In vitro effects of some flavonoids and phenolic acids on human pyruvate kinase isoenzyme M2

Abstract

Pyruvate kinase isoenzyme M2 (PKM2) is one of the most important control point enzyme in glycolysis pathway. Hence, its inhibitors and activators are currently considered as the potential anticancer agents. The effect of 28 polyphenolic compounds on the enzyme activity was investigated in vitro. Among these compounds, neoeriocitrin, (−)-catechin gallate, fisetin, (±)-taxifolin and (−)-epicatechin have the highest inhibition effect with IC₅₀ value within 0.65–1.33 µM range. Myricetin and quercetin 3-β-d-glucoside exhibited the highest activation effect with 0.51 and 1.34 µM AC₅₀ values, respectively. Twelve of the compounds showed inhibition effect within 7–38 µM range of IC₅₀ value. Sinapinic acid and p-coumaric acid showed an activation effect with 26.2 and 22.2 µM AC₅₀ values, respectively. The results propose that the polyphenolics may be the potential PKM2 inhibitors/activators, and they may be used as lead compounds for the synthesis of new inhibitors or activators of this enzyme.

• Activation
• cancer
• flavonoid
• inhibition
• phenolic acid
• PKM2

Introduction

It is a proven fact for years that cancerous tissues produce more glucose than normal cells, and this difference is widely used in diagnosis and treatment of the disease1. Metabolic processes in these cells are revised as over-expression of different isoenyzmes of functioning enzymes2. New approaches targeting these enzymes are developed and such studies yielded positive results3. Pyruvate kinase isoenzyme M2 (PKM2) is the most significant regulatory enzyme functioning in intracellular control of glucose consumption specific to cells with overgrowth tendency. Inactive form of the enzyme is used for the synthesis of biomolecules required for cell growth from glycolysis intermediate products, while active form enables synthesis of molecules, such as pyruvate and lactate for ATP and cell metastasis4–6. Since inhibition and activation of this enzyme controls intracellular consumption of glucose, it is an excellent target for cancer treatment. It takes place in the literature that both its inhibitors and activators can be used as chemotherapeutic agents7.

Moreover, some studies point out that glycolysis products, such as lactate and pyruvate are effective in developing drug resistance3. Hence, as indicated in various studies, inhibitors of this enzyme can be used as combined chemotherapeutic agents to overcome drug resistance in the cell.

Flavonoids are significant sources for pharmacological researches as they are bioactive in a wide area. Some studies have pointed out the regulatory effect of these compounds on some enzyme activities playing critical roles in pathology of cancer8^,9. Moreover, they are significant compounds for design of anticancer drugs as they have huge impact on some molecular structures in cancer metabolism with cardinal roles in drug design. PKM2 is one of the primary glycolytic enzymes selected as the target in cancer treatment10. However, no study on the effect of flavonoids and phenolic acids on this enzyme activity were found in the literature.

This research aims to perform an in-vitro study of the inhibition and activation effect of some flavonoids on PKM2 enzyme activity.

Material and methods

Materials

Recombinant human PKM2 enzyme and other chemicals were purchased from Sigma (Munich, Germany) at the high purity.

Assay of PKM2 activity

The enzyme activity was determined by monitoring the absorbance at 340 nm according to the described method11. All reactions were 1 mL in volume at room temperature (25 °C). Each measurement was repeated three times. Reaction mixture includes recombinant 50 nano gram enzyme, 50 mM Tris pH 7.5, 5 mM MgCl₂, 100 mM KCl, 0.5 mM PEP (Phosphoenol pyruvic acid), 0.6 mM ADP (Adenosine 5′-diphosphate), 180 μM NADH (β-Nicotinamide adenine dinucleotide), 10 μM FBP (D-Fructose 1,6-bisphosphate) and 8 units of lactate dehydrogenase (LDH). One unit of pyruvate kinase activity is designated as the amount of enzyme involved to produce 1.0 µM of pyruvate from PEP in one minute.

In vitro effect of polyphenolic compounds

The AC₅₀/IC₅₀ values as the compound concentration that brought about a 50% raise/reduction in enzyme activity under comparable assay and laboratory conditions were calculated from compound dose response curves (for example Figure 1). To determine the potential effect of polyphenolic, the initial velocity of the PKM2 reaction was measured at saturating substrate concentrations of phosphoenolpyruvic acid (PEP) and ADP with at least five different concentrations of the compounds. For each PKM2 activity assay the experiment was repeated three times.

Figure 1. Activity (%) versus polyphenols concentrations regression analysis graphs for PKM2 in the presence of some polyphenols.

