Urbanavicius J.,Laboratory of Cell Biology |
Scorza C.,Institute Investigaciones Biologicas Clemente Estable
Behavioural Pharmacology | Year: 2014
Melanin-concentrating hormone (MCH) administered within the rat dorsal raphe nucleus (DRN) has been shown to elicit prodepressive behaviors in the forced-swim test. The present study was designed to evaluate the time course (30 and 60min) and dose dependence (25-100ng) of this effect, and whether it would be antagonized by an intra-DRN microinjection of the MCH-1 receptor antagonist ATC0175 (ATC, 1mmol/l) or intraperitoneal pretreatment with the noradrenergic antidepressant nortriptyline (20mg/kg). The results showed that the behavioral effect of MCH was time and dose dependent as immobility was increased, and climbing decreased, only by the 50ng MCH dose at T30. The effect was mediated by MCH-1 receptors as a significant blockade of this behavioral response was observed in ATC-pretreated animals. ATC did not by itself modify animal behavior. Nortriptyline also prevented the prodepressive-like effect of MCH. Concomitantly, the effect of MCH (50ng) at T30 on anxiety-related behaviors was assessed using the elevated plus-maze. Interestingly, these behaviors were unchanged. In conclusion, MCH administration within the DRN elicits, through the MCH-1 receptor, a depression-related behavior that is not accompanied by changes in anxiety and that is prevented by a noradrenergic antidepressant. © 2014 Wolters kluwer Health.
Chen J.-Q.,Laboratory of Cell Biology |
Lee J.-H.,Medical Oncology Branch |
Herrmann M.A.,Laboratory of Cell Biology |
Park K.-S.,Medical Oncology Branch |
And 5 more authors.
Molecular Cancer Therapeutics | Year: 2013
Developing proteomic biomarkers is valuable for evaluating therapeutic effects of drugs and generating better treatment strategies. However, conventional protein analysis is often challenging due to inadequate sample size of clinical specimens, lack of assay reproducibility, accuracy, and sensitivity. A novel capillary isoelectricfocusing (IEF) immunoassay system (NanoPro) was used to study the dynamic phosphorylation status of signaling molecules in non-small cell lung cancer (NSCLC) cells treated with EGFR tyrosine kinase and MEKinhibitors. NanoPro showed the same dynamic ERK phosphorylation as Western blotting with good assay reproducibility using 1,000 times less protein. The IEF separation in NanoPro system enables multiple protein phosphorylation isoforms to be resolved and detected simultaneously. With NanoPro, we identified a specific on-target mitogen-activated protein/extracellular signal-regulated kinase (MEK) response pattern to MEK inhibitor PD325901, which was not detectable by Western blot analysis. We also revealed a MEK2 signal that may be associated with NSCLC cell sensitivity to the EGF receptor inhibitor erlotinib, and distinguished erlotinib-sensitive cells from intrinsic as well as acquired resistant cells to erlotinib. Moreover, NanoPro could differentiate human ERK1 isoforms from the mouse isoforms based on their isoelectric point differences and showed that erlotinib effectively inhibited ERK phosphorylation in targeted human xenograft cancer cells but not in surrounding mouse stromal cells. With 8 mg of tumor aspirates, we precisely quantified the response of 18 signaling molecules to erlotinib andMEK1inhibitor treatments in anNSCLC patient. NanoPro's higher sensitivity, better resolution of protein phosphorylation status, and reduced tissue requirement warrant NanoPro's investigation for future drug development and evaluation of drug effects of targeted therapies. Mol Cancer Ther; 12(11); 2601-13. © 2013 AACR.
Shi Z.,St. John's University |
Shi Z.,Sun Yat Sen University |
Tiwari A.K.,St. John's University |
Shukla S.,Laboratory of Cell Biology |
And 10 more authors.
