Rosário Pinto-Leite

Centro Hospitalar Trás-os-Montes e Alto Douro, Vila Real, Vila Real, Portugal

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Publications (16)25.74 Total impact

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    ABSTRACT: Gemcitabine and cisplatin regimen is an approach currently used in urinary bladder cancer treatment. However, side effects’ arising from its administration is a hard concern. In this study, we evaluated different schedules of gemcitabine and cisplatin to determine the efficacy of this combination together with two mammalian targets of rapamycin (mTOR) inhibitors: temsirolimus and everolimus. The 5637, HT1376 and T24 urinary bladder cancer cell lines were exposed to gemcitabine (72 hours), cisplatin (48 hours), temsirolimus (72 hours) and everolimus (72 hours), in isolation, or in combined schedules (gemcitabine, cisplatin and temsirolimus, or gemcitabine, cisplatin and everolimus). The levels of phosphorylated p70S6 K and 4E-BP1 after treatment with temsirolimus and everolimus were investigated by immunoblotting. The antiproliferative activity, cell cycle distribution, autophagy and apoptosis were analyzed by the MTT assay and immunocytochemistry, flow cytometry, acridine orange staining and M30 CytoDEATH antibody. No significant differences in the expression of P-4E-BP1 and P-p70S6 K after temsirolimus and everolimus exposure were found in the HT1376 and T24 cell line. A statistically significant decrease of phosphorylated 4E-BP1 form was detected in the 5637 cell line (P < 0.05) after everolimus exposure. Temsirolimus and everolimus conjugated with gemcitabine and cisplatin decreased the cell proliferation in all three cell lines. This pattern of response was similar to the other parameters analyzed (reduced Ki-67 expression, increased autophagy and apoptosis). Also, in the combined regimen, an enhanced cell cycle arrest in the G0/G1 phase in the 5637 cell line and in the early S-phase in the HT1376 and T24 cell lines were observed. The muscle invasive HT1376 and T24 cell lines were the most sensitive to both combinations. The combination of gemcitabine, cisplatin and temsirolimus or everolimus yields an enhanced cytotoxicity efficacy, namely in the muscle invasive urinary bladder cancer cell lines. Although further studies are necessary to complement this data, the present results opening new perspectives in muscle invasive urinary bladder cancer treatment.
    Biomedicine & Aging Pathology. 07/2014;
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    ABSTRACT: Several genomic regions are frequently altered and associated with the type, stage and progression of urinary bladder cancer (UBC). We present the characterization of 5637, T24 and HT1376 UBC cell lines by karyotyping, fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH) and multiplex ligation-dependent probe amplification (MLPA) analysis. Some cytogenetic anomalies present in UBC were found in the three cell lines, such as chromosome 20 aneuploidy and the loss of 9p21. Some gene loci losses (e.g. CDKN2A) and gains (e.g. HRAS, BCL2L1 and PTPN1) were coincident across all cell lines. Although some significant heterogeneity and complexity were detected between them, their genomic profiles exhibited a similar pattern to UBC. We suggest that 5637 and HT1376 represent the E2F3/RB1 pathway due to amplification of 6p22.3, concomitant with loss of one copy of RB1 and mutation of the remaining copy. The HT1376 presented a 10q deletion involving PTEN region and no alteration of PIK3CA region which, in combination with the inactivation of TP53, bears more invasive and metastatic properties than 5637. The T24 belongs to the alternative pathway of FGFR3/CCND1 by presenting mutated HRAS and over-represented CCND1. These cell lines cover the more frequent subtypes of UBC and are reliable models that can be used, as a group, in preclinical studies.
    Tumor Biology 01/2014; · 2.52 Impact Factor
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    ABSTRACT: The purpose of this study was to determine the efficacy of naproxen, a nonsteroidal anti-inflammatory drug, on the MG-63 human osteosarcoma cell line. MG-63 cells were exposed to naproxen in a wide range of concentrations of 0.03, 0.05, 0.1, 0.42, 0.83, and 1.67 mg/ml for 72 h. The activity of naproxen was assessed by the following assays: cell morphology; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method; terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay; comet assay; and acridine orange and monodansylcadaverine (MDC) staining. Naproxen exerted a significant inhibitory effect on MG-63 cell proliferation, in a concentration-dependent manner, in all treatment groups compared with untreated cells. An increase in frequency of DNA damage, apoptotic bodies, apoptotic cells, and autophagic vacuoles was observed in MG-63-treated cells. Although future studies are needed, these findings suggest that naproxen may lead to improvements in treatment of patients with osteosarcoma.
