RESUMEN
Insulin-like growth factor I (IGF-I) is implicated in breast cancer development and 1, 25-dihydroxyvitamin D3 (1, 25-D3) has been shown to attenuate prosurvival effects of IGF-I on breast cancer cells. In this study the role of IGF binding protein-3 (IGFBP-3) in 1, 25-D3-induced apoptosis was investigated using parental MCF-7 breast cancer cells and MCF-7/VD(R) cells, which are resistant to the growth inhibitory effects of 1, 25-D3. Treatment with 1, 25-D3 increased IGFBP-3 mRNA expression in both cell lines but increases in intracellular IGFBP-3 protein and its secretion were observed only in MCF-7. 1, 25-D3-induced apoptosis was not associated with activation of any caspase but PARP-1 cleavage was detected in parental cells. IGFBP-3 treatment alone produced cleavage of caspases 7, 8, and 9 and PARP-1 in MCF-7 cells. IGFBP-3 failed to activate caspases in MCF-7/VD(R) cells; however PARP-1 cleavage was detected. 1, 25-D3 treatment inhibited IGF-I/Akt survival signalling in MCF-7 but not in MCF-7/VD(R) cells. In contrast, IGFBP-3 treatment was effective in inhibiting IGF-I/Akt pathways in both breast cancer lines. These results suggest a role for IGFBP-3 in 1, 25-D3 apoptotic signalling and that impaired secretion of IGFBP-3 may be involved in acquired resistance to vitamin D in breast cancer.
RESUMEN
It has been previously demonstrated that 1,25 dihydroxyvitamin D(3) (1,25-D(3)) exerts inhibitory effects in breast cancer cells. The aim of this study was to determine whether mitogen-activated protein kinase (MAPK) pathways are associated with 1,25-D(3)-induced cell death in breast cancer. We used three breast cell lines which have different sensitivities to 1,25-D(3) treatment. Non-malignant MCF-12A cells were more sensitive to 1,25-D(3) treatment than malignant MCF-7 cells (growth inhibition IC(50) 75 nM vs. 100 nM, p<0.001) while malignant MDA-MB-231 cells were resistant. Moreover, 1,25-D(3)-induced apoptosis was caspase-dependent in MCF-12A cells and caspase-independent in MCF-7 cells. Following MAPK activation analysis, we found a significant activation of JNK in MCF-12A cells and malignant MCF-7 cells in response to 1,25-D(3) treatment. Furthermore, 1,25-D(3) treatment stimulated p38 activity in MCF-12A cells and in MCF-7 cells. ERK1/2 activity was unaffected by 1,25-D(3) treatment in all breast cells. Importantly, no increased MAPK activity was observed in MDA-MB-231 breast cancer cells which displayed resistance to 1,25-D(3)-induced apoptosis. Utilising specific pharmacological inhibitors of JNK and p38, it was demonstrated that MCF-12A and MCF-7 cells were protected from death induced by 1,25-D(3). These results implicate JNK and p38 signalling in 1,25-D(3)-induced cancer breast cell death.
Asunto(s)
Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Calcitriol/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Estrés Fisiológico/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacosRESUMEN
Prostate cancer incidence is rising, and represents a major public health issue. Bone is by far the most common site for metastases in this disease, accounting for considerable morbidity. Until recently, there have been few viable options for the treatment of patients with hormone-refractory metastatic disease. This review examines the pathophysiology underlying the development of bone metastases. It also summarises some of the clinical approaches for the management of this common condition, focusing on recent evidence supporting the use of zoledronic acid, a member of one of the most promising groups of pharmacological agents, the third-generation bisphosphonates.