Cancer Research | Year: 2011
Sildenafil is a potent and selective inhibitor of the type 5 cGMP (cyclic guanosine 30,50-monophosphate)- specific phosphodiesterase that is used clinically to treat erectile dysfunction and pulmonary arterial hypertension. Here, we report that sildenafil has differential effects on cell surface ABC transporters such as ABCB1, ABCC1, and ABCG2 that modulate intracompartmental and intracellular concentrations of chemotherapeutic drugs. In ABCB1-overexpressing cells, nontoxic doses of sildenafil inhibited resistance and increased the effective intracellular concentration of ABCB1 substrate drugs such as paclitaxel. Similarly, in ABCG2-overexpressing cells, sildenafil inhibited resistance to ABCG2 substrate anticancer drugs, for example, increasing the effective intracellular concentration of mitoxantrone or the fluorescent compound BODIPY-prazosin. Sildenafil also moderately inhibited the transport of E217βG and methotrexate by the ABCG2 transporter. Mechanistic investigations revealed that sildenafil stimulated ABCB1 ATPase activity and inhibited photolabeling of ABCB1 with [125I]-iodoarylazidoprazosin (IAAP), whereas it only slightly stimulated ABCG2 ATPase activity and inhibited photolabeling of ABCG2 with [125I]-IAAP. In contrast, sildenafil did not alter the sensitivity of parental, ABCB1-, or ABCG2-overexpressing cells to non-ABCB1 and non-ABCG2 substrate drugs, nor did sildenafil affect the function of another ABC drug transporter, ABCC1. Homology modeling predicted the binding conformation of sildenafil within the large cavity of the transmembrane region of ABCB1. Overall, we found that sildenafil inhibits the transporter function of ABCB1 and ABCG2, with a stronger effect on ABCB1. Our findings suggest a possible strategy to enhance the distribution and potentially the activity of anticancer drugs by jointly using a clinically approved drug with known side effects and drug-drug interactions. © 2011 American Association for Cancer Research.
Vasilyev S.A.,Cancer Research Institute |
Vasilyev S.A.,Institute of Medical Genetics |
Kubes M.,Laboratory of Cell Biology |
Markova E.,Cancer Research Institute |
And 2 more authors.
International Journal of Radiation Biology | Year: 2013
Purpose: Human hematopoietic stem cells (HSC) are thought to be a major target of radiation-induced leukemogenesis and also provide a relevant cellular model for assessing cancer risk. Cluster of designation 133+ (CD133+) is a marker found in human progenitor and hematopoietic stem cells. Our study examined the repair of radiation-induced DNA double-strand breaks (DSB) in CD133 + umbilical cord blood cells (UCBC). Materials and methods: After γ-irradiation, endogenous and induced DSB were evaluated in CD133 + UCBC, CD133 - UCBC and peripheral blood lymphocytes (PBL) in terms of phosphorylated histone 2A family member X (γH2AX) and tumor suppressor p53 binding protein 1 (53BP1) foci. Results: We found that repair signaling in CD133 + UCBC is different from CD133 - UCBC and PBL. These differences include lower endogenous DSB levels and higher 53BP1 recruitment. Conclusions: Our data, together with a recent report on radiation-induced γH2AX and 53BP1 foci in CD34 + cells, indicate enhanced DNA repair capacity in HSC as compared to mature lymphocytes. © 2013 Informa UK, Ltd.
Orecchia P.,Laboratory of Immunology |
Conte R.,Laboratory of Immunology |
Balza E.,Laboratory of Cell Biology |
Petretto A.,Instituto Giannina Gaslini |
And 4 more authors.
European Journal of Cancer | Year: 2013
Introduction: Syndecan-1 is a cell membrane protein that, after its shedding by heparanase enzymes, is accumulated in the extracellular matrix of some tumours, e.g. myeloma and lung carcinoma, where it modulates several key processes of tumourigenesis such as cancer cell proliferation and apoptosis, angiogenesis and metastasis. Few studies have focused on syndecan-1 in malignant melanoma, a tumour for which new therapeutic targets are desperately needed. We aimed to investigate the role of syndecan-1 in melanoma and to evaluate the potential therapeutic efficacy of a novel fully human anti-syndecan-1 recombinant antibody in this deadly disease. Methods: The OC-46F2 recombinant antibody was generated by selecting a human antibody phage display library on human melanoma cells and by its expression in mammalian cells. The specific antigen recognised by the antibody was identified by mass spectrometry. Murine models of human melanoma and ovarian carcinoma were used in the pre-clinical in vivo experiments. Results: The fully human antibody OC-46F2, specific for the extracellular domain of syndecan-1, inhibited vascular maturation and tumour growth in an experimental human melanoma model. The therapeutic efficacy of this antibody was also demonstrated in an experimental ovarian carcinoma model. A co-distribution of syndecan-1 with vascular endothelial growth factor receptor 2 (VEGFR2) observed in the intratumour melanoma microenvironment was absent in the tumours from mice treated with OC-46F2 scFv. Conclusion: These findings highlight the role of syndecan-1 as a potential therapeutic target in melanoma and ovarian carcinoma and provide a new tool able to block vessel maturation, one of the mechanisms that underpin the angiogenic process essential for solid tumour growth. © 2012 Elsevier Ltd. All rights reserved.
