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Acidic lysosomes are indispensable for cancer development and linked to chemotherapy resistance. Chloroquine (CQ) and functional analogues have been considered as a potential solution to overcome the cancer progression and chemoresistance by inhibiting the lysosome-mediated autophagy and multidrug exocytosis. However, their anti-cancer efficacy in most clinical trials demonstrated modest improvement. In this study, we investigated the detailed mechanisms underlying the acquired resistance of K562 leukemic cells to CQ treatment. In response to 5-80 µM CQ, the lumen pH of endosomal-lysosomal system immediately increased and gradually reached dynamic equilibrium within 24 h. Leukemic cells produced more acidic organelles to tolerate 5-10 µM CQ. CQ (20-80 µM) concentration-dependently triggered cytosolic pH (pHi) rise, G0/G1 arrest, mitochondrial depolarization/fragmentation, and necrotic/apoptotic cell death. Oxidant induction by CQ was responsible for the mitochondria-dependent cytotoxicity and partial pHi elevation. Cells that survived the CQ cytotoxicity were accompanied with increased mitochondria. Under the 80 µM CQ challenge, co-treatment with the inhibitor of F0 part of mitochondrial H+-ATP synthase, oligomycin (40 nM), prevented the elevation of oxidants as well as pHi, and attenuated stresses on mitochondria, cell survival, and cell proliferation. Besides, oligomycin-treated cells obviously displayed the lysosomal peripheralization and plasma membrane blebbing, suggesting that these cells were in process of lysosomal exocytosis and microvesicle release. Enhanced motion of these secretory processes allowed the cells to exclude CQ and repair necrotic injury. Together, the oxidant production and the proton dynamic interconnection among lysosomes, mitochondria, and cytosol are crucial for leukemic susceptibility to lysosomotropic chemotherapeutics.
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Apoptosis , Cloroquina , Humanos , Cloroquina/farmacología , Necrosis/metabolismo , Línea Celular Tumoral , Lisosomas/metabolismo , Mitocondrias/metabolismo , Oligomicinas , Concentración de Iones de Hidrógeno , AutofagiaRESUMEN
The cell sheet technique is a promising approach for tissue engineering, and the present study is aimed to determine a better configuration of cell sheets for cartilage repair. For stratified chondrocyte sheets (S-CS), articular chondrocytes isolated from superficial, middle, and deep zones were stacked accordingly. Heterogeneous chondrocyte sheets (H-CS) were obtained by mixing zonal chondrocytes. The expressions of chondrocytes, cytokine markers, and glycosaminoglycan (GAG) production were assessed in an in vitro assay. The curative effect was investigated in an in vivo porcine osteochondral defect model. The S-CS showed a higher cell viability, proliferation rate, expression of chondrogenic markers, secretion of tissue inhibitor of metalloproteinase, and GAG production level than the H-CS group. The expressions of ECM destruction enzyme and proinflammatory cytokines were lower in the S-CS group. In the mini-pigs articular cartilage defect model, the S-CS group had a higher International Cartilage Repair Society (ICRS) macroscopic score and displayed a zonal structure that more closely resembled the native cartilage than those implanted with the H-CS. Our study demonstrated that the application of the S-CS increased the hyaline cartilage formation and improved the surgical outcome of chondrocyte implication, offering a better tissue engineering strategy for treating articular cartilage defects.
