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MOLECULAR TARGETS OF EGCG
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- 6.2. Telomerase
- 6.3. Tumor suppressor p53
- 6.4. Vascular endothelial growth factor (VEGF)
- 6.5. Proteasome
6. MOLECULAR TARGETS OF EGCG6.1. CyclooxygenaseOne of the mediators of inflammation is prostaglandins. Cyclooxygenases (COXs) are enzymes that catalyze the rate limiting step during prostaglandin biosynthesis from arachidonic acid. COXs family consists of three enzymes COX-1, COX-2 and COX-3. COX-1 is ubiquitously expressed in mammalian tissues and is involved in homeostasis. COX-3 functions are unknown but it is found in the brain and spinal cord. More recent emphasis has been laid on COX-2 as its expression is found to be up regulated in inflammation and in cancer. In many tumors it has been reported that COX-2 plays a role in tumorigenesis by modulating cell proliferation and apoptosis (116). EGCG inhibited COX-2 expression in colon cancer cells by activating AMPK pathway (117). In androgen-dependent LNCaP cells and androgen-independent PC-3 cells of human prostate cancer, both mRNA and protein levels of COX-2 were inhibited by EGCG (118). Supplementation of GTP and BTP as the sole source of drinking solution to Winstar rats lead to scavenging of ROS by inhibition of COX-2 and inactivation of phosphorylated NF-kappa B and Akt (27). Thus EGCG exerts its antiproliferative effects via COX-2 inhibition. 6.2. TelomeraseA number of different approaches have been developed to inhibit telomerase activity in human cancer cells. It has been shown that telomerase is over expressed in many human cancers and is important for maintaining the telomere nuclear protein endcaps of the chromosome. Telomerase inhibition was seen both in a cell-free system (cell extract) as well as in living cells with a concentration of 1 microM (IC50) and 15 microM of EGCG. In cell-free systems at neutral pH, high nanomolar to low micromolar concentrations of EGCG inhibited telomerase activity. EGCG (5–10 microM) was reported to inhibit telomerase and induce cell senescence after long-term treatment (119). The possible implication was that EGCG decomposes to form a galloyl radical, which can covalently modify telomerase. The effect of EGCG on telomerase activity and growth rate was determined by using two different types of tumor U937 monoblastoid leukemia cells and HT29 colon adenocarcinoma cells. Interestingly, treatment of mice with 1.2 mg of EGCG per day for 80 days bearing telomerase-positive colon cancer xenografts (HCT-L2) resulted in a 50% inhibition in tumor size whereas mice bearing telomerase-negative tumors of the same parent cell line (HCT-S2R) shows no response to EGCG treatment (119). The inhibition of the cancer-associated enzyme telomerase is a key mechanism involved in cancer inhibition by epigallocatechin gallate (EGCG), a major tea polyphenol. In healthy cells telomeres lose up to 300 bp of DNA per cell division that ultimately leads to senescence; though, most cancer cells bypass this lifespan restriction through the expression of telomerase. The telomerase has catalytic subunit, hTERT essential for its proper functioning and has been shown to be expressed in approximately 90% of all cancers. EGCG-treatment repressed telomerase activity and induced apoptosis in laryngeal squamous cell carcinoma Hep 2 cells (120). EGCG can inhibit cell growth by inducing apoptosis in MCF-7 breast cancer cells and is due to decreased hTERT transcription. These conclude that EGCG leads to progressive demethylation of the hTERT promoter including E2F-1 binding sites resulting in an increase in binding of this repressor. While HL60 promyelocytic leukemia cells when exposed to EGCG reduced cellular proliferation and induced apoptosis but no decrease in hTERT mRNA expression (120). The prolonged oral administration of EGCG to nude mice models bearing both telomerase-dependent and -independent xenograft tumors cloned from a single human cancer progeny shows that only the telomerase dependent tumors respond to the treatment (121). 6.3. Tumor suppressor p53Tumor suppressor genes generally follow the two-hit hypothesis that if only one allele for the gene is damaged, the second can still produce the correct protein. p53 is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 mutations are found in almost all tumor types, where they contribute to the complex network of molecular events leading to tumor formation. (−)-EGCG in particular, can stabilize p53, inducing G1 phase cell cycle arrest and apoptosis (122-123). In another similar study 80 mg/ml of -(−) EGCG treatment for 8 hours in bovine aortic smooth muscle cells showed increased nuclear p53 protein levels for up to 9 folds (124). EGCG induced stabilization of p53, which caused an up-regulation in its transcriptional activity, thereby resulting in the activation of its downstream targets such as p21WAF1 and Bax and the induction of apoptosis in prostate carcinoma LNCaP cells. The erlotinib treatment activates p53, which plays a critical role in synergistic growth inhibition by erlotinib and EGCG via inhibiting NF~B signaling pathway in squamous cell carcinoma of the head and neck (125). 