant tumor) provide an excellent research tool for metastasis related studies because these cells are highly aggressive and readily metastasize to the lungs. In this study we determined the effect of Deguelin on in vivo/vitro growth and metastasis of 4T1 cells. Deguelin inhibited the in vitro growth of 4T1 cells in a time and dose dependent manner accompanied with reduced nuclear PCNA immunostaining. In cells treated with Deguelin, reduced expression of nuclear c-Met, and its downstream targets such p-ERK and p-AKT was observed. Deguelin reduced the cell migration in 4T1 cells as determined by scratch wound assay. Combined treatment with Deguelin + ERK or PI3K/AKT inhibitor had no additional effect on cell migration. These results indicated that the action of Deguelin on cell migration may be mediated by AKT and ERK mediated signaling pathways. In vivo, Deguelin treatment significantly inhibited growth of 4T1 cells. Deguelin also reduced the occurrence of metastatic lung lesions by 33 % when cells were injected intravenously into Balb/c female mice. There was no difference in the body weight, nor was there a difference in liver and spleen weights between vehicle treated-control and Deguelin-treated animals, which indicated that Deguelin was nontoxic at the dose used in the present study. These results provide rationale for developing Deguelin as a chemotherapeutic agent for triple negative breast cancer patients.
Assuntos
Metástase Neoplásica/prevenção & controle , Rotenona/análogos & derivados , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Rotenona/farmacologiaRESUMO
The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays a central role in regulating metabolism, including interaction with the estrogen receptor-α (ERα). Significantly, PPARγ activity can be modulated by small molecules to control cancer both in vitro and in vivo (Yin et al., Cancer Res 69:687-694, 2009). Here, we evaluated the effects of the PPARγ agonist GW7845 and the PPARγ antagonist GW9662 on DMBA-induced mammary alveolar lesions (MAL) in a mouse mammary organ culture. The results were as follows: (a) the incidence of MAL development was significantly inhibited by GW 7845 and GW 9662; (b) GW9662 but not GW7845, in the presence of estradiol, induced ER and PR expression in mammary glands and functional ERα in MAL; (c) while GW9662 inhibited expression of adipsin and ap2, GW 7845 enhanced expression of these PPARγ-response genes; and (d) Tamoxifen caused significant inhibition of GW9662 treated MAL, suggesting that GW9662 sensitizes MAL to antiestrogen treatment, presumably through rendering functional ERα and induction of PR. The induction of ERα by GW9662, including newer analogs, may permit use of anti-ER strategies to inhibit breast cancer in ER- patients.
Assuntos
Anilidas/farmacologia , Anticarcinógenos/farmacologia , Receptor alfa de Estrogênio/metabolismo , Glândulas Mamárias Animais/metabolismo , PPAR gama/antagonistas & inibidores , 9,10-Dimetil-1,2-benzantraceno , Animais , Sinergismo Farmacológico , Estradiol/fisiologia , Receptor alfa de Estrogênio/genética , Feminino , Glândulas Mamárias Animais/efeitos dos fármacos , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Oxazóis/farmacologia , PPAR gama/agonistas , PPAR gama/metabolismo , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/metabolismo , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Tamoxifeno/farmacologia , Técnicas de Cultura de Tecidos , Ativação Transcricional/efeitos dos fármacos , Tirosina/análogos & derivados , Tirosina/farmacologiaRESUMO
Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ERα) physically binds to peroxisome proliferator-activated receptor gamma (PPARγ) and inhibits its transcriptional activity. The interaction between PPARγ and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPARγ and VDR signaling, and for the first time we show that PPARγ physically associates with VDR in human breast cancer cells. We found that overexpression of PPARγ decreased 1α,25-dihydroxyvitamin D(3) (1,25D(3)) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPARγ's hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPARγ's AF2 domain attenuated its repressive action on 1,25D(3) transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPARγ was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXRα). Overexpression of RXRα blocked PPARγ's suppressive effect on 1,25D(3) action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPARγ and VDR pathways.
