RESUMEN
INTRODUCTION: Polyamine analogues have demonstrated significant activity against human breast cancer cell lines as single agents as well as in combination with other cytotoxic drugs. This study evaluates the ability of a polyamine analogue N (1),N (11)-bis(ethyl)norspermine (BENSpm) to synergize with six standard chemotherapeutic agents, 5-fluorouracil (FU), fluorodeoxyuridine, cis-diaminechloroplatinum(II) (C-DDP), paclitaxel, docetaxel, and vinorelbine. MATERIALS AND METHODS: Four human breast cancer cell lines (MDA-MB-231, MCF-7, Hs578t, and T47D) and one immortalized, non-tumorigenic mammary epithelial cell line (MCF-10A) were used for in vitro combination studies with BENSpm and cytotoxic drugs. Xenograft mice models generated with MDA-MB-231 cells were used for in vivo studies with BENSpm and paclitaxel. RESULTS AND CONCLUSION: BENSpm exhibited synergistic inhibitory effect on cell proliferation in combination with 5-FU or paclitaxel in human breast cancer cell lines (MDA-MB-231 and MCF-7) and was either antagonistic or less effective in the non-tumorigenic MCF-10A cell line. Synergism was highest with 120 h concomitant treatment or pre-treatment with BENSpm for 24 h followed by concomitant treatment for 96 additional hours. Since the cytotoxic effects of many polyamine analogues and cytotoxic agents are believed to act, in part, through induction of the polyamine catabolic enzymes SSAT and SMO, the role of these enzymes on synergistic response was evaluated in MDA-MB-231 and MCF-7 treated with BENSpm and 5-FU or paclitaxel. Combination treatments of BENSpm with 5-FU or paclitaxel resulted in induction of SSAT mRNA and activity in both cell lines compared to either drug alone, while SMO mRNA and activity were increased only in MDA-MB-231 cells. Induction was greater with BENSpm/paclitaxel combination than BENSpm/5-FU. Further, RNAi studies demonstrated that both SSAT and SMO play a significant role in the response of MDA-MB-231 cells to treatment with BENSpm and 5-FU or paclitaxel. In MCF-7 cells, only SSAT appears to be involved in the response to these treatments. In an effort to translate combination studies from in vitro to in vivo, and to form a basis for clinical setting, the in vivo therapeutic efficacy of BENSpm alone and in combination with paclitaxel on tumor regression was evaluated in xenograft mice models generated with MDA-MB-231 cells. Intraperitoneal exposure to BENSpm or taxol singly and in combination for 4 weeks resulted in significant inhibition in tumor growth. These findings help elucidate the mechanisms involved in synergistic drug response and support combinations of polyamine analogues with chemotherapeutic agents which could potentially be used in the treatment of breast cancer.
Asunto(s)
Acetiltransferasas/metabolismo , Antineoplásicos/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Espermina/análogos & derivados , Acetiltransferasas/genética , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/química , Fluorouracilo/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/enzimología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Estructura Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Paclitaxel/administración & dosificación , Paclitaxel/química , Paclitaxel/farmacología , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermina/administración & dosificación , Espermina/química , Espermina/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Poliamino OxidasaRESUMEN
Because DNA methyltransferase (DNMT) inhibitors like azacytidine and decitabine are known to be effective in the clinic for diseases like myelodysplastic syndromes that may result in part from transcriptional dysregulation due to epigenetic changes, there is interest in developing novel DNMT inhibitors that would be more effective and less toxic. The effects of one such agent, zebularine, which inhibits DNMT and cytidine deaminase, were assessed in two human breast cancer cell lines, MDA-MB-231 and MCF-7. Zebularine treatment inhibited cell growth in a dose and time dependent manner with an IC-50 of approximately 100 microM and 150 microM in MDA-MB-231 and MCF-7 cells, respectively, on 96 h exposure. This was associated with increased expression of p21, decreased expression of cyclin-D, and induction of S-phase arrest. At high doses zebularine induced changes in apoptotic proteins in a cell line specific manner manifested by alteration in caspase-3, Bax, Bcl2 and PARP cleavage. Like other DNMT inhibitors, zebularine decreased expression of DNMTs post-transcriptionally as well as expression of other epigenetic regulators like methyl CpG binding proteins and global acetyl H3 and H4 protein levels. Its capacity to reexpress epigenetically silenced genes in human breast cancer cells at low doses was confirmed by its ability to induce expression of estrogen and progesterone receptor mRNA in association with changes suggestive of active chromatin at the ER promoter as evidenced by ChIP. Finally, its effect in combination with other DNMT or HDAC inhibitors like decitabine or vorinostat was explored. The combination of 50 muM zebularine with decitabine or vorinostat significantly inhibited cell proliferation and colony formation in MDA-MB-231 cells compared with either drug alone. These findings suggest that zebularine is an effective DNMT inhibitor and demethylating agent in human breast cancer cell lines and potentiates the effects of other epigenetic therapeutics like decitabine and vorinostat.
