RESUMEN
Sulfur mustard (SM), a vesicating agent first used during World War I, remains a potent threat as a chemical weapon to cause intentional/accidental chemical emergencies. Eyes are extremely susceptible to SM toxicity. Nitrogen mustard (NM), a bifunctional alkylating agent and potent analog of SM, is used in laboratories to study mustard vesicant-induced ocular toxicity. Previously, we showed that SM-/NM-induced injuries (in vivo and ex vivo rabbit corneas) are reversed upon treatment with dexamethasone (DEX), a US Food and Drug Administration-approved, steroidal anti-inflammatory drug. Here, we optimized NM injuries in ex vivo human corneas and assessed DEX efficacy. For injury optimization, one cornea (randomly selected from paired eyes) was exposed to NM: 100 nmoles for 2 hours or 4 hours, and 200 nmoles for 2 hours, and the other cornea served as a control. Injuries were assessed 24 hours post NM-exposure. NM 100 nmoles exposure for 2 hours was found to cause optimal corneal injury (epithelial thinning [â¼69%]; epithelial-stromal separation [6-fold increase]). In protein arrays studies, 24 proteins displayed ≥40% change in their expression in NM exposed corneas compared with controls. DEX administration initiated 2 hours post NM exposure and every 8 hours thereafter until 24 hours post-exposure reversed NM-induced corneal epithelial-stromal separation [2-fold decrease]). Of the 24 proteins dysregulated upon NM exposure, six proteins (delta-like canonical Notch ligand 1, FGFbasic, CD54, CCL7, endostatin, receptor tyrosine-protein kinase erbB-4) associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, showed significant reversal upon DEX treatment (Student's t test; P ≤ 0.05). Complementing our animal model studies, DEX was shown to mitigate vesicant-induced toxicities in ex vivo human corneas. SIGNIFICANCE STATEMENT: Nitrogen mustard (NM) exposure-induced injuries were optimized in an ex vivo human cornea culture model and studies were carried out at 24 h post 100 nmoles NM exposure. Dexamethasone (DEX) administration (started 2 h post NM exposure and every 8 h thereafter) reversed NM-induced corneal injuries. Molecular mediators of DEX action were associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, indicating DEX aids wound healing via reversing vesicant-induced neovascularization (delta-like canonical Notch ligand 1 and FGF basic) and leukocyte infiltration (CD54 and CCL7).
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Sustancias para la Guerra Química , Lesiones de la Cornea , Gas Mostaza , Animales , Humanos , Conejos , Mecloretamina/toxicidad , Irritantes/efectos adversos , Sustancias para la Guerra Química/toxicidad , Ligandos , Córnea , Lesiones de la Cornea/inducido químicamente , Lesiones de la Cornea/tratamiento farmacológico , Lesiones de la Cornea/metabolismo , Gas Mostaza/toxicidad , Dexametasona/farmacología , Dexametasona/uso terapéuticoRESUMEN
Sulfur mustard (SM) is an ominous chemical warfare agent. Eyes are extremely susceptible to SM toxicity; injuries include inflammation, fibrosis, neovascularization (NV), and vision impairment/blindness, depending on the exposure dosage. Effective countermeasures against ocular SM toxicity remain elusive and are warranted during conflicts/terrorist activities and accidental exposures. We previously determined that dexamethasone (DEX) effectively counters corneal nitrogen mustard toxicity and that the 2-hour postexposure therapeutic window is most beneficial. Here, the efficacy of two DEX dosing frequencies [i.e., every 8 or 12 hours (initiated, as previously established, 2 hours after exposure)] until 28 days after SM exposure was assessed. Furthermore, sustained effects of DEX treatments were observed up to day 56 after SM exposure. Corneal clinical assessments (thickness, opacity, ulceration, and NV) were performed at the day 14, 28, 42, and 56 post-SM exposure time points. Histopathological assessments of corneal injuries (corneal thickness, epithelial degradation, epithelial-stromal separation, inflammatory cell, and blood vessel counts) using H&E staining and molecular assessments (COX-2, MMP-9, VEGF, and SPARC expressions) were performed at days 28, 42, and 56 after SM exposure. Statistical significance was assessed using two-way ANOVA, with Holm-Sidak post hoc pairwise multiple comparisons; significance was established if P < 0.05 (data represented as the mean ± S.E.M.). DEX administration every 8 hours was more potent than every 12 hours in reversing ocular SM injury, with the most pronounced effects observed at days 28 and 42 after SM exposure. These comprehensive results are novel and provide a comprehensive DEX treatment regimen (therapeutic-window and dosing-frequency) for counteracting SM-induced corneal injuries. SIGNIFICANCE STATEMENT: The study aims to establish a dexamethasone (DEX) treatment regimen by comparing the efficacy of DEX administration at 12 versus 8 hours initiated 2 hours after exposure. DEX administration every 8 hours was more effective in reversing sulfur mustard (SM)-induced corneal injuries. SM injury reversal during DEX administration (initial 28 days after exposure) and sustained [further 28 days after cessation of DEX administration (i.e., up to 56 days after exposure)] effects were assessed using clinical, pathophysiological, and molecular biomarkers.
