RESUMEN

OBJECTIVE: Reactive oxygen species (ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H2O2 stimuli. METHODS: Modified microfluidics and imaging techniques were used to determine O2 •- levels and construct an O2 •- reaction network. To elucidate the consequences of increased O2 •- input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca2+ levels, mitochondrial Ca2+ uptake, auto-amplification of intracellular ROS and the intrinsic apoptotic pathway. RESULTS AND CONCLUSIONS: Results from a modified microchip demonstrated that 1 mmol/L H2O2 induced a rapid increase in cellular O2 •- levels (>27 vs. >406 amol in 20 min), leading to increased cellular oxidizing power (evaluated by ROS levels) and decreased reducing power (evaluated by glutathione (GSH) levels). In addition, we examined the dynamics of cytosolic Ca2+ and mitochondrial Ca2+ by confocal laser scanning microscopy and confirmed that Ca2+ stores in the endoplasmic reticulum were the primary source of H2O2-induced cytosolic Ca2+ bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca2+ signals between organelles, ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.

Asunto(s)

Peróxido de Hidrógeno/química , Mitocondrias/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/química , Apoptosis , Calcio/metabolismo , Señalización del Calcio , Caspasas/metabolismo , Línea Celular Tumoral , Linaje de la Célula , Citosol/metabolismo , Glutatión/metabolismo , Humanos , Oxidación-Reducción , Transducción de Señal

RESUMEN

Autophagy has attracted a great deal of interest in tumour therapy research in recent years. However, the anticancer effect of luteoloside, a naturally occurring flavonoid isolated from the medicinal plant Gentiana macrophylla, on autophagy remains poorly understood in human lung cells. In the present study, we have investigated the anticancer effects of luteoloside on non-small cell lung cancer (NSCLC) cells and demonstrated that luteoloside effectively inhibited cancer cell proliferation, inducing G0/G1 phase arrest associated with reduced expression of CyclinE, CyclinD1 and CDK4; we further found that treatment with luteoloside did not strongly result in apoptotic cell death in NSCLC (A549 and H292) cells. Interestingly, luteoloside induced autophagy in lung cancer cells, which was correlated with the formation of autophagic vacuoles, breakdown of p62, and the overexpression of Beclin-1 and LC3-II, but not in a human bronchial epithelial cell line (BEAS-2B). Notably, pretreatment of cancer cells with 3-MA, an autophagy inhibitor, protected against autophagy and promoted cell viability but not apoptosis. To further clarify whether luteoloside-induced autophagy depended on the PI3K/AKT/mTOR/p70S6K signalling pathway, a major autophagy-suppressive cascade, cells were treated with a combination of AKT inhibitor (LY294002) and mTOR inhibitor (Rap). These results demonstrated that luteoloside induced autophagy in lung cancer cell lines by inhibiting the pathway at p-Akt (Ser473), p-mTOR and p-p70S6K (Thr389). Moreover, we observed that luteoloside-induced cell autophagy was correlated with production of reactive oxygen species (ROS). NAC-mediated protection against ROS clearly implicated ROS in the activation of autophagy and cell death. In addition, the results showed that ROS served as an upstream effector of the PI3K/AKT/mTOR/p70S6K pathway. Taken together, the present study provides new insights into the molecular mechanisms underlying luteoloside-mediated cell death in NSCLC cells and supports luteoloside as a potential anti-cancer agent for targeting NSCLC through the induction of autophagy, inhibition of proliferation and PI3K/AKT/mTOR/p70S6K signalling.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Glucósidos/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Luteolina/farmacología , Células A549 , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

Tumor infiltrated type II (M2) macrophages promote tumorigenesis by suppressing immune clearance, promoting proliferation, and stimulating angiogenesis. Interestingly, macrophages were also found to enrich in small foci of altered hepatocytes containing liver tumor-initiating cells (TICs). However, whether and how TICs specifically recruit macrophages and the function of these macrophages in tumor initiation remain unknown due to technical difficulties. In this study, by generating genetically defined liver TICs, we demonstrate that TICs actively recruit M2 macrophages from as early as the single-cell stage. Elimination of TIC-associated macrophages (TICAMs) abolishes tumorigenesis in a manner dependent on the immune system. Mechanistically, activation of the Hippo pathway effector Yes-associated protein (YAP) underlies macrophage recruitment by TICs. These results demonstrate for the first time that macrophages play a decisive role in the survival of single TICs in vivo and provide a proof of principle for TIC elimination by targeting YAP or M2 macrophages.