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Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/fisiopatología , Difosfonatos/uso terapéutico , Imidazoles/uso terapéutico , Neoplasias de la Próstata/patología , Humanos , Masculino , Ácido ZoledrónicoRESUMEN
Although 1,25-dihydroxyvitamin D3 is a potent cell-differentiating agent, its use in cancer prevention or therapy is precluded because it induces hypercalcemia. Synthetic analogs have been developed which inhibit tumor progression in animal models of breast cancer. One analog, Seocalcitol (EB1089) has been shown to be effective in causing regression of N-methyl-nitrosourea-induced rat mammary tumors. However, at the most effective oral dose, a significant increase in serum and urinary calcium levels were observed. In order to compare the efficacy of different dosing schedules of Seocalcitol, rats were treated either 6 times weekly (1 microg/kg) or by intermittent dosing to achieve the same total weekly dose. All dosing schedules of Seocalcitol were effective in inhibiting tumor progression. Once daily dosing was significantly more effective than intermittent dosing but was associated with a greater rise in serum calcium concentration. In order to evaluate alternative treatment strategies to limit calcemic effects, we assessed the efficacy of limiting vitamin D-induced hypercalcemia using bisphosphonates. Seocalcitol (2.5 microg/kg daily p.o. for 4 weeks) alone and in combination with pamidronate (APD 0.4 mg/kg per day s.c.) or the same dose of the bisphosphonate EB 1053 caused substantial tumor regression. No statistically significant difference was seen between combination treatment and Seocalcitol treatment alone. Co-treatment with APD or EB 1053 did not limit the rise in serum calcium induced by Seocalcitol alone. Cessation of treatment or administration of a lower dose (1microg/kg twice weekly) reversed hypercalcemia, hypercalciuria and weight loss induced by high dose Seocalcitol. However, reduction in tumor volume was maintained in the majority of animals.
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Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Calcitriol/análogos & derivados , Calcitriol/efectos adversos , Calcitriol/farmacología , Hipercalcemia/inducido químicamente , Neoplasias Mamarias Animales/tratamiento farmacológico , Alquilantes/efectos adversos , Animales , Relación Dosis-Respuesta a Droga , Femenino , Metilnitrosourea/efectos adversos , Neoplasias Experimentales , Ratas , Ratas WistarRESUMEN
OBJECTIVE: To determine the in vitro and in vivo effects of 1,25-dihydroxyvitamin D3 (calcitriol) and two newer less hypercalcaemic analogues, EB1089 and CB1093 (as the use of calcitriol as a therapeutic agent in humans has been limited by hypercalcaemia) in three rodent models of prostate cancer. MATERIALS AND METHODS: The highly metastatic MAT LyLu Dunning prostate model, PAIII tumours in Lobund-Wistar rats and LNCaP xenografts in nude mice were used. Vitamin D receptor (VDR) expression and binding were assessed in all cell lines. The effects of calcitriol, EB1089 and CB1093 on tumour growth, cell cycle and angiogenesis in vitro, and growth and serum calcium levels in vivo, were assessed. RESULTS: The growth of prostate adenocarcinoma was inhibited by calcitriol, EB1089 and CB1093 in the Dunning prostate model. Although both analogues increased serum calcium levels, the levels were significantly less than in rats treated with calcitriol. Tumour growth was also inhibited in male athymic nu/nu mice with LNCaP tumour xenografts. PAIII cells failed to express functional VDR and were insensitive to calcitriol and its analogues, either in vitro or in vivo. The analogues of calcitriol did not inhibit angiogenesis in a rat aorta assay. CONCLUSION: This is the first report comparing the actions of calcitriol and its analogues in different in vivo models. The results suggest that the newer less hypercalcaemic analogues of calcitriol may offer a novel therapeutic option for treating prostate cancer. VDR-dependent growth inhibition and not the inhibition of angiogenesis is the main mechanism of action of these compounds in vivo.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Calcitriol/análogos & derivados , Calcitriol/uso terapéutico , Agonistas de los Canales de Calcio/uso terapéutico , Hipercalcemia/inducido químicamente , Neoplasias de la Próstata/tratamiento farmacológico , Animales , Western Blotting , Ensayos de Selección de Medicamentos Antitumorales , Masculino , Ratones , Ratones Desnudos , Ratas , Ratas Wistar , Receptores de Calcitriol/metabolismoRESUMEN
All-trans-retinoic acid and 9-cis-retinoic acid have been reported to have inhibitory effects on pancreatic adenocarcinoma cells and we have shown that this is partly due to induction of apoptosis. In this study, the mechanisms whereby 9-cis-retinoic acid induces apoptosis in these cells were investigated. An involvement of the Bcl-2 family of proteins was shown, such that 9-cis-retinoic acid causes a decrease in the Bcl-2/Bax ratio. Overexpression of Bcl-2 also resulted in inhibition of apoptosis induced by 9-cis-retinoic acid. Furthermore, two broad-range caspase inhibitors blocked DNA fragmentation induced by 9-cis-retinoic acid, but had no effect on viability defined by mitochondrial activity. Using synthetic retinoids, which bind selectively to specific retinoic acid receptor subtypes, we further established that activation of retinoic acid receptor-gamma is essential for induction of apoptosis. Only pan-retinoic acid receptor and retinoic acid receptor-gamma selective agonists reduced viability and a cell line expressing very low levels of retinoic acid receptor-gamma is resistant to the effects of 9-cis-retinoic acid. A retinoic acid receptor-beta/gamma selective antagonist also suppressed the cytotoxic effects of 9-cis-retinoic acid in a dose-dependent manner. This study provides important insight into the mechanisms involved in suppression of pancreatic tumour cell growth by retinoids. Our results encourage further work evaluating the clinical use of receptor subtype selective retinoids in pancreatic carcinoma.
