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Treatment of muscle invasive urinary bladders tumors: A potential role of the mTOR inhibitors
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Original article
Treatment of muscle invasive urinary bladders tumors: A potential role of the mTOR inhibitors Rosário Pinto-Leite a , Regina Arantes-Rodrigues b , Rita Ferreira c , Carlos Palmeira d,e , Paula A. Oliveira b,∗ , Lúcio Santos d,e a
Genetic Service, Cytogenetic Laboratory, Hospital Center of Trás-os-Montes and Alto Douro, Vila Real, Portugal Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trás-os-Montes and Alto Douro, Vila Real, Portugal c Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal d Experimental Pathology and Therapeutics Group, Portuguese Institute of Oncology, Porto, Portugal e Health School, University Fernando Pessoa, Porto, Portugal b
a r t i c l e
i n f o
Article history: Received 5 March 2014 Accepted 11 March 2014 Available online xxx Keywords: Gemcitabine Cisplatin Temsirolimus Everolimus Combined antineoplastic chemotherapy Cancer cell lines
a b s t r a c t 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. © 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction Urinary bladder cancer accounts for 90–95% of urothelial carcinomas, being the most frequent malignancy of the urinary system [1,2]. Although the great majority of patients have non-muscle invasive lesions, 20 to 40% either present or later develops muscle invasive tumors [3,4]. Much has changed in the diagnosis and
∗ Corresponding author. Tel.: +35 19 66 47 30 62; fax: +35 12 59 35 04 80. E-mail addresses: [email protected], [email protected], [email protected] (P.A. Oliveira).
management of urinary bladder cancer over the past 5 to 10 years [3]. The standard chemotherapy for the treatment of muscle invasive and metastatic urinary bladder cancer are platinum-based regimens, particularly cisplatin [5,6]. The gemcitabine and cisplatin combination, as well as the methotrexate, vinblastine, adriamycin and cisplatin (MVAC) regimen are commonly used [7]. The combination of gemcitabine and cisplatin has been widely adopted as a platform for antineoplastic chemotherapy in the treatment of muscle invasive urinary bladder cancer, with similar and even superior results in regard to survival and a better toxicity profile compared with the standard MVAC [8,9]. However, this approach has severe side effects (which include ototoxicity, gastrotoxicity,
http://dx.doi.org/10.1016/j.biomag.2014.03.003 2210-5220/© 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Pinto-Leite R, et al. Treatment of muscle invasive urinary bladders tumors: A potential role of the mTOR inhibitors. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.003
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myelosuppression, allergic reactions, and specially nephrotoxicity) and chemoresistance [10]. The response rates remains in the 50% to 70% and the median survival is of only about 13 months [3]. The need for new treatment strategies has led to the research and evaluation of novel drugs that target novel cellular pathways. The mammalian target of rapamycin (mTOR) is frequently deregulated in human cancers, and several studies suggest that it may be involved in the urinary bladder cancer development [11,12]. mTOR is an important pathway controlling cell growth, survival and proliferation [13–15]. Rapamycin and its rapalogs (temsirolimus and everolimus) are well-recognized mTOR inhibitors. Their efficacy in urinary bladder cancer cell lines has already been evaluated as single drugs or in combination with cisplatin or gemcitabine, with promising results [16–18]. Cell lines are important tools in cancer research, their appropriate genomic characterization is essential in preclinical studies. We described similar genetic alterations to ones described by the Cancer Genome Atlas Research Network (2014) [19] in the 5637, HT1376 and T24 cancer cell lines [20] what makes these cell lines a valuable tool to evaluate the efficacy of antineoplastic drugs. The purpose of the present study was to analyze the effects of combining two mTOR inhibitors, temsirolimus and everolimus, with cisplatin and gemcitabine, at lower concentrations, using three urinary bladder cancer cell lines representative of human urinary bladder cancer. 2. Material and methods 2.1. Cell lines and culture conditions The study was performed on a non-muscle invasive urinary bladder cancer cell line (5637) and two muscle invasive urinary bladder cancer cell lines: T24 and HT1376. T24 cell line was provided by DSMZ, Düsseldorf, Germany; 5637 and HT1376 cell lines were kindly provided by Dr. Paula Videira of the Universidade Nova de Lisboa, Lisboa, Portugal. All the cell lines were maintained in RPMI 1640 culture medium (PAA, Pasching, Austria), supplemented with 10% heat-inactivated fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), 100 U/mL penicillin (Biological Industries, Kibbutz Beit Haemek, Israel), 100 g/mL streptomycin (Biological Industries, Kibbutz Beit Haemek, Israel) and 2 mM Lglutamine (Sigma Aldrich, St. Louis, USA). Cells were cultured as a monolayer at 37 ◦ C in 5% CO2 in a humidified atmosphere. 2.2. Drugs treatment To assess dose-response profiles by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, the three urinary bladder cancer cell lines were treated with the four drugs with different times of exposure. In isolation the cells were exposed for 24, 48 and 72 hours to 0.5 M temsirolimus (Sigma Aldrich, Portugal), 0.05 M everolimus (Sigma, St. Louis, USA), 0.1 M gemcitabine (Hospira, Portugal) and 3 M cisplatin (Teva Pharma, Portugal). In the gemcitabine and cisplatin combined schedules, cells were exposed to gemcitabine (72 hours) and cisplatin (24, 48 or 72 hours). In the gemcitabine, cisplatin and temsirolimus; and gemcitabine, cisplatin and everolimus combinations the cells were exposed to gemcitabine (72 hours), cisplatin (48 hours) and it was added temsirolimus or everolimus for 24, 48 or 72 hours. The remaining assays (immunocytochemistry, flow cytometry, acridine orange staining and M30 CytoDEATH antibody) were performed with the combination of gemcitabine (72 hours), cisplatin (48 hours) and temsirolimus (72 hours) or everolimus (72 hours). Controls samples were treated similarly but without the drugs. All the drugs were freshly prepared before each experiment. In each
experiment, triplicate wells were performed for each drug and the assay was repeated in three independent experiments. 2.3. Immunoblotting In order to evaluate the effect of temsirolimus and everolimus on mTOR signaling, the levels of phosphorylated mTOR downstream targets ribosomal protein S kinase (p70S6 K) and 4E-binding protein 1 (4E-BP1) were evaluated. In this sense, whole cell extracts were obtained from mechanical homogenization of cell pellets (with no treatment, with 0.5 M temsirolimus and with 0.05 M everolimus) in phosphate buffer with 1% SDS containing phosphatase inhibitors (P0044 and P5726, Sigma). Protein concentration was assayed by the colorimetric method “RC DC protein assay” (Bio-Rad) using bovine serum albumin (BSA) as protein standard. Samples from each cell line were then diluted in Tris buffered saline (TBS; 100 mM Tris, 1.5 mM NaCl, pH 8.0) to obtain a final protein concentration of 0.4 mg/mL and a volume of 100 l was slot-blotted into a nitrocellulose membrane (Whatman, Protein). Nonspecific binding was blocked with 5% (w/v) BSA in TBS-T (TBS with 0.5% Tween 20) for 1 hour and the membrane was then incubated with primary antibody (1:1000 dilution; rabbit monoclonal phospho-4E-BP1 (Thr37/46) or #2855; phospho-p70 S6 K (Thr389), #9234from Cell Signaling). After 1 hour incubation, the membrane was washed with TBS-T and incubated with anti-rabbit IgG peroxidase secondary antibody (1:1000; Amersham Pharmacia Biotech). Immunoreactivity was detected with enhanced chemiluminescence reagents (ECL, Amersham Pharmacia Biotech) according to the manufacturer’s instructions and images were recorded using X-ray films (Kodak Biomax Light Film, Sigma). Films were scanned in Molecular Imager Gel Doc XR+ System (Bio-Rad) and analyzed with Quantity One software version 4.6.3 (Bio-Rad, Hercules, SA). Protein loading control of blotting membranes was performed by staining with Ponceau S. 2.4. MTT assay The MTT assay was used to assess the relative percentage of metabolically active cells compared to untreated cells. In brief, trypsinized tumor cells were resuspended in a medium at 2-3 × 104 cells/mL based on the growth characteristics of each cell line. One hundred l of cells suspension was seeded in a 96-well culture plate (Sarstedt, USA) and plates were incubated for 24 hours to allow adherent cell growth. After overnight incubation, the medium was removed and 100 l of the different reagent solutions in medium were distributed in each well. After 72 hours incubation, 10 l of MTT (Sigma Aldrich, EUA) dye working solution (5 mg/mL was added to each well and 4 hours later, the supernatant in the wells was removed and replaced by 100 l/well of dimethylsulfoxide (Sigma Aldrich, USA). The absorbance (A) values of each well was recorded at 492 nm on an automatic Elisa plate reader (Multiskan EX. Labsystems). Cell proliferation was calculated as: Aexp group /Acontrol X 100 [21]. 2.5. Immunocytochemistry Immunocytochemistry assay was performed in order to determine the Ki-67 expression, a nuclear protein that is expressed in proliferating cells [22]. Cells were plated in 24-well chamber slides (Sarstedt, USA) and incubated overnight. Drugs were applied in isolation or combined. After being washed with PBS, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed three times in an isotonic PBS buffer. Permeabilization was carried out using 1% Triton X-100 in PBS for 20 minutes at room temperature and internal peroxidase activity was blocked with 3% hydrogen peroxide for 30 minutes. The primary anti-Ki-67 antibody
Please cite this article in press as: Pinto-Leite R, et al. Treatment of muscle invasive urinary bladders tumors: A potential role of the mTOR inhibitors. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.003
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(MIB-1, 1:100) was incubated for 1 hour at room temperature. After incubation of the secondary antibody, positive immunoreactivity was visible by means of the reaction of 3,3-diaminobenzidine chromogen. The samples were washed with water and contrasted with haematoxylin. Negative controls were performed by replacing primary antibody with PBS [16]. Samples were analyzed with a Leica microscope.
