Comparison of Curcumin Inhibition of Rabbit Muscle Phosphofructokinase-1 and Pyruvate Kinase

Percy Russell, Ph.D., Anita Williams, B.S., Rachel Primacio, B.S., Stephanie Castruita, Sean Hui, Mayra Lucas, Mauricio Marquez Palencia, Charlie Millar, Alejandra Perez

University of California, San Diego and Moores UCSD Cancer Center

This research was supported by the following grants: NIH P30CA23100; NIH 5R25CA132699; NIH/NIGMS 2R25GM073590; HCOP D 18HP10623; NIH U54CA132379/U54CA12384; and NIH/NCMHD P60MD000220. The authors would also like to acknowledge the following for their support and contributions: Dr. Georgia Robins Sadler, Dr. Lawrence Alfred, Dr. Percy Russell, Anita Williams, Rachel Primacio, and Stephanie Castruita.

MaterialsPFK-1 was purified from rabbit muscle tissue. The rabbit enzymes were commercially acquired from Sigma-Aldrich Co: Lactate dehydrogenase (LDH), Aldolase (ALD), Pyruvate Kinase (PK).MethodsPurification of PFK-1PFK-1 was purified from rabbit muscle and homogenized in 1 mM EDTA/DTT/NaF, then centrifuged. The pellet was re-suspended in 1mM EDTA/DTT/NaF. The process was then repeated with of 1 mM EDTA/DTT/NaF. The re-suspension was heat-treated and placed into an ice bath until it reached 10ºC. This solution was centrifuged; the supernatant was retained, the pellets were re-suspended and centrifuged again. The supernatant from the last samples were combined and put into a 100 ml ion-exchange column. Two fractions of 40 mM TP8/1 mM DTT were put into the column, followed by fractions of 600 mM TP8/1 mM DTT. Fractions with high PFK-1 activity were combined, then placed in an ice bath with ammonium sulfate per 100 mL of solution. The solution was stirred and then centrifuged; the precipitate is the PFK-1 purified enzyme. PFK-1 Titrations Against Curcumin Fixed [PFK-1] against varying curcumin concentrations were incubated and the PFK-1 activity was determined for the IC50 value. (Figure 1A)PFK-1 Titrations Against a Constant Curcumin ConcentrationFixed concentrations of curcumin with varying [PFK-1] were incubated and the PFK-1 activity was determined. (Figure 2A)Constant PK Concentrations Titration Against Varying Curcumin ConcentrationVarying concentration of curcumin against constant [PK] were used to determine the I50 value. (Figure 1B)PK Titrations Against a Constant Curcumin ConcentrationFixed concentrations of curcumin with varying [PK] were incubated and the PK activity was determined. (Figure 2B)Michaelis – Menten KineticsMichaelis-Menten kinetics determines whether the inhibition is competitive, non-competitive, or uncompetitive. The method for determining the Vmax and KI constant, involves running a series of enzyme assays at increasing substrate concentration. The values can be obtained by plotting 1/[S] vs. 1/v0. (Figure 3)AssaysStandard PFK-1 assaysTwo concentrations of PFK-1 Assays were created: PFK-1 Assay. (0.1 M TP8, 59 mM F6-P, 29 mM ATP, 4.94 mM NADH, GDH/TPI, and aldolase enzyme)ALD AssayAldolase: 0.1 M TP8, 59 mM F1,6-BP, 4.94 mM NADH, and GDH/TPI.LDH AssayLDH solutions: DI-H20, KPO4 buffer, NADH, and pyruvate.

ABSTRACT

INTRODUCTION

MATERIALS & METHODS

ACKNOWLEDGEMENTS

RESULTS

REFERENCES

DISCUSSION

Glycolytic Enzyme

IC-50 Value of Curcumin

Reversible/Irreversible

Type of Reversible Inhibition

PFK-1 0.0025 mM ReversibleNon-

Competitive

PK 0.035 mM ReversibleNon-

Competitive

Glycolysis is a biological process, degradation of glucose that is part of cellular respiration. This process creates 2 molecules of ATP, the major energy source for muscle contraction. Cancer cells depend heavily on glycolysis as an energy source. In this study, we determine whether glycolysis is inhibited by curcumin, a TCM used in the treatment of cancers. Previous research determined that PFK-1, LDH, and adenylate kinase (AK), are inhibited by ascorbate (AA) when muscles are at rest. Aldolase protected these enzymes from AA inhibitions. PFK-1 is considered the controlling enzyme in the glycolytic process. PFK-1 is involved in the first irreversible point in glycolysis, in which ATP is dephosphorylated to adenosine diphosphate, ADP, and fructose 6-phosphate is phosphorylated to fructose 1,6-bisphosphate (F 1,6-BP). The formation of F 1,6-BP commits to the formation of pyruvate. Inhibition of pyruvate kinase by curcumin is tested for the potential of glycolysis inhibition, ultimately discovering ways to discontinue the energy source for cancer cells. This study will test whether curcumin will inhibit glycolysis and serve as an alternate mechanism for cancer chemotherapy.

