RESUMEN
Discovery of CARF (Collaborator of ARF)/CDKN2AIP as an ARF-interacting protein that promotes ARF-p53-p21WAF1 signaling and cellular senescence, initially established its role in genomic stress. Multiple reports further unraveled its role in regulation of senescence, growth arrest, apoptosis, or malignant transformation of cells in response to a variety of stress conditions in cultured human cells. It has been established as an essential protein. Whereas CARF-compromised cells undergo apoptosis, its enrichment has been recorded in a variety of cancer cells and has been associated with malignant transformation. We earlier demonstrated its role in stress-induced cell phenotypes that ranged from growth arrest, apoptosis, or malignant transformation. In the present study, we assessed the molecular mechanism of quantitative impact of change in CARF expression level on these cell fates. Stress-induced changes in CARF expression were assessed quantitatively with proteins involved in proteotoxicity, oxidative, genotoxic, and cytotoxic stress. These comparative quantitative analyses confirmed that (i) CARF responds to diverse stresses in a quantitative manner, (ii) its expression level serves as a reliable predictive measure of cell fates (iii) it correlates more with the DNA damage and MDA levels than the oxidative and proteotoxic signatures and (iv) CARF-expression based quantitative assay may be recruited for stress diagnostic applications.
Asunto(s)
Apoptosis , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/genética , Senescencia Celular/fisiología , Proliferación Celular , Proteínas Reguladoras de la Apoptosis/genética , Transformación Celular Neoplásica , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismoRESUMEN
Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3ß-methoxy withaferin-A (3ßmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3ßmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3ßmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 µM and that 3ßmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [Kd], 5.9 µM). We propose that 3ßmWi-A is a novel modulator of circadian rhythms.
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Relojes Circadianos/efectos de los fármacos , Miembro 1 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Witanólidos/farmacología , Factores de Transcripción ARNTL/metabolismo , Animales , Fibroblastos/efectos de los fármacos , Ratones , Células 3T3 NIH , Extractos VegetalesRESUMEN
Propolis, also known as bee-glue, is a resinous substance produced by honeybees from materials collected from plants they visit. It contains mixtures of wax and bee enzymes and is used by bees as a building material in their hives and by humans for different purposes in traditional healthcare practices. Although the composition of propolis has been shown to depend on its geographic location, climatic zone, and local flora; two largely studied types of propolis: (i) New Zealand and (ii) Brazilian green propolis have been shown to possess Caffeic Acid Phenethyl Ester (CAPE) and Artepillin C (ARC) as the main bioactive constituents, respectively. We have earlier reported that CAPE and ARC possess anticancer activities, mediated by abrogation of mortalin-p53 complex and reactivation of p53 tumor suppressor function. Like CAPE, Artepillin C (ARC) and the supercritical extract of green propolis (GPSE) showed potent anticancer activity. In this study, we recruited low doses of GPSE and ARC (that did not affect either cancer cell proliferation or migration) to investigate their antistress potential using in vitro cell based assays. We report that both GPSE and ARC have the capability to disaggregate metal- and heat-induced aggregated proteins. Metal-induced aggregation of GFP was reduced by fourfold in GPSE- as well as ARC-treated cells. Similarly, whereas heat-induced misfolding of luciferase protein showed 80% loss of activity, the cells treated with either GPSE or ARC showed 60-80% recovery. Furthermore, we demonstrate their pro-hypoxia (marked by the upregulation of HIF-1α) and neuro-differentiation (marked by differentiation morphology and upregulation of expression of GFAP, ß-tubulin III, and MAP2). Both GPSE and ARC also offered significant protection against oxidative stress and, hence, may be useful in the treatment of old age-related brain pathologies.
