The Chemopreventive Ability of EGCG, a Catechin In Green Tea, on Selected Colorectal Carcinoma Cells - A Review
Abstract
Green tea, through tradition and culture, is believed to have various health benefits including being a chemotherapeutic and chemopreventive drug. Relatively recently, various studies have concluded that (-)-Epigallocatechingallate (EGCG), a polyphenol, is the active ingredient in behind the anti-cancerous benefits green tea possess. This paper serves as a review of the various mechanisms, discovered by numerous scientists, in which EGCG works against colorectal cancer. This was done by using the six categories that define a cancer cell from the paper ‘The Hallmarks of Cancer’ by Hanahan and Weinberg. These categories essentially deduce the six general mutations a cell, more specifically a colorectal cell, must undergo to become cancerous. It was concluded that EGCG is effective against all of the six mutations colorectal cancer cells may obtain during tumorgenesis making it a potent and viable anti-cancer drug.
Introduction
“Tea is the second most commonly drank liquid on earth after water.” (1) There are several types of tea including black, white, oolong, and green. Each type is defined by the fermentation process, the age of the tea leaves, and the type of tea plant the leaves are harvested from. Green tea itself, is prepared from unfermented leaves (1). This is an important aspect of green tea because the fermentation process has been known to oxidize the polyphenols and catechins initially present in the plant thereby removing many of the beneficial aspects that could be acquired by drinking it. More specifically, catechins are converted into theaflavins and thearabugins in black tea and polyphenols are oxidized and converted to more complex polyphenols (2). This lack of fermentation makes green tea an undiminished, unadulterated and potent source of the catechins naturally present in the tea plant. Throughout the ages, green tea has been believed to be a very resilient homeopathic cure and preventive measure to a variety of illnesses including oral health, liver disease, arthritis, skin disorder, indigestion and quite interestingly, variable forms of cancer. Due to the wide belief regarding the copious benefits which can be ascertained from drinking green tea, it was a logical step to analyze the tea and find the active ingredients associated with this beverage which many prove to be an effective agent in the prevention of cancer.
During a study conducted which analyzed of the aqueous alcoholic green tea extract, six compounds were found to be present. These are, (+)-gallocatechin (GC) Figure 1, (-)-epicatechin (EC) Figure 2, (-)-epigallocatechin (EGC) Figure 3, (-)-epicatechin gallate (ECG) Figure 4, (-)-epigallocatechin gallate (EGCG) Figure 5 and caffeine Figure 6 (3). Each of these catechins was tested against various forms of cancer cell lines which represent breast (MCF-7), colon (HT-29), lung (A-427) and melanoma to see how each inhibited cellular growth. Through this study, it was found that the IC50 of EGCG was the lowest for three out of four of the cancer cell lines, including the interest of this paper, HT-29 (3). This study is supports the currently accepted notion that EGCG mediates much of the cancer preventive qualities of green tea (2). As further evidence, a study was conducted comparing the potency of EGCG against EC when concerned with the HT116 (colon) cancer cell line. It was found that at low confluency levels, EC and EGCG were equally potent but as the confluency was increased, EC floundered against EGCG (4). These findings, coupled with the dietary aspect and wide consumption associated with green tea, prompted many scientific investigators to look at the anticarcionogenic benefit of EGCG with respect to variable forms of cancer, forms which include the focus of this paper; colon cancer.
To better understand and appreciate the benefits of tea as an antioxidant, the pathological symptoms which a cell must express to be defined as cancerous will be divided into six categories. These six are, “self-sufficiency in growth signals, insensitivity to growth inhibitory signals, ability to evade apoptosis, limitless replicative potential, ability to sustain angiogenesis, and the ability to invade tissues and metastasize (5).” These six symptoms represent the mutated characteristics cells gain during tumorgenesis. Each of these categories represent potent and important targets for anti-carcinogenic drugs. Being so, these categories will be used to gauge the potency of EGCG as an anti-cancer agent.
Self sufficiency in growth signals
Healthy cells which exist in a normal state need to be prompted in order to shift into their proliferative state (6). These prompts are essentially ligands which bind to the receptors present on the surface of the cell. “Many oncogenes in the cancer catalog act by mimicking normal growth signaling in one way or another. (6)” In contrast with healthy cells, malignant cells create their own prompts. This activity essentially removes them from the normal cellular growth regulation and is called acquired GS autonomy. Cells achieve this autonomy through three known ways. These are, the alteration of growth signals, the mutation of intracellular circuits needed to translate the outside signals, and the change of transcellular transducers which are involved in cellular growth (6).
A study conducted by Shimizu et al (7), analyzed that malignant cells cannot be influenced by growth factors if their corresponding receptors were not present in the cell to begin with. Thus it seemed logical to test the potency of EGCG against two known growth factor receptors whose activation has been known to cause cancer. Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor-2 (HER2) both of which belong to the receptor tyrosine kinase super family. “Ligand binding results in homo and heterodimerization leading to phosphorylation of tyrosine residues, activation of downstream signaling pathways, and expression of genes that enhance cell proliferation" (7).Furthermore, abnormalities associated with these complexes, are pivotal in the development of colorectal cancer. Supporting this notion, it is known that EGFR and HER2 proteins are over expressed in colon cancer cell lines. Quantitatively, it was found that there were 8.4 times the amount of EGFR and HER2 present in malignant colorectal cells when compared with FHC (normal) cells. Although the ligand associated with HER2 has yet to be determined, it was found that a prominent ligand associated with EGFR is TGF-α. This was proven experimentally because the introduction of TGF-α caused a correlated cell growth associated with EGFR. An introduction of 50ng/mL TGF-α gave a 3x cell growth in colorectal cells while no stimulation was seen with FHC cell lines. Upon the introduction of EGCG to the HT-29 cell line, it was found that “(-)-Epigallocatechin gallate and polyphenon E cause a decrease in the phosphorylated forms of epidermal growth factor receptor, HER2, extracellular signal regulated kinase, and Akt proteins (7).” This showed even with the introduction of TGF-α showing that EGCG targeted the signaling pathways associated with each growth factor receptor. It is important to note that this inhibition was observed just 6h after the introduction of EGCG. Once treated with EGCG, the cells were arrested in their G1 cycle proving that the phosphorylation of the growth factor receptors halted cellular growth. This is supported by the observation that the amount of cancer cells treated with EGCG that were arrested in the G1 phase upon treatment increased by 26.4% while there was a decrease in S and G2-M phases. Then, EGCG promoted the induction of caspase-3 and -9 which inducted of apoptosis in the HT29 cells thereby destroying the malignant cells (7). The study proved that one of the mechanisms in which EGCG acts as a chemopreventive agent is by phosphorylating HER2 and EGFR thereby biologically inactivating them.