Asunto(s)
Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Ácido Aspártico/análogos & derivados , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes bcl-2 , Proteínas de Neoplasias/biosíntesis , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Receptores de Ácido Retinoico/efectos de los fármacos , Retinoides/farmacología , Tretinoina/farmacología , Adenocarcinoma/genética , Alitretinoína , Clorometilcetonas de Aminoácidos/farmacología , Animales , Ácido Aspártico/farmacología , Inhibidores de Cisteína Proteinasa/farmacología , Fragmentación del ADN/efectos de los fármacos , Resistencia a Medicamentos , Ácidos Grasos Insaturados/farmacología , Humanos , Ratones , Mitocondrias/efectos de los fármacos , Proteínas de Neoplasias/efectos de los fármacos , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/genética , Isoformas de Proteínas/efectos de los fármacos , Isoformas de Proteínas/fisiología , Proteínas Proto-Oncogénicas/genética , Receptores de Ácido Retinoico/fisiología , Proteínas Recombinantes de Fusión/fisiología , Receptores X Retinoide , Retinoides/agonistas , Retinoides/antagonistas & inhibidores , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/fisiología , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Proteína X Asociada a bcl-2 , Receptor de Ácido Retinoico gammaRESUMEN
Bisphosphonates are well established in the management of cancer-induced bone disease. Recent studies have indicated that these compounds have direct inhibitory effects on cultured human breast cancer cells. Nitrogen-containing bisphosphonates including zoledronic acid have been shown to induce apoptosis associated with PARP cleavage and DNA fragmentation. The aim of this study was to identify the signalling pathways involved. Forced expression of the anti-apoptotic protein bcl-2 attenuated bisphosphonate-induced loss of cell viability and induction of DNA fragmentation in MDA-MB-231 cells. Zoledronic acid-mediated apoptosis was associated with a time and dose-related release of mitochondrial cytochrome c into the cytosol in two cell lines. Rescue of cells by preincubation with a caspase-3 selective inhibitor and demonstration of pro-caspase-3 cleavage products by immunoblotting suggests that at least one of the caspases activated in response to zoledronic acid treatment is caspase-3. In both MDA-MB-231 and MCF-7 breast cancer cells, zoledronic acid impaired membrane localisation of Ras indicating reduced prenylation of this protein. These observations demonstrate that zoledronic acid-mediated apoptosis is associated with cytochrome c release and consequent caspase activation. This process may be initiated by inhibition of the enzymes in the mevalonate pathway leading to impaired prenylation of key intracellular proteins including Ras.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Grupo Citocromo c/metabolismo , Difosfonatos/farmacología , Imidazoles/farmacología , Caspasas/farmacología , Membrana Celular/fisiología , Femenino , Genes ras , Humanos , Prenilación de Proteína , Células Tumorales Cultivadas , Ácido Zoledrónico , Proteínas ras/metabolismoRESUMEN
Inoperable cancer of the exocrine pancreas responds poorly to most conventional anti-cancer agents, and new agents are required to palliate this disease. Seocalcitol (EB1089), a vitamin D analogue, can inhibit growth, induce differentiation and induce apoptosis of cancer cell lines in vitro and can also inhibit growth of pancreatic cancer xenografts in vivo. Thirty-six patients with advanced pancreatic cancer received once daily oral treatment with seocalcitol with dose escalation every 2 weeks until hypercalcaemia occurred, following which patients