Anticancer Effects of the Curcumin, Artemisinin, Genistein, and

Anticancer Effects of the Curcumin, Artemisinin, Genistein, and Resveratrol, and
Vitamin C: Free vs. Liposomal Forms
Jerry T. Thornthwaite, Hare Shah, Spencer England, Lee Roland, Seth Thibado, Thomas Ballard
and Brandon Goodman
Cancer Research Institute of West Tennessee, Union University Department of Chemistry,
University of Tennessee Medical Sciences
Send Correspondence to
Dr. Jerry Thornthwaite, Director
Cancer Research Institute of West Tennessee
114 East Main Street
Henderson, TN 38340
[email protected]
Summary
Cancer deaths are projected to double worldwide over the next two decades. Cancer prevention
supplements and more effective treatments with minimal side effects are urgently needed. An
accurate, fast assay system is described that reveals the ability of chemically defined products,
such as Curcumin, Genistein, Resveratrol, Artemisinin, and Vitamin C, are able to kill K562
Erythroleukemic cells in vitro. The cancer cells are added to 48 well plates in 500µL portions
and cultured 48 hrs. to initiate logarithmic growth. All of the free compounds were diluted in
DMSO and diluted sufficiently to minimize the effect of DMSO. In addition, Curcumin and
Vitamin C were encapsulated into fatty acid micelles named NutraNanoSpheres™ (NNS) using
all natural products. A unique viability stain, which allows the rapid staining of dead cells by
membrane penetration using Propidium Iodide, was used to measure the cell viability by flow
cytometry. The control cells were greater than 95% viable during and at the end of the
experiments and were used as a baseline to measure the degree of killing. Curcumin showed
complete cell destruction (zero viable cells out of 7 x 10 5 viable cells in the control wells) at 1.25
µmol/well [LD50 = 41.3 µM] as early as 20 h in culture. Cell death by alteration of the cell
membranes could be seen within 30 s of exposure to curcumin. The other free components
required 0.5-70 h to see maximum killing suggesting a more metabolic and/or apoptotic route of
destruction. Vitamin C up to 1x104 µmol/well did not affect K562 cell viability. The Vitamin
C-NNS (3.2 nm diameter-60 mg/50µL) showed an LD50 = 133 µmol/well ± 11 SD (n=4), which
was over 75 times more potent than the free Vitamin C. The Curcumin-NNS (7.4 nm diameter25 mg/50µL) resulted in an LD50 = 41.3 µmol/well ± 5.6 SD (n=8) and represented a 237 fold
increase in activity to destroy the cancer cells. The clinical goal is to develop water soluble
mixtures of NNS with their high bioavailability (>90%) and without degradation in the stomach
and intestines for the prevention and treatment of cancer.
Introduction
Cancer is one of the major causes of death worldwide and minimal progress has been accomplished
in reducing its morbidity [1]. Cancer is caused by one or a combination of at least three factors:
unhealthy diet, genetic predisposition, and the environment. Estimates by the American Cancer
Society (1) is that over 90% of all cancers are caused by life style and may take as long as 20– 30
years to develop. Current estimates from the American Cancer Society and from the International
Union Against Cancer indicate that 12 million cases of cancer were diagnosed last year, with 7
million deaths worldwide; these numbers are expected to double in 15 years (2). According to
reports from the World Health Organization, more than 80% of world’s populations depend on
traditional medicine for their primary health care needs (3, 4). Plants have a long history of
being used in the treatment of cancer and it is significant that over 60% of currently used anticancer agents come from natural sources (5, 6).
Cancer prevention approaches prescribe natural/synthetic agent(s) with the aim to delay or disrupt
the pathways and processes involved at multiple steps, such as, the initiation, promotion, and
progression of cancer (7) Even our fresh fruits and vegetables are being depleted of essential
minerals and vitamins, requiring supplementation (8). Genetic testing is expensive and causes
unnecessary anxiety that depresses the immune system and increases the likelihood of cancer
developing anyway. Stress hormones, such as adrenaline and cortisol, suppress natural killer cell
function (9). More people are living in large cities with its associated pollution, stress and poor
eating habits.