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OBJECTIVE: Shockwave application is a potential treatment for osteoarthritis (OA), but the underlying mechanism remains unknown. Oxidative stress and a counterbalancing antioxidant system might be the key to understanding this mechanism. We hypothesized that reactive oxygen species (ROS) and the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2),which is an important regulator of cellular redox homeostasis, are plausible elements. DESIGN: Porcine chondrocytes were cultured in a 3-dimensional pellet model and subjected to shockwaves. The effects of shockwaves with various energy-flux densities on optimal extracellular matrix (ECM) synthesis were assessed. ROS, mitogen-activated protein kinase (MAPK) signaling, and the redox activity of Nrf2 were measured. To investigate the signaling mechanism involved in the shockwave treatment in chondrocytes, specific inhibitors of ROS, MAPK signaling, and Nrf2 activity were targeted. RESULTS: Shockwaves increased ECM synthesis without affecting cell viability or proliferation. Furthermore, they induced transient ROS production mainly through xanthine oxidase. The phosphorylation of ERK1/2 and p38 and the nuclear translocation of Nrf2 were activated by shockwaves. By contrast, suppression of ROS signaling mitigated shockwave-induced MAPK phosphorylation, Nrf2 nuclear translocation, and ECM synthesis. Pretreatment of chondrocytes with the specific inhibitors of MEK1/2 and p38, respectively, mitigated the shockwave-induced nuclear translocation of Nrf2 and ECM synthesis. Nrf2 inhibition by both small hairpin RNA knockdown and brusatol reduced the shockwave-enhanced ECM synthesis. CONCLUSIONS: Shockwaves activated Nrf2 activity through the induction of transient ROS signaling and subsequently enhanced ECM synthesis in chondrocytes. This study provided fundamental evidence confirming the potential of shockwaves for OA management.
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Condrocitos , Factor 2 Relacionado con NF-E2 , Animales , Condrocitos/metabolismo , Matriz Extracelular/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/farmacología , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Especies Reactivas de Oxígeno/farmacología , Transducción de Señal , PorcinosRESUMEN
Biochanin A is a major isoflavone in red clover and a potent chemopreventive agent against cancer. However, the effects of biochanin A on human osteosarcoma cells have never been clarified. This study investigated the anti-proliferative potential of biochanin A in osteosarcoma cells. The results indicate that biochanin A inhibited cell growth and colony formation in a dose-dependent manner with a minimal toxicity to normal cells. The combination of doxorubicin and biochanin A could synergistically inhibit osteosarcoma cell growth. The cytotoxic effect of biochanin A via the induction of apoptosis as evidenced by formation of apoptotic bodies, externalization of phosphatidylserine, accumulation of sub-G1 phase cells, caspase 3 activation, and cleavage of PARP. Apoptosis was associated with loss of the mitochondrial membrane potential, release of cytochrome c, caspase 9 activation, increased Bax expression, and reduced Bcl-2 and Bcl-XL expression. Pre-treatment with a caspase-9 specific inhibitor (Z-LEHD-FMK) partially attenuated cell death, suggesting involvement of the intrinsic mitochondrial apoptotic cascade. However, pre-treatment with the JNK inhibitor SP600125, the MEK inhibitor PD-98059, and the p38 MAPK inhibitor SB203580 or the antioxidants vitamin E, N-acetylcysteine, and glutathione failed to prevent biochanin A-induced cell death. Our results suggest that biochanin A inhibits cell growth and induces apoptosis in osteosarcoma cells by triggering activation of the intrinsic mitochondrial pathway and caspase-9 and -3 and increasing the Bax: Bcl-2/Bcl-XL ratio.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Genisteína/farmacología , Mitocondrias/efectos de los fármacos , Osteosarcoma/fisiopatología , Extractos Vegetales/farmacología , Línea Celular Tumoral , Humanos , Mitocondrias/metabolismo , Osteosarcoma/genética , Osteosarcoma/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Trifolium/química , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Benzyl isothiocyanate (BITC) is a cruciferous vegetable-derived compound with anticancer properties in human cancer cells. However, its anticancer potential and underlying mechanisms remain absent in human oral cancer cells. Results indicate that BITC inhibits growth, promotes G2/M phase arrest and triggers apoptosis of OC2 cells with a minimal toxicity to normal cells. BITC-induced cell death was completely prevented by pretreatment with thiol-containing redox compounds including N-acetyl-l-cysteine (NAC), glutathione (GSH), dithiothreitol, and 2-mercaptoethanol, but not free radical scavengers mito-TEMPO, catalase, apocynin, l-NAME and mannitol. BITC rapidly produced reactive oxygen species and nitric oxide, triggered oxidative DNA damage. BITC effectively decreased the intracellular GSH and GSH/GSSG ratio and redox balance recovery by thiol-containing redox compounds, but not by free radical scavengers. Accordingly, redox stresses-DNA damage response (DDR) activated ATM, Chk2, p53, and p21 and subsequently resulted in G2/M phase arrest by inhibiting Cdc2 and cyclin B1. Notably, BITC-induced apoptosis was associated with reduced Mcl-1 and Bcl-2 expression, diminished mitochondrial membrane potential (ΔΨm), and increased PARP cleavage. These BITC-induced redox stress-mediated DDR and apoptosis could be blocked by NAC and GSH. Therefore, BITC can be a rational drug candidate for oral cancer and acted via a redox-dependent pathway.