Assuntos
Neoplasias da Mama/genética , Colestanotriol 26-Mono-Oxigenase/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Receptor Cross-Talk/fisiologia , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Ativação Transcricional , Neoplasias da Mama/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Colestanotriol 26-Mono-Oxigenase/genética , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Células MCF-7 , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptor X Retinoide alfa/genética , Receptor X Retinoide alfa/metabolismo , Deleção de Sequência , Transdução de Sinais , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Células Tumorais Cultivadas , Vitamina D3 24-HidroxilaseRESUMO
CYP24 is a well-established vitamin D receptor (VDR) target gene. The active VDR ligand 1,25(OH)2D3 regulates its own catabolism by increasing CYP24 expression. It is well known that in the presence of 1,25(OH)2D3, VDR binds to VDREs in the promoter region of CYP24 and initiates CYP24 transcription. However, little is known about the role of 1,25(OH)2D3 in the posttranscriptional modulation of CYP24. In this study, we investigated the functional significance of 1,25(OH)2D3 in CYP24 RNA splicing in colon cancer cells. Using RT-PCR, we found that 1,25(OH)2D3 actively induces CYP24 splicing in a time-dependent manner and CYP24 splicing pattern could be cell type or tissue specific. The induction of RNA splicing by 1,25(OH)2D3 was mainly CYP24 selective. Treatment of cells with parathyroid hormone inhibited basal CYP24 splicing, but failed to inhibit 1,25(OH)2D3-induced CYP24 splicing. Further experiments demonstrated that new RNA synthesis was required for the induction of CYP24 splicing by vitamin D. In addition, alteration of multiple signaling pathways also affected CYP24 splicing and cellular sensitivity in response to vitamin D appeared to correlate with the induction of CYP24 splicing. These results suggest that 1,25(OH)2D3 not only regulates CYP24 transcription, but also plays an important role in posttranscriptional modulation of CYP24 by inducing its splicing. Our findings reveal an additional regulatory step that makes the vitamin D mediated action more prompt and efficient.
Assuntos
Calcitriol/metabolismo , Neoplasias do Colo/metabolismo , Proteínas de Neoplasias/metabolismo , Splicing de RNA , Esteroide Hidroxilases/metabolismo , Focos de Criptas Aberrantes/metabolismo , Focos de Criptas Aberrantes/patologia , Biópsia , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/patologia , Humanos , Peso Molecular , Proteínas de Neoplasias/genética , Concentração Osmolar , Hormônio Paratireóideo/análogos & derivados , Hormônio Paratireóideo/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Splicing de RNA/efeitos dos fármacos , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Esteroide Hidroxilases/química , Esteroide Hidroxilases/genética , Vitamina D3 24-HidroxilaseRESUMO
Amino acids 50-77 (p28) of azurin, a 128 aa cupredoxin isolated from Pseudomonas aeruginosa, is essentially responsible for azurin's preferential penetration of cancer cells. We now report that p28 also preferentially penetrates human umbilical vein endothelial cells (HUVEC), co-localized with caveolin-1 and VEGFR-2, and inhibits VEGF- and bFGF-induced migration, capillary tube formation and neoangiogenesis in multiple xenograft models. The antiangiogenic effect of p28 in HUVEC is associated with a dose-related non-competitive inhibition of VEGFR-2 kinase activity. However, unlike other antiangiogenic agents that inhibit the VEGFR-2 kinase, p28 decreased the downstream phosphorylation of FAK and Akt that normally precedes cellular repositioning of the cytoskeletal (F-actin), focal adhesion (FAK and paxillin), and cell to cell junction protein PECAM-1, inhibiting HUVEC motility and migration. The decrease in pFAK and pAkt levels suggests that p28 induces a pFAK-mediated loss of HUVEC motility and migration and a parallel Akt-associated reduction in cell matrix attachment and survival. This novel, direct antiangiogenic effect of p28 on endothelial cells may enhance the cell cycle inhibitory and apoptotic properties of this prototype peptide on tumor cell proliferation as it enters a Phase II clinical trial.