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Sustancias para la Guerra Química , Lesiones de la Cornea , Gas Mostaza , Animales , Conejos , Gas Mostaza/toxicidad , Gas Mostaza/metabolismo , Córnea , Sustancias para la Guerra Química/toxicidad , Lesiones de la Cornea/metabolismo , Lesiones de la Cornea/patología , Dexametasona/farmacologíaRESUMEN
Recent preclinical studies have shown that the intake of nonsteroidal anti-inflammatory drugs (NSAIDs) aspirin and naproxen could be an effective intervention strategy against TMPRSS2-ERG fusion-driven prostate tumorigenesis. Herein, as a follow-up mechanistic study, employing TMPRSS2-ERG (fusion) positive tumors and plasma from TMPRSS2-ERG. Ptenflox/flox mice, we profiled the stage specific proteomic changes (focused on inflammatory circulating and prostate tissue/tumor-specific cytokines, chemokines, and growth factors/growth signaling-associated molecules) that contribute to prostate cancer (PCa) growth and progression in the TMPRSS2-ERG fusion-driven mouse model of tumorigenesis. In addition, the association of the protective effects of NSAIDs (aspirin 1400 ppm and naproxen 400 ppm) with the modulation of these specific molecular pathways was determined. A sandwich Elisa based membrane array-proteome profiler identifying 111 distinct signaling molecules was employed. Overall, the plasma and prostate tissue sample analyses identified 54 significant and differentially expressed cytokines, chemokines, and growth factors/growth signaling-associated molecules between PCa afflicted mice (TMPRSS2-ERG. Ptenflox/flox, age-matched noncancerous controls, NSAIDs-supplemented and no-drug controls). Bioinformatic analysis of the array outcomes indicated that the protective effect of NSAIDs was associated with reduced expression of (a) tumor promoting inflammatory molecules (M-CSF, IL-33, CCL22, CCL12, CX3CL1, CHI3L1, and CD93), (b) growth factors- growth signaling-associated molecules (Chemerin, FGF acidic, Flt-3 ligand, IGFBP-5, and PEDF), and (c) tumor microenvironment/stromal remodeling proteins MMP2 and MMP9. Overall, our findings corroborate the pathological findings that protective effects of NSAIDs in TMPSS2-ERG fusion-driven prostate tumorigenesis are associated with antiproliferative and anti-inflammatory effects and possible modulation of the immune cell enriched microenvironment.
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Antiinflamatorios no Esteroideos , Aspirina , Naproxeno , Neoplasias de la Próstata , Animales , Masculino , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Antiinflamatorios no Esteroideos/farmacología , Aspirina/farmacología , Ratones , Naproxeno/farmacología , Proteómica/métodos , Inflamación/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Próstata/patología , Próstata/metabolismo , Próstata/efectos de los fármacos , Carcinogénesis/efectos de los fármacos , Carcinogénesis/genética , Carcinogénesis/metabolismo , Proteoma/metabolismo , Humanos , Citocinas/metabolismo , Citocinas/sangreRESUMEN
PURPOSE: Sulfur mustard (SM), a bi-functional alkylating agent, was used during World War I and the Iran-Iraq war. SM toxicity is ten times higher in eyes than in other tissues. Cornea is exceptionally susceptible to SM-injuries due to its anterior positioning and mucous-aqueous interphase. Ocular SM exposure induces blepharitis, photosensitivity, dry eye, epithelial defects, limbal ischemia and stem cell deficiency, and mustard gas keratopathy leading to temporary or permanent vision impairments. We demonstrated that dexamethasone (Dex) is a potent therapeutic intervention against SM-induced corneal injuries; however, its mechanism of action is not well known. Investigations employing proteomic profiling (LC-MS/MS) to understand molecular mechanisms behind SM-induced corneal injury and Dex efficacy were performed in the rabbit cornea exposed to SM and then received Dex treatment. PEAKS studio was used to extract, search, and summarize peptide identity. Ingenuity Pathway Analysis was used for pathway identification. Validation was performed using immunofluorescence. One-Way ANOVA (FDR < 0.05; p < 0.005) and Student's t-test (p < 0.05) were utilized for analyzing proteomics and IF data, respectively. Proteomic analysis revealed that SM-exposure upregulated tissue repair pathways, particularly actin cytoskeleton signaling and inflammation. Prominently dysregulated proteins included lipocalin2, coronin1A, actin-related protein2, actin-related protein2/3 complex subunit2, actin-related protein2/3 complex subunit4, cell division cycle42, ezrin, bradykinin/kininogen1, moesin, and profilin. Upregulated actin cytoskeleton signaling increases F-actin formation, dysregulating cell shape and motility. Dex reversed SM-induced increases in the aforementioned proteins levels to near control expression profiles. Dex aids corneal wound healing and improves corneal integrity via actin cytoskeletal signaling and anti-inflammatory effects following SM-induced injuries.