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Apoptosis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Isotiocianatos/farmacología , Neoplasias de la Boca/patología , Estrés Oxidativo/efectos de los fármacos , Enfermedad Aguda , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Glutatión/metabolismo , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Células Tumorales CultivadasRESUMEN
Phenethyl isothiocyanate (PEITC) is a naturally occurring cruciferous vegetable-derived compound that inhibits cell growth and induces apoptosis in oral cancer cells. However, the exact mechanism of PEITC action has not been fully elucidated. This study investigated the molecular mechanism and anticancer potential of PEITC in oral squamous cell carcinoma (OSCC) cells with various p53 statuses. PEITC inhibited the growth of OC2, SCC4, and SCC25 cells (functional p53 mutants) in a dose-dependent manner with low toxicity to normal cells. Treatment with PEITC induced reactive oxygen species production, nitric oxide generation, and GSH depletion and triggered DNA damage response as evidenced by flow cytometry, 8-OHdG formation, and comet assay. Furthermore, the subsequent activation of ATM, Chk2, and p53 as well as the increased expression of downstream proteins p21 and Bax resulted in a G2/M phase arrest by inhibiting Cdc25C, Cdc2, and cyclin B1. The PEITC-induced apoptotic cell death, following a diminished mitochondrial transmembrane potential, reduced the expression of Bcl-2 and Mcl-1, released mitochondrial cytochrome c, and activated caspase 3 and PARP cleavage. The p53 inhibitor pifithrin-α and the antioxidants N-acetylcysteine and glutathione (GSH) protected the cells from PEITC-mediated apoptosis. However, mito-TEMPO, catalase, apocynin, and L-NAME did not prevent PEITC-induced cell death, suggesting that PEITC induced G2/M phase arrest and apoptosis in oral cancer cells via a GSH redox stress and oxidative DNA damage-induced ATM-Chk2-p53-related pathway. These results provide new insights into the critical roles of both GSH redox stress and p53 in the regulation of PEITC-induced G2/M cell cycle arrest and apoptosis in OSCCs.
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Anticarcinógenos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/tratamiento farmacológico , Isotiocianatos/farmacología , Mitocondrias/efectos de los fármacos , Neoplasias de la Boca/tratamiento farmacológico , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Brassicaceae/química , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Quinasa de Punto de Control 2/metabolismo , Daño del ADN/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Inhibidores de Crecimiento/farmacología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Mutación/genética , Óxido Nítrico/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Proteasomes are highly expressed in rapidly growing neoplastic cells and essential for controlling the cell cycle process and mitochondrial homeostasis. Pharmacological inhibition of the proteasome shows a significant anticancer effect on hematopoietic malignancies that is usually associated with the generation of reactive oxygen species. In this study, we comprehensively investigated the role of endogenous oxidants in various cellular events of K562 leukemic cells in response to treatment with MG132, a proteasome inhibitor. MG132 at 1.4 µM potently triggered G2/M arrest, mitochondrial depolarization, and apoptosis. By such treatment, the protein level of inducible nitric oxide synthase (iNOS) was doubled and cellular oxidants, including nitric oxide, superoxide, and their derivatives, were increasingly produced. In MG132-treated cells, the increase in iNOS-derived oxidants was responsible for mitochondrial depolarization and caspase-dependent apoptosis, but was insignificant in G2/M arrest. The amount of iNOS was negatively correlated with that of manganese superoxide dismutase (MnSOD). Whereas iNOS activity was inhibited by aminoguanidine, cellular MnSOD levels as well as mitochondrial membrane potentials were upregulated, and consequentially G2/M arrest and apoptosis were thoroughly reversed. It is suggested that cells rich in functional mitochondria possess improved proteasome activity, which antagonizes the cytotoxic and cytostatic effects of MG132. In contrast to iNOS, endothelial NOS-driven cGMP-dependent signaling promoted mitochondrial function and survival of MG132-stressed cells. In conclusion, the functional interplay of proteasomes and mitochondria is crucial for leukemic cell growth, wherein iNOS plays a key role.