    Journal of toxicology and environmental health. Part A. 01/2014; 77(14-16):916-23.
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    ABSTRACT: To analyze the cytotoxic action of temsirolimus using 3 established human bladder cancer cell lines and to assess whether temsirolimus potentiates the anticancer activity of gemcitabine and cisplatin. Temsirolimus (500, 1,000, 2,000, and 4,000nM), in isolation, and combined with gemcitabine (100nM) and cisplatin (2.5µg/ml), was given to 5637, T24, and HT1376 bladder cancer cell lines. Cell proliferation, autophagy, early apoptosis, and cell cycle distribution were analyzed after a 72-hour period. The expression of mammalian target of rapamycin baseline, Akt, and their phosphorylated forms, before and after treatment with temsirolimus, was evaluated by immunoblotting. Temsirolimus slightly decreased the bladder cancer cell proliferation in all 3 cell lines. No significant differences in the expression of mammalian target of rapamycin, Akt, and their phosphorylated forms because of temsirolimus exposure were found in the 3 cell lines. As part of a combined regime along with gemcitabine, and especially with cisplatin, there was a more pronounced antiproliferative effect. This pattern of response was similar to the other parameters analyzed (increased autophagy and apoptosis). Also, in the combined regime, an enhanced cell cycle arrest in the G0/G1 phase was observed. The non-muscle invasive 5637 bladder cancer cell line was most sensitive to both combinations. Temsirolimus makes a moderate contribution in terms of cell proliferation, apoptosis, and autophagy. However, it does potentiate the activity of gemcitabine and particularly cisplatin. Therefore, cisplatin- or gemcitabine-based chemotherapy regimen used in combination with temsirolimus to treat bladder cancer represents a novel and valuable treatment option, which should be tested for future studies in urinary bladder xenograft models.
    Urologic Oncology 09/2013; · 3.65 Impact Factor
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    ABSTRACT: Chemically-induced urinary bladder cancer in rodents has long been used as a reliable model to study the biopathology of urinary bladder neoplasia and to develop therapeutic strategies against human tumors. Knowledge of the genetic basis underlying carcinogenesis would greatly enhance usability and usefulness of this model for the purposes of comparative pathology. However, little is known about the cytogenetic characteristics of rodent urinary bladder tumors. Accordingly, pathological and negative control specimens were collected for cytogenetic evaluation, from an ongoing mouse urinary bladder N-butyl-N-(4-hydroxybutyl) nitrosamine-induced carcinogenesis study. Histopathological analysis characterized the pathological sample as a papillary urothelial carcinoma. Conventional cytogenetic analysis revealed the presence of 66.3 % tetraploid cells. Fluorescent in situ hybridization using chromosome paint probes allowed the detection of a reciprocal translocation involving chromosomes 4 and 14 (containing the murine homologues to human p16 and retinoblastoma tumor-suppressor genes) in 42 % of tetraploid cells. The control sample showed no histological or cytogenetic changes. CDKN2A and RB1 loss of heterozygosity is associated with human early and advanced urinary bladder cancer, respectively. Thus, the present data paves the way for further studies concerning the molecular mechanisms of urinary bladder carcinogenesis.
    Tumor Biology 05/2013; · 2.52 Impact Factor
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    ABSTRACT: Several drugs have shown in vitro and in vivo pharmacological activity against urinary bladder cancer. This review aims at compiling the different drugs evaluated in in vitro and in vivo models of urinary bladder cancer and to review the advantages and limitations of both types of models, as well as the different methodologies applied for evaluating antineoplastic drug activity.