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Sustancias para la Guerra Química , Lesiones de la Cornea , Gas Mostaza , Animales , Conejos , Gas Mostaza/toxicidad , Sustancias para la Guerra Química/toxicidad , Mediadores de Inflamación/metabolismo , Actinas/metabolismo , Cromatografía Liquida , Proteómica , Espectrometría de Masas en Tándem , Córnea/metabolismo , Lesiones de la Cornea/inducido químicamente , Lesiones de la Cornea/tratamiento farmacológico , Citoesqueleto de Actina/metabolismo , Dexametasona/efectos adversosRESUMEN
Lewisite (LEW) is an arsenical vesicant that can be a potentially dangerous chemical warfare agent (CWA). Eyes are particularly susceptible to vesicant induced injuries and ocular LEW exposure can act swiftly, causing burning of eyes, edema, inflammation, cell death and even blindness. In our previous studies, we developed a LEW exposure-induced corneal injury model in rabbit and showed increased inflammation, neovascularization, cell death, and structural damage to rabbit corneas upon LEW exposure. In the present study, we further assessed the metabolomic changes to delineate the possible mechanisms underlying the LEW-induced corneal injuries. This information is vital and could help in the development of effective targeted therapies against ocular LEW injuries. Thus, the metabolomic changes associated with LEW exposures in rabbit corneas were assessed as a function of time, to delineate pathways from molecular perturbations at the genomic and proteomic levels. New Zealand white rabbit corneas (n = 3-6) were exposed to LEW vapor (0.2 mg/L; flow rate: 300 ml/min) for 2.5 min (short exposure; low dose) or 7.5 min (long-exposure; high dose) and then collected at 1, 3, 7, or 14 days post LEW exposure. Samples were prepared using the automated MicroLab STAR® system, and proteins precipitated to recover the chemically diverse metabolites. Metabolomic analysis was carried out by reverse phase UPLC-MS/MS and gas chromatography (GC)-MS. The data obtained were analyzed using Metabolon's software. The results showed that LEW exposures at high doses were more toxic, particularly at the day 7 post exposure time point. LEW exposure was shown to dysregulate metabolites associated with all the integral functions of the cornea and cause increased inflammation and immune response, as well as generate oxidative stress. Additionally, all important metabolic functions of the cells were also affected: lipid and nucleotide metabolism, and energetics. The high dose LEW exposures were more toxic, particularly at day 7 post LEW exposure (>10-fold increased levels of histamine, quinolinate, N-acetyl-ß-alanine, GMP, and UPM). LEW exposure dysregulated integral functions of the cornea, caused inflammation and heightened immune response, and generated oxidative stress. Lipid and nucleotide metabolism, and energetics were also affected. The novel information about altered metabolic profile of rabbit cornea following LEW exposure could assist in delineating complex molecular events; thus, aid in identifying therapeutic targets to effectively ameliorate ocular trauma.
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Arsenicales , Lesiones de la Cornea , Animales , Conejos , Irritantes/efectos adversos , Irritantes/metabolismo , Cromatografía Liquida , Proteómica , Espectrometría de Masas en Tándem , Córnea/metabolismo , Lesiones de la Cornea/inducido químicamente , Lesiones de la Cornea/metabolismo , Arsenicales/efectos adversos , Arsenicales/metabolismo , Inflamación/metabolismo , Nucleótidos/efectos adversos , Nucleótidos/metabolismo , LípidosRESUMEN
The factors (environmental and genetic) contributing to basal cell carcinoma (BCC) pathogenesis are well-established; however, effective agents for BCC prevention are marred by toxic side-effects. Herein, we assessed the efficacy of flavonolignan silibinin against ultraviolet B (UVB)-induced BCC in Ptch+/- (heterozygous patched homolog 1 gene) mouse model. Both male and female Ptch+/- mice were irradiated with a 240 mJ/cm2 UVB dose 3 times/week for 26 or 46 weeks, with or without topical application of silibinin (9 mg/200 µl in acetone, applied 30 min before or after UVB exposure). Results indicated that silibinin application either pre- or post-UVB exposure for 26 weeks significantly decreased the number of BCC lesions by 65% and 39% (P < 0.001 for both) and the area covered by BCCs (72% and 45%, P < 0.001 for both), respectively, compared to UVB alone. Furthermore, continuous UVB exposure for 46 weeks increased the BCC lesion number and the BCC area covered by ~6 and ~3.4 folds (P < 0.001), respectively. Notably, even in this 46 week prolonged UVB exposure, silibinin (irrespective of pre- or post-UVB treatment) significantly halted the growth of BCCs by 81-94% (P < 0.001) as well as other epidermal lesions; specifically, silibinin treated tissues had less epidermal dysplasia, fibrosarcoma, and squamous cell carcinoma. Immunohistochemistry and immunofluorescence studies revealed that silibinin significantly decreased basal cell proliferation (Ki-67) and the expression of cytokeratins (14 and 15), and Hedgehog signaling mediators Smo and Gli1 in the BCC lesions. Together, our findings demonstrate strong potential of silibinin to be efficacious in preventing the growth and progression of UVB-induced BCC.