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Apoptosis/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Leucemia/enzimología , Leupeptinas/farmacología , Mitocondrias/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Caspasas/metabolismo , GMP Cíclico/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Células K562 , Leucemia/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/fisiología , Óxido Nítrico/metabolismo , Transducción de Señal/efectos de los fármacos , Superóxido Dismutasa/metabolismoRESUMEN
Biochanin A has promising effects on bone formation in vivo, although the underlying mechanism remains unclear yet. This study therefore aimed to investigate whether biochanin A regulates osteogenic and adipogenic differentiation using primary adipose-derived stem cells. The effects of biochanin A (at a physiologically relevant concentration of 0.1-1 µM) were assessed in vitro using various approaches, including Oil red O staining, Nile red staining, alizarin red S staining, alkaline phosphatase (ALP) activity, flow cytometry, RT-PCR, and western blotting. The results showed that biochanin A significantly suppressed adipocyte differentiation, as demonstrated by the inhibition of cytoplasmic lipid droplet accumulation, along with the inhibition of peroxisome proliferator-activated receptor gamma (PPAR γ ), lipoprotein lipase (LPL), and leptin and osteopontin (OPN) mRNA expression, in a dose-dependent manner. On the other hand, treatment of cells with 0.3 µM biochanin A increased the mineralization and ALP activity, and stimulated the expression of the osteogenic marker genes ALP and osteocalcin (OCN). Furthermore, biochanin A induced the expression of runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), and Ras homolog gene family, member A (RhoA) proteins. These observations suggest that biochanin A prevents adipogenesis, enhances osteoblast differentiation in mesenchymal stem cells, and has beneficial regulatory effects in bone formation.
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Upper urinary tract urothelial carcinoma is a relatively uncommon disease and is diagnosed more frequently at advanced stages. The prognosis of these patients mainly has been related to tumor stage and grade. As a result, the definition of prognostic indicators enabling precise patient selection is mandatory for neoadjuvant or adjuvant therapies. The epithelial membrane protein (EMP2) was identified as one of the up-regulated genes by isoflavones. EMP2 overexpression suppressed foci formation, anchorage-independent growth in vitro, and tumorigenicity in severe combined immunodeficiency mice (all P < 0.05). In addition, a cross-talk between EMP2 and integrins αV and ß3 was shown in the regulation of cell adhesion and migration. Higher EMP2 expression was associated with a better progression-free survival (P = 0.008) and cancer-related death (P < 0.001). EMP2 was identified as a tumor-suppressor gene in urinary tract urothelial carcinoma and may be an innovative co-targeting candidate for designing integrin-based cancer therapy.
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Glicoproteínas de Membrana/metabolismo , Neoplasias Urológicas/metabolismo , Neoplasias Urológicas/patología , Urotelio/metabolismo , Urotelio/patología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/metabolismo , Carcinogénesis/patología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Integrinas/metabolismo , Isoflavonas/farmacología , Masculino , Glicoproteínas de Membrana/genética , Ratones , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Transporte de Proteínas/efectos de los fármacos , Neoplasias Urológicas/genética , Urotelio/efectos de los fármacos , Adulto JovenRESUMEN
Caffeine consumption has been related to loss of body weight and modulates lipid metabolism. However, impacts of caffeine on adipogenic differentiation have not been well determined yet. The present study evaluated the effects of caffeine on adipogenesis using primary rat adipose-derived stem cells (ADSCs) and a mouse bone marrow stromal cell line (M2-10B4) in vitro. ADSCs and M2-10B4 were continuously exposed to caffeine (0.1-1mM) during adipogenic differentiation for 7 and 12 days, respectively. Oil red O and Nile red staining showed that caffeine reduced lipid droplet and adipocyte levels in both cell types. In addition, Nile red staining and FACScan flow cytometry showed that caffeine dose-dependently decreased adipocyte differentiation from 20% to 50% of the control ADSCs and M2-10B4 cells. Caffeine decreased the expression of adipogenesis-related genes including peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein-α, adipocyte lipid binding protein, lipoprotein lipase, leptin, and TNFα in a dose-dependent manner. Rather, low concentration of caffeine (0.1mM) significantly increased IL-6 expression, but unexpectedly inhibited that at a concentration more than 0.3mM. Taken together, caffeine was able to effectively inhibit adipogenic differentiation of ADSCs and M2-10B4 cells partly through its inhibition of adipogenesis-related factors.
