Overexpression of P-glycoprotein (P-gp), a plasma membrane transporter which extrudes chemotherapeutic agents out of cells, has been associated with the multidrug resistance of cancer cells. Compounds such as verapamil, dihydropyridine analogs, quinidine and cyclosporin A reversed this P-gp-mediated multidrug resistance (MDR) due to their inhibition of transporter activity. 1,2) In addition to these compounds, it has been revealed that flavonoids and other polyphenols modulate P-gp activity. [3][4][5][6] Although the structure-activity relationships of flavonoids on P-gp functions have been studied, 5,7,8) they are still not clear, especially concerning their effects in whole cells. Recently, we revealed that tea catechins inhibited P-gp function dependent on their chemical structure in multidrug-resistant human epidermal carcinoma cell line KB-C2 cells, 9) which overexpress P-gp. 10)Among the tea catechins, we found the maximum activity in the (Ϫ)epigallocatechin gallate whose chemical structure is shown in Fig. 1. We clarified that the presence of the galloyl moiety in the C ring, and the presence of the trihydric pyrogallol group as the B ring instead of the dihydric catechol group, markedly increased its activity on P-gp, although their effects on the partition coefficients between n-octanol and phosphate-buffered saline (PBS) were opposite to each other. 9)In this study, we examined the structure-activity relationships of seven flavonoids, naringenin (flavanone), baicalein (flavone), kaempferol, quercetin, myricetin, morin, and fisetin (flavonols) (Fig. 1), which do not have large substituents like the galloyl group, as well as two glycosides of quercetin, for their effects on P-gp-mediated drug extrusion in KB-C2 cells. We analyzed the effects from the standpoints of chemical structure, conformation and hydrophobicity. We studied the importance of the 2,3-double bond in the C ring, in relation to the stereoscopic structure of flavonoids. Furthermore, we studied the effects of hydrophobicity of the flavonoids on the inhibitory activity to P-gp function. For this study, we used fluorescent rhodamine-123 and daunorubicin as the P-gp substrates; these substrates have often been used for the study of various P-gp transport modulators including flavonoids.3,6,11) Although daunorubicin has also been revealed to be a substrate of multidrug resistance-associated protein 1 (MRP1), 12) since MRP1 is rarely found in KB-C2 cells, 13) its involvement in substrate efflux is negligible. We studied the effects of flavonoids, naringenin (flavanone), baicalein (flavone), kaempferol, quercetin, myricetin, morin, and fisetin (flavonols) as well as two glycosides of quercetin on P-glycoprotein (P-gp) function in multidrug-resistant P-gp overexpressing KB-C2 cells. Flavonoids such as kaempferol and quercetin increased the accumulation of rhodamine-123 dependent on their chemical structure. Analysis by flow cytometry indicated that the increase in substrate accumulation was due to the inhibition of substrate efflux. Naringenin, w...

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We studied the effects of tannic acid and 1(beta),2,3,4,6-penta-O-galloyl-D-glucose (pentagalloylglucose), one of the components of tannic acid, on the P-glycoprotein (P-gp) function in multidrug-resistant P-gp over-expressing KB-C2 cells. Both tannic acid and pentagalloylglucose markedly elevated the accumulation of P-gp substrates, rhodamine 123 and daunorubicin, by inhibiting their efflux. A 19-fold increase in cellular rhodamine 123 was observed for tannic acid at 60 microM (85 microg mL(-1)) and a 21-fold increase was observed for pentagalloylglucose at 100 microM (94 microg mL(-1)). The increasing effects of these compounds in the accumulation were much larger than that of (-)epigallocatechin-3-O-gallate (EGCG), which has been revealed to have a prominent inhibitory effect on P-gp compared with other flavonoids. Analysis of verapamil-stimulated ATPase activity in membrane vesicles expressing human P-gp suggested that inhibition of P-gp function by tannic acid and pentagalloylglucose was at least partly due to ATPase inhibition of P-gp. The findings also suggested that the presence of a large number of galloyl groups in polyphenols strengthens the interaction with regulatory regions in P-gp.

Overexpression of P-glycoprotein (P-gp), a plasma membrane transporter which extrudes chemotherapeutic agents out of cells, has been associated with the multidrug resistance of cancer cells. Compounds such as verapamil, dihydropyridine analogs, quinidine and cyclosporin A reversed this P-gp-mediated multidrug resistance due to their inhibition of transporter activity.1,2) Phytochemicals such as flavonoids have also been revealed to inhibit transport function of P-gp and the inhibitory mechanism has been studied. 3,4) The root of Panax ginseng C. A. MEYER has been widely used in Eastern Asia as a drug for nourishment, a prophylactic tonic, and as a medicine to maintain perennial youth. Now, its use as a natural medicine has become widespread worldwide.5) Pharmacological effects of ginseng have been demonstrated in cancer, diabetes mellitus, the cardiovascular system, immune system and central nervous system.6) The major components of Panax ginseng are the ginsenosides, a group of saponins with a triterpenoid dammarane structure or oleanolic acid structure. 5) They reduce cell proliferation and induce apotosis. 7) They also inhibit adrenal catecholamine secretion.5) Regarding the effects of ginsenosides on P-gp function, there was a study on the inhibitory effects of ginsenoside Rg3, a red ginseng saponin, in KBV20C cells.8) However, the effects of other saponins and their metabolites, as well as the mechanism of their effects, are unknown.Therefore, we investigated the effects of ginsenoside Rg1, which is a protopanaxatriol-type saponin 5) with two glucose moieties, and its metabolite M4 with no sugar moiety ( Fig. 1) by using colchicine-resistant, P-gp-overexpressing KB-C2 cells. We also studied the effects of two metabolites of protoanaxadiol-type saponin, another dammarane-type saponin, M1 and M12 (Fig. 1). M1 has one glucose moiety and M12 has none. We examined the effects of the structure of the ginsenosides, especially regarding the presence of a sugar moiety. We also examined the mechanism of their effects. For this study, we used fluorescent daunorubicin as the P-gp substrate; this substrate has often been used to study various P-gp transport modulators. Although daunorubicin has also been revealed to be a substrate of multidrug resistance protein 1 (MRP1), 9) since MRP 1 is rarely found in KB-C2 cells, 10) its involvement in substrate efflux is negligible. MATERIALS AND METHODS MaterialsGinsenoside Rg1 was purchased from Funakoshi Co. (Tokyo, Japan). The hydrolyzed metabolites of ginsenosides, M1, M4 and M12, were prepared by fermentation of the protopanazadiol-or protopanaxatriol-type saponins by human intestinal bacteria as described elsewhere.11) Dulbecco's Modified Eagle Medium (D-MEM) and fetal bovine serum were from Invitrogen Co. (Carlsbad, CA, U.S.A.). Daunorubicin hydrochloride and all other reagents were from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). KB-C2 cells and KB3-1 cells were kindly provided by Prof. Shinichi Akiyama (Kagoshima University, Japan) and were cultured in D-MEM cult...

Link Model, a participatory rural appraisal (PRA) programme for micro-infrastructure construction funding aid, has received much attention in Bangladesh over the last few decades. This study aims to contribute to the latest phase of the Link Model project, launched in July 2016 by the Bangladeshi government. To this