[Show abstract][Hide abstract] ABSTRACT: Glioblastoma multiforme (GBM) is the most aggressive type of brain tumor, characterized by excessive cell proliferation, resistance to apoptosis, and invasiveness. Due to resistance to currently available treatment options, the prognosis for patients with GBM is very dismal. The activation of gastrin-releasing peptide receptors (GRPR) stimulates GBM cell proliferation, whereas GRPR antagonists induce antiproliferative effects in in vitro and in vivo experimental models of GBM. However, the role of GRPR in regulating other aspects of GBM cell function related to tumor progression remains poorly understood, and previous studies have not used RNA interference techniques as tools to examine GRPR function in GBM. Here, we found that stable GRPR knockdown by a lentiviral vector using a short hairpin interfering RNA sequence in human A172 GBM cells resulted in increased cell size and altered cell cycle dynamics consistent with cell senescence. These changes were accompanied by increases in the content of p53, p21, and p16, activation of epidermal growth factor receptors (EGFR), and a reduction in p38 content. These results increase our understanding of GRPR regulation of GBM cells and further support that GRPR may be a relevant therapeutic target in GBM.
Full-text · Article · Jan 2016 · Molecular Neurobiology
[Show abstract][Hide abstract] ABSTRACT: Background:
Nanoliposomal irinotecan showed activity in a phase 2 study in patients with metastatic pancreatic ductal adenocarcinoma previously treated with gemcitabine-based therapies. We assessed the effect of nanoliposomal irinotecan alone or combined with fluorouracil and folinic acid in a phase 3 trial in this population.
We did a global, phase 3, randomised, open-label trial at 76 sites in 14 countries. Eligible patients with metastatic pancreatic ductal adenocarcinoma previously treated with gemcitabine-based therapy were randomly assigned (1:1) using an interactive web response system at a central location to receive either nanoliposomal irinotecan monotherapy (120 mg/m(2) every 3 weeks, equivalent to 100 mg/m(2) of irinotecan base) or fluorouracil and folinic acid. A third arm consisting of nanoliposomal irinotecan (80 mg/m(2), equivalent to 70 mg/m(2) of irinotecan base) with fluorouracil and folinic acid every 2 weeks was added later (1:1:1), in a protocol amendment. Randomisation was stratified by baseline albumin, Karnofsky performance status, and ethnic origin. Treatment was continued until disease progression or intolerable toxic effects. The primary endpoint was overall survival, assessed in the intention-to-treat population. The primary analysis was planned after 305 events. Safety was assessed in all patients who had received study drug. This trial is registered at ClinicalTrials.gov, number NCT01494506.
Between Jan 11, 2012, and Sept 11, 2013, 417 patients were randomly assigned either nanoliposomal irinotecan plus fluorouracil and folinic acid (n=117), nanoliposomal irinotecan monotherapy (n=151), or fluorouracil and folinic acid (n=149). After 313 events, median overall survival in patients assigned nanoliposomal irinotecan plus fluorouracil and folinic acid was 6·1 months (95% CI 4·8-8·9) vs 4·2 months (3·3-5·3) with fluorouracil and folinic acid (hazard ratio 0·67, 95% CI 0·49-0·92; p=0·012). Median overall survival did not differ between patients assigned nanoliposomal irinotecan monotherapy and those allocated fluorouracil and folinic acid (4·9 months [4·2-5·6] vs 4·2 months [3·6-4·9]; 0·99, 0·77-1·28; p=0·94). The grade 3 or 4 adverse events that occurred most frequently in the 117 patients assigned nanoliposomal irinotecan plus fluorouracil and folinic acid were neutropenia (32 [27%]), diarrhoea (15 [13%]), vomiting (13 [11%]), and fatigue (16 [14%]).
