RESUMEN

Few studies have explored the effects of omega-3 polyunsaturated fatty acid (n-3 PUFA) supplementation on immune modulation in murine models of mammary carcinogenesis. HER-2/neu and PyMT mice were randomized to 2 dietary interventions: AIN-93G-based diet with 1) 11% of diet (per gram weight) as corn oil (CO) or 2) 10% of diet as menhaden fish oil plus 1% of diet as corn oil (FO). FO significantly reduced the incidence and multiplicity of tumors (P < 0.001) in HER-2/neu, but not PyMT mice. FO-fed mice had significantly larger splenocyte counts than CO-fed mice in both the HER-2/neu and PyMT models; and in both models this was comprised of an increase in most cell types, including Gr-1(+)/CD11b(+) cells. T cells from FO-fed HER-2/neu mice produced significantly more interleukin-2 (P = 0.004) and interferon-γ (P = 0.012) in response to in vitro stimulation with anti-CD3 (0.5 µg/ml). Lastly, FO-fed HER-2/neu mice had significantly more tumor immune infiltrates than CO-fed mice, including NK1.1(+), F4/80(+), and Gr-1(+)/CD11b(+) cells (P ≤ 0.05). Greater Th1 cytokine production and significantly more tumor immune infiltrates in FO-fed Her2/neu mice may account for the cancer prevention effect of fish oil in this model.

Asunto(s)

Aceites de Pescado/farmacología , Neoplasias/prevención & control , Linfocitos T/efectos de los fármacos , Animales , Línea Celular Tumoral , Aceite de Maíz/farmacología , Modelos Animales de Enfermedad , Ácidos Grasos Omega-3/farmacología , Femenino , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Ratones , Ratones Transgénicos , Linfocitos T/metabolismo

RESUMEN

We report here a detailed time course study of the individual and combined chemopreventive effects of Tamoxifen (Tam) and a high fish oil (FO) diet on multiple histologic parameters of mammary carcinogenesis. Groups of female Sprague-Dawley rats were injected ip with 1-methyl-1-nitrosourea at 50 days of age and assigned to either a control diet (20% corn oil [CO]) or a FO-rich diet (10% FO + 10% CO) in the presence and absence of Tam in the diet (0.6 ppm). Rats were sacrificed at weeks 4 (before palpable tumors), 8 and 12 (when ∼90% of control rats had palpable tumors). Our results demonstrate a major effect of Tam in inhibiting the development of early preneoplastic lesions. FO, while having a marginal protective effect of it own, enhanced the antitumor action of Tam on all histologic parameters of carcinogenesis, although the effects of the combination were not statistically different from those of Tam alone. The combination of FO and Tam was the only intervention that induced regression of established preneoplastic lesions. We also found that in contrast to plasma, only target tissue n-3 fatty acids (FAs) levels correlated with select tissue biomarkers of carcinogenesis whose expression was altered in a manner predictive of a protective effect. Our results demonstrating the potentially superior chemopreventive efficacy of Tam and n-3FA have important translational implications. Our data also emphasize the importance of local factors in affecting target tissue levels and biologic effects of n-3FA.

Asunto(s)

Antineoplásicos/farmacología , Carcinogénesis/efectos de los fármacos , Ácidos Grasos Omega-3/farmacología , Neoplasias Mamarias Experimentales/prevención & control , Tamoxifeno/farmacología , Animales , Biomarcadores de Tumor/metabolismo , Carcinogénesis/genética , Carcinogénesis/metabolismo , Quimioprevención/métodos , Dieta , Femenino , Aceites de Pescado/farmacología , Antígeno Ki-67/genética , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Metilnitrosourea , Lesiones Precancerosas/tratamiento farmacológico , Lesiones Precancerosas/genética , Lesiones Precancerosas/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

