Cancer is one of the major causes of death worldwide, and minimal progress has been accomplished in reducing its morbidity  . 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 are that over 90% of all cancers are caused by lifestyle 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  . 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    . Plants have a long history of being used in the treatment of cancer, and it is noteworthy that over 60% of currently used anti-cancer agents come from natural sources      .
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  . Even our fresh fruits and vegetables are being depleted of essential minerals and vitamins, often necessitating supplementation  . Stress hormones such as adrenaline and cortisol suppress natural killer cell function   . More people are living in large cities with its associated pollution, stress and poor eating habits. A partial solution may be that individuals need to make critical lifestyle changes if they want to live long, healthy lives. Nutritional supplements are being increasingly recognized as an essential part of a healthy lifestyle.
The beneficial effects of curcumin on cancer, cardiovascular disease, metabolic disorders, antioxidant capacity, inflammation, and neurological, liver and respiratory disorders have recently been reviewed  -  . This is in spite of the poor bioavailability of curcumin   . Genistein  , resveratrol   , artemisinin  , and vitamin C  also have similar clinical importance but also suffer from low bioavailability.
This study presents importance of supplements using plant derived or synthesized, chemically pure compounds that show direct toxicity to the K562 cancer cells. These supplements include curcumin, genistein, resveratrol, artemisinin, and vitamin C. Furthermore, the greatly increased cytotoxicity of micelle- encapsulated curcumin and vitamin C will be compared to their free forms      .
2. Materials and Methods
Cell Line and Media Production: Experiments were performed using a Chronic Myelogenous Leukemia K-652 cell line purchased from the American Type Culture Collection (ATCC).
The tissue culture media was made by adding 5 mL of 100X penicillin-strep- tomycin (10,000 units penicillin with 10 mg of streptomycin/mL―Sigma- Aldrich), 5 mL of 200 mM sterile-filtered L-Glutamine (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. Jerry Thornthwaite. The viability stain, which uses a special medium and dye exclusion with Propidium Iodide (Sigma-Aldrich), was effective in measuring cell viability by showing a linear decrease in viability as the K562 cells were progressively subjected to a 56 C water bath (data not shown). 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, incubated at room temperature for five min., and analyzed on the Accuri Flow Cytometer (BD Biosciences). Forward Light scatter was used to gate on the K562 cells and analyze the number of viable cells within the established 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.
Figure 1 shows the measurement of cell viability using the viability stains. The K562 cells starting culture was 1 × 105 cells/mL. The right of the vertical line was used as the cutoff of viable (left) and non-viable (right) cells, which was established for the control cultures (95% - 97% viable). The control, viable PI population curve came to a baseline on the left of the curve, which was the beginning of the viable cell population fluorescent 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 × 106 cells/mL. 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). This showed the PI had intercalated into the double-stranded nucleic acids, causing a very large increase in fluorescent yield. At Day 18, all of the cells were dead. There was 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.
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 NutraNanoSpheresTM (NNS) were obtained from Dr. Lothar Haegele, CEO of X-labs (Switzerland- Singapore), and had a concentration of 10.0% (w/v) or 2.5 mg/50 µL for the curcumin and 65 mg/50 µL 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 0.45 µm 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 represented in the respective population.
Sample Preparation and Cell Counting: In all assays, viable cell counts were obtained by mixing 100 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 h. A 10 µL portion
Figure 1. The effects on cell viability of K562 cancer cells vs time in culture showing increased cell death due to overcrowding and depleted nutrients. The vertical lines were used as the cutoff and to the right are the dead cells as measured by the increased uptake of the PI viability stain. The stained cell populations were gated on the light scatter cell population and analyzed in the red fluorescence channel.
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 were then diluted with the media to a concentration of 1 × 105 viable cells/mL. A 500 µL portion of the cells was added to each well of the 48-well plate. The plates incubated in a Forma Scientific CO2 water-jacketed incubator at a temperature of 37.2 C for 48 h to allow the cells to enter the exponential growth phase.
Addition of Compounds: After incubation for 48 h, the stock sample compounds were diluted accordingly, by a factor of two for up to eight dilutions. A 50 µL sample was added to each well, and up to six replicates of each dilution to the wells were prepared. 50 µL of cell culture media was added to each control well. Once finished, every well contained 550 µL. The plates were typically incubated for 48 h.