6.4. Vascular endothelial growth factor (VEGF)One of the important factors for angiogenesis is a vascular endothelial growth factor (VEGF). Overexpression of VEGF can be seen in cancers and enables tumor to grow and metastasize. Neo-vascularization process required for the growth of tumors beyond a few millimeters in diameter, and also for tumor invasion and metastasis (126-127). Jung et al showed that angiogenesis can be inhibited by EGCG through blocking the induction of VEGF in human carcinoma cells. These results showed that (−)-EGCG inhibited tumor volume by 61% compared with (−)-EC treatment, as well as reduction in tumor vessel counts and increase in apoptosis after treatment with (−)-EGCG (128). Tea polyphenols decrease serum levels of prostate-specific antigen, hepatocyte growth factor, and vascular endothelial growth factor in prostate cancer patients. In vitro it also inhibits the production of hepatocyte growth factor and vascular endothelial growth factor. Another study shows that EGCG inhibits the expression of HIF-1-alpha, which strongly activates VEGF expression (129) at both the mRNA and protein levels in colorectal cancer cells. The inhibition of HIF-1-alpha is associated with the inhibition of ERK and Akt phosphorylation, because HIF-1-alpha protein synthesis is regulated by PI3K/Akt and MAPK/ERK pathway activation. Because the noticeable decrease in cellular HIF-1-alpha levels occurred within 3 hours after the addition of EGCG and preceded the inhibition of VEGFR-2 phosphorylation. The author further explained that EGCG inhibits activation of the VEGF/VEGFR axis, by suppressing the expression of HIF-1-alpha and several major growth factors in five different cell lines of colorectal cancer (130). 6.5. ProteasomeThe proteasome is a multicatalytic protease responsible for the degradation of most cellular proteins and now is recognized as a novel and promising target for chemotherapy. The proteasome regulates the cell growth, survival, and metastasis of cancer cells making it an attractive target for new drugs. The eukaryotic proteasome contains three catalytic beta subunits responsible for the chymotrypsin (CT)-like (beta-5), trypsin (T)-like (beta-1), and peptidyl-glutamyl peptide-hydrolyzing (PHGH)-like (beta-2) (131). We and others have reported that inhibition of CT–like activity is associated with induction of apoptosis in various tumors (132-133). We reported that (−)-EGCG potently and specifically inhibited the chymotrypsin-like activity of the proteasome in vitro (IC50 = 86-194 nM) and in vivo (1-10 microM) at the concentrations found in the serum of green tea drinkers and induced tumor cell growth arrest in G1 phase of the cell cycle while having little or no effect on normal, nontransformed cells. We also reported for the first time by using in silico docking methods, that ester bond within (−)-EGCG played a critical role in its inhibitory activity of the proteasome. We found that synthetic (−)-EGCG amides and (−)-EGCG analogs, with modifications in the A-ring, C-ring or ester bond, inhibited the chymotrypsin-like activity of purified 20S proteasome with altered potencies, induced growth arrest in the G1 phase of the cell cycle in leukemia Jurkat T cells, and suppressed colony formation of human prostate cancer LNCaP cells (134). In an effort to discover more stable and potent analogs, we synthesized several (−)-EGCG analogs with −OH groups eliminated from the B- and/or D-rings. We also synthesized the putative prodrugs with −OH groups protected by acetate that can be removed by cellular cytosolic esterases. We then examined the structural activity relationship between the compounds that are protected and those that are not protected. We found that decreasing −OH groups from either the B- or D-ring leads to diminish proteasome-inhibitory activity in vitro. The protected analogs were able to inhibit the proteasomal chymotrypsin-like activity by 97% in cultured tumor cells. In vivo EGCG is subjected to rapid biotransformation, such as methylation by catechol-O-methyltransferase (COMT) that limits its action. The challenge in developing (−)-EGCG for cancer prevention and therapy is its low bioavailability, partly due to its poor absorption and its instability under neutral or alkaline conditions (i.e., physiologic pH) and partly due to biologically inactivating processes such as methylation. We have shown that Pro-EGCG (1), as a novel prodrug, converts to a cellular proteasome inhibitor and anticancer agent in nude mice after treatment for 31 days. Our recent study demonstrate that a pro-drug of a simple synthetic analog 5, the acetate 6, enhanced the cytotoxic activity of 5 just as pro-EGCG (1) enhanced the growth inhibitory activity relative to EGCG. An even simpler analog 7, though not as potent an inhibitor of purified proteasome as EGCG or 5, was found to be a better proteasome inhibitor when incubated with MDA-MB-231 cell extracts (44) (Figure 2). These results also suggest that COMT does play a significant role in affecting the anti proliferative activities of EGCG and its analogs. жүктеу/скачать 1.88 Mb. Достарыңызбен бөлісу:
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