Efficiency Of Transcellular Transport Of Flavonoids From Flavonoids Of Litsea Coreana L.and Its Aglycones In Epithelial Cell Monolayer Models And Reversing The Drug-resistant In Human Hepatocellular Carcinoma Cell Line And Its Mechanisms

Hawk-tea, leaves of Litsea Coreana, which enjoy Anhui region characteristic, is conventionally consumed as healthy tea in southern China for centuries. Is directly used as water drink which has the efficacy of detoxification detumescence, brighten eyes and promote the secretion, etc. Modern pharmacological and clinical studies have shown that its active ingredients are the total flavonoids which exert multiple pharmacological actions, such as hyperglycemic, anti-inflammatory, antioxidant and immunological regulation. Contemporary, its aglycones have the similar pharmacological actions. Although many of the pharmacological activities of these flavonoids are reported, but their absorption characteristics and mechanisms are still unclear.Oral administration is the most common way of drug application in clinical. Absorption from the gastrointestinal tract is the first step covering the access of a drug into the circulation and finally its tissue distribution. A great many of in vitro model systems were employed as models to investigate drug absorption and transport mechanism across intestine. Using the model system, we studied the transport characteristics of the drug, and investigated the influential factors, drug transporters or drug-transporters interaction.ATP-binding cassette(ABC) transporters have been demonstrated to have a well-defined role in a multiple cellular processes, which efflux the drug substrates from the cytoplasm to the extracellular space using ATP driven energy. Among the ABC transporters, two family members of ABC proteins, P-glycoprotein(MDR1/P-gp) and multidrug resistance-associated protein 2(MRP2) play important roles in restricting the permeability and facilitating the efflux of anticancer drugs, which mainly leading to multidrug resistance(MDR). But so far, the detailed mechanisms of MDR in cancer cells are complex and have not been fully elucidated.Therefore, the current study was aimed to investigate the relevant mechanisms governing the oral bioavailability of flavonoids from flavonoids of Litsea coreana L. and its aglycones, and explored the mechanisms involved in drug-transporters interaction using Caco-2、MDCK cell models and human hepatocellular carcinoma MDR variant BEL-7402/5-fluorouracil(BEL/5-FU) cells as follows. 1. Efficiency of transcellular transport and efflux of flavonoids with different glycosidic units from flavonoids of Litsea coreana L. and its aglycones in epithelial cell monolayer modelsThe study was designed to discuss potential transport mechanism of flavonoids from flavonoids of Litsea coreana L. and its aglycones across the Caco-2 and MDCK cell monolayer models. In the concentration range studied(40, 80, 160 μM), except for K-3-rha, transport of Q-3-gal、Q-3-glu、K-3-gal、K-3-glu、Q and K were concentration-dependent in both apical to basolateral and the reverse direction, showing that the absorption of K-3-rha may be passive diffusion, and the absorption of the rest of flavonoids may be complex, both passive diffusion and active transport. Contemporary, the influx and efflux of the flavonoid glycosides were temperature-dependent and pH-dependent at concentration of 80 μM, and transport of flavonoids was obviously decreased when experiments performed in the presence of 1 mM sodium azide(an ATP inhibitor). Uptake of Q-3-glu and K-3-glu was inhibited by phloridzin, a specific and competitive inhibitor of SGLT1. And uptake of Q-3-gal、Q-3-glu、K-3-gal、K-3-glu was inhibited by phloretin, a inhibitor of GLUT2. Moreover, the flavonoids exhibited significantly larger basolateral to apical Papp than that of the reverse direction, especially, the efflux ratio(ER) of flavonoid aglycones quercetin or kaempferol was more than 2, suggesting the existence of efflux mechanisms. Verapamil, a chemical inhibitor of P-glycoprotein(P-gp), had no effect on the transport of four flavonoid glycosides but its aglycones. However, MK-571 or probenecid, MRP2 inhibitors, led to an apparently decrease in the efflux of flavonoids. In addition, drug-drug interaction mediated by transporters could modify the absorption. 2. Selective modulation of ABC transporters by he flavonoids from flavonoids of Litsea coreana L. and its aglycones in Caco-2 and human hepatocellular carcinoma BEL-7402/5-FU cell linesSince ABC transporters play an important role in pharmacokinetics such as absorption, the research aimed to explore the factors regulating their function and expression using Caco-2 and BEL/5-FU cells. In the Caco-2 cell monolayer model, flavonoid aglycones quercetin(40, 80, 160 μM) and kaempferol(80, 160 μM) reduced the efflux of substrates, but flavonoids from flavonoids of Litsea coreana L. have no effect. BEL/5-FU cells pretreated with flavonoid aglycones quercetin(40, 80, 160 μM) down-regulated the mRNA expressions of MDR1 and MRP2 and enhanced the fluorescence intensity of substrates. On the other hand, BEL/5-FU cells pretreated with flavonoid aglycones quercetin(40, 80, 160 μM) increased its sensitivity to 5-FU, thus reversed the multiple drug resistance(MDR). 3. Quercetin modulates multidrug resistance by inhibiting FZD7/β-catenin pathway in human hepatocellular carcinoma BEL-7402/5-FU cell lineNowadays, it is well known that ABC transporters could mediate MDR to antineoplastic drugs with resultant subtherapeutic effect and cause subsequent failure of hepatocellular carcinoma(HCC) treatment. Consequently, approaches undertaken so far to overcome ABC transporter-mediated MDR in HCC will be an effective way. Recent studies indicated that the expressions of ABC transporters were controlled by Wnt/β-catenin signaling in MDR cancer cells. Frizzleds(FZD) 7, which serve as the main receptor of the Wnt pathways, could reverse the MDR in cancer cells. In our previous study, the expressions of FZD7、MDR1/P-gp and MRP2 were all down-regulated in the BEL/5-FU compared with its parental BEL-7402 cell line. It was confirmed that reduction of FZD7 by RNA interference induced inhibitory effects on the expressions of MDR1/P-gp, MRP2, cytoplasmic and nuclear β-catenin, and decreased the IC50 of chemotherapeutic drugs, enhanced the fluorescence intensity of substrates, which similar to quercetin; accordingly overexpression of FZD7 by transfecting with GV144-FZD7 was opposite. Moreover, down-regulation of β-catenin by iCRT-3 obviously suppressed the expressions of MDR1/P-gp and MRP2. Quercetin could concentration-dependent inhibit the function of ABC transporters. Moreover, quercetin could concentration-dependent inhibit the expressions of MDR1/P-gp, MRP2, cytoplasmic and nuclear β-catenin. Subsequently, when treatment with quercetin and siFZD7 together, the expressions of MDR1/P-gp, MRP2, cytoplasmic and nuclear β-catenin were scientifically higher than treatment with quercetin alone. Taken together, our findings suggest that quercetin could function as an inhibitor to restrain expressions of ABC transporters(MDR1/P-gp, MRP2), thus reverse MDR in hepatocellular carcinoma cells by inhibiting the FZD7/β-catenin pathway.In conclusion, the experimental observations in our study showing that the absorption of flavonoids may be closely associated with ABC transporters(MDR1/P-gp, MRP2), among them, quercetin may have an effect on the expressions and functions of these transporters by inhibiting the FZD7/β-catenin pathway.