continued with maintenance therapy. The most frequent toxicity was the anticipated dose-dependent hypercalcaemia, with most patients tolerating a dose of 10-15 microg per day in chronic administration. Fourteen patients completed at least 8 weeks of treatment and were evaluable for efficacy, whereas 22 patients were withdrawn prior to completing 8 weeks' treatment and in 20 of these patients withdrawal was due to clinical deterioration as a result of disease progression. No objective responses were observed, with five of 14 patients having stable disease in whom the duration of stable disease was 82-532 days (median=168 days). The time to treatment failure (n=36) ranged from 22 to 847 days, and with a median survival of approximately 100 days. Seocalcitol is well tolerated in pancreatic cancer but has no objective anti-tumour activity in advanced disease. Further studies are necessary to determine if this agent has any cytostatic activity in this malignancy in minimal disease states.
Asunto(s)
Antineoplásicos/farmacología , Calcitriol/farmacología , Carcinoma/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Administración Oral , Adulto , Anciano , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Calcitriol/administración & dosificación , Calcitriol/efectos adversos , Calcitriol/análogos & derivados , Carcinoma/patología , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/patología , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
It is now well established that, in addition to its central role in the maintenance of extracellular calcium levels and bone mineralization, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the active form of vitamin D, also acts as a modulator of cell growth and differentiation in a number of cell types, including breast cancer cells. The anti-proliferative effects of 1,25(OH)(2)D(3) have been linked to suppression of growth stimulatory signals and potentiation of growth inhibitory signals, which lead to changes in cell cycle regulators such as p21(WAF-1/CIP1) and p27(kip1), cyclins and retinoblastoma protein as well as induction of apoptosis. Such studies have led to interest in the potential use of 1,25(OH)(2)D(3) in the treatment or prevention of certain cancers. Since this approach is limited by the tendency of 1,25(OH)(2)D(3) to cause hypercalcaemia, synthetic vitamin D analogues have been developed which display separation of the growth regulating effects from calcium mobilizing actions. This review examines mechanisms by which 1,25(OH)(2)D(3) and its active analogues exert both anti-proliferative and pro-apoptotic effects and describes some of the synthetic analogues that have been shown to be of particular interest in relation to breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Vitamina D/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , División Celular/efectos de los fármacos , Femenino , Sustancias de Crecimiento/metabolismo , Humanos , Receptores de Calcitriol/efectos de los fármacos , Receptores de Calcitriol/metabolismo , Vitamina D/farmacología , Vitamina D/uso terapéuticoRESUMEN
Skeletal morbidity secondary to metastases and osteoporosis is common in patients with advanced prostate cancer. Despite the typically sclerotic nature of prostate cancer metastases, osteoclast mediated osteolysis may play a significant role. This review addresses the newly recognised antitumour effects of bisphosphonates in addition to their role in inhibiting osteoclast mediated bone resorption. Both preclinical and clinical evidence of a role for bisphosphonates in the treatment and prevention of bone metastases secondary to prostate cancer is assessed.
