Tea drinking is found to be related to the reduced risk of gynecological cancers. A case-control study on Asian-American women in Los Angeles, USA revealed that green tea intake was significantly and negatively correlated to the BC risk (18). Furthermore, a study involving 472 patients with stage I, II, and III BC in Japan showed that green tea consumption was negatively correlated to numbers of axillary lymph node metastases among pre-menopausal patients with stage I and II BC, but positively correlated to the expression of estrogen receptor (ER) and progesterone receptor (PgR) among post-menopausal patients. A 7-year follow-up study on these patients showed that an increase in green tea consumption was negatively correlated to the recurrence of stage I and II BC (p < 0.05), with a recurrence rate being 16.7% among patients who consumed ≥5 cups/day, but being 24.3% among those who consumed ≤4 cups/day. After adjustment for other lifestyle factors, the RR of recurrence among patients with stage I and II BC was 0.564 (95% CI = 0.350∼0.911) (19). In a population-based case-control study involving in 501 BC patients and 594 control subjects among Japanese, Chinese, and Filipino-American women, which was carried out during 1995–1998, showed that the risk of BC decreased with increase in intake of green tea [odds ratio (OR) = 1.00, 95% CI = 0.71 (0.51∼0.99)] in non-tea drinkers (0–85.7 mL/d) and OR = 0.53 (95% CI = 0.35∼0.78) in tea drinkers (>85.7 mL/d) (20). A meta-analysis based on data through literature search in the MEDLINE database from January 1966 to August 2004, which revealed the relationship of black tea or green tea consumption to BC risk among populations from eight countries, showed that high intake of green tea reduced the risk of BC with OR = 0.78 (95% CI = 0.61∼0.98). Black tea consumption showed weakly positive correlation to the risk of BC, with OR = 0.91 (95% CI = 0.84∼0.98), in which the negative correlation was higher in hospital-based studies (OR = 0.77, 95% CI = 0.50∼1.19) than the population-based case-control studies (OR = 0.94, 95% CI = 0.81∼1.09) (21). An investigation on 3,315 women in Singapore showed that mammographic density (PMD) of daily green tea drinkers (19.5%) was significantly lower than that of non-tea drinkers (21.7%, p = 0.002) after the relevant covariates were adjusted, however, the intake of black tea was not associated with the risk of PMD (22). A meta-analysis including 3,323,288 participants showed that heavy tea consumption has a preventing effect against ER-BC, especially in the post-menopausal women. Therefore, tea was considered to be a potentially useful dietary protectant for preventing BC and a recommended dose was ≥5 cups/day (23).

Ovarian cancer risk was found to be associated with tea drinking. Black tea consumption showed linearly suppressive effects on the risk of ovarian cancer (p = 0.03), in which 30% decline in the risk was observed (adjusted OR = 0.70, 95% CI = 0.51∼0.97) among those who consumed ≥2 cups daily (24). Green tea drinking was found significantly to decrease the risk of ovarian tumors (OR = 0.81, 95% CI = 0.73∼0.89, p < 0.0001) in a meta-analysis study including 3,842 cases and 5,271 control cases retrieved from Wanfang, CNKI, CBMdisc, EMbase, and PubMed databases during 2000–2010 (25).

Tea drinking was found to be beneficial to endometrial cancer. A meta-analysis involving 6,797 cases of endometrial cancer and 858,780 normal controls showed that the pooled RR of endometrial cancer for the highest consumption of tea was 0.99 (95% CI = 0.94∼1.04, p = 0.005), compared with the lowest level. Further study showed that the pooled RR was 0.83 (95% CI = 0.73∼0.95) for case-control study but 1.02 (95% CI = 0.96∼1.08) for cohort study, in terms of study design. For various regions, however, the pooled RR was 0.80 (95% CI = 0.69∼0.93) for Asia, 1.06 (95% CI = 0.94∼1.20) for USA/Canada and 1.01 (95% CI = 0.95∼1.08) for Europe. For different kinds of tea, inverse associations of reduced risk of endometrial cancer were found with black tea (RR = 0.65, 95% CI = 0.46∼0.92) and green tea (RR = 0.73, 95% CI = 0.64∼0.84), respectively (26).

Epidemiological investigations showed that tea drinking was correlated with a reduced risk of digestive tract cancers. An 8-year follow-up cohort study on association between tea consumption and cancer incidence involving 35,369 post-menopausal women in Iowa, USA showed that increasing frequency of tea drinking was inversely associated with digestive tract cancer (p = 0.04, RR = 0.68, 95% CI = 0.47∼0.98) for females who drank ≥2 cups (474 mL/cup) daily, in comparison with the women who occasionally or never drank tea (27). A population-based case-control study including 931 colon cancer, 884 rectum cancer, 451 pancreas cancer patients, and 1,552 control residents which was conducted in Shanghai, China during 1990–1993, showed that the amount of green tea consumption was inversely associated with colorectal and pancreatic cancers (28). Green tea drinking reduced by 39, 78, and 81% in the risk of esophageal cancer, liver cancer and gastric cancer were, respectively observed among alcohol drinkers according to a population-based case-control study conducted in Taixing, China (29). Those with a high level of pre-diagnostic urinary epigallocatechin (EGC) (a component of tea catechins) had a lower risk of colon cancer. The ORs for colon cancer in populations with the lowest, intermediate, and highest tertile of EGC detected were 0.64 (95% CI = 0.33∼1.24), 0.60 (95% CI = 0.30∼1.20), and 0.40 (95% CI = 0.19∼0.83) respectively (p < 0.02) in comparison to those without EGC detected. Methylated EGC showed a similar effect on colon cancer (30). A 6-year population-based prospective cohort study involving 14,001 elderly inhabitants with ages ranging from 65 to 84 years in Shizuoka, Japan showed that green tea drinking was negatively correlated with colorectal cancer (CRC) mortality in a mediate dose dependent manner, with hazard ratio (HR) = 0.24 (95% CI = 0.14∼0.40) for total participants, HR = 0.30 (95% CI = 0.15∼0.61) for men, and HR = 0.18 (95% CI = 0.08∼0.40) for women who consumed ≥7 cups/day, compared to those who drank more than one cup daily (31). A pooled analysis of six cohort studies including 219,080 subjects, among which 3,577 cases were gastric cancer, showed a significant risk reduction for gastric cancer for women with consumption of more than 5 cups daily (multivariate-adjusted pooled HR = 0.79, 95% CI = 0.65∼0.96), but no significant risk reduction for gastric cancer in men (32).