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correlation (linear dependence) of the cell cycle and drugs concentration. P values of less than 0.05 were considered to be statistically significant. 3. Results 3.1. Modulation of mTOR pathway by temsirolimus and everolimus
2.6. Flow cytometry Flow cytometry was carried out in order to analyse the distribution of cells by the different cell cycle phases. Cells (1 × 106 cells/mL) were plated in 6-well plates and incubated for 24 hours. Then drugs, in isolation or combined were added to the cells. After 72 hours of incubation, cells were trypsinized, washed with PBS and fixed in ice-cold ethanol 70% for at least 1 hour [23]. Propidium iodide (CycloscopeTM DNA Cytometry, Cytognos) was added in order to label total cellular DNA and cell cycle analysis was carried out using a Coulter® EPICSTM XL-MCLTM flow cytometer. DNA-content histograms were analysed with Modfit LT 3.0 software (VerityTM ) to determine the percentage of cells in each cell cycle phase: G0 /G1 , S and G2 /M. Each independent experiment was performed in triplicate. 2.7. Acridine orange staining The production of acidic vesicular organelles is associated with autophagy. Acridine orange stains the cytoplasm and nucleus of the stained cells by fluoresces bright green, while acidic compartments (including acidic vesicular organelles) fluoresces bright red. Urinary bladder cancer cells were seeded on glass coverslip (8 mm) and incubated overnight. After drug exposure the medium was removed and cells were stained with acridine orange (1 g/mL), for 10 minutes at 37 ◦ C [21]. After washing with PBS, the cells were immediately observed under a fluorescence microscope (Nikon Eclipse E400, Tokyo, Japan). 2.8. M30 CytoDEATH antibody assay M30 CytoDEATH antibody was applied in untreated and treated cells for the determination of early apoptotic events, by the detection of cytokeratin 18 that is exposed in the cellular membrane after cleavage by caspases. Cells were seeded in glass coverslip (8 mm) and incubated overnight. After drug exposure in isolation and combined, cells were washed with PBS and fixed in methanol at -20 ◦ C for 30 minutes. After two washes with 0.1% Tween-20 in PBS, cells were incubated with M30 CytoDEATH antibody working solution (1:10) (Roche Diagnostics, Indianapolis, USA) for 30 minutes, at room temperature and in the dark. Following two washing procedures with buffer, the Anti-Mouse-Ig-Fluorescein (10 g/mL) secondary antibody diluted at 1:100 in incubation buffer was added for 30 minutes. The last washing was performed with PBS. Glass coverslips were mounted in a slide with 4.6-diamidino-2phenylindole (DAPI) (Vysis, Izasa) for visualization of cell nuclei [23]. For the qualitative detection, fluorescence microscopy (Nikon Eclipse E400, Tokyo, Japan) analysis was used. 2.9. Statistical methods Statistical analysis was carried out using the SPSS 17.0 statistical software (SPSS Inc. USA). The equality of variances was tested by Levene F test and the statistical significance of differences between the treatment and control groups were determined by Dunnett’s Multiple Comparison post-hoc test for the MTT assay. The Pearson product-moment correlation coefficient was used to evaluate the
The effect of temsirolimus and everolimus on mTOR pathway was evaluated through the expression analysis of its downstream targets 4E-BP1 and p70S6 K (Fig. 1). We detected a decrease of phosphorylated 4E-BP1 form in the 5637 (P < 0.05) after everolimus incubation. Regarding the effect of temsirolimus, although a decrease of phosphorylated 4E-BP1 form was found in the three cell lines, they were not statistically significant. Concerning the p70S6 K, there were no statistically significant alterations in the three cell lines. 3.2. Cytotoxicity of single gemcitabine, cisplatin, temsirolimus and everolimus Using 3 M of cisplatin, the 5637 cell line did not presented alterations on cell proliferation after 24 hours of treatment, compared with 48 hours of incubation, where it was observed a moderate effect (71.9% of cell proliferation), which was more evident after 72 hours of treatment (30.8% of cell proliferation) (Fig. 2A). In the HT1376 cell line, only in the incubation time of 72 hours an improved effect was observed (76% of cell proliferation) (Fig. 2A). The decrease of cell proliferation in the T24 cell line was observed after 72 hours of cisplatin incubation (58.5% of cell proliferation) (Fig. 2A). Treatment with 0.1 M gemcitabine decrease the 5637 cells proliferation after 48 hours of incubation (56.7% of cell proliferation), being more evident after 72 hours (28.6% of cell proliferation) (Fig. 2B). In the HT1376 and T24 cell lines there was no visible effect after 24 and 48 hours exposure to gemcitabine, and with 72 hours incubation a cell proliferation of 85% and 55% was obtained respectively (Fig. 2B). In previous studies, we already evaluated the activities of temsirolimus (0.5 M) and everolimus (0.05 M) on cell proliferation [18,24] which were confirmed in this study. Temsirolimus induced a slight effect in the three cell lines after 72 hours incubation (Fig. 2C), and only a sensitive activity in the 5637 cell line (IC30 ) was found after everolimus treatment (Fig. 2D). 3.2.1. Cytotoxicity of simultaneous gemcitabine and cisplatin Incubation time of 72 hours gemcitabine was associated with three different times of cisplatin exposure (24, 48 or 72 hours): with 24 hours of cisplatin, only the 5637 cell line presented an evident decrease of cell proliferation (22.1%); with 48 hours of cisplatin the effect remained more visible in the 5637 cell line (12.9%), and the other two cell lines presented a reduction in the cell proliferation similar between them (HT1376: 65.9%; T24: 61.7%); with 72 hours of cisplatin treatment, only in the T24 cell line was observed a marked decreased cell proliferation (40.2% of cell proliferation) (Fig. 2E). 3.2.2. Cytotoxicity of simultaneous gemcitabine, cisplatin and temsirolimus or everolimus In the simultaneous combination of gemcitabine (72 hours) and cisplatin (48 hours), we analyzed three different times of incubation with temsirolimus (24, 48 and 72 hours). A similar pattern response was obtained in the 5637 cell line, this being the most sensitive cell line, with a reduced cell proliferation after treatment (24 hours: 20.47%; 48 hours: 16.1%; 72 hours: 11.8%). The HT1376 cell line was
Please cite this article in press as: Pinto-Leite R, et al. Treatment of muscle invasive urinary bladders tumors: A potential role of the mTOR inhibitors. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.003
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Fig. 1. P-p70S6K (A) and P-4E-BP1 (B) expression evaluated by immunoblotting in the 5637, HT1376 and T24 cell lines. Representative immunoblots are presented below the corresponding graph. Values are expressed as mean ± standard deviation. (*P < 0.05 versus untreated treated cells).
the most resistant, with no differences between 24 hours (41.7%), 48 hours (41.7%) and 72 hours (41.6%). The T24 cell line presented a decrease cell proliferation with the three exposure times tested (24 hours: 44.9%; 48 hours: 36.3%; 72 hours: 30.4%) (Fig. 2F). The same approach was carried out with everolimus. With the three different times of everolimus exposure, a more pronounced effect was detected after 72 hours (5637: 17.5%; HT1376: 31.6%; T24: 30.3%) (Fig. 2G). 3.3. Cell proliferation In all the control groups, a high Ki-67 expression was observed. A decreased expression of this protein was more visible after treatment to either cisplatin or gemcitabine, with a slight effect in temsirolimus or everolimus. This pattern response was maintained for all three cell lines. A lower Ki-67 expression was visualized in the combination of the three drugs and there were not detected differences in the combination with temsirolimus or everolimus (Table 1, Figs. 3–5). 3.4. Cell cycle distribution of single cisplatin, gemcitabine, temsirolimus and everolimus
respectively), HT1376 (7.6% and 15%, respectively) and T24 (43.7% and 16.2%, respectively) cell lines. Concerning the two mTOR inhibitors, in the 5637 cell line, no alterations in the cell cycle was observed; in the HT1376 treated cells an increased cell cycle arrest in the G0 /G1 phase was found (with temsirolimus 74.5% and with everolimus 76.3%); in the T24 cell line a small increase in early S-phase was observed (temsirolimus: 8.7% and everolimus: 7.5%). 3.4.1. Cell cycle distribution of simultaneous and sequential gemcitabine, cisplatin and temsirolimus or everolimus An improved cell cycle arrest in the G0 /G1 phase was found after the treatments with gemcitabine, cisplatin and temsirolimus (73.1%) and gemcitabine, cisplatin and everolimus (66.5%) in the 5637 cell line. A small increase in the early S-phase was more perceptible in the combined schedules, with 26.1% (gemcitabine, cisplatin and temsirolimus) and 25.5% (gemcitabine, cisplatin and everolimus) in the HT1376 treated cells, and with 15.6% (gemcitabine, cisplatin and temsirolimus) and 18.5% (gemcitabine, cisplatin and everolimus) in the T24 treated cells, respectively (Table 2). 3.5. Autophagy
The cell cycle distribution of drug treated cells was investigated by flow cytometry at 48 hours (cisplatin) and 72 hours (gemcitabine, temsirolimus or everolimus) of treatment (Table 2). Untreated cells were mainly distributed in the G0 /G1 phase in the three cell lines (5637: 63%; HT1376: 69.1%; T24: 87.5%). A cell cycle arrest was detected in the early S-phase in cells exposed to gemcitabine or cisplatin in the 5637 (16.9% and 48%, Table 1 Ki-67 immunocytochemical expression in the 5637, HT1376 and T24 urinary bladder cancer cell lines, isolated or in simultaneous schedule after exposure to gemcitabine (G), cisplatin (C), temsirolimus (T) and everolimus (E), in isolation or in combined schedules (TGC, EGC). Drug
5637
HT1376
T24
Control C G T E GCT GCE
++++ ++ ++ +++ +++ + +
++++ ++ ++ +++ +++ + +
++++ ++ ++ +++ +++ + +
–: no staining; +: very weak staining; ++: moderate staining; +++: intense staining; ++++: highest intensity staining.
