Revolving door action of breast cancer resistance protein (BCRP) facilitates or controls the efflux of flavone glucuronides from UGT1A9-overexpressing HeLa cells

Abstract

Cellular production of flavonoid glucuronides requires the action of both UDP-glucuronosyltransferases (UGT) and efflux transporters since glucuronides are too hydrophilic to diffuse across the cellular membrane. We determined the kinetics of efflux of 13 flavonoid glucuronides using the newly developed HeLa-UGT1A9 cells and correlated them with kinetic parameters derived using expressed UGT1A9. The results indicated that, among the seven monohydroxylflavones (HFs), there was moderately good correlation (r(2) ≥ 0.65) between the fraction metabolized (fmet) derived from HeLa-UGT1A9 cells and CLint derived from the UGT1A9-mediated metabolism. However, there was weak or no correlation between these two parameters for six dihydroxylflavones (DHFs). Furthermore, there was weak or no correlation between various kinetic parameters (Km, Vmax, or CLint) for the efflux and the metabolism regardless of whether we were using seven HFs, six DHFs, or a combination thereof. Instead, the cellular excretion of many flavonoid glucuronides appears to be controlled by the efflux transporter, and the poor affinity of glucuronide to the efflux transporter resulted in major intracellular accumulation of glucuronides to a level that is above the dosing concentration of its aglycone. Hence, the efflux transporters appear to act as the "Revolving Door" to control the cellular excretion of glucuronides. In conclusion, the determination of a flavonoid's susceptibility to glucuronidation must be based on both its susceptibility to glucuronidation by the enzyme and resulting glucuronide's affinity to the relevant efflux transporters, which act as the "Revolving Door(s)" to facilitate or control its removal from the cells.

Fig. 1. Structures of seven mono-hydroxyflavones and six dihydroxyflavones

Shown in the scheme are structures of aglycone forms of hydroxyflavone analogs. Glucuronidated metabolites may be formed at the 5, 6, or 7 phenolic group on the A ring, or 2′-OH, 3′-OH or 4′-OH phenolic group on the B ring, or 3-OH group on the C ring. 2′-Hydroxyflavone (2′HF), 3′-Hydroxyflavone (3′HF), 4′-Hydroxyflavone (4′HF), 3-Hydroxyflavone (3HF), 5-Hydroxyflavone (5HF), 6-Hydroxyflavone (6HF), 7-Hydroxyflavone (7HF), 3,2′-Dihydroxyflavone (3,2′DHF), 3,3′-Dihydroxyflavone (3,3′DHF), 3,4′-Dihydroxyflavone (3,4′DHF), 3,5-Dihydroxyflavone (3,5DHF), 3,6-Dihydroxyflavone (3,6DHF), 3,7-Dihydroxyflavone (3,7DHF).

Fig. 2. Kinetics profiles of BCRP-mediated efflux of seven HFs glucuronides (A, 7HF-G; B, 2′HF-G; C, 3′HF-G; D, 3HF-G; E, 4′HF-G; F, 5HF-G; G, 6HF-G) (left) and corresponding Eadie-Hofstee plots for each kinetic profile (right)

For Fig.2 and 6, solid lines (left) denote excretion rate of flavone glucuronides. Each data point represents the average of three replicates, and error bars are the standard deviations of the mean (n=3). “Conc.” is the abbreviation for concentration.

Fig. 2. Kinetics profiles of BCRP-mediated efflux of seven HFs glucuronides (A, 7HF-G; B, 2′HF-G; C, 3′HF-G; D, 3HF-G; E, 4′HF-G; F, 5HF-G; G, 6HF-G) (left) and corresponding Eadie-Hofstee plots for each kinetic profile (right)

For Fig.2 and 6, solid lines (left) denote excretion rate of flavone glucuronides. Each data point represents the average of three replicates, and error bars are the standard deviations of the mean (n=3). “Conc.” is the abbreviation for concentration.

Fig. 3. Intracellular HFs-G concentrations as a function of loading HFs concentration (A, 7HF; B, 2′HF; C, 3′HF; D, 3HF; E, 4′HF; F, 5HF; G, 6HF)

Each bar represents the average of three replicates, and error bars are the standard deviations of the mean (n=3). The symbol “*” means the difference between concentrations for each compound was statistically significant according to a one-way ANOVA test.