Asunto(s)

Carcinoma Hepatocelular/inmunología , Transformación Celular Neoplásica/inmunología , Hepatocitos/inmunología , Neoplasias Hepáticas/inmunología , Macrófagos/inmunología , Células Madre Neoplásicas/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Comunicación Celular/inmunología , Proteínas de Ciclo Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Células Cultivadas , Factor de Crecimiento de Hepatocito/fisiología , Hepatocitos/metabolismo , Hepatocitos/patología , Proteínas de Homeodominio/fisiología , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Macrófagos/citología , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Células Madre Neoplásicas/citología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Serina-Treonina Quinasa 3 , Proteína p53 Supresora de Tumor/fisiología , Proteínas Señalizadoras YAP

RESUMEN

BACKGROUND: Bicyclol, a novel synthetic antihepatitis drug, is widely known to protect against liver injury. However, few reports have focused on the possible effect of bicyclol on anti-proliferation and autophagy induction in cancer cells, particularly hepatocellular carcinoma cells. METHODS: In this study, we investigated the antitumor efficacy of Bicyclol in HepG2 cells and the mechanism of cell growth inhibition. Cell proliferation was analyzed by MTT assay, and the cell cycle and apoptosis were assessed by flow cytometry. And we transfected the cells with the GFP-RFP-LC3 vector to detect the autophagy flux in the cells. Mechanisms of bicyclol-induced cell growth inhibition were probed by western blot analysis. RESULTS: Bicyclol effectively inhibited HepG2 cell proliferation in a dose- and time-dependent manner. In addition, we found that bicyclol inhibited cell cycle progression at G1 phase and induced autophagy in HepG2 cells, which implied that the significant decrease in cell proliferation was mainly induced by autophagy and inhibition of cell proliferation. Furthermore, western blot showed that bicyclol inhibited phosphorylation of Akt and ERK, down-regulated the expressions of cyclin D1, cyclin E2, CDK2, CDK4, p-Rb and p-mTOR. Moreover, AKT or ERK knockdown by siRNA enhanced bicyclol-induced autophagy and inhibition of cell proliferation. CONCLUSION: These results suggest that bicyclol has potent anti-proliferative activity against malignant human hepatoma cells via modulation of the PI3K/AKT pathway and the Ras/Raf/MEK/ERK pathway, and indicate that bicyclol is a potential liver cancer drug worthy of further research and development.

RESUMEN

Oleanolic acid (OA), a plant-derived pentacyclic terpenoid, is known to have hepatoprotective effects. In this study, we found that OA induced autophagic cell death in multiple human gastric cancer cell lines. Moreover, OA-induced autophagy was shown for the first time in human gastric cancer cells, evidenced by the formation of GFP-RFP-LC3 puncta and autophagosomes. OA suppressed phospho-mTOR through inhibition of the PI3 K/AKT and ERK/p38 MAPK signalling pathways and through activation of the AMPK signalling pathway. Furthermore, we found that OA-induced cytotoxicity and autophagy could be blocked by the autophagy inhibitor 3-methyladenine or via siRNA targeting Beclin-1. Our in vivo research showed that OA delayed the formation of MGC-803 tumours in an autophagy-dependent manner. These results reveal a novel mechanism for OA in gastric cancer cells and suggest that OA could be a novel agent in the treatment of gastric cancer.