Asunto(s)
Estrés Oxidativo/efectos de los fármacos , Fenilpropionatos/farmacología , Própolis/química , Própolis/farmacología , Animales , Biomarcadores , Brasil , Fraccionamiento Químico , Humanos , Hipoxia/genética , Hipoxia/metabolismo , Fenilpropionatos/química , Extractos Vegetales , Própolis/aislamiento & purificación , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The anti-metastatic and anti-angiogenic activities of triethylene glycol derivatives have been reported. In this study, we investigated their molecular mechanism(s) using bioinformatics and experimental tools. By molecular dynamics analysis, we found that (i) triethylene glycol dimethacrylate (TD-10) and tetraethylene glycol dimethacrylate (TD-11) can act as inhibitors of the catalytic domain of matrix metalloproteinases (MMP-2, MMP-7 and MMP-9) by binding to the S1' pocket of MMP-2 and MMP-9 and the catalytic Zn ion binding site of MMP-7, and that (ii) TD-11 can cause local disruption of the secondary structure of vascular endothelial growth factor A (VEGFA) dimer and exhibit stable interaction at the binding interface of VEGFA receptor R1 complex. Cell-culture-based in vitro experiments showed anti-metastatic phenotypes as seen in migration and invasion assays in cancer cells by both TD-10 and TD-11. Underlying biochemical evidence revealed downregulation of VEGF and MMPs at the protein level; MMP-9 was also downregulated at the transcriptional level. By molecular analyses, we demonstrate that TD-10 and TD-11 target stress chaperone mortalin at the transcription and translational level, yielding decreased expression of vimentin, fibronectin and hnRNP-K, and increase in extracellular matrix (ECM) proteins (collagen IV and E-cadherin) endorsing reversal of epithelial-mesenchymal transition (EMT) signaling.
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Biología Computacional , Metástasis de la Neoplasia/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Polietilenglicoles/química , Cadherinas/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Metástasis de la Neoplasia/patología , Neoplasias/patología , Polietilenglicoles/uso terapéutico , Transducción de Señal/genéticaRESUMEN
Stress, protein aggregation, and loss of functional properties of cells have been shown to contribute to several deleterious pathologies including cancer and neurodegeneration. The incidence of these pathologies has also been shown to increase with age and are often presented as evidence to the cumulative effect of stress and protein aggregation. Prevention or delay of onset of these diseases may prove to be unprecedentedly beneficial. In this study, we explored the anti-stress and differentiation-inducing potential of two marine bioactive carotenoids (astaxanthin and fucoxanthin) using rat glioma cells as a model. We found that the low (nontoxic) doses of both protected cells against UV-induced DNA damage, heavy metal, and heat-induced protein misfolding and aggregation of proteins. Their long-term treatment in glioma cells caused the induction of physiological differentiation into astrocytes. These phenotypes were supported by upregulation of proteins that regulate cell proliferation, DNA damage repair mechanism, and glial differentiation, suggesting their potential for prevention and treatment of stress, protein aggregation, and age-related pathologies.
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Glioma/tratamiento farmacológico , Agregado de Proteínas/efectos de los fármacos , Agregación Patológica de Proteínas/prevención & control , Xantófilas/farmacología , Animales , Antioxidantes/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Glioma/metabolismo , Glioma/patología , Concentración 50 Inhibidora , Agregación Patológica de Proteínas/tratamiento farmacológico , Agregación Patológica de Proteínas/patología , Ratas , Estrés Fisiológico/efectos de los fármacos , Rayos UltravioletaRESUMEN
Fucoxanthin is commonly found in marine organisms; however, to date, it has been one of the scarcely explored natural compounds. We investigated its activities in human cancer cell culture-based viability, migration, and molecular assays, and found that it possesses strong anticancer and anti-metastatic activities that work irrespective of the p53 status of cancer cells. In our experiments, fucoxanthin caused the transcriptional suppression of mortalin. Cell phenotype-driven molecular analyses on control and treated cells demonstrated that fucoxanthin caused a decrease in hallmark proteins associated with cell proliferation, survival, and the metastatic spread of cancer cells at doses that were relatively safe to the normal cells. The data suggested that the cancer therapy regimen may benefit from the recruitment of fucoxanthin; hence, it warrants further attention for basic mechanistic studies as well as drug development.