We have previously shown that a fish oil (FO)-rich diet increased the chemopreventive efficacy of tamoxifen (Tam) against N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinogenesis. Herein, we provide evidence that Tam treatment modifies gene expression of mammary tumors depending upon the type of dietary fat fed to the animals. Rats initiated with MNU and treated with Tam were fed a diet rich in corn oil or FO. After 8 wk, cribriform tumors were collected and gene expression analysis was performed. Increased RNA expression of genes such as SerpinB10, Wisp2, and Apod in tumors from FO-treated rats is indicative of highly differentiated tumors. Decreased expression of H19 and Igf2 mRNA in Tam-treated groups, and Gamma Synuclein mRNA in the FO + Tam group may be related to tumor growth impairment and lower metastatic capacity. Change in the expression of genes associated with immunity in animals in the FO + Tam group may suggest a shift in the immune response. These data show that, although Tam modulates the expression of genes leading to tumor growth impairment, further modulations of genes are influenced by FO. FO modulation of Tam changes in gene expression accounts for its enhancing chemopreventive effect against MNU-induced mammary carcinogenesis. Supplemental materials are available for this article. Go to the publisher's online edition of Nutrition and Cancer to view the supplemental file.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Aceites de Pescado/administración & dosificación , Neoplasias Mamarias Experimentales/tratamiento farmacológico , ARN Mensajero/genética , Tamoxifeno/farmacología , Animales , Transformación Celular Neoplásica/efectos de los fármacos , Aceite de Maíz/administración & dosificación , Grasas de la Dieta/administración & dosificación , Femenino , Regulación Neoplásica de la Expresión Génica , Inmunidad , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Metilnitrosourea/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Ratas , Reproducibilidad de los Resultados , Transcriptoma

RESUMEN

Epidemiologic studies on the protective role of omega-3 fatty acids (n:3) on breast cancer prevention remain inconclusive but studies in preclinical models provide more positive outcome. However, the mechanisms accounting for the protective effect of n:3 are not defined. In the present study, conducted in the N-methyl-N-nitrosourea-induced rat mammary carcinogenesis model, we examined the effects of n:3 individually and in combination with the anti-estrogen Tamoxifen (Tam) on a comprehensive panel of systemic and preneoplastic mammary gland restricted biomarkers which may be critical in the progression to invasive cancer. We observed that fish oil (FO) rich diets significantly reduced Ki67 expression in hyperplastic lesions, while cleaved caspase-3 expression was not affected. Dietary FO and/or Tam did not have major effects on systemic oxidative stress biomarkers, based on oxidative damage to DNA measured as 8-hydroxy-2-deoxyguanosine (8-OH-dG) and lipid peroxidation assessed as thiobarbituric acid reactive substances (TBARS). Tissue levels of 8-isoprostane, on the other hand, were markedly reduced (p<0.0001) in FO-fed rats, possibly as a result of FO-induced depletion of arachidonic acid in the mammary gland. These results suggest that the protective effect of n:3 in this experimental system is not mediated by changes in the levels of oxidative stress but may result from suppression of arachidonic acid-specific pathways.

Asunto(s)

Aceites de Pescado/farmacología , Glándulas Mamarias Animales/efectos de los fármacos , Neoplasias Mamarias Experimentales/metabolismo , Estrés Oxidativo/efectos de los fármacos , Lesiones Precancerosas/metabolismo , Tamoxifeno/farmacología , Animales , Biomarcadores/metabolismo , Caspasa 3/metabolismo , Transformación Celular Neoplásica/inducido químicamente , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Dieta , Activación Enzimática/efectos de los fármacos , Ácidos Grasos/metabolismo , Femenino , Glutatión/sangre , Glutatión Peroxidasa/metabolismo , Antígeno Ki-67/metabolismo , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Metilnitrosourea , Ratas , Ratas Sprague-Dawley , Factores de Tiempo

RESUMEN

In these experiments, we tested the hypothesis that inhibition of the estrogen receptor (ER) with Tamoxifen and activation of PPARγ with fish oil (FO) rich in omega-3 (n-3; known PPAR agonists) inhibit the development of hormone-independent breast cancer in view of the known crosstalk between the ER and PPARγ pathways. We selected the polyoma middle T transgenic mouse model, since in this system the development of ER- tumors is preceded by ER positive preneoplastic lesions. Tamoxifen admixed with a 20% corn oil (CO) modified AIN-76A diet delayed mammary carcinogenesis and inhibited tumor multiplicity, volume, and weight in a dose-dependent (1, 10, and 100 ppm) fashion. Administration of increasing concentrations of FO in the diet (5%, 10%, and 17%) did not affect any of the tumor parameters. Combined administration of different doses of Tamoxifen and FO delayed carcinogenesis and suppressed tumor multiplicity and volume to the same extent as Tamoxifen alone. Mice fed 10% FO exhibited the expected increase in n-3/n-6 ratio in plasma and tumor based on diet analysis. Further increase in the n-3/n-6 ratio was not observed in mice fed the 17% FO diet. FO reduced tissue levels of arachidonic acid and its metabolite PGF-2α. Our results support the role of ER expression by preneoplastic lesions in the development of hormone-independent tumors and consequently the importance of including ER targeting in combination with mechanistically based novel chemopreventive agents.

