Eon Ji Yeo¹, Myung Chul Cha², Dong Seok Lee^{1, 3,}

The anti-cancer effects of the ethyl acetate (EtOAc) fraction (E-OHS) from the ethanol extract of Opuntia humifusa against A-549 human lung cancer cells were investigated. Selective cytotoxicity was assessed in RAW 264.7 cells and A-549 cells using the MTS assay. After Annexin V-FITC/PI staining, apoptosis induction was confirmed by flow cytometry, and apoptotic nuclear fragmentation was examined using DAPI staining. Furthermore, induction of cell cycle arrest was confirmed by flow cytometry after PI staining, and anti-metastasis was evaluated using wound healing assay. The molecular mechanisms underlying apoptosis, cell cycle arrest, anti-metastasis, and upstream signaling were elucidated using western blot. E-OHS induced anti-cancer activity in a dose-dependent manner in A-549 cells, whereas it did not exhibit cytotoxicity in RAW 264.7 cells. Annexin V-FITC/PI and DAPI assays showed that number of apoptotic bodies increased in a dose-dependent manner. Western blot analysis showed that pro-caspase-9, pro-caspase-3, and PARP decreased, whereas cleaved-caspase-12, Bax, and cleaved PARP increased. This suggests that apoptosis was jointly induced through mitochondrial-mediated intrinsic pathway and endoplasmic reticulum (ER) stress-related pathway. Flow cytometry analysis showed that the distribution of G1/S phase cells increased in the E-OHS-treated group, whereas the distribution of G2/M phase cells decreased, confirming that E-OHS induced cell cycle arrest by inhibiting transition of G1/S phase. Western blot analysis reconfirmed at the molecular level that the cell cycle was arrested in the G1/S phase, as supported by the decrease in CDK2, CDK4, and cyclin A2. Wound-healing assay showed that the migration ability of A-549 cells was inhibited with E-OHS, and western blot analysis confirmed the decrease in integrin β1, MMP-9, and HIF-1α, demonstrating that E-OHS inhibits A-549 cell metastasis. Finally, western blot analysis showed a dose-dependent decrease in ERK1/2, confirming that induction of apoptosis and cell cycle arrest, as well as anti-metastasis, are influenced by upstream signaling pathway.

anti-cancer, ethyl acetate fraction, apoptosis, cell cycle arrest, anti-metastasis, Opuntia humifusa