In vitro cytotoxic activity on KATO-III cancer cell lines of mangiferolic acid purified from Thai Tetragonula laeviceps propolis

Abstract

Gastric cancer is a global health concern, but current treatment with chemotherapy and surgery is often inadequate, prompting the exploration of alternative treatments. Propolis is a natural substance collected by bees known for its diverse properties linked to floral sources. The Dichloromethane Partitioned Extract (DPE) from Tetragonula laeviceps propolis, in Bankha district, Thailand was previously shown to possess significant cytotoxicity against KATO-III gastric cancer cells, while showing lower cytotoxicity toward WI-38 normal fibroblast cells. Here, the DPE was further fractionated by column chromatography, identified active fractions, and subjected to structural analysis using nuclear magnetic resonance spectroscopy. Cytotoxicity against KATO-III cells was reevaluated, and programmed cell death was analyzed using flow cytometry. Expression levels of cancer-related genes were measured using quantitative real-time reverse transcriptase PCR. Cardol C15:2 (compound 1) and mangiferolic acid (MF; compound 2) were discovered in the most active fractions following structural analysis. MF exhibited strong cytotoxicity against KATO-III cells (IC50 of 4.78–16.02 μg/mL), although this was less effective than doxorubicin (IC50 of 0.56–1.55 μg/mL). Morphological changes, including decreased cell density and increased debris, were observed in KATO-III cells treated with 30 μg/mL of MF. Significant induction of late-stage apoptosis and necrosis, particularly at 48 and 72 h, suggested potential DNA damage and cell cycle arrest, evidenced by an increased proportion of sub-G1 and S-phase cells. Doxorubicin, the positive control, triggered late apoptosis but caused more necrosis after 72 h. Furthermore, MF at 30 μg/mL significantly increased the expression level of COX2 and NFκB genes linked to inflammation and cell death pathways. This upregulation was consistent at later time points (48 and 72 h) and was accompanied by increased expression of CASP3 and CASP7 genes. These findings suggest MF effectively induces cell death in KATO-III cells through late apoptosis and necrosis, potentially mediated by upregulated inflammation-related genes.