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BACKGROUND: Enhanced glucose metabolism is a feature of most tumors, but downstream functional effects of aberrant glucose flux are difficult to mechanistically determine. Metabolic diseases including obesity and diabetes have a hyperglycemia component and are correlated with elevated pre-menopausal cancer risk for triple-negative breast cancer (TNBC). However, determining pathways for hyperglycemic disease-coupled cancer risk remains a major unmet need. One aspect of cellular sugar utilization is the addition of the glucose-derived protein modification O-GlcNAc (O-linked N-acetylglucosamine) via the single human enzyme that catalyzes this process, O-GlcNAc transferase (OGT). The data in this report implicate roles of OGT and O-GlcNAc within a pathway leading to cancer stem-like cell (CSC) expansion. CSCs are the minor fraction of tumor cells recognized as a source of tumors as well as fueling metastatic recurrence. The objective of this study was to identify a novel pathway for glucose-driven expansion of CSC as a potential molecular link between hyperglycemic conditions and CSC tumor risk factors. METHODS: We used chemical biology tools to track how a metabolite of glucose, GlcNAc, became linked to the transcriptional regulatory protein tet-methylcytosine dioxygenase 1 (TET1) as an O-GlcNAc post-translational modification in three TNBC cell lines. Using biochemical approaches, genetic models, diet-induced obese animals, and chemical biology labeling, we evaluated the impact of hyperglycemia on CSC pathways driven by OGT in TNBC model systems. RESULTS: We showed that OGT levels were higher in TNBC cell lines compared to non-tumor breast cells, matching patient data. Our data identified that hyperglycemia drove O-GlcNAcylation of the protein TET1 via OGT-catalyzed activity. Suppression of pathway proteins by inhibition, RNA silencing, and overexpression confirmed a mechanism for glucose-driven CSC expansion via TET1-O-GlcNAc. Furthermore, activation of the pathway led to higher levels of OGT production via feed-forward regulation in hyperglycemic conditions. We showed that diet-induced obesity led to elevated tumor OGT expression and O-GlcNAc levels in mice compared to lean littermates, suggesting relevance of this pathway in an animal model of the hyperglycemic TNBC microenvironment. CONCLUSIONS: Taken together, our data revealed a mechanism whereby hyperglycemic conditions activated a CSC pathway in TNBC models. This pathway can be potentially targeted to reduce hyperglycemia-driven breast cancer risk, for instance in metabolic diseases. Because pre-menopausal TNBC risk and mortality are correlated with metabolic diseases, our results could lead to new directions including OGT inhibition for mitigating hyperglycemia as a risk factor for TNBC tumorigenesis and progression.
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NF-κB is a pro-inflammatory transcription factor that critically regulates immune responses and other distinct cellular pathways. However, many NF-κB-mediated pathways for cell survival and apoptosis signaling in cancer remain to be elucidated. Cell cycle and apoptosis regulatory protein 1 (CARP-1 or CCAR1) is a perinuclear phosphoprotein that regulates signaling induced by anticancer chemotherapy and growth factors. Although previous studies have reported that CARP-1 is a part of the NF-κB proteome, regulation of NF-κB signaling by CARP-1 and the molecular mechanism(s) involved are unclear. Here, we report that CARP-1 directly binds the NF-κB-activating kinase IκB kinase subunit γ (NEMO or NF-κB essential modulator) and regulates the chemotherapy-activated canonical NF-κB pathway. Importantly, blockade of NEMO-CARP-1 binding diminished NF-κB activation, indicated by reduced phosphorylation of its subunit p65/RelA by the chemotherapeutic agent adriamycin (ADR), but not NF-κB activation induced by tumor necrosis factor α (TNFα), interleukin (IL)-1ß, or epidermal growth factor. High-throughput screening of a chemical library yielded a small molecule inhibitor of NEMO-CARP-1 binding, termed selective NF-κB inhibitor 1 (SNI)-1). We noted that SNI-1 enhances chemotherapy-dependent growth inhibition of a variety of cancer cells, including human triple-negative breast cancer (TNBC) and patient-derived TNBC cells in vitro, and attenuates chemotherapy-induced secretion of the pro-inflammatory cytokines TNFα, IL-1ß, and IL-8. SNI-1 also enhanced ADR or cisplatin inhibition of murine TNBC tumors in vivo and reduced systemic levels of pro-inflammatory cytokines. We conclude that inhibition of NEMO-CARP-1 binding enhances responses of cancer cells to chemotherapy.