Assuntos
Antineoplásicos/farmacocinética , Azurina/farmacologia , Peptídeos Penetradores de Células/farmacologia , Células Endoteliais/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Neoplasias/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Actinas/metabolismo , Animais , Antineoplásicos/química , Azurina/química , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular , Peptídeos Penetradores de Células/química , Ensaios Clínicos Fase II como Assunto , Células Endoteliais/patologia , Adesões Focais/metabolismo , Humanos , Camundongos , Camundongos Nus , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Fragmentos de Peptídeos/química , Fosforilação/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Pseudomonas aeruginosa/química , Veias Umbilicais/metabolismo , Veias Umbilicais/patologiaRESUMO
Azurin, a member of the cupredoxin family of redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and apoptotic effects. Azurin and amino acids 50-77 (p28) of azurin also produce a dose-dependent reduction in the proliferation of human mammary cancer by increasing the level of the tumor suppressor protein p53 in the cancer cell nucleus. We show that the development of 7,12-dimethylbenz[a]anthracene-induced hormone-dependent premalignant mammary ductal lesions and hormone-independent mammary alveolar lesions in mouse mammary gland organ culture is also significantly reduced by azurin and p28. The dose-dependent reduction in carcinogen-induced mammary cell proliferation by p28 was associated with an increase in the expression of p53. p28 also enhanced the inhibitory effect of a low dose of the antiestrogen tamoxifen on the development of hormone-dependent mammary ductal lesions, but did not enhance the inhibitory activity of fenretinide (N-4-hydroxyphenyl retinamide) on hormone-independent mammary alveolar lesions. These observations suggest that cupredoxins and fragments derived from them can exert a chemopreventive effect on carcinogen-induced mammary gland transformation, irrespective of hormonal environment, and enhance the inhibitory effects of tamoxifen in this model of preneoplastic mammary development.
Assuntos
Antineoplásicos/farmacologia , Azurina/farmacologia , Neoplasias Mamárias Experimentais/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Imuno-Histoquímica , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tamoxifeno/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
We report that amino acids 50 to 77 of azurin (p28) preferentially enter the human breast cancer cell lines MCF-7, ZR-75-1, and T47D through a caveolin-mediated pathway. Although p28 enters p53 wild-type MCF-7 and the isogenic p53 dominant-negative MDD2 breast cancer cell lines, p28 only induces a G(2)-M-phase cell cycle arrest and apoptosis in MCF-7 cells. p28 exerts its antiproliferative activity by reducing proteasomal degradation of p53 through formation of a p28:p53 complex within a hydrophobic DNA-binding domain (amino acids 80-276), increasing p53 levels and DNA-binding activity. Subsequent elevation of the cyclin-dependent kinase inhibitors p21 and p27 reduces cyclin-dependent kinase 2 and cyclin A levels in a time-dependent manner in MCF-7 cells but not in MDD2 cells. These results suggest that p28 and similar peptides that significantly reduce proteasomal degradation of p53 by a MDM2-independent pathway(s) may provide a unique series of cytostatic and cytotoxic (apoptotic) chemotherapeutic agents.