Asunto(s)

Receptor alfa de Estrógeno/antagonistas & inhibidores , Aceites de Pescado/farmacología , Neoplasias Mamarias Experimentales/prevención & control , PPAR gamma/antagonistas & inhibidores , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Animales , Femenino , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Poliomavirus , Infecciones Tumorales por Virus

RESUMEN

The antiestrogen tamoxifen reduces breast cancer incidence in high-risk women but is unable to inhibit the development of hormone-independent tumors. Omega-3 polyunsaturated fatty acids (n-3 PUFA), known ligands of the peroxisome proliferator activated receptor-gamma (PPARgamma), generally exert tumor-suppressive effects. Based on the known crosstalk between the estrogen and the PPARgamma receptors, we tested the hypothesis that the combination of tamoxifen with n-3 PUFA results in a superior antitumor action over the individual interventions. In this study, we report for the first time that the combination of a fish oil diet rich in n-3 PUFA and tamoxifen seemed to inhibit N-methyl-N-nitrosourea-induced mammary carcinogenesis, tumor multiplicity, and volume to a greater extent than the individual interventions. The potential superiority of the combination was particularly evident at a suboptimal dose of tamoxifen, which, by itself, was unable to significantly decrease tumor development. Because activation of PPARgamma is known to inhibit oxidative stress, we examined the effects of our interventions on circulating and tumor levels of glutathione, a major intracellular antioxidant. Our results indicate that reduction in the level of oxidative stress may be a potential mechanism by which the n-3 PUFA-rich diet potentiated the tumor-suppressive effect of tamoxifen. Our interventions were well tolerated without evidence of toxicity. Combined administration of tamoxifen and n-3 PUFA is a promising new approach to breast cancer prevention. Because of its safety, this combination can quickly be translated to the clinic if its superiority can be supported by future studies.

Asunto(s)

Alquilantes/toxicidad , Antineoplásicos Hormonales/uso terapéutico , Aceites de Pescado/uso terapéutico , Neoplasias Mamarias Experimentales/prevención & control , Metilnitrosourea/toxicidad , Lesiones Precancerosas/prevención & control , Tamoxifeno/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Ácidos Grasos/metabolismo , Femenino , Hígado/efectos de los fármacos , Hígado/enzimología , Neoplasias Mamarias Experimentales/inducido químicamente , Lesiones Precancerosas/inducido químicamente , Ratas , Ratas Sprague-Dawley

RESUMEN

We have shown that alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, the first and rate-limiting enzyme in polyamine synthesis, has significant antiproliferative and antiinvasive effects in breast cancer cells. We have also reported that these antitumor effects are associated with activation of multiple signaling pathways, including STAT-3, STAT-1, Jun-N-Terminal kinase (JNK), and Mitogen activated protein kinase (MAPK), the latter being found to mediate its antiinvasive action in MDA-MB-435 cells. The present experiments were designed to test the effect of DFMO on the protein kinase A (PKA) pathway and determine its biological significance. We found that DFMO administration (1 mM) to MDA-MB-435 breast cancer cells significantly increased cAMP response element (CRE)-binding protein (CREB) phosphorylation as well as the transactivation of pCRE-luc, a CREB-dependent promoter activated by PKA. To determine the significance of this biochemical effect of DFMO, we used the PKA inhibitor H89 which, as expected, suppressed in a dose-dependent manner (1 and 10 microM) basal and DFMO-induced CREB phosphorylation in our system. Administration of H89 alone was able to suppress proliferation of MDA-MB-435 cells when used at a concentration (10 microM) shown to completely block basal CREB phosphorylation. At concentrations of 0.5 and 1 muM, H89 treatment, while having no antiproliferative effect of its own, potentiated in a dose-dependent fashion the growth inhibitory action of a suboptimal concentration of DFMO (0.01 mM). Ten micromoles of H89 reduced invasiveness of MDA-MB-435 cells in matrigel by approximately 40% (an effect similar to that of 1 mM DFMO). The combination treatment further reduced invasiveness by approximately 80% (P < 0.01 versus the individual treatments). H89 treatment (10 microM) partially reduced DFMO-induced phosphorylation of STAT-3 but not that of STAT-1, Extracellular regulated kinase (ERK), and JNK. In conclusion, our results indicate that PKA signaling exerts proproliferative and proinvasive effects in our experimental system. Therefore, its activation by DFMO represents a compensatory mechanism which should be blocked in order to maximize the antitumor action of the drug. Our data are also consistent with the notion that STAT-3 activation by DFMO is at least in part mediated through the PKA pathway.