Hoelz A.,California Institute of Technology |
Debler E.W.,Laboratory of Cell Biology |
Blobel G.,Laboratory of Cell Biology |
Blobel G.,Howard Hughes Medical Institute
Annual Review of Biochemistry | Year: 2011
In eukaryotic cells, the spatial segregation of replication and transcription in the nucleus and translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are the sole gateways that facilitate this macromolecular exchange across the nuclear envelope with the help of soluble transport receptors. Whereas the mobile transport machinery is reasonably well understood at the atomic level, a commensurate structural characterization of the NPC has only begun in the past few years. Here, we describe the recent progress toward the elucidation of the atomic structure of the NPC, highlight emerging concepts of its underlying architecture, and discuss key outstanding questions and challenges. The applied structure determination as well as the described design principles of the NPC may serve as paradigms for other macromolecular assemblies. © 2011 by Annual Reviews. All rights reserved.
PubMed | BU Bioscience, Laboratory of Cell Biology, Laboratory of Plant Physiology and Netherlands Metabolomics Center
Type: | Journal: Metabolomics : Official journal of the Metabolomic Society | Year: 2016
Metabolomics has become a valuable tool in many research areas. However, generating metabolomics-based biochemical profiles without any related bioactivity is only of indirect value in understanding a biological process. Therefore, metabolomics research could greatly benefit from tools that directly determine the bioactivity of the detected compounds.We aimed to combine LC-MS metabolomics with a cell based receptor assay. This combination could increase the understanding of biological processes and may provide novel opportunities for functional metabolomics.We developed a flow through biosensor with human cells expressing both the TRPV1, a calcium ion channel which responds to capsaicin, and the fluorescent intracellular calcium ion reporter, YC3.6. We have analysed three contrasting Among the compounds in the crude pepper fruit extracts, we confirmed capsaicin and also identified both nordihydrocapsaicin and dihydrocapsaicin as true agonists of the TRPV1 receptor. Furthermore, the biosensor was able to detect receptor activity in extracts of both Our results demonstrate that the TRPV1 biosensor is suitable for detecting bioactive metabolites. Novel opportunities may lie in the development of a continuous functional assay, where the biosensor is directly coupled to the LC-MS.
News Article | August 23, 2016
Researchers at the University of Massachusetts Medical School have designed a nanoparticle that mimics the bacterium Salmonella and may help to counteract a major mechanism of chemotherapy resistance. Working with mouse models of colon and breast cancer, Beth McCormick, Ph.D., and her colleagues demonstrated that when combined with chemotherapy, the nanoparticle reduced tumor growth substantially more than chemotherapy alone. The results of their research were published in Nature Communications. A membrane protein called P-glycoprotein (P-gp) acts like a garbage chute that pumps waste, foreign particles, and toxins out of cells. P-gp is a member of a large family of transporters, called ATP-binding cassette (ABC) transporters, that are active in normal cells but also have roles in cancer and other diseases. For instance, cancer cells can co-opt P-gp to rid themselves of chemotherapeutic agents, severely limiting the efficacy of these drugs. In previous work, McCormick and her colleagues serendipitously discovered that Salmonella enterica, a bacterium that causes food poisoning, decreases the amount of P-gp on the surface of intestinal cells. Because Salmonella has the capacity to grow selectively in cancer cells, the researchers wondered whether there was a way to use the bacterium to counteract chemotherapy resistance caused by P-gp. “While trying to understand how Salmonella invades the human host, we made this other observation that may be relevant to cancer therapeutics and multidrug resistance,” explains McCormick. To determine the specific bacterial component responsible for reducing P-gp levels, the researchers engineered multiple Salmonella mutant strains and tested their effect on P-gp levels in colon cells. They found that a Salmonella strain lacking the bacterial protein SipA was unable to reduce P-gp levels in the colon of mice or in a human colon cancer cell line. Salmonella secretes SipA, along with other proteins, to