Assuntos
Azurina/química , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Bromodesoxiuridina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclinas/metabolismo , Feminino , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Azurin, a member of the cupredoxin family of copper containing redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and cytotoxic (apoptotic) effects with no apparent activity on normal cells. Amino acids 50 to 77 (p28) of azurin seem responsible for cellular penetration and at least part of the antiproliferative, proapoptotic activity of azurin against a number of solid tumor cell lines. We show by confocal microscopy and fluorescence-activated cell sorting that amino acids 50 to 67 (p18) are a minimal motif (protein transduction domain) responsible for the preferential entry of azurin into human cancer cells. A combination of inhibitors that interfere with discrete steps of the endocytotic process and antibodies for caveolae and Golgi-mediated transport revealed that these amphipathic, alpha-helical peptides are unique. Unlike the cationic cell-penetrating peptides, alpha-helical antennapedia-like, or VP22 type peptides, p18 and p28 are not bound by cell membrane glycosaminoglycans and preferentially penetrate cancer cells via endocytotic, caveosome-directed, and caveosome-independent pathways. Once internalized, p28, but not p18, inhibits cancer cell proliferation initially through a cytostatic mechanism. These observations suggest the azurin fragments, p18 and p28, account for the preferential entry of azurin into human cancer cells and a significant amount of the antiproliferative activity of azurin on human cancer cells, respectively.
Assuntos
Azurina/farmacocinética , Neoplasias/metabolismo , Fragmentos de Peptídeos/farmacocinética , Sequência de Aminoácidos , Azurina/farmacologia , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células HCT116 , Humanos , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fragmentos de Peptídeos/farmacologia , Estrutura Terciária de ProteínaRESUMO
This article comprehensively reviews the clinical trials and considers the future directions of the use of vitamin D and its analogs in the treatment or chemoprevention of breast cancer. Chemopreventive treatment strategies strive to delay the onset of certain cancers, prevent the progression of malignant disease after diagnosis, or delay the advent of recurrence after curative treatment. We first summarize the epidemiological evidence that led to the hypothesis that vitamin D may have an anti-cancer activity. Vitamin D shows great potential as a therapy for breast cancer; however, its use in clinical trials has been hindered by the induction of hypercalcemia at a concentration required to suppress cancer cell proliferation. This has led to the development of less calcemic analogs of vitamin D. We review the clinical trials with breast cancer patients using vitamin D analogs.
Assuntos
Neoplasias da Mama/prevenção & controle , Vitamina D/análogos & derivados , Ensaios Clínicos como Assunto , Feminino , HumanosRESUMO
Several studies have established the active form of vitamin D(3) as an effective tumor-suppressing agent; however, its antitumor activity is achieved at doses that are hypercalcemic in vivo. Therefore, less calcemic vitamin D(3) analog, 1alpha-hydroxy-24-ethyl-cholecalciferol (1alpha[OH]D5), was evaluated for its potential use in breast cancer chemoprevention. Previously, 1alpha(OH)D5 showed anticarcinogenic activity in several in vivo and in vitro models. However, its effects on growth of normal tissue were not known. The present study was conducted to determine the effects of 1alpha(OH)D5 on the growth of normal mouse mammary gland and normal-like human breast epithelial MCF-12F cells and to compare these effects with carcinogen-transformed MCF-12F and breast cancer cells. No significant difference was observed in the growth or morphology of cultured mouse mammary gland and MCF-12F cells in the presence of 1alpha(OH)D5. However, the transformed MCF-12F cells underwent growth inhibition (40-60%, P < 0.05) upon 1alpha(OH)D5 treatment as determined by cell viability assays. Cell cycle analysis showed marked increase (50%) in G-1 phase for cells treated with 1alpha(OH)D5 compared with the controls. Moreover, the percentage of cells in the synthesis (S) phase of cell cycle was decreased by 70% in transformed MCF-12F, BT-474 and MCF-7 cells. The growth arrest was preceded by an increase in expression of cell cycle regulatory proteins p21(Waf-1) and p27(Kip-1). In addition, differential expression studies of parent and transformed MCF-12F cell lines using microarrays showed that prohibitin mRNA was increased 4-fold in the transformed cells. These results indicate that the growth inhibitory effect of 1alpha(OH)D5 was achieved in both carcinogen-transformed MCF-12F and breast cancer cells at a dose that was non-inhibitory in normal-like breast epithelial cells.