Nanoliposomal irinotecan in combination with fluorouracil and folinic acid extends survival with a manageable safety profile in patients with metastatic pancreatic ductal adenocarcinoma who previously received gemcitabine-based therapy. This agent represents a new treatment option for this population.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Medulloblastoma (MB) comprises four distinct molecular subgroups, and survival remains particularly poor in patients with Group 3 tumors. Mutations and copy number variations result in altered epigenetic regulation of gene expression in Group 3 MB. Histone deacetylase inhibitors (HDACi) reduce proliferation, promote cell death and neuronal differentiation, and increase sensitivity to radiation and chemotherapy in experimental MB. Bombesin receptor antagonists potentiate the antiproliferative effects of HDACi in lung cancer cells and show promise as experimental therapies for several human cancers. Here, we examined the viability of D283 cells, which belong to Group 3 MB, treated with an HDACi alone or combined with bombesin receptor antagonists.
D283 MB cells were treated with different doses of the HDACi sodium butyrate (NaB), the neuromedin B receptor (NMBR) antagonist BIM-23127, the gastrin releasing peptide receptor (GRPR) antagonist RC-3095, or combinations of NaB with each receptor antagonist. Cell viability was examined by cell counting.
NaB alone or combined with receptor antagonists reduced cell viability at all doses tested. BIM-23127 alone did not affect cell viability, whereas RC-3095 at an intermediate dose significantly increased cell number.
Although HDACi are promising agents to inhibit MB growth, the present results provide preliminary evidence that combining HDACi with bombesin receptor antagonists is not an effective strategy to improve the effects of HDACi against MB cells.
No preview · Article · Nov 2015 · Child s Nervous System
[Show abstract][Hide abstract] ABSTRACT: To evaluate the impact of CYP3A4*22 in the formation of endoxifen (EDF) and hydroxytamoxifen (HTF), under different CYP2D6 genotypic backgrounds.
178 patients were enrolled in the study. CYP2D6 and CYP3A4 genotyping and tamoxifen (TAM) and metabolites quantification were performed.
EDF concentrations were lower in poor (2.77 ng ml(-1)) and CYP2D6 intermediate metabolizers (5.84 ng ml(-1)), comparing to functional group (EM-F) (10.67 ng ml(-1), p < 0.001). HTF and TAM levels were respectively 47 and 53% higher in CYP3A4*22 carriers compared with *1/*1 patients in the whole group. Patients with impaired CYP2D6 metabolism and carriers of CYP3A4*22 had EDF levels comparable to CYP2D6 EM-F group (9.06 and 10.67 ng ml(-1), p = 0.247).
The presence of CYP3A4*22 might compensate the reduction of EDF concentrations related to CYP2D6 inactivity, especially due to increased HTF concentrations. Original submitted 19 November 2014; Revision submitted 21 January 2015.
[Show abstract][Hide abstract] ABSTRACT: In view of the large variability on therapeutic response and the multiple factors associated to tamoxifen (TAM) metabolic activation, the present study aimed to evaluate the effect of CYP2D6 and CYP3A4 phenotypes, drug interactions and vitamin D exposure on TAM metabolism in a group of breast cancer patients.
Trough blood samples were collected from 116 patients. Tamoxifen and metabolites endoxifen (EDF), N-desmethyltamoxifen (NDT) and 4-hydroxy-tamoxifen (HTF) were measured in plasma by LC-MS/MS. CYP2D6 and CYP3A4 phenotyping were obtained according to [Dextromethorphan/[Dextrophan] and [Omeprazole]/[Omeprazole Sulphone] metabolic ratios, measured by HPLC in plasma collected 3 hours after oral administration of 33 mg of dextromethorphan and 20 mg of omeprazole. Vitamin D3 was measured in plasma by HPLC-UV. Data on concomitant use of drug considered as CYP2D6 and CYP3A4 inhibitor or inducer and vitamin D supplementation were recorded.