Asunto(s)

Neoplasias de la Mama/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Eflornitina/farmacología , Línea Celular Tumoral , Proliferación Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Isoquinolinas/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Sistema de Señalización de MAP Quinasas , Invasividad Neoplásica , Fosforilación , Regiones Promotoras Genéticas , Transducción de Señal , Sulfonamidas/farmacología

RESUMEN

Increased polyamine synthesis has been associated with proliferation and progression of breast cancer, and thus, is a potential target for anticancer therapy. Polyamine depletion by alpha-difluoromethylornithine (DFMO) has been shown to decrease pulmonary and bone metastasis from human breast cancer cell xenografts. Following these observations, this study was designed to test the effects of DFMO on in vitro and in vivo features of the highly invasive and metastatic 4T1 murine mammary cancer cells. DFMO inhibited proliferation, caused G1-S arrest, and suppressed in vitro invasiveness of 4T1 cells. In contrast to our previous findings with MDA-MB-435 cells, DFMO did not affect the activation of signal transducers and activator of transcription 3, c-Jun N-terminal kinase, and extracellular signal-regulated kinase, but decreased phosphorylation of p38. DFMO did not alter the expression of Twist. DFMO delayed the orthotopic growth of 4T1 xenografts in association with suppressed putrescine and spermidine levels but increased levels of spermine. DFMO did not affect pulmonary metastasis when primary tumors of control and DFMO-treated mice were matched for size. Interestingly, DFMO reduced Ki-67 expression only in the primary tumors but did not affect its expression in the metastatic tumors in the lung. Cleaved caspase-3 expression was not affected by DFMO in either the primary tumors or the pulmonary metastasis. In summary, DFMO treatment markedly inhibited in vitro proliferation and invasiveness of 4T1 cells and retarded the growth of orthotopic xenografts in mice. The failure of DFMO to inhibit pulmonary metastasis in this system appears to be due, at least in part, to its lack of antiproliferative effect at the metastatic sites.

Asunto(s)

Eflornitina/metabolismo , Neoplasias Mamarias Animales/metabolismo , Poliaminas/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Técnicas In Vitro , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias Pulmonares/metabolismo , Ratones , Metástasis de la Neoplasia , Trasplante de Neoplasias , Fosforilación , Factor de Transcripción STAT3/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo

RESUMEN

We have shown that administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine (PA) biosynthesis, reduces the invasive and metastatic properties of MDA-MB-435 breast cancer cells while activating multiple signal transduction pathways, including MAPK, Stat3, Stat1, and JNK. Since the activity of these signaling mechanisms is frequently regulated by upstream tyrosine kinases (TKs), we tested whether non-receptor and receptor TKs may be involved in the signaling and biological effects of DFMO in MDA-MB-435 cells. Treatment with DFMO (1 mM for 48 h) did not affect Src phosphorylation (Tyr 416). Administration of the Src-family members inhibitor PP-1 (1 microM), blocked Src phosphorylation in the absence and in the presence of DFMO, but did not block the signaling effects of DFMO (increased phosphorylation of Stat3, Stat1, ERK and JNK). PP-1 treatment, on the other hand, inhibited the invasiveness of MDA-MB-435 cells in matrigel and potentiated the anti-invasive effect of DFMO. Next, we focused on the role of receptor TK. Western analysis of cell lysates from MDA-MB-435 cells failed to show the presence of EGF-R and HER-2neu but demonstrated the expression of c-Met, the receptor for hepatocyte growth factor (HGF). Therefore, we tested the effect of DFMO on the HGF/c-Met pathway which is strongly implicated in the progression of human breast cancer. We found that DFMO treatment blocked HGF-induced c-Met phosphorylation in MDA-MB-435 cells, suggesting that its anti-invasion action may be mediated, at least in part, by blocking c-Met signaling. Next, we showed that 1 mM DFMO suppressed HGF induced invasiveness of MDA-MB-435 cells in matrigel. Combination administration of DFMO with suboptimal doses of PHA-665752, a specific c-Met inhibitor, reduced invasiveness to an even greater extent than the individual treatment. These findings indicate that Src-family members, while not involved in DFMO action, promote invasiveness of breast cancer cells and their inhibition may enhance the antitumor effect of PA depletion. Our data also point to inhibition of HGF/c-Met pathway as a possible novel approach to enhancing the antitumor action of DFMO.