Studies have shown that an increase in IGF-1 and decrease in IGFBP-3 are associated with the development of colorectal cancer .HT-29 has a higher than normal concentration of IGF-1R (membrane RTK) concentration (8). Although IGF-1R is needed for normal cell development, the over expression of this receptor makes the cell overly sensitive to growth factors prompting it to divide when normal cells would not. In another experiment conducted by Shimizu et al the effect of EGCG on this receptor was analyzed. It was found that EGCG did not affect the receptor itself but it increased the concentration of IGFBP-3 which in turn negatively controls the biological activity of IGF-1 and IGF-2, the ligands associated with IGF-1R. Since the regulatory step of this signaling pathway is the binding of IGF-1 to IGF-1R, this is a good target for anticancer drugs. The experiment conducted by Shimizu et al, showed a marked decrease of the p-IGF-1R associated protein within 6 hours of dosing. The concentration of IGF-1 went down after 12 h and drastically decreased after 24h. Conversely, an increase of IGFBP-3 was observed after 6h a trend which was maintained until 48h after dosing. There were no changes in the levels of the other IGF-1Rα and IGF-1Rβ showing the specific targeting of IGF-1R.
Through further analysis, the increased IGFBP-3 is due to increased TGF-β2 which was found by another study to increase due to EGCG. The increased amount of IGFBP-3 was sustained due to the fact that EGCG inhibited MMPs-7 and MMPs-9 (metalloproteases which degrade IGFBP-3). This is good because IGFBP-3 is known to induce apoptosis and inhibit cell proliferation aside from inhibiting the IGF-1R and IGF-1 pathway. Furthermore, there were three mechanisms which were proposed to have led to the inhibition of IGF-1R by EGCG. The first of these was that, EGCG might bind directly to all of these receptors and inhibit their tyrosine kinase activities. The second was that, EGCG may target lipid domains associated with RTKs in the plasma membrane. Lastly, EGCG generates a highly reactive species which attack RTKs at the cell surface thereby inhibiting their function (8).
Adachi et al conducted an experiment to analyze whether or not EGCG disrupts lipid rafts (9). Since it is known that receptor tyrosine kinases including EGFR are present on lipid rafts, the inhibitory effect of EGCG on activation of the EGFR (targeting the membrane lipid order) in HT-29 was analyzed (9). This was done to deduce if the inhibition of was due to the disruption of the lipid rafts or by something else. Their findings suggest that EGCG does alter the organization of the plasma membrane in cancer cells. This is evidenced by the fact that 5μg/mL EGCG caused, within 30 minutes, a reduction in ordered lipid domains which then inactivated EGFR. This inactivation also inhibited the binding of EGF to the surface of these cells which in turn inhibited dimerization and autophosphorylation of the EGFR. This meant that the cell would no longer proliferate due to the outside stimulus EGF. Furthermore, an expreiment using a cholesterol free lipid bilayer was conducted. This is because cholesterol is an integral part of a cell’s lipid bi-layer and there is a possibility that it was cholesterol, not the lipid rafts EGCG targets. Thus Adachi et al experimented on cells without detergent resistant cholesterol and the lack of this compound was found to have no effect on the potency of EGCG. More specifically it was observed that gramicidin channels in a cholesterol free system were disrupted by EGCG. With cholesterol out of the equation, the decrease in EGFR was left to be hypothesized to be caused by the fact that EGCG disrupts the lipid rafts which subsequently inhibits EGFR.
Evades apoptosis
The apoptosis of a cell is a regular and integral occurrence in healthy cell cycle growth. Apoptosis is an ordered process where unwanted and damaged cells are disposed of in the body. “Deregulation of the cell cycle and apoptosis are frequent occurrences in cancer development (10).” This deregulation and evasion of apoptosis are caused by chemical carcinogens which mutate the genes responsible for normal cellular growth and death. Once these genes are mutated, cells with damaged genes are able to survive and prosper. Thus, “uncontrolled cellular growth resulting in cancer development is directly related to repression of apoptosis" (10). Thus, the ability of a drug to induce apoptosis in malignant and defective cells is an important trait.
In a study conducted by Hwang et al EGCG was found to inhibit COX-2, a carcinogenic compound which when inhibited has been shown to sensitize cells to apoptosis (11). Furthermore, this inhibition of COX-2 was found to be caused by the activation of AMPK. This connection was proven by inhibiting the AMPK pathway and showed that this inhibition rendered EGCG impotent in decreasing the amount of COX-2 present. AMPK is an important biological kinase as it down-regulates several anabolic enzymes thereby shutting down the ATP-consuming pathways (11). The activation of AMPK was proposed to be caused by the phosphorylation of AMPK by EGCG. This activation was important not only in the regulation of COX-2 but also because AMPK has been identified as a pivotal point in the cell cycle where it teeters between apoptosis and growth (11). It was further concluded that the AMPK activity was increased due to the ROS (upstream metabolite) produced by EGCG. This conclusion was evidenced by an experiment that was conducted by adding scavenger NAC which abolished AMPK activation and rendering EGCG impotent in causing cellular apoptosis through this pathway. These findings conclude that AMPK is an important path in which EGCG induces apoptosis through COX-2 regulation.
