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5.1. Cell cycle arrest & induction of apoptosis

Apoptosis involves a series of biochemical events, leading to a variety of morphological changes, including changes in the cell membrane. As one of several mechanisms, EGCG has been shown to inhibit the expression of anti-apoptotic proteins Bcl-2 & Bcl-X_L while increasing the expression of Bax and Bak pro-apoptotic proteins (61). It has been reported that EGCG effectively inhibited cellular proliferation and induced apoptosis of SW1353 and CRL-7891 chondrosarcoma cells. Expression levels of Bcl-2 were significantly decreased and the levels of Bax were significantly increased (62). It was also reported that EGCG-induced apoptosis of human laryngeal epidermoid carcinoma Hep2 cells, which proceeds through a caspase-independent, p53-mediated pathway (62). EGCG induced cell cycle arrest and apoptosis in Ewing family tumors (EFT) cells which correlated with altered expression of Bcl-2 family proteins, including increased expression of proapoptotic Bax and decreased expression of prosurvival Bcl2, Bcl-X_L, and Mcl-1 proteins (63). Britschgi et al reported in acute promyelocytic leukaemia (APL), EGCG treatment resulted in a significant increase of death-associated protein kinase 2 (DAPK2) levels and 67 kDa laminin receptor expression, associated with increased cell death. All-trans retinoic acid (ATRA) and EGCG co-treatment significantly boosted neutrophil differentiation, and 67LR expression correlated with susceptibility of acute melocytic leukaemia (AML) cells to EGCG (64).

Human prostate cancer cell lines LNCaP and PC-3 express high levels of a hyperphosphorylated Bcl-X_L protein in mitochondria. Using a combination of nuclear magnetic resonance binding assays, fluorescence polarization assay, and computational docking studies one research group investigated the interaction of tea polyphenols with Bcl-X_L and Bcl-2 proteins. They found that certain green tea catechins such as (−)-EGCG and (−)-ECG were very potent inhibitors (Ki in the nanomolar range) of Bcl-X_L and Bcl-2. This study revealed that tea polyphenols could recognize the BH3 domain on Bcl-2 family of proteins, suggesting that EGCG could inhibit the activity of anti-apoptotic Bcl-2 family of proteins by directly binding to their BH3 domain (30).

It was reported that EGCG triggers the intrinsic apoptosis pathway by regulating the mitochondrial functions, activating caspase-3 and caspase-9 and cleaving PARP (65). In gastrointestinal stromal tumor cell line GIST-T1 including imatinib-resistant cells, it was reported that EGCG inhibits cell growth and causes caspase-dependent cell death (66). In addition Hastak et al reported induction of apoptosis through modulation of p53 stabilization in LNCaP cells by EGCG, consequently activating the downstream targets p21/WAF1 and Bax (67). In human bladder cancer cells, EGCG suppressed cell growth and induced apoptosis in a dose-dependent manner (68). In androgen-independent DU-145 prostate cancer cells, the inhibitor of DNA binding 2 (ID2), known as a dominant anti-retinoblastoma (Rb) helix-loop-helix protein, was found to be down regulated 4-fold by EGCG treatment. Expression of ID2 resulted in increased survival and decreased apoptosis, indicating ID2 as a target in EGCG-mediated apoptosis (69). In cervical cancer cell lines Caski and Hela, EGCG also induced cell cycle arrest and apoptosis (59).

Another promising cancer target is Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In human melanoma A375 cell line EGCG was reported to substantially enhance caspase-8 and TRAIL mediated apoptosis (70). EGCG was also reported to mediate its growth inhibitory effects via pRb-E2F/DP pathway, an important regulator of cell cycle arrest and apoptosis (71).