Figure 1 A. 30 nM Rabbit Muscle PFK-1 Determination of the IC50 value is 0.0025 mM.

Figure 2A. The reversible character of PFK-1 with curcumin inhibition. This is the pattern of convergence at the origin indicating a reversible inhibition. The symbols for the concentration of curcumin are as follows: (), presence; (), absence.

Figure 2B

Figure 3C

This study is base on the variety of explanations for the mechanism of curcumin as a TCM. An alternate possibility is that curcumin inhibits glycolysis, the major energy source of cancer cells. This experiment was performed to demonstrate whether PFK-1 or PK were inhibited by curcumin. These two enzymes are involved in important steps in glycolysis. By inhibiting the enzymes in these two steps, glycolysis is brought to cessation.

In order to perform the enzymatic assays, it is necessary to use pure PFK-1 because, from previous experiments performed with ascorbate, it is shown that other enzymes such as ALD and LDH which protect PFK-1 from any inhibition and vice versa. The analysis of PFK-1 was done with a coupled reaction assay in a spectrophotometer, in which the spectrophotometer measured the oxidation of NADH to NAD+.

The IC50 value was determined from Figure 1A for PFK-1 and Figure 1B for PK to find the concentration of curcumin that inhibits 50% of the activity under a given set of conditions. In this way the inhibition potency can be compared with irreversible inhibitors under the same conditions. The enzyme is then diluted to 30 nM, which decreases the enzyme activity value theoretically to 0.03 eu/mL. This is interpreted as the separation of the tetramer form of PFK-1 to a dimer or monomer form where inhibition occurs. The IC50 value was found to be 0.0025 mM curcumin for PFK-1 and 0.035 mM curcumin for PK. To determine whether inhibition of PFK-1 and PK by curcumin is reversible or irreversible, both enzymes at increasing concentrations of the enzymes were titrated against a constant curcumin concentration. The results from Figure 2A and 2B shows that PFK-1 and PK inhibition, respectively, were indeed reversible because both lines diverge from a single point. Graphically, using Michaelis-Menten kinetics, the type of reversible of inhibition was determined. The graphs, Figure 3A and 3C, shows that both PFK-1 and PK, respectively, are non-competitive because they all intersect at a single point on the x-axis.

This experiment was done to show whether or not curcumin inhibits PFK-1 and PK, and the results shows it does in fact inhibit both. In future experiments, curcumin will need to be used to determine whether other glycolytic enzymes can be inhibited when attached to muscle contractile proteins, and to test if there is a mutual protection of glycolytic enzymes from curcumin inhibition when the muscle is active. By determining the IC50 values, curcumin can be compared with irreversible inhibitors for inhibition potency.

Curcumin can prevent cancer cell growth by stopping the process of glycolysis causing the cancer cells to eventually die, which may be another explanation for the effectiveness of curcumin as a TCM.

Globally, 12.7 million people are diagnosed with cancer per year. A study at Dr. Russell’s biochemistry laboratory at UCSD, tests the inhibition of phosphofructokinase-1 (PFK-1), a glycolytic enzyme, by ascorbic acid when the muscle is at rest and facilitates glycogen storage in muscle cells. Active muscle appears to lead to mutual protection among glycolytic enzymes by forming a complex with muscle contractile proteins permitting glycolysis to resume. In this study curcumin a Traditional Chinese Medicine (TCM) is examined as to whether it inhibits glycolytic enzymes, PFK-1 and pyruvate kinase (PK). Rabbit muscle enzymes, PFK-1 and PK showed a reversible and non-competitive inhibition by curcumin. The IC50 vales for PFK-1 was 0.0025 mM curcumin and for PK was 0.035 mM curcumin. Cancer cells’ main energy source is through glycolysis, so inhibiting this process by curcumin allows us to discover other chemotherapy treatments.

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Figure 3A & Figure 3B. Curcumin inhibition kinetics of rM muscle PFK-1. rM PFK-1 with F6-P as the variable substrate with ATP concentration held constant. The symbols are: 0.5 µM Curcumin (), 0.25 µM Curcumin (), and control (). This graph shows an noncompetitive inhibition, since the trend line of 0.5 µM Curcumin and 0.25 µM curcumin are intersecting at the x-axis with the control. Figure 3B magnifies the 0.5 µM Curcumin (), and control () in Figure 3A.

Figure 1A Figure 1B

Figure 3A

Figure 2A

Figure 3B

Figure 1 B. 30 nM Rabbit Muscle Pyruvate Kinase Determination of the IC50 value is 0.035 mM.

Figure 2B. The reversible character of Pyruvate Kinase with curcumin inhibition. This is the pattern of convergence at the origin indicating a reversible inhibition. The symbols for the concentration of curcumin are as follows: (), presence; (), absence.

Figure 3C. Curcumin Inhibition Kinetics of rM Pyruvate Kinase. rM Pyruvate Kinase with PEP as the variable substrate with ATP concentration held constant. The symbols are: PK + 0.05 mM curcumin (), PK + 0.07 mM curcumin (), and control (). This graph shows curcumin is a non-competitive inhibitor with PK.