Table 1.

Evidence of autophagy modulation induced by EGCG treatment in experimental cancer models.

Cell line/Animal	Principal Techniques	Main Results	Conclusions	Reference
HCT116 human colon carcinoma cell line	Crystal violet staining for cell viability, LDH cytotoxicity assay, western blotting (LC3, p62), RNA interference	EGCG and TRAIL co-treatment: -increased cell viability; -inhibited TRAIL-induced apoptosis by decreasing the level of death receptors DR4 and DR5; -activated autophagic flux. EGCG effect was reversed by using pharmacological and genetic inhibitors of autophagy (chloroquine and ATG5 siRNA).	EGCG can protect against TRAIL-induced cell death by activating autophagy in TRAIL-sensitive cells. Since autophagy activation may prevent cancer cell death, an autophagy inhibitor is recommended in combination with drugs such as TRAIL because of possible autophagic pathway alterations.	[75]
B16-F10 mouse melanoma cells, AML-12 mouse hepatocytes, Male C57BL/6 mice	CCK-8 assay for cell viability, flow cytometric analysis of autophagy flux activation (GFP-LC3, Bafilomycin A1 treatment) and apoptosis, flow cytometry with dihydroethidium for measurement of intracellular ROS, B16-F10 xenograft mouse model for in vivo study	In B16 melanoma cells, the EGC analogue 4-(S)-(2,4,6-trimethylthiobenzyl)-epigallocatechin gallate: activated autophagy and reduced cell viability by inducing apoptosis;selectively induced ROS accumulation with consequent cell damage;suppressed tumor growth in vivo, while inducing ROS accumulation.Pharmacological inhibition of ROS by NAC attenuated induced autophagy and apoptosis.	The EGC analogue has autophagy- and ROS-inducing ability. Induced autophagy may act as a downstream sensor of ROS that sequentially induces cell death.	[76]
Primary effusion lymphoma (PEL) cells (HHV8-positive)	Trypan blue exclusion assay for cell viability, Caspase 3 activity assay, western blotting (LC3, Beclin 1, MAPKs), acridine orange for acidic vesicular organelle staining	EGCG suppressed viral particle production, and inhibited PEL cell line growth. EGCG induced apoptosis and autophagy through ROS generation; 3-MA autophagy inhibitor and Caspase 3 inhibitor failed to rescue the cytotoxic effect of EGCG. NAC co-treatment reduced ROS level, cytotoxicity, Caspase 3 activation, and autophagy in EGCG-treated PEL cells.	In PEL cells, EGCG induces cell death via a mechanism involving ROS generation, leading to autophagy and apoptosis.	[77]
Mouse 4T1 breast cancer cell line, Balb/c mice	CCK-8 assay for cell viability, flow cytometric analysis of cell apoptosis, Caspase activity assay, western blotting (Beclin 1, ATG5, LC3B), glycolysis-related enzyme activity tests	EGCG inhibited the growth of 4T1 cell line by inducing apoptosis and autophagy. EGCG reduced the expression level of HIF1α and GLUT1, and affected the glycolytic pathway by decreasing activity and/or level of HK, PFK and LDH. EGCG reduced breast cancer xenograft growth in mice.	EGCG suppresses glucose metabolism and has antitumor activity through the induction of apoptosis and autophagy. It might be tested as an adjuvant agent against breast cancer.	[78]
HCT-116 colon cancer cell line	CCK-8 assay for cell viability, immunofluorescence microscopy (Nfr2 nuclear translocation), qRT-PCR (LC3 and Caspase 9)	EGCG increased cell sensitivity to X-ray irradiation and reduced proliferation.Combination treatment with EGCG and radiation: -increased nuclear translocation of Nrf2 autophagic signal; -induced LC3 and Caspase 9 mRNA expression.	Combination treatment with EGCG and radiation enhanced the expression of proteins and mRNAs related to autophagy and apoptosis.	[79]
PANC-1 human pancreatic cancer cell line, HepG2 human hepatocellular carcinoma cell line	MTT proliferation assay, flow cytometry with dihydroethidium for measurement of intracellular ROS, MDC staining for autophagic vacuoles detection, western blotting (LC3, pAkt, Caspase 3 and 9)	Application of low strength pulsed electric field and low energy ultrasound enhanced the growth inhibition effect of EGCG on PANC-1 cells. The triple treatment: -initiated the autophagy pathway through ROS increment; -induced autophagy, that cooperatively caused cell death with apoptosis; -induced cytotoxic autophagy through p-Akt down-regulation and LC3-II upregulation; -overcame the cytotoxicity tolerance of HepG2 cells to EGCG.	EGCG combined with non-invasive and mild physical stimulations might be a promising strategy for anticancer treatment.	[80]