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Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quinasa I-kappa B/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas de Ciclo Celular/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Citocinas/metabolismo , Daño del ADN , Doxorrubicina/farmacología , Epítopos/metabolismo , Mediadores de Inflamación/metabolismo , Cinética , Ratones Endogámicos BALB C , Modelos Biológicos , Modelos Moleculares , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Termodinámica , Factor de Transcripción ReIA/metabolismoRESUMEN
Platinum drug-induced cross-link repair requires the concerted activities of translesion synthesis (TLS), Fanconi anemia (FA), and homologous recombination repair pathways. The E2 ubiquitin-conjugating enzyme RAD6 is essential for TLS. Here, we show that RAD6 plays a universal role in platinum-based drug tolerance. Using a novel RAD6-selective small-molecule inhibitor (SMI#9) targeting the RAD6 catalytic site, we demonstrate that SMI#9 potentiates the sensitivities of cancer cells with innate or acquired cisplatin or oxaliplatin resistance. 5-Iododeoxyuridine/5-chlorodeoxyuridine pulse-labeling experiments showed that RAD6 is necessary for overcoming cisplatin-induced replication fork stalling, as replication-restart was impaired in both SMI#9-pretreated and RAD6B-silenced cells. Consistent with the role of RAD6/TLS in late-S phase, SMI#9-induced DNA replication inhibition occurred preferentially in mid/late-S phase. The compromised DNA repair and chemosensitization induced by SMI#9 or RAD6B depletion were associated with decreased platinum drug-induced proliferating cell nuclear antigen (PCNA) and FANCD2 monoubiquitinations (surrogate markers of TLS and FA pathway activation, respectively) and with attenuated FANCD2, RAD6, γH2AX, and POL η foci formation and cisplatin-adduct removal. SMI#9 pretreatment synergistically increased cisplatin inhibition of MDA-MB-231 triple-negative breast cancer cell proliferation and tumor growth. Using an isogenic HCT116 colon cancer model of oxaliplatin resistance, we further show that γH2AX and monoubiquitinated PCNA and FANCD2 are constitutively up-regulated in oxaliplatin-resistant HCT116 (HCT116-OxR) cells and that γH2AX, PCNA, and FANCD2 monoubiquitinations are induced by oxaliplatin in parental HCT116 cells. SMI#9 pretreatment sensitized HCT116-OxR cells to oxaliplatin. These data deepen insights into the vital role of RAD6/TLS in platinum drug tolerance and reveal clinical benefits of targeting RAD6 with SMI#9 for managing chemoresistant cancers.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Reparación del ADN/efectos de los fármacos , ADN de Neoplasias/biosíntesis , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Compuestos Organoplatinos/farmacología , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Replicación del ADN/efectos de los fármacos , ADN de Neoplasias/genética , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Ratones , Ratones Desnudos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Oxaliplatino , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Increasing evidence demonstrates the immunosuppressive kynurenine pathway's (KP) role in the pathophysiology of human gliomas. To study the KP in vivo, we used the noninvasive molecular imaging tracer α-[(11)C]-methyl-l-tryptophan (AMT). The AMT-positron emission tomography (PET) has shown high uptake in high-grade gliomas and predicted survival in patients with recurrent glioblastoma (GBM). We generated patient-derived xenograft (PDX) models from dissociated cells, or tumor fragments, from 5 patients with GBM. Mice bearing subcutaneous tumors were imaged with AMT-PET, and tumors were analyzed to detect the KP enzymes indoleamine 2,3-dioxygenase (IDO) 1, IDO2, tryptophan 2,3-dioxygenase, kynureninase, and kynurenine 3-monooxygenase. Overall, PET imaging showed robust tumoral AMT uptake in PDX mice with prolonged tracer accumulation over 60 minutes, consistent with AMT trapping seen in humans. Immunostained tumor tissues demonstrated positive detection of multiple KP enzymes. Furthermore, intracranial implantation of GBM cells was performed with imaging at both 9 and 14 days postimplant, with a marked increase in AMT uptake at 14 days and a corresponding high level of tissue immunostaining for KP enzymes. These results indicate that our PDX mouse models recapitulate human GBM, including aberrant tryptophan metabolism, and offer an in vivo system for development of targeted therapeutics for patients with GBM.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Quinurenina/metabolismo , Imagen Molecular/métodos , Tomografía de Emisión de Positrones/métodos , Triptófano/farmacocinética , Anciano , Animales , Vías Biosintéticas , Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/patología , Radioisótopos de Carbono/química , Línea Celular Tumoral , Femenino , Glioblastoma/diagnóstico por imagen , Glioblastoma/patología , Humanos , Masculino , Ratones , Persona de Mediana Edad , Trasplante de Neoplasias , Triptófano/químicaRESUMEN
The 3+ and 2+ oxidation states of europium have drastically different magnetic and spectroscopic properties. Electrochemical measurements are often used to probe EuIII/II oxidation state changes, but a full suite of spectroscopic characterization is necessary to demonstrate conversion between these two oxidation states in solution. Here, we report the facile conversion of an europium(III) tetraglycinate complex into its EuII analogue. We present electrochemical, luminescence, electron paramagnetic resonance, UV-visible, and NMR spectroscopic data demonstrating complete reversibility from the reduction and oxidation of the 3+ and 2+ oxidation states, respectively. The EuII-containing analogue has kinetic stability within the range of clinically approved GdIII-containing complexes using an acid-catalyzed dissociation experiment. Additionally, we demonstrate that the 3+ and 2+ oxidation states provide redox-responsive behavior through chemical-exchange saturation transfer or proton relaxation, respectively. These results will be applicable to a wide range of redox-responsive contrast agents and Eu-containing complexes.