help the bacterium invade human cells. The researchers then showed that treatment with SipA protein alone decreased P-gp levels in cell lines of human colon cancer, breast cancer, bladder cancer, and lymphoma. Because P-gp can pump drugs out of cells, the researchers next sought to determine whether SipA treatment would prevent cancer cells from expelling chemotherapy drugs. When they treated human colon cancer cells with the chemotherapy agents doxorubicin or vinblastine, with or without SipA, they found that the addition of SipA increased drug retention inside the cells. SipA also increased the cancer cells’ sensitivity to both drugs, suggesting that it could possibly be used to enhance chemotherapy. “Through millions of years of co-evolution, Salmonella has figured out a way to remove this transporter from the surface of intestinal cells to facilitate host infection,” says McCormick. “We capitalized on the organism’s ability to perform that function.” It would not be feasible to infect people with the bacterium, and SipA on its own will likely deteriorate quickly in the bloodstream, coauthor Gang Han, Ph.D., of the University of Massachusetts Medical School, explained in a press release. The researchers therefore fused SipA to gold nanoparticles, generating what they refer to as a nanoparticle mimic of Salmonella. They designed the nanoparticle to enhance the stability of SipA, while retaining its ability to interact with other proteins. In an effort to target tumors without harming healthy tissues, the researchers used a nanoparticle of specific size that should only be able to access the tumor tissue due to its “leaky” architecture. “Because of this property, we are hoping to be able to avoid negative effects to healthy tissues,” says McCormick. Another benefit of this technology is that the nanoparticle can be modified to enhance tumor targeting and minimize the potential for side effects, she added. The researchers showed that this nanoparticle was 100 times more effective than SipA protein alone at reducing P-gp levels in a human colon cancer cell line. The enhanced function of the nanoparticle is likely due to stabilization of SipA, explained the researchers. The team then tested the nanoparticle in a mouse model of colon cancer, because this cancer type is known to express high levels of P-gp. When they treated tumor-bearing mice with the nanoparticle plus doxorubicin, P-gp levels dropped and the tumors grew substantially less than in mice treated with the nanoparticle or doxorubicin alone. The researchers observed similar results in a mouse model of human breast cancer. There are concerns about the potential effect of nanoparticles on normal tissues. “P-gp has evolved as a defense mechanism” to rid healthy cells of toxic molecules, says Suresh Ambudkar, Ph.D., deputy chief of the Laboratory of Cell Biology in NCI’s Center for Cancer Research. It plays an important role in protecting cells of the blood-brain barrier, liver, testes, and kidney. “So when you try to interfere with that, you may create problems,” he said. The researchers, however, found no evidence of nanoparticle accumulation in the brain, heart, kidney, or lungs of mice, nor did it appear to cause toxicity. They did observe that the nanoparticles accumulated in the liver and spleen, though this was expected because these organs filter the blood, says McCormick. The research team is moving forward with preclinical studies of the SipA nanoparticle to test its safety and toxicity, and to establish appropriate dosage levels. However, Ambudkar notes, “the development of drug resistance in cancer cells is a multifactorial process. In addition to the ABC transporters, other phenomena are involved, such as drug metabolism.” And because there is a large family of ABC transporters, one transporter can compensate if another is blocked, he explained. For the last 25 years, clinical trials with drugs that inhibit P-gp have failed to overcome chemotherapy resistance, Ambudkar says. Tackling the issue of multidrug resistance in cancer, he continues, “is not something that can be solved easily.” McCormick and her team are also pursuing research to better characterize and understand the biology of SipA. “We are not naïve about the complexity of the problem," she says. "However, if we know more about the biology, we believe we can ultimately make a better drug.”
Martina J.A.,U.S. National Institutes of Health |
Martina J.A.,Laboratory of Cell Biology |
Chen Y.,U.S. National Institutes of Health |
Gucek M.,U.S. National Institutes of Health |
And 2 more authors.
