Interactions Between Nutraceutical Supplements and Standard Acute Myeloid Leukemia Chemotherapeutics.

PURPOSE
Concomitant use of nutraceuticals with chemotherapy is very common. Cancer patients self-medicate to relieve the side effects associated with chemotherapy, improve disease outcome and to regain control of their medical care. However, there is limited empirical evidence on potential drug-nutraceutical interactions and their resulting effect on chemotherapy efficacy.


METHOD
To investigate drug-nutraceutical interactions we created and screened a library of commonly used nutraceuticals for their modulatory effects on the activity of cytarabine and daunorubicin, two primary chemotherapeutics used to treat acute myeloid leukemia (AML). Combination screening was performed in 3 AML cell lines (OCI-AML2, KG1a and U937) using the MTS viability assay. Lead compounds were validated using with the Annexin V/ Propidium iodide assay and CalcuSyn drug combination software.


RESULTS
We identified zinc as a nutraceutical that enhanced AML chemotherapy efficacy with combination index (CI) values of 0.649, 0.632 and 0.615 at EC 25, 50 and 75, respectively; CI values <0.9, >1.1 or between 0.9-1.1 denote statistical synergy, antagonism or additivity, respectively. In contrast, we show that echinacea hindered AML chemotherapy efficacy by significantly reducing the ability of cytarabine to induce cell death.


CONCLUSION
Given the positive and negative effects of nutraceuticals, patients undergoing chemotherapy must consult with their oncologist before consuming over-the-counter supplements. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.


INTRODUCTION
At some point after diagnosis, 60-80% of patients suffering from a hematological malignancy consumed an over-the-counter dietary supplement (i.e., nutraceutical, or food-derived bioactive compound) (1). Although the development of methotrexate involved understanding the negative impact of folic acid on acute lymphoblastic leukemia (ALL) cell growth and ALL patient survival, there remains limited clinical evidence supporting or refuting nutraceutical use by cancer patients. Thus, given the importance of understanding the role of nutraceuticals in cancer chemotherapy, we performed a drug combination screen to identify nutraceuticals that enhanced and hindered the efficacy of cytarabine and daunorubicin, two primary therapeutics used in the treatment of acute myeloid leukemia (AML).

Nutraceutical screen
The unique nutraceutical library was created and screened similar to previously described methods (2). Briefly, AML cells (1.5x10 4 /well) were seeded in 96-well plates. After seeding, cells were treated with aliquots (1 mg/ml final concentration) of library compound alone or in combination with 3µM cytarabine and 0.05µM daunorubicin, which correspond to their EC50 values (data not shown). After 72 hours, cell growth and viability were measured by the MTS assay.

Statistical and combination index analysis
Unless otherwise stated, the results are presented as mean ± SD. Data were analyzed using GraphPad Prism 4.0 (GraphPad Software, USA). p<0.05 was accepted as being statistically significant. CalcuSyn software was used to analyze and generate combination index (CI) values, as previously described (3). The software assesses the combined effect of two drugs under fixed molar ratios and generates CI values as a function of cell death. These graphical representations display the potential synergy, antagonism, or additivity of two drugs over a wide range of fixed molar ratio doses.

Nutraceutical screen identifies potential interaction between zinc and cytarabine
The nutraceutical library was screened against 3 AML cell lines following a 72 hour incubation period in the presence or absence of an AML chemotherapeutic. EC50 values for cytarabine and daunorubicin were incubated with 1 mg/ml of an individual nutraceutical and viability was assessed by the MTS assay. Heat map analysis showed that zinc (compound 20) had the greatest effect on enhancing cytarabine and daunorubicin's efficacy ( Figure 1A). Zinc alone had an EC50 of ~0.012 mg/ml when validated with the ANN/PI assay ( Figure 1B, left panel). The ability of zinc to synergistically interact with the clinical therapeutics was next tested using CalcuSyn, where CI values <0.9, >1.1 or between 0.9-1.1 denote statistical synergy, antagonism or additivity, respectively (3). The combination of cytarabine and zinc was synergistic (CI values of 0.649, 0.632 and 0.615 at EC 25, 50 and 75) ( Figure 1C, left panel) while concomitant treatment with daunorubicin displayed an additive relationship ( Figure 1C, right panel).