A partial solution that leads to a complete solution is that individuals need to make critical lifestyle
changes if they want to live a life of quality and quantity. This starts with an appreciation for
supplements. By committing to a daily dosage of supplements that provide protection against
disease, one will naturally develop a healthy lifestyle in their exercise programs, getting enough
sleep, enjoying the blessings in their life, and developing a Spiritual nature, which all minimize
stress in their life.
The beneficial effects of Curcumin on cancer, cardiovascular disease, metabolic disorders,
antioxidant capacity, anti-inflammatory properties, and neurological, liver and respiratory
disorders have recently been reviewed (10). This is in spite of its poor bioavailability (11). The
clinical importance but low bioavailability have also been shown for Genistein (12), Resveratrol
(13), Artemisinin (14), and Vitamin C (15).
This paper will present important supplements using plant derived or synthesized, chemically pure
compounds that show direct toxicity to the K562 cancer cell line. These supplements include
Curcumin, Genistein, Resveratrol, Artemisinin, and Vitamin C. Furthermore, the cytotoxicity of
encapsulated Curcumin and Vitamin C will be compared to their free forms.
Materials and Methods
Cell Line and Media Production: Experiments were performed using a chronic myelogenous
leukemia K-652 cell line was purchased from the American Type Culture Collection (ATCC).
The tissue culture media was made by adding 5 ml of 100x penicillin-streptomycin (10,000 units
penicillin with 10 mg of streptomycin/ml – Sigma-Aldrich), 5 ml of 200 mM sterile-filtered LGlutamine (Sigma-Aldrich), 5 mL of Cellgro sodium 100x bicarbonate solution (Cellgro), and 50
ml of fetal calf serum (Atlanta Biologics) to 500 mL of Minimum Essential Media, Alpha 1X,
with Earle's salts without ribonucleotides, deoxyribonucleotides, and no L-glutamine (Cellgro).
Viability Stain: The viability stain used for analysis with the flow cytometer was developed by
Dr. Thornthwaite. The evidence that showed the viability stain, which uses a special medium and
dye exclusion with Propidium Iodide (Sigma-Aldrich) was effective in measuring cell viability
was shown in two ways. Firstly, 500µl portions of K562 cells (5 x 106 / ml) were heated as
triplicates in a 56 C water bath for up to 20 min. At 5 min. intervals, the timed samples had
500µl of the viability reagent was added at room temperature. All samples were run after a 2
min. incubation at room temperature on the Accuri Flow Cytometer (BD Biosciences). Also,
K562 cell viability was measured directly from cell cultures in which 100 µl samples from the
cell culture were added to 100 µl of the viability stain. After incubation for five minutes at room
temperature, the samples were suspended and analyzed using flow cytometry. Forward Light
scatter was used to gate on the K562 cells and analyze the number of viable cells within the
establish control viability gate in the 585±20 nm red channel. Typical control cultures with 95-
99% viability were used to establish the boundary between the live cells, essentially without
staining, and dead cells, which would take up the dye and fluoresce at larger channel numbers.
Reagents: The highest chemical grades (97-99%) of Curcumin, Genistein, Resveratrol,
Artemisinin and Vitamin C were used in these studies (Sigma-Aldrich). These reagents, except for
the cell culture media soluble Vitamin C, were prepared in 100X concentrations of pure Dimethyl
Sulfoxide [DMSO] (Sigma-Aldrich) and diluted by a factor of 100 in the media sufficient enough
to not have DMSO only being a factor in cell viability.
Encapsulated Reagents: Curcumin and Vitamin C NutraNanoSpheres™ (NNS) and were
obtained from Dr. Lothar Haegeler of X-labs (Switzerland-Singapore) and had a concentration of
10.0% (w/v) or 2.5 mg/50µL for the curcumin and 65mg/50µL for Vitamin C.