Asunto(s)

Ácido Oleanólico/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/efectos de los fármacos , Beclina-1/genética , Línea Celular Tumoral , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , Distribución Aleatoria , Transducción de Señal/efectos de los fármacos , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Serina-Treonina Quinasas TOR/metabolismo , Transfección , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Costunolide is an active sesquiterpene lactone derived from many herbal medicines. It has a broad spectrum of bioactivities, including anti-inflammatory and potential anti-tumor effects. The aims of the present study were to evaluate the inhibitory effects of costunolide on A549 cell growth and to explore the underlying molecular mechanisms. Annexin V-FITC/PI flow cytometry analysis revealed that costunolide induced apoptosis. To study the mechanism, we found that costunolide exposure activated the unfolded protein response (UPR) signaling pathways, as shown by the up-regulation of GRP78 and IRE1α and the activation of ASK1 and JNK. Meanwhile, siRNA knockdown of IRE1α significantly attenuated costunolide-induced apoptosis and partly restored the mitochondrial membrane potential. ER stress-activated JNK phosphorylated Bcl-2 at Ser70, which changes the anti-apoptotic function of Bcl-2, resulting in mitochondrial dysfunction and leading to mitochondrial activation of apoptosis. Furthermore, costunolide induced ROS generation, while the antioxidant N-acetyl cysteine (NAC) effectively blocked ER stress and apoptosis activation, suggesting that ROS acts as an upstream signaling molecule in triggering ER stress and mitochondrial apoptotic pathways. Taken together, our research demonstrates that costunolide exhibits its anti-tumor activity though inducing apoptosis, which is mediated by ER stress. We further confirm that Bcl-2 is a key molecule connecting the ER stress and mitochondrial pathways.

Asunto(s)

Antineoplásicos Fitogénicos/toxicidad , Apoptosis/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Sesquiterpenos/toxicidad , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo

RESUMEN

Organ size determination is one of the most intriguing unsolved mysteries in biology. Aberrant activation of the major effector and transcription co-activator YAP in the Hippo pathway causes drastic organ enlargement in development and underlies tumorigenesis in many human cancers. However, how robust YAP activation is achieved during organ size control remains elusive. Here we report that the YAP signaling is sustained through a novel microRNA-dependent positive feedback loop. miR-130a, which is directly induced by YAP, could effectively repress VGLL4, an inhibitor of YAP activity, thereby amplifying the YAP signals. Inhibition of miR-130a reversed liver size enlargement induced by Hippo pathway inactivation and blocked YAP-induced tumorigenesis. Furthermore, the Drosophila Hippo pathway target bantam functionally mimics miR-130a by repressing the VGLL4 homolog SdBP/Tgi. These findings reveal an evolutionarily conserved positive feedback mechanism underlying robustness of the Hippo pathway in size control and tumorigenesis.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinogénesis , MicroARNs/metabolismo , Fosfoproteínas/metabolismo , Aciltransferasas , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Proteína 61 Rica en Cisteína/antagonistas & inhibidores , Proteína 61 Rica en Cisteína/genética , Proteína 61 Rica en Cisteína/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Células Hep G2 , Humanos , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , Tamaño de los Órganos/genética , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Alineación de Secuencia , Transducción de Señal , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Trasplante Heterólogo , Proteínas Señalizadoras YAP

RESUMEN

Malignant gliomas are the most common primary brain tumors, and novel ways of treating gliomas are urgently needed. Ursolic acid (UA), a pentacyclic triterpenoid, has been reported to exhibit promising antitumor activity. Here, we evaluated the effects of UA on U87MG cells and explored the underlying molecular mechanisms. The results demonstrated that both G1-phase arrest and autophagy were induced by UA in U87MG cells. Evidence of UA-induced autophagy included the formation of acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. UA was also found to induce ER stress and an increase in intracellular calcium accompanied by ROS production. The increase in free cytosolic calcium induced by UA activated the CaMKK-AMPK-mTOR kinase signaling cascade, which ultimately triggered autophagy. Western blot analysis showed that UA promoted the phosphorylation of PERK and eIF2α; this was followed by the upregulation of the downstream protein CHOP, implying the involvement of the ER stress-mediated PERK/eIF2α/CHOP pathway in glioma cells. Meanwhile, UA activated IRE1α and subsequently increased the levels of phosphorylated JNK and Bcl-2, resulting in the dissociation of Beclin1 from Bcl-2. Furthermore, TUDCA and the silencing of either PERK or IRE1α partially blocked the UA-induced accumulation of LC3-II, suggesting that ER stress precedes the process of autophagy. Additionally, NAC attenuated the UA-induced elevation in cytosolic calcium, ER stress markers and autophagy-related proteins, indicating that UA triggered ER stress and autophagy via a ROS-dependent pathway. Collectively, our findings revealed a novel cellular mechanism triggered by UA and provide a molecular basis for developing UA into a drug candidate.

