CURCUMIN AND PYRUVATE KINASE

ABSTRACT

BACKGROUND AND PURPO SE:

Previous studies have identified Curcumin as having anticancer and anti-inflammatory effects, as well as,

being a kinase inhibitor. In contrast, we found stimulation of pyruvate kinase activity with Curcumin.

EXPERI MENTAL APPROAC H:

Homogenised rat l iver used in pyruvate kinase and lactate dehydrogenase coupled enzyme assay both

with and without the presence of Curcumin.

KEY RESULTS:

Curcumin was found to have a stimulatory effect on pyruvate kinase in most of the assays but in some it

was seen as an inhibitory effect this may have been a fluke or the age of the samples may have affected

the results.

CONCLUSI ONS AND I MPLI CATI ONS:

Initial hypothesis rejected, Curcumin could stil l have anticancer effects via the metabolic pathway but

possibly by increasing the pyruvate produced to activate pyruvate dehydrogenase kinase.

I NTRODUCTI ON

Pyruvate kinase (PK) is a an enzyme involved in glycolysis, with phosphoenolpyruvate (PEP)

being the substrate and adenosine diphosphate (ADP) the co-factor, which catalyses the

transfer of a phosphoryl group from the PEP to the ADP to generate one pyruvate and one

adenosine triphosphate (ATP) molecule. Belonging to the pyruvate kinase family there are four

forms of pyruvate kinase in humans depending on the metabolic requirements of the tissue –

M1, M2, L and R, M standing for muscle, L for liver and R for red blood cell , which are encoded

by two separate genes (PK-M AND PK-LR) and expressed in a tissue specific manner (Zanella et

al., 2007). The PK-LR gene, located on chromosome 1 (1q21) codes for the L form of pyruvate

kinase which is found in the liver. In mammalian cells PK activity may be regulated by two

mechanisms; one at the level of gene expression and the second through allosteric regulation.

The enzymes substrate, PEP, and fructose 1, 6-bisphosphate act as enhancers of its activity by

causing a conformational change by changing the orientation of the tetramers by 29 degrees

to change the enzyme from its T – tense – state to the R – relaxed – state (Valentine et al.,

2000).PK is the final enzyme in the glycolysis pathway and is regarded as being critical in the

regulation of glycolysis because of its inhibition by ATP and Acetyl-CoA. PK is thought to have a

large role in cancer metabolism, particularly the PKM2 isomer, it is currently being considered

for rigorous research for various types of cancers.

Scientific interest in medicinal plants has increased considerably over the last decade, making

many efforts to find out if there really is a beneficial effect from the secondary metabolite

phytochemicals derived from these plants and if so how they work. Curcumin

(Diferuloylmethane) a derivative of turmeric, is one of the best studied natural

phytochemicals. Turmeric is derived from the Curcuma longa plant and is used commonly in

Asia for flavouring, food preservation, as a dye, as well as, being used for health care. . Recent

research from the western science has also found beneficial effects on health. These range

from the skin (Nguyen & Friedman, 2013), gastrointestinal systems (Cao et al., 2013), liver

disorders and even cancer (Rivera-Espinoza & Muriel, 2009). Curcumin has been shown to

have an inhibitory effect on a range of proteins from kinases (Liu et al., 1993) (Mahmmoud,

2006) to transcription factors (Choi et al., 2006). Fluorescence emission tests have shown that

the pyrazole ring on Curcumin and the hydroxyl and carboxyl groups present form hydrogen

bonds with the protein residues on the various proteins (Das et al., 2011).

In this study the effects of adding Curcumin to a pyruvate kinase assay were observed, which

showed a stimulatory effect, this being in disagreement with other similar studies showing

inhibitory effects of Curcumin on a range of kinase enzymes (Liu et al., 1993) from protein

kinase C to pyruvate kinase (Das et al., 2011) (Mahmmoud, 2006) )Wong et al., 2008).

Essentially the data from this study disagree with the proposed idea that Curcumin can inhibit

the activity of pyruvate kinase and may therefore have a possible basis in cancer treatment.

MATERI ALS AND METHODS

RAT LI VER HOMOGENI SATI ON

The pH 7.5 homogenisation buffer contained 50 mM tris (hydroxymethyl) aminomethane (Tris

HCl), 0.25 M Sucrose, 1.0 mM Ethylenediaminetetraacetic acid (EDTA), 0.15 mM potassium

chloride and 1 mM of Dithiothreitol (DTT).

