*3.5. MAPK Pathway*

The MAPK pathway (or extensively "Ras-Raf-MEK-ERK pathway") is a key signaling pathway frequently dysregulated in human cancer and can control multiple cell functions, such as proliferation and growth [77]. For these reasons, di fferent chemotherapeutic drugs have been already clinically evaluated or are currently undergoing to specific clinical trials, for example, most promising are the MEK1/2 and Raf inhibitors [78]. In mammalian cells, three major types of MAP kinases are present; ERK, p38 MAP kinase, and JNK, and they can activate c-JNK and ELK [79]. These were the main actors studied and they were associated with EGCG interaction in the MAPK pathway. A recent work studied EGCG and candesartan (anti-hypertensive drug) in a rat model of gentamicin-induced nephrotoxicity [80]. EGCG was administred at 25 mg/kg, intraperitoneally, alone or combined with the above drugs for 21 days. While renal disease revealed increased levels of NF-κB, p38-MAPK, caspase 3, TNF-<sup>α</sup>, and interleukin-1 β, the pre-treatments with EGCG and candesartan (co-treatment had the best results if compared to compounds alone) reduced such key factors, which impacted the MAPK pathway. On the same line, a work with mice of Wang et al. showed that, in animals with artificial unilateral ureteral obstruction, the intraperitoneally administration of EGCG (5 mg/kg) for 14 days inhibited phosphorylation of ERK, JNK and p38 MAPK [81]. The effect of EGCG on mastitis in rats were evaluated by injecting lipopolysaccharide (LPS) into the duct of mammary gland. EGCG was orally given at 25 or 100 mg/kg/day in the morning for 14 days. MAPKs, NFκB-p65 and hypoxia-inducible factor-1α (HIF-1α) were decreased after the treatment with EGCG at both concentrations. The results showed that EGCG suppressed MAPK related oxidative stress and inflammatory responses [82]. In another rat disease model, EGCG exhibited cardioprotective effects in myocardial ischemia/reperfusion (I/R) injuries [83]. The authors showed that EGCG 10mg/kg intravenously administered alone or with wortmannin (PI3K inhibitor) 5 min. before the onset of reperfusion reduced p38 and JNK phosphorylation while enhancing AKT and GSK-3β, but not ERK1/2. Additionally, in artificial induced hypertensive rats, ERK, p38, and JNK phosphorylation were decreased, when the rats were treated with 50 mg/kg EGCG per day, intraperitoneally for 21 days [84]. Previously in human umbilical vein endothelial cell (HUVEC) model, EGCG induced cell proliferation inhibition during ischemia/reperfusion, but, in contrast with previous work, JNK1/2 (and c-Jun) phosphorylation was increased [85]. These data could be imputable to the different model and time of incubation proposed. Also, EGCG >50 μM inhibited angiotensin II- induced phosphorylation of ERK and p38 in HUVEC cells, influencing vascular cell adhesion molecules (VCAMs) and intercellular adhesion molecules (ICAMs) expression [86,87]. In addition, EGCG impacted AKT phosphorylation and its downstream substrates Foxo1, Foxo3a, and ERK1/2. In a prostate cancer mouse model (TRAMP mice: Transgenic Adenocarcinoma Mouse Prostate), Harper and collaborators demonstrated that EGCG given *ad libitum* as 0.06% in water reduced ERK phosphorylation, together with androgen receptor (AR), insulin-like growth factor-1 (IGF-1), IGF-1 receptor (IGF-1R), COX-2, and inducible nitric oxide synthase (iNOS) [88]. The authors claimed that the EGCG dose should mimic a human consumption of six cups of green tea and that the catechin, through the decrease of MAPK signaling, could modulate inflammation biomarkers, reduce cell proliferation, and induce apoptosis. In the same mouse TRAMP model, another work showed a key role for IGF-I/IGFBP-3 signaling pathway, with the modulation of ERK [89]. However, the authors used a solution of 0.1% green tea polyphenols (in which EGCG represented 62%) to experimental animals [89]. The two works are very similar in EGCG dose, but analyzed and demonstrated the implaction of different key factors: they are probably the two sides of the same coin, but more research is needed to confirm this hyphothesis. Moreover, in a NOD mouse, a model for Sjogren's syndrome, oral administration of 0.2% green tea extract in *ad libitum* water for 21 days (in which EGCG represented 40%) reduced the total autoantibody levels in the serum and lymphocytic infiltration of submandibular glands (a key point of Sjogren's syndrome) [90]. The research was also expanded in normal human keratinocytes and immortalized normal human salivary acinar cells, where EGCG was able to protect the cells by specific phosphorylation of p38 MAPK [90–93], contrary to most of works in which there is a de-phosphorylation/deactivation of MAPK pathway. Again, these apparently inconsistent results should be ascribed to different models used and the different human-derived tissue.

