mTOR inhibitors show promising in vitro activity in bladder cancer and head and neck squamous cell carcinoma.

Bladder cancer and head and neck squamous cell carcinoma (HNSCC) are frequent but lack efficient therapies especially in advanced disease. Almost no studies on mTOR function and inhibition in these tumor entities have been reported. We examined the gene and protein expression levels of mTOR and its activated form (pmTOR) in three human bladder carcinoma cell lines (RT-4, T24, EJ28) and three HNSCC cell lines (PCI-1, PCI-13, BHY). Furthermore, the consequences of mTOR inhibition by mTOR-specific siRNAs and the mTOR inhibitor temsirolimus were analysed in vitro using immunohistochemical Ki-67 staining, mTOR and pmTOR western blot analysis, MTT assay, as well as cell cycle analysis with flow cytometry. Especially pmTOR protein expression levels showed marked differences between cell lines. siRNA transfection was associated with dose-dependent target protein reduction but not proliferation inhibition or apoptosis. On the contrary, temsirolimus significantly reduced cell viability and induced apoptosis and cell cycle arrest. According to these data, bladder cancer and HNSCC are promising tumor entities for mTOR inhibition with temsirolimus.

In vivo studies on the availability and toxicity of antisense oligonucleotides in bladder cancer.

BACKGROUND: The urinary bladder is an ideal organ for topical treatment. A substantial number of bladder cancer patients are resistant to conventional intravesical therapy. In search of new agents, antisense oligonucleotides (AS-ON) may be interesting candidates. The availability and toxicity as well as the effectivity of AS-ON after intravesical instillation in different rodent models were examined. MATERIALS AND METHODS: Acute toxicity of AS-ON was tested by intravenous application (215-1,000 mg/kg body weight (bw)) in NMRI mice (n=30). The uptake and distribution of isotope-labelled AS-ON in bladder tissue was determined in Sprague Dawley rats (n=12) by radioactivity after intravesical application (2.5 mg/kg bw 3H-labelled AS-ON). Additionally, uptake and effectivity studies of AS-ON in tumors were performed in MB-49 bladder cancer-bearing C57/B16 mice (n=6) by immunohistochemistry and fluorescence microscopy. RESULTS: No systematic side-effects were noticed after intravenous application of physiological doses of AS-ON in NMRI mice. The mortality rate was 20% at the highest dose of 1,000 mg/kg bw. The highest AS-ON availability after intravesical application in rats was noticed in the bladder wall (12.3 microg/g), while the systemic concentration was low (1.1 microg/g). In fluorescence microscopy analysis, AS-ON were detected in the outer cells of the bladder wall and around vessels. AS-ON accumulated in the cytoplasm and in the nuclei. Immunohistochemical analysis demonstrated a reduction of the Ki-67 positivity after treatment with AS-ON (43%) compared to the untreated controls (58%). CONCLUSION: These preclinical experiments have shown that intravesical antisense oligonucleotides are safe and accumulate in the bladder and in bladder tumors, whereas systemic concentrations remain low. These data are the basis of a first clinical phase I study with intravesical instillation of Ki-67 antisense oligonucleotides.