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A Specific Combination Of Ascorbic Acid, Lysine, Proline And Epigallocatechin Gallate Inhibits Proliferation And Extracellular Matrix Invasion Of Various Human Cancer Cell Lines

Shrirang P Netke, M Waheed Roomi, Vadim Ivanov, Aleksandra Niedzwiecki* and Matthias Rath

Matthias Rath, Inc., Research and Development, Santa Clara, CA 95054
Author for correspondence and reprints. Phone (408) 807-5564; FAX: (408) 986-9403;

EMERGING DRUGS – VOL. II
Copyright © 2003 by
PJD Publications Limited
Research Communications
Westbury, NY 11590-0966 USA

Key Words: Antiproliferative and antimetastatic effect, breast cancer (MDA-MB-231), colon cancer (HCT116), epigallocatechin gallate, gelatinase zymography, matrix metalloproteinases (MMPs) –2 and –9, proliferation of cancer, skin melanoma (A2058), specific combination of ascorbic acid, proline, and lysine.

Abstract

We found that a specific combination of ascorbic acid (AA, 100µM), proline (P, 140 µM) and lysine (L, 400 µM) had a significant antiproliferative and antimetastatic effect against some human cancer cell lines, breast (MDA-MB-231), colon (HCT116) and skin (melanoma, A2058). In the presence of this nutrient combination the invasion of the extracellular matrix by human breast cancer cells, melanoma cells and colon cancer cells was inhibited by 50%, 10% and 30% respectively. Addition of epigallocatechin gallate (EGCG) further enhanced this nutritional synergy producing a more pronounced inhibitory effect on both cellular proliferation and matrix invasion. The proliferation of breast cancer cells MDA-MB-231 in the presence of AA, P, L and 20µg/ml of EGCG was reduced to 74% and colon cancer cells HCT116 to 69% compared to the unsupplemented medium. The increase in concentration of EGCG to 50µg/ml did not cause much further reduction in the number of breast cancer cells. However it reduced proliferation of colon cancer cells to 4,6% and melanoma cells to 30% of the control. Matrigel invasion by breast cancer cells and human melanoma cells in the presence of AA, P, L and 20µg/ml of EGCG was stopped completely. At a similar concentration, invasion by colon cancer cells was reduced to 24,9%. However, the expression of matrix metalloproteinases (MMPs) –2 and –9 was not altered by this nutrient combination in melanoma cells as visualized by gelatinase zymography. MMP-2 and MMP-9 were significantly inhibited by EGCG in a dose dependent manner, and L, P, AA have no additional effect. Thus the combination of AA, P, L and EGCG shows great potential for control of cancer using a safe and multi-targeted approach.

Introduction

One of the key mechanisms that cancer cells use in order to expand and metastasise in the body involves enzymatic destruction of the surrounding connective tissue. Therapeutic approaches to control this process through specific drugs have not been successful and currently there are no means available to control cancer metastasis. Current treatments protocol with chemotherapy and radio-therapy focus on cancer cell destruction in the body, and they do not address metastasis. Moreover, these treatments are toxic, not specific and associated with severe side effects. Chemotherapy and radiotherapy carry a risk of the development of new cancers and through their destruction of connective tissue in the body can facilitate the invasion of cancer. Therefore, there is a need for safe and effective natural approaches that can be used to control the process of cancer expansion in the body.

In order to grow and expand to other parts of the body, cancer cells degrade the extracellular matrix (ECM) through various matrix metalloproteinases (MMPs) and plasmin, whose activities have been correlated with an aggressiveness of tumor growth. Rath and Pauling (1992) postulated that nutrients such as an amino acid, lysine and ascorbic acid can act as natural inhibitors of extracellular matrix proteolysis and as such they have the potential to modulate tumor growth and expansion. These nutrients can exercise their anti-tumor potential through several mechanisms, among them by inhibition of MMPs and by strengthening of the connective tissue surrounding cancer cells (tumor “encapsulating” effect).

In this study, we investigated the effects of lysine, ascorbic acid and proline for their anti-proliferative and anti-invasive potential in various human cancer cell lines. In addition, we also studied the effect of epigallocatechin gallate (EGCG), with the above combination. EGCG is known for its anti-cancer effect in various human and experimental studies (Mukhtar and Ahmed, 2000, Valvic, Timmerman et al., 1996). It was also postulated that such a combination of nutrients would exert a very potent synergistic effect.

Materials and Methods

Human breast cancer cells MDA-MB-231, human colon cancer cells HCT 116, human melanoma cell line A2058 were obtained from ATCC. Normal human dermal fibroblasts were obtained from Gibeo. Where not indicated, the culture media obtained from ATCC were used.