In order to determine K_i constants in the media with inhibitor, substrate (PEP) concentrations were 0.027, 0.048, 0.070, 0.104 and 0.140 mM, respectively. Inhibitor (polyphenolic compounds) solutions were added to the reaction medium, three different inhibitor concentrations were used in 1 mL of total reaction volume. The Lineweaver–Burk graphs were plotted by using 1/V versus 1/[S] values, and K_i constant were calculated from these graphs (Figures 2 and 3). Graphs were plotted using Microsoft Office Excel 2010 (Redmond, WA).

Figure 2. The Lineweaver–Burk graph using three different taxifolin concentrations for determination of K_i.

Figure 3. The Lineweaver–Burk graph using three different neoeriocitrin concentrations for determination of K_i.

Results

The effect of 28 polyphenol compounds on PKM2 enzyme activity was tested. Concentration activity % graphs were drawn to identify the effects of compounds and IC₅₀ and AC₅₀ values were calculated using the graph. Results are given in Table 1. Compounds exhibited different activities on the enzyme. In general terms, flavonols had an inhibition effect of approximately 1 µM IC₅₀ value. Among the flavanones, hesperitin, hesperidin, neohesperidin and naringin showed inhibition effect with IC₅₀ value within 14.20–29.10 µM range, while neoeriocitrin had an inhibition effect at a very low concentration of 0.65 µM. Naringenin is the only flavanone to show activation effect. As seen in the table, phenolic acids showed higher concentrations of inhibition effect compared to flavonoids. IC₅₀ for rutin, galangin, quercetin and kaempferol flavonol compounds is calculated to be approximately 8.5 ± 1.5. Fisetin showed inhibition effect with IC₅₀ value under 1 µM. Myricetin and quercetin 3-β-d-glucoside showed activation effect at very low concentration.

Table 1. PKM2 inhibition or activation data by some polyphenol compounds.

Compounds class	Compounds name	IC50/AC50 µM	Effect
Flavonols	1. Fisetin	0.90	Inhibition
	2. Rutin	7.80	Inhibition
	3. Galangin	8.27	Inhibition
	4. Quercetin	9.24	Inhibition
	5. Kaempferol	9.88	Inhibition
	6. Morin hydrate	37.20	Inhibition
	7. Myricetin	0.51	Activation
	8. Quercetin 3-β-d-glucoside	1.34	Activation
	9. Quercetin 3-d-galactoside	27–107 µM	Ineffective
Flavanons	10. Neoeriocitrin	0.65	Inhibition
	11. Neohesperidin	14.20	Inhibition
	12. Naringin	16.60	Inhibition
	13. Hesperidin	17.30	Inhibition
	14. Hesperitin	29.10	Inhibition
	15. Naringenin	70.80	Activation
Flavanonols	16. (−)-Catechin gallate	0.85	Inhibition
	17. (±)-Taxifolin	1.16	Inhibition
	18. (−)-Epicatechin	1.33	Inhibition
	19. (+)-Gallocatechin	4–16 µM	Ineffective
Phenolic  acids	20. Ferulic	11.4	Inhibition
	21. Syringic and	13.8	Inhibition
	22. Caffeic acid	36.3	Inhibition
	23. 3,4-Dihydroxybenzoic acid	78.7	Inhibition
	24. Gallic acid	332.6	Inhibition
	25. Shikimic acid	990	Inhibition
	26. p-Coumaric acid	22.2	Activation
	27. Sinapinic acids	26.2	Activation
	28. Vanillic	607.9	Activation

The Lineweaver–Burk graphs were drawn to find out K_i values and inhibition types for compounds with IC₅₀ value under 5 µM (Table 2). According to graph results, while (±)-taxifolin perform competitive inhibition on enzyme activity with K_i constant around 0.84 ± 0.25 µM, neoeriocitrin, (−)-catechin gallate, fisetin and (−)-epicatechin showed non-competitive inhibition within 1.99 ± 1.14–4.08 ± 2.27 µM range.

Table 2. K_i values obtained from the Lineweaver–Burk graphs for PKM2 in the presence of three fixed inhibitors and five substrate concentrations for different polyphenol compounds.

Compounds	Inhibition type	Ki constant µM
Taxifolin	Competitive	0.84 ± 0.25
Neoeriocitrin	Non-competitive	1.99 ± 1.14
Fisetin	Non-competitive	3.69 ± 1.89
(−)-Catechin gallate	Non-competitive	2.60 ± 1.89
(−)-Epicatechin	Non-competitive	4.07 ± 2.27

Discussion

Cancer is one of the leading diseases threatening the humanity globally12. Due to the toxic effects of existing treatment methods, various studies are carried to develop new treatment methods with less toxic effects. Cancer cells consume extreme amounts of glucose compared to normal cells. Isoenzymes over-synthesized in cancer cells and functioning in glycolysis pathway, a significant metabolic pathway of glucose, are the new targets in cancer treatment. PKM2 is the most important control point enzyme where glucose consumption is controlled. Thus, it is stated in the literature that inhibitors and activators of the enzyme can be used as drugs13.