Another study done by Chen et Al showed that nuclear fragmentation and condensation, evidence of cellular apoptosis, increased depending on EGCG dosing (12). During their experiment, it was shown that the cellular concentration of Caspase-3 and -9, compounds important in regulating cellular apoptosis, increased after the addition of EGCG which followed the release of cytochrome c leading to the hypothesis that apoptosis was due to oxidative stress. This implies the involvement of the mitochondria and the EGCG induced cellular apoptosis. Furthermore, this oxidative stress was found to cause the activation of the JNK pathway which signaled the release of cytochrome c. Chemically the oxidative stress placed on the cells may have been due to the hydroxyl groups present in EGCG. These groups are important because this allows EGCG to be converted to a phenoxyl radical which would put oxidative stress on the cell which results in cellular apoptosis.
Furthermore, a paper produced by Peng et al observed that EGCG, “inhibits constitutive COX-2 expression observed in cancer cells (13).” Their study proposed another mechanism, when compared with the study done by Hwang et al because they concluded that EGCG inhibits ERK and Akt pathways which essentially decreased the amount of NF-κB activated in the cancer cell line. This decrease then inhibited COX-2 mRNA transcription in the cell (13). Peng et al then went on to say that the inhibition of the Akt pathway happened because EGCG has inhibited the biological activity of the MAPK. Furthermore, it was stated that the ERK pathway was inhibited because EGCG blocks MEK-1 which then does not let it bind with Raf-1, a modulating step needed for the production of ERK. A concern of the paper, which was abated by the specificity of EGCG, was that EGCG would block COX-2 in all cell types there by nullifying the biological activity of murine macrophages (13). The findings of the experiment showed that COX-2 was induced in murine cells yet it blocks COX-2 expression in cancer cells. This means that EGCG was found to promote defensive repairs while killing cancer cells.
An interesting and somewhat controversial study performed by Jeong et al stated that EGCG induced AP-1 concentrations dose dependently in their cancer cell lines which forced the cancer cell to undergo apoptosis (14). This is controversial because AP-1 has been linked to important cellular enzymatic functions which delegate apoptosis, proliferation, transformation, and differentiation. Furthermore, the regulation of AP-1 was found to be stimulated by the increase in activity of MAPK, which as stated by Peng et al was actually inhibited by EGCG. However, it is important to note that it was found that the chemopreventive nature of EGCG through the activation of AP-1 was dependent on the concentration of AP-1. At too high levels, AP-1 in fact induces cancer while at certain concentrations it prevents it by initiating certain anti-cancerous enzymatic pathways. This study shows how the efficacy of EGCG is indeed dose and concentration dependent and proposes a alternate mechanism in which EGCG may be used as an anti-cancer agent.
Insensitivity to antigrowth signals
Akin to the prompts experienced by cells which induce them to proliferate, there exist ligands which induce cells to stay dormant, “these signals induce both soluble growth inhibitors and immobilized inhibitors embedded in the extracellular matrix and on the surface of nearby cells” (6). Once sent, these signals need to be accepted by receptors which then activate a series of enzymatic events preventing the cell from proliferating. In the event of cancer, cells have mutated to become immune to these signals which would otherwise prevent them from proliferation.
In an experiment conducted by Hobson et al the effect of EGCG on heat shock protein 70 (HSP70) was targeted. This protein garnered interest because it was observed that HSP70 is often over expressed in colorectal cancer cells while in normal cells, this protein is not present in high concentration (15). Aside from its high concentration in malignant cells, HSP 70 also allows cancer cells to survive lethal environments by assisting the folding of non-native proteins which allows cancer cells to proliferate even when its environment may be signaling it not to. Chemically, HSP70 activity is dependent on the “functioning of its COOH-(C-) terminal substrate binding domain (SBD) and NH2 (N-) ATPase domain" (15). Through their experiment, it was found that EGCG competes with ATP to bind with HSP70 in its ATPase region. This binding to the ATPase region of HSP 70 by EGCG can activate ASK1. This activation was then observed to activate JNK/p38 which then allows apoptotic signaling to happen. Furthermore, the binding of HSP70 by EGCG will allow p53 or Bax to respond to pro-apoptotic signaling. Once these signals have collected in the mitochondria, the ATPase domain which usually “suppresses mitochondrial permeabilization, cytochrome c release and apoptosome formation (15)” cannot happen. Thus EGCG removed the cancer cells ability to survive when normal cells would undergo apoptosis.
Replicative potential
“Many and perhaps all types of mammalian cells carry an intrinsic, cell-autonomous program that limits their multiplication" (6). This, program is inherent in each cell and is independent of the malfunctions which may occur when concerned with outside signaling. This self regulation has been deemed to put cells into senescence after a few divisions. This self regulation is sometimes overcome and can produce three possible outcomes. These three are characterized by “massive cell death, karyotypic disarray associated with end-to-end fusion of chromosomes, and the occasional emergence of a variant (1 in 107) cell that has acquired the ability to multiply without limit” (6). The last outcome effectively produces cancer cells. Their senescence is never reach thus allowing them to multiply when other cells would have stopped. This state in which cancer cells evade senescence state has been deemed immortalization (6).
In an experiment conducted by Naasani et al it was found that, “EGCG is the strongest telomerase inhibitor among the different catechins tested (16)” which was potent even at 1μM. Consequently, in cells treated with EGCG, telomerase product signals were decreased. Furthermore, it was found that EGCG directly inhibits telomerase which in turn rids the cancer cell of its proliferative capability pushing it towards senescence. This was shown by less cell growth, and morphological augmentations in each treated cell. Furthermore, Naasani et al exemplified the specificity of EGCG. This specificity was construed by the discovery that EGCG only shortened the cellular telomeres by 1kb. This was found to be enough to cause senescence in cancer cells but not in normal cells. Also, aneuploidy was shown and all these link to telomerase shortening. Thus it was proposed that it is through telomerase inhibition EGCG induces its anti-cancer effects.
