TLR4- and TLR9-dependent effects on cytokines, cell viability, and invasion in human bladder cancer cells

Objectives: Adjuvant immunotherapy of bladder cancer by instillation of bacillus Calmette-Guérin (BCG) is highly recommended within certain groups of non-muscle-invasive stages but only partially effective. Toll-like receptors (TLRs) TLR4 and TLR9 likely mediate BCG effects by triggering innate systemic immune cell responses. In addition, TLR4 and TLR9 expressed in bladder cancer cells may contribute to the outcome of BCG treatment. Here, we studied the expression and function of TLR4 and TLR9 in human bladder cancer cell lines.

Methods: TLR4 and TLR9 messenger RNA and protein levels were determined by real-time reverse transcription polymerase chain reaction and Western blot. Selected cell lines were analyzed with respect to cytokine induction, proliferation, and cell invasion after addition of BCG, TLR4-specific agonist lipopolysaccharide (LPS), or TLR9 agonist (CpG-oligodeoxynucleotide [ODN]).

Results: TLR4 and TLR9 were expressed quite heterogeneously in human bladder cancer cells. BCG caused induction of interleukin (IL)-6 or IL-8 in BFTC905 and T24 cells as representatives for TLR4-/TLR9-expressing cells. The study aimed to dissect TLR4- and TLR9-mediated effects. For functional analysis of TLR4 with LPS, we selected T24 and BFTC905 cells with high and undetectable TLR4 levels, respectively. For TLR9 analysis with CpG-ODN, we selected UMUC3 and RT112 cells with high and low TLR9 levels, respectively. Addition of LPS caused significant induction of TNFα and IL-6 messenger RNA in T24 cells but not in BFTC905 cells. Addition of CpG-ODN induced interferon ß (INFß), IL-8, tumor necrosis factor α (TNFα) and the angiogenic factors vascular endothelial growth factor-A and placental growth factor in UMUC3 cells; whereas in RT112 cells, induction of IL-8 and TNFα was noticed. Interestingly, addition of CpG-ODN significantly reduced cell viability and increased cell invasion in UMUC3 and RT112 cells.

Conclusions: Our findings demonstrate that bladder cancer cell lines express functional TLR4 and TLR9 with possible effects on cancer progression and outcome of BCG-based immunotherapy.