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Carcinoma Basocelular , Neoplasias Cutáneas , Animales , Carcinoma Basocelular/tratamiento farmacológico , Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/prevención & control , Femenino , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Masculino , Ratones , Receptor Patched-1/genética , Silibina/farmacología , Silibina/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/prevención & control , Rayos Ultravioleta/efectos adversosRESUMEN
In the present study, we performed a comparative stage-specific pathological and molecular marker evaluation of TMPRSS2-ERG fusion and PTEN loss-driven (TMPRSS2-ERG. Ptenflox/flox ) versus non-fusion-driven prostate tumorigenesis (Hi-Myc) in mice. Anterior, ventral, and dorsolateral prostates were collected from mice at different ages (or time points post-Cre induction). Results indicated that growth and progression of prostatic intraepithelial lesions to adenocarcinoma stages occurred in both mice models albeit at different rates. In the TMPRSS2-ERG. Ptenflox/flox mice, the initiation of tumorigenesis was slow, but subsequent progression through different stages became increasingly faster. Adenocarcinoma stage was reached early on; however, no high-grade undifferentiated tumors were observed. Conversely, in the Hi-Myc+/- mice, tumorigenesis initiation was rapid; however, progression through different stages was relatively slower and it took a while to reach the more aggressive phenotype stage. Nevertheless, at the advanced stages in the Hi-Myc+/- mice, high-grade undifferentiated tumors were observed compared to the later stage tumors observed in the fusion-driven TMPRSS2-ERG. Ptenflox/flox mice. These results were corroborated by the stage specific-pattern in the molecular expression of proliferation markers (PCNA and c-Myc); androgen receptor (AR); fusion-resultant overexpression of ERG; Prostein (SLC45-A3); and angiogenesis marker (CD-31). Importantly, there was a significant increase in immune cell infiltrations, which increased with the stage of tumorigenesis, in the TMPRSS2-ERG fusion-positive tumors relative to fusion negative tumors. Together, these findings are both novel and highly significant in establishing a working preclinical model for evaluating the efficacy of interventions during different stages of tumorigenesis in TMPRSS2-ERG fusion-driven PCa.
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Adenocarcinoma , Neoplasias de la Próstata , Adenocarcinoma/genética , Animales , Carcinogénesis/patología , Humanos , Masculino , Ratones , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Próstata/patología , Neoplasias de la Próstata/patología , Serina Endopeptidasas/metabolismo , Regulador Transcripcional ERG/genética , Regulador Transcripcional ERG/metabolismoRESUMEN
Nitrogen mustard (NM) is an analogue of the potent vesicating agent sulfur mustard, with well-established ocular injury models in rabbit eyes to study vesicant-induced ocular toxicity. The effects of NM-exposure to eyes may include irritation, redness, inflammation, fibrosis, epithelial degradation, blurred vision, partial/complete blindness, which may be temporary or permanent, depending on the route, duration, and dosage of exposure. Effective countermeasures against vesicant exposure are presently not available and are warranted in case of any terrorist activity or accidental leakage from stockpiles. Herein, our focus was to evaluate whether dexamethasone (DEX), an FDA approved potent corticosteroid with documented anti-inflammatory activities, could be an effective treatment modality. Accordingly, utilizing NM-induced corneal injuries in rabbit ocular in vivo model, we examined and compared the efficacy of DEX treatments when administration was started at early (2 h), intermediate (4 h), and late (6 h) therapeutic windows of intervention after NM-exposure and administered every 8 h thereafter. The effects of NM-exposure and DEX treatments were evaluated on clinical (corneal opacity, ulceration, and neovascularization), biological (epithelial thickness, epithelial-stromal separation, blood vessels density, and inflammatory cell and keratocyte counts) and molecular (COX-2 and VEGF expression) parameters, at day 1, 3, 7 and 14. Results indicated that DEX treatment markedly and effectively reversed the NM-induced injury markers in rabbit corneas. Early administration of DEX at 2 h was found to be most effective in reversing NM-induced corneal injuries, followed by DEX 4 h and DEX 6 h administration initiation, indicating that DEX has best efficacy at the early therapeutic window in our study model.
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Antiinflamatorios/uso terapéutico , Lesiones de la Cornea/inducido químicamente , Lesiones de la Cornea/tratamiento farmacológico , Dexametasona/uso terapéutico , Mecloretamina/toxicidad , Animales , Biomarcadores , Irritantes/toxicidad , Masculino , ConejosRESUMEN
Curcumin is a bioactive natural compound with a wide range of pharmacological properties, including antitumor activity; however, its clinical application has been limited because of its low solubility, stability, and bioavailability. In this study, a solid phase approach was proposed for the combinatorial synthesis of a mini library of the mimics of curcumin in good purity and yield. The non-effective findings in pancreatic cancer cells switched to strong growth inhibition and cell death efficacy for PC3 prostate cancer cells, and mimic 9, in which tyrosol (TYR) and homovanillyl alcohol (HVA) units were linked by a phosphodiester bond, was quite effective not only in cell growth inhibition but also in causing strong cell death under the study conditions and treatments that were not effective in PANC1 cells. The results got more exciting when we also consider the findings in SW480 human colorectal carcinoma cell line, where the growth inhibitor effects were more in line with that of the PC3 cells, but the lack of cell death effect was more in line with the PANC1 cells.
