Resveratrol Affects Sphingolipid Metabolism in A549 Lung Adenocarcinoma Cells

Abstract

Resveratrol is a naturally occurring polyphenol which has various beneficial effects, such as anti-inflammatory, anti-tumor, anti-aging, antioxidant, and neuroprotective effects, among others. The anti-cancer activity of resveratrol has been related to alterations in sphingolipid metabolism. We analyzed the effect of resveratrol on the enzymes responsible for accumulation of the two sphingolipids with highest functional activity-apoptosis promoting ceramide (CER) and proliferation-stimulating sphingosine-1-phosphate (S1P)-in human lung adenocarcinoma A549 cells. Resveratrol treatment induced an increase in CER and sphingosine (SPH) and a decrease in sphingomyelin (SM) and S1P. Our results showed that the most common mode of CER accumulation, through sphingomyelinase-induced hydrolysis of SM, was not responsible for a CER increase despite the reduction in SM in A549 plasma membranes. However, both the activity and the expression of CER synthase 6 were upregulated in resveratrol-treated cells, implying that CER was accumulated as a result of stimulated de novo synthesis. Furthermore, the enzyme responsible for CER hydrolysis, alkaline ceramidase, was not altered, suggesting that it was not related to changes in the CER level. The enzyme maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was downregulated, and its expression was reduced, resulting in a decrease in S1P levels in resveratrol-treated lung adenocarcinoma cells. In addition, incubation of resveratrol-treated A549 cells with the SK1 inhibitors DMS and fingolimod additionally downregulated SK1 without affecting its expression. The present studies provide information concerning the biochemical processes underlying the influence of resveratrol on sphingolipid metabolism in A549 lung cancer cells and reveal possibilities for combined use of polyphenols with specific anti-proliferative agents that could serve as the basis for the development of complex therapeutic strategies.

Keywords: ceramide; lung cancer cells; resveratrol; sphingolipid metabolism; sphingosine-1-phosphate.

Figure 1

Sphingomyelin (SM) content in plasma membranes isolated from A549 cells untreated (Control, white bars) and treated with resveratrol (RSV, black bars). Values are expressed as relative percentage participation in the total lipids. Values are means ± SD. * p < 0.01.

Figure 2

Changes in the activity (A) and protein expression (B) of neutral sphingomyelinase (nSMase) in control (white bars) and resveratrol-treated (black bars) A549 cells. Values are expressed as % change of controls (100%). Representative images from Western blot analysis with specific antibodies to neutral sphingomyelinase (nSMase) are shown in the left part of panel B. Reaction with anti-glyceraldehyde-3-phosphate dehydrogenase antibodies (anti-GAPDH) was used as an internal control for loading; (ᵒ) stands for the values from each independent experiment, obtained by pooling of three replicate samples. Values represent means ± SD. The differences between the obtained values were not statistically significant.

Figure 3

Content of sphingomyelin and cholesterol in the incubation medium of A549 cells untreated (Control, white bars) and treated with resveratrol (RSV, black bars). Values are expressed as % of controls (100%). Values are means ± SD. The differences between the values obtained for both SM and CH were statistically significant. * p < 0.01.

Figure 4

Changes in the level of ceramide, sphingosine, and sphingosine-1-phosphate in control (Control, white bars) and resveratrol-treated (RSV, black bars) A549 cells. Values are expressed as % of controls (100%). Values are means ± SD. The differences between the values obtained for ceramide (** p < 0.001), sphingosine (* p < 0.01), and sphingosine-1-phosphate (** p < 0.001) were statistically significant.

Figure 5

Alteration in the activity (A) and the expression (B) of ceramide synthase 6 in control (Control, white bars) and resveratrol-treated (RSV, black bars) A549 cells. Values are expressed as % of controls (100%). Representative images from immunoblotting obtained with antibodies against ceramide synthase (anti-CER synth) are shown on the left part of panel B. Reaction with anti-glyceraldehyde-3-phosphate dehydrogenase antibodies (anti-GAPDH) was used as an internal control for loading. Graphical depiction of the percent change in CER synthase expression is presented on the right part of panel B; (ᵒ) stands for the values from each independent experiment, obtained by pooling of three replicate samples. Values represent means ± S.D. * p < 0.01, ** p < 0.001.

Figure 6

Specific activity (A) and protein expression (B) of alkaline ceramidase in control (Control, white bars) and resveratrol-treated (RSV, black bars) A549 cells. Values are expressed as % change of control (100%). Representative images from Western blot analysis obtained with antibodies against alkaline ceramidase (anti-ALCER) are shown on the left part of panel B. Reaction with anti-glyceraldehyde-3-phosphate dehydrogenase antibodies (anti-GAPDH) was used as an internal control for loading. Graphical depiction of the percent change in alkaline ceramidase expression is presented on the right part of panel B; (ᵒ) stands for the values from each independent experiment, obtained by pooling of three replicate samples. Values are means ± SD. The differences between the obtained values were not statistically significant.

Figure 7

Inhibition of the activity (A) and the expression (B) of sphingosine kinase 1 in control (Control, white bars) and resveratrol-treated (RSV, black bars) A549 cells. Values are expressed as % of controls (100%). Representative images from Western blot analysis obtained with antibodies against sphingosine kinase 1 (anti-SK1) are shown on the left part of panel B. Reaction with anti-glyceraldehyde-3-phosphate dehydrogenase antibodies (anti-GAPDH) was used as control for loading. Graphical depiction of the percent change in SK1 expression is presented on the right part of panel B; (ᵒ) stands for the values from each independent experiment, obtained by pooling of three replicate samples. Values represent means ± S.D. * p < 0.01.

Figure 8

Alterations in the activity (A) and expression (B) of sphingosine kinase 1 in A549 cells incubated only with resveratrol (RSV, black bars), resveratrol and DMS (RSV+DMS, dark gray bars), and resveratrol plus fingolimod (RSV+FTY, light gray bars). Illustrative images from Western blots, obtained with antibodies against sphingosine kinase 1 (anti-SK1), are shown on the left part of panel B. Reaction with anti-glyceraldehyde-3-phosphate dehydrogenase antibodies (anti-GAPDH) was used as internal control for loading. Graphical representation of the percent change in SK1 expression is presented on the right part of panel B. Values are expressed as % of RSV-treated A549 cells serving as controls; (ᵒ) stands for the values from each independent experiment, obtained by pooling of three replicate samples. Values are means ± SD. * p < 0.01.

Figure 9

Schematic presentation of the effects of resveratrol on sphingolipid metabolism in lung cancer A549 cells. SM—sphingomyelin; CER—ceramide; SPH—sphingosine; S1P—sphingosine-1-phosphate; nSMase—neutral sphingomyelinase; ALCER—alkaline ceramidase; SK1—sphingosine kinase 1; CER synthase—ceramide synthase. (*) indicates the presence in the literature of information about resveratrol-induced changes in HepG2 cells [30]; (#)—in breast cancer cells [27]; (^)—in prostate cancer cells [28]; and (•)—in a mouse model of acute peritonitis [29].