Go to research.

Results

The combined treatment with EPA and lovastatin enhanced the regulatory effect on gene expression of HMGCoA reductase and LDL receptor in HepG2 cell line. Moreover, we detected a synergistic effect on the inhibition of cancer cell proliferation obtained by combination of EPA and Lovastatin.

Introduction

In the present study we investigated in vitro whether combined treatment with Eicosapentaenoic acid (EPA), belonging to omega-3 family, and lovastatin enhanced the regulatory effect on gene expression of HMGCoA reductase and LDL receptor in HepG2 cell line.

Cell culture conditions

HepG2, a cell line derived from human liver tissue with a well differentiated hepatocellular carcinoma, were obtained from the ICLC (IST, Genoa, Italy).

Lovastatin and EPA treatment

To examine the response to EPA, HepG2 cells were treated for 48 h with culture medium supplemented with various concentrations of EPA (1, 10, 25, 50 and 100 μM). Each experiment included a control without EPA and a control with the same amount of DMEM -BSA used for dissolving the fatty acid.

Lovastatin and EPA treatment

For the experiment testing the effect of combined treatment of Lovastatin and EPA on subconfluent HepG2 cells two sets of experiments were prepared. In the first set, HepG2 cells were incubated with 50 μM of EPA and increasing concentrations of Lovastatin (1,10, 25, 50 and 100 μM). The second set of experiments were performed by incubating HepG2 with Lovastatin at the concentration of 50 μM with increasing concentrations of EPA (1,10, 25, 50 and 100 μM) (data not shown). In these experimental conditions, HepG2 cells were allowed to growth for 48 h. Each experiment included an untreated control and a control with the equivalent concentration of solvent used for adding Lovastatin and EPA.

LDL receptor and HMG-CoA reductase gene expression

Analysis of gene expression was performed in HepG2 cells treated with 1, 10, 25, 50 and 100 μM of EPA and Lovastatin alone and with both compounds at established concentrations for 48 hours.

Methods

Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 μM EPA, or 50 μM DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits.

Results

The fat accumulation in 3T3-L1 adipocytes was positively correlated with their age; however, EPA and DHA did not affect lipid accumulation on any stage of maturation. EPA and DHA increased the concentration of secreted adiponectin when compared with control, but only in the case of young adipocytes (58% and 35%, respectively). Moreover, EPA supplementation increased interleukin 6 concentration in conditioned medium, while DHA exerted an opposite effect on all stages of cellular maturation. Furthermore, EPA treatment increased leptin release from young cells, while DHA did not affect the secretion of this adipokine. In mature 3T3-L1 adipocytes both experimental factors decreased synthesis of leptin; however, in old cells no impact of these PUFA was noted.

Methods

Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 μM EPA, or 50 μM DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits.

Results

The fat accumulation in 3T3-L1 adipocytes was positively correlated with their age; however, EPA and DHA did not affect lipid accumulation on any stage of maturation. EPA and DHA increased the concentration of secreted adiponectin when compared with control, but only in the case of young adipocytes (58% and 35%, respectively). Moreover, EPA supplementation increased interleukin 6 concentration in conditioned medium, while DHA exerted an opposite effect on all stages of cellular maturation. Furthermore, EPA treatment increased leptin release from young cells, while DHA did not affect the secretion of this adipokine. In mature 3T3-L1 adipocytes both experimental factors decreased synthesis of leptin; however, in old cells no impact of these PUFA was noted.

Methods

Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 μM EPA, or 50 μM DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits.

Results

The fat accumulation in 3T3-L1 adipocytes was positively correlated with their age; however, EPA and DHA did not affect lipid accumulation on any stage of maturation. EPA and DHA increased the concentration of secreted adiponectin when compared with control, but only in the case of young adipocytes (58% and 35%, respectively). Moreover, EPA supplementation increased interleukin 6 concentration in conditioned medium, while DHA exerted an opposite effect on all stages of cellular maturation. Furthermore, EPA treatment increased leptin release from young cells, while DHA did not affect the secretion of this adipokine. In mature 3T3-L1 adipocytes both experimental factors decreased synthesis of leptin; however, in old cells no impact of these PUFA was noted.

Methods

Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 μM EPA, or 50 μM DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits.

Results

The fat accumulation in 3T3-L1 adipocytes was positively correlated with their age; however, EPA and DHA did not affect lipid accumulation on any stage of maturation. EPA and DHA increased the concentration of secreted adiponectin when compared with control, but only in the case of young adipocytes (58% and 35%, respectively). Moreover, EPA supplementation increased interleukin 6 concentration in conditioned medium, while DHA exerted an opposite effect on all stages of cellular maturation. Furthermore, EPA treatment increased leptin release from young cells, while DHA did not affect the secretion of this adipokine. In mature 3T3-L1 adipocytes both experimental factors decreased synthesis of leptin; however, in old cells no impact of these PUFA was noted.

Methods

Young, mature and old differentiated 3T3-L1 adipocytes were cultured for 48 h in the presence of 100 μM EPA, or 50 μM DHA complexed to albumin, whereas in control conditions only albumin was added to the medium. The Oil Red O staining was used to confirm adipocytes differentiation, and measure triglycerides content in cells. The concentration of adipokines (interleukin 6, adiponectin and leptin) in conditioned media was measured using mouse-specific ELISA kits.

Effects of EPA and DHA on lipid accumulation and lipolysis

Oil Red O was extracted from adipocytes and absorbance was measured to evaluate triglyceride contents in cells. The accumulation of lipids in differentiated 3T3-L1 cells was positively correlated with the day of culture (Figure ). However, examined polyunsaturated fatty acids EPA and DHA did not affect lipid accumulation on any stage of maturation. Glycerol release was measured to determine the influence of EPA and DHA on the intensity of lipolysis in cultured adipocytes. Experimental factors had no impact on lipolysis in used concentrations (that constituted the highest concentrations showing no toxic effect in the cells, as evaluated in a preliminary study – data not shown).