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Adenocarcinoma/secundario , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/secundario , Resorción Ósea/tratamiento farmacológico , Difosfonatos/uso terapéutico , Neoplasias de la Próstata/patología , Adenocarcinoma/irrigación sanguínea , Adenocarcinoma/complicaciones , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/fisiopatología , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Densidad Ósea/efectos de los fármacos , Neoplasias Óseas/irrigación sanguínea , Neoplasias Óseas/complicaciones , Neoplasias Óseas/fisiopatología , Calcio/metabolismo , Difosfonatos/farmacología , Progresión de la Enfermedad , Homeostasis , Humanos , Masculino , Neovascularización Patológica/tratamiento farmacológico , Osteoclastos/efectos de los fármacos , Osteólisis/tratamiento farmacológico , Osteólisis/etiología , Osteólisis/prevención & control , Osteoporosis/tratamiento farmacológico , Osteoporosis/etiología , Osteoporosis/prevención & control , Cuidados Paliativos , Ensayos Clínicos Controlados Aleatorios como Asunto , RatasRESUMEN
INTRODUCTION: Retinoids, which are derivatives of vitamin A, are important factors involved in the control of biologic functions such as cell growth and differentiation, development, and carcinogenesis. We have shown previously that the naturally occurring retinoids all-trans-retinoic acid (ATRA) and 9-cisretinoic acid (9cRA) induce growth inhibition followed by apoptosis in pancreatic adenocarcinoma cells in vitro. AIM: To evaluate the efficacy of retinoids in combination with the chemotherapeutic drugs gemcitabine and cisplatin. METHODOLOGY: In vitro growth inhibition and induction of apoptosis by different combinations of retinoids and cytotoxic drugs were studied by using the T3M-4 and BxPc-3 cell lines. For in vivo studies, T3M-4 cells were injected subcutaneously in nude mice. RESULTS: Pre-treatment of pancreatic adenocarcinoma cells with ATRA or 9cRA before the addition of the drugs resulted in significant reduction in cell number compared with treatment with the drugs alone. Pre-treatment with 9cRA followed by gemcitabine or cisplatin alone also resulted in a strong increase in the percentage of cells undergoing programmed cell death, or apoptosis. Furthermore, there was an indication that the combination of ATRA and gemcitabine caused increased apoptosis in vivo. CONCLUSION: Our results clearly suggest the need for additional studies exploring the potential role of the combination of retinoids and gemcitabine in the management of pancreatic cancer.
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Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Tretinoina/uso terapéutico , Adenocarcinoma/patología , Alitretinoína , Animales , Apoptosis/efectos de los fármacos , Cisplatino/administración & dosificación , Cisplatino/uso terapéutico , Desoxicitidina/administración & dosificación , Desoxicitidina/uso terapéutico , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Pancreáticas/patología , Tretinoina/administración & dosificación , Células Tumorales Cultivadas , GemcitabinaRESUMEN
Several in vitro and in vivo experiments have demonstrated potent cell regulatory effects of vitamin D compounds in cancer cells. Moreover, a promising phase I study with the vitamin D analogue Seocalcitol (EB 1089) in patients with advanced breast and colon cancer has already been carried out and more clinical trials evaluating the clinical effectiveness of EB 1089 in other cancer types are in progress (Mørk Hansen et al. [2000a]). However, only little is known about the mechanisms underlying the actions of vitamin D or about the possible development of drug resistance in the patients. Therefore, in an attempt to gain more insight into these aspects, we have developed the MCF-7/VD(R) cell line, a stable subclone of the human MCF-7 breast cancer cell line, which is resistant to the growth inhibitory and apoptosis inducing effects of 1alpha,25(OH)(2)D(3). Despite this characteristic, receptor studies on the VDR have clearly demonstrated that the MCF-7/VD(R) cells contain fully functional VDRs, although in a lower number than seen with the parental MCF-7 cells. The regulation of the 24-hydroxylase enzyme appeared to be intact in the MCF-7/VD(R) cells and no differences with regard to growth rate and morphological appearance between the MCF-7/VD(R) cells and the parental MCF-7 cells were observed. Interestingly, however, the sensitivity of the MCF-7/VD(R) cells to the pure anti-estrogen ICI 182,780 was found to be increased. The MCF-7/VD(R) cell line shows characteristics different from those of previously described vitamin D resistant breast cancer cell lines but also some similarities. Together such vitamin D resistant cell lines therefore serve as a useful tool for studying the exact mechanism of action of vitamin D and the development of vitamin D resistance.
