Stat3 Inhibitors TTI-101 and SH5-07 Suppress Bladder Cancer Cell Survival in 3D Tumor Models

• Published in: Cells (Basel, Switzerland) Switzerland), 2024-08, Vol.13 (17), p.1463
• Main Authors: Singh, Surya P, Pathuri, Gopal, Asch, Adam S, Rao, Chinthalapally V, Madka, Venkateshwar
• Format: Article
• Language: English
• Subjects: 3D models; Animal models; Animals; Apoptosis; Apoptosis - drug effects; Bladder cancer; Calcium (intracellular); Calcium (mitochondrial); Carcinogens; Caspase-3; CD44 antigen; Cell activation; Cell culture; Cell death; Cell Line, Tumor; Cell proliferation; Cell Proliferation - drug effects; Cell survival; Cell Survival - drug effects; Cell viability; Chemoresistance; Chemotherapy; Cyclin D1; Drug development; FDA approval; Humans; Intracellular signalling; Malignancy; Medical prognosis; Metastasis; Mice; Microscopy; Neoplastic Stem Cells - drug effects; Neoplastic Stem Cells - metabolism; Neoplastic Stem Cells - pathology; Nitrosamines; Organoids; Organoids - drug effects; Organoids - metabolism; Organoids - pathology; Pharmacodynamics; Phosphorylation; preclinical; Rats; Signal transduction; Signal Transduction - drug effects; Spheroids; Spheroids, Cellular - drug effects; Spheroids, Cellular - metabolism; Spheroids, Cellular - pathology; STAT3 inhibitor; Stat3 protein; STAT3 Transcription Factor - metabolism; Stem cells; Urinary Bladder Neoplasms - drug therapy; Urinary Bladder Neoplasms - metabolism; Urinary Bladder Neoplasms - pathology
• ISSN: 2073-4409; 2073-4409
• DOI: 10.3390/cells13171463

Bladder cancer (BCa) is one of the most lethal genitourinary malignancies owing to its propensity for recurrence and poor survival. The biochemical pathway, signal transducer and activator of transcription 3 (STAT3), has gained significance as a molecular pathway that promotes proliferation, invasion, and chemoresistance. In this study, we explored the targeting of STAT3 with TTI-101 and SH5-07 in BCa and elucidated the mechanisms in three-dimensional (3D) spheroid and organoid models. We optimized the growth of spheroids from human, rat, and mouse BCa cell lines (J82, NBT-II, and MB49 respectively) and organoids from BBN (N-butyl-N-(4-hydroxybutyl)-nitrosamine)-induced rat bladder tumors. Cell viability was assessed using MTT and trypan blue assays. Intracellular ATP production, ROS production, and calcium AM (CA)/EtBr staining were used to measure the spheroid and organoid inhibition and mitochondrial function. Western blot analysis was performed to evaluate the pharmacodynamic markers involved in cell proliferation, apoptosis, cancer stem cells (CSCs), and STAT3 signaling in BCa. We found that targeting STAT3 (using TTI-101 and SH5-07) significantly reduced the proliferation of BCa spheroids and organoids, which was accompanied by decreased expression of pSTAT3, Cyclin D1, and PCNA. Our data also demonstrated that treatment with STAT3 inhibitors induced ROS production and cell death in BCa spheroids and organoids. STAT3 inhibition-induced cell death was associated with the activation of caspase 3/7 and PARP cleavage. Moreover, TTI-101 and SH5-07 target cancer stem cells by downregulating the expression of CD44 and CD133 in 3D models. This study provides the first evidence for the prevention of BCa with small-molecule inhibitors TTI-101 and SH5-07 via suppression of CSCs and STAT3 signaling.