Effect of Quercetin and Fingolimod, Alone or in Combination, on the Sphingolipid Metabolism in HepG2 Cells

Abstract

Combinations of anti-cancer drugs can overcome resistance to therapy and provide new more effective treatments. In this work we have analyzed the effect of the polyphenol quercetin and the anti-cancer sphingosine analog fingolimod on the sphingolipid metabolism in HepG2 cells, since sphingolipids are recognized as mediators of cell proliferation and apoptosis in cancer cells. Treatment of hepatocellular carcinoma HepG2 cells with quercetin and fingolimod, alone or in combination, induced different degrees of sphingomyelin (SM) reduction and a corresponding activation of neutral sphingomyelinase (nSMase). Western blot analysis showed that only treatments containing quercetin induced up-regulation of nSMase expression. The same treatment caused elevation of ceramide (CER) levels, whereas the observed alterations in sphingosine (SPH) content were not statistically significant. The two tested drugs induced a reduction of the pro-proliferative sphingolipid, sphingosine 1 phosphate (S1P), in the following order: quercetin, fingolimod, quercetin + fingolimod. The activity of the enzyme responsible for CER hydrolysis, alkaline ceramidase (ALCER) was down-regulated only in the incubations involving quercetin and fingolimod did not affect this activity. The enzyme, maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was down-regulated by incubations in the following order: quercetin, fingolimod, quercetin + fingolimod. Western blot analysis showed down-regulation in SK1 expression upon quercetin but not upon fingolimod treatment. Studies on the effect of quercetin and fingolimod on the two proteins associated with apoptotic events, AKT and Bcl-2, showed that only quercetin, alone or in combination, down-regulated the activity of the two proteins. The reported observations provide information which can be useful in the search of novel anti-tumor approaches, aiming at optimization of the therapeutic effect and maximal preservation of healthy tissues.

Keywords: HepG2 cells; ceramide; fingolimod; quercetin; sphingolipid metabolism; sphingosine-1-phosphate.

Figure 1

Viability of HepG2 cells treated for 24 h with quercetin in a concentration range of 20–120 μM (A) and fingolimod (B) in a concentration range of 1–80 μM. Statistical significance is presented as comparison of treated cells with controls. * p< 0.05; ** p< 0.001.

Figure 2

Sphingomyelin content in HepG2 cells untreated (Control) and treated with quercetin (Q) and fingolimod (F). Values are expressed as relative percentage participation in the total lipids. Values are means ± SD. Statistical significance is presented as comparison of treated cells with controls. * p < 0.01.

Figure 3

Changes in the activity (A) and protein expression (B) of neutral sphingomyelinase (nSMase) in control and treated with quercetin (Q) and fingolimod (F) HepG2 cells. Values are expressed as % of controls (100%). Representative images from Western blot analysis with specific antibodies to neutral sphingomyelinase (anti-nSMase) are shown at the lowermost of panel B. Reaction with anti-glyceraldehyde-3-phosphate dehydrogenase antibodies (anti-GAPDH) was used as an internal control for loading. Values are means ± SD. Statistical significance is presented as comparison of treated cells with controls. * p < 0.05; ** p < 0.01.

Figure 4

Changes in the level of ceramide (CER), sphingosine (SPH) and sphingosine-1-phosphate (S1P) in control and treated with quercetin (Q) and fingolimod (F) HepG2 cells. Values are expressed as % of controls. Values are means ± SD. Statistical significance is presented as comparison of treated cells with controls. * p < 0.01.

Figure 5

Alteration in the activity (A) and protein expression (B) of alkaline ceramidase (ALCER) in control and treated with quercetin (Q) and fingolimod (F) HepG2 cells. Values are expressed as % of controls (100%). Representative images from Western blot analysis with specific antibodies to alkaline ceramidase (anti-ALCER) are shown at the bottom of panel B. Reaction with anti-glyceraldehyde-3-phosphate dehydrogenase antibodies (anti-GAPDH) was used as an internal control for loading. Values are means ± SD. Statistical significance is presented as comparison of treated cells with controls. * p < 0.01.

Figure 6

Changes in the activity (A) and protein expression (B) of sphingosine kinase 1 (SK1) in control and treated with quercetin (Q) and fingolimod (F) HepG2 cells. Values are expressed as % of controls (100%). Representative images from Western blot analysis with specific antibodies to sphingosine kinase 1 (anti-SK1) are shown at the lowermost of panel B. Reaction with anti-glyceraldehyde-3-phosphate dehydrogenase antibodies (anti-GAPDH) was used as an internal control for loading. Values are means ± SD. Statistical significance is presented as comparison of treated cells with controls. * p < 0.05; ** p < 0.01.

Figure 7

Changes in Akt phosphorylation (A) and Bcl-2 expression (B) in control and treated with quercetin (Q) and fingolimod (F) HepG2 cells. Values are expressed as % of controls. Representative images from Western blot analysis with specific antibodies to phosphorylated Akt (anti-pAkt) and B cell lymphoma 2 protein (anti-Bcl-2) are shown at the lowermost of the panels. Reactions with antibodies against total Akt (tAkt) and anti-glyceraldehyde-3-phosphate dehydrogenase antibodies (anti-GAPDH) were used as internal controls for loading. Values are means ± SD. Statistical significance is presented as comparison of treated cells with controls. * p < 0.05; ** p < 0.01.

Figure 8

Apoptotic response of HepG2 cells after treatment with quercetin (Q), fingolimod (F) or combination of both drugs (Q + F). Apoptotic (white arrows in the insets) and non-apoptotic (gray arrows in the insets) nuclei were scored, and the percentage of apoptotic cells in each sample was determined. Bar, 100 µm. * p < 0.001.