Asunto(s)

Antineoplásicos Fitogénicos/farmacología , Autofagia/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Triterpenos/farmacología , Western Blotting , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Microscopía Electrónica de Transmisión , Estructura Molecular , Ácido Ursólico

RESUMEN

Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Bufalin is the major component of Chan-Su (a traditional Chinese medicine) extracts from the venom of Bufo gargarizan. In this study, we evaluated the growth inhibitory effect of bufalin on glioma cells and explored the underlying molecular mechanisms. Our results showed that bufalin inhibited the growth of glioma cells significantly. Mechanistic studies demonstrated that bufalin induced apoptosis through mitochondrial apoptotic pathway. In addition, bufalin was also found to induce ER stress-mediated apoptosis, which was supported by the up- regulation of ER stress markers, CHOP and GRP78, and augmented phosphorylation of PERK and eIF2α as well as cleavage of caspase-4. Downregulation of CHOP using siCHOP RNA attenuated bufalin-induced apoptosis, further confirming the role of ER stress response in mediating bufalin-induced apoptosis. Evidence of bufalin-induced autophagy included formation of the acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. Further experiments showed that the mechanism of bufalin-induced autophagy associated with ATP deleption involved an increase in the active form of AMPK, decreased phosphorylation levels of mTOR and its downstream targets 4EBP1 and p70S6K1. Furthermore, TUDC and silencing of eIF2α or CHOP partially blocked bufalin-induced accumulation of LC3-II, which indicated that ER stress preceded bufalin-induced autophagy and PERK/eIF2α/CHOP signaling pathway played a major part in the process. Blockage of autophagy increased expression of ER stress associated proteins and the ratio of apoptosis, indicating that autophagy played a cytoprotective role in bufalin induced ER stress and cell death. In conclusion, bufalin inhibits glioma cell growth and induces interplay between apoptosis and autophagy through endoplasmic reticulum stress. It will provide molecular bases for developing bufalin into a drug candidate for the treatment of maglinant glioma.

Asunto(s)

Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Bufanólidos/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Glioma/patología , Antineoplásicos/uso terapéutico , Bufanólidos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/tratamiento farmacológico , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Mitocondrias/efectos de los fármacos , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/metabolismo

RESUMEN

OBJECTIVE: To investigate the effect of luteolin on cell growth and apoptosis of HepG2 cells in vitro. METHODS: Cultured HepG2,HL60,A549 and LO2 cells were treated with luteolin for different doses (0 µg/ml,2.5 µg/ml,10 µg/ml and 20 µg/ml) and varied times (0 h,24 h,48 h and 72 h). Cell viability was measured with MTT assay and IC50 was calculated. The reactive oxygen species (ROS) levels in HepG2 cells treated with luteolin for 6 h and 12 h were measured with flow cytometry. Cell apoptosis of HepG2 cells treated with luteolin for 24h was examined with flow cytometry and Annexin V-FITC/PI. Expression levels of apoptosis pathway proteins (p53,ASPP2 and iASPP) in HepG2 cells were detected with western blot and the dose and time-effect was analyzed. RESULTS: Luteolin effectively inhibited tumor cell proliferation in a dose-and time-dependent manner,and the inhibition rates of 20 µg/ml Luteolin for 72 h were 39.34%,62.90%,57.57% and 62.90% to LO2,HepG2, HL60 and A549 cells,respectively. The intracellular ROS level was decreased in HepG2 cells by 13.88% and 41.11% after being treated with luteolin for 6 h and 12 h,respectively. The apoptosis rate of HepG2 cells treated with luteolin for 24 h was 14.43%,and western blot showed that luteolin reduced the expression level of iASPP by 77.07% and up-regulated the expression of p53 by 179.37% and ASPP2 by 725.02% in HepG2 cells treated with luteolin for 12 h. CONCLUSION: Luteolin has ant-proliferative and pro-apoptotic activity on hepatoma HepG2 cells, which is associated with the altered expression of pro-apoptotic factors and decreased ROS level in HepG2 cells.