Asunto(s)

Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Eflornitina/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Antineoplásicos/farmacología , Proliferación Celular , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Indoles/farmacología , Invasividad Neoplásica , Fosforilación , Poliaminas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal , Sulfonas/farmacología , Familia-src Quinasas/metabolismo

RESUMEN

We have previously observed that inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) upregulates production of thrombospondin-1 (TSP-1), an extracellular matrix protein with potent anti-angiogenic and antimetastatic properties, by MDA-MB-435 human breast cancer cells in culture. The present experiments were designed to investigate the mechanisms by which DFMO regulates TSP-1 production in this system. 35S-methionine pulse chase experiments indicated that DFMO administration increased TSP-1 synthesis by approximately 6-fold, while it slightly but significantly decreased protein half-life from 35 to 28 min. DFMO treatment increased steady state TSP-1 mRNA levels by 2-fold in MDA-MB-435 cells. TSP-1 promoter reporter studies indicated that this increase was largely due to activation of transcription. Analysis of distribution of TSP-1 mRNA levels between non-polysomal, subpolysomal and polysomal fractions in control and DFMO-treated cells suggested a major stimulatory effect of the drug on TSP-1 translation. A similar increase in TSP-1 transcription and translation in response to DFMO treatment was also observed in vivo in MDA-MB-435 breast cancer xenografts. Surprisingly however, we failed to detect an increase in TSP-1 protein as assessed by Western blot analysis. The reason for this unexpected finding is unknown but may be due to DFMO-induced stimulation of TSP-1 secretion into the systemic circulation, thus preventing its accumulation within the tumor.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Eflornitina/farmacología , Inhibidores Enzimáticos/farmacología , Trombospondina 1/biosíntesis , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , ARN Mensajero/análisis , Trombospondina 1/genética

RESUMEN

Increased polyamine synthesis has been associated with proliferation and progression of breast cancer, and thus, is a potential target for anti-cancer therapy. Polyamine depletion by DFMO has been shown to decrease pulmonary and bone metastasis from human breast cancer cell xenografts. Following these observations, this study was designed to test the effects of DFMO on in vitro and in vivo features of the highly invasive and metastatic 4T1 murine mammary cancer cells. DFMO inhibited proliferation, caused G1-S arrest, and suppressed in vitro invasiveness of 4T1 cells. In contrast to our previous findings with MDA-MB-435 cells, DFMO did not affect the activation of STAT3, JNK, and ERK, but decreased phosphorylation of p38. DFMO did not alter the expression of Twist. DFMO delayed the orthotopic growth of 4T1 xenografts in association with suppressed putrescine and spermidine levels but increased levels of spermine. DFMO did not affect pulmonary metastasis when primary tumors of control and DFMO-treated mice were matched for size. Interestingly, DFMO reduced Ki-67 expression only in the primary tumors but did not affect its expression in the metastatic tumors in the lung. Cleaved caspase-3 expression was not affected by DFMO in either the primary tumors or pulmonary metastasis. In summary, DFMO treatment markedly inhibited in vitro proliferation and invasiveness of 4T1 cells and retarded the growth of orthotopic xenografts in mice. The failure of DFMO to inhibit pulmonary metastasis in this system appears to be due, at least in part, to its lack of anti-proliferative effect at the metastatic sites.

Asunto(s)

Eflornitina/química , Poliaminas/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis , Línea Celular Tumoral , Colágeno/química , Combinación de Medicamentos , Antígeno Ki-67/biosíntesis , Laminina/química , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Trasplante de Neoplasias , Proteoglicanos/química , Putrescina/química , Espermidina/química , Espermina/química

RESUMEN

Inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) has been shown to inhibit proliferation of breast cancer cells although its mechanism of action has not been fully elucidated. To address this issue, we tested the effects of DFMO on cell cycle variables of MDA-MB-435 human breast cancer cells in culture. We also focused on the possible mediatory role of the mitogen-activated protein kinase (MAPK) pathway on the cell cycle effects of DFMO because this compound has been shown to activate MAPK signaling. We found that DFMO caused a p53-independent increase in p21 and its association with cyclin-dependent kinase (cdk)-2 and decreased cdk-2 protein as well as its phosphorylation on Thr160. In addition, DFMO markedly suppressed the expression of the full-length and low molecular weight forms of cyclin E. These effects of DFMO were reversible with exogenous putrescine, thus indicating that they are specifically mediated through polyamine depletion. Cdk-2 activity was drastically reduced in DFMO-treated breast cancer cells which exhibited a reduction in retinoblastoma (Rb) phosphorylation and protein. As a predictable consequence of these effects, DFMO caused a G1-S block. In addition, DFMO inhibited G2-M transition, most likely as a result of its induction of p21 expression. Inhibition of the MAPK pathway with PD98059 or U0126 blocked the DFMO-induced induction of p21 and the reduction of cdk-2 protein. PD98059 reversed the G2-M block induced by DFMO (probably as a result of suppression of p21) but not the G1-S arrest. MDA-MB-435 cells treated with PD98059 or U0126 in the presence and absence of DFMO exhibited a marked increase in the expression of p27 and its association with cdk-2, a decrease in phosphorylation of cdk-2 on Thr160, and a decrease in cyclin E expression. As predicted, PD98059 treatment reduced cdk-2 activity and Rb phosphorylation while reversing the decrease in Rb protein induced by DFMO. Neither DFMO nor PD98059, either alone or in combination, reduced cdk-4 activity despite a marked induction in p15 expression caused by DFMO. Our results indicate that activation of the MAPK pathway accounts for some of the effects of DFMO on cell cycle events of breast cancer cells. Inhibition of the MAPK pathway, however, does not reverse the cell cycle arrest induced by DFMO because of activation of alternative mechanisms leading to suppression of cdk-2 activity.

