Effect of sirolimus on urinary bladder cancer T24 cell line

Bladder cancer is the second most common urologic malignancy and accounts for approximately 90% of cancers of the urinary tract. Is the fourth most incident cancer in male and ninth in females [1]. In industrialized countries, more than 90% of cases are originate in the urothelial epithelial cells (called urothelial cell carcinoma) [2]. At diagnosis, 75% are non-invasive bladder cancer. The invasive bladder cancers may spread outside the bladder and affect other organs. Bladder cancer's staging, treatment and prognosis depend on how deeply it has invaded urinary bladder [3].

Fortunately, about 80% of patients with non-muscle invasive disease can be successfully treated using the surgery. Historically, two-thirds of patients have tumour recurrence within 5 years. High-grade tumours have a significantly worse prognosis. Both high-grade T1 tumours and carcinoma in situ have the potential to progress and even metastasize [4]. Patients with invasive bladder cancer require a radical cystectomy. Controversy exists as to whether neoadjuvant or adjuvant chemotherapy improves survival in patients with invasive bladder cancer, despite a number of randomised controlled trials. So far there are no data to confirm what is the best combination of treatments (neoadjuvant chemotherapy, adjuvant with or without radiotherapy) to treat invasive bladder cancer [5]. The modest results with currently drugs, suggest the urgent need to identify new agents [6]. Sirolimus is a macrocyclic lactone that was first discovered as a product of the soil bacteria Streptomyces hygroscopicus. It was originally used as an immunosuppressant drug to help prevent rejection in organ transplantation, particularly in kidney transplant operations, but the authors of a number of recent reports have indicated that it may have other potential biological effects as an anti-cancer medicine [7, 8]. Both the immunosuppressive and anti-cancer properties of sirolimus are due to the inhibition of the mammalian target of the sirolimus (mTOR) signalling pathway, which controls mRNA translation and induces angiogenesis and cell proliferation. Angiogenesis and a high proliferative index correspond to a poor prognosis for urothelial bladder cancer patients [9, 10]. Sirolimus forms a complex with the immunophilin prolyl isomerase FK binding protein complex (FKBP-12) that binds with high affinity to mTOR [11, 12]. This interaction inhibits mTOR kinase activity and subsequently decreases the phosphorylation of 4E binding protein-1 and the inhibition of the 40S ribosomal protein p70 S6 kinase [13–15]. Sirolimus's antineoplasic effects have been related to its capacity to inhibit the translation machinery involved in the regulation of G1- to S-phase transition in cell cycle [16, 17]. Cell growth and proliferation in numerous cancer types are often regulated by the mammalian target of sirolimus (mTOR) pathway through p7056 kinase, ribosomal S6 protein, and eukaryotic initiation factor 4 E-binding protein 1 [18]. Recently there has been an enormous increase in our understanding of the molecular mechanisms underlying sirolimus's therapeutic anti-cancer properties. Alterations in the pathway regulating mTOR occur in many solid malignancies including bladder cancer. In vitro and in vivo models of bladder cancer have established the importance of the mTOR pathway in controlling cancer progression and metastasis [19]. The T24 cell line has been established from a highly malignant grade III human urinary bladder carcinoma [20]. This cell line can be easily grown in vitro and has been extensively used to evaluate the therapeutic effects of several anticancer drugs. Here, we describe the preliminary results of the study of the therapeutic effect of sirolimus against human T24 bladder cancer cell line in vitro using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for assessing cell proliferation and Trypan blue for assessing cell viability.

The findings of the present study revealed that sirolimus inhibits T24 bladder cancer cell proliferation and decrease the cell viability including in clinical dose of this mTOR inhibitor. These data may be relevant if we remember the action of the mTOR pathway. mTOR is a 290 kDa serine-threonine kinase that regulates both cell growth and cell cycle progression through its ability to integrate signals from nutrient and growth factor stimuli [24]. Tumour angiogenesis may depend on mTOR signalling. Hypoxia induces production of vascular endothelial cell growth factor (VEGF) by tumour and stromal cells which may be partly controlled by mTOR signaling and through PI3K-Akt-mTOR pathway [25]. Thus, the anti-tumour effects noted by inhibiting mTOR may be related to antiproliferative effects within tumour cells as well endothelial cells. Upstream effectors that signal through mTOR may up-regulate mTOR gene. Wu X et al (2004) showed that a specific inhibitor of PI3 kinase enzyme activity, Ly294002, potently suppressed the invasive properties of three highly invasive bladder tumour cell lines and 55% of primary tumours from patients with bladder cancer had markedly high levels of phosphorylated Akt [26]. Thus, the inhibition of mTOR may inhibit abnormal cell proliferation, tumour angiogenesis, and abnormal cell metabolism and potentially enhance the efficacy of other cancer treatments. The biological mechanisms responsible for anti-proliferative effect of sirolimus and the role of PI3K-Akt-mTOR pathway are under investigation [27]. Tanaka and Grossman (2003) demonstrate that PTEN can induce growth suppression and increase sensitivity to doxorubicin in bladder cancer cells and suggest that the PTEN gene and its pathways can be therapeutic targets for bladder cancer. To emphasize that, no results have been yet published on the activity of mTOR inhibitors against T24, or other, bladder cancer cell lines. Luan FL et al. (2002) showed that, sirolimus treatment alone, or with cyclosporine, prolonged the survival of mice inoculated with renal cancer cells or T24 human bladder cancer cells [28]. This is an indirect assumption of sirolimus effect against T24. The present study is the first address this issue. In the other hand, our team observed similar results, when we studied the effects of sirolimus in chemical induced urothelial cancers in ICR mice (data submitted). Sirolimus has been shown to inhibit the proliferation of various tumour cell lines including rhabdomyosarcoma, neuroblastoma, glioblastoma, small cell lung cancer, osteosarcoma, pancreatic cancer, breast cancer, prostate cancer, murine melanoma, leukaemia, and B-cell lymphoma [29–33].

Sirolimus enhances the anti-tumour effect of gemcitabine [34]. Now we intend to verify the efficacy of sirolimous mTOR inhibition, in other bladder cancer cell lines (5637, HT1376 and MC). Clinical results show that mTOR inhibitors are well tolerated and may induce prolonged stable disease and tumour regression in cancer patients [24]. Urgent research is needed to evaluate the real place of sirolimus or similar drugs in urothelial bladder cancer therapeutic.

Sirolimus inhibits T24 bladder cancer cell proliferation and decrease cell viability including in clinical dose, therefore should be considered to be a promising agents against bladder cancer. However, more positive data will be necessary.