Average Diameter Measurements of the NNS: The samples were diluted by volume in a ratio
of 1:6 with DI Water and filtered by a 0.45um Nylon membrane to remove any dust
contaminants. The Zetasizer ZSP (Malvern Instruments) was used with a backscattering angle of
173 degrees to measure the particle size by dynamic light scattering. A non-negative least squares
algorithm was used to generate the size distribution by intensity, which indicated the diameter of
the major population for the Curcumin and Vitamin C NNS. The intensity data was then converted
to a mass or volume distribution to compare relative amounts of each size population, which
indicated the percentage of the sample represent in the respective population.
Sample Preparation and Cell counting: In all assays, viable cell counts were obtained by
mixing100 µL of a cell culture with 100 µL of viability stain. All samples were analyzed within
an hour after room temperature incubation for at least 5 min. The percentage viability was stable
for at least 2 hr. A 10µl portion of each sample was run through the Accuri C6 Flow Cytometer at
a medium flow setting. The resulting number was multiplied by 200 to determine the number of
viable cells/ml.
Procedures for Sterilization: All reagent samples were 0.22 µm sterile filtered and diluted with
media in a sterile biologic safety cabinet.
Cell Growth Plate Preparation: The cells that were counted would then be diluted with the media
to a concentration of 1 x 105 viable cells/ml. A 500 µl portion of the cells were added to each well
of the 48-well plate. The plates incubated in a Forma Scientific CO2 water-jacketed incubator with
a temperature of 37.2 C for 48hr to allow the cells to enter the exponential growth phase.
Addition of Compounds: After incubation for 48 hours, the stock sample compounds were
diluted accordingly, using by a factor of two for up to eight dilutions. A 50µl sample was added to
each well up to six replicates of each dilution to the wells. The control wells had 50µl of cell
culture media added to their wells. Once finished, every well contained 550 µL. The plates were
typically incubated for 48hr.
Cell processing, Staining, and Analysis: Up to six replicates at each concentration, starting with
the controls that were used to set the gates for viability, were suspended with a 500µl pipet, and
100 µl portions were added to 2 ml 96 well analysis tubes. After all of the samples were added to
the tubes, 100µl of the viability stain were added using an 8-channel multipipetor and the tray
shaken slightly and incubated for at least 5 min. All samples were analyzed within an hour after
room temperature incubation for at least 5 min. The viability stained cells were stable for at least
2 hr. at room temperature. A 10µl portion of each sample was run through the Accuri C6 Flow
Cytometer at a medium flow setting. The resulting number was multiplied by 200 to determine the
number of viable cells/ml.
Data Analysis: The data collected was then graphed using PSI-Plot and their standard procedure
for graphing. The data was graphed in the form of percentage inhibition v concentration of each
component. Proof of the quantitative ability of the viability stain is shown in Figures 1 and 2. To
calculate percentage inhibition, first the cell counts were multiplied by 200 to account for the 1:1
dilution of adding the Propidium Iodide stain, as well as the volume of the analyzed sample being
10 µl. Multiplication by 200 resulted in cells/ml. The values for the viable cells/ml were
𝑥
incorporated into the equation, %Inhibition = (1 − (𝑦)) ∗ 100%, where x was equal to the
cells/ml in that particular well, and y was the average number of cells/ml in the control ± the
Standard Deviation (SD).
The LD50values were report in Molar (M=moles/liter) concentrations using the following formula:
M = µmol of supplement/550µl culture media, where 500 µl of the tumor cells had 50µL of the
respective supplements added after a 48-hr incubation starting with 5x104 cells and ended with
about 5-6 x 107cells in the controls after a total of 72-96 hrs. incubation after the initial addition.
Results
Cytotoxicity of “Free” supplements: Curcumin, Genistein, Resveratrol, Artemisinin and
Vitamin C. In the process of developing the viability assay used in this paper, K562 cells were
heated at 56 C in 500 µl aliquots at 5 min. intervals up to 20 min. Each sample had 500µl of the
PI-viability stain added and immediate analyzed by flow cytometry. By gating on the K562 cancer
cell forward light scatter and analyzing by red fluorescence channel (585±20 nm), the percentage
of dead cells positive for PI staining increased as a function of the exposure time at 56 C (Fig. 1).
