Everolimus Enhances Gemcitabine-Induced Cytotoxicity in Bladder-Cancer Cell Lines

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.

Phospho-mTOR in non-tumour and tumour bladder urothelium: Pattern of expression and impact on urothelial bladder cancer patients

“…In accordance with this hypothesis, mTOR inhibition through rapamycin or rapamycin analogues has been shown to reduce proliferation in in vitro and in vivo UBC models, with corresponding diminished levels of p-S6 (31,32,41). Notably, treatment with mTOR inhibitors has been observed to enhance the therapeutic efficacy of cisplatin and gemcitabine in bladder cancer cell lines (32,42,43), and impair tumour progression when administered intravesically in a bladder cancer mouse model (44). In a phase II study of everolimus treatment in patients with locally advanced or metastatic UBC, clinical activity was demonstrated, and the profile of plasma angiogenesis-related proteins suggested that everolimus exhibits antiangiogenic properties that have a significant function in disease control (45).…”

“…Fahmy et al (56) have recently reported that activation of the mTOR pathway may be used as a predictor of recurrence among patients with high-risk NMI (56). It was shown by Pinto-Leite et al (43) that the effect of everolimus in bladder cancer cell lines, alone or in combination with gemcitabine treatment, produced a significant antiproliferative effect for everolimus in an NMI cell line (5637), while an MI cell line (T24) demonstrated marked resistance. These results, together with the results from the present study, suggest that interfering with the mTOR pathway may represent an appealing approach for therapeutic intervention in patients with NMI tumours.…”

Temsirolimus improves cytotoxic efficacy of cisplatin and gemcitabine against urinary bladder cancer cell lines

“…Concerning its phosphorylation at Ser473, only in the 5637 cell line was a slight increase found. The average IC35 of gemcitabine in the 3 cell lines (100 nM) was determined in our previous investigation [29]. The antiproliferative activity of the combined treatment (gemcitabine at 100 nM plus 500, 1,000, 2,000, and 4,000 nM temsirolimus) was statistically significant in all 3 cell lines at all concentrations tested, when compared with the untreated cells (P o 0.05).…”

Ruthenium porphyrin-induced photodamage in bladder cancer cells

“…Preclinical studies also play an important role in this aspect [32]. Investigations on bladdercancer cell lines is a natural first step to determine the properties of newly developed drugs and to test various treatment strategies [6,[33][34][35][36].…”