Untreated cells did not show positive staining. In cisplatin or gemcitabine treated cells, the incidence of acidic vesicles was moderately higher in the three cell lines. Temsirolimus and everolimus induce a small incidence of acidic vesicles in the 5637 and T24 cells. However, in the HT1376 cells treated with temsirolimus in isolation, a small increase of the acidic vesicles was observed when compared with everolimus treated cells. A more pronounced effect was observed in cells treated with gemcitabine, cisplatin and temsirolimus, and gemcitabine, cisplatin and everolimus, with a high incidence of acidic vesicles in the three cell lines (Figs. 3–5). 3.6. Apoptosis Using the M30 CytoDEATH antibody, untreated cells showed no positive staining in the three cell lines (Figs. 3–5). Regarding the 5637 cell line, one to two positive cells per field of visualization were detected after treatment with temsirolimus or everolimus. Treatment with gemcitabine slightly increased apoptosis, with approximately five cells per field of visualization. In the presence of cisplatin, two cells per field of visualization were visualized in the 5637 cell line (Fig. 3). The HT1376 cells showed a similar pattern of response when they were treated with temsirolimus, everolimus, gemcitabine and cisplatin, in isolation, with only one
Please cite this article in press as: Pinto-Leite R, et al. Treatment of muscle invasive urinary bladders tumors: A potential role of the mTOR inhibitors. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.003
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* * *
0 0
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G(72h)+C(48h)+E(72h)
Fig. 2. Evaluation of cell proliferation in the 5637, HT1376 and T24 human urinary bladder cancer cell lines. A: cells were treated with 3 M cisplatin (C) for 24, 48 and 72 hours; B: cells were treated with 0.1 M gemcitabine (G) for 24, 48 and 72 hours; C: cells were treated with 0.5 M temsirolimus (T) for 24, 48 and 72 hours; D: cells were treated with 0.05 M everolimus (E) for 24, 48 and 72 hours. E: cells were treated with gemcitabine (72 hours) and cisplatin (for 24, 48 or 72 hours); F: cells were treated with gemcitabine (72 hours), cisplatin (48 hours) and temsirolimus (for 24, 48 or 72 hours); G: cells were treated with gemcitabine (72 hours), cisplatin (48 hours) and everolimus (for 24, 48 or 72 hours). The data shown and bars represent the mean value ± standard deviation. *P < 0.05 versus untreated cells.
to three apoptotic cells per field of visualization (Fig. 4). The T24 cell line showed a lower incidence of apoptotic cells when exposed to temsirolimus and everolimus, in isolation, with approximately two positive cells per field of visualization. This incidence doubled when cells were treated with gemcitabine and cisplatin, also in isolation (Fig. 5). An increased number of apoptotic cells were
found with both simultaneous schedules (gemcitabine, cisplatin and temsirolimus, and gemcitabine, cisplatin and everolimus), with approximately eight positive cells per field of visualization in the 5637 cell line (Fig. 3), five to six apoptotic cells per field of visualization in the HT1376 cell line (Fig. 4) and approximately eleven positive cells in the T24 cell line (Fig. 5).
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Fig. 3. The 5637 human urinary bladder cancer cell line was incubated in the absence (control) or in the presence of cisplatin (C) (3 M, 48 hours), gemcitabine (G) (0.1 M, 72 hours), temsirolimus (T) (0.5 M, 72 hours) and everolimus (E) (0.05 M, 72 hours), in isolation or combined: gemcitabine, cisplatin and temsirolimus (GCT), and gemcitabine, cisplatin and everolimus (GCE). After incubation, cells were treated with Ki-67 antibody (proliferating cells stain the nuclei in brown), acridine orange staining (green: cytoplasm and nucleus cells; red: acidic compartments) and M30 CytoDEATH antibody (cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole; apoptotic cells are visible due to their green colour). Original magnification: Ki-67 400 ×; acridine orange 400 ×; M30 CytoDEATH 200 ×.
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Fig. 4. The HT1376 human urinary bladder cancer cell line was incubated in the absence (control) or in the presence of cisplatin (C) (3 M, 48 hours), gemcitabine (G) (0.1 M, 72 hours), temsirolimus (T) (0.5 M, 72 hours) and everolimus (E) (0.05 M, 72 hours), in isolation or combined: gemcitabine, cisplatin and temsirolimus (GCT), and gemcitabine, cisplatin and everolimus (GCE). After incubation, cells were treated with Ki-67 antibody (proliferating cells stain the nuclei in brown), acridine orange staining (green: cytoplasm and nucleus cells; red: acidic compartments) and M30 CytoDEATH antibody (cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole; apoptotic cells are visible due to their green colour). Original magnification: Ki-67 400 ×; acridine orange 400 ×; M30 CytoDEATH 200 ×.
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Fig. 5. The T24 human urinary bladder cancer cell line was incubated in the absence (control) or in the presence of cisplatin (C) (3 M, 48 hours), gemcitabine (G) (0.1 M, 72 hours), temsirolimus (T) (0.5 M, 72 hours) and everolimus (E) (0.05 M, 72 hours), in isolation or combined: gemcitabine, cisplatin and temsirolimus (GCT), and gemcitabine, cisplatin and everolimus (GCE). After incubation, cells were treated with Ki-67 antibody (proliferating cells stain the nuclei in brown), acridine orange staining (green: cytoplasm and nucleus cells; red: acidic compartments) and M30 CytoDEATH antibody (cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole; apoptotic cells are visible due to their green colour). Original magnification: Ki-67 400 ×; acridine orange 400 ×; M30 CytoDEATH 200 ×.