DNMT, on the other hand, is important in the hypermethylation of newly synthesized DNA as it supports the hypermethylation of CoG islands. Blocking DNMT would result in the reversal of hypermethylation which would unsilence the genes that are needed for cell cycle regulation, receptors, DNA repair and apoptosis (17). In a study done by Fang et al, it was proven that EGCG does in fact suppress DNMT activity (17). This also causes CpG demethylation and the reactivation of silenced genes. It was found that EGCG suppresses DNMT activity through competitive inhibition with a Ki of 6.89μM (17). This competitive inhibition was proven by finding that EGCG effectively bound itself within the hydrophilic region of DNMT1. EGCG maintained its grasp by 4 hydrogen bonds with a possible presence of one more. This bonding scheme is further proven by using 5 different analogues of EGCG and each maintained its grasp on DNMT. This proposed mechanism of EGCG inhibition of DNMT is supports the findings that ECG (EGCG with a lacking 4’’ hydroxyl group in MeEGCG) is not as potent as EGCG as it lacked a hydrogen bond. Once DNMT was inhibited, all the genes tested in this study were unsilenced after 48h of treatment. This means that the previously silenced apoptotic genes became functional. However, it is important to note that although DNMT inhibition proved to have good results, over inhibition would cause hypomethylation thus moderation in dosing must be followed.
Sustain angiogenesis
In order to survive, cells need to be close to a capillary. If they are not, these cells will die off due to the lack of nutrients and oxygen supplied to them by the blood. Thus, cells need to possess an angiogenic ability which allows them to induce blood vessel growth in their surroundings. This is an essential ability which is not present in most normal functioning cells and it is needed in order to sustain cellular growth. Thus, malignant tumors need to acquire this attribute. Critical players in the development of this ability include cell surface receptors and their ligands, an example of this is vascular endothelial growth factor (VEGF). Other factors like “integrins and adhesion molecules mediating cell-matrix and cell-cell association also play critical roles. (6)”
As aforementioned, vascular endothelial growth factor (VEGF) is a main factor in the maintenance of the angiogenesis of cancer cells. It was found that the activation of Erk-1 and Erk-2 up-regulate VEGF mRNA. This was discovered through the alteration of AP-2 and Sp1 transcription factors. In a study performed by Jung et al, it was found that Erk-1 and Erk-2 are both activated in HT-29 serum starved cells (18). Consequently it was determined that 30μM of EGCG markedly inhibits Erk-1 and Erk-2 activation which in turn decreased the expression of VEGF mRNA (18). It was then hypothesized that this inhibition was caused by the fact that EGCG is a strong metal ion chelator and that EGCG could be chelating the divalent cations. Upon the introduction of EGCG, xenographed human tumors in mice decreased its growth, vascularity and proliferation. Since EGCG effectively inhibits VEGF it decreases the possibility that the cancerous cells would form new blood vessels which would then disallow its proliferation.
Basic fibrolast growth factor (bFGF) is another one of the most potent angiogenic compounds. It is synthesized in the malignant cell and secreted into the tissue around it to induce the production of capilliaries. It is important to note that, bFGF was found to be expressed in high concentrations in LoVo (a colon carcinoma cell line) cells when compared to healthy cell lines. In an experiment performend by Sukhthankar et al EGCG was shown to suppress the biological activity of bFGF (19). Because the malignant cells could no longer induce vascular growth tumor growth and metastasis was compromised. Mechanistically EGCG suppression of bFGF was found to occur specifically through posttranslational modification (19). This was concluded because it was found that EGCG does not affect the stability of bFGF mRNA level or stability in LoVo cells which means that the inhibition could have happened only after translation. Furthermore, it was found that EGCG increased the ubiquitin presence and activity in a tumor cell (19). This is good because ubiquitin, one of the most scarse eukaryoutic proteins, conjugates itself with other proteins through an enzymatic pathway (19). This pathway is called the ubiquitin-proteasome pathway and it is being recognized as an important regulatory system in normal cellular function. This heightened activity of this pathway is pertinent because when bFGF is ubiquitinated it, “triggers a trypsin-like activity of the 20S proteosome" (19). This then consequently degrades bFGF. This degradation ultimately realizes depressed angiogenesis.
Invade tissues and metastisize
The ability for cancerous cells to invade other tissues and cause them to become malignant is not essential for the survival of the cancer cell itself but it is quintessential in the development of a deadly cancer. So much so, that “metastases- are the cause of 90% of human cancer deaths" (6). Once the cancer cell acquires the ability to metastasize, it is no longer bound by the nutritional supply and space constriction which may have limited it before. This then spreads the mutated gene developed by the successful yet somewhat isolated cancer cell to the rest of the afflicted body. The prevention of metastases is a important area in which anticarcinogenic drugs should be active because then the cancerous cell can be isolated and dealt with.
The formation of spheroids is considered to predict malignancy as it allows the cell to grow independently. Furthermore, this suggests a mutation in cellular proliferation and adhesion. Spheroids are not controlled by the same restrictions as normal body cells in terms of cellular adhesion and contact inhibition which allows these cells to invade and metastisize. It was discovered through experimental procedure by McLoughlin et al that EGCG decreased the spheroid formation and the size of each spheroid in a dose dependent fashion (5). More specifically it was determined that the genes that are most responsive to EGCG include galectin-2 and annexin A13. Both of which are known to be involved in cellular adhesion were subsequently down regulated. This down regulation leads EGCG to have an increase in sensitivity to contact inhibition which ultimately means that the cancerous cells cannot grow or invade other tissues due to crowding, a limitation which was not set before.
Conclusion
EGCG prevents cancer in all six categories used to determine cellular malignancy, based on the article Hallmarks of Cancer by Hanahan and Weinberg. The mechanisms in which EGCG is proven to work range from lipid disruption to competitive inhibition each a novel mechanism which serves to battle several distinct mutations in a cancerous cell. If nothing else, these studies and findings show that EGCG is not just a potent drug but it is also resilient and multifaceted one. Moreover, in some cases, EGCG has shown differentiation in activity between cancer cells and normal functioning cells, an important aspect when selecting a chemopreventive drug. Furthermore, although this paper focused on EGCG in green tea it is important to note that polyphenols from red wine have been proven to induce cellular apoptosis selectively in teratocarcinoma cells (20). This proves that EGCG and other polyphenols from other sources prove to be viable chemotherapeutic drugs. It would be of great interest if further studies were performed to gauge the dosing requirements of this drug and whether or not the actual drinking of green tea possesses a legitimate health benefit.