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Supervivencia Celular/efectos de los fármacos , Xantófilas/farmacología , Antineoplásicos/farmacología , Organismos Acuáticos/química , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , HumanosRESUMEN
INTRODUCTION: Cancer is one of the leading causes of mortality worldwide that requires attention in terms of extensive study and research. Eradication of mortalin-p53 interaction that leads to the inhibition of transcriptional activation or blocking of p53 from functioning as a suppressor and induction of nuclear translocation of p53 can prove to be one of the useful approaches for cancer management. RESULTS: In this study, we used structure-based approach to target the p53-binding domain of mortalin in order to prevent mortalin-p53 complex formation. We screened compounds from ZINC database against the modeled mortalin protein using Glide virtual screening. The top two compounds, DTOM (ZINC 28639308) and TTOM (ZINC 38143676) with Glide score of -12.27 and -12.16, respectively, were identified with the potential to abrogate mortalin-p53 interaction. Finally, molecular dynamics simulations were used to analyze the dynamic stability of the ligand-bound complex and it was observed that residues Tyr196, Asn198, Val264 and Thr267 were involved in intermolecular interactions in both the simulated ligand-bound complexes, and thus, these residues may have a paramount role in stabilizing the binding of the ligands with the protein. CONCLUSION: These detailed insights can further facilitate the development of potent inhibitors against mortalin-p53 complex.
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Antineoplásicos/farmacología , Productos Biológicos/farmacología , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas Mitocondriales/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Antineoplásicos/química , Productos Biológicos/química , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
2,3-Dihydro-3ß-methoxy withaferin-A (3ßmWi-A) is a natural withanolide that is structurally close to withaferin-A (Wi-A), is cytotoxic to human cancer cells, and is a candidate anticancer natural compound. Using cell-based biochemical, molecular, and imaging assays, we report that Wi-A and 3ßmWi-A possess contrasting activities. Whereas Wi-A caused oxidative stress to normal cells, 3ßmWi-A was well tolerated at even 10-fold higher concentrations. Furthermore, it promoted survival and protected normal cells against oxidative, UV radiation, and chemical stresses. We provide molecular evidence that 3ßmWi-A induces antistress and pro-survival signaling through activation of the pAkt/MAPK pathway. We demonstrate that 3ßmWi-A (i) contrary to Wi-A is safe and possesses stress-relieving activity, (ii) when given subsequent to a variety of stress factors including Wi-A, protects normal cells against their toxicity, and (iii) is a vital compound that may guard normal cells against the toxicity associated with various targeted therapeutic regimes in clinical practice.
Asunto(s)
Citoprotección/efectos de los fármacos , Witanólidos/farmacología , Potenciales de Acción , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Estructura Molecular , Estrés Oxidativo , Transducción de Señal , Witanólidos/químicaRESUMEN
Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.
Asunto(s)
Proteínas HSP70 de Choque Térmico/genética , Enfermedad de Parkinson/genética , Mutación Puntual , Transporte Activo de Núcleo Celular , Carcinogénesis/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proliferación Celular , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/fisiología , Humanos , Proteínas Mitocondriales/fisiología , Mutación MissenseRESUMEN
BACKGROUND AIMS: Internalizing quantum dots (i-QDs) are a useful tool for tracking cells in vivo in models of tissue regeneration. We previously synthesized i-QDs by conjugating QDs with a unique internalizing antibody against a heat shock protein 70 family stress chaperone. In the present study, i-QDs were used to label rabbit mesenchymal stromal cells (MSCs) that were then transplanted into rabbits to assess differentiation potential in an osteonecrosis model. METHODS: The i-QDs were taken up by bone marrow-derived MSCs collected from the iliac of 12-week-old Japanese white rabbits that were positive for cluster of differentiation (CD)81 and negative for CD34 and human leukocyte antigen DR. The average rate of i-QD internalization was 93.3%. At 4, 8, 12, and 24 weeks after transplantation, tissue repair was evaluated histologically and by epifluorescence and electron microscopy. RESULTS: The i-QDs were detected at the margins of the drill holes and in the necrotized bone trabecular. There was significant colocalization of the i-QD signal in transplanted cells and markers of osteoblast and mineralization at 4, 8, and 12 weeks post-transplantation, while i-QDs were detected in areas of mineralization at 12 and 24 weeks post-transplantation. Moreover, i-QDs were observed in osteoblasts in regenerated tissue by electron microscopy, demonstrating that the tissue was derived from transplanted cells. CONCLUSION: These results indicate that transplanted MSCs can differentiate into osteoblasts and induce tissue repair in an osteonecrosis model and can be tracked over the long term by i-QD labeling.