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Table 2 Cell cycle analysis of the 5637, HT1376 and T24 urinary bladder cancer cell lines after exposure to gemcitabine (G), cisplatin (C), temsirolimus (T) and everolimus (E), in isolation or in combined schedules (GCT, GCE). The percentages of cells in each cell cycle phases are mean ± SD of 3 independent experiments. SD = standard deviation. 5637
HT1376
G0 /G1 Control C G T E GCT GCE
63.0 21.8 42.7 61.9 60.8 73.1 66.5
± ± ± ± ± ± ±
S 1.2 0.9 1.6 0.4 1.6 1.1 1.7
8.8 48.0 16.9 11.1 9.9 10.1 15.4
G2 /M ± ± ± ± ± ± ±
0.2 1.7 2.1 0.1 1.4 0.1 0.4
8.6 14.4 5.0 10.0 8.3 6.9 9.0
G0 /G1 ± ± ± ± ± ± ±
1.8 1.2 0.4 0.6 2.8 1.1 0.5
69.1 67.2 64.3 74.5 76.3 48.9 51.1
± ± ± ± ± ± ±
T24 S
1.5 1.1 1.2 1.3 0.3 0.1 1.2
4. Discussion In the last years, mTOR inhibitors have raised many perspectives in cancer treatment [25,26]. The temsirolimus and everolimus shared the same sirolimus mechanism of action, specifically binding to the abundant intracellular protein FK506-binding protein 12 (FKBP12), inhibiting the activation of mTORC1 complex [27]. Subsequently, the phosphorylation of the downstream targets p70S6 K and 4E-BP1 is also inhibited [11,28]. Inhibition of these protein complexes ultimately results in decreased cell growth and proliferation, cellular metabolism and angiogenesis, leading to a cell cycle blockage at the G0 /G1 phase [29]. Park et al. (2011) revealed that mTOR pathway activation, as assessed by 4E-BP1 and/or S6K1 phosphorylation, is involved in urinary bladder cancer tumorigenesis and is a predictor of disease progression and poor cancer-specific survival [30]. We have previously evaluated everolimus and temsirolimus actions on mTOR and Akt, as well as on their phosphorylated forms, in the three cell lines, without encouraging results [18,24]. Therefore, we decided to analyze, the p70S6 K and 4E-BP1 and its phosphorylated forms. We have found a decrease of the phosphorylated 4E-BP1 statistically significant in the 5637 cell line with everolimus. The T24 and HT1376 cell lines did not presented any statistically significant alterations of the expression of the two targets analyzed. It has already been described that the dephosphorylation of S6K1 results in a feedback loop that results in up-regulation of receptor tyrosine kinases or insulin receptor substrate proteins, which then activate the PI3 K and consequently mTOR [31,32]. These results, to some extent, justify the results obtained when we have previously analyzed rapalogs effects in the same three cell lines. We have observed in previous studies and confirmed in this investigation, that everolimus [24] and temsirolimus [18] in isolation induce a slight interference on proliferation, apoptosis and autophagy in the three cell lines used. The one that was slightly sensitive was the 5637 non-muscle invasive cancer cell line. According to the currently accepted knowledge, mTOR pathway interferes with the non-muscle invasive urinary bladder tumors [33] and the 5637 cell line fits in this type of tumors. In several clinical trials, it was found that rapalogs have predominantly led to disease stabilization rather than inhibition of tumor progression [34]. Indeed, the heterogeneity of the results with everolimus has been described [14,24,35]. This drug is still under investigation and in a clinical trial performed by Yao et al. (2013) with advanced pancreatic neuroendocrine tumors, the results obtained suggest that everolimus delays disease progression in the patients analyzed. In patients who experience disease progression, with everolimus, they do not appear to have a more aggressive metastatic phenotype than those patients with placebo [36]. For temsirolimus, the results were also poorly encouraging and some disagreement on the effects obtained with its application has been described [37–39]. However, to enhance the efficacy of rapalogs, evaluation for potential effect with other classes of antineoplastic
G2 /M
5.4 15.0 7.6 5.8 5.4 26.1 25.5
± ± ± ± ± ± ±
0.2 0.6 0.7 0.3 0.3 0.5 0.7
15.2 10.7 19.0 11.9 10.5 17.1 16.2
G0 /G1 ± ± ± ± ± ± ±
0.9 0.5 0.7 0.9 1 0.4 0.7
87.5 67.8 34.1 81.2 83.1 72.6 66.1
± ± ± ± ± ± ±
S 1.7 1.2 1.1 2.9 0.1 0.4 1.9
4.3 16.2 43.7 8.7 7.5 15.6 18.5
G2 /M ± ± ± ± ± ± ±
0.7 0.3 0.3 1.2 0.1 0.3 1.2
31.2 29.1 30.6 30.9 30.8 31.2 31.1
± ± ± ± ± ± ±
0.8 2.1 0.6 1.1 0.2 0.3 1.1
drugs has been suggested [40]. Multidrug combination is the corner stone of treatment for widely neoplastic diseases. We already know that these two mTOR inhibitors (temsirolimus and everolimus), when combined with cisplatin or gemcitabine improved their effects, concerning proliferation inhibition, increased apoptosis and autophagy in the three urinary bladder cancer cell lines [16,17]. As we found that the simultaneous combination of gemcitabine (72 hours) and cisplatin (24, 48 and 72 hours) were effective in reducing the cell proliferation in the cell lines tested, we decided to analyze the effect of 48 hours cisplatin incubation (an average of IC55 in the three cell lines) with gemcitabine (72 hours) and temsirolimus or everolimus (24, 48 and 72 hours). As far as we know, this is the first study combining these three drugs in urinary bladder cancer cell lines. Concerning the gemcitabine, cisplatin and temsirolimus conjugation an enhanced inhibition of cell proliferation was obtained in the three cell lines, with the three different incubation temsirolimus times, being the 72 hours incubation the most effective one. With the gemcitabine, cisplatin and everolimus approach, a similar pattern response was obtained in muscle invasive urinary bladder cancer cell lines (HT1376 and T24). Indeed, in the 5637 non-muscle invasive urinary cell line, the addition of everolimus to gemcitabine and cisplatin did not increase the antiproliferative effects of the antineoplastic drugs conjugation. Analyzing the cell cycle distribution, a pronounced cell cycle arrest in the G0 /G1 phase was found in the 5637 cell line, and an accumulation of early S-phase was observed in the HT1376 and T24 cell lines after exposure to gemcitabine, cisplatin and temsirolimus or everolimus. With the others technical approaches used (immunocytochemistry, flow cytometry, acridine orange staining and M30 CytoDEATH antibody) the results obtained were in agreement. There was a reduced Ki-67 expression, a higher percentage of apoptotic and autophagic cells either in the gemcitabine, cisplatin and temsirolimus or in gemcitabine, cisplatin and everolimus combination. Despite the many studies that have been conducted, the results have not been promising and have demonstrated that rapalogs, as single drugs, have not been very effective in the treatment of urinary bladder cancer. Although the apparent lack of cellular response to the rapalogs application, when cells are also exposed to antineoplastic drugs, the effect is remarkable. It seems that the rapalogs incubation in combination with antineoplastic drugs, the cells became more susceptible and with a lower ability to resist. For some patients the side effects of getting more than one antineoplastic drug might be too much to handle. Chemotherapy for urinary bladder cancer can be hard to tolerate, especially for older patients who have other serious medical complications. On the other, it should be considered that the rapalogs may cause some unwanted effects. In the treatment of advanced urinary bladder cancer, gemcitabine in low dose and prolonged infusion in combination with cisplatin is not inferior to high-dose short infusion gemcitabine and cisplatin in terms of overall survival, time to disease progression, and response rates with favorable toxicity profile.
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The great heterogeneity response detected in the muscle invasive urinary bladder cancer treatment, is a consequence of the complexity of the molecular pathways involved in urinary bladder cancer onset, combined with the several genetic and epigenetic events that occur during tumor progression [41–43]. According to the Cancer Genome Atlas Research Network (2014) [19], 42% of the tumours have mutations, copy number alterations or RNA expression changes affecting the PI(3)K/AKT/mTOR pathway including mutation or deletion of TSC1 or TSC2. These specific gene alterations will be responsible for the 9% of potential responses to mTOR inhibitors. Based on this data we intend to analyze TSC1 and TSC2 expression, hoping that this will explain the behavior of the cell lines tested. 5. Conclusions The results presented in this study are very encouraging, since the simultaneous combination of mTOR inhibitors (either with temsirolimus or everolimus) with classical antineoplastic drugs used in the treatment of muscle invasive urinary bladder cancer, by using lowest concentration of each drug, induced an increased sensibility in the cancer cell lines to the antineoplastic drugs. This positive response can be useful to decrease the toxicity of the therapy keeping, or even improving, the effectiveness in treatment of muscle invasive urinary bladder cancer patients. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments The authors express their deepest appreciation to Professor Ana Maria Nazaré Pereira for the availability of the Elisa reader. References [1] Mitra AP, Castelao JE, Hawes D, Tsao-Wei DD, Jiang X, Shi SR, et al. Combination of molecular alterations and smoking intensity predicts bladder cancer outcome: a report from the Los Angeles Cancer Surveillance Program. Cancer 2013;119:756–65. [2] Rouprêt M, Babjuk M, Compérat E, Zigeuner R, Sylvester R, Burger M, et al. European guidelines on upper tract urothelial carcinomas: 2013 update. Eur Urol 2013;63:1059–71. [3] Cheung G, Saha A, Billia M, Dasgupta P, Khan MS. Recent advances in the diagnosis and treatment of bladder cancer. BMC Med 2013;1:13. [4] Eldefrawy A, Soloway MS, Katkoori D, Singal R, Pan D, Manoharan M. Neoadjuvant and adjuvant chemotherapy for muscle invasive bladder cancer: the likelihood of initiation and completion. Indian J Urol 2012;28:424–6. [5] Ismaili N, Amzerin M, Flechon A. Chemotherapy in advanced bladder cancer: current status and future. J Hematol Oncol 2011;4:35. [6] Kim JJ. Recent advances in treatment of advanced urothelial carcinoma. Curr Urol Rep 2012;2012:147–52. [7] Witjes JA, Compérat E, Cowan NC, De Santis M, Gakis G, Lebret T, et al. EUA Guidelines on muscle invasive and metastatic bladder cancer: summary of the 2013 Guidelines. Eur Urol 2014;65:778–92. [8] Siefker-Radtke A. Bladder cancer: can we move beyond chemotherapy? Curr Oncol Rep 2010;12:278–83. [9] Edeline J, Loriot Y, Culine S, Massard C, Albiges L, Blesius A, et al. Accelerated MVAC chemotherapy in patients with advanced urinary bladder cancer previously treated with a platinum-gemcitabine regimen. Eur J Cancer 2012;48:1141–6. [10] Miller RP, Tadagavadi RK, Ramesh G, Brian W. Mechanisms of cisplatin nephrotoxicity. Reeves Toxins 2012;2:2490–518. [11] Ching CB, Hansel DE. Expanding therapeutic targets in bladder cancer: the PI3 K/Akt/mTOR pathway. Lab Invest 2010;90:1406–14. [12] Sun CH, Chang YH, Pan CC. Activation of the PI3 K/Akt/mTOR pathway correlates with tumor progression and reduced survival in patients with urothelial carcinoma of the urinary bladder. Histopathology 2011;58:1054–63. [13] Strimpakos AS, Karapanagiotou EM, Saif MW, Syrigos KN. The role of mTOR in the management of solid tumors: an overview. Cancer Treat Rev 2009;35:148–59. [14] Garcia JA, Danielpour D. Mammalian target of rapamycin inhibition as a therapeutic strategy in the management of urologic malignancies. Mol Cancer Ther 2008;27:1347–54.