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BACKGROUND: There is a paucity of effective therapies for recurrent/aggressive meningiomas. Establishment of improved in vitro and in vivo meningioma models will facilitate development and testing of novel therapeutic approaches. METHODS: A primary meningioma cell line was generated from a patient with an olfactory groove meningioma. The cell line was extensively characterized by performing analysis of growth kinetics, immunocytochemistry, telomerase activity, karyotype, and comparative genomic hybridization. Xenograft models using immunocompromised SCID mice were also developed. RESULTS: Histopathology of the patient tumor was consistent with a WHO grade I typical meningioma composed of meningothelial cells, whorls, and occasional psammoma bodies. The original tumor and the early passage primary cells shared the standard immunohistochemical profile consistent with low-grade, good prognosis meningioma. Low passage KCI-MENG1 cells were composed of two cell types with spindle and round morphologies, showed linear growth curve, had very low telomerase activity, and were composed of two distinct unrelated clones on cytogenetic analysis. In contrast, high passage cells were homogeneously round, rapidly growing, had high telomerase activity, and were composed of a single clone with a near triploid karyotype containing 64-66 chromosomes with numerous aberrations. Following subcutaneous and orthotopic transplantation of low passage cells into SCID mice, firm tumors positive for vimentin and progesterone receptor (PR) formed, while subcutaneous implant of high passage cells yielded vimentin-positive, PR-negative tumors, concordant with a high-grade meningioma. CONCLUSIONS: Although derived from a benign meningioma specimen, the newly-established spontaneously immortal KCI-MENG1 meningioma cell line can be utilized to generate xenograft tumor models with either low- or high-grade features, dependent on the cell passage number (likely due to the relative abundance of the round, near-triploid cells). These human meningioma mouse xenograft models will provide biologically relevant platforms from which to investigate differences in low- vs. high-grade meningioma tumor biology and disease progression as well as to develop novel therapies to improve treatment options for poor prognosis or recurrent meningiomas.
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Neoplasias Meníngeas/patología , Meningioma/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Forma de la Célula , Bandeo Cromosómico , Hibridación Genómica Comparativa , Femenino , Humanos , Inmunohistoquímica , Cariotipificación , Ratones SCID , Persona de Mediana Edad , Clasificación del Tumor , NeuroimagenRESUMEN
The Eu(II) ion rivals Gd(III) in its ability to enhance contrast in magnetic resonance imaging. However, all reported Eu(II)-based complexes have been studied inâ vitro largely because the tendency of Eu(II) to oxidize to Eu(III) has been viewed as a major obstacle to inâ vivo imaging. Herein, we present solid- and solution-phase characterization of a Eu(II)-containing cryptate and the first inâ vivo use of Eu(II) to provide contrast enhancement. The results indicate that between one and two water molecules are coordinated to the Eu(II)â core upon dissolution. We also demonstrate that Eu(II)-based contrast enhancement can be observed for hours in a mouse.
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Europio/química , Imagen por Resonancia Magnética/métodos , Neoplasias Mamarias Experimentales/patología , Animales , Cristalografía por Rayos X , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Oxidación-ReducciónRESUMEN
In response to antigens, B cells undergo affinity maturation and class switching mediated by activation-induced cytidine deaminase (AID) in germinal centers (GCs) of secondary lymphoid organs, but uncontrolled AID activity can precipitate autoimmunity and cancer. The regulation of GC antibody diversification is of fundamental importance but not well understood. We found that autoimmune regulator (AIRE), the molecule essential for T cell tolerance, is expressed in GC B cells in a CD40-dependent manner, interacts with AID and negatively regulates antibody affinity maturation and class switching by inhibiting AID function. AIRE deficiency in B cells caused altered antibody repertoire, increased somatic hypermutations, elevated autoantibodies to T helper 17 effector cytokines and defective control of skin Candida albicans. These results define a GC B cell checkpoint of humoral immunity and illuminate new approaches of generating high-affinity neutralizing antibodies for immunotherapy.