Green tea intake was shown to be negatively correlated with stomach cancer risk (RR/OR = 0.86, 95% CI = 0.74∼1.00). The group with green tea consumption >5 cups/day showed significantly preventive effect on stomach cancer (RR/OR = 0.68, 95% CI = 0.53∼0.87) (33). An 11-year follow-up study involving 1,255 persons suffering from digestive system cancers including esophagus, stomach, liver, pancreas, colorectal and gallbladder/bile duct cancers from 69,310 non-alcohol-drinking and non-smoking women in Shanghai, China showed that normal tea drinking (more than three times per week for more than 6 months) was showed suppressive effects on the overall digestive system cancers (HR = 0.86, 95% CI = 0.74∼0.98, p = 0.01). In comparison with women who never drank tea, and the risk decreased with increase in duration and amount of tea drinking (p < 0.01) (34). A cohort study involving a total of 65,042 Japanese residents aging from 40 to 79 years revealed that green tea consumption showed a protective effect against hematologic neoplasms, with multivariate HR = 0.65 (95% CI = 0.42∼1.00) for leukemia among those who consumed 2 cups of green tea daily or less, 0.73 (95% CI = 0.47∼1.13) for those who drank 3–4 cups of green tea daily, and 0.63 (95% CI = 0.42∼0.96) for those who drank 5 cups of green tea daily (35). An investigation involving 7,355 eligible subjects who were classified into polyp-free, low-risk colorectal adenomas and high-risk colorectal adenomas based on health check-ups with colonoscopies showed that tea drinking was inversely associated with low-risk colorectal adenomas. A larger cumulative dose (≥42 cup-year) was negatively associated with high-risk colorectal adenomas, especially adenomas with villous-rich pathology and when three or more adenomas were present (36).

Tea consumption was revealed to be negatively correlated to the risk of glioblastoma (HR = 0.93, 95% CI = 0.89∼0.98) in a prospective cohort study involving 379,259 UK Biobank participants among which 487 were incident glioma cases. Consuming ≥4 cups/day was decreased the risk of glioma in comparison of no tea consumption (HR = 0.69, 95% CI = 0.51∼0.94) (37). A study on relationship of recent (up to 12 years) or average long-term (up to 30 years) dietary flavonoid intake to the risks of incident glioma based on data from the male Health Professionals Follow-Up Study (1986–2014, n = 49,885), Nurses’ Health Study II (1991–2017, n = 95,228) and the female Nurses’ Health Study (1984–2014, n = 81,688) showed that long-term tea catechins intake was associated with reduced risks of glioma in pooled analysis with comparison of the highest quintile to the lowest quintile of tea consumption (HR = 0.76, 95% CI = 0.57∼1.01, p = 0.04). The association with recent intake was weaker (38). Meta-regression analysis showed that higher tea consumption was related to a reduced glioma risk (RR = 0.84, 95% CI = 0.71∼0.98, p = 0.030), and sensitivity study through removing case-control studies revealed that more consumption of tea was associated with lower risk of glioma (RR = 0.81, 95% CI = 0.70∼0.93, p = 0.004). Daily 1 cup of tea reduced the glioma risk by 3% (RR = 0.97, 95% CI = 0.94∼1.00, p = 0.048) (39).

A population-based case-control study including 1,037 cases with biliary stones, 627 incident cases with biliary tract cancer (BTC) and 959 randomly selected controls of biliary tract disease in Shanghai, China, showed that female tea drinkers have significantly decreased gallbladder cancer risk (OR = 0.56, 95% CI = 0.38∼0.83) and biliary stone risks (OR = 0.73, 95% CI = 0.54∼0.98). However, these risk assessments among male tea drinkers who were more likely to be cigarette smokers were not significantly lower than the non-tea drinkers (40). A 9-year follow-up cohort study involving in 41,761 Ohsaki residents with ages from 40- to 79-year old in Japan showed that green tea drinking was negatively correlated to the liver cancer risk. The multivariate-adjusted HRs for the risk of liver cancer among women were 0.68 (95% CI = 0.35∼1.31) for those who drank 2 cups or less daily, 0.79 (95% CI = 0.44∼1.44) for those who drank 3–4 cups daily, and 0.50 (95% CI = 0.27∼0.90) for those who drank >5 cups daily, compared with the group drinking less than 1 cup/day. The corresponding HRs among men were 0.83 (95% CI = 0.53∼1.30) for 1–2 cups/day, 1.11 (95% CI = 0.73∼1.68) for 3–4 cups/day, and 0.63 (95% CI = 0.41∼0.98) for >5 cups/day (41). Studies on tea consumption in relation to primary liver cancer from 1979 to 2009 in China, which included 13 epidemiological investigations consisting of seven prospective cohort and six case-control studies, showed that consumption of polyphenols abundant tea was negatively correlated with the risk of primary hepatic cell carcinoma (HCC) (RR = 0.77, 95% CI = 0.57∼1.03). Tea drinking showed preventive effects on the development of primary liver HCC both among women (RR = 0.54, 95% CI = 0.37∼0.79) and men (RR = 0.86, 95% CI = 0.77∼0.95) (42). Larger quantities and longer duration of green tea drinking were negatively correlated with primary HCC, among which individuals who consumed green tea more than 30 years showed the lowest risk (adjusted OR = 0.44, 95% CI = 0.19∼0.96) in comparison with non-tea drinkers (43).