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Cafeína/toxicidad , Células Madre/efectos de los fármacos , Adipocitos/citología , Adipocitos/efectos de los fármacos , Tejido Adiposo/citología , Animales , Proteína alfa Potenciadora de Unión a CCAAT/genética , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión a Ácidos Grasos/genética , Interleucina-6/genética , Leptina/genética , Lipoproteína Lipasa/genética , Ratones , Ratas , Ratas Sprague-Dawley , Células Madre/citología , Células Madre/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Biochanin A (BCA) is a major isoflavone abundant in red clover (Trifolium pretense). The protective effect of BCA on bone loss in an ovariectomized (OVX) animal model has never been clarified. The objective of this study was to investigate the biological effects of BCA on bone loss in OVX rats in vivo and on the development of osteoblasts and osteoclasts in vitro. Ovariectomy resulted in a marked increase in body weight and a decrease in femoral bone mineral density and trabecular bone volume that was prevented by BCA or 17 ß -estradiol (E2) treatment. However, an increase in uterine weight was observed in E2-treated OVX rats, but not in response to BCA treatment. Treatment with BCA increased the mRNA expression of osterix, collagen type I, alkaline phosphatase (ALP), and osteocalcin and decreased the mRNA expression of tartrate-resistant acid phosphatase (TRAP) and the receptor activator of nuclear factor- κ B ligand (RANKL)/osteoprotegerin (OPG) ratio in the femur of OVX rats. Treatment with BCA or E2 prevented the OVX-induced increase in urinary deoxypyridinoline (DPD) and serum tumor necrosis factor α (TNF- α ) and interleukin-1 ß (IL-1 ß ). In vitro, BCA induced preosteoblasts to differentiate into osteoblasts and increased osteoblast mineralization. BCA inhibited preosteoclasts and osteoclast proliferation and decreased osteoclast bone resorption. These findings suggest that BCA treatment can effectively prevent the OVX-induced increase in bone loss and bone turnover possibly by increasing osteoblastic activities and decreasing osteoclastic activities.
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Caffeine consumption reportedly influences bone mineral density and body weight. However, the effects of caffeine on bone metabolism are still controversial, and whether the dosage of caffeine influences osteogenic differentiation is yet to be clarified. In the present study, we cultured primary adipose-derived stem cells (ADSCs) and a bone marrow stromal cell line (M2-10B4) in osteogenic differentiation media containing varying concentrations of caffeine. Caffeine had biphasic effects: 0.1 mM caffeine significantly enhanced mineralization and alkaline phosphatase (ALP) activity. Consistent with these observations, a caffeine concentration of 0.1 mM upregulated the osteogenic differentiation marker genes ALP and osteocalcin (OCN), and elevated osteoprotegerin (OPG), Runt-related transcription factor 2 (RUNX2) and Sirtuin 1 (SIRT1) levels. However, a concentration of caffeine greater than 0.3 mM suppressed the differentiation of both the cell types. These findings indicate that caffeine has a beneficial effect on ADSCs and bone marrow stromal cells, enhancing differentiation to osteoblasts; this effect, which is mediated via RUNX2 activation at low doses is significantly suppressed at high doses.