Additionally, EGCG exerts growth inhibitory effects and disruption of cell cycle regulation in androgen-dependent and androgen-independent prostate cancer cells (9). Recent finding demonstrates that addition of cupric acid to one of the catechins [(2R,3S)-3′,4′,5,7-Tetrahydroxyflavan-3-yl decanoate; catechin-C10] induces significant apoptosis in U937 cells. In addition to this C10 was found to induce apoptosis in human colon cancer (HCT116) cells while it showed resistance to human keratinocytes (HaCat) (72). In breast carcinoma cell line resistant to tamoxifen (MCF-7Tam cells), EGCG down-regulated the activity of two molecules Pg-p and BCRP that play a key role in drug metabolism and transport and that are highly expressed in MCF-7Tam cells (73).

5.2. EGCG affects Ras/MAPK pathway

Ras is a small GTPase associated with multiple signaling pathways (74). Mutations in the ras gene and constitutively active ras lead to cellular transformations by activating the growth signal transduction pathways (75). Oncogenic ras is observed frequently in many cancers. Mitogen activated protein kinases (MAPK) are a group of serine/threonine kinases that act as signal transducers of cell proliferation that are activated by various extra cellular stimuli. Three well known MAPKs are extracellular signal regulated kinases (ERKs), stress activated or c-Jun N-terminak kinase (JNKs/SAPKs) and p38 kinases (76-77). EGCG has been reported to regulate various molecules in the MAPK pathway thereby inhibiting cancer cell survival. Chung et al in 2001 (78) reported that EGCG inhibited Ras/MAPK pathway. In HT1080 cells EGCG inhibited the phosphorylation of extracellular regulated kinases 1 and 2 (ERK1/2) (79). In human epidermal keratinocytes pretreatment with EGCG inhibited the phosphorylation of ERK1/2, JNK and p38 induced by H₂O₂ (80). EGCG inhibits UVB-induced c-fos expression in a human keratinocyte cell line HaCaT by significantly inhibiting the activation of p38 MAPK (81). EGCG also triggers JNK mediated apoptosis in p57 negative oral carcinoma cells (82). It was also reported that EGCG inhibited the activation of beta1 integrin-downstream molecules such as FAK, AKT and ERK as well as the expression of beta1 integrin itself (83). Shim et al. found that EGCG interacted with the Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) with high binding affinity (Kd = 0.4 micromol/L) and interfered with the interaction of G3BP1 and the Ras-GTPase-activating protein and further suppressed the activation of Ras. Their results revealed that EGCG suppresses lung tumorigenesis by effectively attenuating G3BP1 downstream signaling, including ERK and MAPK/ERK kinase, in wild-type H1299 and shMock H1299 cells which contain an abundance of the G3BP1 protein but had little effect on knockdown shG3BP1-transfected H1299 cells (84).

5.3. Phosphoinositide 3 kinase (PI3 kinase)/Akt pathway

PI3kinase/Akt is a pro-survival signaling pathway. Three different isoforms of Akt are present in mammalian cells. Growth factor receptors activate PI3 kinase which phosphorylates the inositol ring of phosphoinositol to give PIP3. PIP3 binding to Akt and subsequent translocation to plasma membrane assists in phosphorylation of Akt by PI3 dependent kinases (PDK) 1 and 2. Active Akt inhibits apoptosis by phosphorylation of apoptotic proteins like Bad and caspase-9 (85-86).