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Submitted on: 12/05/2009
Word Count: 4507
The Chemopreventive Ability of EGCG, a Catechin In Green Tea, on Selected Colorectal Carcinoma Cells - A Review
Abstract
Green tea, through tradition and culture, is believed to have various health benefits including being a chemotherapeutic and chemopreventive drug. Relatively recently, various studies have concluded that (-)-Epigallocatechingallate (EGCG), a polyphenol, is the active ingredient in behind the anti-cancerous benefits green tea possess. This paper serves as a review of the various mechanisms, discovered by numerous scientists, in which EGCG works against colorectal cancer. This was done by using the six categories that define a cancer cell from the paper ‘The Hallmarks of Cancer’ by Hanahan and Weinberg. These categories essentially deduce the six general mutations a cell, more specifically a colorectal cell, must undergo to become cancerous. It was concluded that EGCG is effective against all of the six mutations colorectal cancer cells may obtain during tumorgenesis making it a potent and viable anti-cancer drug.
Introduction
“Tea is the second most commonly drank liquid on earth after water.” (1) There are several types of tea including black, white, oolong, and green. Each type is defined by the fermentation process, the age of the tea leaves, and the type of tea plant the leaves are harvested from. Green tea itself, is prepared from unfermented leaves (1). This is an important aspect of green tea because the fermentation process has been known to oxidize the polyphenols and catechins initially present in the plant thereby removing many of the beneficial aspects that could be acquired by drinking it. More specifically, catechins are converted into theaflavins and thearabugins in black tea and polyphenols are oxidized and converted to more complex polyphenols (2). This lack of fermentation makes green tea an undiminished, unadulterated and potent source of the catechins naturally present in the tea plant. Throughout the ages, green tea has been believed to be a very resilient homeopathic cure and preventive measure to a variety of illnesses including oral health, liver disease, arthritis, skin disorder, indigestion and quite interestingly, variable forms of cancer. Due to the wide belief regarding the copious benefits which can be ascertained from drinking green tea, it was a logical step to analyze the tea and find the active ingredients associated with this beverage which many prove to be an effective agent in the prevention of cancer.
During a study conducted which analyzed of the aqueous alcoholic green tea extract, six compounds were found to be present. These are, (+)-gallocatechin (GC) Figure 1, (-)-epicatechin (EC) Figure 2, (-)-epigallocatechin (EGC) Figure 3, (-)-epicatechin gallate (ECG) Figure 4, (-)-epigallocatechin gallate (EGCG) Figure 5 and caffeine Figure 6 (3). Each of these catechins was tested against various forms of cancer cell lines which represent breast (MCF-7), colon (HT-29), lung (A-427) and melanoma to see how each inhibited cellular growth. Through this study, it was found that the IC50 of EGCG was the lowest for three out of four of the cancer cell lines, including the interest of this paper, HT-29 (3). This study is supports the currently accepted notion that EGCG mediates much of the cancer preventive qualities of green tea (2). As further evidence, a study was conducted comparing the potency of EGCG against EC when concerned with the HT116 (colon) cancer cell line. It was found that at low confluency levels, EC and EGCG were equally potent but as the confluency was increased, EC floundered against EGCG (4). These findings, coupled with the dietary aspect and wide consumption associated with green tea, prompted many scientific investigators to look at the anticarcionogenic benefit of EGCG with respect to variable forms of cancer, forms which include the focus of this paper; colon cancer.
To better understand and appreciate the benefits of tea as an antioxidant, the pathological symptoms which a cell must express to be defined as cancerous will be divided into six categories. These six are, “self-sufficiency in growth signals, insensitivity to growth inhibitory signals, ability to evade apoptosis, limitless replicative potential, ability to sustain angiogenesis, and the ability to invade tissues and metastasize (5).” These six symptoms represent the mutated characteristics cells gain during tumorgenesis. Each of these categories represent potent and important targets for anti-carcinogenic drugs. Being so, these categories will be used to gauge the potency of EGCG as an anti-cancer agent.
Self sufficiency in growth signals
Healthy cells which exist in a normal state need to be prompted in order to shift into their proliferative state (6). These prompts are essentially ligands which bind to the receptors present on the surface of the cell. “Many oncogenes in the cancer catalog act by mimicking normal growth signaling in one way or another. (6)” In contrast with healthy cells, malignant cells create their own prompts. This activity essentially removes them from the normal cellular growth regulation and is called acquired GS autonomy. Cells achieve this autonomy through three known ways. These are, the alteration of growth signals, the mutation of intracellular circuits needed to translate the outside signals, and the change of transcellular transducers which are involved in cellular growth (6).
A study conducted by Shimizu et al (7), analyzed that malignant cells cannot be influenced by growth factors if their corresponding receptors were not present in the cell to begin with. Thus it seemed logical to test the potency of EGCG against two known growth factor receptors whose activation has been known to cause cancer. Epidermal Growth Factor Receptor (EGFR) and Human Epidermal Growth Factor Receptor-2 (HER2) both of which belong to the receptor tyrosine kinase super family. “Ligand binding results in homo and heterodimerization leading to phosphorylation of tyrosine residues, activation of downstream signaling pathways, and expression of genes that enhance cell proliferation" (7).Furthermore, abnormalities associated with these complexes, are pivotal in the development of colorectal cancer. Supporting this notion, it is known that EGFR and HER2 proteins are over expressed in colon cancer cell lines. Quantitatively, it was found that there were 8.4 times the amount of EGFR and HER2 present in malignant colorectal cells when compared with FHC (normal) cells. Although the ligand associated with HER2 has yet to be determined, it was found that a prominent ligand associated with EGFR is TGF-α. This was proven experimentally because the introduction of TGF-α caused a correlated cell growth associated with EGFR. An introduction of 50ng/mL TGF-α gave a 3x cell growth in colorectal cells while no stimulation was seen with FHC cell lines. Upon the introduction of EGCG to the HT-29 cell line, it was found that “(-)-Epigallocatechin gallate and polyphenon E cause a decrease in the phosphorylated forms of epidermal growth factor receptor, HER2, extracellular signal regulated kinase, and Akt proteins (7).” This showed even with the introduction of TGF-α showing that EGCG targeted the signaling pathways associated with each growth factor receptor. It is important to note that this inhibition was observed just 6h after the introduction of EGCG. Once treated with EGCG, the cells were arrested in their G1 cycle proving that the phosphorylation of the growth factor receptors halted cellular growth. This is supported by the observation that the amount of cancer cells treated with EGCG that were arrested in the G1 phase upon treatment increased by 26.4% while there was a decrease in S and G2-M phases. Then, EGCG promoted the induction of caspase-3 and -9 which inducted of apoptosis in the HT29 cells thereby destroying the malignant cells (7). The study proved that one of the mechanisms in which EGCG acts as a chemopreventive agent is by phosphorylating HER2 and EGFR thereby biologically inactivating them.