Asunto(s)
Adenocarcinoma/patología , Neoplasias de la Mama/patología , Citidina/análogos & derivados , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Adenocarcinoma/enzimología , Proteínas Reguladoras de la Apoptosis/biosíntesis , Proteínas Reguladoras de la Apoptosis/genética , Azacitidina/análogos & derivados , Azacitidina/farmacología , Neoplasias de la Mama/enzimología , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/enzimología , Inmunoprecipitación de Cromatina , Citidina/farmacología , Citidina Desaminasa/antagonistas & inhibidores , Decitabina , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ácidos Hidroxámicos/farmacología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Receptores de Estrógenos/biosíntesis , Receptores de Estrógenos/genética , Fase S/efectos de los fármacos , Ensayo de Tumor de Célula Madre , VorinostatRESUMEN
One of the most common cancers in women world wide, breast cancer is classically an endocrine-dependent cancer. It has been known for over a century that development, progression and metastasis of breast cancer are strongly influenced by hormonal factors. Indeed about two-thirds of breast cancers express the estrogen receptor α (ERα) protein, a key predictor of prognosis and response to endocrine therapy. These cancers are frequently amenable to therapies that target estrogen signaling pathways, including selective estrogen receptor modulators like tamoxifen, selective estrogen receptor downregulators like fulvestrant; and agents that reduce estrogen ligand like aromatase inhibitors and ovarian suppression through luteinizing hormone-releasing hormone (LHRH) agonists. It is likely that these approaches, especially adjuvant tamoxifen, have contributed to the reduction in breast cancer mortality that has been observed in recent years. However, data from clinical studies have suggested that only about 60% of ERα-positive breast cancers respond to hormonal therapy. Further, those tumors that lack expression of ERα and the estrogen-regulated progesterone receptor (PgR) are unresponsive to hormone therapy. Thus the problem of acquired or de novo endocrine resistance is a substantial one. Recent molecular and biological advances have contributed to our understanding about potential underlying mechanisms. Here we will focus especially on silencing the expression of ERα as one such endocrine-resistance mechanism and how it might be exploited clinically.
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Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Epigénesis Genética/efectos de los fármacos , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Moduladores Selectivos de los Receptores de Estrógeno/uso terapéutico , Transducción de Señal/efectos de los fármacosRESUMEN
Microcystin-LR (MCLR) is the most common hepatotoxic cyanotoxin produced primarily by Microcystis aeruginosa. In this study, young F344 rats were intraperitoneally injected with a single dose (25, 50, 100, and 150 microg/kg) of MCLR to explore possible toxic effect and toxic response indicators. Acute toxic symptoms, including body weight loss and death, were monitored for 7 days. Mortality reached 100% (9/9) in rats treated with a single MCLR dose of 150 microg/kg. Histopathological examination showed spot necrosis in the liver of animals treated at low doses, while massive hemorrhage and widespread necrotic foci occurred at higher doses, indicating extensive liver damage. Protein phosphatase 2A (PP2A) expression showed a dose-dependent decrease in the liver. Immunohistochemical localization indicated that nuclear PP2A was affected first, followed by cytoplasmic PP2A. In addition, there was a significant increase in sphingolipid levels at higher doses, indicating the involvement of a ceramide-mediated apoptotic pathway. Expression of apoptosis and cell cycle regulatory proteins like Bax, Bcl2, and Bad showed a dose-dependent decrease. This study demonstrated that treatment with a single dose of MCLR caused liver damage, increased sphingolipid levels, and decreased PP2A expression, which ultimately down-regulated the expression of Bcl2 family proteins.