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Original article
Treatment of muscle invasive urinary bladders tumors: A potential role of the mTOR inhibitors Rosário Pinto-Leite a , Regina Arantes-Rodrigues b , Rita Ferreira c , Carlos Palmeira d,e , Paula A. Oliveira b,∗ , Lúcio Santos d,e a
Genetic Service, Cytogenetic Laboratory, Hospital Center of Trás-os-Montes and Alto Douro, Vila Real, Portugal Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trás-os-Montes and Alto Douro, Vila Real, Portugal c Department of Chemistry, QOPNA, Mass Spectrometry Center, University of Aveiro, Aveiro, Portugal d Experimental Pathology and Therapeutics Group, Portuguese Institute of Oncology, Porto, Portugal e Health School, University Fernando Pessoa, Porto, Portugal b
a r t i c l e
i n f o
Article history: Received 5 March 2014 Accepted 11 March 2014 Available online xxx Keywords: Gemcitabine Cisplatin Temsirolimus Everolimus Combined antineoplastic chemotherapy Cancer cell lines
a b s t r a c t 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. © 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction Urinary bladder cancer accounts for 90–95% of urothelial carcinomas, being the most frequent malignancy of the urinary system [1,2]. Although the great majority of patients have non-muscle invasive lesions, 20 to 40% either present or later develops muscle invasive tumors [3,4]. Much has changed in the diagnosis and
∗ Corresponding author. Tel.: +35 19 66 47 30 62; fax: +35 12 59 35 04 80. E-mail addresses: [email protected], [email protected], [email protected] (P.A. Oliveira).
management of urinary bladder cancer over the past 5 to 10 years [3]. The standard chemotherapy for the treatment of muscle invasive and metastatic urinary bladder cancer are platinum-based regimens, particularly cisplatin [5,6]. The gemcitabine and cisplatin combination, as well as the methotrexate, vinblastine, adriamycin and cisplatin (MVAC) regimen are commonly used [7]. The combination of gemcitabine and cisplatin has been widely adopted as a platform for antineoplastic chemotherapy in the treatment of muscle invasive urinary bladder cancer, with similar and even superior results in regard to survival and a better toxicity profile compared with the standard MVAC [8,9]. However, this approach has severe side effects (which include ototoxicity, gastrotoxicity,
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myelosuppression, allergic reactions, and specially nephrotoxicity) and chemoresistance [10]. The response rates remains in the 50% to 70% and the median survival is of only about 13 months [3]. The need for new treatment strategies has led to the research and evaluation of novel drugs that target novel cellular pathways. The mammalian target of rapamycin (mTOR) is frequently deregulated in human cancers, and several studies suggest that it may be involved in the urinary bladder cancer development [11,12]. mTOR is an important pathway controlling cell growth, survival and proliferation [13–15]. Rapamycin and its rapalogs (temsirolimus and everolimus) are well-recognized mTOR inhibitors. Their efficacy in urinary bladder cancer cell lines has already been evaluated as single drugs or in combination with cisplatin or gemcitabine, with promising results [16–18]. Cell lines are important tools in cancer research, their appropriate genomic characterization is essential in preclinical studies. We described similar genetic alterations to ones described by the Cancer Genome Atlas Research Network (2014) [19] in the 5637, HT1376 and T24 cancer cell lines [20] what makes these cell lines a valuable tool to evaluate the efficacy of antineoplastic drugs. The purpose of the present study was to analyze the effects of combining two mTOR inhibitors, temsirolimus and everolimus, with cisplatin and gemcitabine, at lower concentrations, using three urinary bladder cancer cell lines representative of human urinary bladder cancer. 2. Material and methods 2.1. Cell lines and culture conditions The study was performed on a non-muscle invasive urinary bladder cancer cell line (5637) and two muscle invasive urinary bladder cancer cell lines: T24 and HT1376. T24 cell line was provided by DSMZ, Düsseldorf, Germany; 5637 and HT1376 cell lines were kindly provided by Dr. Paula Videira of the Universidade Nova de Lisboa, Lisboa, Portugal. All the cell lines were maintained in RPMI 1640 culture medium (PAA, Pasching, Austria), supplemented with 10% heat-inactivated fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), 100 U/mL penicillin (Biological Industries, Kibbutz Beit Haemek, Israel), 100 g/mL streptomycin (Biological Industries, Kibbutz Beit Haemek, Israel) and 2 mM Lglutamine (Sigma Aldrich, St. Louis, USA). Cells were cultured as a monolayer at 37 ◦ C in 5% CO2 in a humidified atmosphere. 2.2. Drugs treatment To assess dose-response profiles by the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, the three urinary bladder cancer cell lines were treated with the four drugs with different times of exposure. In isolation the cells were exposed for 24, 48 and 72 hours to 0.5 M temsirolimus (Sigma Aldrich, Portugal), 0.05 M everolimus (Sigma, St. Louis, USA), 0.1 M gemcitabine (Hospira, Portugal) and 3 M cisplatin (Teva Pharma, Portugal). In the gemcitabine and cisplatin combined schedules, cells were exposed to gemcitabine (72 hours) and cisplatin (24, 48 or 72 hours). In the gemcitabine, cisplatin and temsirolimus; and gemcitabine, cisplatin and everolimus combinations the cells were exposed to gemcitabine (72 hours), cisplatin (48 hours) and it was added temsirolimus or everolimus for 24, 48 or 72 hours. The remaining assays (immunocytochemistry, flow cytometry, acridine orange staining and M30 CytoDEATH antibody) were performed with the combination of gemcitabine (72 hours), cisplatin (48 hours) and temsirolimus (72 hours) or everolimus (72 hours). Controls samples were treated similarly but without the drugs. All the drugs were freshly prepared before each experiment. In each
experiment, triplicate wells were performed for each drug and the assay was repeated in three independent experiments. 2.3. Immunoblotting In order to evaluate the effect of temsirolimus and everolimus on mTOR signaling, the levels of phosphorylated mTOR downstream targets ribosomal protein S kinase (p70S6 K) and 4E-binding protein 1 (4E-BP1) were evaluated. In this sense, whole cell extracts were obtained from mechanical homogenization of cell pellets (with no treatment, with 0.5 M temsirolimus and with 0.05 M everolimus) in phosphate buffer with 1% SDS containing phosphatase inhibitors (P0044 and P5726, Sigma). Protein concentration was assayed by the colorimetric method “RC DC protein assay” (Bio-Rad) using bovine serum albumin (BSA) as protein standard. Samples from each cell line were then diluted in Tris buffered saline (TBS; 100 mM Tris, 1.5 mM NaCl, pH 8.0) to obtain a final protein concentration of 0.4 mg/mL and a volume of 100 l was slot-blotted into a nitrocellulose membrane (Whatman, Protein). Nonspecific binding was blocked with 5% (w/v) BSA in TBS-T (TBS with 0.5% Tween 20) for 1 hour and the membrane was then incubated with primary antibody (1:1000 dilution; rabbit monoclonal phospho-4E-BP1 (Thr37/46) or #2855; phospho-p70 S6 K (Thr389), #9234from Cell Signaling). After 1 hour incubation, the membrane was washed with TBS-T and incubated with anti-rabbit IgG peroxidase secondary antibody (1:1000; Amersham Pharmacia Biotech). Immunoreactivity was detected with enhanced chemiluminescence reagents (ECL, Amersham Pharmacia Biotech) according to the manufacturer’s instructions and images were recorded using X-ray films (Kodak Biomax Light Film, Sigma). Films were scanned in Molecular Imager Gel Doc XR+ System (Bio-Rad) and analyzed with Quantity One software version 4.6.3 (Bio-Rad, Hercules, SA). Protein loading control of blotting membranes was performed by staining with Ponceau S. 2.4. MTT assay The MTT assay was used to assess the relative percentage of metabolically active cells compared to untreated cells. In brief, trypsinized tumor cells were resuspended in a medium at 2-3 × 104 cells/mL based on the growth characteristics of each cell line. One hundred l of cells suspension was seeded in a 96-well culture plate (Sarstedt, USA) and plates were incubated for 24 hours to allow adherent cell growth. After overnight incubation, the medium was removed and 100 l of the different reagent solutions in medium were distributed in each well. After 72 hours incubation, 10 l of MTT (Sigma Aldrich, EUA) dye working solution (5 mg/mL was added to each well and 4 hours later, the supernatant in the wells was removed and replaced by 100 l/well of dimethylsulfoxide (Sigma Aldrich, USA). The absorbance (A) values of each well was recorded at 492 nm on an automatic Elisa plate reader (Multiskan EX. Labsystems). Cell proliferation was calculated as: Aexp group /Acontrol X 100 [21]. 2.5. Immunocytochemistry Immunocytochemistry assay was performed in order to determine the Ki-67 expression, a nuclear protein that is expressed in proliferating cells [22]. Cells were plated in 24-well chamber slides (Sarstedt, USA) and incubated overnight. Drugs were applied in isolation or combined. After being washed with PBS, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed three times in an isotonic PBS buffer. Permeabilization was carried out using 1% Triton X-100 in PBS for 20 minutes at room temperature and internal peroxidase activity was blocked with 3% hydrogen peroxide for 30 minutes. The primary anti-Ki-67 antibody
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(MIB-1, 1:100) was incubated for 1 hour at room temperature. After incubation of the secondary antibody, positive immunoreactivity was visible by means of the reaction of 3,3-diaminobenzidine chromogen. The samples were washed with water and contrasted with haematoxylin. Negative controls were performed by replacing primary antibody with PBS [16]. Samples were analyzed with a Leica microscope.