In cancer cell proliferation studies each treatment was replicated eight times. In the invasion assays, each treatment was performed in three or four replicates.

Cell proliferation studies

In these studies 5X10 breast cancer cells were grown in Liebovitz`s medium with 10% fetal bovine serum (FBS) in 24 well-plates. The medium was used as such (basal) or with designated supplements. Plates were incubated in an air circulating incubator (without supplemental CO2) for four days. The colon cancer cells HCT116 were grown in McCoy`s 5A media and maintained in a 5% CO2-air circulating incubator. At the end of the incubation period, the media were withdrawn and the cells in the wells were washed with PBS followed by incubation for 3 hours with MTT stain. Dimethylsulfoxide (DMSO, 1 ml) was added to each well. The plates with DMSO were allowed to stand at room temperature for 15 minutes with gently agitation and then OD of the solution in each well was measured at 550 nm. The OD550 of the DMSO solution in the well was considered to be directly proportional to the number of cells. The OD550 of treatment that did not contain any supplement (Basal) was considered as 100.

Matrigel Invasion Studies

The studies were conducted using Matrigel (Becton Dickinson) inserts in compatible 24 well plates. Fibroblasts were seeded and grown in the wells of the plate using DMEM. When fibroblasts reached confluence, the medium was withdrawn and replaced with 750 µl of the media designated for the treatment. The cancer cells (5X 104) suspended in 250 µl of the 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 air circulating incubator for MDA-MB-231 cells and 5% CO2 incubator for colon cancer cells and melanoma cells) for 18-20 hours. After incubation, the media from the wells was withdrawn. The cells on the supper surface of the inserts were gently scrubbed away with cotton swab. The cells that had penetrated the Matrigel membrane and had migrated on the lower surface of the Matrigel were stained with hemacolor stain (EM Sciences) and were visually counted under the microscope. The results were subjected to ANOVA and all the possible pairs were tested for significance at p<0.05.

The media in different studies were supplemented with ascorbic acid, proline, lysine and EGCG at concentrations as indicated in the Results and Discussion.

Gelatinase Zymography

Gelatinase zymography was performed in 10% Novex pre-cast polyacrylamide gel (Invitrogen) in the presence of 0.1% gelatin. Culture media (20µl) was loaded and SDS-PAGE was performed with tris-glysine SDS buffer. After electrophoresis, the gels were washed with 5% Triton X-100 for 30 minutes and stained. Protein standards were run concurrently and approximate molecular weights were determined.

Results and Discussion

(A) Cancer Cells Proliferation Study

Melanoma A2058 Cells

Figure I shows the effect of 10, 20 and 50 µg/ml of EGCG without and with L, P, and AA supplementation on the proliferation of melanoma cells. Neither L, P, and AA nor EGCG at 10 and 20 µg/ml had any significant effect on cell proliferation. However, EGCG at 50 µgm/ml significantly reduced the cell number to 30%. A similar effect was observed with L, P and AA supplementation.

Figure 1: Effect of supplementation of Basal with 100µM ascorbic acid (AA), 140µM proline (P), 400µM lysine (L) and various levels of epigallocatechin gallate (EGCG) on cell proliferation of melanoma A2058 cells

Breast Cancer MDA-MB-231 Cell

In these experiments, the basal media was supplemented with 0, 10, 20, 50, 100 or 200 µg/ml of EGCG (Figure 2). The results show that supplementation of the basal media with 50, 100 and 200 µg/ml of EGCG significantly reduced the cell number to 66.1 ± 5.3, 33.6 ± 2 and 29.6 ± 0.8% compared to unsupplemented controls respectively. EGCG concentrations in the cellular media up to 20µg/ml did not have any significant inhibitory effect on cell proliferation.

Figure 2: Effect of epigallocatechin gallate (EGCG) on cell proliferation of breast cancer cells MDA-MB 231

We also studied the effect of AA, L, P and different concentrations of EGCG on proliferation of cancer cells. Figure 3 shows non-significantly reduced the cell number to 86.1 + 1.93% with AA, L, and P. Further addition of 20, 50 and 100µg EGCG to this combination significantly reduced the cell number to 74 ± 5.8, 64.8 ± 1.6 and 22 ± 5% compared to the control group respectively.

Figure 3: Effect of supplementation of Basal with 100µM ascorbic acid (AA), 140µM proline (P), 400µM lysine (L) and various levels of epigallocatechin gallate (EGCG) on cell proliferation of breast cancer cells MDA-MB 231

Colon cancer HCT116 cells

While the inhibitory effect of AA, P and L on cell proliferation of colon cancer cells was not pronounced, the combination of AA, P and L with 20µg/ml EGCG significantly decreased the cell number to 69 ± 0.5 (figure 4). A higher level of EGCG in this combination (50µg/ml) drastically reduced the cell number to 4.6 ± 0.3%.