Heavy drinking of tea (more than 2 cups per day) was found to be negatively correlated with the risk of lung cancer in a case-control study including 428 hospitalized controls and 427 lung cancer cases in Uruguay (RR = 0.34, 95% CI = 0.14∼0.84) (44). Green tea drinking was found to have a reduced primary lung cancer risk in a case-control study including 649 female patients with primary lung cancer based on the data from Shanghai Cancer Registry from February 1992 to January 1994. However, the effect was dependent on smoking. The risks reduced with the increase in green tea consumption among non-smoking women (OR = 0.65, 95% CI = 0.45∼0.93), but little association was found among female smokers (OR = 0.94, 95% CI = 0.40∼2.22) (45). An investigation on the relationship of diet and physical activity to the risk of lung cancer in the Czechia revealed that black tea drinking had preventive effect on lung cancer among non-smokers (OR = 0.67, 95% CI = 0.46∼0.99) (46). The frequency of black tea drinking (daily or several times per week) showed a reduced risk of lung cancer female non-smokers (OR = 0.65, 95% CI = 0.43∼0.99) (47).

Consumption of hot black tea was found to significantly reduce skin squamous cell carcinoma (SCC) risk (OR = 0.22; 95% CI = 0.10∼0.51) in a population-based case-control study conducted by the Southeastern Arizona Health Study (SEAHS) from 1994 to 1996 (48). Prostate cancer risk was significantly decreased with an increase in quantity, duration and frequency of green tea drinking in a case-control study including 130 incidental prostate cancer cases confirmed histologically and 274 cases without cancer matched by age in southeast China (49). A study in Italy revealed that orally administrating green tea catechins (600 mg/day for 1 year) showed cancer chemoprevention effect on prostate cancer among patients who were at high risk to develop prostate cancer and had pre-malignant lesions (50). A study based on 3 nutrient-specific databases developed by the United States Department of Agriculture which included 466 cases of non-Hodgkin lymphoma (NHL) and 390 controls showed that higher intakes of green tea components including epicatechin, flavonols, proanthocyanidins and anthocyanidins significantly reduced NHL risk (51).

It was found in a case-control study including 107 leukemia cases and 110 orthopedic controls in China mainland that the risk of leukemia decreased with the increase in frequency, quantity and duration of green tea consumption (52). An investigation including 252 cases and 637 controls in China Taiwan revealed that green tea consumption showed significantly negative correlation with leukemia risk among persons from 16- to 29-year-old (OR = 0.49, 95% CI = 0.27∼0.91). The adjusted OR for heavy tea drinking group (more than 550 cups annually) was 0.47 (95% CI = 0.23∼0.97), compared with non-tea drinking group. Large amounts of tea catechins intake showed a reduced risk of leukemia (OR = 0.49, 95% CI = 0.27∼0.91) after adjusting for smoking status and medical irradiation exposure (53). A prospective UK Biobank cohort study involving more than 500,000 participants with ages ranging from 38 to 73 years at enrollment (2006–2010) revealed that tea drinking showed significantly negative correlation to cancer mortality in both male and female (54).

Major components of green tea polyphenols (GTPs) are catechins which include at least eight compounds, i.e., EC, ECG, EGC, EGCG, (+)-catechin-3-gallate (CG), (+)-gallocatechin-3-gallate (GCG), (+)-gallocatechin (GC), and (+)-catechin (GC). The content of EGCG accounts for more than 40% of total content of catechins and so it is the most abundant one (8–10). The research on anticarcinogenic activity of catechins has started since the 1980s (55). EGCG showed a dose-dependent inhibitory effect on human papillomavirus type 16 (HPV-16) induced cervical cancer cell CaSki, in which the inhibitory dose (ID) was 35 μM approximately. Cell cycles were arrested at the G1 phase when incubated with 35 μM EGCG and EGCG-induced apoptosis was occurred after incubation at 100 μM EGCG for 24 h (56). When non-small cell lung cancer A549 cells were incubated with catechin at 600 μmol/L for 24 h, inhibition rate of the cell proliferation was 19.76% (57). The inhibitory effects of catechins on proliferation of cancer cells depend on the components of catechins and the types of cancer cells. For the HSC-2 carcinoma cells, ECG, CG, and EGCG were grouped as highly toxic, EGC as moderately toxic, and C and EC as least toxic. For the HGF-2 fibroblasts, ECG and CG were grouped as highly toxic, EGCG as moderately toxic, and EGC, C, and EC as least toxic (58).

The mechanism inhibiting cancer cell proliferation involves in several aspects, such as cancer cell growth arrest and cancer cell death (Figure 1). Catechin inhibits A549 cells by regulating its cell cycle arrest, increasing the expressions of p21 and p27 and inhibiting the expressions of p-AKT (phosphorylated protein kinase B) and cyclin E1 in a dose-dependent manner in the cancer cells, which also contributed to the suppression of cancer cell proliferation (57). EGCG can trigger cell growth arrest pathways at the G1 stage of cell cycle by regulating p27/KIP1, p21/WAF1/CIP1, cdk4, cdk6, and cyclin D1 (58, 59). Catechin pyrogallol inhibits G2-M transition of human lung cancer cells in cell cycling, resulting in tumor growth suppression (60). In BC T47D cell line, catechin activates the phosphorylation of p38 and JNK/SAPK, and the former suppresses the phosphorylation of cell division control protein 2 (CDC2) and regulates the expression of cyclin dependent kinase (CDK), cyclin A and cyclin B1 proteins, leading to G2 arrest (61). EGCG inhibited the proliferation of human lung cancer cells through targeting the epidermal growth factor receptor (EGFR) signaling pathway (62). EGCG displays anticarcinogenic effects in human BTC cell lines by activating caspase and inducing cell arrest in the sub-G1 phase in cell cycling (63).

