Anticancer Effects of Curcumin, Artemisinin, Genistein, and Resveratrol, and Vitamin C: Free Versus Liposomal Forms

Cancer prevention supplements, which also provide effective treatment 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, to kill K562 Erythroleukemic cells in vitro. 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. 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 cancer cell destruction. Vitamin C up to 1 × 10 μmol/well did not affect K562 cell viability. The vitamin C-NNS (3.2 nm diameter-60 mg/50 μL) 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 diameter-25 mg/50 μL) resulted in an LD50 = 41.3 μmol/well ± 5.6 SD (n = 8) and represented a 264 fold increase in activity to destroy the cancer cells. The clinical goal is to develop water-soluble mixtures of anti-cancer compounds in the NNS with their high bioavailability (>90%) and without degradation in the stomach for preventing and curing 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 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 [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] [5]. 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 [6] [7] [8] [9] [10].
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 [11]. Even our fresh fruits and vegetables are being depleted of essential minerals and vitamins, often necessitating supplementation [12]. Stress hormones such as adrenaline and cortisol suppress natural killer cell function [13] [14]. 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.
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 micelleencapsulated curcumin and vitamin C will be compared to their free forms [29] [30] [31] [32] [33].

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).
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

Results
Cytotoxicity of "Free" Supplements: Curcumin, Genistein, Resveratrol, Artemisinin and Vitamin C.   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 LD 50 = 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 10 4 nm/well. This can be seen in Figure 4(a), Figure 4  The curcumin NNS (7 nm diameter-25 mg/50 µL) resulted in a mean LD 50 = 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 LD 50 for the free curcumin was 10,900 nm/mL, while the NNS curcumin averaged LD 50

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 TM (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 × 10 4 µmol/well. The solubility of vitamin C is 1 g/3 mL of water or 1.89 M [34] [35]. However, there was concern that going much above 1 × 10 4 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 LD 50 = 133 nmol/well average, which makes the anti-cancer toxic measurements even possible.
The LD 50 of curcumin in the "free" form was 10,900 nmol/well (Figure 2 The free and NNS curcumin showed complete cell destruction (zero viable cells out of 7 × 10 5 viable cells in the control wells) at the LD 50 = 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.

Conclusions
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 [37] [38]. 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 [39] [40] [41].
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 [42] using curcumin [43] [44], genistein [45], resveratrol [46] [47] [48] [49] and at least a general immune stimulatory role with artemisinin and its derivatives [50] [51]. 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.