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 ARTICLE IN PRESS G Model BIOMAG-139; No. of Pages 10 Biomedicine & Aging Pathology xxx (2014) xxx–xxx Available online at ScienceDirect www.sciencedirect.com 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 b 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 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 dif- ferent 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 phos- phorylated 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 M 30 CytoDEATH antibody. No significant differences in the expression of P-4E-BP1 and P-p70S6 K after tem- sirolimus 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 ana- lyzed (reduced Ki-67 expression, increased autophagy and apoptosis). Also, in the combined regimen, an enhanced cell cycle arrest in the G 0 /G 1 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 carci- nomas, 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 mus- cle 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: pamo@utad.pt, pamooliveira28@gmail.com, paulaalexandra1301@gmail.com (P.A. Oliveira). management of urinary bladder cancer over the past 5 to 10 years [3]. The standard chemotherapy for the treatment of muscle inva- sive 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 com- bination 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.