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Antineoplásicos/farmacología , Curcumina/farmacología , Técnicas de Síntesis en Fase Sólida , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Curcumina/síntesis química , Curcumina/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Approximately, 30 000 men die from prostate cancer (PCa) every year in the United States, mainly due to the metastasis. Thus, the key events associated with PCa metastasis are under rigorous investigation, with recent studies showing that preparation of pre-metastatic niches (PMN) in distant organs is an important step. However, the molecular basis for PMN preparation is still unclear. Hypoxia in primary tumors promotes aggressiveness; however, its precise role in metastasis is not clear. We recently reported that exosomes secreted by PCa cells under hypoxia promote stemness and invasiveness in naïve PCa cells; however, whether these extracellular vesicles also influence PMN remains unknown. In the present study, we isolated exosomes from human PCa PC3 cells under normoxic (21% O2 , exosomes secreted under normoxic condition [ExoNormoxic ]) and hypoxic (1% O2 , exosomes secreted under hypoxic condition [ExoHypoxic ]) conditions, and characterized their effect (10 µg exosomes, intraperitoneal (IP) treatment every 48 hours for 4 weeks) on key biomarkers associated with PMN in nude mice. Whole animal fluorescence imaging showed that ExoHypoxic treatment promotes matrix metalloproteinases (MMPs) activity in several putative metastatic sites. Histological studies confirmed that ExoHypoxic treatment enhanced the level of MMP2, MMP9, and extracellular matrix proteins (fibronectin and collagen) as well as increased the number of CD11b+ cells at selective PMN sites. Furthermore, proteomic profiling of exosomes by liquid chromatography/mass spectrometry identified cargo proteins in ExoNormoxic and ExoHypoxic as well as distinct canonical pathways targeted by them. These results suggest that exosomes secreted by PCa cells under hypoxia plausibly remodel distant PMN, and thus, could be a potential target to control metastatic PCa.
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Exosomas/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Activación Enzimática , Exosomas/patología , Humanos , Masculino , Ratones Desnudos , Metástasis de la Neoplasia/patología , Células PC-3 , Próstata/citología , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/patología , Hipoxia TumoralRESUMEN
Chemoresistance to gemcitabine (GEM)-a frontline chemotherapeutic, resulting from its dysfunctional uptake and metabolism in cancer cells, is a major contributing factor for failed therapy in pancreatic cancer (PanC) patients. Therefore, there is an urgent need for agents that could reverse GEM resistance and allow continued chemosensitivity to the drug. We employed natural nontoxic agent (with anti-PanC potential) bitter melon juice (BMJ) and GEM to examine their combinatorial benefits against tumorigenesis of PanC patient-derived xenograft (PDX)-pancreatic ductal adenocarcinomas explants PDX272 (wild-type KRAS), PDX271 (mutant KRAS and SMAD4), and PDX266 (mutant KRAS). Anti-PanC efficacy of single agents vs combination in the three tumor explants, both at the end of active dosing regimen and following a drug-washout phase were compared. In animal studies, GEM alone treatment significantly inhibited PDX tumor growth, but effects were not sustained, as GEM-treated tumors exhibited regrowth posttreatment termination. However, combination-regimen displayed enhanced and sustained efficacy. Mechanistic assessments revealed that overcoming GEM resistance by coadministration with BMJ was possibly due to modulation of GEM transport/metabolism pathway molecules (ribonucleotide reductase regulatory subunit M1, human equilibrative nucleoside transporter 1, and deoxycytidine kinase). Study outcomes, highlighting significantly higher and sustained efficacy of GEM in combination with BMJ, make a compelling case for a clinical trial in PanC patients, wherein BMJ could be combined with GEM to target and overcome GEM resistance. In addition, given their specific effectiveness against KRAS-mutant tumors, this combination could be potentially beneficial to a broader PanC patient population.
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Carcinoma Ductal Pancreático/tratamiento farmacológico , Desoxicitidina/análogos & derivados , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Momordica charantia/química , Neoplasias Pancreáticas/tratamiento farmacológico , Extractos Vegetales/farmacología , Animales , Antimetabolitos Antineoplásicos/farmacología , Apoptosis , Carcinoma Ductal Pancreático/patología , Proliferación Celular , Desoxicitidina/farmacología , Femenino , Humanos , Ratones , Ratones Desnudos , Invasividad Neoplásica , Neoplasias Pancreáticas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
The established role of bitter melon juice (BMJ), a natural product, in activating master metabolic regulator adenosine monophosphate-activated protein kinase in pancreatic cancer (PanC) cells served as a basis for pursuing deeper investigation into the underlying metabolic alterations leading to BMJ efficacy in PanC. We investigated the comparative metabolic profiles of PanC cells with differential KRAS mutational status on BMJ exposure. Specifically, we employed nuclear magnetic resonance (NMR) metabolomics and in vivo imaging platforms to understand the relevance of altered metabolism in PanC management by BMJ. Multinuclear NMR metabolomics was performed, as a function of time, post-BMJ treatment followed by partial least square discriminant analysis assessments on the quantitative metabolic data sets to visualize the treatment group clustering; altered glucose uptake, lactate export and energy state were identified as the key components responsible for cell death induction. We next employed PANC1 xenograft model for assessing in vivo BMJ efficacy against PanC. Positron emission tomography ([18FDG]-PET) and magnetic resonance imaging on PANC1 tumor-bearing animals reiterated the in vitro results, with BMJ-associated significant changes in tumor volumes, tumor cellularity and glucose uptake. Additional studies in BMJ-treated PanC cells and xenografts displayed a strong decrease in the expression of glucose and lactate transporters GLUT1 and MCT4, respectively, supporting their role in metabolic changes by BMJ. Collectively, these results highlight BMJ-induced modification in PanC metabolomics phenotype and establish primarily lactate efflux and glucose metabolism, specifically GLUT1 and MCT4 transporters, as the potential metabolic targets underlying BMJ efficacy in PanC.