Asunto(s)
Células Tumorales Cultivadas/efectos de los fármacos , Vitamina D/farmacología , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Resistencia a Medicamentos , Moduladores de los Receptores de Estrógeno/farmacología , Femenino , Humanos , Ligandos , Microscopía Fluorescente , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Calcitriol/metabolismo , Receptores de Estrógenos/metabolismo , Esteroide Hidroxilasas/genética , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
There is increasing evidence that vitamin D can protect against breast cancer. The actions of vitamin D are mediated via the vitamin D receptor (VDR). We have investigated whether polymorphisms in the VDR gene are associated with altered breast cancer risk in a UK Caucasian population. We recruited 241 women following a negative screening mammogram and 181 women with known breast cancer. The VDR polymorphism Bsm I, an intronic 3' gene variant, was significantly associated with increased breast cancer risk: odds ratio bb vs BB genotype = 2.32 (95% CI, 1.23-4.39). The Bsm I polymorphism was in linkage disequilibrium with a candidate translational control site, the variable length poly (A) sequence in the 3' untranslated region. Thus, the 'L' poly (A) variant was also associated with a similar breast cancer risk. A 5' VDR gene variant, Fok I, was not associated with breast cancer risk. Further investigations into the mechanisms of interactions of the VDR with other environmental and/or genetic influences to alter breast cancer risk may lead to a new understanding of the role of vitamin D in the control of cellular and developmental pathways.
Asunto(s)
Neoplasias de la Mama/genética , Polimorfismo Genético , Receptores de Calcitriol/genética , Población Blanca/genética , Adulto , Anciano , Neoplasias de la Mama/epidemiología , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Reino Unido/epidemiologíaRESUMEN
Induction of apoptosis is a feature of the anti-tumor effects of certain vitamin D analogs. The aim of this study was to identify if common effectors are involved in cell death mediated by serum starvation, vitamin D analogs and tumor necrosis factor (TNF) alpha in 3 human breast cancer cell lines: MCF-7, T47-D and Hs578T. Incubation of cells in serum-free medium induced apoptosis as assessed by loss of cell viability and increased DNA fragmentation. Addition of IGF-I (30 ng/ml) protected against loss of cell viability in MCF-7 cells and co-treatment with two synthetic analogs (CB1093 and EB1089, 50 nM for 4 days) prevented these anti-apoptotic effects of IGF-I. Pretreatment of MCF-7 and Hs578T cells with the vitamin D analogs substantially potentiated the cytotoxic effects of TNFalpha. This cytokine was not cytotoxic for T47-D cells but co-incubation with CB1093 led to loss of cell viability. Potentiation by CB1093 of TNFalpha-induced apoptosis in MCF-7 cells was accompanied by increased activation of cytosolic phospholipase A2 and arachidonic acid release, which was partially inhibited by AACOCF3, a specific cPLA2 inhibitor. The broad-spectrum caspase inhibitor z-VAD-fmk prevented TNFalpha but not CB1093 mediated cell death and activation of cPLA2. Serum starvation induced apoptosis was accompanied by cPLA2 activation, which was inhibited by IGF-I and by z-VAD-fmk. However, the ability of these agents to suppress cPLA2 activation was abrogated by co-treatment with CB1093, suggesting a role for arachidonic acid release in the caspase-independent mechanism by which vitamin D analogs prevent the protective effects of IGF-I on breast cancer cell survival.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Transducción de Señal/efectos de los fármacos , Vitamina D/análogos & derivados , Vitamina D/fisiología , Neoplasias de la Mama/fisiopatología , Inhibidores de Caspasas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Factor I del Crecimiento Similar a la Insulina/farmacología , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Células Tumorales Cultivadas/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Mechanisms by which vitamin D analogues promote apoptosis in tumour cells are unclear. In this study we have examined possible interactions between the synthetic vitamin D analogue CB1093 and two other known mediators of apoptosis, TNFalpha and ceramide, in MCF-7, T47D and Hs578T breast cancer cells. These studies indicated that cytosolic phospholipase A(2) (cPLA(2)) is involved in CB1093 as well as TNFalpha-mediated cell death. CB1093 promoted both TNFalpha and ceramide-induced c-PLA(2) activation, which was inversely related to loss of cell viability in MCF-7 and Hs578T cells. TNFalpha alone (5-20 ng/ml) failed to induce