Asunto(s)

Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Eflornitina/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Putrescina/farmacología , Butadienos/farmacología , Proteínas de Unión al Calcio/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/biosíntesis , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Interacciones Farmacológicas , Flavonoides/farmacología , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neoplasias Hormono-Dependientes/enzimología , Neoplasias Hormono-Dependientes/patología , Nitrilos/farmacología , Fosforilación , Proteína de Retinoblastoma/metabolismo

RESUMEN

INTRODUCTION: Polyamines affect proliferation, differentiation, migration and apoptosis of cells, indicating their potential as a target for cancer chemotherapy. Ornithine decarboxylase converts ornithine to putrescine and is the rate-limiting step in polyamine synthesis.alpha-Difluoromethylornithine (DFMO) irreversibly inhibits ornithine decarboxylase and MDA-MB-435 human breast cancer metastasis to the lung without blocking orthotopic tumor growth. This study tested the effects of DFMO on orthotopic tumor growth and lung colonization of another breast cancer cell line (MDA-MB-231) and the effects on bone metastasis of MDA-MB-435 cells. METHODS: MDA-MB-231 cells were injected into the mammary fat pad of athymic mice. DFMO treatment (2% per orally) began at the day of tumor cell injection or 21 days post injection. Tumor growth was measured weekly. MDA-MB-231 cells were injected into the tail vein of athymic mice. DFMO treatment began 7 days prior to injection, or 7 or 14 days post injection. The number and incidence of lung metastases were determined. Green fluorescent protein-tagged MDA-MB-435 cells were injected into the left cardiac ventricle in order to assess the incidence and extent of metastasis to the femur. DFMO treatment began 7 days prior to injection. RESULTS: DFMO treatment delayed MDA-MB-231 orthotopic tumor growth to a greater extent than growth of MDA-MB-435 tumors. The most substantial effect on lung colonization by MDA-MB-231 cells occurred when DFMO treatment began 7 days before intravenous injection of tumor cells (incidence decreased 28% and number of metastases per lung decreased 35-40%). When DFMO treatment began 7 days post injection, the incidence and number of metastases decreased less than 10%. Surprisingly, treatment initiated 14 days after tumor cell inoculation resulted in a nearly 50% reduction in the number of lung metastases without diminishing the incidence. After intracardiac injection, DFMO treatment decreased the incidence of bone metastases (55% vs 87%) and the area occupied by the tumor (1.66 mm2 vs 4.51 mm2, P < 0.05). CONCLUSION: Taken together, these data demonstrate that DFMO exerts an anti-metastatic effect in more than one hormone-independent breast cancer, for which no standard form of biologically-based treatment exists. Importantly, the data show that DFMO is effective against metastasis to multiple sites and that treatment is generally more effective when administered early.

Asunto(s)

Antineoplásicos/uso terapéutico , Neoplasias Óseas/prevención & control , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Eflornitina/uso terapéutico , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Metástasis de la Neoplasia/prevención & control , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Trasplante Heterólogo

