In Vitro Stabilities And Antitumor Activities Against Human Gastric Cancer AGS Cells Of Fisetin And Quercetin

Flavonoids are important phytochemicals rich in plant-derived foods. They can afford a variety of biological activities. Epidemiological investigation, in vitro and in vivo experiments have indicated that diets intake of flavonoids can reduce the risk of human cancer and have important effects on cancer chemoprevention and therapy. However, the molecular mechanism of the effect of flavonoids on cancers remained to be elucidated. Flavonoids contain sensitive chemical groups and structural element in their molecules, and plant foods usually undergo necessary thermal processing and storage before eating. Thus they are susceptible to degradation and their anticancer activity is affected by degradation. In addition, proteins in plant foods can interact with flavonoids, and the antitumor activity of flavonoids in vitro against human cancer can be affected by the interaction of flavonoids and proteins. Therefore, factors that affect chemical stability of flavonoids are the premise that study on antitumor activity of flavonoids. In the present study, factors that affect chemical stability in solutions, anticancer activity and molecular mechanism on cancer of naturally occurring flavonoids fisetin and quercetin were investigated. The effect of interaction of flavonoids and proteins on anticancer activity of fisetin and quercetin were also investigated.Impacts of medium p H, temperature and coexisted proteins on the degradation of two flavonoids fisetin and quercetin were assessed by spectroscopic method in the present study. Based on the measured degradation rate constants(k), fisetin was more stable than quercetin in all cases. Increasing medium p H from 6.0 to 7.5 at 37 ?C enhanced respective k values of fisetin and quercetin from 2.81?10-2 and 8.30?10-3 to 0.375 and 0.202 h-1(P<0.05). In comparison with their degradation at 37 ?C, fisetin and quercetin showed larger k values at higher temperature(0.124 and 0.245 h-1 at 50 ?C, or 0.490 and 1.42 h-1 at 65 ?C). Five protein products in medium stabilized the two flavonoids(P<0.05), as these proteins at 0.10 g L-1 decreased respective k values of fisetin and quercetin to 2.28?10-2-2.98?10-2 and 4.37?10-2-5.97?10-2 h-1. Casein, soybean protein and bovine serum albumin provided greater stabilization than whey protein isolate. Hydrophobic interaction between the proteins and the two flavonoids was evidenced responsible for the stabilization, as sodium dodecyl sulfate could destroy the stabilization significantly(P<0.05).CCK-8 assay results showed that fisetin and quercetin could inhibit AGS cells proliferation in a dose-time-dependent manner, and fisetin was more powerful than quercetin in growth inhibition. Hoechst 33258 staining results indicated that fisetin and quercetin could induce apoptosis of AGS cells effectively. AGS cells treated with fisetin and quercetin displayed typical characteristics of apoptosis. The results of flow cytometry analysis also showed that fisetin and quercetin could induce apoptosis of AGS cells, and fisetin was more powerful than quercetin. Potency of fisetin and quercetin on induction of apoptosis was also similar to proliferation inhibition. It was suggested that fisetin and quercetin inhibited proliferation of AGS cells by inducing apoptosis. Real-time PT-PCR and Western blot assay were used to figure out the mechanism of anticancer activity of querctin and fisetin. Fisetin and quercetin induced PIG3 expression, and PIG3 catalyzed the oxidation of fisetin and quercetin and induced ROS generation, which resulted in the loss of mitochondrial membrane potential and an increase of Bax/Bcl-2 ratio. The loss of mitochondrial membrane potential stimulated the release of AIF from mitochondria, and eventually induced apoptosis of AGS cells. However, Activities of fisetin and quercetin in inhibition proliferation and inducing apoptosis were inhibited by coexisted proteins(bovine serum albumin and casein).It is thus concluded that higher temperature and alkaline p H can enhance flavonoid loss, whereas coexisted proteins as flavonoid stabilizers can inhibit degradation and anticancer activity of flavonoids. PIG3 may be a new regulator involved in quercetin- and fisetin-induced apoptosis through the ROS-mediated mitochondrial apoptotic pathway.