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

Abstract

Objectives

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.

Methods

Temsirolimus (500, 1,000, 2,000, and 4,000 nM), in isolation, and combined with gemcitabine (100 nM) and cisplatin (2.5 µg/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.

Results

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.

Conclusions

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.

Introduction

Temsirolimus belongs to the second generation of rapamycin derivatives (rapalogs), which has a better bioavailability than rapamycin, and this facilitates its clinical use [1], [2]. It is a selective inhibitor of the mammalian target of rapamycin (mTOR) [3]. This biological pathway regulates some vital cellular processes, such as cell survival, growth, protein synthesis, cellular metabolism, and angiogenesis, all of which are crucial to urothelial bladder cancer progression [4], [5], [6], [7].

In 2007, the US Food and Drug Administration approved temsirolimus as the first-in-class mTOR inhibitors in oncology and as a first-line treatment for renal cell cancer (RCC). In 2012, Sun et al. [8] presented a study in which they tested temsirolimus in phase II studies in East Asian patients with advanced RCC, having obtained promising results. This drug has also been used to treat cancer in multiple clinical studies, such as breast cancer [9], glioma [10], endometrial [11], and mantle cell lymphoma [12], as well as in preclinical studies, such as ovarian, lung, colon, breast, and prostate cell lines [13]. To enhance its action, combinations of temsirolimus with other antineoplastic drugs, such as sorafenib, bevacizumab, and tivozanib, have been explored in clinical trials in patients with metastatic RCC [14]. Temsirolimus and gemcitabine have already been tested in xenograft models of human pancreatic cancer in a promising therapeutic strategy [15]. However, a randomized phase III placebo-controlled trial of letrozole plus oral temsirolimus as a first-line endocrine therapy in postmenopausal women with locally advanced or metastatic breast cancer concluded that adding temsirolimus to letrozole did not improve progression-free survival, as first-line therapy in patients with nonsteroidal aromatase inhibitor-naive advanced breast cancer [16].

In bladder carcinoma, Gerullis et al. [17] applied a combined treatment of temsirolimus and vinflunine with a long-term response in a patient with bladder cancer. Rapamycin alone seems to be an effective drug in urothelial cell lines [18], [19] and its action when used in combination with cisplatin exhibited potential results [20]. Previous studies with other rapalog drugs, such as everolimus, on bladder cancer cell lines showed heterogeneous effects [21], [22]. In addition, rapalogs’ activity has been discussed and it has been suggested that its effectiveness may depend on the drug concentration (nanomolar vs. micromolar) [13] and on the genetic tumor profile, such as a loss of phosphatase and tensin homolog (PTEN) or of von Hippel-Lindau tumor suppressor genes, as well as the overexpression of Akt signaling [23], [24], [25].

In this study, we analyzed the effect of temsirolimus, in isolation and in combination, with cisplatin and gemcitabine (the cytotoxic backbone drugs of several chemotherapeutic treatments against bladder cancer) on 5637, T24, and HT1376 human bladder cancer cell lines, evaluating their action during cell proliferation, apoptosis, autophagy, and cell cycle distribution.