*3.8. VEGF Pathway*

Angiogenesis is known to play a major role in the pathogenesis of many cancer types [127]. Given its involvement in metastasis and growth of tumor cells, angiogenesis possesses a key player in vascular endothelial growth factor (VEGF, also known as VEGF-A) and it has been intensively studied as a therapeutic target [128]. Consequently, EGCG has been investigated in the VEGF pathway, discovering its capacity to modulate angiogenesis, with stimutating e ffects in nervous tissue and inhibiting e ffects in tumor tissue. For example, in a mouse model of transient middle cerebral artery occlusion (MCAO), the authors showed that EGCG injected intraperitoneally daily for seven days could stimulate angiogenesis. EGCG had a neuroprotective e ffect, indeed treated mice had better neurologic outcome, a decreased infarct volume, greater vascular density, with the modulation of VEGF signaling pathway and increased Nrf2 expression (fundamental role in against ischemic stroke) [129]. Similarly, hepatic tissue from mice that were treated with EGCG showed increased heme oxygenase-1 (HO-1) and decreased vascular cell adhesion molecule (VCAM)-1 expression, with an increased expression of Nrf-2 (mediated by p38) that drives the expression of HO-1, as also demonstrated in human aortic endothelial cells (HAEC) [130]. Another group used the same mouse model (MCAO) and demonstrated the involvement of the PI3K/AKT/eNOS signaling pathway with a concomitant reduction of apoptotic rate and caspase-3 in neurons, while Bcl-2 increased in the ECGC treated set (EGCG intraperitoneally injected at 20 mg/kg for 24 h) [125]. Similarly, it was studied on the e ffects of EGCG on murine dendritic cells (DC), with a reduction of ERK1/2, p38, JNK, and blockade of NF-κB p65 translocation [131]. EGCG was given to cells at di fferent doses, but 100 μM was the most e ffective with suppression of the phenotypic and functional maturation of DC. Di fferently in tumor tissue EGCG acted in a opposite way. For example, in human gastric cancer SGC7901 cells (in hypoxic state) EGCG (5, 10, 20, 40 μM), reduced expressions of HIF-1 α (a subunit of a heterodimeric transcription factor hypoxia-inducible factor 1-HIF1) and VEGF with a concentration-dependent e ffect. In addition, in SGC-7901 mouse xenografts, 1.5 mg/day EGCG intraperitoneally injected reduced VEGF, tumor microvessel density, endothelial cell proliferation, migration, and tube formation, substantially blocking tumor growth [132]. Similarly, in gastric cancer 5-fluorouracil (5-FU) resistant cells (MGC803/FU treated with 100 μg/mL and SGC7901/FU treated with 20 μM), EGCG inhibited VEGF and HIF-1 expression, reversing 5-FU resistance that is mediated by VEGF pathway [133,134]. HIF-1 is a master regulator of cell homeostatic response to hypoxia by activating the transcription of numerous genes that are implicated in growth, apoptosis, angiogenesis, and metabolism [135]. HIF-1 is strictly connected with VEGF (it is a primary target of HIF-1) and, thus, understanding the regulation of HIF-1 will o ffer new insight into VEGF pathway and new potential therapeutic targets. In human gastric cancer cells (AGS) EGCG >10 μM reduce dose-dependently VEGF expression and secretion mediated by signal transducer and activator of transcription 3 (Stat3) activity [136]. In colon cancer cells (Caco2, HCT116, HT29, SW480, and SW837 lines) and SW837 mouse xenograft, EGCG impacted on VEGF/VEGFR axis suppressing VEGFR2 (a primary responder to VEGF signal), together with HIF-1 α, ERK, and AKT [137]. Moreover in a di fferent human colon cancer cell line (HT29) and derived mouse xenografts, EGCG lessened VEGF expression and its promoter activity, while decreasing the tumour growth and microvessel density in mice (treated daily intraperitoneally with 1.5 mg EGCG), and modulating cell proliferation and apoptosis in HT29 cells [138]. In breast cancer cells (MCF-7), HIF-1 α and VEGF was decreased by EGCG in a dose-dependent manner (25, 50, 100 mg/L) [139], similarly to non-small lung cancer cells (A549), in which the catechin (25, 50, 100 μM) was able both to inhibit HIF-1 α protein expression (stimulated by IGF-I, insulin growth factor-1, a growth hormone mediating cell growth) and the formation of capillary tube-like structures on a model of extracellular matrix [140]. In head and neck squamous cell carcinoma (YCU-H891) and breast carcinoma cell lines (MDA-MB-231), EGCG blocked VEGF activity, thanks to the inhibition of constitutive activation of Stat3 and NF-κB [141]. The results showed that EGCG could modulate a