Catechins anticarcinogenic effects by inducing apoptosis of various types of tumor cells. EGCG inhibited the growth and induced apoptosis (programmed cell death) of human stomach cancer cell line KATO III (64). EGCG triggers the Fas-mediated apoptosis in LNCaP cells by reducing Fas activity, presumably on the cell surface (65). EGCG induces apoptosis in human prostate cancer cells via changing nuclear morphology and DNA fragmentation (66). The EGCG-induced apoptosis and inhibition of EGCG-mediated Bcl2-associated x protein (Bax) expression were partially blocked by pre-treatment with PD169316 [a p38 mitogen-activated protein kinase (MAPK) inhibitor] (67). Similarly, the EGCG-induced apoptosis, and inhibition of EGCG-mediated expression of p-p38 alpha MAPK and Bax were partially blocked by pre-treatment with SHP-1 inhibitor NSC87877. Therefore, it is considered that EGCG induced apoptosis in NB4 cells via the SHP-1-p38 alpha MAPK-Bax cascade (68). EGCG promoted the growth inhibition and apoptosis of nasopharyngeal carcinoma (NPC) cell lines CNE-2 and 5-8F through inhibition of the SIRT1-p53 signaling pathway (69). EGCG induced inhibition of growth migration, invasion and apoptosis of human osteosarcoma OS cells through regulating the Wnt/beta-catenin pathway (70). Copper dynamics and mobilization played an important role in the EGCG-induced cancer cell apoptosis through regulating cellular copper transporters CTR1 and ATP7A (71).

The necrosis is thought to be a result of ATP depletion to a level unsuitable for cell survival (72). EGCG induced necrosis-like cell apoptosis through a caspase-independent mechanism in C2F8, K562, and CML cell lines. Furthermore, EGCG promoted cell death induced by imatinib (p < 0.01), leading to increased K562 cell death and suppressing the imatinib-resistant viability of cell line K562 (p < 0.01) (72). Green tea extract induced necrosis of cancer cell lines K-562U-937, Clone E6-1, and Jurkat, with half maximal inhibitory concentrations, with IC50 = 88 ± 1.89, 98 ± 1.96, and 205 ± 2.23 μg/mL, respectively (73). Topical (+)-catechin prevents DMBA/TPA-induced carcinoma of the skin squamous cells by modulating antioxidants and tumor necrosis factor α (TNF-α) in BALB/c mice (74). ECG reduced tumor viability and induced necrosis in HT29 cells by inactivating glucose-6-phosphate dehydrogenase and transketolase, the key pentose phosphate pathway enzymes (75).

Combination treatment of EGCG with a low energy ultrasound and a low strength pulsed electric field (PEF) for 72 h induced autophagy of human pancreatic cancer cell PANC-1 (76). Autophagy and apoptosis of glioblastoma cells were strongly induced by 500 μM EGCG treatment (77). Also, EGCG induced autophagy by activating light chain 3 (LC3) and generating reactive oxygen species (ROS) in tumor cells. By enhancing autophagy, the sensitivity of cancer cells had been improved, that’s how EGCG can improve the efficacy of anti-cancer drugs (78).

(−)-Epigallocatechin gallate is crucial factor in governing secretion of α-fetal protein (AFP) modulating autophagic activity of cells HepG(2). EGCG reduced AFP secretion and simultaneously induce AFP aggregation in human HCC HepG(2) cells. EGCG-stimulated autophagy induces the degradation of AFP aggregates in HepG(2) cells, and it directly interacted with protein LC3-I, leading to exposure of the eminent Gly-120 site of LC3-I to the other pivotal binding partners such as promoting the synthesis of LC3-II and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (79). EGCG induced cisplatin-resistant oral cancer CAR cell autophagy in a time- and concentration-dependent manner, during which the AKT/signal transducer and activator of transcription 3 (STAT3) pathway was stimulated and the expression of multidrug resistance 1 (MDR1) was dose-dependently inhibited, suggesting the alteration of AKT/STAT3 signaling and downregulation of MDR1 were partially responsible for the EGCG-induced CAR cell autophagy and apoptosis (80). EGCG promoted nuclear factor-erythroid 2-related factor 2 nuclear translocation and autophagy, enhanced the sensitivity of CRC cells to radiation and inhibited CRC cell proliferation (81).

Reactive oxygen species is a crucial signaling substance that play important roles in carcinogenesis. ROS is a group of highly sensitive molecules containing oxygen which are continually generated by metabolizing organelles such as endoplasmic reticulum, peroxisomes and mitochondria (82). ROS appears to elicit both pro-malignant and anti-malignant effects with an abnormal spectrum of actions. Excessive accumulation of ROS in cells activates the signaling pathways precipitating DNA damage or inducing mutagenesis, resulting in carcinogenicity. Regulation of ROS levels in cells seems to be an encouraging therapeutic measure, especially for screening anticarcinogenic effects of natural products with strong antioxidant properties (83).

Antioxidants are substances that protect interior molecules from oxidative damages (84). Catechins play a role as reducing agent antioxidants in many reactions owing to their abundant hydroxyl groups (85), and have been attracting enormous attention as possible compounds to be used in prevention and treatment of ROS-targeted cancers. The mechanisms underlying the anticarcinogenic potential of catechins include chelating trace metals, inhibiting oxidases responsible for producing the superoxide anion, scavenging ROS, and activating antioxidant enzymes (86, 87). During the DPPH scavenging behavior, an electron on B-ring in catechins molecules is oxidized by DPPH radical, which generates a catechin phenoxyl radical to be tautomerized to a corresponding o-quinone, then comes the nucleophilic attack by the reactive C-8 or C-6 carbon of other catechins molecules in a Michael-type addition reaction to a quinone on B-ring. Both B-ring and A-ring, but not the gallate moiety, can be the antioxidant site of catechins (87–89). The oligomers of C, EC, and resveratrol have shown anticarcinogenic properties on T24 human urinary bladder cancer cells due to scavenge ROS and chelate metal ions Fe²⁺ and Cu²⁺, the antioxidant capacity of which is significantly higher than their preceding monomers (90). In vitro study showed catechin increased manganese superoxide dismutase (MnSOD) gene expression after being incubated with pheochromocytoma cells (PC-12) for 2 days, which can lead to growth inhibition of the cancer cells (91). EGCG can also covalently interact with cysteinyl thiol residues in protein molecules via autoxidation, resulting in functional modulation which may be related to the anticancer effects (92).