about 20% of patients had reduced CYP2D6 metabolic activity and 7% CYP3A4 impaired metabolism. EDF levels diminished proportionally to the reduction of CYP2D6 metabolic activity (PM 2.79 ng mL, IM 5.36 ng mL and EM 10.65 ng mL, P<0.01). Median plasma levels of TAM (161.50 ng mL) and HTF (1.32 ng mL) in CYP2D6 IM/CYP3A4 PM patients were higher (P<0.05) than those from CYP2D6 IM/CYP3A4 EM patients (122.07 ng mL and 0.61 ng mL, respectively). Seasons contributed to the interpatient variability of EDF and HTF levels, summer concentrations were 24% and 42% higher compared to winter. Vitamin D3 was not associated to CYP3A4 metabolic activity, indicating that other mechanisms might be involved in the relation between TAM metabolism and vitamin D exposure.
CYP3A4 contributes to the bioactivation of TAM through formation of HTF and becomes increasingly important in case of reduced or absent CYP2D6 activity. EDF and HTF exposure were associated to seasonal variations, with considerable higher plasma concentrations during summer.
Full-text · Article · Apr 2015 · Therapeutic drug monitoring
[Show abstract][Hide abstract] ABSTRACT: RC-3095, a selective GRPR antagonist, has been shown to have anti-inflammatory properties in different models of inflammation. However, its protective effect on lungs submitted to lung ischemia-reperfusion injury has not been addressed before. Then, we administrated RC-3095 intravenously before and after lung reperfusion using an animal model of lung ischemia-reperfusion injury (LIRI) by clamping the pulmonary hilum. Twenty Wistar rats were subjected to an experimental model in four groups: SHAM, ischemia-reperfusion (IR), RC-Pre, and RC-Post. The final mean arterial pressure significantly decreased in IR and RC-Pre compared to their values before reperfusion (). The RC-Post group showed significant decrease of partial pressure of arterial oxygen at the end of the observation when compared to baseline (). Caspase-9 activity was significantly higher in the RC-Post as compared to the other groups (). No significant differences were observed in eNOS activity among the groups. The groups RC-Pre and RC-Post did not show any significant decrease in IL- () and TNF- (), as compared to IR. The histological score showed no significant differences among the groups. In conclusion, RC-3095 does not demonstrate a protective effect in our LIRI model. Additionally, its use after reperfusion seems to potentiate cell damage, stimulating apoptosis.
Full-text · Article · Mar 2015 · BioMed Research International
[Show abstract][Hide abstract] ABSTRACT: TP53 mutation is a common event in many cancers, including pancreatic adenocarcinoma, where it occurs in 50-70 % of cases. In an effort to reactivate mutant p53 protein, several new drugs are being developed, including PRIMA-1 and PRIMA-1(Met)/APR-246 (p53 reactivation and induction of massive apoptosis). PRIMA-1 has been shown to induce apoptosis in tumor cells by reactivating p53 mutants, but its effect in pancreatic cancer remains unclear. Here we investigated the effects of PRIMA-1 on cell viability, cell cycle and expression of p53-regulated proteins in PANC-1 and BxPC-3 (mutant TP53), and CAPAN-2 (wild-type TP53) pancreatic cell lines. Treatment with PRIMA-1 selectively induced apoptosis and cell cycle arrest in p53 mutant cells compared to CAPAN-2 cells. The growth suppressive effect of PRIMA-1 was markedly reduced in p53 mutant cell lines transfected with p53 siRNA, supporting the role of mutant p53 in PRIMA-1 induced cell death. Moreover, treatment with the thiol group donor N-acetylcysteine completely blocked PRIMA-1-induced apoptosis and reinforced the hypothesis that thiol modifications are important for PRIMA-1 biological activity. In combination treatments, PRIMA-1 enhanced the anti-tumor activity of several chemotherapic drugs against pancreatic cancer cells and also exhibited a pronounced synergistic effect in association with the Mdm2 inhibitor Nutlin-3. Taken together, our data indicate that PRIMA-1 induces apoptosis in p53 mutant pancreatic cancer cells by promoting the re-activation of p53 and inducing proapoptotic signaling pathways, providing in vitro evidence for a potential therapeutic approach in pancreatic cancer.
