Ganoderic acid a potentiates cisplatin's cytotoxicity on gallbladder cancer cells by promoting DNA damage and inhibiting cell stemness.

BACKGROUND: Ganoderma acid A (GAA), a triterpenoid compound from Ganoderma lucidum, has gained attention for its anti-tumor properties. Herein, we hypothesized that GAA may enhance cisplatin's (DDP) anticancer effect in gallbladder cancer (GBC) cells by promoting DNA damage response, particularly through upregulation of DNA damage markers such as γH2AX, p-ATM, p-ATR, and p-p53, and reducing cell stemness by downregulating stemness markers like SOX2, Oct4, and NANOG. MATERIALS AND METHODS: The human GBC cell line GBC-SD and human gallbladder epithelial cell line HGBEC were cultured in RPMI-1640 and DMEM/F12 media with 10% fetal bovine serum. Cells were treated with 2 µM DDP and 60 µM GAA for 24 h. To evaluate the toxicity of GAA in normal cells, HGBEC cells were treated under the same conditions. Cell viability was assessed by CCK-8 assay, and colony formation was measured in 6-well plates. Apoptosis was evaluated by TUNEL assay, and DNA damage was assessed using comet assay. Stemness was analyzed by spheroid formation and CD44 immunofluorescence staining. Western blot analysis was performed to evaluate the expression of apoptotic, stemness, and DNA damage markers (Bax/Bcl-2, cleaved-caspase 3, SOX2, Oct4, NANOG, γH2AX, p-ATM, p-ATR, p-p53). RESULTS: The results showed that GAA significantly reduced GBC-SD cell viability in a concentration-dependent manner (p < 0.05). The combined treatment of GAA and DDP further decreased cell viability, with the DDP IC50 value reduced from 8.98 µM to 4.07 µM (p < 0.05). Colony formation was significantly inhibited (p < 0.05), and apoptosis increased, as assessed by TUNEL assay (p < 0.05). Western blot analysis revealed increased pro-apoptotic proteins Bax/Bcl-2 and cleaved-caspase 3(p < 0.05). The expression of stemness markers SOX2, Oct4, NANOG, and DNA damage markers γH2AX, p-ATM, p-ATR, and p-p53 was significantly altered (p < 0.05). Specifically, p53 expression was significantly increased, indicating enhanced DNA damage response (p < 0.05). CONCLUSION: GAA can significantly enhance the anticancer effects of DDP on GBC cells by inhibiting DNA damage response and cell stemness, supporting GAA as an adjuvant treatment for GBC and warrants further validatory preclinical studies.