Figure 4: Effect of supplementation of Basal with 100µM ascorbic acid (AA), 140µM proline (P), 400µM lysine (L) and various levels of epigallocatechin gallate (EGCG) on cell proliferation of colon cancer HCT116

The antiproliferative activity of the nutrient combinations used in these studies varied with the type of cancer cells. In breast cancer cells, the combination of AA, P and L with EGCG had higher anti-proliferative effects than when these nutrients were used individually. In melanoma and colon cancer cells exposed to the combination of AA, P and L did not affect their proliferation. However, combining these nutrients with 20 µg/ml EGCG resulted in a significant reduction in the number of colon cancer cells but not in melanoma cells. The colon cancer cells appeared to be more sensitive than the breast cancer cells and melanoma cells the least to the combination of AA, P, L and EGCG. The proliferation of the colon cancer cells was almost completely reduced (4.6%) when AA, P and L was given with 50µg/ml of EGCG.

(B) Geltinase Zymograph Studies

The effect of EGCG on melanoma cells with basal and AA, P and L supplementation on expression of MMPs is depicted in figure 5 by gelatinase zymography. Melanoma cells showed two bands corresponding to MMP-2 and MMP-9. AA, L and P combination have no effect on the expression of MMPs bands as compared to the basal. However, EGCG inhibited the expression of both MMP-2 and MMP-9 in a dose dependent manner. The intensity of bands for basal and AA, L and P combination were the same.

Figure 5: Effect of supplementation of Basal with 100µM ascorbic acid (AA), 140µM proline (P), 400µM lysine (L) and various levels of epigallocatechin gallate (EGCG) on expression of matrix metalloproteinases (MMP) by melanoma cells.
1. Markers; 2. Basal; 3. AA+P+L; 4. EGCG 10µg/ml; 5. AA+P+L+10µg/ml EGCG; 6. EGCG 20µg/ml; 7. AA+P+L+EGCG 20µg/ml; 8. EGCG 50µg/ml; 9. AA+P+L+EGCG 50µg/ml

(C ) Extracellular Matrix Invasion and Migration studies

We investigated inhibitory effects of a combination of AA, P and L used separately and together with various concentrations of EGCG on the extracellular matrix using pre-formed Matrigel matrices, routinely used to assess invasive potential of various cancer cell lines.

Breast cancer MDA-MB-231 cells

Figure 6 shows the results of Matrigel invasion of breast cancer cells incubated in the presence of AA, P and L. Invasion of cancer cells incubated in a combination of AA, P and L was reduced to 48.1 ± 22.1% compared to cells incubated in un-supplemented media. In the media supplemented with 20 µg/ml of EGCG only, the number of invading cells decreased to 69.5 ± 27.4%. The complete inhibitation of matrix invasion by breast cancer cells was achieved in the presence of higher EGCG concentrations (50µg/ml and 100 µg/ml).

Figure 6: Effect of supplementation of Basal with 100µM ascorbic acid (AA), 140µM proline (P), 400µM lysine (L) and various levels of epigallocatechin gallate (EGCG) on Matrigel invasion by breast cancer cells MDA-MB 231

Figure 6 also shows that the combination of AA, P and L and 20 µg/ml of EGCG was effective in a complete stopping of the invasion of cancer cells through the extracellular matrix. This combination made it possible to achieve the maximum inhibitory effect on cancer cells invasion without the necessity of using high concentrations of individual nutrients. As such, AA, P and L along with EGCG made it possible to stop matrix invasion of breast cancer cells completely at lower level of EGCG (20µg/ml).

Colon cancer HCT 116 cells

Figure 7 shows that the combination of AA, P and L significantly reduced the invasion of colon cancer cells to 67.2 ± 3.7%. The EGCG used alone at 20µg/ml reduced the invasion to 44.9 ± 3.3%, while the combination of AA, P, L and 20µg/ml of EGCG had a synergistic effect reducing colon cancer cells invasion to 24.9 ± 4.6%.

Figure 7: Effect of supplementation of Basal with 100µM ascorbic acid (AA), 140µM proline (P), 400µM lysine (L) and various levels of epigallocatechin gallate (EGCG) on Matrigel invasion by colon cancer cells HCT116

Melanoma A2058 cells

Figure 8 shows that the combination of AA, P and L was effective in reducing the number of invading cells to 88.2 ± 4%, however this decrease was not statistically significant. Combining these nutrients with as little as 20µg/ml of EGCG was effective in reducing the number of invading cells to zero.