cytotoxicity and activation of cPLA(2) in T47D cells. However, pretreatment of these cells with CB1093 potentiated C(2)-ceramide-induced cPLA(2) activation and cell death. Treatment with CB1093 alone induced loss of cell viability and DNA fragmentation in all three cell lines by 5 days and these effects were accompanied by activation of cPLA(2). Furthermore, co-treatment with the cPLA(2) inhibitor AACOCF(3) led to partial protection against loss of cell viability induced by CB1093 in Hs578T and T47D cells as well as MCF-7 cells. The broad-spectrum caspase inhibitor z-VAD-fmk prevented TNFalpha but not C(2)-ceramide and CB1093-mediated release of arachidonic acid and cell death in MCF-7 cells. These results indicate that CB1093 potentiates responsiveness of breast cancer cells to TNFalpha and suggest that ceramide and/or cPLA(2) might be involved as downstream effectors in vitamin D-mediated caspase-independent cell death.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Calcitriol/farmacología , Ceramidas/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Antineoplásicos/farmacología , Calcitriol/análogos & derivados , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Fosfolipasas A/efectos de los fármacos , Fosfolipasas A/metabolismo , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Retinoids and vitamin D are known to exert important anti-tumour effects in a variety of cell types. In this study the effects of 9-cis-retinoic acid (9cRA) the vitamin D analogues EB1089 and CB1093 on three pancreatic adenocarcinoma cell lines were investigated. All compounds caused inhibition of in vitro growth but the vitamin D analogues were generally the more potent growth inhibitors. They were also more effective on their own than in combination with 9cRA. Growth arrest correlated with an increased proportion of cells in the G0/G1 phase. Apoptosis was induced in the three cell lines by 9cRA, whereas neither EB1089 nor CB1093 had this effect. Furthermore, addition of EB1089 or CB1093 together with 9cRA resulted in significantly reduced apoptosis. Our results show that retinoic acids as well as vitamin D analogues have inhibitory effects on pancreatic tumour cells but different and antagonistic mechanisms seem to be employed.
Asunto(s)
Antineoplásicos/farmacología , Calcitriol/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Tretinoina/farmacología , Vitamina D/análogos & derivados , Alitretinoína , Antineoplásicos/antagonistas & inhibidores , Apoptosis , Calcitriol/farmacología , División Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Neoplasias Pancreáticas/patología , Tretinoina/antagonistas & inhibidores , Células Tumorales Cultivadas , Vitamina D/antagonistas & inhibidores , Vitamina D/farmacologíaRESUMEN
Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50 values of 15, 20 and 3 microM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 microM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells.
Asunto(s)
Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Difosfonatos/toxicidad , División Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/patología , Supervivencia Celular/efectos de los fármacos , Ácido Clodrónico/toxicidad , Fragmentación del ADN/efectos de los fármacos , Femenino , Humanos , Imidazoles/toxicidad , Cinética , Pamidronato , Pirrolidinas/toxicidad , Células Tumorales Cultivadas , Ácido ZoledrónicoRESUMEN
Synthetic analogs of vitamin D induce apoptosis in cultured breast cancer cells and cause regression of experimentally-induced rat mammary tumors. To further elucidate the mechanisms involved, we have examined interactions between two vitamin D analogs (CB1093 and EB1089) and known mediators of apoptosis, TNFalpha and ceramide. Pretreatment of MCF-7 breast cancer cells with CB1093 and EB1089 substantially potentiated cytotoxic effects of TNFalpha as assessed by cell viability assay, DNA fragmentation and videomicroscopy. No significant changes in the levels of TNFalpha or TNF-RI transcripts were detected. CB1093 primed cells demonstrated enhanced responsiveness to cell permeable C2-ceramide in terms of increased DNA fragmentation and loss of cell viability. Activation of cytosolic phospholipase A2 (cPLA2) has been implicated in TNFalpha-mediated apoptosis. As assessed by [3H]-arachidonic acid release, cells primed for 48 h with CB1093 (50 nM) showed enhanced cPLA2 activation in response to TNFalpha or ceramide. CB1093 treatment alone led to cPLA2 activation and loss of cell viability which was inhibited by the specific inhibitor AACOCF3. These results suggest that TNFalpha and vitamin D analogs share a common pathway leading to apoptosis involving cPLA2 activation and/or ceramide generation.