Assuntos
Neoplasias da Mama/prevenção & controle , Mama/citologia , Ciclo Celular/efeitos dos fármacos , Hidroxicolecalciferóis/farmacologia , Neoplasias Mamárias Animais/prevenção & controle , Animais , Neoplasias da Mama/patologia , Carcinógenos/farmacologia , Transformação Celular Neoplásica , Quimioprevenção , Células Epiteliais , Feminino , Humanos , Neoplasias Mamárias Animais/patologia , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
Vitamin D shows significant potential as a therapy for prostate cancer. However, its use in clinical trials has been hampered by its induction of hypercalcemia at serum concentrations required to suppress cancer cell proliferation. This has spurred the development of less calcemic analogs of vitamin D. In this article, we review the clinical trials and consider the future directions of the use of vitamin D and its analogs in the treatment or chemoprevention of prostate cancer. First, we summarize the epidemiological evidence leading to the hypothesis that vitamin D has anticancer activity. We then review the clinical trials using vitamin D analogs that involve patients with prostate cancer and conclude with a brief overview of our planned study with vitamin D5, [1alpha(OH)D5], which will begin shortly. Data for this review were identified by searches of PubMed, the Cochrane Library, Biosis, and references from relevant articles, using the search terms "vitamin D," "prostate cancer," "chemoprevention" and "vitamin D analog." Abstracts from recent international meetings were also reviewed but were only included when they were the only known reference to the clinical trial or the research mentioned. Only papers published in English were included.
Assuntos
Neoplasias da Próstata/tratamento farmacológico , Vitamina D/análogos & derivados , Vitamina D/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Hidroxicolecalciferóis/uso terapêutico , Masculino , Neoplasias da Próstata/epidemiologia , Resultado do TratamentoRESUMO
We previously showed that a new vitamin D analog, 1alpha(OH)D5 (D5), induced differentiation and inhibited the growth of breast cancer cells. In this report, we examined whether D5 specifically delivered to breast cancer cells could have any therapeutic effect. D5 was linked to Her-2 antibody using sulfosuccinimidyl 6-4 azido nitrophenylamido hexanode (SANPAH) as a linker. The Her-2 antibody selected in our study had no significant effect on the in vitro or in vivo growth of breast cancer cells; however, it had cell-differentiating action. In vitro, D5-Her-2 antibody conjugate (IMC) showed the ability to specifically bind to Her-2-expressing cells, to compete with Her-2 antibody for surface receptor and to cause internalization. IMC (equivalent to 5 microg Her-2 antibody given intraperitoneally once weekly for 6 weeks) significantly inhibited the growth of BT-474 cells transplanted into athymic mice. The in vivo growth-inhibitory effect of IMC treatment was similar to that observed in animals receiving D5 continuously as a dietary supplement. These results show that the targeted delivery of D5 by immunoconjugation to cell surface receptor antibodies may be of potential therapeutic value for the treatment of Her-2 positive breast cancer.
Assuntos
Neoplasias da Mama/imunologia , Genes erbB-2/genética , Hidroxicolecalciferóis/farmacologia , Vitamina D/análogos & derivados , Animais , Antígenos de Neoplasias , Azidas/farmacologia , Neoplasias da Mama/química , Cálcio/sangue , Cálcio/metabolismo , Caseínas/metabolismo , Diferenciação Celular , Divisão Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Feminino , Antígeno Ki-67/biossíntese , Metabolismo dos Lipídeos , Camundongos , Camundongos Nus , Ligação Proteica , Succinimidas/farmacologia , Temperatura , Fatores de TempoRESUMO
Prostate cancer continues to be a significant source of morbidity and mortality among older men. One possible means of reducing its impact on overall health and vitality is via cancer chemoprevention, both in the population that is unaffected but at some risk and in those who have undergone some form of curative therapy after the onset of the disease. Chemoprevention holds significant promise, but large phase III clinical trials evaluating chemopreventive agents in prostate cancer can require vast numbers of enrollees and require the commitment of significant financial resources and time before any therapeutic benefit may become apparent. One technique to shorten the time required for chemoprevention clinical trials is to use surrogate endpoint biomarkers in place of the currently used actual endpoints of cancer incidence or survival. The validation of such surrogate endpoint biomarkers will require small, well-designed phase I and/or II trials to accumulate data on the modulation of the surrogate biomarkers and the endpoints of cancer incidence or survival by the chemopreventive agent. Careful statistical correlation and clinical validation of the data will then allow us to justify the use such surrogates in place of the actual endpoint in large, randomized trials, potentially shortening trial duration, improving financial efficiency, and accelerating approval of the chemopreventive agent. To that end, we first review the theoretical construct of cancer chemoprevention trials with particular reference to prostate cancer. We thereafter describe the design of a small, randomized, double-blinded, placebo-controlled phase I/II clinical trial of an analogue of vitamin D, vitamin D5, which we believe could serve as a model for data accumulation on surrogate biomarkers and correlation with other clinical endpoints.
