RESUMEN
BACKGROUND: Mitochondrial dysfunction and bioenergetic stress play an important role in the etiology of alcoholic liver disease. Previous studies from our laboratory show that the primary methyl donor S-Adenosylmethionine (SAM) minimizes alcohol-induced disruptions in several mitochondrial functions in the liver. Herein, we expand on these earlier observations to determine whether the beneficial actions of SAM against alcohol toxicity extend to changes in the responsiveness of mitochondrial respiration to inhibition by nitric oxide (NO), induction of the mitochondrial permeability transition (MPT) pore, and the hypoxic state of the liver. METHODS: For this, male Sprague-Dawley rats were pair-fed control and alcohol-containing liquid diets with and without SAM for 5 weeks and liver hypoxia, mitochondrial respiration, MPT pore induction, and NO-dependent control of respiration were examined. RESULTS: Chronic alcohol feeding significantly enhanced liver hypoxia, whereas SAM supplementation attenuated hypoxia in livers of alcohol-fed rats. SAM supplementation prevented alcohol-mediated decreases in mitochondrial state 3 respiration and cytochrome c oxidase activity. Mitochondria isolated from livers of alcohol-fed rats were more sensitive to calcium-mediated MPT pore induction (i.e., mitochondrial swelling) than mitochondria from pair-fed controls, whereas SAM treatment normalized sensitivity for calcium-induced swelling in mitochondria from alcohol-fed rats. Liver mitochondria from alcohol-fed rats showed increased sensitivity to NO-dependent inhibition of respiration compared with pair-fed controls. In contrast, mitochondria isolated from the livers of SAM treated alcohol-fed rats showed no change in the sensitivity to NO-mediated inhibition of respiration. CONCLUSION: Collectively, these findings indicate that the hepato-protective effects of SAM against alcohol toxicity are mediated, in part, through a mitochondrial mechanism involving preservation of key mitochondrial bioenergetic parameters and the attenuation of hypoxic stress.
Asunto(s)
Hígado Graso Alcohólico/metabolismo , Hipoxia/metabolismo , Hígado/metabolismo , Mitocondrias Hepáticas/metabolismo , Biogénesis de Organelos , S-Adenosilmetionina/metabolismo , Animales , Biomarcadores , Respiración de la Célula , Modelos Animales de Enfermedad , Complejo I de Transporte de Electrón/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Etanol/efectos adversos , Etanol/metabolismo , Hígado Graso Alcohólico/patología , Hígado/efectos de los fármacos , Hígado/patología , Mitocondrias Hepáticas/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Óxido Nítrico/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismo , S-Adenosilmetionina/farmacologíaRESUMEN
S-adenosylmethionine (SAM) minimizes alcohol hepatotoxicity; however, the molecular mechanisms responsible for SAM hepatoprotection remain unknown. Herein, we use proteomics to determine whether the hepatoprotective action of SAM against early-stage alcoholic liver disease is linked to alterations in the mitochondrial proteome. For this, male rats were fed control or ethanol-containing liquid diets +/- SAM and liver mitochondria were prepared for proteomic analysis. Two-dimensional isoelectric focusing (2D IEF/SDS-PAGE) and blue native gel electrophoresis (BN-PAGE) were used to determine changes in matrix and oxidative phosphorylation (OxPhos) proteins, respectively. SAM coadministration minimized alcohol-dependent inflammation and preserved mitochondrial respiration. SAM supplementation preserved liver SAM levels in ethanol-fed rats; however, mitochondrial SAM levels were increased by ethanol and SAM treatments. With use of 2D IEF/SDS-PAGE, 30 proteins showed significant changes in abundance in response to ethanol, SAM, or both. Classes of proteins affected by ethanol and SAM treatments were chaperones, beta oxidation proteins, sulfur metabolism proteins, and dehydrogenase enzymes involved in methionine, glycine, and choline metabolism. BN-PAGE revealed novel changes in the levels of 19 OxPhos proteins in response to ethanol, SAM, or both. Ethanol- and SAM-dependent alterations in the proteome were not linked to corresponding changes in gene expression. In conclusion, ethanol and SAM treatment led to multiple changes in the liver mitochondrial proteome. The protective effects of SAM against alcohol toxicity are mediated, in part, through maintenance of proteins involved in key mitochondrial energy conserving and biosynthetic pathways. This study demonstrates that SAM may be a promising candidate for treatment of alcoholic liver disease.