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Background: The aryl hydrocarbon receptor (AhR) is expressed in gliomas and the highest staining is observed in glioblastomas. A recent study showed that the AhR exhibited tumor suppressor-like activity in established and patient-derived glioblastoma cells and genomic analysis showed that this was due, in part, to suppression of CXCL12, CXCR4 and MMP9. Methods: Selective AhR modulators (SAhRMs) including AhR-active pharmaceuticals were screened for their inhibition of invasion using a spheroid invasion assay in patient-derived AhR-expressing 15-037 glioblastoma cells and in AhR-silenced 15-037 cells. Invasion, migration and cell proliferation were determined using spheroid invasion, Boyden chambers and scratch assay, and XTT metabolic assays for cell growth. Changes in gene and gene product expression were determined by real-time PCR and Western blot assays, respectively. In vivo antitumorigenic activity of omeprazole was determined in SCID mice bearing subcutaneous patient-derived 15-037 cells. Results: Results of a screening assay using patient-derived 15-037 cells (wild-type and AhR knockout) identified the AhR-active proton pump inhibitor omeprazole as an inhibitor of glioblastoma cell invasion and migration only AhR-expressing cells but not in cells where the AhR was downregulated. Omeprazole also enhanced AhR-dependent repression of the pro-invasion CXCL12, CXCR4 and MMP9 genes, and interactions and effectiveness of omeprazole plus temozolomide were response-dependent. Omeprazole (100 mg/kg/injection) inhibited and delayed tumors in SCID mice bearing patient-derived 15-037 cells injected subcutaneously. Conclusion: Our results demonstrate that omeprazole enhances AhR-dependent inhibition of glioblastoma invasion and highlights a potential new avenue for development of a novel therapeutic mechanism-based approach for treating glioblastoma.
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Obesity is a risk factor for triple-negative breast cancer (TNBC) incidence and poor outcomes, but the underlying molecular biology remains unknown. We previously identified in TNBC cell cultures that expression of epigenetic reader methyl-CpG-binding domain protein 2 (MBD2), specifically the alternative mRNA splicing variant MBD variant 2 (MBD2_v2), is dependent on reactive oxygen species (ROS) and is crucial for maintenance and expansion of cancer stem cell-like cells (CSCs). Because obesity is coupled with inflammation and ROS, we hypothesized that obesity can fuel an increase in MBD2_v2 expression to promote the tumor-initiating CSC phenotype in TNBC cells in vivo. Analysis of TNBC patient datasets revealed associations between high tumor MBD2_v2 expression and high relapse rates and high body mass index (BMI). Stable gene knockdown/overexpression methods were applied to TNBC cell lines to elucidate that MBD2_v2 expression is governed by ROS-dependent expression of serine- and arginine-rich splicing factor 2 (SRSF2). We employed a diet-induced obesity (DIO) mouse model that mimics human obesity to investigate whether obesity causes increased MBD2_v2 expression and increased tumor initiation capacity in inoculated TNBC cell lines. MBD2_v2 and SRSF2 levels were increased in TNBC cell line-derived tumors that formed more frequently in DIO mice relative to tumors in lean control mice. Stable MBD2_v2 overexpression increased the CSC fraction in culture and increased TNBC cell line tumor initiation capacity in vivo. SRSF2 knockdown resulted in decreased MBD2_v2 expression, decreased CSCs in TNBC cell cultures, and hindered tumor formation in vivo. This report describes evidence to support the conclusion that MBD2_v2 expression is induced by obesity and drives TNBC cell tumorigenicity, and thus provides molecular insights into support of the epidemiological evidence that obesity is a risk factor for TNBC. The majority of TNBC patients are obese and rising obesity rates threaten to further increase the burden of obesity-linked cancers, which reinforces the relevance of this report.
