Antitumoral effect of Ocoxin on acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy whose incidence is growing in developed countries. In the relapse setting, very limited therapeutic options are available and in most cases only palliative care can be offered to patients. The effect of a composite formulation that contains several antioxidants, Ocoxin Oral solution (OOS), was tested in this condition. When analyzed in vitro, OOS exhibited anti-AML action that was both time and dose dependent. In vivo OOS induced a ralentization of tumor growth that was due to a decrease in cell proliferation. Such effect could, at least partially, be due to an increase in the cell cycle inhibitor p27, although other cell cycle proteins seemed to be altered. Besides, OOS induced an immunomodulatory effect through the induction of IL6. When tested in combination with other therapeutic agents normally used in the treatment of AML patients, OOS demonstrated a higher antiproliferative action, suggesting that it may be used in combination with those standard of care treatments to potentiate their antiproliferative action in the AML clinic.

Keywords: acute myeloid leukemia; antioxidants; cell cycle; p27.

Conflict of interest statement

CONFLICTS OF INTEREST

E.S. is an employee of Catalysis S.L. The research costs of this work were partially supported by Catalysis S.L.

Figures

Figure 1. Efficacy of OOS on AML cell lines in vitro

Dose-dependent effect of OOS on the proliferation of HEL, KG1 or HL60 AML cells was assessed in vitro. Cells were incubated with OOS at the indicated dilution factors and MTT metabolization was measured at 24 (A), 48 (B), 72 (C) or 96 (D) hours. Mean absorbance values of untreated samples were taken as 100% and then mean values referred to that. Data are represented as mean ± SD of quadruplicates of an experiment that was repeated at least twice.

Figure 2. OOS is more effective in combination with standard of care treatments

The effect of OOS alone (1:50) or in combination with Ara C (250 nM, A), Doxorubicin (DXR, 100 nM, B) or Fludarabine (Fluda, 250 nM, C) were determined in MTT assays. The mean absorbance values of untreated samples from each were considered as 100%. Data are represented as mean ± SD of quadruplicates of an experiment that was repeated at least twice.

Figure 3. Efficacy of OOS on AML models in vivo

(A) OOS interferes with tumor growth. Female CB17-SCID athymic mice were injected with HEL cells. When tumors became palpable and were growing, they were randomized to different groups that were orally treated 5 days per week (Monday to Friday) with 100μl OOS/animal (■) or vehicle alone (water,♦), and tumor volumes were measured twice a week. Data are represented as mean tumor volume ± SEM of the animals on each group. (B) Effect of OOS on animal weight. Statistical significant differences are shown (*p

Figure 4. Action of OOS on immune system cytokines

At the time of sacrifice, blood from the animals under the different treatments was collected by intracardiac injection, and sera clarified and frozen. The amount of the indicated cytokines was determined by flow cytometry using a BD Cytometric Bead Array as described in the materials and methods section. The amount for each molecule and experimental condition are shown in the corresponding boxplots, as indicated. Statistical significant differences are shown (*p

Figure 5. OOS induces a decrease in tumor proliferation

(A) OOS does not induce apoptotic cell death on AML tumors. For each experimental condition, two tumors were randomly processed for IHQ analysis and apoptotic cells were detected by tunnel staining. The number of apoptotic cells/field was quantified for each condition and its mean number ± SD is shown in the graph. Pictures of representative fields stained for this marker are shown on the right. (B) Similarly the number or endothelial cells was measured by CD31 staining. (C) Besides, to establish the proliferative status of the tumors, Ki67 marker was used. Pictures of representative fields stained for Ki-67 are shown on the right upper row and lower pictures show part of them (inset) at higher magnification. Statistical significant differences are shown (*p

Figure 6. OOS causes accumulation of p27 and G1 arrest

(A) Quantitation of the in vivo levels of cell cycle related proteins. The amount of the indicated proteins in the tumors was assessed by conventional WB. (B) The amount of p27 was quantified using the NIH image software, and the mean ± SD of such amount is represented in the graph. (C) Cell cycle analysis of HEL cells treated for 24 hours with OOS (1/50 dilution). The percentage of cells in the different phases of the cell cycle is shown. (D) Action of OOS (1/50) on p27 levels. HEL cells were treated for 24 hours with OOS and cell extracts prepared to analyze p27 by WB. GAPDH was used as a loading control.

Figure 7. In vivo effect of OOS on gene expression profiles

(A) Hierarchical clustering of the 6 tumors and the 37 genes deregulated after OOS treatment. Each row represents a gene and each column represents a tumor (1- control, 2- OOS treated). The expression level of each gene in each tumor is relative to its medium abundance across all the tumors and is depicted according to the color scale shown. Red and green indicate high or low expression levels, respectively. (B) The gene expression profile of three control tumors was compared to that of three tumors treated with 100 μl of OOS. A list with those genes whose expression changed above 2 times with the treatment is shown in the figure.