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Rastreo Celular/métodos , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Osteonecrosis/terapia , Puntos Cuánticos/metabolismo , Animales , Antígenos CD34/metabolismo , Médula Ósea , Células de la Médula Ósea/citología , Huesos/citología , Diferenciación Celular/fisiología , Humanos , Conejos , Tetraspanina 28/metabolismo , Trasplante AutólogoRESUMEN
Collaborator of ARF (CARF) has been shown to directly bind to and regulate p53, a central protein that controls tumor suppression via cellular senescence and apoptosis. However, the cellular functions of CARF and the mechanisms governing its effect on senescence, apoptosis, or proliferation are still unknown. Our previous studies have shown that (i) CARF is up-regulated during replicative and stress-induced senescence, and its exogenous overexpression caused senescence-like growth arrest of cells, and (ii) suppression of CARF induces aneuploidy, DNA damage, and mitotic catastrophe, resulting in apoptosis via the ATR/CHK1 pathway. In the present study, we dissected the cellular role of CARF by investigating the molecular pathways triggered by its overexpression in vitro and in vivo. We found that the dosage of CARF is a critical factor in determining the proliferation potential of cancer cells. Most surprisingly, although a moderate level of CARF overexpression induced senescence, a very high level of CARF resulted in increased cell proliferation. We demonstrate that the level of CARF is crucial for DNA damage and checkpoint response of cells through ATM/CHK1/CHK2, p53, and ERK pathways that in turn determine the proliferative fate of cancer cells toward growth arrest or proproliferative and malignant phenotypes. To the best of our knowledge, this is the first report that demonstrates the capability of a fundamental protein, CARF, in controlling cell proliferation in two opposite directions and hence may play a key role in tumor biology and cancer therapeutics.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Proliferación Celular , Daño del ADN , Neoplasias/metabolismo , Neoplasias/fisiopatología , Proteínas de Unión al ARN/metabolismo , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Humanos , Neoplasias/genética , Proteínas de Unión al ARN/genética , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The Hsp70 family protein mortalin is an essential chaperone that is frequently enriched in cancer cells and exists in various subcellular sites, including the mitochondrion, plasma membrane, endoplasmic reticulum, and cytosol. Although the molecular mechanisms underlying its multiple subcellular localizations are not yet clear, their functional significance has been revealed by several studies. In this study, we examined the nuclear fractions of human cells and found that the malignantly transformed cells have more mortalin than the normal cells. We then generated a mortalin mutant that lacked a mitochondrial targeting signal peptide. It was largely localized in the nucleus, and, hence, is called nuclear mortalin (mot-N). Functional characterization of mot-N revealed that it efficiently protects cancer cells against endogenous and exogenous oxidative stress. Furthermore, compared with the full-length mortalin overexpressing cancer cells, mot-N derivatives showed increased malignant properties, including higher proliferation rate, colony forming efficacy, motility, and tumor forming capacity both in in vitro and in vivo assays. We demonstrate that mot-N promotes carcinogenesis and cancer cell metastasis by inactivation of tumor suppressor protein p53 functions and by interaction and functional activation of telomerase and heterogeneous ribonucleoprotein K (hnRNP-K) proteins.