Figure 1. Percentage of viable
K562 cells as a function of time
exposure to 56 C. Standard
Deviation (SD) bars (n=3) are
shown
Furthermore, the viability of expended cells in long term cultures showed two major populations
of dead cells (Fig. 2), those where the cellular membranes were compromised and second very
intense population where the nuclear membranes were compromised and intercalation of the PI
into double stranded DNA took place. The percentage viability was determined after subtraction
of the both cellular and nuclear membrane compromised populations.
Figure 2. K562 cell viability at
various days after the initial culture
of 1x 105 cells/ml. Vertical lines
show the cutoff between viable and
non-viable cells as measured by
the uptake of Pl.
K562 cells were cultured at 1 x 105 cells/ml in the media. The vertical line was used as the cutoff
of viable cells to the left of the vertical line that showed very small background staining. The
dead cells were to the right of the vertical line. At Day 6, the cells were 98% viable with up to
1.5 -2.0 x 106/mL of cells. At Day 10, dead cells were detected in the positive PI fluorescence
portion of the histogram. By Days 14-18, two distinct populations of dead cells were seen, which
showed the cells where the cell membrane was compromised (peak to the left of the vertical line)
and cells where the nuclear membrane was compromised (peak to the right of the vertical line),
showing the PI had intercalated into the double strained nucleic acids causing a very large
increase in fluorescent yield. At Day 18, all of the cells were dead. There is over a 1000 fold
increase in fluorescence, which is indicative of the high fluorescence yield due to the
intercalation of the Pl into the double stained nucleic acids.
Several supplement compounds were tested for their ability to kill cancer cells as measured by the
viability assay after 24-48 hrs. incubation at 37 C. Figure 3 shows percentage of inhibition of cell
growth when µmol additions of either Genistein or Artemisinin were added to the cell culture
wells. The lethal dosage where the supplements kill 50% of the cancer cells (LD50) for Genistein
was 26 µmol/well while the LD50 for Artemisinin was 36 µmol/well. In Figure 4, the LD50 = 12.5
and 12.4 µmol/well for Resveratrol and Curcumin, respectively.
Figure 3. The percentage inhibition
of the growth of K562 cells in the
presence of Genistein or
Artemisinin from 0 to 400
µmol/500 µL after 48 hr. The
lethal dosages that kill half of the
K562 cells (𝐿𝐷50 ) are shown. SD
bars (n=6) are shown.
Figure 4. The percentage
inhibition of the growth of K562
cells in the presence of Curcumin
or Resveratrol from 0 to 250
µmol/well after 48 hr. exposure.
The lethal dosages that kill half of
the K562 cells (𝐿𝐷50 ) are shown.
SD bars are shown (n=6).
K562 Cancer cell Survival kinetic experiments (data not shown) were conducted from 30 min to
70.3 hr. For Genistein, a three stage of maximum % Inhibition took place at 0.5 hr, 1.0-9.8 hr and
23.3-70.3 hr. Artemisinin and Resveratrol showed maximum % Inhibition at 70.3 hr in what may
represent multistage apoptotic events.
In contrast, a closer exam of the Curcumin concentration between 0 and 160 µmol/well, showed
the LD50 = 9.8 µmol/well. A very dramatic dose response was seen as early as 30 min., which was
near equivalent to what was seen in Figure 4. Importantly, Figure 5 revealed significant membrane
damage to the K562 cancer cells within 30 sec of exposure to (LD50=50 nmol/well) Curcumin,
where the membrane fluorescence increased by 10 fold. A 30-sec exposure, whether the Curcumin
was “free” or encapsulated (Fig. 5), was the minimal time from sample preparation and running
on the flow cytometer. The destruction of the cancer cells by exposure to curcumin could very well
be instantaneous.