Assuntos
Antineoplásicos/uso terapêutico , Ensaios Clínicos Fase I como Assunto/métodos , Ensaios Clínicos Fase II como Assunto/métodos , Hidroxicolecalciferóis/uso terapêutico , Neoplasias da Próstata/prevenção & controle , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos , Antineoplásicos/farmacologia , Biomarcadores Tumorais/análise , Método Duplo-Cego , Humanos , Hidroxicolecalciferóis/farmacologia , Masculino , Seleção de PacientesRESUMO
It is now well established that the active metabolite of vitamin D3, 1alpha,25(OH)2D3, regulates cell growth and differentiation in various in vitro cancer models. However, its clinical use is precluded due to its hypercalcemic activity in vivo. Hence, several less calcemic vitamin D analogs have been synthesized and evaluated for their chemopreventive and therapeutic efficacy in experimental carcinogenesis models. A novel analog of vitamin D3, 1alpha-hydroxy-24-ethyl-cholecalciferol (1alpha[OH]D5), has currently been under investigation in our laboratory for its application in breast cancer prevention and therapy. 1alpha(OH)D5 had been shown to inhibit development of estrogen- and progesterone-dependent ductal lesions as well as steroid hormone-independent alveolar lesions in a mammary gland organ culture (MMOC) model. Moreover, the inhibitory effect was more significant if 1alpha(OH)D5 was present during the promotional phase of the lesion development. The growth inhibitory effect of 1alpha(OH)D5 has also been manifested in several breast cancer cell lines, including BT-474 and MCF-7. Breast cancer cell lines that responded to 1alpha(OH)D5 treatment were vitamin D receptor positive (VDR+). Vitamin D receptor-negative (VDR-) cell lines, such as MDA-MB-231 and MDA-MB-435, did not show growth inhibition upon incubation with 1alpha(OH)D5. This suggests the requirement of VDR in 1alpha(OH)D5-mediated growth effects. Interestingly, breast cancer cells that were VDR+ as well as estrogen receptor positive (ER+) showed cell cycle arrest and apoptosis, while VDR+ but ER- cells (UISO-BCA-4 breast cancer cells) showed enhanced expression of various differentiation markers with la(OH)D5 treatment. Transcription and expression of estrogen-inducible genes, progesterone receptor (PR) and trefoil factor 1 (pS2), were significantly down-regulated in ER+ BT-474 cells with 1alpha(OH)D5 treatment. This implies a differential effect of 1alpha(OH)D5 on ER+ vs. ER- cells. Additionally, comparison between the effects of 1alpha(OH)D5 on normal vs. transformed cells indicated that 1alpha(OH)D5 does not suppress cell prolifera-
Assuntos
Anticarcinógenos/farmacologia , Neoplasias da Mama/prevenção & controle , Hidroxicolecalciferóis/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , CamundongosRESUMO
Numerous analogs of Vitamin D have been synthesized in recent years with the hope of generating a compound that retains the anticarcinogenic activity of Vitamin D without causing any toxicity. We synthesized such an analog, 1alpha-hydroxy-24-ethylcholecalciferol [1alpha-hydroxyvitamin D(5) or 1alpha(OH)D(5)], and showed that it was tolerated by rats and mice at a much higher dose than 1alpha,25 dihydroxy cholecalciferol [1alpha,25(OH)(2)D(3)]. This property makes it a prime candidate for chemoprevention studies. In the mouse mammary gland organ culture (MMOC), 1alpha(OH)D(5) inhibited carcinogen-induced development of both mammary alveolar and ductal lesions. In vivo carcinogenesis study showed statistically significant reduction of tumor incidence and multiplicity in