Asunto(s)
Núcleo Celular/metabolismo , Transformación Celular Neoplásica/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Neoplasias/metabolismo , Animales , Western Blotting , Línea Celular , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Células HCT116 , Proteínas HSP70 de Choque Térmico/genética , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Humanos , Inmunohistoquímica , Células MCF-7 , Ratones Endogámicos BALB C , Ratones Desnudos , Mutación , Metástasis de la Neoplasia , Neoplasias/genética , Neoplasias/patología , Estrés Oxidativo , Telomerasa/genética , Telomerasa/metabolismo , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
CARF is an ARF-binding protein that has been shown to regulate the p53-p21-HDM2 pathway. CARF overexpression was shown to cause growth arrest of human cancer cells and premature senescence of normal cells through activation of the p53 pathway. Because replicative senescence involves permanent withdrawal from the cell cycle in response to DNA damage response-mediated signaling, in the present study we investigated the relationship between CARF and the cell cycle and whether it is involved in the DNA damage response. We demonstrate that the half-life of CARF protein is less than 60 min, and that in cycling cells CARF levels are highest in G2 and early prophase. Serially passaged normal human skin and stromal fibroblasts showed upregulation of CARF during replicative senescence. Induction of G1 growth arrest and senescence by a variety of drugs was associated with increase in CARF expression at the transcriptional and translational level and was seen to correlate with increase in DNA damage response and checkpoint proteins, ATM, ATR, CHK1, CHK2, γH2AX, p53 and p21. Induction of growth arrest by oncogenic RAS and shRNA-mediated knockdown of TRF2 in cancer cells also caused upregulation of CARF. We conclude that CARF is associated with DNA damage response and checkpoint signaling pathways.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Puntos de Control del Ciclo Celular/fisiología , Daño del ADN/fisiología , Fibroblastos/metabolismo , Proteínas de Unión al ARN/metabolismo , Piel/metabolismo , Células del Estroma/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Senescencia Celular/fisiología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citología , Piel/efectos de los fármacos , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Proteína 2 de Unión a Repeticiones Teloméricas/antagonistas & inhibidores , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
Withania somnifera is one of the most valued plants and is extensively used in Indian, Unani, and African systems of traditional medicine. It possess a wide array of therapeutic properties including anti-arthritic, anti-aging, anti-cancer, anti-inflammatory, immunoregulatory, chemoprotective, cardioprotective, and recovery from neurodegenerative disorders. With the growing realization of benefits and associated challenges in the improvement of W. somnifera, studies on exploration of genetic and chemotypic variations, identification and characterization of important genes, and understanding the secondary metabolites production and their modulation has gained significant momentum. In recent years, several in vitro and in vivo preclinical studies have facilitated the validation of therapeutic potential of the phytochemicals derived from W. somnifera and have provided necessary impetus for gaining deeper insight into the mechanistic aspects involved in the mode of action of these important pharmaceutically active constituents. The present review highlights some of the current developments and future prospects of biotechnological intervention in this important medicinal plant.
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Fitoquímicos/farmacología , Sustancias Protectoras/farmacología , Withania/genética , Witanólidos/metabolismo , Vías Biosintéticas , Humanos , Fitoquímicos/uso terapéutico , Proteínas de Plantas/genética , Plantas Medicinales/genética , Plantas Medicinales/metabolismo , Regiones Promotoras Genéticas , Sustancias Protectoras/uso terapéutico , Metabolismo Secundario , Withania/metabolismoRESUMEN
Cancer is a leading cause of death and still awaits effective therapies. Rapid industrialization has contributed to increase in incidence of cancer. One of the reasons why most of the cancers fail therapy is due to their metastatic property. Hence identification of factors leading to metastasis is highly important to design effective and novel anti-cancer therapeutics. In our earlier study (Inoue, A., Sawata, S. Y., Taira, K., and Wadhwa, R. (2007) Loss-of-function screening by randomized intracellular antibodies: identification of hnRNP-K as a potential target for metastasis. Proc. Natl. Acad. Sci. U.S.A. 104, 8983-8988), we had reported that the involvement of heterogeneous nuclear ribonucleoprotein K (hnRNP-K) in metastasis. Here, we established hnRNP-K-overexpressing and -underexpressing derivative cell lines and examined their proliferation and metastatic properties in vitro and in vivo. Whereas hnRNP-K compromised cells showed delayed tumor growth, its overexpression resulted in enhanced malignancy and metastasis. Molecular basis of the hnRNP-K induced malignant and metastatic phenotypes was dissected by cDNA microarray and pathway analyses. We found that the hnRNP-K regulates extracellular matrix, cell motility, and angiogenesis pathways. Involvement of the selected genes (Cck, Mmp-3, Ptgs2, and Ctgf) and pathways was validated by gene-specific expression analysis. Our results demonstrated that the hnRNP-K is a potential target for metastasis therapy.