Natural compounds, especially the phenolic compounds, are significant sources for pharmacological researches in the discovery of active drug substances14–16. Due to their structural properties, phenolic compounds can have non-covalent interactions with proteins. The polyphenols were reported as the inhibitors of many enzymes, such as glucosidases, P-glycoprotein and xanthine oxidase in low micromolar ranges17–19. Similarly, the recent studies showed that the carbonic anhydrase isoenzymes were significantly inhibited by natural phenolic compounds and their derivatives20–24. In this study, the effects of some phenolic compounds on PKM2 activity were investigated, and their impact order was determined as flavanonol > flavonol > flavanon > phenolic acids. Flavonoids were generally found to be more active compounds compared to the phenolic acids.

The highest inhibition effect was measured with neoeriocitrin, fisetin and (−)-catechin gallate under 1 µM IC₅₀ value. Moreover, (−)-epicatechin and (±)-taxifolin showed inhibition effect at the value of approximately 1 µM. Flavonoids exhibited high inhibitory effect on enzyme activity having hydroxyl group substitution at position 3′,4′. These groups are considered to be effective in the interaction between compounds and proteins. Given the enzyme kinetics of these compounds, (±)-taxifolin and neoeriocitrin inhibit the enzyme by binding to the active site where the enzyme catalyzes PEP, while other compounds bind to the different amino acid residues outside the active site for inhibition.

Flavonols, such as quercetin, galangin and kaempferol that include 3, 5, 7 OH groups showed a similar inhibition effect. These compounds can interact with the enzyme in these OH groups25^,26. In contrast to the inhibition effect of quercetin, its analogues, myricetinin and quercetin 3-β-d-glucoside showed activation effect with 0.5 and 1.34 µM AC₅₀ values, respectively. However, quercetin 3-d-galactoside, with d-galactose at its three positions, did not show any effect on PKM2 enzyme activity within 27–107 µM range.

It is considered that flavonoids can be potential inhibitors or activators in low concentrations. The changes in substituted groups result in significant differences in activity; therefore, flavonoids can be used as lead compounds in synthesis and design of the inhibitors and activators of this enzyme.

Phenolic acids exhibited different effects within a wide range. Substituents over the benzene ring and acrylic functional groups play an important role in activity differences. Inhibition effect of phenolic acids containing acryl group, such as shikimic acid and 3,4-dihydroxybenzoic acid, is higher than those containing carboxylic acid, such as gallic acid and caffeic acid. Comparison of the effects of syringic-ferulic acid and shikimic-caffeic acid reveals that methoxy groups improve the inhibition effect of the compounds. The change over benzene ring of p-coumaric acid and sinapinic acids did not significantly change the activation effect of the compound.

Drug resistance is an important problem in cancer treatment27. The PKM2 enzyme inhibition causes a reduction in the amount of metabolites, such as pyruvate and lactate, which involves in drug resistance. Therefore, the compounds inhibiting the enzyme can be used as combined chemotherapeutic agents28. Moreover, the lactate participates in the formation of micro-acidic environment around the cell, and contributes to metabolic symbiosis as a significant factor in permanence of the other cancer cells29. Reduction of the lactate amount is a new therapeutic target for cancer treatment. These metabolic processes can be prevented by PKM2 enzyme inhibitors.

Biological, pharmacological and drug properties of polyphenols are widely studied in previous studies9. It is reported that these compounds affect metabolic pathways and show anticancer activity with action mechanisms, such as inhibition of protein, DNA and RNA synthesis, reduction in cAMP level, and protein kinase and topoisomerase enzyme inhibition in cell metabolism. Likewise, PKM2 enzyme is considered as a potential target in cancer treatment since its inhibition or activation significantly affects the glucose consumption. Therefore, we propose that the inhibition and activation effect of these compounds on PKM2 may have a contribution to their anticancer property.

In conclusion, the present in vitro study reports the effects of some flavonoids and phenolic acids on PKM2 enzyme. A substantial part of these compounds showed inhibition effect, while some compounds exhibited powerful activation effect. These compounds can be considered as potential inhibitors or activators due to their significant effects on the enzyme activity. The results contribute to the understanding of anticancer action mechanisms of these compounds. Further studies might be performed on the effect of new phenolic compounds on the enzyme activity, and additional active compounds may be synthesized using these compounds.

Declaration of interest

This research was financed by TUBITAK (Project No: 112T781).