Figure 5. The percentage inhibition
of the growth of K562 Cells in the
presence of NutraNanoSphere™
Curcumin from 0 to 450
nmol/well. The viability of the
K562 cancer cells dramatically
diminishes even after a little as 30
seconds of exposure to the
NutraNanoSphere™ Curcumin.
The synergism of Genistein, Artemisinin, Resveratrol and Curcumin was determined by adding
the LD50 dosages of each in all combinations. In Figure 6, the normalized percentage inhibition
was based on the relative maximum inhibition combination, Genistein and Curcumin. The
individual near LD50 values of Genistein (55%), Artemisinin (45%), Resveratrol (44%) and
Curcumin (18%) were used to show the synergism. For example, the synergistic effect of Genistein
+ Curcumin was 27% higher than the additive effect of the combination. The synergistic increase
could also be seen for Resveratrol and Curcumin (10%). The other combinations were not as clear,
but the apoptotic and direct membrane damage effects may be synergistic in vivo, which would
increase a synergistic effect for all combinations.
Figure 6. The normalized percentage
inhibition using Artemisinin (A),
Curcumin (C). Genistein (G) and
Resveratrol (R). SD bars shown (n=3)
Two antioxidant compounds showed no inhibition of tumor growth. N-acetyl Cysteine up to 400
µmol (not shown) and Vitamin C up to 10,000 µmol (Fig. 7), were not cytotoxicity against the
K562 Cancer cells.
Figure 7. At three different
curcumin concentrations, the free
Vitamin C had no effect on the %
Inhibition up to 1X106 nm Vitamin C.
The greatly enhanced anticancer effects of encapsulating Curcumin and Vitamin C in the
NutraNanoSpheres™ (NNS). The Curcumin and Vitamin C NNS had a concentration 2.5
mg/50µL for the curcumin and 65mg/50µL for Vitamin C. Measurements of their respective LD50
determinations showed a greatly enhanced potent activity when these compounds were
encapsulated in the liposomal structure.
Figures 8 and 9 show the size distributions for the Curcumin and Vitamin C NNS samples. The
samples were diluted by volume in a ratio of 1:6 with DI Water and filtered by a 0.45um Nylon
membrane to remove any dust contaminants. These samples were run on the Malvern Zetasizer
ZSP with a backscattering angle of 173 degrees to measure the particle size by dynamic light
scattering. A non-negative least squares algorithm was used to generate the size distribution by
intensity. Figure 8 shows the diameter measurements for the curcumin samples, while Figure 9
shows the mean diameters for the Vitamin C.
Figure 10. The percentage Inhibition of the growth of K562 cells in the presence of
NutraNanoSphere™ Vitamin C from 0 to 200 µmol/well. The 𝐿𝐷50 =141 and 125
µmol/well levels example data are shown. The “free” vitamin C had no effect on the K562
viability up to 1 𝑥104 µmol/well.
Figure 11. The percentage Inhibition of the growth of K562 cells in the presence of
NutraNanoSphere™ Curcumin from 0 to 180 µmol/well. The 𝐿𝐷50 = 37.5 and 30.5
µmol/well levels example data are shown.
Discussion
The ability to inhibit the viability of the K562 cancer cells was greatly enhanced by the fatty acid
micelle encapsulation of curcumin and Vitamin C in the form of NutraNanoSpheres™ (NNS).
In the case of Vitamin C, no inhibition of cancer cell growth was seen with the “free” Vitamin C
up to 10mM. The solubility of Vitamin C is 1 g/ 3 mL of water or 1.89M (16, 17). However, in
this study going above 1x106 nmol/well may cause osmotic toxicity to the K562 cells. The
encapsulated NNS Vitamin C allows for the measurement of Vitamin C toxicity against the K562
cells at a LD50= 133 nmol/well in which makes the anti-cancer toxic measurements even possible.
