Effects of 2-Methoxyestradiol, a Main Metabolite of Estradiol on Hepatic ABCA1 Expression in HepG2 Cells.

ATP-binding cassette transporter A1 (ABCA1) is a key regulator of lipid efflux, and the absence of ABCA1 induces hepatic lipid accumulation, which is one of the major causes of fatty liver. 2-Methoxyestradiol (2-ME2) has been demonstrated to protect against fatty liver. In this study, we investigated the effects of 2-ME2 on the hepatic lipid content and ABCA1 expression. We found that 2-ME2 dose-dependently increased ABCA1 expression, and therefore, the lipid content was significantly decreased in HepG2 cells. 2-ME2 enhanced the ABCA1 promoter activity; however, this effect was reduced after the inhibition of the PI3K pathway. The overexpression of Akt or p110 induced ABCA1 promoter activity, while dominant-negative Akt diminished the ability of 2-ME2 on ABCA1 promoter activity. Further, 2-ME2 stimulated the rapid phosphorylation of Akt and FoxO1 and reduced the nuclear accumulation of FoxO1. Chromatin immunoprecipitation confirmed that FoxO1 bonded to the ABCA1 promoter region. The binding was reduced by 2-ME2, which facilitated ABCA1 gene transcription. Furthermore, mutating FoxO1-binding sites in the ABCA1 promoter region or treatment with FoxO1-specific siRNA disrupted the effect of 2-ME2 on ABCA1 expression. All of our results demonstrated that 2-ME2 might upregulate ABCA1 expression via the PI3K/Akt/FoxO1 pathway, which thus reduces the lipid content in hepatocytes.

Arsenic inhibited cholesterol efflux of THP-1 macrophages via ROS-mediated ABCA1 hypermethylation.

Growing evidences indicate that epigenetic modification involves in the mechanisms of atherosclerosis, which intersects with oxidative stress pathway. Arsenic is an important environmental contaminant and has been linked to atherosclerosis. However, the exact mechanism is not well understood. In the present study, we analyzed the effect of arsenic on oxidative stress, ABCA1 promoter methylation and cholesterol efflux of THP-1 macrophages. Results showed that arsenic could induce ROS-mediated DNA methyltransferase 1 (DNMT1) transcription and activity up-regulation, causing ABCA1 promoter to be hypermethylated with repressed expression. In addition, arsenic depleted the methyl donor S-adenosylmethionine (SAM) and induced global DNA hypomethylation. Arsenic inhibited cholesterol efflux of THP-1 macrophages, which could be attenuated after pretreatment with NAC or DNMT inhibitor 5-Aza-2'-deoxycytidine, but not with SAM. All of the findings suggest that arsenic inhibit cholesterol efflux of THP-1 macrophages via ROS-mediated ABCA1 hypermethylation.

Efflux inhibition by H2S confers sensitivity to doxorubicin-induced cell death in liver cancer cells.

AIMS: Hydrogen sulfide (H2S), an important gasotransmitter, is involved in a variety of cellular functions and pathophysiologic processes. Drug resistance due to alterations in drug trafficking and metabolism severely limits the effectiveness of cancer therapy. This study examined the role of H2S in drug resistance in liver cancer cells. MATERIALS AND METHODS: Human primary hepatocellular carcinoma cell line (HepG2) and doxorubicin (Dox)-resistant cells were used in this study. Cell survival was analyzed by MTT, Annexin V-FITC/propidium iodide staining and clonogenic assay. Western blotting was used for analysis of protein expression, and immunoprecipitation was used to determine interactions of LXR/RXR. KEY FINDINGS: The expression of H2S-generating enzyme cystathionine gamma-lyase (CSE) was inhibited by doxorubicin treatment in HepG2 cells, and H2S sensitized Dox-inhibited cell survival and colony formation. In addition, H2S promoted cellular retention of Dox by suppressing the expressions of ABCA1 and ABCG8. H2S significantly blocked Dox-induced heterodimer formation between LXRα and RXRß and attenuated the binding of LXRα/RXRß to the promoters of ABCA1 and ABCG8 genes. RXRß but not LXRα was S-sulfhydrated by H2S, and blockage of RXRß S-sulfhydration abolished the inhibitory role of H2S on LXRα/RXRß heterodimer formation. CSE expression was reduced in Dox-resistant cells in comparison with their parental cells, while H2S could reverse drug resistance in Dox-resistant cells. SIGNIFICANCE: Our study provides a novel solution for reversing drug resistance in cancer cells by targeting H2S signalling.