Asunto(s)
Apoptosis , Calcitriol/análogos & derivados , Fosfolipasas A/metabolismo , Esfingosina/análogos & derivados , Factor de Necrosis Tumoral alfa/metabolismo , Antígenos CD/genética , Apoptosis/efectos de los fármacos , Calcitriol/metabolismo , Calcitriol/farmacología , Supervivencia Celular/efectos de los fármacos , Citosol , Citotoxicidad Inmunológica , Sinergismo Farmacológico , Activación Enzimática , Humanos , Fosfolipasas A2 , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Esfingosina/metabolismo , Esfingosina/farmacología , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
1. The conventional approach to treatment of acute myeloid leukemia has been the use of chemotherapy, which although being cytotoxic to malignant clones, is also cytodestructive to normal cells. In addition, some leukemia cells develop resistance to chemotherapy and are therefore difficult to eradicate. 2. Differentiation therapy, whereby immature cells are induced to attain a mature phenotype by differentiation agents, has provided an alternative strategy in the treatment of hyperproliferative disorders. This has been highlighted by the use of all-trans retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL). 3. Another differentiation agent, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), directs monocytic maturation of normal and leukemic cells. Cellular studies have revealed that combinations of vitamin D derivatives and retinoids such as ATRA and 9-cis retinoic acid (9-cis RA) exhibit cooperative effects on differentiation in established leukemia cell lines such as HL-60, U937, and NB4. Furthermore, vitamin D compounds, although not able to induce apoptosis when used alone, potentiate apoptosis induced by 9-cis RA in HL-60 cells and differentially regulate the expression of the apoptosis-related gene products bcl-2 and bax. The molecular mechanisms involved in regulating differentiation and apoptosis by these agents are mediated through the interactions of the nuclear receptors for vitamin D (VDR), ATRA (RAR), and 9-cis RA (RXR), which are able to form homo- or heterodimeric complexes and transcriptionally activate or repress target gene expression. 4. There is evidence to suggest that nitric oxide may also play a role in leukemic cell differentiation and that 1,25(OH)2D3 may influence endogenous nitric oxide production either by directly increasing tumor necrosis factor-alpha (TNF-alpha) or through a secondary mediator such as the C-type lectin CD23.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Leucemia/tratamiento farmacológico , Retinoides/farmacología , Vitamina D/farmacología , Enfermedad Aguda , Apoptosis/efectos de los fármacos , Calcitriol/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Leucemia/patología , Proteínas de Neoplasias/fisiología , Óxido Nítrico/fisiología , Proteínas de Fusión Oncogénica/fisiología , Retinoides/uso terapéutico , Vitamina D/uso terapéuticoRESUMEN
Survival factors are known to promote cell viability, and factor deprivation can be a potent apoptotic signal. Insulin-like growth factors are potent mitogens and inhibitors of apoptosis for many normal and neoplastic cells with insulin-like growth factor-I (IGF-I) being the most effective in many breast cancer cell lines. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and its analogues inhibit IGF-I-stimulated growth of MCF-7 human breast cancer cells. The aim of this study was to determine the relationship between inhibition of IGF-I responsiveness and induction of apoptosis by vitamin D analogues in breast cancer cells. Vitamin D analogues EB1089 and CB1093 inhibited autonomous and IGF-I-stimulated growth of MCF-7 and T47D cells and autonomous growth of IGF-I-insensitive Hs578T cells. In MCF-7 cells, IGF-I alone (4 nM) protected against apoptosis mediated by serum deprivation. Co-treatment with vitamin D analogues prevented the anti-apoptotic effects of IGF-I. In T47D cells, IGF-I treatment provided only partial protection against apoptosis induced by serum deprivation and co-incubation of serum-deprived cells with 100 nM CB1093 and IGF-I abrogated this partial protection. In Hs578T cells, addition of IGF-I did not prevent apoptosis induced by serum deprivation. However, treatment with CB1093 attenuated the protective effect of the serum in these cells. Our findings suggest that vitamin D analogues inhibit IGF-I signalling pathways to promote apoptosis in breast cancer cells.