    Anticancer research 04/2013; 33(4):1273-96. · 1.71 Impact Factor
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    ABSTRACT: Cisplatin (CDDP)-based chemotherapy is a commonly treatment for advanced urothelial carcinoma. However, episodes of cisplatin resistance have been referenced. Recently it has been reported that everolimus (RAD001) could have an important role to play in bladder-cancer treatment and that mTOR inhibitors may restore chemosensitivity in resistant tumours. The aim of this study was to assess RAD001 in vitro ability to enhance CDDP cytotoxicity in three human bladder-cancer cell lines. Over the course of 72h, the cells were exposed to different concentrations of CDDP and RAD001, isolated or combined. Treatment with CDDP statistically (P<0.05) decreased cell proliferation in cell lines in a dose-dependent manner. The anti-proliferative activity of CDDP used in combination with RAD001 was statistically significant (P<0.05) in the cell lines at all concentrations tested. RAD001 had a therapeutic effect when used in combination with CDDP and could therefore be a useful anti-cancer drug combination for patients with bladder cancer.
    Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 03/2013; 67(2):116-21. · 2.24 Impact Factor
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    ABSTRACT: To assess the efficacy of meloxicam, a non-steroidal anti-inflammatory drug (NSAID), on three human urinary bladder-cancer cell lines (HT1376, T24 and 5637) and on mice urinary bladder cancer chemically induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). The in vitro effects of meloxicam were assessed by optical microscopy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method, flow cytometry and comet assay. In vivo, Hsd:ICR male mice were exposed to BBN in drinking water, over the course of 12 weeks. Subsequently, animals were treated with meloxicam by intraperitoneal route, for 6 consecutively weeks. Tumour development was evaluated by haematoxylin and eosin staining. Renal and hepatic functions, interleucin-6 (IL-6), C-reactive protein (CRP) and tumour necrosis factor (TNFα) were also evaluated. In vitro, meloxicam induced a significant (P<0.05) decrease of cell proliferation. A significant (P<0.05) cell cycle arrest on G0/G1 phase was also detected in all the cell lines, with a slight but significant increase of sub-G0/G1 fraction on T24 (P=0.006) and 5637 (P<0.001) cells. Also a significant (P<0.05) increase in DNA damage was found on meloxicam-treated cells. In vivo, the incidence of pre-neoplastic lesions induced by BBN was not affected by meloxicam treatment. However, although not statistically significant, the development of neoplastic lesions was inhibited by meloxicam treatment without significant alterations of renal or hepatic parameters. Meloxicam is effective on in vitro and in vivo models of urinary bladder cancer. These findings support that meloxicam deserves more attention on urinary bladder cancer study.
    Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie 02/2013; · 2.24 Impact Factor
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    ABSTRACT: The aim of this paper is to analyse sunitinib malate in vitro ability to enhance cisplatin cytotoxicity in T24, 5637, and HT1376 human urinary bladder-cancer cell lines. Cells were treated with cisplatin (3, 6, 13, and 18 μ M) and sunitinib malate (1, 2, 4, 6, and 20 μ M), either in isolation or combined, over the course of 72 hours. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, acridine orange, and monodansylcadaverine staining and flow cytometry were performed. The combination index (CI) was calculated based on the Chou and Talalay method. In isolation, cisplatin and sunitinib malate statistically (P < 0.05) decrease cell viability in all cell lines in a dose-dependent manner, with the presence of autophagic vacuoles. A cell cycle arrest in early S-phase and in G0/G1-phase was also found after exposure to cisplatin and sunitinib malate, in isolation, respectively. Treatment of urinary bladder-cancer cells with a combination of cisplatin and sunitinib malate showed a synergistic effect (CI < 1). Autophagy and apoptosis studies showed a greater incidence when the combined treatment was put into use. This hints at the possibility of a new combined therapeutic approach. If confirmed in vivo, this conjugation may provide a means of new perspectives in muscle-invasive urinary bladder cancer treatment.
    BioMed research international. 01/2013; 2013:791406.
  • Journal of applied biomedicine 01/2013; 11(2):79-92. · 0.98 Impact Factor
  • Journal of applied biomedicine 01/2013; 11:79-92. · 0.98 Impact Factor
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    ABSTRACT: The purpose of this study was to determine whether everolimus, a rapamycin derivative, might significantly enhance the cytotoxicity of gemcitabine, an antitumor drug, in two human bladder-cancer cell lines. Human bladder-cancer T24 and 5637 cells were incubated with gemcitabine and everolimus in a range of concentrations either alone or in combination for 72 h. Flow cytometry, comet assay, MTT method and optical microscopy were used to assess cell proliferation, cell cycle, DNA damage, and morphological alterations. Gemcitabine exerted an inhibitory effect on T24 and 5637 cell proliferation, in a concentration-dependent manner. Everolimus significantly reduced proliferation of 5637 bladder cancer cells (IC₃₀) at 1 μM), whereas T24 demonstrated marked resistance to everolimus treatment. A significant antiproliferative effect was obtained combining gemcitabine (100 nM) with everolimus (0.05-2 μM) with an arrest of cell cycle at S phase. Furthermore, an increase in frequency of DNA damage, apoptotic bodies, and apoptotic cells was observed when T24 and 5637 cancer cells were treated simultaneously with both drugs. Data show that in vitro combination produced a more potent antiproliferative effect when compared with single drugs.