The LD50 of Curcumin in the “free” form was 9,800 nm/well compared to the Curcumin NNS of
only 41.3 nm/well. The LD50=41.3 nmol/well is equal to 41.3 nmol/550 µL, which in turn is 75.1
µM. The average 70Kg human has about 4.7 liters of blood (18). In order to have an equivalent
LD50= 75.1µM in 4.7 L of blood, one would need at least 353 µmol of curcumin. Since the NNS
contain 65 mg of Curcumin per drop (50µL) or 65 mg/0.36838 mg/mol= 176.4 µmol. Therefore,
to provide an LD50 would require (353 µmol/176.4 µmol) = 2.0 drops of the Curcumin NNS
assuming 100% bioavailability, which is not too far from the 90.2% that appears in the serum
within 30 min. in patients orally ingesting the encapsulated curcumin as reported by X-Labs.
The free Curcumin showed complete cell destruction (zero viable cells out of 7 x 105 viable cells
in the control wells) at the LD50 = 90-120 µmol/well as early as 20 h in culture. Cell death by
alteration of the cell membranes could be seen within 30 s of exposure to curcumin. The other
components required at least 48 hrs. for maximum killing, suggesting a more metabolic and/or
apoptotic route of destruction using the other free supplements.
The free Vitamin C up to 1x106 nmol/well did not affect K562 cell viability. The Vitamin CNNS (3.2 nm diameter-65 mg/50µL) showed an LD50 = 133 µM ± 11 SD (n=4), which was over
75 times more potent than the free Vitamin C. The Curcumin-NNS (7 nm diameter-25
mg/50µL) resulted in an LD50 = 41.3 nmol/well ± 5.6 SD (n=8) and represented a 237 fold
increase in activity to destroy the cancer cells. The clinical goal is to develop water soluble
mixtures of NNS with their high bioavailability (>90%) and without degradation in the stomach
and intestines for the prevention and treatment of cancer.
Genistein, Resveratrol, and Artemisinin may also be encapsulated in the NNS and be used to
produce a “cocktail” of components that would have different mechanisms of action, which
include metabolic, apoptotic, and direct cytotoxicity for the prevention and treatment of cancer.
These supplements may also show a significant increase in their anti-cancer activity as seen with
NNS Curcumin and Vitamin C.
These supplements, which are effective and safe to use as a preventative combinations, in the NNS
form would provide a simple, water soluble droplet formulation, which could be added to water or
juice and taken on a daily basis. The high bioavailability that results from encapsulation would
assure effective dosages to be determined in clinical trials for both the prevention and treatment of
cancer.
The supplements presented in this paper have functions that affect virtually every aspect of cellular
development. For example Vitamin C has been shown to be a cofactor for methyl cytosine
dioxygenases, which are enzymes for DNA demethylation, an important crossroad in epigenetic
regulation (19, 20). Curcumin is a complementary therapy that may be helpful for the treatment
of a variety of diseases because of its anti-inflammatory, antiangiogenic, antioxidant, and
antiproliferative effects (21).
Finally, the possibility of being able to use a combination of NNS types for cancer preventative
and treatment protocols will revolutionize our options in treating cancer. The advantages of using
this proposed NNS cancer formulation include safety, efficacy without immunosuppression and
actual facilitation of stimulation of the Natural Killer Cell activity (22) using Curcumin (23, 24),
Genistein (25), Resveratrol (26) and at least a general immune stimulatory role with Artemisinin
and its derivatives (27, 28).
Acknowledgements
All work was done at the Cancer Research Institute of West Tennessee, under the direction of Dr.
Jerry Thornthwaite Ph.D., who provided all of the materials and equipment used. This research
was supported in part by generous donations from Mr. Henry Respess, The Shumard Foundation
and the Carter Family Trust. We thank Dr. Tony Kirk, Edra Shalla, and Bonita Thornthwaite for
reviewing this manuscript. Special thanks goes to Dr. Lothar Haegele of X-Labs (Singapore) for
supplying the Curcumin and Vitamin C NutraNanoSpheres™ preparations for this study.
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