MP06-03: Discovery and Validation of Nitroxoline as a Novel STAT3 Inhibitor in Drug-resistant Urothelial Bladder Cancer

Peng HUANG*, Wenfeng Lin, Motoo Araki, Masami Watanbe, Yasutomo Nasu, Okayama, Japan

Introduction: Repeated cycles of first-line chemotherapy drugs such as doxorubicin (DOX) and cisplatin (CIS) trigger frequent chemoresistance in recurrent urothelial bladder cancer (UBC), posing a threat to the prognosis of patients. However, there remains lack of effective agents clinically approved for the treatment of chemoresistant UBC. Nitroxoline (NTX), an antibiotic to treat urinary tract infections, has been recently repurposed for the treatment of multiple tumors. Here we aimed to investigate whether NTX suppresses the growth of drug-resistant UBC in vitro and in vivo and the underlying mechanism at the molecular level.

Methods: The drug-resistant cell lines T24/DOX and T24/CIS were established by continual exposure of parental cell line T24 to DOX and CIS, respectively. The drug effects on cell proliferation were measured by XTT assay. Cell cycle distribution was analyzed by PI/RNase staining, while apoptosis was assessed by Hoechst 33342 and Annexin V-FITC/PI staining. The expressions of proteins related to STAT3 signaling, cell cycle, and apoptosis were detected by western blotting. Subcutaneous xenograft models of T24/DOX and T24/CIS were established to determine the in vivo anti-tumor effect of NTX.

Results: T24/DOX and T24/CIS cells were resistant to DOX and CIS respectively, but they were sensitive to NTX in a time- and dose-dependent manner. Overexpressions of STAT3 and P-glycoprotein (P-gp) were identified in T24/DOX and T24/CIS, which could be reversed by NTX. Furthermore, western blotting showed that NTX downregulated the expressions of phosphorylated STAT3, c-Myc, Cyclin D1, CDK4, CDK6, Bcl-xL, Mcl-1, and Survivin, which were further confirmed by Stattic. In vivo, NTX exhibits the significant anti-tumor effect in T24/DOX and T24/CIS tumor-bearing mice. These results suggested that NTX-induced G0/G1 arrest, apoptosis, and P-gp reversal in drug-resistant UBC were mediated by inhibition of STAT3 signaling.

Conclusions: Our findings repurpose NTX as a novel STAT3 inhibitor to induce P-gp reversal, G0/G1 arrest, and apoptosis in drug-resistant UBC.

Source of Funding: This research was funded by the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant No. 17K11138, 21K09371),