The whole liver was quickly removed from the rat and divided by four, this was then whilst

constantly being kept on ice to prevent any decaying of the tissue. The four different

homogenates produced were used as counterweights for the centrifuge. First centrifuge was

carried out at 4000xg for 10 mins. The supernatant fluid was collected then centrifuged again

at 13000xg for 15 minutes. The second supernatant fluid was collected and frozen to be used

for the enzymatic assay.

BRADFORD REAGENT.

The concentration of the protein in the supernatant was measured using the method of

Bradford (1976) with bovine serum albumin (BSA) used as the standard

PYRUVATE KI NASE ASSAY

The assay was performed essentially as described by Worthington Biochemical Corporation.

The assay was altered slightly bringing the final volume down to 1ml from 3ml. The assay

medium contained 0.05 M Imidazole⋅HCl buffer, pH 7.6, containing 0.12 M potassium chloride

and 0.062 M magnesium sulphate, 45 mM Adenosine diphosphate, 45 mM

Phosphoenolpyruvate, 6.6 mM NADH, and diluted lactate dehydrogenase at a concentration of

1300-1400 units/ml in the imidazole buffer. These solution were then used to make the

following preparations

To prepare the Curcumin solution a 2.7 mM solution was produced using 10ml ethanol and

10mg pure Curcumin

RESULTS

DATA COMPARI SON FROM AVERAGE DATA OF ALL FOUR WEEKS

Table 1 - Average Moles per min produced from all four weeks

PEP (mM) No Curcumin (M/min) Curcumin (M/min)

2.64 0.041 0.069

3.96 0.051 0.094

5.28 0.061 0.109

6.6 0.068 0.132

Figure 1

Figure 2

Table 2 - Km and Vmax for the average data of all four weeks

No Curcumin Curcumin

Km (µM) 0.1242 0.3262

Vmax (mM/min) 37.45 68.00

An average was calculated from both sets of all four weeks’ worth of data and used to produce

a Michaelis-Menten and a Lineweaver-Burk plot. The data were normally distributed (P=0.956)

and a paired-samples t-test was conducted to compare the effects of no Curcumin and

Curcumin. There was a significant difference in the production of pyruvate for no Curcumin

(M=0.055, SD=0.011) and Curcumin (M=0.101, SD=0.026) conditions; t (3) =-6.165, p = 0.009.

These results suggest that Curcumin does have a statistically significant effect on pyruvate

kinase activity.

ANOVA OF THE FOUR WEEKS OF NO CURCUMI N D ATA

Table 3 - Moles per min produced from all four weeks - control

PEP (mM) Week 1 (M/min) Week 2 (M/min) Week 3 (M/min) Week 4 (M/min)

2.64 0.036 0.035 0.038 0.056

3.96 0.037 0.035 0.040 0.093

5.28 0.049 0.049 0.041 0.106

6.6 0.061 0.056 0.048 0.108

A one-way between subjects ANOVA was conducted to compare the effect of time on the

substrate produced by pyruvate kinase, a time of one week between each sample. There was a

significant effect of time on substrate produced (F (3, 12) = 10.466, p = 0.001). Tukey post-hoc

comparisons of the four sets of results indicates that the fourth week results (M = 0.090,

SD=0.024) gave significantly different moles per min than the data from the other three weeks,

week 1 (M=0.045, SD= 0.011), week 2 (M=0.043, SD=0.010, week 3 (M=0.041, SD=0.004). The

comparison between the other three weeks were not statistically significant (P=>0.05)

ANOVA OF THE FOUR WEEKS OF CURCUMI N DATA

Table 4 - Moles per min produced from all four weeks - Curcumin

PEP (mM) Week 1 (M/min) Week 2 (M/min) Week 3 (M/min) Week 4 (M/min)

2.64 0.135 0.046 0.046 0.050

3.96 0.143 0.127 0.049 0.056

5.28 0.165 0.132 0.056 0.084

6.6 0.193 0.142 0.096 0.096

A one-way between subjects ANOVA was conducted to compare the effect of time on the

substrate produced by pyruvate kinase, a time of one week between each sample. There was a

significant effect of time on substrate produced (F (3, 12) = 8.583, p = 0.003). Tukey post-hoc

comparisons of the four sets of results indicates that the first week results (M = 0.159,