    Journal of Toxicology and Environmental Health Part A 07/2012; 75(13-15):788-99. · 1.73 Impact Factor
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    ABSTRACT: Oral gavage is a widely used method for administering substances to animals in pharmacological and toxicological studies. The authors evaluated whether oral gavage causes behavioral indicators of stress, increased mortality rate, alterations in food and water consumption and body weight or histological lesions in CD-1 mice. Gavage was carried out once per d for 5 d per week over 6 consecutive weeks. The mortality rate of mice in this study was 15%. Mice subjected to gavage did not undergo changes in food or water consumption during the study, and their mean body weights and relative organ weights were similar to those of mice in the control group. Serum cortisol levels at the time of euthanasia in mice in both groups were within the normal range. Histopathology showed acute esophagitis and pleurisy, indicative of perforation of the esophagus, in the two mice that died but no abnormalities in the other mice. The results suggest that animal stress and mortality related to oral gavage can be minimized when the procedure is carried out by an experienced technician.
    Lab Animal 05/2012; 41(5):129-34. · 0.47 Impact Factor
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    ABSTRACT: OBJECTIVE: To evaluate the influence of Everolimus (RAD001) on chemically induced urothelial lesions in mice and its influence on in vitro human bladder cancer cell lines. METHODS: ICR male mice were given N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water for a period of 12 weeks. Subsequently, RAD001 was administered via oral gavage, for 6 weeks. At the end of the experiment, all the animals were sacrificed and tumor development was determined by means of histopathologic evaluation; mammalian target of rapamycin (mTOR) expressivity was evaluated by immunohistochemistry. Three human bladder cancer cell lines (T24, HT1376, and 5637) were treated using a range of RAD001 concentrations. MTT assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cytometry were used to assess cell proliferation, apoptosis index, and cell cycle analysis, respectively. Immunoblotting analysis of 3 cell line extracts using mTOR and Akt antibodies was performed in order to study the expression of Akt and mTOR proteins and their phosphorylated forms. RESULTS: The incidence of urothelial lesions in animals treated with RAD001 was similar to those animals not treated. RAD001 did not block T24 and HT1376 cell proliferation or induce apoptosis. A reduction in cell proliferation rate and therefore G(0)/G(1) phase arrest, as well as a statistically significant induction of apoptosis (P = 0.001), was only observed in the 5637 cell line. CONCLUSION: RAD001 seems not to have a significant effect on chemically induced murine bladder tumors. The effect of RAD001 on tumor proliferation and apoptosis was achieved only in superficial derived bladder cancer cell line, no effect was observed in invasive cell lines.
    Urologic Oncology 12/2011; · 3.65 Impact Factor
  • BMC proceedings 01/2010; 4(Suppl 2).
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    ABSTRACT: Sirolimus is recently reported to have antitumour effects on a large variety of cancers. The present study was performed to investigate sirolimus's ability to inhibit growth in T24 bladder cancer cells. T24 bladder cancer cells were treated with various concentrations of sirolimus. MTT assay was used to evaluate the proliferation inhibitory effect on T24 cell line. The viability of T24 cell line was determined by Trypan blue exclusion analysis. Sirolimus inhibits the growth of bladder carcinoma cells and decreases their viability. Significant correlations were found between cell proliferation and sirolimus concentration (r = 0.830; p < 0.01) as well as between cell viability and sirolimus concentration (r = -0.896; p < 0.01). Sirolimus has an anti-proliferation effect on the T24 bladder carcinoma cell line. The information from our results is useful for a better understanding sirolimus's anti-proliferative activity in the T24 bladder cancer cell line.
    Journal of Experimental & Clinical Cancer Research 01/2009; 28:3. · 3.07 Impact Factor