There was evidence showing EGCG attenuates DNA damage mediated by ROS in pre-malignant cells, resulting in inhibition of tumor initiation (93). These effects were confirmed in cells hepatocytes, lymphocytes, and colonocytes (94, 95). Carcinogen-induced activation of NOX1/ROS is a signaling pathway in malignant and pre-malignant cells, and treating these cells using EGCG markedly suppressed NOX1/ROS signaling and interrupted the carcinogenesis (96). ROS inhibited MAPK phosphatases, which negatively regulate MAPK, it can be speculated that decreasing ROS levels and subsequent phosphorylation blocking of extracellular signal-regulated ERK1/2 (kinases 1 and 2) contribute partially to the antioxidation activity of EGCG in treating and preventing fibrosarcoma (97).

The invasion and metastasis of cancer cells are the main reasons for cancer recurrence and treatment failure. The metastatic process of cancer cells includes loss of cell adhesion, increased cell motility and invasiveness, entry into the blood circulation, and spread to distant tissues (98). Several in vitro cell experiments and in vivo animal experiments have shown that catechins, especially EGCG, could negatively regulate these steps to effectively suppressed the metastasis and invasion of various cancer cells (Figure 2). Specifically, they included inhibiting metastasis by regulating proteolytic enzyme activity, inhibiting the epithelial-mesenchymal transition (EMT) process, and inhibiting the formation of blood vessels or lymphatic vessels.

The anti-metastatic activity of catechins has been confirmed by in vivo experiments in mouse models. Different doses of EGCG given by gavage administration inhibited liver and lung metastasis of colon tumors implanted orthotopically in the cecum of nude mice to different degrees, high-dose EGCG had a potent inhibitory effect (99). A study using an in vivo mouse model of lung metastasis showed that EGCG treatment significantly reduced the size and number of metastatic nodules in the lungs (100).

Matrix metalloproteinases (MMPs) degrade various protein components in the extracellular matrix (ECM) and inhibit the histological barrier of tumor cell invasion, which plays a key role in tumor invasion and metastasis. Studies on renal carcinoma cells 786-0 and ACHN (101), bladder cancer cell SW780 (102), oral cancer cell OC2 (103), three types of NPC cancer cells (104), hepatocellular carcinoma cell LM6 (105), fibrosarcoma cell HT1080 (106), lung cancer cell CL1-5 (107) showed that EGCG treatment downregulated mRNA or protein expression of MMP-2 or MMP-9, and decreased the ability of the cancer cells to metastasize. Tissue inhibitor of metalloproteinase-3 (TIMP-3) downregulates MMPs activity. Abnormal epigenetic silencing of the TIMP-3 has been associated with the carcinogenesis and metastasis of BC. GTPs or EGCG mediated epigenetic induction of TIMP-3 activity and played a crucial role in blocking invasiveness and gelatinolytic activity of MMP-9 and MMP-2 in BC cells (108). The same effect was found in polycystic ovary syndrome (PCOS) mice treated with oolong tea (109).

Urokinase plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor-1 (PAI-1) are also associated with ECM degradation and are considered biomarkers for metastasis of cancer cells. GTPs inhibited the uPA secretion and suppressed the metastatic behavior of BC cell malondialdehyde (MDA)-MB-231 by inhibiting activator protein-1 (AP-1) and nuclear factor κB (NF-κB) (110), in which an EGCG derivative 3e was able to inhibit the expression of both uPA and PAI-1, resulting in inhibition of the cancer cell metastasis (111). The inhibitive effect of EGCG on the metastasis of prostate cancer cell LNCaP (112) and the above-mentioned oral cancer cell OC2 (103) is also involved in the inhibition of uPA expression.

Epithelial-mesenchymal transition of cancer cells is considered a prerequisite for the acquisition of an invasive/migratory phenotype and subsequent metastasis. EGCG inhibited the in vitro proliferation, migration, and invasion of pancreatic cancer cells, and suppressed mouse xenograft pancreatic cancer in vivo. EGCG halted the “Cadherin switch” and suppressed the expression of mesenchymal markers vimentin and β-catenin (113). Similarly, EGCG suppressed the expression of vimentin in non-small cell lung cancer H1299 cells (114), decreased TGF-β1-induced EMT in anaplastic thyroid carcinoma 8505C cells (115), downregulated the expression of EMT phenotypes of cancer stem cells (CSCs) (116), and inhibited these cancer cells invasion and migration. Catechins containing galloyl moiety, or gallated catechins (such as EGCG, GCG, ECG, and CG) potently suppressed the EMT and migration of ovarian cancer cell ES-2 induced by TGF-β (117, 118), indicating they play a role in inhibiting the metastasis of cancer cells by regulating EMT markers and inhibiting EMT. In addition to the molecular phenotypes of EMT mentioned above, cell motility and cell stiffness are considered to be the mechanical phenotypes of EMT and are also closely related to the metastatic activity of cancer cells. Lower stiffness of cancer cells is connected with stronger metastatic potential in many types of cancers. EGCG and green tea extract increased the mean level of Young’s modulus of cancer cells, resulting in increased cell hardness and stiffness and enhanced inhibition of cell motility (118, 119), leading to the suppression of cancer cell metastasis.