Akt is frequently found activated in many human cancers (87). Activation of Akt is associated with increased proliferation and resistance to apoptosis. EGCG has been shown to inhibit PI3K/Akt pathway in many cancers like breast, prostate and cervical cancers. In the benign skin tumor keloid fibroblasts, EGCG inhibited phosphorylation of PI3K, ERK1/2, and STAT3 (Tyr705 and Ser727). This study demonstrated that EGCG suppresses the pathological characteristics of keloids through inhibition of the STAT3-signaling pathway and proposed the potential of EGCG in the treatment and prevention of keloids (88). Cheng et al reported that EGCG inhibits both tyrosine and serine phosphorylation of STAT1 and its translocation into the nucleus in IFN-gamma-stimulated human oral cancer cells. Phosphorylation of PKC-delta, JAK-1, and JAK-2, which are the upstream events for the activation of STAT1, were also inhibited by EGCG in IFN-gamma-stimulated human oral cancer cells (89). In pancreatic cancer EGCG has been reported to inhibit downstream signaling and activation of PI3K/Akt pathway by inhibiting tyrosine kinase phosphorylation of PDGF receptor (90). In MDA-MB-231 breast cancer cell line and YCU-H891 head and neck squamous cell carcinoma cell line, EGCG could inhibit the constitutive activation of EGFR, Stat3 and Akt (91). In T24 bladder cancer cell line, EGCG treatments lead to enhanced apoptosis by modulation of Bcl-2 family and inhibiting PI3K/Akt pathway (61). In prostate cancer DU154 and LNCaP cells, both EGCG and TF treatment were found to (i) decrease the levels of PI3K and phospho-Akt and (ii) increase Erk1/2 in both cells. Modulation of the constitutive activation of PI3K/Akt and Erk1/2 pathways by EGCG as well as TF could be one of the anti-cancer effects of EGCG (92). EGCG inhibited mouse mammary tumor virus (MMTV)-Her-2/neu NF639 cell growth by reducing the signaling via PI3K/Akt to NF-kappa B pathway because of inhibition of basal Her-2/neu receptor tyrosine phosphorylation (93).

5.4. Inhibition of insulin like growth factor (IGF)-1 pathway

IGF pathway operates via peptide hormones, their cell surface receptors, and a family of six distinct IGF binding proteins (IGFBPs) that determine the activity by modulating the bioavailability of IGFs in circulation. Heterotetrameric IGF-R1 transmembrane glycoprotein has an intracellular tyrosine kinase domain that phosphorylates various targets within the cell. IGF-R1 initiates a signaling cascade in response to the mitogens IGF-1 and IGF-2 that regulate cell proliferation, differentiation and apoptosis. IGFBPs and IGF-R2 neutralize IGF action by regulating the internalization and degradation of IGF-2. Increased expression of IGF-R1 and low levels of IGFBPs are associated with an increased risk of cancer and are thus considered as potential therapeutic targets (94).

Several investigators thus focused on the effects of GTPs on IGF signaling pathway. Effects of GTPs, specifically EGCG, on IGF-R1 tyrosine kinase activity in malignant cell growth were found to be highly inhibitory (95). In TRAMP mice there was a significant increase in IGFBP-3 while decreased levels of IGF-1 were observed in response to GTP (96). It was also reported that EGCG caused a transient increase in TGF-beta, an inducer of IGFBP-3, and decreased the mRNA levels of proteins that proteolyses IGFBP-3 in human colon cancer HT29 cells (97). Kang et al showed that treatment of Ewing family tumors (EFT) cell lines with EGCG blocked the autophosphorylation of IGFIR tyrosine residues and inhibited its downstream pathways including phosphoinositol 3-kinase-Akt, Ras-Erk, and Jak-Stat cascades. Effects of EGCG were associated with dose- and time-dependent inhibition of cellular proliferation, viability, and anchorage-independent growth, as well as with the induction of cell cycle arrest and apoptosis (63). Thus EGCG has been reported to exert its anticancer effects by inhibiting IGF signaling pathway.

5.5. Inhibition of metastasis

Degradation of the extracellular matrix by proteolytic enzymes is crucial for tumor invasion and metastasis. Urokinase Plasminogen activator (uPA) catalyzes the cleavage of plasminogen to plasmin, which in turn facilitates the release of several proteolytic enzymes (98). It has been shown that GTPs could modulate the secretion of uPA and inhibit the invasive behaviour of breast cancer cells by suppressing the constitutively active transcription factors AP-1 and NF-kappa B (99).