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Empalme Alternativo/genética , Carcinogénesis/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Células Madre Neoplásicas/patología , Obesidad/patología , Neoplasias de la Mama Triple Negativas/patología , Empalme Alternativo/efectos de los fármacos , Animales , Antioxidantes/farmacología , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Dieta , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
The triple-negative breast cancer (TNBC) subtype, regardless of their BRCA1 status, has the poorest outcome compared with other breast cancer subtypes, and currently there are no approved targeted therapies for TNBC. We have previously demonstrated the importance of RAD6-mediated translesion synthesis pathway in TNBC development/progression and chemoresistance, and the potential therapeutic benefit of targeting RAD6 with a RAD6-selective small-molecule inhibitor, SMI#9. To overcome SMI#9 solubility limitations, we recently developed a gold nanoparticle (GNP)-based platform for conjugation and intracellular release of SMI#9, and demonstrated its in vitro cytotoxic activity toward TNBC cells. Here, we characterized the in vivo pharmacokinetic and therapeutic properties of PEGylated GNP-conjugated SMI#9 in BRCA1 wild-type and BRCA1-mutant TNBC xenograft models, and investigated the impact of RAD6 inhibition on TNBC metabolism by 1H-NMR spectroscopy. GNP conjugation allowed the released SMI#9 to achieve higher systemic exposure and longer retention as compared with the unconjugated drug. Systemically administered SMI#9-GNP inhibited the TNBC growth as effectively as intratumorally injected unconjugated SMI#9. Inductively coupled mass spectrometry analysis showed highest GNP concentrations in tumors and liver of SMI#9-GNP and blank-GNP-treated mice; however, tumor growth inhibition occurred only in the SMI#9-GNP-treated group. SMI#9-GNP was tolerated without overt signs of toxicity. SMI#9-induced sensitization was associated with perturbation of a common set of glycolytic pathways in BRCA1 wild-type and BRCA1-mutant TNBC cells. These data reveal novel SMI#9 sensitive markers of metabolic vulnerability for TNBC management and suggest that nanotherapy-mediated RAD6 inhibition offers a promising strategy for TNBC treatment.
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Reparación del ADN , Tiazinas/uso terapéutico , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Enzimas Ubiquitina-Conjugadoras/metabolismo , Animales , Proteína BRCA1/metabolismo , Línea Celular Tumoral , Reparación del ADN/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Femenino , Oro/química , Oro/farmacocinética , Humanos , Redes y Vías Metabólicas/efectos de los fármacos , Nanopartículas del Metal/química , Ratones Desnudos , Mutación/genética , Polietilenglicoles/química , Tiazinas/farmacología , Distribución Tisular/efectos de los fármacosRESUMEN
Drug resistance is one of the significant clinical burden in renal cell carcinoma (RCC). The development of drug resistance is attributed to many factors, including impairment of apoptosis, elevation of carbonic anhydrase IX (CA IX, a marker of tumor hypoxia), and infiltration of tumorigenic immune cells. To alleviate the drug resistance, we have used Sorafenib (Sor) in combination with tumor hypoxia directed nanoparticle (NP) loaded with a new class of apoptosis inducer, CFM 4.16 (C4.16), namely CA IX-C4.16. The NP is designed to selectively deliver the payload to the hypoxic tumor (core), provoke superior cell death in parental (WT) and Everolimus-resistant (Evr-res) RCC and selectively downmodulate tumorigenic M2-macrophage. Copper-free 'click' chemistry was utilized for conjugating SMA-TPGS with Acetazolamide (ATZ, a CA IX-specific targeting ligand). The NP was further tagged with a clinically approved NIR dye (S0456) for evaluating hypoxic tumor core penetration and organ distribution. Imaging of tumor spheroid treated with NIR dye-labeled CA IX-SMA-TPGS revealed remarkable tumor core penetration that was modulated by CA IX-mediated targeting in hypoxic-A498 RCC cells. The significant cell killing effect with synergistic combination index (CI) of CA IX-C4.16 and Sor treatment suggests efficient reversal of Evr-resistance in A498â¯cells. The CA IX directed nanoplatform in combination with Sor has shown multiple benefits in overcoming drug resistance through (i) inhibition of p-AKT, (ii) upregulation of tumoricidal M1 macrophages resulting in induction of caspase 3/7 mediated apoptosis of Evr-res A498â¯cells in macrophage-RCC co-culturing condition, (iii) significant in vitro and in vivo Evr-res A498 tumor growth inhibition as compared to individual therapy, and (iv) untraceable liver and kidney toxicity in mice. Near-infrared (NIR) imaging of CA IX-SMA-TPGS-S0456 in Evr-res A498 RCC model exhibited significant accumulation of CA IX-oligomer in tumor core with >3-fold higher tumor uptake as compared to control. In conclusion, this proof-of-concept study demonstrates versatile tumor hypoxia directed nanoplatform that can work in synergy with existing drugs for reversing drug-resistance in RCC accompanied with re-education of tumor-associated macrophages, that could be applied universally for several hypoxic tumors.