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Huesos/efectos de los fármacos , Cafeína/farmacología , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Células Madre/efectos de los fármacos , Tejido Adiposo/citología , Fosfatasa Alcalina/metabolismo , Animales , Densidad Ósea/efectos de los fármacos , Densidad Ósea/genética , Conservadores de la Densidad Ósea/administración & dosificación , Conservadores de la Densidad Ósea/farmacología , Huesos/metabolismo , Huesos/fisiología , Cafeína/administración & dosificación , Calcificación Fisiológica/genética , Técnicas de Cultivo de Célula , Diferenciación Celular/genética , Línea Celular , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Hormesis , Células Madre Mesenquimatosas/enzimología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Osteoblastos/efectos de los fármacos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogénesis/genética , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Extractos Vegetales/farmacología , Ratas , Ratas Sprague-Dawley , Sirtuina 1/genética , Sirtuina 1/metabolismo , Células Madre/enzimología , Células Madre/metabolismo , Regulación hacia ArribaRESUMEN
OBJECTIVE: Several studies have shown that soy isoflavones have estrogen-like activities and might constitute an alternative to hormone replacement treatment. The present study investigated the effects of soy isoflavones alone and combined with vitamin D3 on prevention of bone loss. METHODS: Sprague-Dawley rats were sham-operated (n = 8) or ovariectomized (OVX; n = 40), and then the OVX rats were randomly assigned to five groups that were untreated or treated for 14 wk with vitamin D3, 17ß-estradiol, soy isoflavone extract (SIE), or vitamin D3 plus SIE. The effects of the isoflavones and 1α,25(OH)(2)D(3) on cultured osteoblasts and osteoclasts also were investigated. RESULTS: In OVX rats, the bone mineral density and trabecular bone volume loss were improved by 17ß-estradiol, SIE, or SIE plus vitamin D3 treatment. SIE treatment was more effective than vitamin D3 or 17ß-estradiol in inhibiting increases in serum tumor necrosis factor-α levels and osteoblast osteoprotegerin expression. SIE plus vitamin D3 was more effective in increasing osterix expression than each alone. Bone cell cultures showed that the isoflavones induced preosteoblasts to differentiate into osteoblasts and increased osteoblast mineralization. Isoflavones inhibited preosteoclasts and osteoclast proliferation and decreased osteoclast resorption. The combination of isoflavones plus 1α,25(OH)(2)D(3) showed additive effects on the increase in cell proliferation of cultured preosteoblasts. CONCLUSION: Treatment with soy isoflavones might be an alternative to hormone replacement therapy in decreasing bone loss from postmenopausal estrogen deficiency. In addition, there are further effects on increasing transcription factor osterix expression and preosteoblast proliferation when these were combined with vitamin D3.
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Colecalciferol/administración & dosificación , Isoflavonas/administración & dosificación , Osteoporosis/tratamiento farmacológico , Fosfatasa Alcalina/sangre , Animales , Densidad Ósea/efectos de los fármacos , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Estradiol/administración & dosificación , Femenino , Humanos , Interleucina-1beta/sangre , Osteoblastos/efectos de los fármacos , Osteoblastos/patología , Osteocalcina/sangre , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Osteoporosis/metabolismo , Osteoporosis/patología , Ovariectomía , Fitoestrógenos/administración & dosificación , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Glycine max/química , Factor de Necrosis Tumoral alfa/sangreRESUMEN
BACKGROUND: Arecoline, the most abundant areca alkaloid, has been reported to stimulate reactive oxygen species (ROS) production in several cell types. Overproduction of ROS has been implicated in atherogenesis. Hemeoxygenase-1 (HO-1) has cytoprotective activities in vascular tissues. This study investigated the effect of arecoline on adhesion molecule expression and explored the role of HO-1 in this process. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with arecoline, then ROS levels and the expression of adhesion molecules and HO-1 were analyzed and potential signaling pathways investigated. RESULTS: After 2h of arecoline treatment, ROS production was stimulated and reached a maximum at 12h. Expression of the adhesion molecules ICAM and VCAM was also induced. Glutathione pretreatment completely blocked arecoline-stimulated ROS production and VCAM expression, but not ICAM expression. Arecoline also induced HO-1 expression and this effect was partly due by ROS stimulation. Inhibition of c-jun N-terminal kinase (JNK) by SP600125, p38 by SB 203580, or tyrosine kinase by genistein reduced arecoline-induced HO-1 expression. In contrast, inhibition of ERK (extracellular signal-related MAP kinase) by PD98059 had no effect. Transfection of HUVECs with the GFP/HO-1 gene, which resulted in a 5-fold increase in HO-1 activity, markedly, but not completely, inhibited the decrease in cell viability caused by arecoline. CONCLUSIONS: This study demonstrates that, in HUVECs, arecoline stimulates ROS production and ICAM and VCAM expression. HO-1 expression is also upregulated through the ROS, tyrosine kinase, and MAPK (JNK and p38) signaling pathways.
