Luteolin-7- O-β-d-Glucoside Inhibits Cellular Energy Production Interacting with HEK2 in Keratinocytes

Abstract

Flavonoids have been demonstrated to affect the activity of many mammalian enzyme systems. Their functional phenolic groups are able to mediate antioxidant effects by scavenging free radicals. Molecules of this class have been found able to modulate the activity of kinases, phospholipase A2, cyclooxygenases, lipoxygenase, glutathione S-transferase, and many others. Recently, it has been demonstrated that luteolin, in the form of Luteolin-7-O-β-d-glucoside (LUT-7G) is able to induce the keratinocyte differentiation process in vitro. This flavonoid is able to counteract the proliferative effects of IL-22/IL6 pathway by the inhibition of STAT3 activity also in vivo in a psoriatic mouse model. Observations on energy metabolism changes of differentiating cells led us to perform a complete metabolomics analysis using human primary keratinocytes treated with LUT-7G. Our results show that LUT-7G, is not only able to impair the nuclear translocation of STAT3, but it also blocks the energy metabolism pathway, depressing the glycolytic and Krebs pathway by the inhibition of hexokinase 2 activity. These data confirm that LUT-7G can be proposed as a potential candidate for the treatment of inflammatory and proliferative diseases, but its role as a hexokinase 2 (HEK2) inhibitor opens new perspectives in nutritional science, and especially in cancer therapy, in which the inhibition of the Warburg effect could be relevant.

Keywords: glycolysis; hexokinase inhibitor; luteolin-7-O-β-d-glucoside.

Figure 1

(A) Cellular ATP content in keratinocyte treated with LUT-7G showing a reduction of about 60%. (B) ADP content. (C) Metabolic analysis showing the decrease of G6P, F6P, and 3PG, as well of phosphoenolpyruvate (PEP). The cellular glucose (first upper panel) remains high inside cells. The data are represented as a boxplot from nine replicate samples. (D) ATP content of HaCaT cells (immortalized keratinocytes) showing a reduction of about 25% of ATP after LUT-7G treatment. * p < 0.05.

Figure 2

(A) Metabolomic analysis of tricarboxylic acid cycle or krebs cycle (TCA) intermediates showing the depression of citrate, succinate, fumarate, and malate in LUT-7G treated keratinocytes. (B) Analysis of intermediate of pentose phosphate pathway also shows a downregulation. (C) The analysis of important vitamin cofactors shows increased availability.

Figure 3

(A) Clustering and annotation analysis (methods) from RNAseq experiment in differentiated keratinocytes. The nodes are the processes where most regulated gene cluster, the arrows represent the connections between the processes. As is visible, there is a preponderant association with epidermis differentiation, cornification, and peptide crosslinking. (B) The same analysis showed another cluster (of downregulated genes) acknowledging downregulation of energy pathway metabolism. (C–E) Analysis of metabolic intermediate of glycolysis and Krebs cycle in differentiating keratinocytes, showing depression of G6P and citrate. In this case (first panel), a strong decrease of cellular content of glucose is visible.

Figure 4

Top, best docking complexes between HEK2 and LUT-7G. The luteolin molecules, hosted in the active sites of HEK2, are depicted by spacefill representations colored by atom type, while HEK2 is shown as a green cartoon. (A) Site 1 and (B) site 2, detailed views of the best docking solutions. The LUT-7G molecules are depicted by stick representations, while the protein is shown as a green cartoon. The molecular views have been produced by using the program UCSF Chimera [25]. (C) Spectrophotometric analysis for NADH measuring. The sample containing the reaction mixture without glucose is indicated by a black curve, the sample containing LUT-7G shows a characteristic absorbance curve of luteolin with a peak around 350 nm (red curve). When the LUT-7G was added to the reaction mixture (green line) the absorbance peaks disappeared. (D) The presence of NADH was evaluated measuring absorbance at 340 nm. The data are expressed as fold over control (absorbance of reaction mix), in the left group a concentration of glucose of 0.28 mM was used, and two concentrations of LUT-7G of 0.028 and 0.055 mM, respectively. In the right-hand group of columns, a concentration of glucose of 0.55 mM was used. In both experiments a reduction of NADH synthesis is visible. t-test, * p < 0.05.

Figure 5

(A) Schematic view of the best HEK2 active site docking solution with LUT-7G for site 1. The 2D depiction shows hydrogen bonds as green dashed lines between the interaction partners. The residues that are in proximity of the ligand are indicated. This image was produced using the LigPlot+ software [26]. (B) A schematic view of the best HEK2 active site docking solution with LUT-7G for site 2.

Figure 6

Side view of HEK2 active site 1 showing the bound LUT-7G molecule located by the docking procedure at the bottom of the active site crevice. The spacefill representation colored by atom type indicates the LUT-7G molecule, while the enzyme was rendered as a red solid surface. This picture was produced by using the program PyMol [27].