Asunto(s)

Apoptosis/efectos de los fármacos , Luteolina/farmacología , Células Hep G2 , Humanos , Luteolina/administración & dosificación , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

HS002-II, a novel protein-bound polysaccharide with 44kDa molecular weight, was fractionated from submerged cultures of Hirsutella sinensis Liu, Guo, Yu & Zeng by DEAE-Sepharose and Sephacryl S200 chromatography. Based on the results of infrared spectroscopy, high performance liquid chromatography, methylation, amino acid analysis, NMR spectroscopy and atomic force microscopy, the polysaccharide moieties of HS002-II mainly contained a long backbone of (1→3)-linked α-d-ribofuranosyl units (1→4)-linked α-d-xylopyranosyl units and (1→4)-linked ß-d-glucopyranosyl units, which was substituted at C-6. The two branches were ß-d-mannopyranosyl residues and ß-d-galactopyranosyl residues terminated with α-l-arabinopyranosyl residues, respectively. HS002-II consisted of 57.9% polysaccharide and 42.1% protein with the existence of N-type carbohydrate-protein linkage. In terms of the pro-inflammatory cytokines assay (NO, TNF-α, IL-1ß and NF-κB) using murine macrophages cell line (RAW264.7), HS002-II exhibited significant immunomodulatory activity by stimulating the IκB-NF-κB pathway.

Asunto(s)

Adyuvantes Inmunológicos/química , Hypocreales/química , Polisacáridos/química , Polisacáridos/farmacología , Proteínas/química , Adyuvantes Inmunológicos/farmacología , Animales , Conformación de Carbohidratos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Espectroscopía de Resonancia Magnética , Ratones , Peso Molecular , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

Nutlin3, a non-genotoxic agonist of p53, is currently in phase II clinical trials for cancer treatment. However, its effects on normal tissues and cell types remain largely to be determined. Drugs that can selectively target cancer cells as well as cooperate with the p53 pathway are thus greatly needed. Iron-superoxide dismutase (Fe-SOD) is a potential candidate as it selectively targets cancer cells by eliminating the abnormally high levels of reactive oxygen species (ROS) in cancer cells; it also inhibits cancer cell growth by induction of p27. Here, we show evidence that modulating redox and ROS homeostasis cooperates with Nutlin3 to selectively inhibit cancer cells in vitro and in vivo. Co-treatment of Fe-SOD and Nutlin3 showed synergistic inhibition on cancer cells in vitro, and the induction of p27 appeared to be involved. No effects were observed on normal cells. In addition, such co-treatment further exhibited synergistic inhibition on tumor growth in vivo in a murine B16 xenograft model, while the individual treatments only achieved very limited inhibition. Thus, Fe-SOD cooperated with Nutlin3 to selectively inhibit cancer cells in vitro and in vivo.

Asunto(s)

Imidazoles/administración & dosificación , Neoplasias/tratamiento farmacológico , Piperazinas/administración & dosificación , Superóxido Dismutasa/administración & dosificación , Animales , Proliferación Celular/efectos de los fármacos , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/biosíntesis , Sinergismo Farmacológico , Femenino , Células Hep G2 , Humanos , Liposomas , Células MCF-7 , Melanoma Experimental/tratamiento farmacológico , Ratones , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Sarsasapogenin is a sapogenin from the Chinese medical herb Anemarrhena asphodeloides Bunge. In the present study, we revealed that sarsasapogenin exhibited antitumor activity by inducing apoptosis in vitro as determined by Hoechst staining analysis and double staining of Annexin V-FITC/PI. In addition, cell cycle arrest in G2/M phase was observed in sarsasapogenin-treated HeLa cells. Moreover, the results revealed that perturbations in the mitochondrial membrane were associated with the deregulation of the Bax/Bcl-2 ratio which led to the upregulation of cytochrome c, followed by activation of caspases. Meanwhile, treatment of sarsasapogenin also activated Unfolded Protein Response (UPR) signaling pathways and these changes were accompanied by increased expression of CHOP. Salubrinal (Sal), a selective inhibitor of endoplasmic reticulum (ER) stress, partially abrogated the sarsasapogenin-related cell death. Furthermore, sarsasapogenin provoked the generation of reactive oxygen species, while the antioxidant N-acetyl cysteine (NAC) effectively blocked the activation of ER stress and apoptosis, suggesting that sarsasapogenin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways. Taken together, the results demonstrate that sarsasapogenin exerts its antitumor activity through both reactive oxygen species (ROS)-mediate mitochondrial dysfunction and ER stress cell death.

