Inhibitory Effects of Propolis Flavonoids on Migration and Invasion of Laryngeal Cancer Cell and Analysis of Related Signal Pathways

Abstract

Laryngeal cancer (LGC) is a malignant tumor that occurs in the larynx, and it is mainly treated through chemotherapy, radiotherapy, and surgery. Nevertheless, the five-year survival rate for patients is poor. Bee propolis contains various bioactive compounds and abundant anti-tumor active ingredients. Nevertheless, research on the use of propolis extracts for the treatment of LGC is relatively limited. This research aimed to demonstrate the inhibitory effects of ethanol extracts of propolis on migration (Mig) and invasion (Inv ) of LGC cells, as well as the related signaling pathways. The effects of graded ethanol extraction of propolis on the proliferation (Pro), Inv, Mig, apoptosis (Apo), and related signaling pathways of Hep-2 cells were analyzed. Propolis was extracted using ethanol (0%, 25%, 50%, 75%, and 100%) for the graded extraction of crude propolis. The flavonoid content and yield of the extracts were determined. The effects of various concentrations of propolis flavonoids on the clearance of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, O2- radicals, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radicals were evaluated, as well as their effects on the Pro inhibition of normal human pancreatic ductal epithelial (hTERT-HPNE) cells. Hep-2 cells of LGC were cultured using media containing 0, 25, 50, and 100 μmol/L propolis flavonoids. The cell Pro activity, Inv, Mig, Apo, and expression of PI3K/Akt pathway-related proteins were evaluated using CCK-8 assay, Transwell chamber assay, acridine orange/ethidium bromide (AO/EB) double staining method, and Western blotting, respectively. It was revealed that extraction with 50% ethanol solution yielded a higher content and yield of flavonoids, which were 51.20% and 7.42%, respectively. As the concentration of propolis flavonoids increased, the clearance rates of DPPH, O2-, and ABTS radicals, as well as the inhibition of hTERT-HPNE Pro, gradually increased. The maximum clearance rates were 84.1%, 26.6%, and 92.3%, respectively, while the maximum cell Pro inhibition rate was only 8.6%. Relative to the 0 μmol/L propolis flavonoid treatment group, the Hep-2 cells treated with 25, 50, and 100 μmol/L propolis flavonoids exhibited decreased cell Pro activity, reduced number of invasive and migratory cells, increased Apo rate, decreased PI3K and p-Akt proteins, and demonstrated a concentration-dependent effect (P < 0.05). In summary, the extraction with 50% ethanol solution resulted in a higher yield of flavonoids. Propolis flavonoids demonstrated marked antioxidant activity and did not cause damage to normal hTERT-HPNE cells. They exhibited inhibitory effects on the Pro, Inv, and Mig of Hep-2 cells in LGC, and promoted cell Apo. These effects may be associated with PI3K/Akt signaling inhibition.