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Movimiento Celular , Matriz Extracelular/metabolismo , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo K/biosíntesis , Proteínas de Neoplasias/biosíntesis , Neoplasias Experimentales/metabolismo , Neovascularización Patológica/metabolismo , Animales , Línea Celular Tumoral , Matriz Extracelular/genética , Matriz Extracelular/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células 3T3 NIH , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Neoplasias Experimentales/genética , Neoplasias Experimentales/patología , Neovascularización Patológica/genética , Neovascularización Patológica/patologíaRESUMEN
BACKGROUND: Embelin, a quinone derivative, is found in the fruits of Embelia ribes Burm (Myrsinaceae). It has been shown to have a variety of therapeutic potentials including anthelmintic, anti-tumor, anti-diabetic, anti-bacterial and anti-inflammation. Inflammation is an immunological response to external harmful stimuli and is regulated by an endogenous pyrogen and pleiotropic pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-α). TNF-α production has been implicated in a variety of other human pathologies including neurodegeneration and cancer. Several studies have shown that the anti-inflammatory activity of embelin is mediated by reduction in TNF-α. The latter is synthesized as a membrane anchored protein (pro-TNF-α); the soluble component of pro-TNF-α is then released into the extracellular space by the action of a protease called TNF-α converting enzyme (TACE). TACE, hence, has been proposed as a therapeutic target for inflammation and cancer. METHODS: We used molecular docking and experimental approaches to investigate the docking potential and molecular effects of embelin to TACE and human cancer cell characteristics, respectively. RESULTS: We demonstrate that embelin is a potential inhibitor of TACE. Furthermore, in vitro studies revealed that it inhibits malignant properties of cancer cells through inactivation of metastatic signaling molecules including MMPs, VEGF and hnRNP-K in breast cancer cells. CONCLUSION: Based on the molecular dynamics and experimental data, embelin is proposed as a natural anti-inflammatory and anticancer drug.
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Proteínas ADAM/antagonistas & inhibidores , Benzoquinonas/farmacología , Neoplasias/metabolismo , Neoplasias/patología , Proteínas ADAM/química , Proteína ADAM17 , Benzoquinonas/química , Dominio Catalítico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Ligandos , Modelos Moleculares , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Metástasis de la Neoplasia , Neoplasias/tratamiento farmacológico , Unión Proteica , Ensayo de Tumor de Célula MadreRESUMEN
The role of tumor stroma in regulation of breast cancer growth has been widely studied. However, the details on the type of heterocellular cross-talk between stromal and breast cancer cells (BCCs) are still poorly known. In the present study, in order to investigate the intercellular communication between human mesenchymal stromal cells (hMSCs) and breast cancer cells (BCCs, MDA-MB-231), we recruited cell-internalizing quantum dots (i-QD) generated by conjugation of cell-internalizing anti-mortalin antibody and quantum dots (QD). Co-culture of illuminated and color-coded hMSCs (QD655) and BCCs (QD585) revealed the intercellular transfer of QD655 signal from hMSCs to BCCs. The amount of QD double positive BCCs increased gradually within 48h of co-culture. We found prominent intercellular transfer of QD655 in hanging drop co-culture system and it was non-existent when hMSCs and BBCs cells were co-cultured in trans-well system lacking imminent cell-cell contact. Fluorescent and electron microscope analyses also supported that the direct cell-to-cell interactions may be required for the intercellular transfer of QD655 from hMSCs to BCCs. To the best of our knowledge, the study provides a first demonstration of transcellular crosstalk between stromal cells and BCCs that involve direct contact and may also include a transfer of mortalin, an anti-apoptotic and growth-promoting factor enriched in cancer cells.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Neoplasias de la Mama/metabolismo , Comunicación Celular , Proteínas HSP70 de Choque Térmico/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Puntos Cuánticos , Neoplasias de la Mama/patología , Línea Celular , Línea Celular Tumoral , Técnicas de Cocultivo , Citosol/metabolismo , Femenino , Humanos , Células Madre Mesenquimatosas/ultraestructura , Microscopía Electrónica de Transmisión , Coloración y EtiquetadoRESUMEN