RESUMEN

We have previously reported that inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) reduces pulmonary metastasis from MDA-MB-435 human breast cancer xenografts without affecting the volume of the primary tumors (Manni et al. Clin Exp Mets 20:321, 2003). In these experiments, we show that DFMO treatment (2% in drinking H(2)O) reduced the growth fraction of the primary tumors by 60%. However, this effect was counter-balanced by a similar reduction in non-apoptotic necrosis, thus accounting for the preservation of tumor volume in DFMO-treated mice. DFMO treatment caused a 4-fold increase in cytoplasmic staining for cleaved caspase-3 (as opposed to the nuclear staining observed in control tonsil tissue) in the absence of histologic evidence of apoptosis. DFMO treatment reduced the number of mice with pulmonary metastasis by approximately 80% and the number of metastasis per mouse by >90% in association with a reduction in invasiveness of the primary tumor in the surrounding dermis and muscle by approximately 30%. DFMO treatment increased ERK phosphorylation in the tumors, an effect that has been found by us in vitro to be causally linked to the anti-invasive effect of the drug (Manni et al. Clin Exp Metast 2004; 21: 461]. DFMO also increased tyrosine phosphorylation of STAT-3 and expression of STAT-1 and JNK proteins. Administration of SAM486A (1 mg/kg/i.p. daily), an inhibitor of S-adenosylmethionine decarboxylase, either individually or in combination with DFMO, was not found to exert any biological or biochemical effects, most likely as a result of its failure to suppress tissue polyamine levels under these experimental conditions.

Asunto(s)

Neoplasias de la Mama/metabolismo , Eflornitina/farmacología , Neoplasias Pulmonares/metabolismo , Metástasis de la Neoplasia/prevención & control , Poliaminas/metabolismo , Amidinas/administración & dosificación , Amidinas/farmacología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/secundario , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Eflornitina/administración & dosificación , Femenino , Humanos , Indanos/administración & dosificación , Indanos/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Ratones , Ratones Desnudos , Invasividad Neoplásica , Trasplante de Neoplasias , Poliaminas/química , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo

RESUMEN

The antitumor activity of the S-adenosylmethionine decarboxylase (SAMDC) inhibitor SAM486A in human breast cancer cells was investigated. Our in vitro study focused on testing the effects of SAM486A on the proliferation, clonogenicity, invasiveness, cell signaling and PA levels of hormone-independent MDA-MB-435 human breast cancer cells. We also investigated the antitumor action, effects on polyamine pools and tolerability of SAM486A administered to nude mice carrying MDA-MB-435 xenografts. SAM486A suppressed anchorage-independent and -dependent growth and invasiveness of breast cancer cells and the inhibition of cell growth was associated with suppression of spermine synthesis. Combined administration of SAM486A and alpha-difluoromethylornithine (DFMO), a selective inhibitor of ornithine decarboxylase (ODC), exerted greater antiproliferative and anti-invasive effects and induced an overall greater suppression of cellular PA levels than the individual treatments. Both SAM486A and DFMO increased phosphorylation of STAT-1, -3, ERK1/2 and p38, thus indicating activation of both STAT signaling and the MAPK pathway. SAM486A (1 mg/kg) significantly suppressed the growth and spermine levels of established MDA-MB435 breast tumors in nude mice. SAM486A exerts a potent antitumor action in MDA-MB-435 breast cancer cells both in vitro and in vivo. Inhibition of cellular spermine is consistently observed with SAM486A treatment and may mediate its antitumor action. Combination treatment with DFMO may allow the use of lower and, hence, less toxic doses of each compound with preservation of optimal therapeutic effect. The role of activation of STAT signaling and the MAPK pathway in the antitumor action of SAM486A remains to be determined.

Asunto(s)

Adenosilmetionina Descarboxilasa/antagonistas & inhibidores , Amidinas/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Indanos/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/farmacología , Invasividad Neoplásica , Transducción de Señal/efectos de los fármacos , Trasplante Heterólogo , Células Tumorales Cultivadas

RESUMEN

We have shown that inhibition of polyamine biosynthesis with alpha-difluoromethylornithine (DFMO) reduces in vitro invasiveness and metastatic capacity of MDA-MB-435 breast cancer cells. These experiments investigated the mechanisms mediating the anti-invasive properties of DFMO. DFMO did not affect phosphorylation of FAK or Akt, but increased ERK phosphorylation by approximately threefold. To test the biologic significance of this finding, we tested the effect of the MEK inhibitor PD98059 on in vitro invasiveness of MDA-MB-435 breast cancer cells, both in the absence and in the presence of the proinvasive peptide hepatocyte growth factor (HGF) as a chemoattractant. We observed that PD98059 treatment reversed the anti-invasive effect of DFMO under both experimental conditions. Next, we tested the influence of DFMO on the production of the prometastatic peptide osteopontin (OPN) and the anti-metastatic protein thrombospondin-1 (TSP-1). DFMO treatment, while not affecting OPN production, markedly increased the TSP-1 level in the conditioned media. This effect was abolished by putrescine administration, thus indicating the specificity of the DFMO action through the polyamine pathway. PD98059 completely blocked the stimulatory effect of DFMO on TSP-1 production, which supports a mediatory role for activation of the MAPK pathway in the upregulation of this anti-metastatic peptide by DFMO. In summary, our results show that the increase in ERK phosphorylation induced by DFMO plays a critical role in the anti-invasive action of the drug and in its ability to upregulate TSP-1 production.