In SKH-1 hairless mouse model, the topical application of green tea polyphenols (in which EGCG represented 18.8%) showed a decrease in UVB-induced thickness, skin edem, a and infiltration of leukocytes, processes mediated by inhibition of ERK, c-JUN, and p38, as well as NF-kB [94]. Before the animal model, the authors demonstrated the same results in cell models in which EGCG was used at 10–40 μM [92,95]. In addition, EGCG diminished the negative effects of ultraviolet irradiation in human dermal fibroblasts, reducing the phosphorylation of MAPK, JNK, p38 MAPK, and ERK1/2 [95,96]. Collagen destruction was reduced by inhibiting matrix metalloproteinases (MMP-1, MMP-8, and

MMP-13). In human AC16 cardiomyocytes, EGCG was examined for alleviating cigarette smoke medium (CSM)-induced inflammation: EGCG protected from oxidative stress, inflammation, and apoptosis, modifying interleukin (IL)-8 productions via the inhibition of ERK1/2, p38 MAPK, and NF-κB pathways [97]. In the human retinal endothelial cell (HREC) line, a preclinical model for diabetic retinopathy, EGCG was able to decrease phosphorylated p38, ERK, VEGF, and the expression of inflammatory markers, such as TNFα, IL-6, and ICAM-1 [98]. In addition, EGCG amended the negative e ffect of high glucose concentrations acting on the MAPK pathway. Furthermore, EGCG in chronic myeloid leukaemia cells inhibited cell growth and induced apoptosis through the inhibition of Bcr/Abl oncoprotein and regulation of its downstream signals, such as p38-MAPK and JNK (in addition to involvement of JAK2/STAT3/AKT pathway) [99]. In a di fferent cellular context, JB6 mouse epidermal cell line, it has been shown that EGCG could inhibit AP-1(activating protein-1)-dependent transcriptional activity through the inhibition of a c-JUN, but not ERK [100]. AP-1 is a transcription factor involved that can be stimulated by cytokines, growth factors, stress, and, consequently, implicated in proliferation and apoptosis, with overlapping role to MAPK pathway. Successively, another work remarked the importance of AP-1 (and MAPK), with the inhibition of c-JUN, and also ERK by EGCG, di fferently from previous research [101]. Of note that a di fferent cell line was used (human gastric AGS cells). EGCG also impacted endothelial cell activation, where the catechin could downregulate endothelial inflammation by acting on MAPK signaling through a caveolae-regulated cell mechanism [102]. Endothelial cells are fundamental in atherosclerosis: EGCG demonstrated antiatherogenic e ffect in reducing oxidized LDL upregulated by c-Jun phosphorylation and blocking oxidized LDL-induced endothelial apoptosis [103]. In the pancreatic alpha TC1-6 ( αTC1-6) cell line, EGCG confirmed its anti-hyperglycemia and antioxidant potential [104]. Indeed, EGCG re-established glucagon secretion and reduced ROS, while inhibiting JNK and p38 and activating AKT signaling. It is of note that a higher concentration of EGCG (100 μM) was used, if compared to previous studies, and this can potentially reduce EGCG e fficacy when moved to clinical trials. Moreover, in a cell model of insulin-stimulated mitogenesis (3T3-L1 preadipocytes), it has been shown that EGCG inhibited the insulin e ffects, switching o ff signaling through both insulin receptor-β, insulin receptor substrates 1 and 2 (IRS1 and IRS2) and RAF1, MEK1/2, and ERK1/2, but not JNK [105].

