Abstract 1678: Overcoming treatment resistance in advanced bladder cancers using a neo-adjuvant combination with a natural product analog
Authors: Seremak Juliette R. , Bonigala Sunilkanth , Gupta Kunj B. , Panda Siva S. , Lokeshwar Bal L. 
Published: April 2023

Abstract
Urinary bladder cancer (BC) is a common cancer worldwide, with approximately 573,278 new cases and 212,536 deaths arising annually. Tumors detected beyond the transitional zone of the bladder (advanced BC) account for most deaths. In advanced BC, tumor heterogeneity and resistance to successive chemotherapy regimens are major contributors to morbidity and mortality. Toxicity associated with the combination of Gemcitabine and Cisplatin (G+C) treatment limits the efficacy of chemotherapy. We tested the potential antitumor activity of a novel combination therapy involving a semisynthetic derivative of the pentacyclic triterpene (PTA) Ursolic Acid (N-methyl piperazine conjugated Ursolate, UA4) and Gemcitabine (Gem) in two Gem resistant BC cell lines developed in the laboratory. UA4 was synthesized from the PTA Ursolic Acid and Gem was purchased from LC-Labs. We tested the response to Gem and UA4 on naïve (WT) and Gem Resistant (GemR) cells. We generated Gem resistant BC cell lines HTB-4 (T24) and HTB-9 (5637) by repeated selection in increased Gem doses in vitro. The surviving cells were tested for a stable resistant phenotype. We measured the sensitivity to Gem and UA4 alone, and in adjuvant and neo-adjuvant combinations by colorimetric, cell counting, or clonogenic assays. The antitumor efficacy of UA4 and Gem were tested in vivo by xenograft studies on Fox N1-/- athymic mice (both sexes). The expression of markers associated with drug resistance was determined by qPCR, immunoblotting, and immunofluorescence studies. The 50% growth inhibition (IC50) dose in Gem treated cells increased significantly in 5637 and T24 GemR cells - 10-fold higher in GemR (100nM) than in WT cells (10 nM). Both WT and GemR cells showed equal sensitivity to UA4 (~5 µM). GemR cells showed elevated mRNA expression of a pyrimidine salvaging enzyme, cytidine deaminase (CDA), which degrades intracellular Gem, MRP2, and ENT1, all related to limiting intracellular drug availability. Tetrahydrouridine (THU), a competitive inhibitor of CDA, reduced the resistance significantly, indicating CDA as the principal mediator of resistance in GemR cells. Oral administration of UA4 was highly effective in reducing tumor growth of 5637 WT and GemR xenografts, whereas Gem (25mg/kg, 2x per week, i.p.) did not significantly reduce growth of GemR xenografts. Gem antitumor activity on 5637 WT tumors was predominantly due to apoptosis. Increased expression of autophagy markers, but not markers of apoptosis, was evident in vitro and in vivo treatments with UA4. Overall, treatment with UA4 was equally effective in decreasing viability in both WT and GemR advanced BC cells in vitro and reducing their tumor growth in vivo. Unlike Gem, UA4 did not induce significant levels of apoptosis but mainly autophagy-induced cell death. Ongoing studies indicate potential to use UA4 as an adjuvant or neo-adjuvant therapy in the treatment of advanced BC.
Citation Format: Juliette R. Seremak, Sunilkanth Bonigala, Kunj B. Gupta, Siva S. Panda, Bal L. Lokeshwar. Overcoming treatment resistance in advanced bladder cancers using a neo-adjuvant combination with a natural product analog [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1678.