Asunto(s)

Neoplasias de la Mama/metabolismo , Eflornitina/farmacología , Inhibidores Enzimáticos/farmacología , Invasividad Neoplásica/prevención & control , Inhibidores de la Ornitina Descarboxilasa , Neoplasias de la Mama/patología , Flavonoides/farmacología , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteopontina , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Putrescina/administración & dosificación , Sialoglicoproteínas/metabolismo , Trombospondina 1/metabolismo , Células Tumorales Cultivadas

RESUMEN

We have recently shown that administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine (PA) biosynthesis reduces pulmonary metastasis from MDA-MB-435 breast cancer xenografts in nude mice. The present experiments were designed to further explore PA involvement in breast cancer metastasis, using GFP-tagged MDA-MB-435 cells that can be tracked at the single cell level. Administration of DFMO significantly reduced the number of mice with pulmonary metastasis as well as the number of metastases per mouse. Both single-cell and multicellular metastatic deposits were similarly suppressed, thus suggesting that DFMO was inhibiting lung colonization by tumor cells rather than preventing progression of single-cell deposits to overt metastasis. DFMO administration also significantly reduced local recurrences following removal of the primary tumor. Prolongation of DFMO treatment to 14 weeks did not yield a superior antimetastatic effect beyond that provided by a 10-week course of therapy. Discontinuation of DFMO, on the other hand, was associated with local regrowth of the tumors and, possibly, recurrence of pulmonary metastasis. These data provide a rationale for testing the efficacy of anti-PA treatment within the context of adjuvant therapy of breast cancer.

Asunto(s)

Eflornitina/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias Pulmonares/prevención & control , Neoplasias Mamarias Experimentales/prevención & control , Recurrencia Local de Neoplasia/prevención & control , Animales , Femenino , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Desnudos , Inhibidores de la Ornitina Descarboxilasa , Trasplante Heterólogo , Células Tumorales Cultivadas

RESUMEN

Increased cellular activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine (PA) synthesis, is an independent adverse prognostic factor for overall survival in human breast cancer, thus suggesting an important role for PA in tumor progression. The experiments presented here were designed to investigate the role of PA in invasion and metastasis, using the highly aggressive MDA-MB-435 and MDA-MB-231 human breast cancer cell lines. Administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, significantly reduced, in a dose-dependent manner, the invasiveness in matrigel of both MDA-MB-435 and MDA-MB-231 cells by approximately 70%. DFMO treatment also inhibited (P < 0.0001) 'stellate' colony formation (an indicator of aggressive phenotype) by MDA-MB-435 cells plated in the matrigel outgrowth assay. Administration of DFMO (2% in drinking water) reduced the growth rate of both cell lines implanted orthotopically in nude mice. To evaluate metastasis while minimizing effects on proliferation, DFMO-treated mice were sacrificed later to allow their tumors to reach the same size of the tumors in the control mice. The most striking finding was that DFMO, while ineffective in reducing local invasion, nearly totally abolished (P = 0.0152) pulmonary metastasis in mice bearing MDA-MB-435 xenografts. These results support a role of PA in promoting breast cancer aggressiveness, particularly with regard to the development of distant metastasis. Furthermore, the data suggest that PA involvement is distal to local invasion in the metastatic cascade.

Asunto(s)

Poliaminas Biogénicas/farmacología , Neoplasias de la Mama/patología , Neoplasias Pulmonares/prevención & control , Neoplasias Mamarias Experimentales/prevención & control , Proteínas de Neoplasias , Nucleósido-Difosfato Quinasa , Inhibidores de la Ornitina Descarboxilasa , Animales , Northern Blotting , Colágeno , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Eflornitina/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Laminina , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundario , Ganglios Linfáticos/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos CBA , Ratones Desnudos , Proteínas de Unión al GTP Monoméricas/genética , Nucleósido Difosfato Quinasas NM23 , Invasividad Neoplásica , Ornitina Descarboxilasa/metabolismo , Proteínas/genética , Proteoglicanos , Proteínas Represoras , Factores de Transcripción/genética , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patología , Vimentina/metabolismo