Cancer metastasis, a highly complex process wherein cancer cells move from the primary site to other sites in the body, is a major hurdle in its therapeutics. A large array of synthetic chemotherapeutic molecules used for the treatment of metastatic cancers, besides being extremely expensive and unaffordable, are known to cause severe adverse effects leading to poor quality of life (QOL) of the patients. In this premise, natural compounds (considered safe, easily available and economic) that possess the potential to inhibit migration of cancer cells are deemed useful and hence are on demand. Cucurbitacin-B (19-(10â9ß)-abeo-10-lanost-5-ene triterpene, called Cuc-B) is a steroid mostly found in plants of Cucurbitaceae family. It has been shown to possess anticancer activity although the molecular mechanism remains poorly defined. We present evidence that Cuc-B has the ability to interact with mortalin and HDM2 proteins that are enriched in cancer cells, suppress wild type p53 function and promote cancer cell migration. Computational analyses showed that Cuc-B interacts with mortalin similar to MKT077 and Withanone, both have been shown to reactivate p53 function and inhibit cell migration. Furthermore, Cuc-B interacted with HDM2 similar to Y30, a well-known inhibitor of HDM2. Experimental cell and molecular analyses demonstrated the downregulation of several proteins, critically involved in cell migration in Cuc-B (low non-toxic doses)-treated cancer cells and exhibited inhibition of cell migration. The data suggested that Cuc-B is a potential natural drug that warrants further mechanistic and clinical studies for its use in the management of metastatic cancers.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Proteínas HSP70 de Choque Térmico , Neoplasias , Triterpenos , Humanos , Cucurbitacinas/farmacología , Calidad de Vida , Proteína p53 Supresora de Tumor , Neoplasias/tratamiento farmacológico , Triterpenos/farmacología , Movimiento CelularRESUMEN
Mortalin, a member of the Hsp70 family of proteins, is commonly enriched in many types of cancers. It promotes carcinogenesis and metastasis in multiple ways of which the inactivation of the tumor suppressor activity of p53 has been firmly established. The downregulation of mortalin and/or disruption of mortalin-p53 interactions by small molecules has earlier been shown to activate p53 function yielding growth arrest/apoptosis in cancer cells. Mortaparibs (Mortaparib, MortaparibPlus, and MortaparibMild) are chemical inhibitors of mortalin isolated by cell-based two-way screening involving (i) a shift in the mortalin staining pattern from perinuclear (characteristics of cancer cells) to pancytoplasmic (characteristics of normal cells) and (ii) the nuclear enrichment of p53. They have similar structures and also cause the inhibition of PARP1 and hence were named Mortaparibs. In the present study, we report the anticancer and anti-metastasis activity of MortaparibMild (4-[(4-amino-5-thiophen-2-yl-1,2,4-triazol-3-yl)sulfanylmethyl]-N-(4-methoxyphenyl)-1,3-thiazol-2-amine) in p53-null cells. By extensive molecular analyses of cell proliferation, growth arrest, and apoptosis pathways, we demonstrate that although it causes relatively weaker cytotoxicity compared to Mortaparib and MortaparibPlus, its lower concentrations were equally potent to inhibit cell migration. We developed combinations (called MortaparibMix-AP, MortaparibMix-AM, and MortaparibMix-AS) consisting of different ratios of three Mortaparibs for specifically enhancing their anti-proliferation, anti-migration, and antistress activities, respectively. Based on the molecular analyses of control and treated cells, we suggest that the three Mortaparibs and their mixtures may be considered for further laboratory and clinical studies validating their use for the treatment of cancer as well as prevention of its relapse and metastasis.