SD=0.026) gave significantly different moles per min than the data from weeks three and four,

week 3 (M=0.061, SD=0.023, week 4 (M=0.071, SD=0.002). The comparison between the other

three weeks were not statistically significant (P=>0.05)

DATA COMPARI SON FROM ONLY WEEK 4 DATA

Table 5 - Moles per min produced from week four supernatant

PEP (mM) No Curcumin (M/min) Curcumin (M/min)

2.64 0.056 0.050

3.96 0.093 0.056

5.28 0.106 0.084

6.6 0.108 0.096

Figure 3

Figure 4

Table 6 - Km and Vmax for the data from week 4 only

No Curcumin Curcumin

Km (µM) 0.2335 0.4019

Vmax (mM/min) 47.28 143.3

The results from week 4 were used to produce a Michaelis-Menten and a Lineweaver-Burk

plot. The data were normally distributed (P=0.766) and a paired-samples t-test was conducted

to compare the effects of no Curcumin and Curcumin. There was no significant difference in

the production of pyruvate for no Curcumin (M=0.090, SD=0.024) and Curcumin (M=0.071,

SD=0.022) conditions; t (3) =2.841, p = 0.066. These results suggest that Curcumin does not

have a statistically significant effect on pyruvate kinase activity

DI SCUSSI ON

By using a tried and tested pyruvate kinase assay evidence has been provided to show that

Curcumin does have an interaction with the enzyme but this particular experiment suggests

that Curcumin leads to an increase in the activity of the enzyme and therefore the activity of

glycolysis. When looking at figure 1 and 2, which show an average of all 4 weeks’ worth of

results, it can be seen that in the presence of Curcumin a stimulatory effect can be seen with

the rate of reaction almost doubling at the higher concentration of PEP (control M= 0.068,

Curcumin M= 0.132) and these results were seen to be significantly different based on a paired

t test. It is also seen that the Km and Vmax increase in the presence of Curcumin as seen in

table 1. However, when the results from the most recent supernatant sample , week 4, were

looked at an inhibitory effect could be seen across the board, from the lowest PEP

concentration (control M=0056. Curcumin M=0.050) through to the highest (control M=0.108

Curcumin M=0.096), however, these results were not seen to be significantly different when a

t test was carried out. The age of the samples which range from a month old to a week old

seem to have a statistically significant effect on the rate of reaction as seen from the results of

the ANOVA and the Tukeys post hoc test which indicate that the results from week 4 are

statistically different to the results of weeks 1, 2 and 3. Due to the results of the ANOVA it was

deemed important to carry out the Michaelis-Menten and Lineweaver-Burk plot just for week

4 which can be seen in figure 5 and 7 and 6 and 8 respectively. For week 4 it can be seen that

the inhibitory effect of Curcumin can be seen throughout the different concentrations as

opposed to just at high concentrations as seen in the 4 week average on the direct results, but

the graphs again show an increase in Km and Vmax as can be seen in table 6.

The main issue with the results from this experiment was that there were no repeats carried

out, although the four different weeks can in essence be regarded as repeats they were

essentially from different livers and therefore could have a different concentration of protein

in the enzyme so it can’t really be seen as a repeat, the idea to do repeats was there but

because of the issue with oxidisation of the NADH it wasn’t possible to do these and make the

results more reliable and judge their accuracy based on their similarity.

This decrease in the activity of pyruvate kinase in week 4 in comparison to week 1 could be

explained by the findings of Katunuma et al. (1972) of a class of group specific proteinases

which attack enzymes via proteolysis, hydrolysing the peptide bonds which link the amino

acids of the protein together. These proteinases if functional below the temperature at which

the samples were frozen could account for the loss of activity. It is also possible that a more

simple reason maybe the cause of this change in activity, the alteration of the water

environment could have caused an irreversible conformational change in the protein leading

to a loss of activity (Cryer and Bartley, 1974). To test the significance of this idea in the future it

is suggested that a test with a fresh sample be carried out also.

The use of ethanol could also have influenced the results. The effect of alcohol on the liver is

well documented so it would be important to consider the effects of it when it is used in an

experiment. Ethanol is a small molecule which may interact with different proteins and not

necessarily with alcohol dehydrogenase. In rats it was shown to change the expression levels

of 646 different genes within 3 hours of admission (Li et al., 2010). In general, glycolysis is

thought to be inhibited indirectly during alcohol consumption. The metabolism of ethanol is

such that the end product is Acetyl-CoA as can be seen in figure 9. This results in most of the

Acetyl-CoA entering the citric acid cycle to be derived from ethanol rather than glucose. It is

thought that Acetyl-CoA is a pyruvate kinase inhibitor and this therefore may have had a role

in the inhibition seen in the lower PEP/higher Curcumin concentration in the results seen in

the average and across the board in just week 4. To see if this is true it would be apt to repeat

the test using just ethanol and no Curcumin to see if these results correlate.