Studies have shown that an increase in IGF-1 and decrease in IGFBP-3 are associated with the development of colorectal cancer .HT-29 has a higher than normal concentration of IGF-1R (membrane RTK) concentration (8). Although IGF-1R is needed for normal cell development, the over expression of this receptor makes the cell overly sensitive to growth factors prompting it to divide when normal cells would not. In another experiment conducted by Shimizu et al the effect of EGCG on this receptor was analyzed. It was found that EGCG did not affect the receptor itself but it increased the concentration of IGFBP-3 which in turn negatively controls the biological activity of IGF-1 and IGF-2, the ligands associated with IGF-1R. Since the regulatory step of this signaling pathway is the binding of IGF-1 to IGF-1R, this is a good target for anticancer drugs. The experiment conducted by Shimizu et al, showed a marked decrease of the p-IGF-1R associated protein within 6 hours of dosing. The concentration of IGF-1 went down after 12 h and drastically decreased after 24h. Conversely, an increase of IGFBP-3 was observed after 6h a trend which was maintained until 48h after dosing. There were no changes in the levels of the other IGF-1Rα and IGF-1Rβ showing the specific targeting of IGF-1R.
Through further analysis, the increased IGFBP-3 is due to increased TGF-β2 which was found by another study to increase due to EGCG. The increased amount of IGFBP-3 was sustained due to the fact that EGCG inhibited MMPs-7 and MMPs-9 (metalloproteases which degrade IGFBP-3). This is good because IGFBP-3 is known to induce apoptosis and inhibit cell proliferation aside from inhibiting the IGF-1R and IGF-1 pathway. Furthermore, there were three mechanisms which were proposed to have led to the inhibition of IGF-1R by EGCG. The first of these was that, EGCG might bind directly to all of these receptors and inhibit their tyrosine kinase activities. The second was that, EGCG may target lipid domains associated with RTKs in the plasma membrane. Lastly, EGCG generates a highly reactive species which attack RTKs at the cell surface thereby inhibiting their function (8).
Adachi et al conducted an experiment to analyze whether or not EGCG disrupts lipid rafts (9). Since it is known that receptor tyrosine kinases including EGFR are present on lipid rafts, the inhibitory effect of EGCG on activation of the EGFR (targeting the membrane lipid order) in HT-29 was analyzed (9). This was done to deduce if the inhibition of was due to the disruption of the lipid rafts or by something else. Their findings suggest that EGCG does alter the organization of the plasma membrane in cancer cells. This is evidenced by the fact that 5μg/mL EGCG caused, within 30 minutes, a reduction in ordered lipid domains which then inactivated EGFR. This inactivation also inhibited the binding of EGF to the surface of these cells which in turn inhibited dimerization and autophosphorylation of the EGFR. This meant that the cell would no longer proliferate due to the outside stimulus EGF. Furthermore, an expreiment using a cholesterol free lipid bilayer was conducted. This is because cholesterol is an integral part of a cell’s lipid bi-layer and there is a possibility that it was cholesterol, not the lipid rafts EGCG targets. Thus Adachi et al experimented on cells without detergent resistant cholesterol and the lack of this compound was found to have no effect on the potency of EGCG. More specifically it was observed that gramicidin channels in a cholesterol free system were disrupted by EGCG. With cholesterol out of the equation, the decrease in EGFR was left to be hypothesized to be caused by the fact that EGCG disrupts the lipid rafts which subsequently inhibits EGFR.
Evades apoptosis
The apoptosis of a cell is a regular and integral occurrence in healthy cell cycle growth. Apoptosis is an ordered process where unwanted and damaged cells are disposed of in the body. “Deregulation of the cell cycle and apoptosis are frequent occurrences in cancer development (10).” This deregulation and evasion of apoptosis are caused by chemical carcinogens which mutate the genes responsible for normal cellular growth and death. Once these genes are mutated, cells with damaged genes are able to survive and prosper. Thus, “uncontrolled cellular growth resulting in cancer development is directly related to repression of apoptosis" (10). Thus, the ability of a drug to induce apoptosis in malignant and defective cells is an important trait.
In a study conducted by Hwang et al EGCG was found to inhibit COX-2, a carcinogenic compound which when inhibited has been shown to sensitize cells to apoptosis (11). Furthermore, this inhibition of COX-2 was found to be caused by the activation of AMPK. This connection was proven by inhibiting the AMPK pathway and showed that this inhibition rendered EGCG impotent in decreasing the amount of COX-2 present. AMPK is an important biological kinase as it down-regulates several anabolic enzymes thereby shutting down the ATP-consuming pathways (11). The activation of AMPK was proposed to be caused by the phosphorylation of AMPK by EGCG. This activation was important not only in the regulation of COX-2 but also because AMPK has been identified as a pivotal point in the cell cycle where it teeters between apoptosis and growth (11). It was further concluded that the AMPK activity was increased due to the ROS (upstream metabolite) produced by EGCG. This conclusion was evidenced by an experiment that was conducted by adding scavenger NAC which abolished AMPK activation and rendering EGCG impotent in causing cellular apoptosis through this pathway. These findings conclude that AMPK is an important path in which EGCG induces apoptosis through COX-2 regulation.
