Center for Cancer Prevention and Drug Development, Stephenson Cancer Center, Hem-Onc Section, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Stephenson Cancer Center, Hem-Onc Section, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Cells. 2024 Aug 31;13(17):1463. 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.
膀胱癌（BCa）是最致命的泌尿生殖系统恶性肿瘤之一，因其易于复发和生存不良。生化途径信号转导和转录激活因子 3（STAT3）已成为促进增殖、侵袭和化疗耐药性的分子途径而受到重视。在这项研究中，我们探索了 TTI-101 和 SH5-07 对 BCa 中 STAT3 的靶向作用，并在三维（3D）球体和类器官模型中阐明了机制。我们优化了来自人、大鼠和小鼠 BCa 细胞系（分别为 J82、NBT-II 和 MB49）的球体和来自 BBN（N-丁基-N-（4-羟基丁基）-亚硝胺）诱导的大鼠膀胱癌的类器官的生长。使用 MTT 和台盼蓝测定法评估细胞活力。使用细胞内 ATP 产生、ROS 产生和钙 AM（CA）/EtBr 染色来测量球体和类器官的抑制作用和线粒体功能。通过 Western blot 分析评估与细胞增殖、细胞凋亡、癌症干细胞（CSCs）和 BCa 中 STAT3 信号相关的药效标志物。我们发现，靶向 STAT3（使用 TTI-101 和 SH5-07）可显著降低 BCa 球体和类器官的增殖，同时降低 pSTAT3、Cyclin D1 和 PCNA 的表达。我们的数据还表明，用 STAT3 抑制剂处理会在 BCa 球体和类器官中诱导 ROS 产生和细胞死亡。STAT3 抑制剂诱导的细胞死亡与 caspase 3/7 和 PARP 切割的激活有关。此外，TTI-101 和 SH5-07 通过下调 3D 模型中 CD44 和 CD133 的表达来靶向癌症干细胞。这项研究首次提供了使用小分子抑制剂 TTI-101 和 SH5-07 通过抑制 CSCs 和 STAT3 信号来预防 BCa 的证据。