Fig. 4. Correlation between loading concentrations and metabolic efficiency or f_met of HeLa-UGT1A9 cells or CL_int of UGT1A9

Fig.4A plots f_met values for seven HFs at six loading concentrations to contrast the effects of structural changes on f_met values. For Fig. 4A, each bar represents the average of three replicates, and error bars are the standard deviations of the mean (n=3). Fig. 4B plots correlation between f_met values derived from UGT1A9-overexpressing HeLa cell and Log(CL_int)′ derived from commercial UGT1A9 of seven HFs (2′HF, 3′HF, 4′HF, 3HF, 5HF, 6HF, 7HF). Correlation coefficients were calculated for each of the tested concentrations. Each data point represents the average of three replicates (n=3). The symbol “*” means the difference between compounds within each concentration was statistically significant according to a one-way ANOVA test.

Fig.5. Kinetic profiles of BCRP-mediated efflux of seven HF glucuronides

The concentration versus rate plots are on the left and corresponding Eadie-Hofstee plots for each kinetic profile are on the right. Each data represents the average of three replicates, and error bars are the standard deviations of the mean (n=3).

Fig.5. Kinetic profiles of BCRP-mediated efflux of seven HF glucuronides

The concentration versus rate plots are on the left and corresponding Eadie-Hofstee plots for each kinetic profile are on the right. Each data represents the average of three replicates, and error bars are the standard deviations of the mean (n=3).

Fig. 6. Kinetics profiles of BCRP-mediated efflux of six DHFs glucuronides (A,3,7DHF-3-O-G; B, 3,5DHF-3-O-G; C, 3,3′DHF-3-O-G; D, 3,2′DHF-3-O-G, E, 3,4′DHF-3-O-G and F, 3,6DHF-3-O-G) (left) and corresponding Eadie-Hofstee plots for each kinetic profile (right)

Each data represents the average of three replicates, and error bars are the standard deviations of the mean (n=3).

Fig. 6. Kinetics profiles of BCRP-mediated efflux of six DHFs glucuronides (A,3,7DHF-3-O-G; B, 3,5DHF-3-O-G; C, 3,3′DHF-3-O-G; D, 3,2′DHF-3-O-G, E, 3,4′DHF-3-O-G and F, 3,6DHF-3-O-G) (left) and corresponding Eadie-Hofstee plots for each kinetic profile (right)

Each data represents the average of three replicates, and error bars are the standard deviations of the mean (n=3).

Fig. 7. Intracellular concentration of DHF glucuronides as a function of concentrations (A,3,7DHF; B, 3,5DHF; C, 3,3′DHF; D, 3,2′DHF; E, 3,4′DHF and F, 3,6DHF)

Each bar represents the average of three replicates, and error bars are the standard deviations of the mean (n=3). The symbol “*” means the difference between concentrations for each compound was statistically significant according to a one-way ANOVA test.

Fig. 8. Correlation between loading concentrations and metabolic efficiency or f_met derived from HeLa-UGT1A9 or CL_int of UGT1A9

Fig.8A plots f_met values for six DHFs at five loading concentrations to contrast the effects of structural changes on f_met values. Fig. 8B plots correlation between f_met values derived from UGT1A9-overexpressing HeLa cell and Log(CL_int)′ derived from commercial UGT1A9 of six DHFs (3,2′-, 3,3′-, 3,4′-, 3,5-, 3,6- and 3,7DHF). Correlation coefficients were calculated for each of the tested concentrations. Each bar or point represents the average of three replicates, and error bars are the standard deviations of the mean (n=3). The symbol “*” means the difference between compounds within each concentration was statistically significant according to a one-way ANOVA test.

Fig. 9. Scatter plots of kinetic parameters derived from BCRP-mediated efflux (K_m, V_max and Log(CL_int)) of HFs-G and DHFs-3-O-G and those derived from UGT1A9-mediated glucuronidation (K_m′, V_max′ and Log(CL_int)′) of HFs and DHFs

I, K_m-BCRP vs. K_m‘-UGT1A9; II, V_max-BCRP vs. V_max'-UGT1A9; III, Log(CL_int)-BCRP vs. Log(CL_int)'-UGT1A9.

Fig. 10. Correlation between Rates of Glucuronide Efflux and f_met at Similar Intracellular Concentration of Glucuronides

The rates of efflux at 50 μM were calculated for the plot because each glucuronide tested had an intracellular concentration (measured experimentally) that was close to an intracellular concentration of 50 μM. Once the concentration was selected, we were then able to find the f_met value for that compound for use in the plot. At this condition, none of the compound was exhausted. Different loading concentrations were used for different compounds and a table that details the actual concentrations of each flavonoid used is provided in the Supplemental Materials. The outlier compound (in circle) was 3HF in Fig.10A, or 3,6DHF in Fig.10B.