Investigation of the coordinated functional activities of cytochrome P450 3A4 and P-glycoprotein in limiting the absorption of xenobiotics in Caco-2 cells

The coordination of the functional activities of intestinal CYP3A4 and P-gp in limiting the absorption of xenobiotics in Caco-2 cells was investigated. Growing Caco-2 cells were exposed to increasing concentrations of doxorubicin (1-2 μM) in plastic flasks to encourage a subpopulation of cells, that displayed an intrinsically higher multidrug resistance (mdr) phenotype than the parent cells, to survive and grow. Doxorubicin-exposed (hereinafter referred to as type I cells) and nonexposed Caco-2 cells (parent cells) on collagen-coated inserts were also treated with either 0 (control) or 0.25 μM 1α,25-dihydroxyvitamin D3 to promote cellular CYP3A4 expression. Increased P-gp protein expression, as detected by Western blotting, was noted in type I cells (213±54.35%) compared to that of parent cells (100±6.05%). Furthermore, they retained significantly less [3H]vincristine sulphate (p<0.05), a P-gp substrate, after efflux (272.89±11.86 fmol/mg protein) than the parent cells (381.39±61.82 fmol/mg protein). The expression of CYP3A4 in parental cells after 1α,25-dihydroxyvitamin D3 treatment was quantified to be 76.2±7.6 pmol/mg protein and comparable with that found in human jejunal enterocytes (70.0±20.0 pmol/mg protein). Type I cells, however, expressed a very low quantity of CYP3A4 both before and after the treatment that was beyond the minimum detection limit of Western blotting. Functionally, the rates of 1-hydroxylation of midazolam by CYP3A for both cell types ranged from 257.0±20.0 to 1057.0±46.0 pmol/min/mg protein. Type I cells, although having a higher P-gp expression and activity comparatively, metabolized midazolam less extensively than the parent cells. The results suggested that there were noncoordinated functional activities of intestinal CYP3A4 and P-gp in Caco-2 cells, although they both functioned independently to minimize intestinal epithelial absorption of xenobiotics. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association.

Intestinal absorption barriers as modelled by p-glycoprotein and cytochrome p450 a34 in caco2 cells

The passage number and origin of two populations of Caco-2 cells influence their enterocyte-like characteristics. Caco-2 cells of passage number >90 from Novartis pharmaceutical company possess higher levels of expression of alkaline phosphatase and P-glycoprotein and a greater cellular uptake of Gly-1.-Pro than those of passage number <40 from the American Type Tissue Culture collection. High P-gp expressing Caco-2 cells have been developed through stepwise selection of the cells with doxonibicin. This newly-developed cell line (hereafter referred to as Type I) possesses approximately twice as much P-gp protein than non-exposed cells, restricts the transepithelial transport of vincristine in the apical-to-basolateral direction whilst facilitating its transport in the reverse direction and accumulates less vincristine than non-exposed cells. There is no apparent evidence of the co-existence of the multidrug resistance protein (MIT) in Type I cells to account for the above-listed observations. Stopping the exposure for more than 28 days decreases the P-gp protein expression in previously doxorubicin-exposed Type I Caco-2 cells and reduces the magnitude of vincristine transepithelial fluxes in both directions to the levels that are almost similar to those of non-exposed cells. Exposing Caco-2 cells to 0.25 JAM la, 25-dihydroxyvitamin D3 induces their expression of cytochrome P450 3A4 protein to the level that is equivalent to that from isolated human jejunal cells. Under the same treatment, doxorubiein-exposed (Type I) cells metabolise naidazolam poorly and less extensively compared to non-exposed cells, suggesting that there is no such co-regulation of P-gp and CYP3A4 in Caco-2 cells. However, there is evidence which suggests CYP3A metabolises mida_zolam into 1- and 4-hydroxymidazolam, the latter may possibly be a P-gp substrate and is transported extracellularly by P-gp, supporting the hypothesis of P-gp-CYP3A4 synergistic roles in keeping xenobiotics out of the body. Doxoru.bicin-exposed (Type I) cells are less effective in translocating L-proline and glycyl-L-proline across the cell mono layers.

Mechanistic differences between GSH transport by multidrug resistance protein 1 (MRP1/ABCC1) and GSH modulation of MRP1-mediated transport

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent polytopic membrane protein that transports many anticancer drugs and organic anions. Its transport mechanism is multifaceted, especially with respect to the participation of GSH. For example, vincristine is cotransported with GSH, estrone sulfate transport is stimulated by GSH, or MRP1 can transport GSH alone, and this can be stimulated by compounds such as verapamil or apigenin. Thus, the interactions between GSH and MRP1 are mechanistically complex. To examine the similarities and differences among the various GSH-associated mechanisms of MRP1 transport, we have measured first the effect of GSH and several GSH-associated substrates/modulators on the binding and hydrolysis of ATP by MRP1 using 8-azidoadenosine-5'-[(32)P]-triphosphate ([(32)P]azidoATP) analogs, and second the initial binding of GSH and GSH-associated substrates/modulators to MRP1. We observed that GSH or its nonreducing derivative S-methylGSH (S-mGSH), but none of the GSH-associated substrate/modulators, caused a significant increase in [gamma-(32)P]azidoATP labeling of MRP1. Moreover, GSH and S-mGSH decreased levels of orthovanadate-induced trapping of [alpha-(32)P]azidoADP. [alpha-(32)P]azidoADP.Vi trapping was also decreased by estone sulfate, whereas vincristine, verapamil, and apigenin had no apparent effects on nucleotide interactions with MRP1. Furthermore, estrone sulfate and S-mGSH enhanced the effect of each other 15- and 10-fold, respectively. Second, although GSH binding increased the apparent affinity of MRP1 for all GSH-associated substrates/modulators tested, only estrone sulfate had a reciprocal effect on the apparent affinity of MRP1 for GSH. Overall, these results indicate significant mechanistic differences between MRP1-mediated transport of GSH and the ability of GSH to modulate MRP1 transport.

Role of GSH in estrone sulfate binding and translocation by the multidrug resistance protein 1 (MRP1/ABCC1)

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-dependent efflux pump that can confer resistance to multiple anticancer drugs and transport conjugated organic anions. Unusually, transport of several MRP1 substrates requires glutathione (GSH). For example, estrone sulfate transport by MRP1 is stimulated by GSH, vincristine is co-transported with GSH, or GSH can be transported alone. In the present study, radioligand binding assays were developed to investigate the mechanistic details of GSH-stimulated transport of estrone sulfate by MRP1. We have established that estrone sulfate binding to MRP1 requires GSH, or its non-reducing analogue S-methyl GSH (S-mGSH), and further that the affinity (Kd) of MRP1 for estrone sulfate is 2.5-fold higher in the presence of S-mGSH than GSH itself. Association kinetics show that GSH binds to MRP1 first, and we propose that GSH binding induces a conformational change, which makes the estrone sulfate binding site accessible. Binding of non-hydrolyzable ATP analogues to MRP1 decreases the affinity for estrone sulfate. However, GSH (or S-mGSH) is still required for estrone sulfate binding, and the affinity for GSH is unchanged. Estrone sulfate affinity remains low following hydrolysis of ATP. The affinity for GSH also appears to decrease in the post-hydrolytic state. Our results indicate ATP binding is sufficient for reconfiguration of the estrone sulfate binding site to lower affinity and argue for the presence of a modulatory GSH binding site not associated with transport of this tripeptide. A model for the mechanism of GSH-stimulated estrone sulfate transport is proposed.