plethora a key factors, such as TNFα, Rantes, monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), nitric oxide (NO), VEGF, and MMP-2, through the inhibition of NF-κB and MAPK signaling pathways (downregulation of p-IκBα, p65, p-p65, p-p38, p-ERK1/2, and p-AKT). EGCG repressed hypoxia- and serum-induced HIF-1 α and VEGF, through MAPK and PI3K/AKT pathways, in another hepatoma cell line (HepG2) and in human cervical carcinoma cell line (HeLa) [142]. In a group of acute myeloid leukemia (AML) cell lines harboring FLT3 mutation (FLT3 is a commonly mutated gene in AML), EGCG 60 μM (and (−)-epigallocatechin (EGC), (−)-epicatechin-3-gallate (ECG), and (+)-Catechin (C)) showed reduced levels of FLT3 and the suppression of phosphorylated MAPK, AKT, and STAT5 [143]. Moreover, in B-cell chronic lymphocytic leukemia (B-CLL), EGCG 3–25 μM reduced VEGF-R1 and VEGF-R2 phosphorylation (fundamental in B-CLL survival and growth) and induced apoptosis by caspase-3 activation, poly-adenosine diphosphate ribose polymerase (PARP) cleavage, and B-cell leukemia/lymphoma-2 protein (Bcl-2) [144,145].

Additionally, in non-tumoral cell lines, EGCG demonstrated a concrete role. In human microvascular endothelial cells (HMVEC) used as a model of angiogenesis, EGCG inhibited VEGF-induced tube formation through the suppression of vascular endothelial-cadherin phosphorylation and AKT activation [146]. In normal human keratinocytes (NHK) that were stimulated by TNF α, EGCG at di fferent concentrations (0.1 to 10 μM) decreased VEGF and interleukin-8 (proinflammatory cytokine involved in skin inflammation) [147]. Similarly, in human umbilical vein endothelial cells (HUVEC), EGCG inhibited VEGF in a concentration dependent manner and reduced cell proliferation [148]. On the same line, two works from the same research group used HUVEC cells (and MDA-MB231 breast cancer cell) and found that EGCG decreased VEGF production in a concentration-dependent manner (6.25, 25, 100 μM) [149,150]. They also demonstrated that, in HUVEC and MDA-MB231 mouse xenografts, in addition to cell line, green tea extract reduced the proliferation, tumor size, and tumor vessel density [150]. Mice fed with green tea extract in water *ad libitum* with a concentration of 0.62, 1.25, and 2.5 g/<sup>L</sup> (in which EGCG represented 46.8% weight). The e ffects were both time and concetration dependent. In human vascular smooth muscle cells (hVSMCs), EGCG was investigated after cell stimulation with platelet-derived growth factor (PDGF), which is known to induce VEGF expression [151]. EGCG blocked dose-dependently VEGF expression and PDGF receptor activation, in addition to ERK1/2 and AP-1. Additionally, in livestock ovarian cell model (swine granulosa cells), EGCG was evaluated as a food supplement. The catechin reduced VEGF production independently of oxygen condition and decreased cell growth, which suggested a role in angiogenic process, fundamental for follicle development [152].
