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Effect Of Nutrient Synergy on Human Osteosarcoma Cells U-2 OS

M.Waheed Roomi, Vadim Ivanov, Matthias Rath and Aleksandra Niedzwiecki

Matthias Rath, Inc., R&D, Cancer Division, Santa Clara, CA 95050

Running Title: Anti-Tumor Effect of Nutrient Synergy on Osteosarcoma Cells
Keywords: osteosarcoma, MMP, nutrient synergy, antitumor effect

Corresponding Author – Alexandra Niedzwiecki, Ph.D.
1260 Memorex Drive
Santa Clara, CA, 94050 USA
Phone – 408-567-5564
Fax – 408-986-9403
Email – a.niedz@drrath.com

Abstract

Introduction:
Current treatment of steosarcoma, a primary malignant tumor of bone or soft parts that arises from bone-forming mesenchymal cells, is associated with poor prognosis, especially due to the increased risk of developing other cancers with chemotherapy. Therefore, new treatment strategies that are effective without toxic side effects are needed. Recent in vitro studies carried out in our laboratory have demonstrated that Nutrient Synergy (NS), a unique formulation composed of lysine, proline, arginine, ascorbic acid, and epigallocatechin gallate exerts a chemo preventive effect on several types of cancer. NS demonstrated a potential synergistic anticancer activity by inhibiting matrix metalloproteinases, key enzymes in invasion and metastasis.

Objective:
We investigated the effect of Nutrient Synergy, on human osteosarcoma cells U-2OS by measuring: cytotoxicity, modulation of matrix metalloproteinases (MMPs): MMP-2 and MMP-9, and invasive potential.

Methods and Materials:
Human osteosarcoma cells U-2 Os were cultured in McCoy medium supplemented with 10% fetal bovine serum and antibiotic in 24 well-culture plates. At near confluence, the cells were treated with Nutrient Synergy at 0, 10, 50, 100, 500 and 1000µg/ml in triplicate at each dose. Cytotoxicity was evaluated by cell number measured by MTT assay after 24 hours, MMP expression by gelatinase zymography in condition media, and invasion through Matrigel.

Results:
Nutrient Synergy was not toxic at 10µg/ml, and exhibited dose response toxicity with maximum of 60% over the control at 1000µg/ml. Zymography showed dose dependent inhibition of MMP-2 and MMP-9 expression by Nutrient Synergy, with virtual total inhibition at 500µg/ml concentration. The invasion of osteosarcoma cells through Matrigel was significantly reduced at 50µg/ml (74%) and totally inhibited at 100µg/ml concentration.

Conclusion:
Our results suggest that Nutrient Synergy is an excellent candidate for therapeutic use in the treatment of osteosarcoma, by inhibiting MMPs expression, angiogenesis and invasion –all important promising parameters for cancer prevention.

Introduction

Osteosarcoma, a primary malignant tumor of bone or soft parts that arises from bone-forming mesenchymal cells, most commonly develops in the distal femur, the proximal tibia, the proximal humerus and the distal radius. Classic osteosarcoma demonstrates aggressive, rapid growth with a high risk of local, “skip” metastases and early, pulmonary metastasis. It is the most common bone cancer and the sixth most common cancer in children, typically developing in adolescents experiencing their growth spurt, and is more frequent in males than females. Most osteosarcomas arise from non-inherited errors in the DNA of growing bone cells. Because these errors occur randomly and unpredictably during intense bone growth there is currently no effective way to prevent this type of cancer (1).