Another study done by Chen et Al showed that nuclear fragmentation and condensation, evidence of cellular apoptosis, increased depending on EGCG dosing (12). During their experiment, it was shown that the cellular concentration of Caspase-3 and -9, compounds important in regulating cellular apoptosis, increased after the addition of EGCG which followed the release of cytochrome c leading to the hypothesis that apoptosis was due to oxidative stress. This implies the involvement of the mitochondria and the EGCG induced cellular apoptosis. Furthermore, this oxidative stress was found to cause the activation of the JNK pathway which signaled the release of cytochrome c. Chemically the oxidative stress placed on the cells may have been due to the hydroxyl groups present in EGCG. These groups are important because this allows EGCG to be converted to a phenoxyl radical which would put oxidative stress on the cell which results in cellular apoptosis.
Furthermore, a paper produced by Peng et al observed that EGCG, “inhibits constitutive COX-2 expression observed in cancer cells (13).” Their study proposed another mechanism, when compared with the study done by Hwang et al because they concluded that EGCG inhibits ERK and Akt pathways which essentially decreased the amount of NF-κB activated in the cancer cell line. This decrease then inhibited COX-2 mRNA transcription in the cell (13). Peng et al then went on to say that the inhibition of the Akt pathway happened because EGCG has inhibited the biological activity of the MAPK. Furthermore, it was stated that the ERK pathway was inhibited because EGCG blocks MEK-1 which then does not let it bind with Raf-1, a modulating step needed for the production of ERK. A concern of the paper, which was abated by the specificity of EGCG, was that EGCG would block COX-2 in all cell types there by nullifying the biological activity of murine macrophages (13). The findings of the experiment showed that COX-2 was induced in murine cells yet it blocks COX-2 expression in cancer cells. This means that EGCG was found to promote defensive repairs while killing cancer cells.
An interesting and somewhat controversial study performed by Jeong et al stated that EGCG induced AP-1 concentrations dose dependently in their cancer cell lines which forced the cancer cell to undergo apoptosis (14). This is controversial because AP-1 has been linked to important cellular enzymatic functions which delegate apoptosis, proliferation, transformation, and differentiation. Furthermore, the regulation of AP-1 was found to be stimulated by the increase in activity of MAPK, which as stated by Peng et al was actually inhibited by EGCG. However, it is important to note that it was found that the chemopreventive nature of EGCG through the activation of AP-1 was dependent on the concentration of AP-1. At too high levels, AP-1 in fact induces cancer while at certain concentrations it prevents it by initiating certain anti-cancerous enzymatic pathways. This study shows how the efficacy of EGCG is indeed dose and concentration dependent and proposes a alternate mechanism in which EGCG may be used as an anti-cancer agent.
Insensitivity to antigrowth signals
Akin to the prompts experienced by cells which induce them to proliferate, there exist ligands which induce cells to stay dormant, “these signals induce both soluble growth inhibitors and immobilized inhibitors embedded in the extracellular matrix and on the surface of nearby cells” (6). Once sent, these signals need to be accepted by receptors which then activate a series of enzymatic events preventing the cell from proliferating. In the event of cancer, cells have mutated to become immune to these signals which would otherwise prevent them from proliferation.
In an experiment conducted by Hobson et al the effect of EGCG on heat shock protein 70 (HSP70) was targeted. This protein garnered interest because it was observed that HSP70 is often over expressed in colorectal cancer cells while in normal cells, this protein is not present in high concentration (15). Aside from its high concentration in malignant cells, HSP 70 also allows cancer cells to survive lethal environments by assisting the folding of non-native proteins which allows cancer cells to proliferate even when its environment may be signaling it not to. Chemically, HSP70 activity is dependent on the “functioning of its COOH-(C-) terminal substrate binding domain (SBD) and NH2 (N-) ATPase domain" (15). Through their experiment, it was found that EGCG competes with ATP to bind with HSP70 in its ATPase region. This binding to the ATPase region of HSP 70 by EGCG can activate ASK1. This activation was then observed to activate JNK/p38 which then allows apoptotic signaling to happen. Furthermore, the binding of HSP70 by EGCG will allow p53 or Bax to respond to pro-apoptotic signaling. Once these signals have collected in the mitochondria, the ATPase domain which usually “suppresses mitochondrial permeabilization, cytochrome c release and apoptosome formation (15)” cannot happen. Thus EGCG removed the cancer cells ability to survive when normal cells would undergo apoptosis.
Replicative potential
“Many and perhaps all types of mammalian cells carry an intrinsic, cell-autonomous program that limits their multiplication" (6). This, program is inherent in each cell and is independent of the malfunctions which may occur when concerned with outside signaling. This self regulation has been deemed to put cells into senescence after a few divisions. This self regulation is sometimes overcome and can produce three possible outcomes. These three are characterized by “massive cell death, karyotypic disarray associated with end-to-end fusion of chromosomes, and the occasional emergence of a variant (1 in 107) cell that has acquired the ability to multiply without limit” (6). The last outcome effectively produces cancer cells. Their senescence is never reach thus allowing them to multiply when other cells would have stopped. This state in which cancer cells evade senescence state has been deemed immortalization (6).
In an experiment conducted by Naasani et al it was found that, “EGCG is the strongest telomerase inhibitor among the different catechins tested (16)” which was potent even at 1μM. Consequently, in cells treated with EGCG, telomerase product signals were decreased. Furthermore, it was found that EGCG directly inhibits telomerase which in turn rids the cancer cell of its proliferative capability pushing it towards senescence. This was shown by less cell growth, and morphological augmentations in each treated cell. Furthermore, Naasani et al exemplified the specificity of EGCG. This specificity was construed by the discovery that EGCG only shortened the cellular telomeres by 1kb. This was found to be enough to cause senescence in cancer cells but not in normal cells. Also, aneuploidy was shown and all these link to telomerase shortening. Thus it was proposed that it is through telomerase inhibition EGCG induces its anti-cancer effects.