Full-text · Article · May 2014 · Investigational New Drugs
[Show abstract][Hide abstract] ABSTRACT: To develop and validate a method for determination of dextromethorphan (DMT) and dextrorphan (DTP) in plasma samples using HPLC-FL and to apply it to CYP2D6 phenotyping of a population from the south of Brazil.
Samples were prepared by hydrolysis and liquid-liquid extraction. Analysis was conducted in a reversed phase column, with isocratic elution and fluorescence detection. One hundred and forty patients being treated with tamoxifen were given 30mg of dextromethorphan and their CYP2D6 phenotypes were determined on the basis of [DMT]/[DTP] metabolic ratios in plasma samples collected after 3hours.
total chromatography running time was 12min. Precision (CV %) was below 9.7 % and accuracy was between 92.1 and 106.9 %. The lower limits of quantification were 1ngmL(-1) for DMT and 10ngmL(-1) for DTP. Mean extraction yield of analytes was 86.6 %. Mean age of patients was 55.7years. Phenotype frequencies were as follows: 7.1% poor metabolizers, 13.6% intermediate metabolizers, 77.1 % extensive metabolizers and 2.1 ultra-rapid metabolizers. Metabolic ratios for patients on strong (n=11) and weak (n=16) CYP2D6 activity inhibitors were different from each other and also different from ratios for patients not taking enzyme inhibitors (n=113).
A sensitive method for determination of dextromethorphan and its metabolite in plasma samples was developed and successfully applied, providing evidence of the impact of that CYP2D6 inhibitors have on the enzyme's metabolic capacity.
[Show abstract][Hide abstract] ABSTRACT: Research endeavours require the collaborative effort of an increasing number of individuals. International scientific collaborations are particularly important for HIV and HPV co-infection studies, since the burden of disease is rising in developing countries, but most experts and research funds are found in developed countries, where the prevalence of HIV is low. The objective of our study was to investigate patterns of international scientific collaboration in HIV and HPV research using social network analysis. Through a systematic review of the literature, we obtained epidemiological data, as well as data on countries and authors involved in co-infection studies. The collaboration network was analysed in respect to the following: centrality, density, modularity, connected components, distance, clustering and spectral clustering. We observed that for many low- and middle-income countries there were no epidemiological estimates of HPV infection of the cervix among HIV-infected individuals. Most studies found only involved researchers from the same country (64%). Studies derived from international collaborations including high-income countries and either low- or middle-income countries had on average three times larger sample sizes than those including only high-income countries or low-income countries. The high global clustering coefficient (0.9) coupled with a short average distance between researchers (4.34) suggests a "small-world phenomenon." Researchers from high-income countries seem to have higher degree centrality and tend to cluster together in densely connected communities. We found a large well-connected community, which encompasses 70% of researchers, and 49 other small isolated communities. Our findings suggest that in the field of HIV and HPV, there seems to be both room and incentives for researchers to engage in collaborations between countries of different income-level. Through international collaboration resources available to researchers in high-income countries can be efficiently used to enroll more participants in low- and middle-income countries.
[Show abstract][Hide abstract] ABSTRACT: Established fear-related memories can undergo phenomena such as extinction or reconsolidation when recalled. Extinction probably involves the creation of a new, competing memory trace that decreases fear expression, whereas reconsolidation can mediate memory maintenance, updating, or strengthening. The factors determining whether retrieval will initiate extinction, reconsolidation, or neither of these two processes include training intensity, duration of the retrieval session, and age of the memory. However, previous studies have not shown that the same behavioral protocol can be used to induce either extinction or reconsolidation and strengthening, depending on the pharmacological intervention used. Here we show that, within an experiment that leads to extinction in control rats, memory can be strengthened if rolipram, a selective inhibitor of phosphodiesterase type 4 (PDE4), is administered into the dorsal hippocampus immediately after retrieval. The memory-enhancing effect of rolipram lasted for at least 1 week, was blocked by the protein synthesis inhibitor anisomycin, and did not occur when drug administration was not paired with retrieval. These findings indicate that the behavioral outcome of memory retrieval can be pharmacologically switched from extinction to strengthening. The cAMP/protein kinase A (PKA) signaling pathway might be a crucial mechanism determining the fate of memories after recall.
