BFTC-909

AF Interferes with Cell Cycle Progression in HT 1376 and BFTC 909 Cells

Urothelial carcinoma (UC) remains a prevalent urinary tract cancer with low five-year survival rates because patients develop resistance to cisplatin and experience cancer recurrence following surgical intervention. Drugs that generate reactive oxygen species (ROS) show potential because cancer cells with high ROS concentrations become susceptible to oxidative stress. Auranofin (AF), which works as an antirheumatic drug, shows anticancer potential through redox balance disruption. The effects of AF treatment on cell cycle progression were assessed by applying 1.5 and 3 µM AF to HT 1376 cells and 2 and 4 µM AF to BFTC 909 cells followed by flow cytometry analysis. Treatment of HT 1376 cells with AF resulted in a G0/G1 phase arrest which was evidenced by the concentration-dependent growth in the number of cells within the G0/G1 phase (Fig. 1A). The G0/G1 phase cell percentage rose from 66.06% to 71.05% at 8 hours while treatment with AF concentrations of 1.5 and 3 µM and then increased from 65.47% to 69.74% at 12 hours and from 57.00% to 65.67% at 24 hours (Fig. 2B). The application of AF resulted in increased levels of p21 and p27 while simultaneously decreasing CDK2, CDK4, cyclin D1, and cyclin E2 in HT 1376 cells (Fig. 2C) AF treatment leads to G0/G1 arrest in the examined cells.

Fig. 1. AF induces G0/G1 cell cycle arrest in HT 1376 urothelial carcinoma cells (Chen S, Chao C, et al., 2021).

Downregulation of GAL1 in UC Cell Lines

UTUC, though rare, is more aggressive than bladder cancer and particularly prevalent in Taiwan. Galectin-1 (GAL1), a protein involved in tumorigenesis, is linked to poorer outcomes in various cancers, but its full role in UTUC is unexplored. This study aims to uncover GAL1's impact on UTUC survival and its oncological pathways. In vitro, GAL1's effect on tumor growth and metastasis was evaluated across four UC cell lines (BFTC-909, T24, J82, and RT4). Figure 2A, B shows that GAL1 mRNA levels were significantly high in BFTC-909, T24, and J82 cells, correlating with elevated GAL1 protein levels, while RT4 cells showed low expression. We chose BFTC-909 and T24, with the highest GAL1 levels, to create GAL1 knockdown lines (sh-Gal1). Figure 2C, D demonstrates significantly reduced GAL1 protein and mRNA in sh-GAL1-T24 and sh-GAL1-BFTC-909 cells, confirming effective LGALS1 gene silencing. Stable knockdown lines were established via lentivirus transfection. Figure 3E–H presents that GAL1 protein and mRNA were notably downregulated in sh-GAL1-RNAi-A and sh-GAL1-RNAi-B BFTC-909 and T24 cells, with RNAi-A achieving higher knockdown efficiency than RNAi-B.

Fig. 2. Evaluation of GAL1 expression in UC cell lines and verification of downregulation efficiency through Western blotting and q-PCR analyses (Su Y-L, Luo H-L, et al., 2020).