Novel Anticancer Mechanism of Chamuangone through the Inhibition of Oncogenic Protein-Driven Oxidative Stress

To evaluate the anticancer mechanisms of Chamuangone against cholangiocarcinoma (CCA) cells.

Chamuangone was tested for cytotoxicity against KKU-100 and KKU-452 cells for 24 and 48 h. Apoptosis, cell proliferation, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels were assessed using Annexin V, Ki-67, JC-1 assays, and DCFH-DA fluorescence probe, respectively. Oncology proteins expression was measured.

Chamuangone inhibited CCA cell growth in a dose- and time- dependent manner, with IC50 values in KKU- 100 cells of 1.175 and 0.331 μg/mL at 24 and 48 hours, respectively; in KKU- 452 cells, the IC50 values were 1.208 and 0.428 μg/mL. Consequently, Chamuangone at 1.5 and 3.0 μg/mL effectively induced both early and late apoptosis in a statistically significant manner, which correlated with a marked reduction in cell proliferation, as evidenced by the decrease in Ki-67 positive populations to 49.04% and 17.02%, respectively. Chamuangone at concentrations of 0.75, 1.5, and 3.0 μg/mL significantly induced mitochondrial dysfunction by reducing the Red/Green fluorescence ratio across all time points (3-24 h), indicating a loss of mitochondrial membrane potential that triggers apoptosis. The induction of intracellular oxidative stress was  indicated by a significant increase in the high-dose group of Chamuangone. Moreover, it also suppressed the expression of key ROS- and oxidative stress-associated oncogenic proteins, including Carbonic Anhydrase IX, Enolase2, CXCL8/IL-8, Galectin-3, EGFR/ErbB1, Progranulin, FGF basic, Dkk-1, p27/kip1, Mesothelin, Survivin, leading to redox imbalance and apoptosis in KKU-100 cells.

Chamuangone inhibits CCA cell proliferation by inducing apoptosis through mechanisms involving the suppression of the Ki67, loss of mitochondrial membrane potential, intracellular ROS accumulation, and downregulation of oncogenic-related proteins involved in proliferation, survival, angiogenesis, and oxidative stress. Thus, Chamuangone has significant potential as a lead compound for the development of novel CCA therapeutics.