Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression

Abstract

Background: Curcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1) protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells.

Methods: Caco-2 cells were cultured to confluence. The micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. We first incubated the cells with the micelles in the presence and absence of ezetimibe, the specific inhibitor of NPC1L1, to see whether the uptake of the cholesterol in the cells was mediated by NPC1L1. We then pretreated the cells with curcumin at different concentrations for 24 h followed by examination of the changes of cholesterol uptake in these curcumin-treated cells. Finally we determined whether curcumin affects the expression of NPC1L1 by both Western blot analysis and qPCR quantification.

Results: We found that the uptake of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in a dose-dependent manner. The results indicate that the uptake of cholesterol in this study was mediated by NPC1L1. We then pretreated the cells with 25-100 muM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 muM curcumin. In addition, we found that the curcumin-induced inhibition of cholesterol uptake was associated with significant decrease in the levels of NPC1L1 protein and NPC1L1 mRNA, as analyzed by Western blot and qPCR, respectively.

Conclusion: Curcumin inhibits cholesterol uptake through suppression of NPC1L1 expression in the intestinal cells.

Figure 1

Cholesterol uptake by Caco-2 cells is mediated by NPC1L1. The cells were cultured to 100% confluence and pretreated with ezetimibe at different concentrations for 2 h. The cells were then incubated with radioactive micellar cholesterol for 2 h. The cells were then lysed and the uptake of radioactive cholesterol was quantified by liquid scintillation. Results are mean ± SEM of duplicate determinations in three separate experiments. * P < 0.05, ** P < 0.01.

Figure 2

Inhibitory effect of curcumin on micellar cholesterol absorption in Caco-2 cells. The cells were pretreated with curcumin at different concentrations for 24 h, then incubated with radioactive micellar cholesterol for 2 h. The uptake of cholesterol in the absence of curcumin was normalized to 100%. Results are mean ± SEM from triplicate determinations in three separate experiments.

Figure 3

The effect of curcumin on NPC1L1 protein expression in Caco-2 cells. In the upper panel, the cells were treated with curcumin at different concentrations for 24 h, and the whole-cell lysates were analyzed by Western blot. The results are representative of three independent experiments. In the lower panel, NPC1L1 mRNA abundance was determined by real-time RT-PCR as described in Methods. Expression values were normalized to housekeeping genes, and expression in untreated cells was set to 1. Values shown represent means ± SEM of three independent experiments, * P < 0.01, ** P < 0.001 compared to untreated cells.