Nutraceutical screen identifies Echinacea as a compound
that hinders cytarabine and daunorubicin activity. Heat map analysis of our drug combination screen showed that compound 23 and echinacea (compound 21), had the greatest effect on hindering cytarabine and daunorubicin's cytotoxicity ( Figure 1A). However, compound 23 was a false positive and was not assessed further (validation results not shown). Echinacea's inhibitory activity was confirmed in secondary assays and since it was not cytotoxic to AML cells at the concentrations used ( Figure 1D), we were unable to use CalcuSyn software to determine CI values (i.e., it requires EC50 values). Thus, we incubated echinacea with cytarabine or daunorubicin in equal molar ratio concentrations, as described in the methods, and used the ANN/PI assay to measure cell viability. Echinacea significantly inhibited cytarabine and daunorubicin's cytotoxicity in AML cells ( Figure 1D).

DISCUSSION
A high throughput screen of a nutraceutical library has shown that zinc is able to enhance and echinachea is able to hinder the efficacy of AML chemotherapy. Given the high rate of nutraceutical consumption, this information is critical for accurate patient recommendations.
Zinc is an essential trace element in humans and supplementation (2mg/kg/day for 60 days) in children and adolescents with acute leukemia was effective in relieving chemotherapy-associated side effects (weight loss, malnutrition, nausea, and vomiting) (4). This study demonstrated the safety and physiological benefit of zinc supplementation but did not evaluate supplementation effects on chemotherapy efficacy. Marginal zinc deficiencies can have deleterious effects on DNA integrity and repair (5) and zinc supplementation in zinc deficient patients has been shown to increase DNA repair to normal levels (5). Thus, when in combination with cytarabine, which can impart its anti-cancer activity by incorporating metabolites into DNA during DNA synthesis, zinc may be potentiating cytarabine's activity by increasing the rate of DNA repair thereby incorporating more arabinofuranosylcytosine triphosphate (i.e., araCTP, the active form of AraC) into DNA.
None-the-less, future detailed mechanistic studies are needed to confirm the exact mechanism of this synergistic interaction.
Zinc is tightly regulated and supplementation will only marginally increase total zinc plasma levels. Supplementation to alleviate chemotherapy side effects, were demonstrated at plasma concentrations of 1.0 ± 0.07 mg/L zinc after 60 days (4). In our study, zinc was used in the form of zinc gluconate (MW 455.685 g/mol) in which zinc is found at 12.9% (as formulated by Jamieson). In our study, in vitro synergistic interactions were seen at zinc concentrations of 4.0mg/L ( Figure 1C). Therefore, in order to achieve relevant physiological levels, zinc would need to be administered systemically with cytarabine, as oral supplementation may not achieve the plasma concentrations required to impart synergy. Recently it was shown that nanoparticle zinc (ZnO-NPs) administered intravenously in rats resulted in zinc plasma levels of 1000 mg/L with no associated toxicity (6). None-the-less, future pharmacokinetic studies with oral zinc supplementation or intravenous zinc are needed to support clinical efficacy.
Through this screen we have also demonstrated that Echinacea, among the most purchased nutraceuticals, hindered the efficacy of AML chemotherapy. Echinacea has been shown to increase the cellular concentration of CYP3A, a potent drug metabolizer, which could decrease plasma concentrations of anticancer drugs that are CYP3A substrates such as cytarabine (7,8). Therefore, echinacea could be hindering cytarabine by inducing CYP3A enzymes. In addition, lymphocytes harvested from echinacea supplemented mice showed up-regulation of the antiapoptotic Bcl-2 protein (9). This provides evidence that echinacea can have anti-apoptotic in vivo modulatory effects, which could be of great importance to patient outcome as Bcl-2 is commonly overexpressed in AML.
Cytarabine and daunorubicin can cause AML cell apoptosis through this pathway and thus, echinacea may interfere with these chemotherapeutics through Bcl-2 modifications.
Due to the multiple formulations and sources of echinacea in supplements, it is difficult to define physiologically relevant doses and accurately assess pharmacokinetics. In this study, a maximum of 2 mg/ml final concentration of Echinacea Purpurea (Jamieson Inc., Echinacea Purpurea (4:1) extract 87.5 mg root, NPN# 80010071) was used, which is similar to previous reports (10). Multiple studies administered echinacea at high doses in humans (i.e., 1500 mg/day for 28 days) and mice (30 -100 mg/kg for 14 days) with no signs of toxicity (7) (9). Therefore, given echinacea's ability to reduce chemotherapy efficacy, cancer patients undergoing chemotherapy should avoid echinacea until further studies can confirm its safety.
In summary, cytarabine and daunorubicin's activity were enhanced by the nutraceutical zinc and hindered by echinacea. These results provide seminal evidence that define the role of nutraceuticals in AML chemotherapy and provide the basis for future studies that will lead to important clinical recommendations for AML patients.