Current treatments for osteosarcoma consist of surgery (amputation or limb salvage surgery) and chemotherapy. These treatments are associated with poor prognosis, especially due to the highly invasive nature of this cancer and the increased risk of metastasis and development of other cancers associated with chemotherapy. For example, of 31 patients studied with localized osteosarcoma (2) and treated with conventional chemotherapy (high-dose methotrexate and leucovarin rescue in 3 patients and intra-arterial cisplatin in 28 patients) at the Anderson Cancer Center, only 3 patients did not experience local recurrence or pulmonary metastases during the follow-up period of 225+ months. Side effects of chemotherapy also include: anemia, abnormal bleeding, increased risk of infection due to destruction of bone marrow, liver and kidney damage, menstrual irregularities, bladder inflammation and bleeding into the urine, skin and heart problems, and hearing loss. Approximately 20% of children diagnosed with osteosarcoma have advanced stage of osteosarcoma, osteosarcoma that has metastasized to the lungs, brain and other bones (1).

One of the key mechanisms that cancer cells use to spread and metastasize in the body involves enzymatic destruction of the surrounding connective tissue. Therapeutic approaches for controlling this process with specific drugs have not been successful and currently there are no means available to control cancer metastasis. For decades, standard treatment for osteosarcoma has consisted of surgery and chemotherapy, which focus on cancer cell destruction, but do not address metastases. Radiation and chemotherapy have not only been ineffective in providing a cure, but also indiscriminately attack all cells – causing cellular damage and destruction of the body's connective tissue, and thus facilitate cancer metastasis. There is obviously a need for safe and effective natural approaches that can be used to control the process of cancer metastasis.

Objective

We investigated the effect of Nutrient Synergy (NS) on human osteosarcoma cells U-2OS by measuring: cytotoxicity, modulation of matrix metalloproteinases (MMPs): MMP-2 and MMP-9, and invasive potential.

Materials and Methods

Cell Culture
Human osteosarcoma cells U -2 OS were obtained from ATCC (American Type Culture Collection, Rockville, MD) and grown in McCoy medium supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) in 24-well tissue culture plates (Costar, Cambridge, MA). Cells were incubated with 1ml of media at 37 0 C in a tissue culture incubator equilibrated with 95% air and 5% C0 2 . At near confluence, the cells were treated with Nutrient Synergy, dissolved in media and tested at 0, 10, 100, and 1000 m M in triplicate at each dose. The plates were then returned to the incubator. The cytotoxicity was evaluated after 24 hrs following incubation with test reagents.

MTT Assay
Viability/cytotoxicity was evaluated based on cell proliferation by MTT assay both in serum-free media and media supplemented with 10% FBS. Serum-free media from cells were saved and assayed for MMPs. The cells in the wells were washed with phosphate buffered saline (PBS) and 500µl of MTT (Sigma #M-2128) 5mg/ml in PBS was added to each well. The culture plates were incubated at 37 0 C for an additional 2 hours. The media was carefully aspirated and 1ml of DMSO (dimethly sulfoxide) was added to each well to dissolve the blue formazan crystals that formed. The optical density was measured at 590nThe OD 550 of DMSO solution in the well was considered to be directly with BioSpec 1601, Shimadzu spectrophotometer. The OD 590 of the DMSO solution in each well was considered to be proportional to the number of cells. The OD 590 of the control (treatment without supplement) was considered 100%.

Gelatinase Zymography
MMP expression in condition media was determined by gelatinase zymography. Gelatinase zymography was performed in 10% polyacrylamide gel (Bio-Rad, Hercules, CA) in the presence of 0.1% gelatin. Culture media (20µl) was loaded and SDS-PAGE was performed with a tris-glycine SDS buffer. After electrophoresis, the gels were washed with 5% Triton X-100 for 30 minutes. After washing, the gels were incubated for 24 hours at 37 0 C in the presence of 50mM Tris-HCl, 5mM CaCl 2 , 5µM, ZnCl 2 , PH 7.5 and stained with Coomassie Blue R 0.5% for 30 minutes and destained. Protein standards were run concurrently and approximate molecular weights were determined.