Asunto(s)

Antineoplásicos/farmacología , Medicamentos Herbarios Chinos/farmacología , Estrés del Retículo Endoplásmico/fisiología , Mitocondrias/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Espirostanos/farmacología , Neoplasias del Cuello Uterino/metabolismo , Anemarrhena/química , Puntos de Control del Ciclo Celular , Cinamatos/farmacología , Citocromos c/biosíntesis , Estrés del Retículo Endoplásmico/efectos de los fármacos , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G1 del Ciclo Celular/fisiología , Células HeLa , Humanos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Tiourea/análogos & derivados , Tiourea/farmacología , Factor de Transcripción CHOP/biosíntesis , Respuesta de Proteína Desplegada , Neoplasias del Cuello Uterino/patología , Proteína X Asociada a bcl-2/biosíntesis

Asunto(s)

Nefropatías Diabéticas/tratamiento farmacológico , Podocitos/fisiología , Proteínas/metabolismo , Transducción de Señal/fisiología , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/metabolismo , Animales , Nefropatías Diabéticas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Proteínas de Unión al GTP Monoméricas/metabolismo , Complejos Multiproteicos , Neuropéptidos/metabolismo , Podocitos/metabolismo , Proteínas/antagonistas & inhibidores , Proteína Homóloga de Ras Enriquecida en el Cerebro , Sirolimus/uso terapéutico , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo

RESUMEN

FLZ is a synthetic novel squamosamide derivative and has previously been proved to be a potential drug for Parkinson's disease and Alzheimer's disease. FLZ has strong antioxidant activity, which implies that FLZ could eliminate excessive intracellular reactive oxygen species (ROS) in tumor cells and induce a pathway related to low cellular ROS levels, thereby inhibiting tumor cells proliferation. However, few reports have focused on the antitumor effects of FLZ. In this study, we investigated the antitumor efficacy of FLZ in HepG2 cells and the mechanism of cell growth inhibition. FLZ effectively inhibited HepG2 cell proliferation in a dose- and time-dependent manner; meanwhile, it was minimally toxic to normal cells. FLZ induced a significant decrease in oxidative stress through elimination of excessive intracellular ROS and strengthening of the glutathione antioxidant system. In addition, FLZ can effectively attenuate redundant [Ca(2+)](i), thereby avoiding uncontrolled amplification by Ca(2+)/ROS positive feedback. Furthermore, Western blot showed that FLZ inhibited phosphorylation of Akt and retinoblastoma protein (Rb), down-regulated the expressions of cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2), and enhanced the expression of CDK inhibitor p27(kip1), while did not affect CDK4 expression. These results suggest that FLZ has potent anti-proliferative activity against malignant human hepatoma cells via modulation of the expression or activation of cell-cycle regulatory proteins, which are associated with decreased Ca(2+)/ROS levels, and indicate that FLZ is a potential liver cancer drug worthy of further research and development.

