Antiandrogen Therapy with Hydroxyflutamide or Androgen Receptor Degradation Enhancer ASC-J9 Enhances BCG Efficacy to Better Suppress Bladder Cancer Progression

Abstract

Recent studies suggest that the androgen receptor (AR) might play important roles in influencing bladder cancer progression, yet its clinical application remains unclear. Here, we developed a new combined therapy with Bacillus Calmette-Guérin (BCG) and the AR degradation enhancer ASC-J9 or antiandrogen hydroxyflutamide (HF) to better suppress bladder cancer progression. Mechanism dissection revealed that ASC-J9 treatment enhanced BCG efficacy to suppress bladder cancer cell proliferation via increasing the recruitment of monocytes/macrophages that involved the promotion of BCG attachment/internalization to the bladder cancer cells through increased integrin-α5β1 expression and IL6 release. Such consequences might then enhance BCG-induced bladder cancer cell death via increased TNFα release. Interestingly, we also found that ASC-J9 treatment could directly promote BCG-induced HMGB1 release to enhance the BCG cytotoxic effects for suppression of bladder cancer cell growth. In vivo approaches also concluded that ASC-J9 could enhance the efficacy of BCG to better suppress bladder cancer progression in BBN-induced bladder cancer mouse models. Together, these results suggest that the newly developed therapy combining BCG plus ASC-J9 may become a novel therapy to better suppress bladder cancer progress.

Figure 1

HF/ASC-J9^® promotes BCG attachment/internalization through regulating the integrin-α5β1 pathway in BCa cells. (A) Structure of ASC-J9^®.(B&C) We seeded 4×10⁵ T24 and 253J cells into plates. Cells were treated with 5 μmol/L and/or 5 μmol ASC-J9^® for 12 h, then incubated with BCG (2×10⁷ CFU) for 2 h. Excess BCG was washed away and genomic DNA was extracted to perform PCR using BCG primers. (D&E) The T24 and 253J cells were treated as in Fig. 1 B&C, RNA was extracted, and integrin-α5β1 expression was determined using Q-PCR. (F&G) We seeded 4×10⁵ T24 and 253J cells into the plates. Cells were treat with 5μM HF and ASC-J9^® for 12 h, then incubated with BCG (2×10⁷ CFU) and integrin-α5β1 neutralizing antibody for 2h. After incubation, excess BCG was washed out by 1xPBS 3 times and genomic DNA collected to perform PCR using BCG primers. Each experiment was performed in triplicate. *P<0.05(Student’s t test)

Figure 2

HF/ASC-J9^® promotes monocyte migration toward BCG treated bladder cancer cells. (A) 5×10⁴ BCa cells were seeded into the bottom chambers of transwells and treated with HF or ASC-J9^® for 12 h, then BCG was added and incubated for additional 2 h. After washing out excess BCG, 4×10⁵ THP-1 cells were added into the upper chambers and then co-incubated with BCa cells for 2 h. The media of bottom chambers were collected to count the migrated THP1 cells. (B&C) THP-1 cell migration assay were detected in T24 and 253J cell. Each experiment was performed in triplicate. (D–G) HF and ASC-J9^® increased BCG induced IL-6 release from BCa cells. We seeded 4×10⁵ T24 and 253J cell into the plates. Cells were treated with 5μM HF or 5μM ASC-J9^® in the presence of 10 nM DHT for 12 h, and then incubated with BCG(2×10⁷ CFU) for 2 h. After washing away excess BCG, mRNA was extracted from BCa cells to determine IL-6 expression using Q-PCR. Each experiment was performed in triplicate. *P<0.05(Student’s t test)

Figure 3

Monocytes/macrophages recruitment to BCa cells under BCG plus ASC-J9^®/HF treatment lead to decreased proliferation of BCa cells through TNF-α production. (A) 10⁶ THP-1 cells were cultured in 6-well plates, treated with BCG only, BCG+ASC-J9^®, or BCG+HF for 48 h, Those 3 different conditioned media (CM) were collected to detect TNF-α production by Elisa. (B&C) The above condition mediums were used to treat T24 and 253J cells co-cultured with the THP-1 cells and cell viabilities were detected by MTT. Each experiment was performed in triplicate. *P<0.05(Student’s t test)

Figure 4

HF and ASC-J9^® potentiates the antitumor effects of BCG in vitro.(A–D) Four different BCa cells were seeded in the 24-well plates, treated with BCG only, HF, ASC-J9^®, or BCG plus HF or ASC-J9^®, then collected the cells, then MTT assay were performed. (E) Above CM were collected and used to detect HMGB1 release by Elisa. *P<0.05(Student’s t test)

Figure 5

ASC-J9^® potentiates the antitumor effects of BCG in BBN-induced mouse BCa model. 12 weeks old FVB female mice were divided into 4 groups (10 mice/group) and treated with 0.05% BBN in drinking water for 12 weeks. Mice were then injected with 1) vehicle control, 2) BCG (2×10⁶ CFU/mouse, intravesical injection weekly) alone, 3) ASC-J9^® (75 mg/kg body weight, every other day, IP) alone and 4) BCG (2×10⁶ CFU/mouse, intravesical injection weekly)+ASC-J9^® (75 mg/kg body weight, every other day, IP). Mice were then sacrificed 48 h after the 4^th injection and bladders collected for further examination including (A)H&E section and (B) BrdU stain for the BCa cell proliferation.(C)We used monocytes/macrophages marker F4/80 antibody to assay monocyte/macrophage cells infiltrate. (D&E) The positive expression cells of BrdU and F4/80 were quantified in three random fields. Bule arrows mean positive cells. *P<0.05(Student’s t test)