Asunto(s)
Etanol/farmacología , Hepatopatías Alcohólicas/prevención & control , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/metabolismo , Proteoma/efectos de los fármacos , S-Adenosilmetionina/farmacología , Animales , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Masculino , Mitocondrias Hepáticas/química , Proteínas Mitocondriales/análisis , Consumo de Oxígeno/efectos de los fármacos , Proteómica , Ratas , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Sulfasalazine (SAZ), a prescribed drug for inflammatory bowel disease, is a potent scavenger of reactive oxygen species. The present study was undertaken to ascertain its ability to protect against gamma radiation-induced damage. Acute toxicity of the drug was studied taking 24-h, 72-h and 30-day mortality after a single intraperitoneal injection of 400-1200 mg/kg body weight (b.wt.) of the drug. The drug LD(50) for 24- and 72-h/30-day survival were found to be 933 and 676 mg/kg b.wt., respectively. The optimum time of drug administration and drug dose-dependent effect on in vivo radiation protection of bone marrow chromosomes was studied in mice. Injection of 30-180 mg/kg SAZ 30 min before gamma irradiation (RT) with 4 Gy produced a significant dose-dependent reduction in the RT-induced percent aberrant metaphases and in the frequency of micronucleated erythrocytes at 24 h after exposure, with a corresponding decrease in the different types of aberrations. The optimum dose for protection without drug toxicity was 120 mg/kg b.wt. At this dose, SAZ produced >60% reduction in the RT-induced percent aberrant metaphases and micronucleated erythrocytes. SAZ also produced a significant increase in the ratio of polychromatic erythrocytes to normochromatic erythrocytes from that of irradiated control. Injection of 120 mg/kg of the drug 60 or 30 min before or within 15 min after 4 Gy whole-body RT resulted in a significant decrease in the percent of aberrant metaphases and in the frequency of micronucleated erythrocytes at 24 h post-irradiation; the maximum effect was seen when the drug was administered 30 min before irradiation. These results show that SAZ protect mice against RT-induced chromosomal damage and cell cycle progression delay. SAZ also protected plasmid DNA (pGEM-7Zf) against Fenton's reactant-induced breaks, suggesting free radical scavenging as one of the possible mechanism for radiation protection.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Cromosomas/efectos de los fármacos , Cromosomas/efectos de la radiación , Protectores contra Radiación/farmacología , Sulfasalazina/farmacología , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/efectos de la radiación , Cromosomas/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Rayos gamma , Masculino , Ratones , Protectores contra Radiación/efectos adversos , Sulfasalazina/efectos adversos , Factores de TiempoRESUMEN
The strategies available for the treatment of metastatic breast cancer are limited. Dietary botanicals may have a better protective effect on this disease. We therefore investigated the effects of grape seed proanthocyanidins (GSPs) on a highly metastatic mouse mammary carcinoma cell line. In vitro treatment of breast cancer cells, 4T1, MCF-7 and MDA-MB-468, with GSPs resulted in significant inhibition of cellular proliferation and viability, and induction of apoptosis in 4T1 cells in a time- and dose-dependent manner. Further analysis indicated an alteration in the ratio of Bax/Bcl-2 proteins in favor of apoptosis, and the knockdown of Bax using Bax siRNA transfection of 4T1 cells resulted in blocking of GSPs-induced apoptosis. Induction of apoptosis was associated with the release of cytochrome c, increased expression of Apaf-1 and activation of caspase 3 and poly (ADP-ribose) polymerase. Treatment with the pan-caspase inhibitor (Z-VAD-FMK) resulted in partial but significant inhibition of apoptosis in 4T1 cells suggesting the involvement of both caspase activation-dependent and activation-independent pathways in the apoptosis of 4T1 cells induced by GSPs. The effects of dietary GSPs were then examined using an in vivo model in which 4T1 cells were implanted subcutaneously in Balb/c mice. Dietary GSPs (0.2 and 0.5%, w/w) significantly inhibited the growth of the implanted 4T1 tumor cells and increased the ratio of Bax:Bcl-2 proteins, cytochrome c release, induction of Apaf-1 and activation of caspase 3 in the tumor microenvironment. Notably, the metastasis of tumor cells to the lungs was inhibited significantly and the survival of the mice enhanced. These data suggest that GSPs possess chemotherapeutic efficacy against breast cancer including inhibition of metastasis.
Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/patología , Proantocianidinas/uso terapéutico , Vitis/química , Animales , Caspasa 3 , Caspasas/metabolismo , Proliferación Celular/efectos de los fármacos , Citocromos c/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Interferente Pequeño/farmacología , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2/antagonistas & inhibidores , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Berberine, a naturally occurring isoquinoline alkaloid, has been shown to possess anti-inflammatory and antitumor properties in some in vitro systems. Here, we report that in vitro treatment of androgen-insensitive (DU145 and PC-3) and androgen-sensitive (LNCaP) prostate cancer cells with berberine inhibited cell proliferation and induced cell death in a dose-dependent (10-100 micromol/L) and time-dependent (24-72 hours) manner. Treatment of nonneoplastic human prostate epithelial cells (PWR-1E) with berberine under identical conditions did not significantly affect their viability. The berberine-induced inhibition of proliferation of DU145, PC-3, and LNCaP cells was associated with G1-phase arrest, which in DU145 cells was associated with inhibition of expression of cyclins D1, D2, and E and cyclin-dependent kinase (Cdk) 2, Cdk4, and Cdk6 proteins, increased expression of the Cdk inhibitory proteins (Cip1/p21 and Kip1/p27), and enhanced binding of Cdk inhibitors to Cdk. Berberine also significantly (P < 0.05-0.001) enhanced apoptosis of DU145 and LNCaP cells with induction of a higher ratio of Bax/Bcl-2 proteins, disruption of mitochondrial membrane potential, and activation of caspase-9, caspase-3, and poly(ADP-ribose) polymerase. Pretreatment with the pan-caspase inhibitor z-VAD-fmk partially, but significantly, blocked the berberine-induced apoptosis, as also confirmed by the comet assay analysis of DNA fragmentation, suggesting that berberine-induced apoptosis of human prostate cancer cells is mediated primarily through the caspase-dependent pathway. The effectiveness of berberine in checking the growth of androgen-insensitive, as well as androgen-sensitive, prostate cancer cells without affecting the growth of normal prostate epithelial cells indicates that it may be a promising candidate for prostate cancer therapy.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis , Berberina/uso terapéutico , Carcinoma/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Andrógenos/uso terapéutico , Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Berberina/farmacología , Carcinoma/metabolismo , Caspasa 3 , Inhibidores de Caspasas , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citocromos c/metabolismo , Activación Enzimática , Células Epiteliales/efectos de los fármacos , Fase G1/efectos de los fármacos , Humanos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasas/metabolismo , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Green tea polyphenols (GTPs) show promise as anticarcinogenic agents and may prevent the development of solar UV radiation-induced skin cancer. Here we investigated the mechanisms by which GTPs prevent UVB-induced skin cancer in mice. Two groups of 6- to 7-wk-old female SKH-1 hairless mice were UVB irradiated (180 mJ/cm(2)) 3 times each week for 24 wk. One group consumed water and the other, water containing 2 g/L GTPs. A control group drank water and was not exposed to UVB radiation. UVB-induced tumors and skin biopsies from the control group were analyzed using immunostaining, Western blotting, and gelatinolytic zymography. Oral administration of GTPs reduced UVB-induced tumor incidence (35%), tumor multiplicity (63%), and tumor growth (55%). The GTPs+UVB group had reduced expression of the matrix metalloproteinases (MMP)-2 and MMP-9, which have crucial roles in tumor growth and metastasis, and enhanced expression of tissue inhibitor of MMP in the tumors compared with mice that were treated with UVB alone. The GTPs+UVB group also had reduced expressions of CD31 and vascular endothelial growth factor, which are essential for angiogenesis, and inhibited expression of proliferating cell nuclear antigen in the tumors compared with the UVB group. Additionally, there were more cytotoxic CD8(+) T cells in the tumors of the GTPs+UVB group than in the UVB group and their tumor cells exhibited greater activation of caspase-3, indicating the apoptotic death of the tumor cells. Taken together, these data suggest that in mice, administration of GTPs affects several biomarkers that are involved in UV-carcinogenesis, including inhibition of angiogenic factors and recruitment of cytotoxic T cells in the tumor microenvironment.
Asunto(s)
Flavonoides/administración & dosificación , Neoplasias Inducidas por Radiación/prevención & control , Neovascularización Patológica/prevención & control , Fenoles/administración & dosificación , Neoplasias Cutáneas/diagnóstico por imagen , Neoplasias Cutáneas/prevención & control , Té , Rayos Ultravioleta , Administración Oral , Animales , Modelos Animales de Enfermedad , Femenino , Flavonoides/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/uso terapéutico , Ratones , Ratones Pelados , Neoplasias Inducidas por Radiación/irrigación sanguínea , Fenoles/farmacología , Polifenoles , Cintigrafía , Neoplasias Cutáneas/irrigación sanguíneaRESUMEN
There has been considerable interest in the use of botanical supplements to protect skin from the adverse effects of solar UV radiation, including photocarcinogenesis. We and others have shown that topical application of (-)-epigallocatechin-3-gallate (EGCG) from green tea prevents photocarcinogenesis in mice; however, the chemopreventive mechanism of EGCG in an in vivo tumor model is not clearly understood. In this study, UV-B-induced skin tumors with and without treatment of EGCG ( approximately 1 mg/cm(2)) and age-matched skin biopsies from SKH-1 hairless mice were used to identify potential molecular targets of skin cancer prevention by EGCG. These biopsies were analyzed for various biomarkers of angiogenesis and antitumor immune response using immunostaining, Western blotting and gelatinolytic zymography. We report that compared to non-EGCG-treated tumors, topical application of EGCG in UV-induced tumors resulted in inhibition of protein expression and activity of matrix metalloproteinase (MMP)-2 and MMP-9, which play crucial roles in tumor growth and metastasis. In contrast, tissue inhibitor of MMP-1 (TIMP-1), which inhibits MMP activity, was increased in tumors. With respect to the tumor vasculature, EGCG decreased the expression of CD31, a cell surface marker of vascular endothelial cells, and inhibited the expression of vascular endothelial growth factor in tumors, which are essential for angiogenesis. EGCG inhibited proliferating cell nuclear antigen in UV-B-induced tumors as well. Additionally, higher numbers of cytotoxic T lymphocytes (CD8(+) T cells) were detected in EGCG-treated tumors compared with non-EGCG-treated tumors. Together, these in vivo tumor data suggested that inhibition of photocarcinogenesis in mice by EGCG is associated with inhibition of angiogenic factors and induction of antitumor immune reactivity.