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correlation (linear dependence) of the cell cycle and drugs concentration. P values of less than 0.05 were considered to be statistically significant. 3. Results 3.1. Modulation of mTOR pathway by temsirolimus and everolimus
2.6. Flow cytometry Flow cytometry was carried out in order to analyse the distribution of cells by the different cell cycle phases. Cells (1 × 106 cells/mL) were plated in 6-well plates and incubated for 24 hours. Then drugs, in isolation or combined were added to the cells. After 72 hours of incubation, cells were trypsinized, washed with PBS and fixed in ice-cold ethanol 70% for at least 1 hour [23]. Propidium iodide (CycloscopeTM DNA Cytometry, Cytognos) was added in order to label total cellular DNA and cell cycle analysis was carried out using a Coulter® EPICSTM XL-MCLTM flow cytometer. DNA-content histograms were analysed with Modfit LT 3.0 software (VerityTM ) to determine the percentage of cells in each cell cycle phase: G0 /G1 , S and G2 /M. Each independent experiment was performed in triplicate. 2.7. Acridine orange staining The production of acidic vesicular organelles is associated with autophagy. Acridine orange stains the cytoplasm and nucleus of the stained cells by fluoresces bright green, while acidic compartments (including acidic vesicular organelles) fluoresces bright red. Urinary bladder cancer cells were seeded on glass coverslip (8 mm) and incubated overnight. After drug exposure the medium was removed and cells were stained with acridine orange (1 g/mL), for 10 minutes at 37 ◦ C [21]. After washing with PBS, the cells were immediately observed under a fluorescence microscope (Nikon Eclipse E400, Tokyo, Japan). 2.8. M30 CytoDEATH antibody assay M30 CytoDEATH antibody was applied in untreated and treated cells for the determination of early apoptotic events, by the detection of cytokeratin 18 that is exposed in the cellular membrane after cleavage by caspases. Cells were seeded in glass coverslip (8 mm) and incubated overnight. After drug exposure in isolation and combined, cells were washed with PBS and fixed in methanol at -20 ◦ C for 30 minutes. After two washes with 0.1% Tween-20 in PBS, cells were incubated with M30 CytoDEATH antibody working solution (1:10) (Roche Diagnostics, Indianapolis, USA) for 30 minutes, at room temperature and in the dark. Following two washing procedures with buffer, the Anti-Mouse-Ig-Fluorescein (10 g/mL) secondary antibody diluted at 1:100 in incubation buffer was added for 30 minutes. The last washing was performed with PBS. Glass coverslips were mounted in a slide with 4.6-diamidino-2phenylindole (DAPI) (Vysis, Izasa) for visualization of cell nuclei [23]. For the qualitative detection, fluorescence microscopy (Nikon Eclipse E400, Tokyo, Japan) analysis was used. 2.9. Statistical methods Statistical analysis was carried out using the SPSS 17.0 statistical software (SPSS Inc. USA). The equality of variances was tested by Levene F test and the statistical significance of differences between the treatment and control groups were determined by Dunnett’s Multiple Comparison post-hoc test for the MTT assay. The Pearson product-moment correlation coefficient was used to evaluate the
The effect of temsirolimus and everolimus on mTOR pathway was evaluated through the expression analysis of its downstream targets 4E-BP1 and p70S6 K (Fig. 1). We detected a decrease of phosphorylated 4E-BP1 form in the 5637 (P < 0.05) after everolimus incubation. Regarding the effect of temsirolimus, although a decrease of phosphorylated 4E-BP1 form was found in the three cell lines, they were not statistically significant. Concerning the p70S6 K, there were no statistically significant alterations in the three cell lines. 3.2. Cytotoxicity of single gemcitabine, cisplatin, temsirolimus and everolimus Using 3 M of cisplatin, the 5637 cell line did not presented alterations on cell proliferation after 24 hours of treatment, compared with 48 hours of incubation, where it was observed a moderate effect (71.9% of cell proliferation), which was more evident after 72 hours of treatment (30.8% of cell proliferation) (Fig. 2A). In the HT1376 cell line, only in the incubation time of 72 hours an improved effect was observed (76% of cell proliferation) (Fig. 2A). The decrease of cell proliferation in the T24 cell line was observed after 72 hours of cisplatin incubation (58.5% of cell proliferation) (Fig. 2A). Treatment with 0.1 M gemcitabine decrease the 5637 cells proliferation after 48 hours of incubation (56.7% of cell proliferation), being more evident after 72 hours (28.6% of cell proliferation) (Fig. 2B). In the HT1376 and T24 cell lines there was no visible effect after 24 and 48 hours exposure to gemcitabine, and with 72 hours incubation a cell proliferation of 85% and 55% was obtained respectively (Fig. 2B). In previous studies, we already evaluated the activities of temsirolimus (0.5 M) and everolimus (0.05 M) on cell proliferation [18,24] which were confirmed in this study. Temsirolimus induced a slight effect in the three cell lines after 72 hours incubation (Fig. 2C), and only a sensitive activity in the 5637 cell line (IC30 ) was found after everolimus treatment (Fig. 2D). 3.2.1. Cytotoxicity of simultaneous gemcitabine and cisplatin Incubation time of 72 hours gemcitabine was associated with three different times of cisplatin exposure (24, 48 or 72 hours): with 24 hours of cisplatin, only the 5637 cell line presented an evident decrease of cell proliferation (22.1%); with 48 hours of cisplatin the effect remained more visible in the 5637 cell line (12.9%), and the other two cell lines presented a reduction in the cell proliferation similar between them (HT1376: 65.9%; T24: 61.7%); with 72 hours of cisplatin treatment, only in the T24 cell line was observed a marked decreased cell proliferation (40.2% of cell proliferation) (Fig. 2E). 3.2.2. Cytotoxicity of simultaneous gemcitabine, cisplatin and temsirolimus or everolimus In the simultaneous combination of gemcitabine (72 hours) and cisplatin (48 hours), we analyzed three different times of incubation with temsirolimus (24, 48 and 72 hours). A similar pattern response was obtained in the 5637 cell line, this being the most sensitive cell line, with a reduced cell proliferation after treatment (24 hours: 20.47%; 48 hours: 16.1%; 72 hours: 11.8%). The HT1376 cell line was
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Fig. 1. P-p70S6K (A) and P-4E-BP1 (B) expression evaluated by immunoblotting in the 5637, HT1376 and T24 cell lines. Representative immunoblots are presented below the corresponding graph. Values are expressed as mean ± standard deviation. (*P < 0.05 versus untreated treated cells).
the most resistant, with no differences between 24 hours (41.7%), 48 hours (41.7%) and 72 hours (41.6%). The T24 cell line presented a decrease cell proliferation with the three exposure times tested (24 hours: 44.9%; 48 hours: 36.3%; 72 hours: 30.4%) (Fig. 2F). The same approach was carried out with everolimus. With the three different times of everolimus exposure, a more pronounced effect was detected after 72 hours (5637: 17.5%; HT1376: 31.6%; T24: 30.3%) (Fig. 2G). 3.3. Cell proliferation In all the control groups, a high Ki-67 expression was observed. A decreased expression of this protein was more visible after treatment to either cisplatin or gemcitabine, with a slight effect in temsirolimus or everolimus. This pattern response was maintained for all three cell lines. A lower Ki-67 expression was visualized in the combination of the three drugs and there were not detected differences in the combination with temsirolimus or everolimus (Table 1, Figs. 3–5). 3.4. Cell cycle distribution of single cisplatin, gemcitabine, temsirolimus and everolimus
respectively), HT1376 (7.6% and 15%, respectively) and T24 (43.7% and 16.2%, respectively) cell lines. Concerning the two mTOR inhibitors, in the 5637 cell line, no alterations in the cell cycle was observed; in the HT1376 treated cells an increased cell cycle arrest in the G0 /G1 phase was found (with temsirolimus 74.5% and with everolimus 76.3%); in the T24 cell line a small increase in early S-phase was observed (temsirolimus: 8.7% and everolimus: 7.5%). 3.4.1. Cell cycle distribution of simultaneous and sequential gemcitabine, cisplatin and temsirolimus or everolimus An improved cell cycle arrest in the G0 /G1 phase was found after the treatments with gemcitabine, cisplatin and temsirolimus (73.1%) and gemcitabine, cisplatin and everolimus (66.5%) in the 5637 cell line. A small increase in the early S-phase was more perceptible in the combined schedules, with 26.1% (gemcitabine, cisplatin and temsirolimus) and 25.5% (gemcitabine, cisplatin and everolimus) in the HT1376 treated cells, and with 15.6% (gemcitabine, cisplatin and temsirolimus) and 18.5% (gemcitabine, cisplatin and everolimus) in the T24 treated cells, respectively (Table 2). 3.5. Autophagy
The cell cycle distribution of drug treated cells was investigated by flow cytometry at 48 hours (cisplatin) and 72 hours (gemcitabine, temsirolimus or everolimus) of treatment (Table 2). Untreated cells were mainly distributed in the G0 /G1 phase in the three cell lines (5637: 63%; HT1376: 69.1%; T24: 87.5%). A cell cycle arrest was detected in the early S-phase in cells exposed to gemcitabine or cisplatin in the 5637 (16.9% and 48%, Table 1 Ki-67 immunocytochemical expression in the 5637, HT1376 and T24 urinary bladder cancer cell lines, isolated or in simultaneous schedule after exposure to gemcitabine (G), cisplatin (C), temsirolimus (T) and everolimus (E), in isolation or in combined schedules (TGC, EGC). Drug
5637
HT1376
T24
Control C G T E GCT GCE
++++ ++ ++ +++ +++ + +
++++ ++ ++ +++ +++ + +
++++ ++ ++ +++ +++ + +
–: no staining; +: very weak staining; ++: moderate staining; +++: intense staining; ++++: highest intensity staining.