N-methyl-N-nitrosourea (MNU)-treated rats that were fed 25-50 microg 1alpha(OH)D(5)/kg diet. There were no adverse effects on plasma calcium concentrations. In order to determine if the effect of 1alpha(OH)D(5) would be selective in suppressing proliferation of transformed cells, its effects on cell growth and proliferation were compared between BT474 (cancer) and MCF12F (non-tumorigenic) human breast epithelial cells. Results showed that 1alpha(OH)D(5) induced apoptosis and cell cycle G1 phase arrest in BT474 breast cancer cells without having any effects on proliferation of the MCF12F cells. In addition, in MMOC it had no growth inhibitory effects on normal epithelial cell proliferation in the absence of carcinogen. Similarly, non-tumorigenic human breast epithelial cells in explant culture did not respond to 1alpha(OH)D(5), whereas treatment with 1alpha(OH)D(5) induced cell death in the explants of cancer tissue. These results collectively indicate that 1alpha(OH)D(5) selectively induced apoptosis only in transformed cells but not in normal breast epithelial cells. Interestingly, the growth inhibitory effects of 1alpha(OH)D(5) were observed in Vitamin D receptor positive (VDR(+)) breast cancer cells, but not in highly metastatic VDR(-) breast cancer cells, such as MDA-MB-435 and MDA-MB-231, suggesting that 1alpha(OH)D(5) action may be mediated, in part, by VDR.
Assuntos
Anticarcinógenos/farmacologia , Neoplasias da Mama/prevenção & controle , Hidroxicolecalciferóis/farmacologia , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Apoptose/efeitos dos fármacos , Carcinógenos/toxicidade , Ciclo Celular/efeitos dos fármacos , Feminino , Humanos , Glândulas Mamárias Animais/efeitos dos fármacos , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Células Tumorais CultivadasRESUMO
Vitamin D, a steroid hormone and exerts its biological effects through its active metabolite 1alpha, 25 dihydroxyvitamin D3 [1,25(OH)2D3]. Like steroid hormones, 1,25(OH)2D3 is efficacious at very low concentrations and serves as a ligand for vitamin D receptors (VDR), associating with VDR very high affinity. Despite its potent property as a differentiating agent, its use in the clinical practice is hampered by the induction of hypercalcemia at a concentration required to suppress cancer cell proliferation. Therefore nearly 400 structural analogs of vitamin D3 have been synthesized and evaluated for their efficacy and toxicity. Among these analogs, relatively less toxic but highly efficacious analogs, EB1089, RO24-5531, 1alpha-hydroxyvitamin D5 and a few others have been evaluated in a preclinical toxicity and in Phase I clinical trials for dose tolerance in advanced cancer patients. Clinical trials using vitamin D analogs for prevention or therapy of cancer patients are still in their infancy. Vitamin D mediates its action by two independent pathways. Genomic pathway involves nuclear VDR and induces biological effects by interactions with hormone response elements and modulation of differential gene expressions. Evidence also suggests that vitamin D analogs also interact with steroid hormone(s) inducible genes. The non-genomic pathway is characterized by rapid actions of vitamin D. It involves interactions with membrane-VDR interactions and its interactions with protein kinase C and by altering intracellular calcium channels. Thus, the development of nontoxic analogs of vitamin D analogs and understanding of their molecular mechanism(s) of action are of significant importance in the prevention and treatment of cancer by vitamin D.