Cancer cell metastasis requires angiogenesis and lymphangiogenesis, which involve hematogenous and lymphatic metastasis of cancer cells, respectively. The inhibitory effect of EGCG in synergy with TNF-related apoptosis-inducing ligand (TRAIL) on migration and invasion of prostate cancer LNCaP cells was achieved by inhibiting the protein expression of vascular endothelial growth factor (VEGF) and angiopoietin 1 and 2 (112). VEGF-D is one of the major lymphangiogenic secretory factors, and it induced lymphatic invasion and metastatic spread of cancer cells (119). EGCG treatment reduced VEGF-D secretion in inflammatory breast cancer cells SUM-149 and SUM-190, resulting in reduction of lymphatic endothelial cell migration and tube formation, leading to inhibition of metastasis of cancer cells (120). EGCG has also been shown to reduce the expression of vascular endothelial growth factor receptor 2 (VEGFR2) in CRC cell line RKO (121). Interleukin IL-6 and IL-8 are confirmed to promote angiogenesis in tumor tissues, and treatment of tumor-bearing mice using EGCG suppressed the expression of IL-6 and IL-8 in a concentration-dependent manner, suggesting that IL-6 and IL-8 might mediate the anti-metastatic activity of EGCG (122). EGCG inhibited the angiogenesis of human thyroid cancer xenograft tumors in nude mice (123).

The immune response to abnormal cells in the body is crucial for maintaining homeostasis. The development of cancer usually triggers a series of immune responses. Tumors can escape immune system attack and migrate by regulating the production of cytokines. Therefore, bioactive substances that target the immune system often have unexpected effects on cancer control, among which catechins are included. Meanwhile, catechins have a significant regulatory effect on multiple sites in the immune system, which has an innate advantage in tumor immunotherapy (Figure 3).

The attack on immune checkpoint is one of the main strategies of tumor cells to escape. The immune checkpoint consists of programmed death factor 1 (PD-1), a receptor molecule secreted by T-cells, and programmed death factor ligand (PD-L1) secreted by tumor cells on the cell membrane. The binding of PD-1/PD-L1 can inhibit the activity of cytotoxic T-cells (CTLs) and even cause T-cell failure, thus protecting tumor cells from immune system attack (124). PD-L1 was induced by epidermal growth factor (EGF) and interferon γ (IFN-γ). Studies were showing that EGCG could regulate the expression of PD-L1 by inhibiting the phosphorylation of STAT and preventing the Janus kinase (JAK)-STAT signaling pathway from activating the transcription factor of PD-L1/PD-L2 (125). PD-L1 mRNA level can also be controlled through the EGFR-Akt signaling pathway (124). IFN-γ is an important factor to stimulate cell expression of PD-L1, so the inhibition of EGCG on IFN-γ could effectively reduce PD-L1 (126). These effects eventually relieve the inhibition of T-cells and improve their attack effect on tumors (Figure 3A).

In addition to PD-1, toll-like receptor 4 (TLR-4) is also a catechin-acting receptor molecule, which may be a target for inhibiting chronic inflammation. Studies have shown that 20% of cancer-related deaths were directly due to TLR-induced cancer cachexia, in which cancer cells released heat shock proteins that acted as TLR-4 agonists in macrophages, skeletal muscle, and fat cells, causing downstream signal transduction. EGCG could effectively downregulate the TLR-4 signal pathway (127). Toll interaction protein (Tollip) is a strong suppressor of this signaling pathway and can directly interact with the ligand IL-1R or TLR4 to inhibit the TLR-induced immune response and negatively regulate signal transduction. Elf-1 is the blocking protein that can inhibit Tollip expression. EGCG achieved the inhibitory regulation of TLR by increasing the level of cGMP in PP2A/cGMP pathway in macrophages to inhibit Elf-1 expression (128). It has also been found that EGCG could inhibit TLR-4/MyD88-mediated NF-κB activation by downregulating both mRNA and protein levels of MyD88 (129) (Figure 3B).

There is an abnormal tumor microenvironment (TME) in cancer patients that causes different immune responses. This low-grade inflammatory state, activated under long-term, low-dose stimulation of specific antigens, often leads to the depletion of immune cells and induces immune tolerance of cancer cells (130). Therefore, some inflammatory factors such as interleukin-2 (IL-2), interleukin-6 (IL-6), and PGE2 are needed to initiate immunosuppression, antagonize this low-grade inflammation and improve the attack of lymphocytes on tumors. EGCG could effectively inhibit inflammation by interrupting the binding of pro-inflammatory factors to receptors and blocking signal transduction such as NF-κB and STAT (131). And the cancer-associated adipocyte (CAA)-like phenotype generated by cells in human triple-negative breast cancer (TNBC) in response to TME. CAA induced cells to secrete many pro-inflammatory factors including CCL2, CCL5, IL-1β, IL-6, and some immune modulators such as COX-2, HIF-1α, VEGF, and PD-L1, which caused carcinogenic through pro-inflammatory effects. EGCG could effectively ameliorate CAA by inhibiting the expression of CAA-related genes and blocking the activation of Smad2 and NF-κB, to alleviate these pro-inflammatory factors and further induces cancer progression (132) (Figure 3C).

Another target of catechins is to regulate immune cell response. First, dendritic cells (DCs), the largest antigen-presenting cells in the immune system, recognize tumor-specific antigens and present them to T-cells (Figure 3D). T-cells initiate a series of responses, typically a Th1 response. Th1-polarized CD4+ T-cells secreted IL-2, IFN-γ, and TNF-α. Activated by IFN-γ and TLRs, macrophages transformed into M1-like macrophages, which can secrete ROS, TNF-α, IL-12, and Nitric oxide (NO) to kill the abnormal cells. At the same time, the cytokines secreted by CD4+ T could further enhance the cytotoxic activity of macrophages and the antigen presentation function of DCs. However, in the TME, Th1 responses were converted to Th2 responses. In this case, Th2 cells secreted IL-4 and IL-10, meanwhile, TGF-β was expressed in large quantities. Simultaneously Foxp3+ B regulatory (Breg) cells and T regulatory (Treg) cells were recruited to initiate humoral immunity. At the same time, m2-polarized macrophages participated in the response to secrete VEGF, IL-10, and TGF-β, which promotes angiogenesis and migration of cancer cells (133, 134). A clinical grade catechin mixture, Polyphenon E, containing about 50% EGCG interacts with the 67 kD laminin receptor (67LR) and promotes the release of granulocyte colony-stimulating factor (G-CSF). The G-CSF then induces the molecular and phenotypic maturation of myeloid-derived suppressor cells (MDSCs) and suppresses their migratory capacity, leading to markedly suppression of their immunosuppressive functions. It was considered that the Polyphenon E could potentially be beneficial to cancer patients through antagonizing cells which interfere with immunotherapy induced antitumor immune responses (135).