Untreated cells did not show positive staining. In cisplatin or gemcitabine treated cells, the incidence of acidic vesicles was moderately higher in the three cell lines. Temsirolimus and everolimus induce a small incidence of acidic vesicles in the 5637 and T24 cells. However, in the HT1376 cells treated with temsirolimus in isolation, a small increase of the acidic vesicles was observed when compared with everolimus treated cells. A more pronounced effect was observed in cells treated with gemcitabine, cisplatin and temsirolimus, and gemcitabine, cisplatin and everolimus, with a high incidence of acidic vesicles in the three cell lines (Figs. 3–5). 3.6. Apoptosis Using the M30 CytoDEATH antibody, untreated cells showed no positive staining in the three cell lines (Figs. 3–5). Regarding the 5637 cell line, one to two positive cells per field of visualization were detected after treatment with temsirolimus or everolimus. Treatment with gemcitabine slightly increased apoptosis, with approximately five cells per field of visualization. In the presence of cisplatin, two cells per field of visualization were visualized in the 5637 cell line (Fig. 3). The HT1376 cells showed a similar pattern of response when they were treated with temsirolimus, everolimus, gemcitabine and cisplatin, in isolation, with only one
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0 0
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Fig. 2. Evaluation of cell proliferation in the 5637, HT1376 and T24 human urinary bladder cancer cell lines. A: cells were treated with 3 M cisplatin (C) for 24, 48 and 72 hours; B: cells were treated with 0.1 M gemcitabine (G) for 24, 48 and 72 hours; C: cells were treated with 0.5 M temsirolimus (T) for 24, 48 and 72 hours; D: cells were treated with 0.05 M everolimus (E) for 24, 48 and 72 hours. E: cells were treated with gemcitabine (72 hours) and cisplatin (for 24, 48 or 72 hours); F: cells were treated with gemcitabine (72 hours), cisplatin (48 hours) and temsirolimus (for 24, 48 or 72 hours); G: cells were treated with gemcitabine (72 hours), cisplatin (48 hours) and everolimus (for 24, 48 or 72 hours). The data shown and bars represent the mean value ± standard deviation. *P < 0.05 versus untreated cells.
to three apoptotic cells per field of visualization (Fig. 4). The T24 cell line showed a lower incidence of apoptotic cells when exposed to temsirolimus and everolimus, in isolation, with approximately two positive cells per field of visualization. This incidence doubled when cells were treated with gemcitabine and cisplatin, also in isolation (Fig. 5). An increased number of apoptotic cells were
found with both simultaneous schedules (gemcitabine, cisplatin and temsirolimus, and gemcitabine, cisplatin and everolimus), with approximately eight positive cells per field of visualization in the 5637 cell line (Fig. 3), five to six apoptotic cells per field of visualization in the HT1376 cell line (Fig. 4) and approximately eleven positive cells in the T24 cell line (Fig. 5).
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Fig. 3. The 5637 human urinary bladder cancer cell line was incubated in the absence (control) or in the presence of cisplatin (C) (3 M, 48 hours), gemcitabine (G) (0.1 M, 72 hours), temsirolimus (T) (0.5 M, 72 hours) and everolimus (E) (0.05 M, 72 hours), in isolation or combined: gemcitabine, cisplatin and temsirolimus (GCT), and gemcitabine, cisplatin and everolimus (GCE). After incubation, cells were treated with Ki-67 antibody (proliferating cells stain the nuclei in brown), acridine orange staining (green: cytoplasm and nucleus cells; red: acidic compartments) and M30 CytoDEATH antibody (cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole; apoptotic cells are visible due to their green colour). Original magnification: Ki-67 400 ×; acridine orange 400 ×; M30 CytoDEATH 200 ×.
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Fig. 4. The HT1376 human urinary bladder cancer cell line was incubated in the absence (control) or in the presence of cisplatin (C) (3 M, 48 hours), gemcitabine (G) (0.1 M, 72 hours), temsirolimus (T) (0.5 M, 72 hours) and everolimus (E) (0.05 M, 72 hours), in isolation or combined: gemcitabine, cisplatin and temsirolimus (GCT), and gemcitabine, cisplatin and everolimus (GCE). After incubation, cells were treated with Ki-67 antibody (proliferating cells stain the nuclei in brown), acridine orange staining (green: cytoplasm and nucleus cells; red: acidic compartments) and M30 CytoDEATH antibody (cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole; apoptotic cells are visible due to their green colour). Original magnification: Ki-67 400 ×; acridine orange 400 ×; M30 CytoDEATH 200 ×.
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Fig. 5. The T24 human urinary bladder cancer cell line was incubated in the absence (control) or in the presence of cisplatin (C) (3 M, 48 hours), gemcitabine (G) (0.1 M, 72 hours), temsirolimus (T) (0.5 M, 72 hours) and everolimus (E) (0.05 M, 72 hours), in isolation or combined: gemcitabine, cisplatin and temsirolimus (GCT), and gemcitabine, cisplatin and everolimus (GCE). After incubation, cells were treated with Ki-67 antibody (proliferating cells stain the nuclei in brown), acridine orange staining (green: cytoplasm and nucleus cells; red: acidic compartments) and M30 CytoDEATH antibody (cell nuclei were counterstained with 4’,6-diamidino-2-phenylindole; apoptotic cells are visible due to their green colour). Original magnification: Ki-67 400 ×; acridine orange 400 ×; M30 CytoDEATH 200 ×.
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Table 2 Cell cycle analysis of the 5637, HT1376 and T24 urinary bladder cancer cell lines after exposure to gemcitabine (G), cisplatin (C), temsirolimus (T) and everolimus (E), in isolation or in combined schedules (GCT, GCE). The percentages of cells in each cell cycle phases are mean ± SD of 3 independent experiments. SD = standard deviation. 5637
HT1376
G0 /G1 Control C G T E GCT GCE
63.0 21.8 42.7 61.9 60.8 73.1 66.5
± ± ± ± ± ± ±
S 1.2 0.9 1.6 0.4 1.6 1.1 1.7
8.8 48.0 16.9 11.1 9.9 10.1 15.4
G2 /M ± ± ± ± ± ± ±
0.2 1.7 2.1 0.1 1.4 0.1 0.4
8.6 14.4 5.0 10.0 8.3 6.9 9.0
G0 /G1 ± ± ± ± ± ± ±
1.8 1.2 0.4 0.6 2.8 1.1 0.5
69.1 67.2 64.3 74.5 76.3 48.9 51.1
± ± ± ± ± ± ±
T24 S
1.5 1.1 1.2 1.3 0.3 0.1 1.2
4. Discussion In the last years, mTOR inhibitors have raised many perspectives in cancer treatment [25,26]. The temsirolimus and everolimus shared the same sirolimus mechanism of action, specifically binding to the abundant intracellular protein FK506-binding protein 12 (FKBP12), inhibiting the activation of mTORC1 complex [27]. Subsequently, the phosphorylation of the downstream targets p70S6 K and 4E-BP1 is also inhibited [11,28]. Inhibition of these protein complexes ultimately results in decreased cell growth and proliferation, cellular metabolism and angiogenesis, leading to a cell cycle blockage at the G0 /G1 phase [29]. Park et al. (2011) revealed that mTOR pathway activation, as assessed by 4E-BP1 and/or S6K1 phosphorylation, is involved in urinary bladder cancer tumorigenesis and is a predictor of disease progression and poor cancer-specific survival [30]. We have previously evaluated everolimus and temsirolimus actions on mTOR and Akt, as well as on their phosphorylated forms, in the three cell lines, without encouraging results [18,24]. Therefore, we decided to analyze, the p70S6 K and 4E-BP1 and its phosphorylated forms. We have found a decrease of the phosphorylated 4E-BP1 statistically significant in the 5637 cell line with everolimus. The T24 and HT1376 cell lines did not presented any statistically significant alterations of the expression of the two targets analyzed. It has already been described that the dephosphorylation of S6K1 results in a feedback loop that results in up-regulation of receptor tyrosine kinases or insulin receptor substrate proteins, which then activate the PI3 K and consequently mTOR [31,32]. These results, to some extent, justify the results obtained when we have previously analyzed rapalogs effects in the same three cell lines. We have observed in previous studies and confirmed in this investigation, that everolimus [24] and temsirolimus [18] in isolation induce a slight interference on proliferation, apoptosis and autophagy in the three cell lines used. The one that was slightly sensitive was the 5637 non-muscle invasive cancer cell line. According to the currently accepted knowledge, mTOR pathway interferes with the non-muscle invasive urinary bladder tumors [33] and the 5637 cell line fits in this type of tumors. In several clinical trials, it was found that rapalogs have predominantly led to disease stabilization rather than inhibition of tumor progression [34]. Indeed, the heterogeneity of the results with everolimus has been described [14,24,35]. This drug is still under investigation and in a clinical trial performed by Yao et al. (2013) with advanced pancreatic neuroendocrine tumors, the results obtained suggest that everolimus delays disease progression in the patients analyzed. In patients who experience disease progression, with everolimus, they do not appear to have a more aggressive metastatic phenotype than those patients with placebo [36]. For temsirolimus, the results were also poorly encouraging and some disagreement on the effects obtained with its application has been described [37–39]. However, to enhance the efficacy of rapalogs, evaluation for potential effect with other classes of antineoplastic