Full-text · Article · Mar 2014 · Frontiers in Behavioral Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Epigenetic alterations have been increasingly implicated in glioblastoma (GBM) pathogenesis, and epigenetic modulators including histone deacetylase inhibitors (HDACis) have been investigated as candidate therapies. GBMs are proposed to contain a subpopulation of glioblastoma stem cells (GSCs) that sustain tumor progression and therapeutic resistance and can form tumorspheres in culture. Here, we investigate the effects of the HDACi trichostatin A (TSA) in U87 GBM cultures and tumorsphere-derived cells. Using approaches that include a novel method to measure tumorsphere sizes and the area covered by spheres in GBM cultures, as well as a nuclear morphometric analysis, we show that TSA reduced proliferation and colony sizes, led to G2/M arrest, induced alterations in nuclear morphology consistent with cell senescence, and increased the protein content of GFAP, but did not affect migration, in cultured human U87 GBM cells. In cells expanded in tumorsphere assays, TSA reduced sphere formation and induced neuron-like morphological changes. The expression of stemness markers in these cells was detected by reverse transcriptase polymerase chain reaction. These findings indicate that HDACis can inhibit proliferation, survival, and tumorsphere formation, and promote differentiation of U87 GBM cells, providing further evidence for the development of HDACis as potential therapeutics against GBM.
No preview · Article · Jan 2014 · Journal of Molecular Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Gastrin releasing peptide, the mammalian counterpart of the amphibian peptide, Bombesin, has been increasingly implicated in regulating normal brain function as well as in the pathogenesis of psychiatric and/or neurodevelopmental disorders. We have previously shown that the neonatal blockade of the gastrin-releasing peptide receptor (GRPr) in rats produces long-lasting consequences during central nervous system development that are commonly observed in neurodevelopmental disorders such as autism spectrum disorders. The present investigation assessed in further detail, long-term behavioral effects of neonatal GRPr blockade. During postnatal days 1 to10, male Wistar rat pups (n=5-10/litter) were injected (subcutaneously) with the GRPr antagonist, RC-3095 (1mg/kg), or a vehicle (control), twice daily. Following the drug treatment regimen, several behaviors were assessed (starting on postnatal day 14) including specific social behaviors (namely, group huddling characteristics, social interaction, and social approach), restrictive/repetitive and stereotyped behaviors (y-maze, repetitive novel objection contact task, observation for stereotypies) and anxiety/fear-related responses (open field, elevated plus maze and contextual fear conditioning). Rats treated neonatally with RC-3095 showed reduced sociability, restrictive interests, motor stereotypies and enhanced learned fear response compared to the controls (vehicle-treated rats). These behavioral abnormalities are consistent with those observed in autism spectrum disorders and provide further evidence that neonatal blockade of GRPr could potentially serve as a useful model to gain a better understanding of the underlying neurodevelopmental disruptions contributing to the expression of autism-relevant phenotypes.
No preview · Article · Jan 2014 · Behavioural brain research
[Show abstract][Hide abstract] ABSTRACT: Context: The spinocerebellar ataxia type 2 (SCA-2) is a progressive neurodegenerative disorder without specific therapy identified, and it is related to the loss of function in the cerebellum, mitochondrial dysfunction, oxidative stress and neurotoxic processes. Scientific evidence indicates that Mangifera indica L. aqueous extract (MiE) and its major constituent (mangiferin) display antioxidant, anti-inflammatory and neuroprotective actions.
Aims: To investigate the MiE and mangiferin effects on behavioral outcomes of neurological function in SCA-2 transgenic mice.
