BCG-induced cytokine release in bladder cancer cells is regulated by Ca2+ signaling

Abstract

Bacillus Calmette-Guérin (BCG) is widely used in the clinic to effectively treat superficial urinary bladder cancer. However, a significant proportion of patients who fail to respond to BCG risk cystectomy or death. Though more than 3 million cancer treatments with BCG occur annually, surprisingly little is known about the initial signaling cascades activated by BCG. Here, we report that BCG induces a rapid intracellular Ca²⁺ (calcium ion) signal in bladder cancer cells that is essential for activating the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and for synthesizing and secreting proinflammatory cytokines, including interleukin 8 (IL-8). A similar Ca²⁺ response was observed when cells were exposed to the supernatant of BCG. Studying cellular molecular mechanisms involved in the BCG signaling event, we found pivotal roles for phospholipase C and the Toll-like receptor 4. Further assessment revealed that this signaling pathway induces synthesis of IL-8, whereas exocytosis appeared to be controlled by global Ca²⁺ signaling. These results shed new light on the molecular mechanisms underlying BCG treatment of bladder cancer, which can help in improving therapeutic efficacy and reducing adverse side effects.

Keywords: BCG; TLR4; calcium signaling; urinary bladder cancer.

Figure 1

BCG evokes intracellular Ca²⁺ signaling in bladder cancer cells. Primary human bladder cancer cells (A) and human T24 cells (B) exposed to BCG (4 × 10⁶–6 × 10⁷ cfu·mL⁻¹) in a Ca²⁺‐containing buffer exhibit Ca²⁺ signaling. Primary human bladder cancer cells (C) and human T24 cells (D) exposed to BCG (4 × 10⁶–6 × 10⁷ cfu·mL⁻¹) in a Ca²⁺‐free buffer also exhibit Ca²⁺ signaling. (E) The number of human T24 cells responding to BCG with Ca²⁺ signaling was significantly reduced by the inhibitors CPA, 2APB, U73122, ET‐18‐OCH3 (ET‐18), and PTX, whereas wortmannin failed to significantly reduce the number of active cells. Results are means ± SEM of measurements from at least three separate cell cultures. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t‐test).

Figure 2

BCG triggers cytokine release in bladder cancer cells. Primary human bladder cancer cells derived from one male tumor (A) and one female tumor (B) or mouse MB49 cells (C) exposed to BCG (4 × 10⁶–6 × 10⁷ cfu·mL⁻¹) secrete multiple cytokines, as compared to a control group. Results are means ± SEM of measurements from four separate cell cultures. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t‐test)

Figure 3

BCG activates NF‐κB and IL‐8. (A) BCG‐stimulated IL‐8 secretion is reduced when Ca²⁺ signaling is inhibited by 2APB or U73122 (U73). (B) NF‐κB reporter gene assay shows that NF‐κB is fully activated after 6 h of BCG treatment. (C) NF‐κB reporter gene assay shows that the NF‐κB activation is reduced when Ca²⁺ signaling is inhibited by 2APB or U73122 (U73). (D) BCG‐stimulated IL‐8 transcription is blocked when Ca²⁺ signaling is inhibited by 2APB. (E) BCG‐stimulated IL‐8 secretion levels are NF‐κB dependent as the inhibitor Ro106‐9920 hampers IL‐8 secretion in a dose‐dependent manner. Results are means ± SEM of measurements from at least three separate cell cultures. *P < 0.05, **P < 0.01, ***P < 0.001 (one‐way ANOVA)

Figure 4

The BCGsn and TLR4 are key for BCG‐induced Ca²⁺ signaling. Assessment of Ca²⁺ signaling in mouse MB49 cells exposed to BCG preparations BCGsn (A), BCG pellet (B), fBCGsn (C), bBCGsn (D), ppBCGsn (E), or various weight fractions (F). (G) The BCG preparations that triggers Ca²⁺ signaling also stimulate IL‐8 secretion. (H) BCG at dilutions of 1/16, 1/8, 1/4, 1/2, and 1/1 activate IL‐8 in a dose‐dependent manner. TLR4 knockdown with shRNA (shTLR4) abolishes BCG‐induced IL‐8 transcription (I) but not IL‐8 secretion (J), compared to scramble shRNA controls (shControl). Results are means ± SEM of measurements from at least three separate cell cultures. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t‐test).