Interestingly, EGCG was associated with sunitinib, which is a multi-target receptor tyrosine kinase inhibitor approved for renal carcinoma and gastrointestinal stromal tumor, in MCF-7 (breast cancer) and H460 (lung cancer) mouse xenograft tumors [106]. The authors found that synergize with sunitinib due to its ability to suppress both insulin receptor substrate-1 pathway and MAPK signaling induced by sunitinib. In another breast cancer cell line T47D, EGCG induced the phosphorylation of JNK/SAPK and p38 and modulated cyclin A, cyclin B1, and cdk proteins, triggering G2 arrest [107]. In a human colon cancer cell line (HT-29), it was found that EGCG activated phospho-AKT, phospho-ERK, phospho-JNK, and phospho-p38 α/γ/δ and, consequently, induced cell death, a potential mechanism for anti-cancer effect of EGCG [108]. Previously with same cell line, Chen et al. showed the activation of caspase-3 and -9, cytochrome *c*, mitochondria, c-Jun, and MAPK pathway after treatment with EGCG, with the end result of apoptosis induction [109]. In another colon cancer cell line (SW480), EGCG induced the phosphorylation of p38, which mediated the phosphorylation of EGFR at Ser1046/1047, leading to a downregulation of the EGFR/MAPK pathway [110]. The author claimed that this phosphorylation could play a pivotal role in EGFR downregulation that is induced by EGCG. Furthermore, in HCT116 colorectal carcinoma cells, EGCG triggered the phosphorylation of ERK, JNK, and p38, which, in turn, conducted to apoptosis [111]. In human lung cancer cell line PC-9, EGCG was associated with celecoxib (a cyclooxygenase-2 selective inhibitor) provoking an activation of ERK1/2 and p38 regulated by GADD153 (DNA damage-inducible 153) expression [112]. EGCG induced apoptosis and the authors suggested that GADD153 could be a new molecular target for cancer prevention in combination with EGCG. Similarly, EGCG was found to suppress cell proliferation by upregulating BTG2 (B-cell translocation gene 2) expression via MAPK pathway in oral squamous cell carcinoma (OSCC), in particular with the phosphorylation of p38, JNK, and ERK [113].

Moreover, in anaplastic thyroid cell carcinoma (ARO cells), EGCG 50 μM showed anti-proliferative and pro-apoptotic effects, being mediated by the suppression of phosphorylated EGFR, ERK1/2, JNK, and p38 and p21, caspase-3, and cleaved PARP increase and cyclin B1/CDK1 expression decline [114]. In a previous work, human hepatoma HepG2-C8 cells and human cervical squamous carcinoma HeLa cells were used to evaluate EGCG [115]. The results indicated that catechin induced the activation of ERK, JNK, and p38 in a dose- and time-dependent manner, with stimulation of apoptosis (mediated by caspase-3) and antioxidant response element (ARE). In DU-145 prostate cancer cells, EGCG associated with ibuprofen reduced >90% tumor cell growth through the activation of both MAPK and caspases [116]. Similarly, human prostate cancer androgen-unresponsive and responsive cells (DU145 and LNCaP, respectively) that were treated with EGCG showed an increase of ERK1/2 with concomitant decrease of phosphorylated AKT and the level of PI3K, again underlining the anticancer effect of the catechin [117].