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 500 µL pipet, and 100 µL portions were added to 2 mL 96 well analysis tubes. After all of the samples were added to the tubes, 100 µL of the viability stain were added using an 8-channel multipipetor, and the tray was 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 h at room temperature. A 10 µL portion of each sample was run through the Accuri C6 Flow Cytometer (BD Biosciences) using the fluorescence red channel (585 ± 20 nm) at a medium flow setting (200 - 300 cells/sec). Again the resulting number was multiplied by 200 to determine the number of viable cells/mL.
Percentage Cell Viability: Control cells were used to set the forward angle light scatter gate for the entire cell population gate for the cells less debris to the left of the scatter peak. The background fluorescent peak population of cells comprised the viable cells (95% - 97% viable). Any fluorescent cells to the right of the right baseline of the viable cell population comprised the dead cell population. The percentage viable cells were determined by dividing the number cells in the viable fluorescent cell population by the total number of viable + nonviable cells and multiplying by 100%.
Data Analysis: The data collected was then graphed using PSI-Plot. The data was graphed in the form of percentage inhibition vs. concentration of each component. To calculate percentage inhibition, the values for the viable cells/mL were incorporated into the equation, % Inhibition = (1-X/Y) * 100%, where X was equal to the cells/mL in a particular well, and Y was the average number of cells/mL in the control. The mean of multiple replicates (4-6) ± the Standard Deviation (SD) were then determined.LD50 is the concentration, either in nmol or µmol per well, of the supplement of interest that causes 50% cell death.
Cytotoxicity of “Free” Supplements: Curcumin, Genistein, Resveratrol, Artemisinin and Vitamin C.
Figure 2(a) shows percentage of inhibition of cell growth when µmol additions of either genistein or artemisinin were added to the cell culture wells. The
Figure 2. (a) 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 h. The lethal dosages that kill half of the K562 cells (LD50) are shown. SD bars (n = 6) are shown. The mean LD50 ± SD µmol/well for eight experiments were 24.1 ± 4.6 SD for Genistein and 22.3 ± 5.8 for Artemisinin. (b) The percentage inhibition of the growth of K562 cells in the presence of Curcumin or Resveratrol from 0 to 250 µmol/well after 48 h exposure. The lethal dosages that kill half of the K562 cells (LD50) are shown. SD bars are shown (n = 6). The mean LD50 ± SD µmol/well for eight experiments were 10.9 ± 2.6 SD for Curcumin and 15.9 ± 6.7 SD for Resveratrol.
lethal dosages in eight experiments, where the supplements kill 50% of the cancer cells (LD50), for genistein was 24.1 µmol/well ± 4.6 SD, while the LD50 for artemisinin was 22.3 µmol/well ± 5.8 SD. In Figure 2(b), the LD50 = 15.9 µmol/well ± 6.7 SD for resveratrol and 10.9 µmol/well ± 2.6 SD for curcumin.
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 (not shown), were not cytotoxic against the K562 cancer cells.
The Greatly Enhanced Anticancer Effects of Encapsulating Curcumin and Vitamin C in the NutraNanoSpheresTM (NNS). The curcumin and vitamin C NNS had a concentration 2.5 mg/50 µL for the curcumin and 65 mg/50 µL for vitamin C. Measurements of their respective LD50 determinations showed a greatly enhanced potent activity when these compounds were encapsulated in the liposomal structure.
Figure 3 shows the size distributions for the curcumin and vitamin C NNS samples, respectively. The samples were diluted by volume in a ratio of 1:6 with DI Water and filtered by a 0.45 µm 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
Figure 3. Volume distribution of the NutraNanoSpheresTM using the Zetasizer ZSP (Malvern Instruments) to measure the sphere sizes in nm diameter. Samples were run as six replicates. Standard deviation (SD) and mean diameters are shown. (a), (b) Representative Curcumin and (c) Vitamin C size distributions are shown.
scattering. A non-negative least squares algorithm was used to generate the size distribution by intensity. Figure 3(a), Figure 3(b) reveals the diameter measurements (7.552 ± 7.303SD nm) for the curcumin NNS for two separate preparations, while the mean diameter for a typical preparation for NNS vitamin C is 3.168 nm is shown in Figure 3(c).
The vitamin C NNS (3.2 nm diameter-65 mg/50 µL) showed a mean LD50 = 133 µmol/well ± 11 SD (n = 4) was greater than 75 times more potent than the free vitamin C, which showed no inhibition of K562 cell viability to at least 104 nm/well. This can be seen in Figure 4(a), Figure 4(b) where two typical Percentage Inhibition vitamin C NNS curves are shown.