Figure 8: Effect of supplementation of Basal with 100µM ascorbic acid (AA), 140µM proline (P), 400µM lysine (L) and various levels of epigallocatechin gallate (EGCG) on Matrigel

These results show that the use of AA, P and L with EGCG enabled us to obtain drastic reduction in the number of cells invading and migrating through Matrigel membrane at lower levels of EGCG. The invasion was reduced to zero using as low a level as 20µg/ml EGCG with AA, P and L, in breast cancer cells and melanoma cells. The benefits of the combination results were not as spectacular with colon cancer cells as obtained with breast cancer cells. The level of EGCG had to be at 50µg/ml to obtain 90% reduction in invasion by these cells. In the present study, we did not observe any alteration in MPs expression in melanoma cells, although EGCG has an inhibitory effect on their expression in a dose dependent fashion.

The aim of this study was to identify nutrient combination with the most effective anti-proliferative and anti-invasive effects on cancer cells, leaving the investigation of molecular mechanisms involved as a second step. Here we can postulate several mechanisms by which the inhibitory effect of involved nutrients can be investigated.

MMPs activity can be affected by lysine through plasmin –mediated mechanisms although other mechanisms are not excluded. The MMPs are secreted as proenzymes and their activation is mediated partially by plasmin and its completion requires active forms of MMP-3. The mechanism of activation of various MMPs detailed by Nagase (1997) indicates that MMP-3 also requires the conversion of plasminogen to its active form, plasmin. Plasminogen active binding center has the sites where lysine gets specifically attached. Therefore lysine can interfere with activation of plasminogen into plasmin by plasminogen activator (Rath and Pauling, 1992), by binding to plasminogen active sites. Tranexamic acid, a synthetic lysine analog has been used to inhibit plasmin induced proteolysis through this mechanism.

Since plasmin activity is essential to induce several tissue MMPs, lysine can intefere with conversion of plasminogen to plasmin and thereby it can inhibit the activation of almost all MMPs. In addition, EGCG has been shown to have an inhibitory effect on extracellular matrix degradation through inhibition of MMP-2 (Demeule et al., 2000).

It is also possible to affect cancer cell matrix invasion by increasing the stability and strength of the connective tissue surrounding cancer cells, and contributing to the “encapsulation” of the tumor. This requires optimizing the synthesis and structure of collagen fibrils, for which the hydroxylation of hydroxyproline and hydroxylysine residues in collagen fibers is essential. It is well known that ascorbic acid is essential for hydroxylation of these amino acids. Ascorbic acid and L-lysine are not normally produced in the human body, therefore, suboptimal levels of these nutrients is possible in various pathological stages as well as through inadequate diets. Although proline can be synthesized from arginine, its sythesis and or hydroxylation may be affected at pathological conditions. As such, it has been shown that hydroxyproline content of metastatic tumor tissue is much lower than non-metastatic tumor tissue (Chubainskaia et al., 1989). A variety of drugs that reduced metastasis also increased the hydroxyproline content of the tissues (Chubinskaia et al., 1989). The urinary hydroxyproline content of cancer patients has been found to be higher than that in healthy persons or non-cancer patients (Okazaki et al., 1992). All these findings suggest adverse effects of cancer cells on metabolism of proline and possible conditioned deficiency of proline in cancer patients.

Lower levels of ascorbic acid have been reported in cancer patients. (Anthony and Schorah, 1982, Nunez et al., 1995, Kurbacher et al., 1996). Ascorbic acid is very cytotoxic to malignant cell lines (Koh et al., 1998, Roomi et al., 1998) and exerts antimetastatic action (Liu et al., 2000). Our studies using the combination of ascorbic acid, proline and lysine indicated that this combination exerts a potent antiproliferative and antimetastatatic effect on several cancer cell lines in tissue culture studies. The effects however fell short of complete inhibition of proliferataion and metastasis.

EGCG, one of the catechins in green tea extract is the most potent anti cancer agent (Valcic et al., 1996, Mukhtar and Ahmed, 2000). It has been reported to have growth inhibitory effects against certain human cancer cell lines. (Ahmad et al., 1997, Liang et al., 1997, Yang et al., 1998). There is thus quite a synergy in the anticancer activities of the combination of AA, P and L and EGCG. It was therefore hypothesized that this combination along with EGCG would exert synergistic anti-cancer activity.

It is clear that the combination of AA, P and L with EGCG holds the potential of providing substantial relief in all the three types of cancers investigated in these studies. In the case of breast cancer and melanoma relief will be obtained mostly by drastic reduction in cell metastasis. In respect to colon cancer cell drastic reduction both in proliferation and metastasis can be achieved with higher level of EGCG.

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