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Acetazolamida/química , Carcinoma de Células Renales/terapia , Portadores de Fármacos/química , Resistencia a Antineoplásicos , Neoplasias Renales/terapia , Nanopartículas/química , Hipoxia Tumoral/fisiología , Animales , Antineoplásicos/farmacología , Apoptosis , Anhidrasa Carbónica IX/antagonistas & inhibidores , Anhidrasa Carbónica IX/metabolismo , Hipoxia de la Célula , Línea Celular Tumoral , Supervivencia Celular , Reprogramación Celular , Terapia Combinada , Everolimus/farmacología , Femenino , Humanos , Macrófagos/metabolismo , Ratones , Ratones Desnudos , Nanopartículas/metabolismo , Permeabilidad , Prueba de Estudio Conceptual , Sorafenib/farmacología , Nanomedicina TeranósticaRESUMEN
Non-small cell lung cancers (NSCLC) account for 85% of all lung cancers, and the epidermal growth factor receptor (EGFR) is highly expressed or activated in many NSCLC that permit use of EGFR tyrosine kinase inhibitors (TKIs) as frontline therapies. Resistance to EGFR TKIs eventually develops that necessitates development of improved and effective therapeutics. CARP-1/CCAR1 is an effector of apoptosis by Doxorubicin, Etoposide, or Gefitinib, while CARP-1 functional mimetic (CFM) compounds bind with CARP-1, and stimulate CARP-1 expression and apoptosis. To test whether CFMs would inhibit TKI-resistant NSCLCs, we first generated and characterized TKI-resistant NSCLC cells. The GI 50 dose of Erlotinib for parental and Erlotinib-resistant HCC827 cells was â¼0.1 µM and ≥15 µM, respectively. While Rociletinib or Ocimertinib inhibited the parental H1975 cells with GI 50 doses of ≤0.18 µM, the Ocimertinib-resistant pools of H1975 cells had a GI50 dose of â¼12 µM. The GI50 dose for Rociletinib-resistant H1975 sublines ranged from 4.5-8.0 µM. CFM-4 and its novel analog CFM-4.16 attenuated growth of the parental and TKI-resistant NSCLC cells. CFMs activated p38/JNKs, inhibited oncogenic cMet and Akt kinases, while CARP-1 depletion blocked NSCLC cell growth inhibition by CFM-4.16 or Erlotinib. CFM-4.16 was synergistic with B-Raf-targeting in NSCLC, triple-negative breast cancer, and renal cancer cells. A nano-lipid formulation (NLF) of CFM-4.16 in combination with Sorafenib elicited a superior growth inhibition of xenografted tumors derived from Rociletinib-resistant H1975 NSCLC cells in part by stimulating CARP-1 and apoptosis. These findings support therapeutic potential of CFM-4.16 together with B-Raf targeting in treatment of TKI-resistant NSCLCs.
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Abnormal tryptophan metabolism via the kynurenine pathway is involved in the pathophysiology of a variety of human diseases including cancers. α-11C-methyl-l-tryptophan (11C-AMT) PET imaging demonstrated increased tryptophan uptake and trapping in epileptic foci and brain tumors, but the short half-life of 11C limits its widespread clinical application. Recent in vitro studies suggested that the novel radiotracer 1-(2-18F-fluoroethyl)-l-tryptophan (18F-FETrp) may be useful to assess tryptophan metabolism via the kynurenine pathway. In this study, we tested in vivo organ and tumor uptake and kinetics of 18F-FETrp in patient-derived xenograft mouse models and compared them with 11C-AMT uptake. METHODS: Xenograft mouse models of glioblastoma and metastatic brain tumors (from lung and breast cancer) were developed by subcutaneous implantation of patient tumor fragments. Dynamic PET scans with 18F-FETrp and 11C-AMT were obtained for mice bearing human brain tumors 1-7 d apart. The biodistribution and tumoral SUVs for both tracers were compared. RESULTS: 18F-FETrp showed prominent uptake in the pancreas and no bone uptake, whereas 11C-AMT showed higher uptake in the kidneys. Both tracers showed uptake in the xenograft tumors, with a plateau of approximately 30 min after injection; however, 18F-FETrp showed higher tumoral SUV than 11C-AMT in all 3 tumor types tested. The radiation dosimetry for 18F-FETrp determined from the mouse data compared favorably with the clinical 18F-FDG PET tracer. CONCLUSION: 18F-FETrp tumoral uptake, biodistribution, and radiation dosimetry data provide strong preclinical evidence that this new radiotracer warrants further studies that may lead to a broadly applicable molecular imaging tool to examine abnormal tryptophan metabolism in human tumors.