Asunto(s)

Acrilamidas/farmacología , Antineoplásicos/farmacología , Ácidos Cafeicos/farmacología , Calcio/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Proteínas de Ciclo Celular/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Especies Reactivas de Oxígeno/metabolismo , Acrilamidas/química , Acrilamidas/uso terapéutico , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Ácidos Cafeicos/química , Ácidos Cafeicos/uso terapéutico , Carcinoma Hepatocelular/metabolismo , Proliferación Celular/efectos de los fármacos , Ciclina D1/metabolismo , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Fase G1 , Glutatión/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína de Retinoblastoma/metabolismo

RESUMEN

A novel water-soluble polysaccharide pMTPS-3, obtained from Melia toosendan Sieb. Et Zucc fruit by hot-water extraction and ethanol precipitation, was fractionated by DEAE-52 cellulose anion-exchange and Sephadex G-100 gel filtration chromatography. Its primary structural features and molecular weight were characterized by Fourier infrared spectrometry (FTIR), gel permeation chromatography (GPC) and gas chromatography (GC). And the antioxidant activities of pMTPS-3 in vitro were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay, superoxide radical scavenging assay and hydroxyl radical scavenging assay. The results suggested that pMTPS-3 was a heteropolysaccharide, composed of arabinose, glucose, mannose, and galactose in the molar ratio of 17.3:28.3:41.6:12.6 with molecular weight 26100Da. The purified pMTPS-3 was revealed to have notable scavenging activity against DPPH radical in a concentration-dependent manner and present a moderate inhibition of superoxide radicals with an IC(50) (5.6mg/ml), and potent inhibiting power for hydroxyl radical compared with crude polysaccharide. Further, it exhibited strong inhibition effect in vitro on the growth of human gastric cancer BGC-823 cells. It is strongly evidenced that pMTPS-3 purified from the crude polysaccharides of Melia toosendan Sieb. Et Zucc could be explored as a potential antioxidant and therapeutics.

Asunto(s)

Fenómenos Químicos , Frutas/química , Melia/química , Polisacáridos/química , Polisacáridos/farmacología , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Compuestos de Bifenilo/química , Línea Celular Tumoral , Cromatografía en Gel , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/aislamiento & purificación , Depuradores de Radicales Libres/farmacología , Humanos , Radical Hidroxilo/química , Peso Molecular , Monosacáridos/análisis , Picratos/química , Polisacáridos/aislamiento & purificación , Superóxidos/química

RESUMEN

BACKGROUND: Many cancer cells develop resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, necessitating combination with chemotherapy, and normal cells manifest side effects due to the combined treatment regimen of TRAIL and chemotherapeutic drugs. A novel cancer therapy utilizing TRAIL is thus urgently needed. RESULTS: In this study, we exploited TRAIL receptor-mediated endocytosis for the first time to produce a cell-permeable molecule, soluble forms of recombinant TRAIL:iron superoxide dismutase (sTRAIL:FeSOD), which possesses sTRAIL-induced apoptotic ability and FeSOD antioxidant activity. The FeSOD component was rapidly introduced into the cell by sTRAIL and intracellular superoxide radical (O2-), which have been implicated as potential modulators of apoptosis in cancer cells, was eliminated, resulting in a highly reduced cellular environment. The decrease in cellular O2-, which was accompanied by a brief accumulation of H2O2 and downregulation of phosphorylated Akt (p-Akt) and cellular FLICE-inhibitory protein, sensitized K562 leukemia cells and human promyelocytic leukemia (HL-60) cells to TRAIL-induced apoptosis. The low H2O2 levels protected human LO2 hepatocytes from sTRAIL:FeSOD-induced apoptosis despite downregulation of p-Akt. We also obtained evidence that the lack of response to sTRAIL:FeSOD in normal T cells occurred because sTRAIL:FeSOD shows much stronger shifts of redox state in erythroleukemia (K562) and HL-60 cells compared to that in normal T cells. K562 and HL-60 cells underwent sTRAIL:FeSOD-induced apoptosis without the dysfunction of mitochondria. CONCLUSIONS: The fusion protein overcomes the inability of FeSOD to permeate the cell membrane, exhibits synergistic apoptotic effects on K562 and HL-60 cells and demonstrates minimal toxicity to normal T cells and the normal liver cell line LO2, indicating its potential value for the treatment of leukemia.