Methods: The SCA-2 transgenic mice were daily and orally administered during 12 months with MiE (10, 50, and 100 mg/kg), mangiferin (10 mg/kg) or vehicle. It was evaluated locomotion (open-field), aversive memory (inhibitory avoidance) and declarative memory (object recognition). To explore possible cellular mechanisms underlying the in vivo effects was also evaluated their effects on nerve grow factor (NGF) and tumor necrosis factor-α (TNF-α) levels in the human glioblastoma cell line U138-MG supernatant.
Results: MiE administration did not affect the object recognition memory, but mangiferin did. The natural extract improved selectively the aversive memory in SCA-2 mice, indicating that MiE can affect behavioral parameters regarding fear-related memory. MiE also induced a significant increase in supernatant levels of NGF and TNF-α in vitro in human U138-MG glioblastoma cells.
Conclusions: The results suggest that MiE enhances the aversive memory through a mechanism that might involve an increase in neurotrophin and cytokine levels. These findings constitute the basis for the use of the natural extract in the prevention/treatment of memory deficits in SCA-2.
[Show abstract][Hide abstract] ABSTRACT: Context.-Gastrin-releasing peptide receptors (GRPRs) activate mitogen-activated protein kinase signaling pathway primarily through epidermal growth factor receptor activation and are under investigation as a molecular target because they are overexpressed in several solid tumors. Objective.-To determine GRPR expression in both non-small cell lung carcinoma and small cell lung carcinoma, comparing results with clinical stages and demographic data. Design.-We analyzed the immunohistochemical expression of GRPR in 200 non-small cell lung carcinoma and 38 small cell lung carcinoma archival cases from 2004 to 2008. Results.-Non-small cell lung carcinoma cases tended to be higher GRPR expressers at a rate of 62.5% (weak, moderate, and strong expression in 41.5%, 13.5%, and 7.5%, respectively), compared with 52.62% in small cell lung carcinoma cases (weak, moderate, and strong expression in 34.21%, 15.78%, and 2.63%, respectively; P = .30). In non-small cell lung carcinoma there was a trend for higher percentages of strong expression in adenocarcinoma cases (10%; P = .67), and in patients with advanced stages (III and IV; 9.43% and 6.9%; P = .01). Conclusions.-To the best of our knowledge, this is the first study to demonstrate GRPR tissue expression in a large population of patients with lung cancer. Although GRPR expression was similar in small cell and non-small cell carcinoma, the expression was more pronounced in an advanced-stage lung cancer, particularly in adenocarcinoma cases, and may represent a potential target for the development of new treatment approaches in this population.
No preview · Article · Jan 2014 · Archives of pathology & laboratory medicine
[Show abstract][Hide abstract] ABSTRACT: Medulloblastoma is the most common malignant childhood brain tumor for which the development of new molecularly targeted therapies is needed. Novel therapeutic targets under investigation include growth factor receptors. Here, we show that the combined inhibition of the epidermal growth factor receptor (EGFR) and neuromedin B receptor (NMBR, BB1) results in increased cell death in human medulloblastoma cell lines.
DAOY and D283 human medulloblastoma cells were treated with human recombinant neuromedin B (NMB, an NMBR agonist), the NMBR antagonist BIM-23127, the anti-EGFR monoclonal antibody cetuximab, or BIM-23127 combined with cetuximab. Cell death was examined with trypan blue cell counting.
Both cell lines expressed mRNA for EGFR, NMB, and NMBR detected by reverse transcriptase polymerase chain reaction. Cetuximab at 10 μg/ml significantly reduced the number of DAOY cells, but did not affect D283 cells. NMB and BIM-23127 did not change cell number when used alone. However, cetuximab, at a dose that did not have an effect by itself, was able to reduce the number of DAOY cells when combined with BIM-23127.
These results provide preliminary evidence that NMBR blockade can potentiate the antitumor effect of anti-EGFR therapy in medulloblastoma.
Preview · Article · Oct 2013 · Child s Nervous System