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Neoplasias Encefálicas/diagnóstico por imagen , Neoplasias Encefálicas/metabolismo , Imagen Molecular/métodos , Tomografía de Emisión de Positrones/métodos , Triptófano/farmacocinética , Tirosina/análogos & derivados , Animales , Biomarcadores de Tumor/metabolismo , Radioisótopos de Carbono/farmacocinética , Línea Celular Tumoral , Femenino , Humanos , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Especificidad de Órganos , Radiofármacos/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Distribución Tisular , Tirosina/farmacocinéticaRESUMEN
Current treatments for Renal Cell Carcinoma (RCC) include a combination of surgery, targeted therapy, and immunotherapy. Emergence of resistant RCCs contributes to failure of drugs and poor prognosis, and thus warrants development of new and improved treatment options for RCCs. Here we generated and characterized RCC cells that are resistant to Everolimus, a frontline mToR-targeted therapy, and tested whether our novel class of CARP-1 functional mimetic (CFM) compounds inhibit parental and Everolimus-resistant RCC cells. CFMs inhibited RCC cell viability in a dose-dependent manner that was comparable to Everolimus treatments. The GI50 dose of Everolimus for parental A498 cells was â¼1.2µM while it was <0.02µM for the parental UOK262 and UOK268 cells. The GI50 dose for Everolimus-resistant A498, UOK262, and UOK268 cells were ≥10.0µM, 1.8-7.0µM, and 7.0-≥10.0µM, respectively. CFM-4 and its novel analog CFM-4.16 inhibited viabilities of Everolimus resistant RCC cells albeit CFM-4.16 was more effective than CFM-4. CFM-dependent loss of RCC cell viabilities was due in part to reduced cyclin B1 levels, activation of pro-apoptotic, stress-activated protein kinases (SAPKs), and apoptosis. CFM-4.16 suppressed growth of resistant RCC cells in three-dimensional suspension cultures. However, CFMs are hydrophobic and their intravenous administration and dose escalation for in-vivo studies remain challenging. In this study, we encapsulated CFM-4.16 in Vitamin-E TPGS-based- nanomicelles that resulted in its water-soluble formulation with higher CFM-4.16 loading (30% w/w). This CFM-4.16 formulation inhibited viability of parental and Everolimus-resistant RCC cells in vitro, and suppressed growth of parental A498 RCC-cell-derived xenografts in part by stimulating apoptosis. These findings portent promising therapeutic potential of CFM-4.16 for treatment of RCCs.
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Preterm birth (PTB) is a leading cause of neonatal death worldwide. Intrauterine and systemic infection and inflammation cause 30-40% of spontaneous preterm labor (PTL), which precedes PTB. Although antibody production is a major immune defense mechanism against infection, and B cell dysfunction has been implicated in pregnancy complications associated with PTL, the functions of B cells in pregnancy are not well known. We found that choriodecidua of women undergoing spontaneous PTL harbored functionally altered B cell populations. B cell-deficient mice were markedly more susceptible than wild-type (WT) mice to PTL after inflammation, but B cells conferred interleukin (IL)-10-independent protection against PTL. B cell deficiency in mice resulted in a lower uterine level of active progesterone-induced blocking factor 1 (PIBF1), and therapeutic administration of PIBF1 mitigated PTL and uterine inflammation in B cell-deficient mice. B cells are a significant producer of PIBF1 in human choriodecidua and mouse uterus in late gestation. PIBF1 expression by B cells is induced by the mucosal alarmin IL-33 (ref. 9). Human PTL was associated with diminished expression of the α-chain of IL-33 receptor on choriodecidual B cells and a lower level of active PIBF1 in late gestation choriodecidua. These results define a vital regulatory cascade involving IL-33, decidual B cells and PIBF1 in safeguarding term pregnancy and suggest new therapeutic approaches based on IL-33 and PIBF1 to prevent human PTL.
Asunto(s)
Linfocitos B/metabolismo , Decidua/metabolismo , Interleucina-33/metabolismo , Trabajo de Parto Prematuro/metabolismo , Proteínas Gestacionales/metabolismo , Adulto , Animales , Linfocitos B/inmunología , Western Blotting , Decidua/citología , Decidua/inmunología , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Proteína 1 Similar al Receptor de Interleucina-1/inmunología , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Interleucina-33/inmunología , Ratones , Trabajo de Parto Prematuro/inmunología , Embarazo , Proteínas Gestacionales/inmunología , Adulto JovenRESUMEN
Eu(II) -based contrast agents offer physiologically relevant, metal-based redox sensing that is unachievable with Gd(III) -based contrast agents. To evaluate the in vivo contrast enhancement of Eu(II) as a function of injection type, we performed intravenous, intraperitoneal, and subcutaneous injections in mice. Our data reveal a correlation between reported oxygen content and expected rates of diffusion with the persistence of Eu(II) -based contrast enhancement. Biodistribution studies revealed europium clearance through the liver and kidneys for intravenous and intraperitoneal injections, but no contrast enhancement was observed in organs associated with clearance. These data represent a step toward understanding the behavior of Eu(II) -based complexes in vivo. Copyright © 2016 John Wiley & Sons, Ltd.