Further to uPA, matrix metalloproteinases (MMP’s) are structurally related zinc-dependent endopeptidases that are involved in ECM degradation and remodeling. Several investigations have reported that MMPs have anti-apoptotic properties, and aid in tumor progression and metastasis by promoting angiogenesis, tumor cell proliferation and differentiation. Thus MMPs and MAPK pathway that regulates the expression of MMPs are considered as a potential targets for chemotherapy (100). Not surprisingly cancer-preventive effects of EGCG have been reported in human salivary gland adenocarcinoma cells. EGCG inhibits the invasion and migration by inhibiting MMP-2 and MMP-9 expression and enzymatic activity in a dose-dependent manner (96). It is also reported to inhibit MMP-9, thereby preventing the metastasis of lung cancer cells (101). In rat hepatic stellate cells EGCG was reported to cause a decrease in MMP-2 mRNA and protein levels (102). In human tendon derived fibroblasts, IL-1B induced expression of collagenases, MMP-1, MMP-3, MMP-13 and stomelysin was inhibited by EGCG and EGC (103). Activities of secreted MMP-2 and MMP-9 were also inhibited by EGCG (104). In human gastric cancer AGS cells, activation of phorbol myristate 13-acetate (PMA) induced upstream regulators of AP-1, ERK and JNK was abrogated by EGCG thus inhibiting the invasiveness and MMP-9 expression of these cells (105). In the prostate of TRAMP mice oral administration of GTP significantly reduced the expression of MMP-2 and MMP-9 (106). MMP suppression mechanisms of EGCG also include increasing the expression of tissue inhibitors of MMPs (TIMPs) (107). Jackson et al reported that tannic acid, EGCG and ECG (but not GA) strongly inhibited collagen degradation at low micromolar concentrations by strongly binding and stabilizing collagen via extensive hydrogen bonding, augmented by some hydrophobic interactions and preventing the free access of collagenase to active sites on the collagen chains (108). Thus regulation of AP-1, MAPK pathway and inhibition of MMPs in several cancers by EGCG contributes to its overall cancer preventive effects.

5.6. EGCG affects transcription factors

5.6.1. Nuclear factor kappa B (NF-kappa B)

NF-kappa B is a sequence-specific transcription factor sensitive to oxidative stress. Under resting conditions I-kappa B binds NF-kappa B and localizes it in the cytoplasm of the cell. NF-kappa B inducing kinase/I-kappa B kinase regulates I-kappa B phosphorylation. Phosphorylation of I-kappa B releases active NF-kappa B that translocates to the nucleus and induces the expression of over 200 genes. Many of these genes suppress apoptosis and can induce proliferation. Deregulation of the pathway and aberrant activation of NF-kappa B or constitutively active NF-kappa B is frequently found in many human cancers (109). Hence inhibiting NF-kappa B has potential for cancer prevention. EGCG inhibited NF-kappa B activation in UV induced normal human epidermal keratinocytes (110). In A431 epidermoid cancer cells I-kappa B levels were increased in a dose/time dependent manner of EGCG while inhibiting the nuclear translocation of NF-kappa B (111). In human bronchial epithelial cells EGCG could suppress NF-kappa B activation along with other pro-survival pathways (112).

5.6.2. Activator protein 1 (AP-1)

c-Jun and c-fos are oncogenes that encode for AP-1, a transcription factor, whose activity has been associated with invasive and metastatic characteristics of cancer cells. They are immediate-early genes, whose transcription is induced rapidly in response to external stimuli. These oncogenes are components of signal transduction pathways that function to stimulate cell proliferation. Researchers have shown that EGCG inhibits the activity of AP-1 through the inhibition of mitogen-activated protein kinase (MAPK), specifically, through inhibition of c-Jun NH2-terminal kinase (JNK) dependent activity in JB6 cells and H-ras transformed JB6 cells (113). In mouse epidermal JB6 cell line with mutant H-ras gene, both the galloyl structure on the B ring and the gallate moiety of catechins inhibited cell growth by exerting inhibitory effects on AP-1 activity (114). In Ha-ras-transformed (21BES) human bronchial epithelial cells EGCG and theaflavin digallate (TFdiG) are reported to induce rapid apoptosis by decreasing c-jun protein phosphorylation resulting in lowered AP-1 activity, which may contribute to the growth inhibitory activity of tea polyphenols (115). Chen et al (81) reported that in human keratinocyte cell line HaCaT cells, EGCG could inhibit UVB-induced c-fos expression, a major component of AP-1, and prevent AP-1 activation which is important for tumor promotion.

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