Anti-androgen Therapy With Hydroxyflutamide Or Androgen Receptor Degradation Enhancer ASC-J9(?) Enhances BCG Efficacy To Better Suppress Bladder Cancer Progression

Objective:Intravesical instillation of BCG has been a standard immunotherapy for patients with high-risk non-muscular invasion bladder cancer since 1976,but the BCG therapy still has its limitations.A recent study suggested the potential linkage of androgen receptor(AR)and AR signaling with BCa initiation and progression.Here we found a new potential therapy with combination of BCG and anti-androgen better suppressed BCa progression. Methods:We try to verify our purpose with in vitro and in vivo experiments.In vitro: 1) The expression level of AR in T24 and 253 J measured by western-blot analysis. 2) Set up the experimental groups: the BCG group, the BCG with HF group, the BCG with ASC-J9? group.Treated the BCa cell lines with differernt experiment conditions. Measured the expression level of BCG in BCa cell lines by PCR, and the mRNA expression level of intergin α5β1 in BCa cell lines by Q-PCR. 3)Found the differences in the recruitment of THP-1 by BCa cells using the Migration assay. BCa cells were seeded into the bottom well treated with HF or ASC-J9? for 12 hours, treated with BCG for 2 hours. Then the excess BCG was removed and THP-1 cells placed into the uppe transwell, incubated for 2 hours, then we collected the THP-1 cells migrated into the bottom wells for counting. And next we detected the secretion of immune cell cytokines, such as IL-2, IL-4, IL-6, IL-10, CCL-2 by the different treated BCa cells. 4) Detected the secretion level of TNF-α of THP-1 treated with different drugs by Elisa assay. Collected the conditioned medium(CM) of THP-1 cells, using th CM treat BCa cells, and measured the vability of BCa cells by the MTT assay.In vivo: Set up the experimental groups: the control group, the BCG group, the ASC-J9? group, the BCG with ASC-J9? group. The BBN-induced BCa mice were divided into 4 groups(10 mice/group). Mice were then injected with different drugs as aforesaid. Mice were then sacrificed 48 h after 4th injection and bladders collected for further examination including H&E and BrdU stain and so on. wo also calculated the lifetime of mice in differrnt groups. Results:1. We detected the expression of AR in both T24 and 253 J cells, and the expression of AR decreased when we treated cells by 5μM ASC-J9?.2. We found addition of either 5 μM ASC-J9? or 5μM HF increased BCG internalization near 2 fold. And we found HF or ASC-J9? enhanced significantly the integrin-α5β1 mRNA expression in the two BCa urothelial cell lines.3. We found addition of BCG increased THP-1 cells migration to BCa cells, and addition of HF or ASC-J9? further increased this ability. And addition HF or ASC-J9? further enhanced IL-6 expression in both T24 and 253 J cells.4. Adding BCG to THP-1 cells released more TNF-α compared to THP-1 only, and adding HF or ASC-J9? released even more TNF-α compared to BCG only. The CM collected from THP-1 with HF or ASC-J9? could lower the vibility of BCa cells best.5. Using H&E and BrdU stain, immunohistochemical assay, we found ASC-J9? could enhanced the ability of recruitment of macrophage and suppressed the ability of cell proliferation in BBN-induced mouse model.And ASC-J9? could extend the survival time of mice. Conclusions:1. ASC-J9? and HF enhance BCG attachment/internalization to better suppress the BCa cells.2. ASC-J9? and HF treatment increases IL-6 expression to enhance the recruitmrnt of monocytes/macrophages to the BCa cells.And more recruited monocytes/ macrophages to BCa cells leads to more TNF-α secretion to kill more BCa cells.3. ASC-J9? enhances the BCG efficacy to suppress BCa development in BBN-induced mouse BCa model.