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Medios de Contraste/farmacocinética , Europio/farmacocinética , Inyecciones/métodos , Animales , Medios de Contraste/administración & dosificación , Medios de Contraste/análisis , Europio/análisis , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Inyecciones Subcutáneas , Riñón/metabolismo , Hígado/metabolismo , Tasa de Depuración Metabólica , Ratones , Distribución TisularRESUMEN
The triple negative breast cancer (TNBCs) and non-small cell lung cancers (NSCLCs) often acquire mutations that contribute to failure of drugs in clinic and poor prognosis, thus presenting an urgent need to develop new and improved therapeutic modalities. Here we report that CARP-1 functional mimetic (CFMs) compounds 4 and 5, and 4.6, a structurally related analog of CFM-4, are potent inhibitors of TNBC and NSCLC cells in vitro. Cell growth suppression by CFM-4 and -4.6 involved interaction and elevated expression of CARP-1/CCAR1 and Death Effector Domain (DED) containing DNA binding (DEDD)2 proteins. Apoptosis by these compounds also involved activation of pro-apoptotic stress-activated kinases p38 and JNK1/2, cleavage of PARP and loss of mitotic cyclin B1. Both the CFMs inhibited abilities of NSCLC and TNBC cells to migrate, invade, and form colonies in suspension, while disrupting tubule formation by the human umbilical vein endothelial cells (HUVECs). Nano-lipid formulation of CFM-4 (CFM-4 NLF) enhanced its serum bioavailability when compared with the free CFM-4. Oral administration of CFM-4 NLF reduced weights and volume of the xenografted tumors derived from A549 NSCLC and MDA-MB-231 TNBC cells. Although no gross tissue or histological toxicities were noticed, the immuno-histochemical analysis revealed increased CARP-1 and DNA fragmentation in tumors of the CFM-4 NLF-treated animals. In conclusion, while stimulation of pro-apoptotic CARP-1 and DEDD2 expression and their binding underscore a novel mechanism of apoptosis transduction by CFM compounds, our proof-of-concept xenograft studies demonstrate therapeutic potential of CFM-4 for TNBC and NSCLC.
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Proteínas Reguladoras de la Apoptosis/administración & dosificación , Proteínas Reguladoras de la Apoptosis/farmacocinética , Proteínas de Ciclo Celular/administración & dosificación , Proteínas de Ciclo Celular/farmacocinética , Nanopartículas/administración & dosificación , Nanopartículas/química , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/síntesis química , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/química , Materiales Biomiméticos/administración & dosificación , Materiales Biomiméticos/síntesis química , Proteínas de Ciclo Celular/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Composición de Medicamentos/métodos , Femenino , Ratones , Ratones Desnudos , Ratones SCID , Nanopartículas/ultraestructura , Neoplasias Experimentales/patología , Resultado del TratamientoRESUMEN
Dithiocarbamate compound Disulfiram (DSF) that binds with copper and functions as an inhibitor of aldehyde dehydrogenase is a Food and Drug Administration approved agent for treatment of alcoholism. Copper complexed DSF (DSF-Cu) also possesses anti-tumor and chemosensitizing properties; however, its molecular mechanisms of action remain unclear. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of DSF-Cu and the molecular mechanisms involved. DSF-Cu inhibited growth of the murine as well as human MPM cells in part by increasing levels of ubiquitinated proteins. DSF-Cu exposure stimulated apoptosis in MPM cells that involved activation of stress-activated protein kinases (SAPKs) p38 and JNK1/2, caspase-3, and cleavage of poly-(ADP-ribose)-polymerase, as well as increased expression of sulfatase 1 and apoptosis transducing CARP-1/CCAR1 protein. Gene-array based analyses revealed that DSF-Cu suppressed cell growth and metastasis-promoting genes including matrix metallopeptidase 3 and 10. DSF inhibited MPM cell growth and survival by upregulating cell cycle inhibitor p27Kip1, IGFBP7, and inhibitors of NF-κB such as ABIN 1 and 2 and Inhibitory κB (IκB)α and ß proteins. DSF-Cu promoted cleavage of vimentin, as well as serine-phosphorylation and lysine-63 linked ubiquitination of podoplanin. Administration of 50 mg/kg DSF-Cu by daily i.p injections inhibited growth of murine MPM cell-derived tumors in vivo. Although podoplanin expression often correlates with metastatic disease and poor prognosis, phosphorylation of serines in cytoplasmic domain of podoplanin has recently been shown to interfere with cellular motility and migration signaling. Post-translational modification of podoplanin and cleavage of vimentin by DSF-Cu underscore a metastasis inhibitory property of this agent and together with our in vivo studies underscore its potential as an anti-MPM agent.