Matrigel Invasion Studies
Invasion studies were conducted using Matrigel (Becton Dickinson) inserts in 24-well plates. Osteosarcoma cells, suspended in medium supplemented with nutrients, as specified in the design of the experiment, were seeded on the insert in the well. Thus both the medium on the insert and in the well contained the same supplements. The plates with the inserts were then incubated in a culture incubator equilibrated with 95% air and 5% C0 2 for 24 hours. After incubation, the media from the wells were withdrawn. The cells on the upper surface of the inserts were gently scrubbed away with cotton swabs. The cells that had penetrated the Matrigel membrane and migrated onto the lower surface of the Matrigel were stained with Hematoxylin and Eosin and visually counted under the microscope.

Table 1 - Composition of Nutrient Synergy
A stock solution of Nutrient Synergy, prepared in the proportions indicated below, was dissolved in media as indicated in the experimental design to treat cells.

Statistical Analysis
The results were expressed as means + SE for the groups. Data was analyzed by independent sample “t” test.

Results

1. Osteosarcoma Cytotoxicity/Proliferation Study
Nutrient Synergy was not cytotoxic at 10 µg/ml and exhibited a dose-response toxicity with maximum toxicity of 60% over the control at 1000 µg/ml, as shown in Figure 1. Results were statistically significant (P < 0.01)

2. Gelatinase Zymography Study
As shown in Figure 2, zymography demonstrated expression of MMP-2 and MMP-9 by human osteosarcoma U-2OS cells; Nutrient Synergy inhibited the expression of both MMPs in a dose-dependent fashion with virtual total inhibition at 500µg/ml concentration.

3. Invasion Study
The invasion of osteosarcoma cells through Matrigel was significantly reduced at 50µg/ml (74%) and totally inhibited at 100µg/ml concentration.

Figure 4 — Invasion Study: Hematoxylin & Eosin Stained Micrographs
A. Control		B. 10µg/ml NS		C. 50µg/ml NS		D. 100µg/ml NS

Discussion

The results of this study showed substantial anti-proliferative (~60% inhibition at 1000 µg/ml) without morphological changes, and drastic anti-invasive effects of nutrient synergy in vitro on human osteosarcoma cell line U-2OS. Matrigel invasion and expressions of MMP-2 and MMP-9 by osteosarcoma cancer cells decreased in a dose-dependent fashion with complete inhibition of invasion and MMP expression at 100µg/ml and 500 µg/ml respectively.

Matrix invasion can be controlled by inhibition of MMP expression as well as by increasing connective tissue strength and stability, contributing to the “encapsulation” of the tumor. In this study, the dose-dependent inhibitory effect of Nutrient Synergy on MMP-9 and MMP-2 expression of the osteosarcoma cells was consistent with its dose-dependent inhibition of matrix invasion. In addition, matrix invasion was probably also modulated by enhanced stability and strength of the connective tissue secondary to the activity of the nutrients provided in Nutrient Synergy. Optimization of synthesis and structure of collagen fibrils depends upon hydroxylation of hydroxyproline and hydroxylysine residues in collagen fibers . It is well known that ascorbic acid is essential for the hydroxylation of these amino acids. Ascorbic acid and lysine are not produced in the human body, therefore sub-optimal levels of these nutrients is possible in various pathological stages, as well as through inadequate diets. Although proline can be synthesized from arginine, its synthesis and /or hydroxylation may be affected in pathological conditions.

The inhibitory effects of the individual nutrients composing Nutrient Synergy have been reported to have inhibitory effects in clinical as well as experimental studies. Ascorbic acid has been reported to have cytotoxic and antimetastatic actions on malignant cell lines (5)(6)(7); in addition, low levels of ascorbic acid have been reported in cancer patients (8) (9) (10). ECGC is a potent anticancer agent that has been reported to have a growth inhibitory effect against certain human cancer cell lines.(11,12, 13).

Conclusions

Our results suggest that Nutrient Synergy is an excellent candidate for therapeutic use in the treatment of the highly aggressive osteosarcoma cancer, by inhibiting cell proliferation, MMP expression, and invasion.

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