Asunto(s)

Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Superóxido Dismutasa/farmacología , Superóxidos/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Apoptosis/efectos de los fármacos , Células HL-60 , Humanos , Peróxido de Hidrógeno/metabolismo , Células K562 , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo

RESUMEN

3-(4-(Benzo[d]thiazol-2-yl)-1-phenyl-1H-pyrazol-3-yl) phenyl acetate (DPB-5) is a synthetic benzothiazole derivative. In the present study, we revealed that DPB-5 had strong cytotoxicity to induce cell apoptosis, which was mediated by ROS. And DPB-5 was more cytotoxic toward hepatoma cells than toward normal hepatic cells, which was resulted from the greater susceptibility of the malignant cells to ROS. DBP-5 caused massive ROS accumulation and GSH decrease, which lead to MMP disruption, caspase activation and finally induced cell apoptosis. Additionally, rotenone, an inhibitor of mitochondria electron transport system, effectively blocked the ROS elevated effect of DPB-5, which suggested that DPB-5-induced ROS generated from the mitochondria. Further studies showed that DPB-5-induced cell apoptosis through caspases-cascade, but failed to activate caspase-9. Hence, we concluded that DPB-5-induced Hep G2 cells apoptosis via a ROS-mediated pathway which was caspase-dependent but did not rely on caspase-9.

Asunto(s)

Apoptosis , Benzotiazoles/toxicidad , Pirazoles/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Benzotiazoles/química , Caspasa 9/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Glutatión/metabolismo , Células Hep G2 , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Pirazoles/química

RESUMEN

Reactive oxygen species (ROS) are constantly generated and eliminated in the biological system and play important roles in a variety of physiological and pathological processes. Previous studies indicate that modulation of cellular ROS affects cell proliferation. Thymosin alpha 1 (Talpha1) is a naturally occurring thymic peptide and has previously been shown to be a potential therapy for some immunodeficiencies, malignancies, and infections. However, few reports have focused on manipulation of cellular ROS level effects of Talpha1. In this study, the Talpha1-treated leukomonocytes, which were isolated from mice spleens, exhibited a higher ROS level and a lower reduced glutathione (GSH) level; however, HepG2 cells treated with Talpha1 exhibited lower ROS level and higher GSH level. In addition, after treatment with Talpha1, the population of leukomonocytes in the G(2) phase increased, resulting in a slight increase in viability. However, in Talpha1-treated HepG2 cells, the cell cycle was delayed in the G(1) phase, thereby inhibiting tumor cell proliferation; in addition, dephosphorylation of the serine/threonine kinase Akt was detected. In conclusion, we show that Talpha1 has potent anti-proliferative activity against malignant human hepatoma cells and proliferative activity against leukomonocytes associated with manipulation of oxidative stress levels which indicates the potential of Talpha1 as an antitumor drug.

Asunto(s)

Antineoplásicos/farmacología , Especies Reactivas de Oxígeno/metabolismo , Timosina/análogos & derivados , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fase G1 , Fase G2 , Glutatión/metabolismo , Humanos , Ratones , Monocitos/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Timalfasina , Timosina/farmacología

RESUMEN

Exogenous oxidative stress induces cell death, but the upstream molecular mechanisms involved of the process remain relatively unknown. We determined the instant dynamic reactions of intracellular reactive oxygen species (ROS, including hydrogen peroxide (H(2)O(2)), superoxide radical (O(2)(*)(-)), and nitric oxide (NO)) in cells exposed to exogenous oxidative stress by using a confocal laser scanning microscope. Stimulation with extracellular H(2)O(2) significantly increased the production of intracellular H(2)O(2), O(2)(*)(-), and NO (P<0.01) through certain mechanisms. Increased levels of intracellular ROS resulted in mitochondrial dysfunction, involving the impairment of mitochondrial activity and the depolarization of mitochondrial membrane potential. Mitochondrial dysfunction significantly inhibited the proliferation of human hepatoblastoma G2 (HepG2) cells and resulted in mitochondrial cytochrome c (cyt c) release. The results indicate that upstream ROS signals play a potential role in exogenous oxidative stress-induced cell death through mitochondrial dysfunction and cyt c release.

Asunto(s)

Mitocondrias/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Apoptosis , Línea Celular Tumoral , Citocromos c/metabolismo , Humanos , Potenciales de la Membrana/fisiología , Microscopía Confocal , Transducción de Señal