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Arecolina/farmacología , ADN/genética , Células Endoteliales/enzimología , Regulación de la Expresión Génica , Hemo-Oxigenasa 1/genética , Estrés Oxidativo/efectos de los fármacos , Venas Umbilicales/enzimología , Western Blotting , Células Cultivadas , Agonistas Colinérgicos/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Hemo-Oxigenasa 1/biosíntesis , Humanos , Líquido Intracelular/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/patologíaRESUMEN
Arginine is a physiological substrate for nitric oxide synthase to generate nitric oxide (NO), which can influence tumor cell survival, while ascorbic acid is selectively toxic for cancer cells. This study explored the effect of an arginine/ascorbic acid combination on human cancer cell lines. The hepatoma cell line HA22T/VGH was the most sensitive of the tested cells to combination treatment. A combination of 5.74 mM of arginine and 0.57 mM of ascorbic acid induced HA22T/VGH cell death through apoptosis and an increase in levels of reactive oxygen species and NO, as well as its stable products NO(2)(-) and NO(3)(-). The combination also reduced the activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and transaldolase in the pentose phosphate pathway, a major mechanism for producing NADPH, resulting in a marked decrease in intracellular NADPH levels. A dramatic decrease in intracellular glutathione (GSH) levels, a decrease in the mitochondrial membrane potential, ATP depletion and release of cytochrome c were also seen. Caspase-9 and caspase-3 were activated, apoptotic protein Bax expression increased and the expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL decreased. These results suggest that this combination induced HA22T/VGH cell death by interfering with redox state regulation by a reduction in pentose phosphate pathway activity and increasing oxidative and nitrosative stress.
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Apoptosis/efectos de los fármacos , Arginina/farmacología , Ácido Ascórbico/farmacología , Vía de Pentosa Fosfato/efectos de los fármacos , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Citocromos c/metabolismo , Glutatión/metabolismo , Humanos , Potencial de la Membrana Mitocondrial , Nitrógeno/metabolismo , Oxidación-Reducción , Oxígeno/metabolismo , Fosfogluconato Deshidrogenasa/metabolismo , Transaldolasa/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Dengue virus (DV) infections cause mild dengue fever or severe life-threatening dengue haemorrhagic fever (DHF)/ dengue shock syndrome (DSS). DV-infected patients have high plasma concentrations of plasminogen activator inhibitor type I (PAI-1). However, the mechanism to cause haemorrhage in DV infections remains poorly understood. In this study, investigation was carried out on the purified recombinant domain III of the envelope glycoprotein of DV serotypes 2 (EIII) and the signalling pathways of EIII leading to PAI-1 gene expression were measured by RT-PCR, Western blot, and immunofluorescence stain. Reporter gene constructs containing serially 5'-deleted sequences of the proximal human PAI-1 promoter region were constructed and then transfected to Huh7 cells, a human hepatoma cell line, prior to EIII treatment. EIII increased the PAI-1 mRNA and protein levels in a dose-dependent manner in Huh7 cells. Results showed that U0126, an inhibitor of extracellular signal-regulated kinase (ERK) kinase (MEK), almost completely suppressed EIII-induced PAI-1 expression. The results suggest that the MEK/ERK signalling pathways mediate the EIII-dependent induction of PAI-1 gene expression via the proximal promoter region.