Figure 9

The effects of Curcumin have been found to be remarkably similar to the drug dichloroacetate

(DCA) but without the harmful side effects which accompany the drug such as nerve, kidney

and liver damage, as well as, impaired heart and immune function (bonnet et al., 2007)(Wong

et al., 2008). There are major changes in the way cancer cells produce energy for themselves in

comparison to normal healthy cells. Normal cells use mitochondria which carry out oxidative

phosphorylation, which, as the name suggests, require oxygen, giving rise to a maximum of 36

molecules of ATP. Cancer cells on the other hand utilise just glycolysis, rendering the

mitochondria useless, and produce just two molecules of ATP. This is called the Warburg

effect, as described by Otto Warburg in 1956. In cancer cells, the enzyme pyruvate

dehydrogenase kinase (PDK) is more active than in normal cells because of the high presence

of ATP, it uses this ATP to phosphorylate the enzyme pyruvate dehydrogenase , a complex of

three enzyme which convert pyruvate to Acetyl-CoA with the help of coenzyme A and NAD.

Curcumin and DCA are found to inhibit PDK as opposed to activate it, allowing for pyruvate to

be converted to Acetyl-CoA and continue down the oxidative phosphorylation pathway and

allowing the mitochondria to work effectively. DCA activating the enzyme directly and

Curcumin, indirectly, by behaving as an activator for pyruvate kinase and increasing the

Ethanol

Acetaldehyde

Acetic Acid

Acetyl-CoA

Alcohol Dehydrogenase

Acetaldehyde Dehydrogenase

Acetyl CoA synthase-1

production of pyruvate which is one of the inhibitory molecule for PDK. The mitochondria is

seen as the primary regulator of apoptosis. Many pro-apoptotic signals gather at the

mitochondria and are stored there, being prevented to leave by the membrane permeability of

the mitochondria. The pro-apoptotic molecules released from the mitochondria include:

cytochrome c, caspase, the second mitochondrial activator of caspases (SMAC), apoptosis-

inducing factor (AIF) and endonuclease G (Gulbins et al., 2003). In the last two decades it has

been found that the mitochondria from cancer cells are resistant against the induction of

mitochondrial outer membrane permeabilisation (MOMP), a process which mediates the

intrinsic pathway of apoptosis (Green and Kroemer, 2004.) This is most likely just because the

mitochondria are inactive in cancer cells, if they can be reactivated it is possible that they

could carry out the programmed cell death as they should and therefore kill the cancer cells.

This warrants further research into the indirect effects of Curcumin on the enzyme PDK in

cancer cells as opposed to normal cells to see if the ideas suggested above do have any

scientific significance.

In the future because this assay is expensive to run it might be worth trying it in a micro titre

plate, but this leads to the other issue that the actual reaction occurs very quickly , so unless a

way can be thought of to counteract the issue of the speed, the cost may just have to be

absorbed for the assay. It is also worth trying a run without Curcumin present to see if ethanol

itself has an effect on the reaction.

CONCLUSI ON

The experiment didn’t agree with the original hypothesis that Curcumin has an inhibitory

effect on pyruvate kinase and therefore cancer, on the contrary it worked as an activator for

the enzyme. It did shed light on the activation being more important in the prevention of

cancer by possibly inducing apoptosis by activating the mitochondria rather than using

inhibition to starve the cancer cells.

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x 1 = 3.77x104 = 0.038 moles/min

APPENDI X

Interpreting the graph for reaction rate

A= ε.C.L

0.2345 = 6.22X103 x C x L

C = 0.2345

6.22X103

Δ A = 0.34

87mm = 87s Δ A/min = 0.34x60

87

Change in absorption per min

Δ a/min = 0.2345

Making up the final solution

ADP

Final Conc 45mm

Final Vol – 0.033 µl

45mM = 45mMoles/L =45µmoles/ml

Conc of stock should be 45µmoles/33µl = 45mmoles/ml

33ml enough for 100 assays

Too much!

6 cuvettes x 0.033 = 0.198ml

Round up to 2 ml