(−)-Epigallocatechin gallate plays an efficient regulatory role in this process (Figures 3D,F). EGCG to downregulate the expression of genes necessary for DC presenting antigen, induce the secretion of immunosuppressive factor IL-10, prevent cell surface molecules from binding to receptors, inhibit the low-grade inflammation and prevent the consumption of immune cells (134). Furthermore, EGCG could upregulate the G-CSF secreted by mononuclear MDSCs, a cytokine stimulating the directed proliferation and differentiation of hematopoietic stem cells (133). It also significantly inhibited the accumulation of MDSCs, leading to restoration of the IFN-γ level inhibited by MDSCs, enhancement the activity of CD8+ T-cells, and improvement of the ratio of CD4(+) to CD8(+) T-cells (136), which is beneficial to the improvement of the immune system’s attack on tumor cells. In addition, a phytochemical mixture including EGCG could exert anti-tumor activity by repolarization of m2-polarized macrophages and induce the production of IL12, which could recruit CTLs and natural killer cells (NK) inside tumor cells (137). The combination of EGCG and vitamin A/E increased the adaptive B-cell activity by enhancing adaptive antibody responses and improving CD4(+) and CD8(+) T-cell responses, in which TNF, Il-6, and IL-17 were decreased but IL-15 increased (138). There were studies revealing that EGCG could induce lymphocyte migration in the presence of macrophages, suggesting that the anticancer effect of EGCG depended on the existence of the immune system to a certain extent (139).

It is shown that catechins could act on most of the pathways related to immune regulation (140), among which the star molecules NF-κB and STATS were the targeting signaling pathway of EGCG. EGCG contributes to immunosuppression by inhibiting NF-κB and STATS phosphorylation activation and blocking nuclear translocation to inhibit downstream gene expression (129, 136, 141). EGCG targeting Arg-1/iNOS/NOX2/NF-κB/STAT3 signaling pathway has also been demonstrated in detail, and its activation can enhance immune function (136). Molecular docking experiments proved that catechin has high affinity hydrogen binding ability with NF-κB p52 and TNF-α active site, but has no H-binding effect with anti-inflammatory factor IL 10 (142). Interestingly, EGCG may inhibit the ERK/JUK pathway rather than the p38 MAPK pathway by inhibiting the AP-1 in stimulated human T-cells. EGCG selectively activates signaling pathways (143).

In addition, EGCG also regulated many immune cytokines. For example, TNF-α, an important pro-inflammatory factor in the body, which activates endogenous inflammatory cascades. EGCG antagonized inflammation induced by TNF-α by suppressing the expression of pro-inflammatory cytokines IL-12, IL-6, IL-1β, IL-1α, inflammatory enzymes COX-2 and iNOS, and stimulating anti-inflammatory cytokines IL-10 and IL-4 expression (144). It has also been proved that tea polyphenols could reduce the destruction of the keratinocyte tight junction barrier induced by TNF-α (145). Tea catechins could significantly reduce the expression of TNF-α (142, 143, 146). Chemokines are also a kind of cytokines related to immune cell metastasis. After receiving the antigen information, the immune cells with a specific response will make directional movement along the concentration gradient of the chemical stimulus, which is the chemotaxis of immune cells, and the chemical stimulus that plays locating role is called chemokine. EGCG reduced the migration of neutrophils induced by the chemokine IL-8, inhibited neutrophils recruitment at inflammatory sites, and reduced the expression of pro-inflammatory factors such as MCP-1 (chemokine monocyte chemotactic protein 1) and its receptor (CCR2) (147). Other catechin components have been shown to have immunosuppressive activity. EC could inhibit cell proliferation by inhibiting the AMPK pathway, reducing Akt phosphorylation and mechanistic target of rapamycin (mTOR) expression (148). Like EGCG, EC reduced NF-κB p65 phosphorylation and blocked nuclear translocation, as well as inhibitor levels. By lowering the phosphorylation levels of MAPK members such as P38, JNK, and ERK1/2, EC blocked the signaling pathway (149). EC could inhibit the upregulation of TLR4 and activation of NOX to inhibit downstream events and achieve a preventive effect (150). Immunoactivity of ECG has also been reported, suggesting that ECG inhibited lymphocyte proliferation in an apoptosis-specific manner (151).

In general, catechins maintain the balance of the immune system and have a significant antagonistic effect against most abnormal responses caused by tumors. Catechins have bilateral regulatory effects, i.e., inhibiting the overreaction of the immune system and restoring the immune tolerance caused by tumor cells, in which secretion of cytokines is regulated by their targeting key signaling pathways to maintain a stable and orderly operation of the intracellular immune system.

The advent of a variety of anticancer drugs can be beneficial to controlling cancers to a certain extent, resulting in an improvement in life quality for the patients. However, problems in clinical chemotherapy still exist, such as low bioavailability, cell resistance, and strong toxic and side effects, which greatly limit the use of the drugs. Studies were showing that catechins would play a role in alleviating these side effects induced by chemotherapy, owing to their diversely biological activities including antagonistic effects on oxidative stress. Due to their effects on the important signaling pathways in vivo, catechins are often used as sensitizing agents in combination with drugs. The multiple phenolic hydroxyl groups in catechins can bind with various proteins through hydrogen and ionic bonds, regulating the functions of the proteins. The combination of anti-cancer drugs with catechins, whether before or after drug administration, would alleviate the toxic and side effects induced by the drugs to a certain extent, resulting in apoptosis acceleration of cancer cells and efficacy enhancement of the drugs.

The signaling pathway is the core system in which cells regulate various physiological processes and respond to external stimuli. Normally, cells have a complete set of regulatory mechanisms for initiating and/or inhibiting signal reception, cascade transmission, and ultimately gene expression, but in cancer cells, the signaling pathway is usually overactivated, and the balance is broken. Catechins played an anticarcinogenic role by promoting and/or inhibiting signal transmission through the targeted regulation of multiple links in the signal pathways.