Asunto(s)
Coagulación Sanguínea , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas , Inhibidor 1 de Activador Plasminogénico/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Western Blotting , Butadienos/farmacología , Línea Celular Tumoral , Quinasas MAP Reguladas por Señal Extracelular , Técnica del Anticuerpo Fluorescente , Humanos , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Nitrilos/farmacología , Inhibidor 1 de Activador Plasminogénico/genética , Regiones Promotoras Genéticas , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción AP-1/metabolismo , Transfección , Regulación hacia Arriba , Proteínas del Envoltorio Viral/genéticaRESUMEN
Dengue virus (DV) causes a non-specific febrile illness known as Dengue fever (DF), and a severe life-threatening illness, Dengue hemorrhagic fever/Dengue shock syndrome (DHF/DSS). Hemostatic changes induced by this virus involve three main factors: thrombocytopenia, endothelial cell damage, and significant abnormalities of the coagulation and fibrinolysis systems. The pathogenesis of bleeding in DV infections remains unknown. In this article, we focused on the DV activating endothelial cells and altering the parameters of hemostasis system. The expression of hemostasis-related factors, Thrombomodulin, TF, TFPI, t-PA, and PAI-1, in DV-infected cells were determined by RT-PCR. Flow cytometry analysis and immunofluorescence staining confirmed that the expression levels of TM in the DV-infected HMEC-1 and THP-1 cells were increased. In addition, the purified recombinant domain III of the envelope glycoprotein of DV (EIII) could induce the expression of TM in the HMEC-1 cells and THP-1 cells. The TM expression induced by DV or EIII in the endothelial cells and monocytic cells suggests that the EIII of DV plays an important role in the pathogenesis of DHF/DSS.
Asunto(s)
Anticoagulantes/metabolismo , Virus del Dengue/fisiología , Dengue/metabolismo , Endotelio Vascular/metabolismo , Leucemia Monocítica Aguda/metabolismo , Venas Umbilicales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Biomarcadores/metabolismo , Western Blotting , Dengue/patología , Dengue/virología , Endotelio Vascular/citología , Endotelio Vascular/virología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Hemostasis , Humanos , Leucemia Monocítica Aguda/patología , Leucemia Monocítica Aguda/virología , ARN Mensajero/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Venas Umbilicales/citología , Venas Umbilicales/virología , Proteínas del Envoltorio Viral/genéticaRESUMEN
BACKGROUND: Our previous study showed that, in basal cell carcinoma cells, arecoline reduces levels of the tumor cell survival factor interleukin-6 (IL-6), increases levels of tumor suppressor factor p53, and elicits cell cycle arrest, followed by apoptosis. In preliminarily studies, we observed that arecoline induces detachment of the human-derived hepatoma cell line HA22T/VGH from the extracellular matrix. In the present study, we explored the fate of the detached HA22T/VGH cells and investigated the underlying mechanism. METHODS: HA22T/VGH cells or primary cultured rat hepatocytes were treated with arecoline, then changes in morphology, viability, apoptosis, and the expression of surface beta1-integrin, apoptosis-related proteins, and IL-6 were examined. Furthermore, activation of the signal transducer and activator of transcription 3 (STAT3) pathway and the RhoA/Rock signaling pathway, including p190RhoGAP and Src homology-2 domain-containing phosphatase SHP2, was examined. RESULTS: A low concentration of arecoline (Asunto(s)
Anoicis/efectos de los fármacos
, Antineoplásicos/farmacología
, Arecolina/farmacología
, Carcinoma Hepatocelular/metabolismo
, Activación Enzimática/efectos de los fármacos
, Neoplasias Hepáticas/metabolismo
, Animales
, Western Blotting
, Carcinoma Hepatocelular/patología
, Adhesión Celular/efectos de los fármacos
, Línea Celular Tumoral
, Separación Celular
, Fragmentación del ADN/efectos de los fármacos
, Citometría de Flujo
, Técnica del Anticuerpo Fluorescente
, Humanos
, Etiquetado Corte-Fin in Situ
, Neoplasias Hepáticas/patología
, Ratas
, Ratas Sprague-Dawley
, Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
, Factor de Transcripción STAT3/efectos de los fármacos
, Factor de Transcripción STAT3/metabolismo
, Transducción de Señal/efectos de los fármacos
, Transducción de Señal/fisiología
, Proteína de Unión al GTP rhoA/efectos de los fármacos
, Proteína de Unión al GTP rhoA/metabolismo