Probucol-Oxidized Products, Spiroquinone and Diphenoquinone, Promote Reverse Cholesterol Transport in Mice.

OBJECTIVE: Oxidized products of probucol, spiroquinone and diphenoquinone, were shown to increase cell cholesterol release and plasma high-density lipoprotein (HDL) by inhibiting degradation of ATP-binding cassette transporter A1. We investigated whether these compounds enhance reverse cholesterol transport in mice. APPROACH AND RESULTS: Spiroquinone and diphenoquinone increased ATP-binding cassette transporter A1 protein (2.8- and 2.6-fold, respectively, P<0.01) and apolipoprotein A-I-mediated cholesterol release (1.4- and 1.4-fold, P<0.01 and P<0.05, respectively) in RAW264.7 cells. However, diphenoquinone, but not spiroquinone, enhanced cholesterol efflux to HDL (+12%, P<0.05), whereas both increased ATP-binding cassette transporter G1 protein, by 1.8- and 1.6-fold, respectively. When given orally to mice, both compounds significantly increased plasma HDL-cholesterol, by 19% and 20%, respectively (P<0.05), accompanied by an increase in hepatic and macrophage ATP-binding cassette transporter A1 but not ATP-binding cassette transporter G1. We next evaluated in vivo reverse cholesterol transport by injecting RAW264.7 cells labeled with (3)H-cholesterol intraperitoneally into mice. Both spiroquinone and diphenoquinone increased fecal excretion of the macrophage-derived (3)H-tracer, by 25% and 28% (P<0.01 and P<0.05), respectively. spiroquinone/diphenoquinone did not affect fecal excretion of HDL-derived (3)H-cholesterol, implying that macrophage-to-plasma was the most important step in spiroquinone/diphenoquinone-mediated promotion of in vivo reverse cholesterol transport. Finally, spiroquinone significantly reduced aortic atherosclerosis in apolipoprotein E null mice when compared with the vehicle. CONCLUSIONS: Spiroquinone and diphenoquinone increase functional ATP-binding cassette transporter A1 in both the macrophages and the liver, elevate plasma HDL-cholesterol, and promote overall reverse cholesterol transport in vivo. These compounds are promising as therapeutic reagents against atherosclerosis.

Rosuvastatin activates ATP-binding cassette transporter A1-dependent efflux ex vivo and promotes reverse cholesterol transport in macrophage cells in mice fed a high-fat diet.

OBJECTIVE: It is controversial whether statins improve high-density lipoprotein (HDL) function, which plays an important role in reverse cholesterol transport in vivo. The aim of the present study was to clarify the effects of rosuvastatin and atorvastatin on reverse cholesterol transport in macrophage cells in vivo and their underlying mechanisms. APPROACH AND RESULTS: Male C57BL mice were divided into 3 groups (rosuvastatin, atorvastatin, and control groups) and orally administered rosuvastatin, atorvastatin, or placebo for 6 weeks under feeding with a 0.5% cholesterol+10% coconut oil diet. After administration, although there were no changes in plasma HDL cholesterol levels among the groups, plasma from the rosuvastatin group showed an increased ability to promote ATP-binding cassette transporter A1-mediated cholesterol efflux ex vivo. In addition, capillary electrophoresis revealed a shift in HDL toward the pre-ß HDL fraction only in the rosuvastatin group. Mice in all 3 groups were intraperitoneally injected with (3)H-cholesterol-labeled and cholesterol-loaded macrophages and then were monitored for the appearance of (3)H-tracer in plasma and feces. The amount of (3)H-tracer excreted into feces during 48 hours in the rosuvastatin group was greater than that in the control group. Finally, (3)H-cholesteryl oleate-HDL was intravenously injected into all groups, blood samples were taken, and the count of (3)H-cholesterol was analyzed. Plasma (3)H-cholesteryl oleate-HDL changed similarly, and no differences in fractional catabolic rates were observed. CONCLUSIONS: Rosuvastatin enhanced the ATP-binding cassette transporter A1-dependent HDL efflux function of reverse cholesterol transport, and this finding highlights the potential of rosuvastatin for the regression of atherosclerosis.