(−)-Epigallocatechin gallate regulates signaling pathways by interacting with membrane receptors. Signal transduction usually begins when an external stimulus activates a receptor on the cell membrane. Membrane target protein 67LR, an important membrane glycoprotein to communicate with the ECM, is widely expressed in tumor cells, which in turn can affect tumor metastasis and regulate many other signal pathways. EGCG matched to 67LR at residues 161–170 (205, 206) and bound to 67LR via lipid rafts (207), blocking the activation of downstream pathways by preventing the combination of the receptors and ligands. In the PKA/PP2A pathway, EGCG binding to 67LR induced the activation of PKA, which dephosphorylates related proteins such as the tumor suppressor Merlin and inhibited the proliferation of cancer cells (208) (Figure 4A). VEGF receptor is a high affinity receptor binding specifically to VEGF. Inactivation of the VEGF signaling pathway suppresses angiogenesis, a common strategy for inhibiting carcinogenesis. EGCG significantly inhibited the expression of VEGF and reduced VEGF receptors (209).

(−)-Epigallocatechin gallate regulates signaling pathways by inhibiting phosphorylation of signaling molecules (Figures 4B–D). STAT is a transcription factor family that combines with DNA to initiate downstream genes transcript. STAT3 suppresses anti-tumor immune responses and promotes the proliferation and migration of cancer cells. EGCG inhibited STAT3 phosphorylation by blocking JAK2 phosphorylation (210) (Figure 4B). The mTOR is a serine/threonine kinase belonging to the PI3K related kinase (PIKK) family, which can regulate cell growth and proliferation (211). EGCG blocked AKT phosphorylation at Ser473, and also acted as an ATP competition inhibitor competing for ATP binding sites of PI3K and mTOR to block the mTOR signal pathway (212) (Figure 4C). MAPK signaling pathway is a core pathway in the cellular regulatory network. EGCG inhibited the MAPK signaling by competing for the phosphorylation sites of downstream proteins (213). Wnt (Wingless-type mice mammary tumor virus integration site family) pathway is a highly conserved signal pathway in species evolution, which plays an important role in early embryo development, organogenesis, and tissue regeneration. The key factor in this pathway is β-catenin, a class of transcription factors that activate the cell division related gene expression. β-catenin binds to the destruction complex when the Wnt ligand does not bind to Frizzled family of proteins (FZD). CK1 and GSK3 in destruction complex can phosphorylate β-catenin, leading to its degradation by E3 ubiquitin ligase. When Wnt is combined with FZD, destruction complex will be confined to the cell membrane and cannot degrade β-catenin, resulting in accumulation of β-catenin in the cytoplasm (214). EGCG could inhibit the Wnt pathway by phosphorylating β-catenin and promoting its degradation (215) (Figure 4D).

(−)-Epigallocatechin gallate suppressed some signaling pathways by reducing the related bioactive proteins (216). Sonic hedgehog (SHH) pathway involves in regulating cell proliferation and differentiation. Excessive activation of SHH leads to carcinogenesis. The downstream transcription factor Gli1 is the key factor of the SHH pathway and it is regulated by membrane receptors Patched (Ptc) and Smoothened (Smo). Normally, Ptc inhibits Smo, and Gli1 binds to the protein kinase inhibitor SUFU and is trapped in the cytoplasm. When SHH binds to Ptc1, the inhibition of Smo is relieved and SUFU is disconnected from Gli1. Then Gli1 will enter the nucleus to initiate the expression of downstream genes (217). EGCG inhibited the expression of Smo, SHH, Gli1, and Gli2 (193) (Figure 5A).

(−)-Epigallocatechin gallate regulates some signaling pathways by interacting with specific inhibitors in the pathway. Nrf2 pathway involves in a variety of diseases and Kelch-like ECH-associated protein 1 (Keap1) is its inhibitor which acts as a bridge between Nrf2 and E3 ubiquitin ligases. When Nrf2 is inactive, it will be degraded by ubiquitination. When Keap1’s key cysteine is modified upon stimulation, it will lose its inhibitive effect and release the Nrf2. The released Nrf2 is then transferred to the nucleus to bind with antioxidant responsive element (ARE), which activates downstream transcription factors. EGCG could inhibit Keap1 and activate Nrf2, resulting in the expression of the downstream antioxidant related genes (218) (Figure 5B). Furthermore, EGCG could induce the re-expression of secreted frizzled-related protein 1 (SFRP1), an inhibitor of the Wnt pathway, which prevents ligand-receptor interaction and is silenced in cancer cells (215).

(−)-Epigallocatechin gallate can regulate some signaling pathways by controlling downstream genes expression in the signal pathway. EGCG could act on the MMP associated with cell apoptosis (207, 219). These enzymes are secreted by tumor cells and can degrade various proteins in the ECM and are the main proteolytic enzymes in tumor invasion and metastasis. EGCG inhibited the activities of MMP2 and MMP9, and promoted the expression of tissue inhibitor of MMPs (TIMp1/2) to suppress the invasion and metastasis of tumor cells (107). Peptidyl-prolyl isomerase NIMA-interacting 1 (PIN1) is a specially phosphorylated prolyl isomerase necessary for mitotic regulation and a direct target of EGCG. EGCG inhibited the activity of PPIase (207) and reduced the expression of PIN, inducing apoptosis (220). NF-κB is normally inactivated when it binds to the inhibitor IkBα. However, when upstream signal factor TNF binds to membrane receptors, IkB kinase (IKK) is activated, which phosphorylates IkBα and dissociates NF-κB. Free NF-κB is released into the nucleus and binds with nuclear DNA to initiate the transcription of genes such as CyclinD1, C-MYC, MMP-9, and VEGF (221). EGCG could significantly downregulate NF-κB activity, inhibiting these gene transcription (222) (Figure 5C).