Autophagy | Year: 2012
The mammalian target of rapamycin (MTOR) protein kinase complex is a key component of a pathway that regulates cell growth and proliferation in response to energy levels, hypoxia, nutrients and insulin. Inhibition of MTORC1 strongly induces autophagy by regulating the activity of the ULK protein kinase complex that is required for the formation of autophagosomes. However, the participation of MTORC1 in the expression of autophagy genes has not been characterized. Here we show that MTORC1 regulates nuclear localization and activity of the transcription factor EB (TFEB), a member of the bHLH leucine-zipper family of transcription factors that drives expression of autophagy and lysosomal genes. Under normal nutrient conditions, TFEB is phosphorylated in Ser211 in an MTORC1-dependent manner. This phosphorylation promotes association of TFEB with members of the YWHA (14-3-3) family of proteins and retention of the transcription factor in the cytosol. Pharmacological or genetic inhibition of MTORC1 causes dissociation of the TFEB/ YWHA complex and rapid transport of TFEB to the nucleus where it increases transcription of multiple genes implicated in autophagy and lysosomal function. Active TFEB also associates with late endosomal/lysosomal membranes through interaction with the LAMTOR/RRAG/MTORC1 complex. Our results unveil a novel role for MTORC1 in the maintenance of cellular homeostasis by regulating autophagy at the transcriptional level. © 2012 Landes Bioscience.
Azuma N.,Laboratory of Cell Biology |
Ito M.,Laboratory of Cell Biology |
Yokoi T.,Laboratory of Cell Biology |
Nakayama Y.,Laboratory of Cell Biology |
Nishina S.,Laboratory of Cell Biology
JAMA Ophthalmology | Year: 2013
Importance Aggressive posterior retinopathy of prematurity (AP-ROP) rapidly progresses to retinal detachment despite application of photocoagulation. Early vitreous surgery might achieve prompt regression of neovascular activity and a high incidence of retinal reattachment. Objective To evaluate visual outcomes in eyes with AP-ROP after early vitreous surgery. Design Retrospective nonrandomized study of patients who underwent early vitreous surgery with lensectomy when retinal detachment developed despite photocoagulation. Aphakic correction with spectacles or contact lenses and the use of orthoptics were continued postoperatively. The best-corrected visual acuity (VA) was measured in eyes with a total retinal reattachment using the preferential looking technique in patients ranging in age from 8 months to no more than 3 years and a VA chart with Landolt rings or pictures for older children. The VA findings were converted to Snellen lines. Setting Institutional ophthalmology practice. Participants Of the 103 eyes (57 patients) that underwent early vitreous surgery for AP-ROP, the VA was measured in 58 (32 patients) at a corrected age ranging from 8 months to 4 years. Interventions Early vitreous surgery and VA measurement using the preferential looking technique and a VA chart. Main Outcomes And Measures Postoperative VA, ROP stage, extent of fibrovascular tissue (FT) growth, and laterality of the eyes that underwent surgery. RESULTS The VAs ranged from 20/2000 to 20/40. The VAmay not be related to the preoperative ROP stage 4A or 4B but may depend on the preoperative extent of FT growth. In 39 of 58 eyes (67.2%), the FT had not reached the vitreous base preoperatively, and foveal formation occurred postoperatively with nearly age-appropriate VA (range, 20/250 to 20/40). In 17 of 58 eyes (29.3%), the FT had reached the vitreous base, and no fovea formed (VA range, 20/2000 to 20/250). Two of 58 eyes (3.4%) had deprivation amblyopia with a VA worse than 20/1600. The difference in VA between both eyes of patients who underwent bilateral vitreous surgery depended on ROP progression; patients who underwent a unilateral procedure in which the fellow eyes with ROP stabilized after photocoagulation tended to have poor vision because of deprivation amblyopia. Conclusions And Relevance Early vitreous surgerymay be beneficial for AP-ROP and should be performed before the FT reaches the vitreous base to facilitate foveal formation and good VA outcomes. The roles of photocoagulation, vitreous surgery, and anti-vascular endothelial growth factor therapy in the treatment of AP-ROP should be investigated in randomized trials regarding efficacy, safety, convenience, and cost.