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Apoptosis/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Toxinas Marinas/toxicidad , Microcistinas/toxicidad , Animales , Apoptosis/fisiología , Peso Corporal/fisiología , Proteínas de Ciclo Celular/metabolismo , Relación Dosis-Respuesta a Droga , Hepatocitos/metabolismo , Masculino , Mortalidad , Proteína Fosfatasa 2/metabolismo , Ratas , Ratas Endogámicas F344 , Factores de TiempoRESUMEN
Seasonal variations in the concentration of microcystin-LR (MCLR) in Buffalo Springs Lake (BSL) and Lake Ransom Canyon (LRC; both locations in Lubbock, TX, USA) were monitored from 2003 to 2004. In BSL, the average concentrations of MCLR were 1.78 +/- 1.43 microg/L (mean +/- SD; range, 0.177-4.914 microg/L) in spring, 0.41 +/- 0.096 microg/L (range, 0.191-0.502 microg/L) in summer, 0.46 +/- 0.41 microg/L (range, 0.205-1.598 microg/L) in fall, and 1.04 +/- 0.71 microg/L (range, 0.096-2.428 microg/L) in winter. In LRC, the corresponding concentrations were 1.08 +/- 1.29 microg/L (range, 0.2-5.83 microg/L) in spring, 0.83 +/- 0.46 microg/L (range, 0.315-1.671 microg/L) in summer, 0.44 +/- 0.03 microg/L (range, 0.368-0.555 microg/L) in fall, and 0.78 +/- 0.52 microg/L (range, 0.225-2.130 microg/L) in winter. In both lakes, the seasonal fluctuation of MCLR concentrations correlated positively with dissolved oxygen and negatively with temperature and pH.
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Inhibidores Enzimáticos/análisis , Péptidos Cíclicos/análisis , Monitoreo del Ambiente , Toxinas Marinas , Microcistinas , Estaciones del Año , Texas , Agua/química , Abastecimiento de AguaRESUMEN
Modulation of urinary excretion of green tea polyphenols (GTPs) and oxidative DNA damage biomarker, 8-hydroxydeoxyguanosine (8-OHdG), were assessed in urine samples collected from a randomized, double-blinded and placebo-controlled phase IIa chemoprevention trial with GTP in 124 individuals. These individuals were sero-positive for both HBsAg and aflatoxin-albumin adducts, and took GTP capsules daily at doses of 500 mg, 1000 mg or a placebo for 3 months. Twenty-four hour urine samples were collected before the intervention and at the first and third month of the study. Urinary excretion of 8-OHdG and GTP components was measured by HPLC-CoulArray electrochemical detection. The baseline levels of 8-OHdG and GTP components among the three groups showed homogeneity (P > 0.70), and a non-significant fluctuation was observed in the placebo group over the 3 months (P > 0.30). In GTP-treated groups, epigallocatechin (EGC) and epicatechin (EC) levels displayed significant and dose-dependent increases in both the 500 mg group and 1000 mg group (P < 0.05). The 8-OHdG levels did not differ between the three groups at the 1 month collection, with medians of 1.83, 2.08 and 1.86 ng/mg-creatinine for placebo, 500 and 1000 mg group, respectively (P = 0.999). At the end of the 3 months' intervention, 8-OHdG levels decreased significantly in both GTP-treated groups, with medians of 2.02, 1.03 and 1.15 ng/mg-creatinine for placebo, 500 mg and 1000 mg group, respectively (P = 0.007). These results suggest that urinary excretions of EGC and EC can serve as practical biomarkers for green tea consumption in human populations. The results also suggest that chemoprevention with GTP is effective in diminishing oxidative DNA damage.
Asunto(s)
Quimioprevención , Desoxiguanosina/análogos & derivados , Flavonoides/orina , Neoplasias Hepáticas/prevención & control , Fenoles/orina , Té/química , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Biomarcadores , Catequina/análogos & derivados , Catequina/farmacología , Catequina/orina , Daño del ADN , Desoxiguanosina/orina , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Flavonoides/farmacología , Humanos , Masculino , Estrés Oxidativo , Fenoles/farmacología , Polifenoles , Factores de RiesgoRESUMEN
Aflatoxin B1 (AFB1) and T-2 toxin (T-2) are important food-borne mycotoxins that have been implicated in human health and as potential biochemical weapons threats. In this study the acute and combinative toxicity of AFB1 and T-2 were tested in F-344 rats, mosquitofish (Gambusia affinis), immortalized human hepatoma cells (HepG2) and human bronchial epithelial cells (BEAS-2B). Preliminary experiments were conducted in order to assess the acute toxicity and to obtain LD50, LC50 and IC50 values for individual toxins in each model, respectively. This was followed by testing combinations of AFB1 and T-2 to obtain LD50, LC50 and IC50 values for the combination in each model. All models demonstrated a significant dose response in the observed parameters to treatment. The potency of the mixture was gauged through the determination of the interaction index metric. The results of this study demonstrate that these two toxins interacted to produce alterations in the toxic responses generally classifiable as additive; however, a synergistic interaction was noted in the case of BEAS-2B. It can be gathered that this combination may pose a significant threat to public health and further research needs to be completed addressing alterations in metabolism and detoxification that may influence the toxic manifestations in combination.