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Around 80% of nonmelanoma skin cancers (NMSCs) are basal cell carcinoma (BCC), still studies evaluating the efficacy of chemopreventive agents during early stage/s of BCC development are lacking. Accordingly, utilizing the well-established patched (Ptch)+/- mouse model of ultraviolet B (UVB) radiation-induced BCC formation, we excised skin samples from UVB exposed Ptch+/- and Ptch+/+ mice before tumor formation to study the promotion/progression of BCC and to determine the efficacy and target/s of silibinin, a well-known skin cancer chemopreventive agent. UVB exposure for 1 month increased the number of mast cells in Ptch+/- mice by ~48% (P < 0.05), which was completely inhibited by silibinin. Polymerase chain reaction profiler array analysis of skin samples showed strong molecular differences between Ptch+/+ and Ptch+/- mice which were either unexposed or UVB irradiated+/- silibinin treatment. Most notably, silibinin treatment significant decreased the expression of BMP-2, Bbc3, PUMA, and Ccnd1 in Ptch+/- mice irradiated with silibinin + UVB. Additional studies showed that silibinin targets UVB-induced expression of bone morphogenetic protein 2 (BMP-2) in Ptch+/- mouse skin. Last, our studies found that silibinin strongly attenuates UVB-induced BMP-2 expression and DNA damage in Ptch+/- mouse skin ex vivo only after single UVB exposure. Together, our results suggest a possible role of mast cell recruitment and BMP-2 activation in the early stages of BCC development; these are strongly inhibited by silibinin suggesting its possible chemopreventive efficacy against BCC formation in long-term UVB exposure regimen.
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Antineoplásicos Fitogénicos/farmacología , Proteína Morfogenética Ósea 2/biosíntesis , Carcinoma Basocelular/tratamiento farmacológico , Mastocitos/efectos de la radiación , Receptor Patched-1/genética , Silibina/farmacología , Neoplasias Cutáneas/tratamiento farmacológico , Rayos Ultravioleta/efectos adversos , Animales , Proteínas Reguladoras de la Apoptosis/biosíntesis , Carcinoma Basocelular/patología , Quimioprevención , Ciclina D1/biosíntesis , Daño del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Mastocitos/patología , Ratones , Ratones Transgénicos , Transducción de Señal , Piel/patología , Neoplasias Cutáneas/patología , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
The present study aimed to determine whether grape seed extract (GSE) procyanidin mix, and its active constituent procyanidin B2 3,3â³-di-O-gallate (B2G2) have the potential to target cancer stem cells (CSCs) in prostate cancer (PCa). The CSC populations were isolated and purified based on CD44+ -α2ß1high surface markers in PCa cell lines LNCaP, C4-2B, 22Rv1, PC3, and DU145, and then subjected to prostasphere formation assays in the absence or presence of GSE or B2G2. Results indicated that at lower doses (<15 µg) , the GSE procyanidin mix produced activity in unsorted prostate cancer antigen (PCA) cells, but not in sorted; however, multiple treatments with low dose GSE over a course of time inhibited sphere formation by sorted PCA CSCs. Importantly, B2G2 demonstrated significant potential to target both unsorted and sorted CSCs at lower doses. As formation of spheroids, under specific in vitro conditions, is a measure of stemness, these results indicated the potential of both GSE and B2G2 to target the self-renewal of CSC in PCa cell lines, though B2G2 was more potent in its efficacy. Subsequent mechanistic studies revealed that both GSE procyanidins and B2G2 strongly decreased the constitutive as well as Jagged1 (Notch1 ligand)-induced activated Notch1 pathway. In totality, these in vitro studies warrant extensive dose-profiling-based assessments in vivo settings to conclusively determine the impact on CSC pool kinetics on the efficacy of both GSE and B2G2 to target PCa growth as well as tumor relapse.
Asunto(s)
Antocianinas/farmacología , Antineoplásicos Fitogénicos/farmacología , Biflavonoides/farmacología , Catequina/farmacología , Extracto de Semillas de Uva/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Proantocianidinas/farmacología , Neoplasias de la Próstata/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Proteína Jagged-1/metabolismo , Masculino , Células Madre Neoplásicas/patología , Células PC-3 , Próstata/patología , Neoplasias de la Próstata/patología , Receptor Notch1/metabolismo , Esferoides Celulares/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Neoplastic cells exhibit higher oxidative stress compared to normal cells; however, antioxidants based clinical trials have mostly failed. Another attractive therapeutic approach is to further increase the oxidative stress in cancer cells leading to cell death. Herein, we show that Procyanidin B2 3,3â³-di-O-gallate (B2G2), the most active constituent of grape seed extract, treatment causes cell death in human prostate cancer (PCa) cells (LNCaP and 22Rv1) via increasing the reactive oxygen species (ROS) generation. Mechanistically, B2G2 treatment decreased the mitochondrial electron transport chain complex III activity leading to enhanced mitochondrial superoxide generation and decreased ATP production in LNCaP cells. Additional molecular studies revealed that B2G2-induced cell death was mediated mainly through ROS-induced sustained activation of ERK1/2, which was due to inhibition of MAP kinase phosphatase (MKP) activity as over-expression of MKP3 in LNCaP cells conferred significant protection against B2G2-induced cell death. Along with ERK1/2, AMP-activated protein kinase α (AMPKα) was also activated by B2G2 treatment, and pre-treatment with AMPKα inhibitor compound C significantly reversed the cytotoxic effects of B2G2 in LNCaP cells. Furthermore, pre-treatment of MKP3 over-expressing LNCaP cells with compound C further reduced the B2G2-induced cell death, suggesting the involvement of AMPKα along with MKP3 and ERK1/2 in the biological effects of B2G2. Together, these results for the first time identified that oxidative stress and MKP3 inhibition play a critical role in B2G2-induced cell death in PCa cells through sustained activation of both ERK1/2 and AMPKα. These results offer a unique opportunity to control this deadly malignancy through B2G2 use.