Figure 4. The percentage Inhibition of the growth of K562 cells in the presence of NutraNanoSphereTM Vitamin C from (a) 0 to 200 and (b) 0 to 350 µmol/well. The LD50 = 125 and 141 µmol/well levels example data are shown.
The curcumin NNS (7 nm diameter-25 mg/50 µL) resulted in a mean LD50 = 41.3 nmol/well ± 5.6 SD (n = 8). Two representative percentage inhibition experiments are shown in Figure 5. In Figure 1(b) the average LD50 for the free curcumin was 10,900 nm/mL, while the NNS curcumin averaged LD50 was 41.3 nmol/well. The ratio of the LD50 values (10,900/41.3) revealed there was a 264- fold increase in toxicity when the curcumin was encapsulated.
In Figure 6, cancer cell death by alteration of the cell membranes could be seen within 30 s of exposure to the NNS curcumin. The free curcumin showed a similar rapid killing (not shown). The killing process was probably near instantaneous, since the time from the addition of the viability stain and analyzing on the flow cytometer was about 30 s.
Figure 5. The percentage Inhibition of the growth of K562 cells in the presence of NutraNanoSphereTM Curcumin from 0 to 180 µmol/well. The LD50 = 37.5 and 30.5 nmol/well levels example data are shown.
Figure 6. The percentage inhibition of the growth of K562 Cells in the presence of NutraNanoSphereTM Curcumin from 0 to 450 nmol/well. The LD50 values per well were 20 nmol after a 48 hr exposure and 50 nmol for a 30 sec exposure. The viability of the K562 cancer cells dramatically diminishes as early as 30 seconds of exposure to the NutraNanoSphereTM Curcumin.
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 NutraNanoSpheresTM (NNS). 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.
In the case of vitamin C, no inhibition of cancer cell growth was seen with the “free” vitamin C up to 1 × 104 µmol/well. The solubility of vitamin C is 1 g/3 mL of water or 1.89 M   . However, there was concern that going much above 1 × 104 µmol/well would 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 average, which makes the anti-cancer toxic measurements even possible.
The LD50 of curcumin in the “free” form was 10,900 nmol/well (Figure 2(b)) compared to the curcumin NNS (Figure 5), which was averaged only 41.3 nmol/well. The LD50 = 41.3 nmol/well is equal to 41.3 nmol/550 µL well volume, which in turn is 75.1 µM. The average 70 kg human has about 4.7 liters of blood  . In order to have an equivalent LD50 = 75.1 µM in 4.7 L of blood, one would need at least 353 µmol of curcumin. The NNS contain 65 mg of curcumin per drop (50 µL) 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 and NNS curcumin showed complete cell destruction (zero viable cells out of 7 × 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 (Figure 6). The other components required at least 48 h for maximum killing, suggesting a more metabolic and/or apoptotic route of destruction using the other free supplements.
The freeform vitamin C did not affect the K562 cell viability. However, the NNS-vitamin C exhibited a greatly enhanced cytotoxicity against cancer cells. The same was seen for the NNS-curcumin. High dose NNS-curcumin above 1 - 2 mL per day may be very effective in treating advanced cancers, and warrant clinical trials, because of its relatively low cytotoxicity to normal cells.
Genistein, resveratrol, and artemisinin may also be encapsulated in the NNS and be used to produce a “cocktail” of components that would show synergism or additive effects with 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.
In conclusion, these supplements are safe and effective in combination, especially in the NNS form, to 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 NNS Vitamin C and Curcumin 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 methylcytosine dioxygenases, which are enzymes for DNA demethylation, an important crossroad in epigenetic regulation   . 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    .
Our next goal is to encapsulate the other free supplements that show anticancer activity, such as genistein, resveratrol, and artemisinin. Therefore, the possibility of being able to use a combination of NNS supplement 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  using curcumin   , genistein  , resveratrol     and at least a general immune stimulatory role with artemisinin and its derivatives   . By simply adding NNS supplement drops together in a cancer prevention or treatment protocol in a drinkable form, would eliminate individuals having to swallow pills and greatly enhance bioavailability.
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. Carrie Schindler for the Malvern ZetasizerNano System analyses of the sizing of the NutraNanoSpheres. We thank Dr. Tony Kirk, Jacob Robertson, Edra Shalla, Mark Scott, Patrick Bolton, and Bonita Thornthwaite for reviewing this manuscript. We thank Special thanks goes to Dr. Lothar Haegele of X-Labs (Singapore) for supplying the curcumin and vitamin C NutraNanoSpheres™ preparations for this study.