Immune System

Mechanism of the anti‑angiogenic effect of Avemar on tumor cells

Introduction

Angiogenesis, the physiological formation of new blood vessels

from pre-existing ones (1), serves a central role in human

physiology during fetal development, wound healing, tissue

repair following surgery or trauma, menstruation, cancer, and

various ischemic and inflammatory diseases (2). However,

unregulated angiogenesis may result in angiogenic diseases,

including diabetic retinopathy, rheumatoid arthritis, inflammatory diseases, or tumor growth and metastasis (3,4). As

cancer growth is associated with angiogenesis, the inhibition

of angiogenesis is a promising therapeutic strategy in cancer treatment. Furthermore, understanding the mechanisms of

angiogenesis inhibition well enough to manipulate it may lead

to numerous therapeutic possibilities. 

Introduction

Angiogenesis, the physiological formation of new blood vessels

from pre-existing ones (1), serves a central role in human

physiology during fetal development, wound healing, tissue

repair following surgery or trauma, menstruation, cancer, and

various ischemic and inflammatory diseases (2). However,

unregulated angiogenesis may result in angiogenic diseases,

including diabetic retinopathy, rheumatoid arthritis, inflammatory diseases, or tumor growth and metastasis (3,4). As

cancer growth is associated with angiogenesis, the inhibition

of angiogenesis is a promising therapeutic strategy in cancer treatment. Furthermore, understanding the mechanisms of

angiogenesis inhibition well enough to manipulate it may lead

to numerous therapeutic possibilities. 

Mechanism of the anti‑angiogenic effect of Avemar on tumor cells

Avemar (fermented wheat germ extract) is produced

by the industrial fermentation of wheat germ. Avemar is a

completely natural and non-toxic compound that is used

clinically as a dietary supplement for cancer patients undergoing chemotherapy and radiotherapy (5-9). It is known to

have certain biological effects due its major components,

2-methoxy-benzoquinone and 2,6-dimethoxy-benzoquinone.

Additionally, Avemar has been demonstrated to be associated

with anaerobic glycolysis, the pentose cycle and ribonucleotide reductase enzymes; to exert significant anti‑proliferative

effects in a broad spectrum of tumor cell lines; and to possess

the ability to kill tumor cells by inducing apoptosis through

the caspase-poly ADP-ribose polymerase pathway (5,10).

Furthermore, Avemar was reported to be an effective adjuvant agent in cancer treatment for several types of cancer.

such as breast, colon, lung and prostate cancer (11). However,

the mechanism of the anti-angiogenic effect of Avemar

is unclear. Numerous studies have investigated cytotoxic

effects of Avemar on nearly all types of cancers that's why

we did not do any cell viability in this project. Therefore, the

Avemar concentrations were determined according to the

literature (11-14). The main aim of the present study was to

determine the effects of Avemar on angiogenesis. Therefore,

the present study focused on molecular target genes associated

with angiogenesis, vascular endothelial growth factor (VEGF)

and cyclooxygenase-2 (Cox-2), to evaluate the anti-angiogenic

effect of Avemar on tumor cells.

Materials and methods

Cell lines and Avemar. The human gastric carcinoma cell line

NCI-N87, human prostate cancer cell line PC3, human cervical

carcinoma cell line HeLa, and human lung adenocarcinoma

cell line A549 were purchased from ATCC (Manassas, VA,

USA). All cells were grown in Dulbecco's modified Eagle's

medium (Gibco; Thermo Fisher Scientific, Inc., Waltham,

MA, USA), supplemented with 10% heat-inactivated fetal

bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 2 mM

glutamine, and 1% penicillin/streptomycin/neomycin in a

humidified incubator (5% CO2 in air at 37˚C). Avemar was

donated by Biropharma USA Inc. (New York, NY, USA). The

Avemar was stored as dried powder at 4˚C in a bottle until use. Prior to use, it was freshly prepared in sterile water to a final

concentration of 400 µg/ml. The solution was centrifuged to

remove indissoluble materials and then filtered with a 0.22‑µm

filter. Determination of VEGF and Cox‑2 levels by ELISA. A549,

PC3 and NCI-N87 cells release VEGF protein constitutively.

The augmented release of VEGF protein after 48 h was determined in response to serum starvation in PC3 and NCI-N87

cells, and in response to 1,000 U/ml tumor necrosis factor α

(TNF-α) in A549 cells (15). In our previous study, the basal

VEGF protein levels were determined at 24, 48 and 72 h

following seeding of HeLa cells (5x103 cells/well) without any

stimuli (16).

A human VEGF ELISA kit (cat. no., ENZ‑KIT156;

Enzo Life Sciences, Inc., Farmingdale, NY, USA) was used

according to the manufacturer's protocols in order to determine

the possible effects of Avemar on VEGF levels in tumor cells.

Briefly, 5x103

 cells were plated in each well of a 96-well plate

and were treated with various concentrations (400, 800, 1,600

or 3,200 µg/ml) of Avemar for 24 or 48 h. Samples (100 µl)

were then added to the microplates containing VEGF‑specific

monoclonal antibodies, and the mixtures were incubated for

2 h at room temperature. The plates were then washed three

times to remove any unbound substances. Enzyme-linked

polyclonal antibodies specific for VEGF were then added to

the wells, and the mixtures were incubated for 2 h at room

temperature, prior to a further wash to remove any unbound

antibody or enzyme reagent. The substrate solution was

subsequently added to the wells, and the reaction resulted in

the development of a blue color, the intensity of which was

proportionate to the amount of VEGF bound in the initial step.

Following quenching to cease color development, the intensity

of the color was measured at 450 nm with a Multiskan GO

Microplate Spectrophotometer (Thermo Fisher Scientific,

Inc.) and compared to a standard curve.


The Cox-2 concentration was measured using a human

Cox-2 ELISA kit (cat. no., ADI-900-094) provided by Enzo

Life Sciences, Inc. Samples were prepared by extracting Cox-2

from the cells and stock solutions were prepared according

the manufacturer's protocol. Briefly, the cells were harvested

and medium was removed. The cells were re-suspended in

radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris,

pH 7.4, 0.15 M KCl, 1% NP‑40, 5 mM EDTA, 0.5% Sodium

deoxycholate, 0.1% SDS). Samples were prepared by sonicating cells in RIPA buffer for 5 cycles of 30 sec, in 1 min

intervals on ice. Samples were then added the microplate. The

plate was incubated at 37˚C for 1 h, then washed prior to the

addition of a labeled antibody. The plate was incubated at 4˚C

for 30 min, washed, and a substrate solution was added. The

reaction was stopped and absorbance was measured at 450 nm

using a Multiskan GO Microplate Spectrophotometer. Protein

concentrations were calculated with reference to the standard

curve.

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