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Antocianinas/farmacología , Fosfatasa 6 de Especificidad Dual/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Immunoblotting , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Pancreatic cancer (PanC) is one of the deadliest malignancies worldwide and frontline treatment with gemcitabine becomes eventually ineffective due to increasing PanC resistance, suggesting additional approaches are needed to manage PanC. Recently, we have shown the efficacy of bitter melon juice (BMJ) against PanC cells, including those resistant to gemcitabine. As cancer stem cells (CSCs) are actively involved in PanC initiation, progression, relapse and drug-resistance, here we assessed BMJ ability in targeting pancreatic cancer-associated cancer stem cells (PanC-CSCs). We found BMJ efficacy against CD44+ /CD24+ /EpCAMhigh enriched PanC-CSCs in spheroid assays; BMJ also increased the sensitivity of gemcitabine-resistant PanC-CSCs. Exogenous addition of BMJ to PanC-CSC generated spheroids (not pre-exposed to BMJ) also significantly reduced spheroid number and size. Mechanistically, BMJ effects were associated with a decrease in the expression of genes and proteins involved in PanC-CSC renewal and proliferation. Specifically, immunofluorescence staining showed that BMJ decreases protein expression/nuclear localization of CSC-associated transcription factors SOX2, OCT4 and NANOG, and CSC marker CD44. Immunohistochemical analysis of MiaPaCa2 xenografts from BMJ treated animals also showed a significant decrease in the levels of CSC-associated transcription factors. Together, these results show BMJ potential in targeting PanC-CSC pool and associated regulatory pathways, suggesting the need for further investigation of its efficacy against PanC growth and progression including gemcitabine-resistant PanC.
Asunto(s)
Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Momordica charantia/química , Células Madre Neoplásicas/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Antígeno CD24/análisis , Línea Celular Tumoral , Molécula de Adhesión Celular Epitelial/análisis , Jugos de Frutas y Vegetales/análisis , Humanos , Receptores de Hialuranos/análisis , Ratones , Células Madre Neoplásicas/patología , Neoplasias Pancreáticas/patologíaRESUMEN
New silibinin phosphodiester glyco-conjugates were synthesized by efficient phosphoramidite chemistry and were fully characterized by 2D-NMR. A wide-ranging study focused on the determination of their pKa and E° values as well as on their radical scavenging activities by different assays (DPPH, ABTS+ and HRSA) was conducted. The new glyco-conjugates are more water-soluble than silibinin, and their radical scavenging activities are higher than those of silibinin. The conjugation therefore improves both the water solubilities and antioxidant activities of the flavonolignan moieties. The serum stability was evaluated under physiological conditions, and the glyco-conjugates degraded with half-lives of 40-70â¯h, making them useful in pro-drug approaches. We started by treating androgen-dependent prostate cancer (PCa) LNCaP cells and then expanded our studies to androgen-independent PCa PC3 and DU145 cells. In most cases, the new derivatives significantly reduced both total and live cell numbers, albeit at different levels. Anti-HIV activities were evaluated and the glucosamine-phosphate silibinin derivative showed higher activity (IC50â¯=â¯73⯵M) than silibinin.
Asunto(s)
Antivirales/farmacología , Glicoconjugados/farmacología , VIH/efectos de los fármacos , Organofosfatos/farmacología , Silibina/farmacología , Antivirales/síntesis química , Antivirales/química , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glicoconjugados/síntesis química , Glicoconjugados/química , Humanos , Estructura Molecular , Organofosfatos/química , Oxidación-Reducción , Células PC-3 , Silibina/química , Relación Estructura-ActividadRESUMEN
Non-melanoma skin cancers (NMSC) are a growing problem given that solar ultraviolet B (UVB) radiation exposure is increasing most likely due to depletion of the atmospheric ozone layer and lack of adequate sun protection. Better preventive methods are urgently required to reduce UV-caused photodamage and NMSC incidence. Earlier, we have reported that silibinin treatment activates p53 and reduces photodamage and NMSC, both in vitro and in vivo; but whether silibinin exerts its protective effects primarily through p53 remains unknown. To address this question, we generated p53 heterozygous (p53+/-) and p53 knockout (p53-/-) mice on SKH-1 hairless mouse background, and assessed silibinin efficacy in both short- and long-term UVB exposure experiments. In the chronic UVB-exposed skin tumorigenesis study, compared to p53+/+ mice, p53+/- mice developed skin tumors earlier and had higher tumor number, multiplicity and volume. Silibinin topical treatment significantly reduced the tumor number, multiplicity and volume in p53+/+ mice but silibinin' protective efficacy was significantly compromised in p53+/- mice. Additionally, silibinin treatment failed to inhibit precursor skin cancer lesions in p53-/- mice but improved the survival of the mice. In short-term studies, silibinin application accelerated the removal of UVB-induced DNA damage in p53+/+ mice while its efficacy was partially compromised in p53-/- mice. Interestingly, silibinin treatment also inhibited the UVB-induced inflammatory markers in skin tissue. These results further confirmed that absence of the p53 allele predisposes mice to photodamage and photocarcinogenesis, and established that silibinin mediates its protection against UVB-induced photodamage, inflammation and photocarcinogenesis partly through p53 activation.
