Abstract:

Objective  To observe the regulations of rapamycin (Rapa) on UDP-glucuronosyltransferase (UGT) 1A1, UGT1A8, UGT1A10 and cytochrome P450 (CYP) 3A4 induced by sulforaphane (SFN) in human colon cancer Caco-2 cells and to explore the influence of Rapa on chemopreventive effect induced by SFN. Methods  Experiments were divided into control group, 10nmol/L Rapa group, 25μmol/L SFN group, and 25μmol/L SFN+10nmol/L Rapa group. The ultrastructures of Caco-2 cells were observed by transmission electron microscope. Western blotting was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3) and NF-E2-related factor 2 (Nrf2) proteins. Quantitative real-time RT-PCR was employed to examine the mRNA expression of UGT1A1, UGT1A8, UGT1A10 and CYP3A4. Immunocytochemistry was used to observe the nuclear localization of Nrf2. Results  Autophagysomes and autolysosomes could be found in the 10nmol/L Rapa, 25μmol/L SFN and 25μmol/L SFN+10nmol/L Rapa groups by transmission electron microscope. In comparison to the control group, the proteins of LC3-II and Nrf2 and the mRNA of UGT1A1, UGT1A8 and UGT1A10 were increased significantly in the 25μmol/L SFN group and 25μmol/L SFN+10nmol/L Rapa group, and 25μmol/L SFN+10nmol/L Rapa group possessed the highest level. An intense nuclear labeling of Nrf2 could also be observed in SFN-treated cells, especially in 25μmol/L SFN+10nmol/L Rapa group. CYP3A4 mRNA expression could be inhibited in the 10nmol/L Rapa, 25μmol/L SFN and 25μmol/L SFN+10nmol/L Rapa groups. Conclusion  Rapa can enhance SFN-induced autophagy, and improve SFN-induced mRNA expression of UGT1A1, UGT1A8 and UGT1A10 through the Nrf2 signaling pathway up-regulated by Rapa. There is no effect of Rapa on CYP3A4 mRNA down-regulated by SFN.

Key words: Sulforaphane; Rapamycin; Glucuronosyltransferase 1A; Cytochrome P450 3A4