Puerarin 6-O-xyloside suppressed HCC via regulating proliferation, stemness, and apoptosis with inhibited PI3K/AKT/mTOR

Puerarin 6 ''-O-xyloside is a tumor suppressive derivate of Puerarin that is recently characterized as a lysine-specific demethylase 6B inhibitor. Here we investigated the effects of Puerarin 6 ''-O-xyloside in hepatocellular carcinoma (HCC) cell lines SMMC-7721 and HepG2. Cell viability, proliferation, stemness, protein expression, and autophagy were tested by CCK-8, colony formation, sphere formation, western blotting, and LC3B GFP puncta per cell, respectively. Apoptosis, CD133-positive cells, and JC-1-labeled mitochondrial membrane potential were measured by flow cytometry. The effects of Puerarin 6 ''-O-xyloside in vivo were explored in HepG2 xenograft mice. Puerarin 6 ''-O-xyloside inhibited cell viability, proliferation, and stemness, and promoted apoptosis in both SMMC-7721 and HepG2 cells. Further experiments showed promoted autophagy and decreased mitochondrial membrane potential, and decreased expression of p-PI3K, p-AKT, and p-mTOR in HepG2 cells. Co-administration of 3-MA with Puerarin 6 ''-O-xyloside obviously augmented these effects including inhibited protein expression of p-PI3K, p-AKT, and p-mTOR, and inhibited proliferation, promoted apoptosis, and decreased stemness. In HepG2 xenograft mice, 100 mg/kg/d Puerarin 6 ''-O-xyloside significantly suppressed tumor growth, stemness, and apoptosis. In conclusion, our study indicated that Puerarin 6 ''-O-xyloside decreased cell viability, proliferation, and stemness, and promoted autophagy and mitochondria-dependent apoptosis of HCC, at least partly through inhibiting PI3K/AKT/mTOR. These results highlighted Puerarin 6 ''-O-xyloside as a promising prodrug that could inhibit both PI3K/AKT/mTOR and epigenetic demethylation.