DNMT, on the other hand, is important in the hypermethylation of newly synthesized DNA as it supports the hypermethylation of CoG islands. Blocking DNMT would result in the reversal of hypermethylation which would unsilence the genes that are needed for cell cycle regulation, receptors, DNA repair and apoptosis (17). In a study done by Fang et al, it was proven that EGCG does in fact suppress DNMT activity (17). This also causes CpG demethylation and the reactivation of silenced genes. It was found that EGCG suppresses DNMT activity through competitive inhibition with a Ki of 6.89μM (17). This competitive inhibition was proven by finding that EGCG effectively bound itself within the hydrophilic region of DNMT1. EGCG maintained its grasp by 4 hydrogen bonds with a possible presence of one more. This bonding scheme is further proven by using 5 different analogues of EGCG and each maintained its grasp on DNMT. This proposed mechanism of EGCG inhibition of DNMT is supports the findings that ECG (EGCG with a lacking 4’’ hydroxyl group in MeEGCG) is not as potent as EGCG as it lacked a hydrogen bond. Once DNMT was inhibited, all the genes tested in this study were unsilenced after 48h of treatment. This means that the previously silenced apoptotic genes became functional. However, it is important to note that although DNMT inhibition proved to have good results, over inhibition would cause hypomethylation thus moderation in dosing must be followed.
Sustain angiogenesis
In order to survive, cells need to be close to a capillary. If they are not, these cells will die off due to the lack of nutrients and oxygen supplied to them by the blood. Thus, cells need to possess an angiogenic ability which allows them to induce blood vessel growth in their surroundings. This is an essential ability which is not present in most normal functioning cells and it is needed in order to sustain cellular growth. Thus, malignant tumors need to acquire this attribute. Critical players in the development of this ability include cell surface receptors and their ligands, an example of this is vascular endothelial growth factor (VEGF). Other factors like “integrins and adhesion molecules mediating cell-matrix and cell-cell association also play critical roles. (6)”
As aforementioned, vascular endothelial growth factor (VEGF) is a main factor in the maintenance of the angiogenesis of cancer cells. It was found that the activation of Erk-1 and Erk-2 up-regulate VEGF mRNA. This was discovered through the alteration of AP-2 and Sp1 transcription factors. In a study performed by Jung et al, it was found that Erk-1 and Erk-2 are both activated in HT-29 serum starved cells (18). Consequently it was determined that 30μM of EGCG markedly inhibits Erk-1 and Erk-2 activation which in turn decreased the expression of VEGF mRNA (18). It was then hypothesized that this inhibition was caused by the fact that EGCG is a strong metal ion chelator and that EGCG could be chelating the divalent cations. Upon the introduction of EGCG, xenographed human tumors in mice decreased its growth, vascularity and proliferation. Since EGCG effectively inhibits VEGF it decreases the possibility that the cancerous cells would form new blood vessels which would then disallow its proliferation.
Basic fibrolast growth factor (bFGF) is another one of the most potent angiogenic compounds. It is synthesized in the malignant cell and secreted into the tissue around it to induce the production of capilliaries. It is important to note that, bFGF was found to be expressed in high concentrations in LoVo (a colon carcinoma cell line) cells when compared to healthy cell lines. In an experiment performend by Sukhthankar et al EGCG was shown to suppress the biological activity of bFGF (19). Because the malignant cells could no longer induce vascular growth tumor growth and metastasis was compromised. Mechanistically EGCG suppression of bFGF was found to occur specifically through posttranslational modification (19). This was concluded because it was found that EGCG does not affect the stability of bFGF mRNA level or stability in LoVo cells which means that the inhibition could have happened only after translation. Furthermore, it was found that EGCG increased the ubiquitin presence and activity in a tumor cell (19). This is good because ubiquitin, one of the most scarse eukaryoutic proteins, conjugates itself with other proteins through an enzymatic pathway (19). This pathway is called the ubiquitin-proteasome pathway and it is being recognized as an important regulatory system in normal cellular function. This heightened activity of this pathway is pertinent because when bFGF is ubiquitinated it, “triggers a trypsin-like activity of the 20S proteosome" (19). This then consequently degrades bFGF. This degradation ultimately realizes depressed angiogenesis.
Invade tissues and metastisize
The ability for cancerous cells to invade other tissues and cause them to become malignant is not essential for the survival of the cancer cell itself but it is quintessential in the development of a deadly cancer. So much so, that “metastases- are the cause of 90% of human cancer deaths" (6). Once the cancer cell acquires the ability to metastasize, it is no longer bound by the nutritional supply and space constriction which may have limited it before. This then spreads the mutated gene developed by the successful yet somewhat isolated cancer cell to the rest of the afflicted body. The prevention of metastases is a important area in which anticarcinogenic drugs should be active because then the cancerous cell can be isolated and dealt with.
The formation of spheroids is considered to predict malignancy as it allows the cell to grow independently. Furthermore, this suggests a mutation in cellular proliferation and adhesion. Spheroids are not controlled by the same restrictions as normal body cells in terms of cellular adhesion and contact inhibition which allows these cells to invade and metastisize. It was discovered through experimental procedure by McLoughlin et al that EGCG decreased the spheroid formation and the size of each spheroid in a dose dependent fashion (5). More specifically it was determined that the genes that are most responsive to EGCG include galectin-2 and annexin A13. Both of which are known to be involved in cellular adhesion were subsequently down regulated. This down regulation leads EGCG to have an increase in sensitivity to contact inhibition which ultimately means that the cancerous cells cannot grow or invade other tissues due to crowding, a limitation which was not set before.
Conclusion
EGCG prevents cancer in all six categories used to determine cellular malignancy, based on the article Hallmarks of Cancer by Hanahan and Weinberg. The mechanisms in which EGCG is proven to work range from lipid disruption to competitive inhibition each a novel mechanism which serves to battle several distinct mutations in a cancerous cell. If nothing else, these studies and findings show that EGCG is not just a potent drug but it is also resilient and multifaceted one. Moreover, in some cases, EGCG has shown differentiation in activity between cancer cells and normal functioning cells, an important aspect when selecting a chemopreventive drug. Furthermore, although this paper focused on EGCG in green tea it is important to note that polyphenols from red wine have been proven to induce cellular apoptosis selectively in teratocarcinoma cells (20). This proves that EGCG and other polyphenols from other sources prove to be viable chemotherapeutic drugs. It would be of great interest if further studies were performed to gauge the dosing requirements of this drug and whether or not the actual drinking of green tea possesses a legitimate health benefit.
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