G2 /M
5.4 15.0 7.6 5.8 5.4 26.1 25.5
± ± ± ± ± ± ±
0.2 0.6 0.7 0.3 0.3 0.5 0.7
15.2 10.7 19.0 11.9 10.5 17.1 16.2
G0 /G1 ± ± ± ± ± ± ±
0.9 0.5 0.7 0.9 1 0.4 0.7
87.5 67.8 34.1 81.2 83.1 72.6 66.1
± ± ± ± ± ± ±
S 1.7 1.2 1.1 2.9 0.1 0.4 1.9
4.3 16.2 43.7 8.7 7.5 15.6 18.5
G2 /M ± ± ± ± ± ± ±
0.7 0.3 0.3 1.2 0.1 0.3 1.2
31.2 29.1 30.6 30.9 30.8 31.2 31.1
± ± ± ± ± ± ±
0.8 2.1 0.6 1.1 0.2 0.3 1.1
drugs has been suggested [40]. Multidrug combination is the corner stone of treatment for widely neoplastic diseases. We already know that these two mTOR inhibitors (temsirolimus and everolimus), when combined with cisplatin or gemcitabine improved their effects, concerning proliferation inhibition, increased apoptosis and autophagy in the three urinary bladder cancer cell lines [16,17]. As we found that the simultaneous combination of gemcitabine (72 hours) and cisplatin (24, 48 and 72 hours) were effective in reducing the cell proliferation in the cell lines tested, we decided to analyze the effect of 48 hours cisplatin incubation (an average of IC55 in the three cell lines) with gemcitabine (72 hours) and temsirolimus or everolimus (24, 48 and 72 hours). As far as we know, this is the first study combining these three drugs in urinary bladder cancer cell lines. Concerning the gemcitabine, cisplatin and temsirolimus conjugation an enhanced inhibition of cell proliferation was obtained in the three cell lines, with the three different incubation temsirolimus times, being the 72 hours incubation the most effective one. With the gemcitabine, cisplatin and everolimus approach, a similar pattern response was obtained in muscle invasive urinary bladder cancer cell lines (HT1376 and T24). Indeed, in the 5637 non-muscle invasive urinary cell line, the addition of everolimus to gemcitabine and cisplatin did not increase the antiproliferative effects of the antineoplastic drugs conjugation. Analyzing the cell cycle distribution, a pronounced cell cycle arrest in the G0 /G1 phase was found in the 5637 cell line, and an accumulation of early S-phase was observed in the HT1376 and T24 cell lines after exposure to gemcitabine, cisplatin and temsirolimus or everolimus. With the others technical approaches used (immunocytochemistry, flow cytometry, acridine orange staining and M30 CytoDEATH antibody) the results obtained were in agreement. There was a reduced Ki-67 expression, a higher percentage of apoptotic and autophagic cells either in the gemcitabine, cisplatin and temsirolimus or in gemcitabine, cisplatin and everolimus combination. Despite the many studies that have been conducted, the results have not been promising and have demonstrated that rapalogs, as single drugs, have not been very effective in the treatment of urinary bladder cancer. Although the apparent lack of cellular response to the rapalogs application, when cells are also exposed to antineoplastic drugs, the effect is remarkable. It seems that the rapalogs incubation in combination with antineoplastic drugs, the cells became more susceptible and with a lower ability to resist. For some patients the side effects of getting more than one antineoplastic drug might be too much to handle. Chemotherapy for urinary bladder cancer can be hard to tolerate, especially for older patients who have other serious medical complications. On the other, it should be considered that the rapalogs may cause some unwanted effects. In the treatment of advanced urinary bladder cancer, gemcitabine in low dose and prolonged infusion in combination with cisplatin is not inferior to high-dose short infusion gemcitabine and cisplatin in terms of overall survival, time to disease progression, and response rates with favorable toxicity profile.
Please cite this article in press as: Pinto-Leite R, et al. Treatment of muscle invasive urinary bladders tumors: A potential role of the mTOR inhibitors. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.003
G Model BIOMAG-139; No. of Pages 10 10
ARTICLE IN PRESS R. Pinto-Leite et al. / Biomedicine & Aging Pathology xxx (2014) xxx–xxx
The great heterogeneity response detected in the muscle invasive urinary bladder cancer treatment, is a consequence of the complexity of the molecular pathways involved in urinary bladder cancer onset, combined with the several genetic and epigenetic events that occur during tumor progression [41–43]. According to the Cancer Genome Atlas Research Network (2014) [19], 42% of the tumours have mutations, copy number alterations or RNA expression changes affecting the PI(3)K/AKT/mTOR pathway including mutation or deletion of TSC1 or TSC2. These specific gene alterations will be responsible for the 9% of potential responses to mTOR inhibitors. Based on this data we intend to analyze TSC1 and TSC2 expression, hoping that this will explain the behavior of the cell lines tested. 5. Conclusions The results presented in this study are very encouraging, since the simultaneous combination of mTOR inhibitors (either with temsirolimus or everolimus) with classical antineoplastic drugs used in the treatment of muscle invasive urinary bladder cancer, by using lowest concentration of each drug, induced an increased sensibility in the cancer cell lines to the antineoplastic drugs. This positive response can be useful to decrease the toxicity of the therapy keeping, or even improving, the effectiveness in treatment of muscle invasive urinary bladder cancer patients. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments The authors express their deepest appreciation to Professor Ana Maria Nazaré Pereira for the availability of the Elisa reader. References [1] Mitra AP, Castelao JE, Hawes D, Tsao-Wei DD, Jiang X, Shi SR, et al. Combination of molecular alterations and smoking intensity predicts bladder cancer outcome: a report from the Los Angeles Cancer Surveillance Program. Cancer 2013;119:756–65. [2] Rouprêt M, Babjuk M, Compérat E, Zigeuner R, Sylvester R, Burger M, et al. European guidelines on upper tract urothelial carcinomas: 2013 update. Eur Urol 2013;63:1059–71. [3] Cheung G, Saha A, Billia M, Dasgupta P, Khan MS. Recent advances in the diagnosis and treatment of bladder cancer. BMC Med 2013;1:13. [4] Eldefrawy A, Soloway MS, Katkoori D, Singal R, Pan D, Manoharan M. Neoadjuvant and adjuvant chemotherapy for muscle invasive bladder cancer: the likelihood of initiation and completion. Indian J Urol 2012;28:424–6. [5] Ismaili N, Amzerin M, Flechon A. Chemotherapy in advanced bladder cancer: current status and future. J Hematol Oncol 2011;4:35. [6] Kim JJ. Recent advances in treatment of advanced urothelial carcinoma. Curr Urol Rep 2012;2012:147–52. [7] Witjes JA, Compérat E, Cowan NC, De Santis M, Gakis G, Lebret T, et al. EUA Guidelines on muscle invasive and metastatic bladder cancer: summary of the 2013 Guidelines. Eur Urol 2014;65:778–92. [8] Siefker-Radtke A. Bladder cancer: can we move beyond chemotherapy? Curr Oncol Rep 2010;12:278–83. [9] Edeline J, Loriot Y, Culine S, Massard C, Albiges L, Blesius A, et al. Accelerated MVAC chemotherapy in patients with advanced urinary bladder cancer previously treated with a platinum-gemcitabine regimen. Eur J Cancer 2012;48:1141–6. [10] Miller RP, Tadagavadi RK, Ramesh G, Brian W. Mechanisms of cisplatin nephrotoxicity. Reeves Toxins 2012;2:2490–518. [11] Ching CB, Hansel DE. Expanding therapeutic targets in bladder cancer: the PI3 K/Akt/mTOR pathway. Lab Invest 2010;90:1406–14. [12] Sun CH, Chang YH, Pan CC. Activation of the PI3 K/Akt/mTOR pathway correlates with tumor progression and reduced survival in patients with urothelial carcinoma of the urinary bladder. Histopathology 2011;58:1054–63. [13] Strimpakos AS, Karapanagiotou EM, Saif MW, Syrigos KN. The role of mTOR in the management of solid tumors: an overview. Cancer Treat Rev 2009;35:148–59. [14] Garcia JA, Danielpour D. Mammalian target of rapamycin inhibition as a therapeutic strategy in the management of urologic malignancies. Mol Cancer Ther 2008;27:1347–54.
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Please cite this article in press as: Pinto-Leite R, et al. Treatment of muscle invasive urinary bladders tumors: A potential role of the mTOR inhibitors. Biomed Aging Pathol (2014), http://dx.doi.org/10.1016/j.biomag.2014.03.003