Asunto(s)
Transformación Celular Neoplásica/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Inflamación/prevención & control , Silimarina/farmacología , Neoplasias Cutáneas/prevención & control , Proteína p53 Supresora de Tumor/fisiología , Rayos Ultravioleta/efectos adversos , Animales , Antioxidantes/farmacología , Transformación Celular Neoplásica/patología , Transformación Celular Neoplásica/efectos de la radiación , Daño del ADN/efectos de la radiación , Femenino , Inflamación/etiología , Inflamación/patología , Masculino , Ratones , Ratones Pelados , Ratones Endogámicos C57BL , Ratones Noqueados , Silibina , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/patologíaRESUMEN
Hypoxia is associated with aggressive phenotype and poor prognosis in prostate cancer (PCa) patients suggesting that PCa growth and progression could be controlled via targeting hypoxia-induced signaling and biological effects. Here, we analyzed silibinin (a natural flavonoid) efficacy to target cell growth, angiogenesis, and metabolic changes in human PCa, LNCaP, and 22Rv1 cells under hypoxic condition. Silibinin treatment inhibited the proliferation, clonogenicity, and endothelial cells tube formation by hypoxic (1% O2 ) PCa cells. Interestingly, hypoxia promoted a lipogenic phenotype in PCa cells via activating acetyl-Co A carboxylase (ACC) and fatty acid synthase (FASN) that was inhibited by silibinin treatment. Importantly, silibinin treatment strongly decreased hypoxia-induced HIF-1α expression in PCa cells together with a strong reduction in hypoxia-induced NADPH oxidase (NOX) activity. HIF-1α overexpression in LNCaP cells significantly increased the lipid accumulation and NOX activity; however, silibinin treatment reduced HIF-1α expression, lipid levels, clonogenicity, and NOX activity even in HIF-1α overexpressing LNCaP cells. In vivo, silibinin feeding (200 mg/kg body weight) to male nude mice with 22Rv1 tumors, specifically inhibited tumor vascularity (measured by dynamic contrast-enhanced MRI) resulting in tumor growth inhibition without directly inducing necrosis (as revealed by diffusion-weighted MRI). Silibinin feeding did not significantly affect tumor glucose uptake measured by FDG-PET; however, reduced the lipid synthesis measured by quantitative 1 H-NMR metabolomics. IHC analyses of tumor tissues confirmed that silibinin feeding decreased proliferation and angiogenesis as well as reduced HIF-1α, FASN, and ACC levels. Together, these findings further support silibinin usefulness against PCa through inhibiting hypoxia-induced signaling. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Antineoplásicos/administración & dosificación , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lipogénesis/efectos de los fármacos , Metabolómica/métodos , Neovascularización Patológica/tratamiento farmacológico , Neoplasias de la Próstata/tratamiento farmacológico , Silimarina/administración & dosificación , Animales , Antineoplásicos/farmacología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Masculino , Ratones , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Transducción de Señal/efectos de los fármacos , Silibina , Silimarina/farmacología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor microenvironment plays an essential role in prostate carcinogenesis and offers novel opportunities to prevent and treat prostate cancer (PCA). Here, we investigated the ability of cancer-associated fibroblasts (CAFs) to promote PCA progression, and silibinin efficacy to target this response. We collected conditioned media from CAFs treated with vehicle or silibinin, and labeled as control conditioned media (CCM) or silibinin-treatment conditioned media (SBCM), respectively. Next, we characterized the effect of CCM and SBCM treatment in several PCA cell lines (RWPE-1, WPE-1 NA-22, WPE-1 NB-14 and PC3). Result showed that compared with SBCM, CCM significantly reduces E-cadherin expression and increases invasiveness and clonogenicity in PCA cells. Further molecular studies identified monocyte chemotactic protein-1 (MCP-1) as the key component of CCM that promotes PCA invasiveness, whereas silibinin treatment strongly reduced MCP-1 expression in CAFs by inhibiting the DNA-binding activity of MCP-1 transcriptional regulators-nuclear factor-kappaB and AP-1. In vivo, silibinin feeding (200mg/kg body weight) strongly reduced TRAMPC1 allografts growth (by 68%) in syngeneic C57Bl/6 mice. TRAMPC1 tumor analysis showed that silibinin reduced MCP-1 and CAFs' biomarkers (fibroblast activation protein, α-smooth muscle actin, transforming growth factor beta 2, vimentin etc.) and significantly modulated the recruitment of immune cells in the tumor microenvironment. Similar inhibitory effects of silibinin on MCP-1 and immune cells recruitment were also observed in TRAMP PCA tissues with reported silibinin efficacy. Overall, our data suggest that silibinin can target CAF-mediated invasiveness in PCA by inhibiting MCP-1 secretion. This, in turn, was associated with a reduction in immune cell recruitment in vivo along with a marked reduction in tumor growth.