Transport characteristics of tryptanthrin and its inhibitory effect on P-gp and MRP2 in Caco-2 cells.

PURPOSE. Tryptanthrin, an indole quinazoline alkaloid with multiple medical activities, has been recently under preclinical development as an anti-tuberculosis and anti-tumor drug. The aims of this study are to characterize the intestinal transport of tryptanthrin in Caco-2 cells, to determine whether P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) are involved in this issue, and to evaluate the potential influence of tryptanthrin on the function of P-gp and MRP2. METHODS. Transport assays of tryptanthrin were performed in Caco-2 monolayers with or without the supplement of P-gp and MRP2 inhibitors. Transport assays of P-gp and MRP2 substrates were also performed in the presence of tryptanthrin. The effect of tryptanthrin on the expression of P-gp and MRP2 was analyzed by reverse transcriptase-PCR. RESULTS. Both absorption and secretion of tryptanthrin were concentration-independent at a low concentration range of 0.8-20 µM. The apparent permeability (Papp) for the apical (AP) to basolateral (BL) was 6.138 ± 0.291 × 10-5. The ratio of Papp (BL→AP) to Papp (AP→BL) was 0.77, suggesting greater permeability in the absorptive direction. Both the P-gp inhibitor, verapamil, and the MRP2 inhibitor, glibenclamide, didn't affect the efflux transport of tryptanthrin. The efflux transport of the P-gp substrate, digoxin, and the MRP2 substrate, pravastatin sodium, decreased when tryptanthrin was present. However, tryptanthrin didn't change the expression of P-gp and MRP2. CONCLUSIONS. Tryptanthrin was well absorbed across the Caco-2 monolayers, and its transepithelial transports were dominated by passive diffusion. Tryptanthrin was not a substrate, but a potential inhibitor of P-gp and MRP2. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

The effect of sphingomyelin synthase 2 (SMS2) deficiency on the expression of drug transporters in mouse brain.

Sphingomyelin synthase (SMS), the last enzyme involved in the biosynthesis of sphingomyelin (SM), plays a critical role in the constitution of cell membrane and has impact on the expression of membrane proteins. SMS2, one of two SMS enzymes, is predominantly located in the plasma membrane, and is mainly expressed in the brain. Therefore, it is conceivable that SMS2 deficiency may have impact on expression of some membrane proteins, such as membrane-bound drug transporters. Using SMS2 gene deficient mouse brain tissues, we studied the gene and protein expression profiles of drug transporters, ERM proteins (ezrin/radixin/moesin) and the cytoskeleton protein, ß-actin, in mouse brain by RT-PCR, western blot and immunohistochemistry analysis. We found that the mRNA expression of Mdr1 rather than the other drug transporters was significantly decreased in the SMS2 deficient brain. Accordingly, the expression and the function of Pgp (Mdr1/P-glycoprotein) were significantly downregulated in brain. In addition, the substantially downregulated expression of ezrin and ß-actin was also observed in the SMS2 deficient brain. The immunohistochemistry analysis further revealed the suppressed expression of Pgp, ezrin and ß-actin in both cortex and paraventricular areas of SMS2 knockout mice. Furthermore, both Pgp and ß-actin were found to be co-immunoprecipitated with ezrin from the total brain lysate, suggesting the association between Pgp, ezrin and ß-actin in the brain. These results indicate that SMS2 participates in the expression regulation of drug transporters, particularly Pgp, and suggest that SMS2 may be a potential target for enhancing drug access to the brain.

ERM stable knockdown by siRNA reduced in vitro migration and invasion of human SGC-7901 cells.

Three closely related proteins, ezrin, radixin, and moesin (ERM), which primarily act as a linker between the plasma membrane and the cytoskeleton, are involved in many cellular functions, including regulation of actin cytoskeleton, control of cell shape, adhesion and motility, and modulation of signaling pathways. Although, ezrin is now recognized as a key component in tumor metastasis, the functional role of the radixin and moesin in tumor metastasis has not been established. In the present study, we chose highly metastatic human gastric carcinoma SGC-7901 cells, which express all of the ERM proteins as a model to examine the functional roles of these proteins in tumor metastasis. Ezrin, radixin or moesin stable knockdown SGC-7901 cell lines were established using siRNA methodology. In vitro cell migration and invasion studies showed that either ezrin, radixin or moesin specific deficiency in the cells caused the substantial reduction of the cell migration and invasion. Western blotting and immunofluorescence analysis showed that the expression of E-cadherin was also significantly increased when any member of ERM proteins was downregulated. Our results indicated that these three ERM proteins play similar roles in the SGC-7901 cell metastatic potential and their roles of upregulating the expression of E-cadherin may be important in tumor progression.