HT-29 human colorectal adenocarcinoma cell line	MTT proliferation assay, flow cytometry and TUNEL staining for cell apoptosis, MDC staining for autophagic vacuoles detection, western blotting (LC3B, Beclin1, Caspase 3 and 9), transcriptomics, and metabolomics analyses	EGCG inhibited cell proliferation, and induced apoptosis and autophagy in HT-29 cells. EGCG treatment was associated with significant changes in gene-expression and metabolic profile. Differential metabolites of CRC are involved in the metabolism of glutathione, glycerophospholipids, starch, and sucrose, among others.	The anti-proliferative activity of EGCG is closely related to apoptosis and autophagy. Transcriptome and metabolome analyses reveal that the anti-CRC effect of EGCG may depend on its modulation of glycerophospholipids metabolism.	[81]
HeLa cell line, HEK293 cell line	MTT proliferation assay, DCFDA ROS assay, flow cytometric analysis of cell apoptosis, mRFP-GFP-LC3 plasmid transfection and confocal microscopy, MDC staining for autophagic vacuoles detection, western blotting (LC3, Beclin 1, Caspase 3 and 9)	EGCG-palmitate remained stable in DMEM medium for a longer time than EGCG. In cancerous cells, EGCG-palmitate induced a lower cell proliferation rate, as compared with normal cells, and promoted apoptosis and autophagy, both resulting from excess of ROS generation.	EGCG-palmitate displayed improved stability and targeted cytotoxicity for cancerous cells. EGCG palmitate expresses its pro-oxidative bioactivity when working as an anticancer drug, and its antioxidant potential in normal cells.	[82]
HT93, OCI/AML2, MOLM-13 and NB4 human AML cell lines	Western blotting (FASN, LC3B, p-mTOR), shRNA transfection for FASN knockdown, acridine orange for acidic vesicular organelle staining, immunofluorescence microscopy	FASN is upregulated in tumor-associated myeloid cells and becomes a target for autophagic degradation during all-trans retinoic acid-induced differentiation of APL cells. Co-treatment with EGCG improved the response to all-trans retinoic acid in NB4 cells, and enhanced FASN protein degradation by autophagy. Lowering FASN expression is associated to mTOR pathway inhibition, promoting autophagy.	Differentiation therapy holds great promise for cancer treatment. Co-treatment with EGCG improves the response to all-trans retinoic acid in APL cell lines and significantly re-sensitizes refractory non-APL AML cells.	[83]
T24 and 5637 human bladder transitional cell carcinoma cell lines	MTT proliferation assay, flow cytometric analysis of cell apoptosis, western blotting (LC3B, Beclin 1, mTOR/p-mTOR, Caspase 3 and 9), shRNA transfection for ATG5 knockdown	EGCG inhibited proliferation and induced apoptosis in T24 and 5637 cells EGCG regulated apoptosis- and autophagy-related protein expression, and significantly increased autophagosome formation in T24 and 5637 cells. In 5637 cells: -knockdown of ATG5 reversed EGCG-induced apoptosis; -co-treatment with EGCG and PI3K/AKT inhibitor LY294002 synergically enhanced apoptosis via activation of autophagy.	EGCG treatment inactivates PI3K/Akt/mTOR pathway, resulting in cancer cell growth inhibition. EGCG inhibits bladder cancer cells proliferation by facilitating crosstalk between apoptosis and autophagy.	[84]
A549 human lung carcinoma cell line, BALB/C male nude mice	MTS proliferation assay, GFP-LC3 plasmid transfection and confocal microscopy, flow cytometry and TUNEL staining for cell apoptosis, western blotting (LC3, ATG5, pERK, p-MEK)	EGCG and Gef synergized in inhibiting the proliferation of Gef-resistant NSCLC cell; the synergy was confirmed also in A549 mouse xenograft models. EGCG inhibited Gef-induced pro-survival autophagy and ERK phosphorylation in A549 cells, thus promoting cell death by apoptosis. EGCG alleviated Gef resistance by inhibiting Raf/MEK/ ERK pathway.	EGCG overcomes A549 Gef resistance by inhibiting Gef-induced autophagy and induced cell death by targeting ERK pathway. This study presents an effective strategy to overcome acquired Gef resistance in NSCLC.	[85]
SaoS2 and U2OS osteosarcoma cell lines	MTT proliferation assay, qRT-PCR (Atg5 and Beclin 1), LC3 immunofluorescence staining, western blotting (LC3), MDC staining for autophagic vacuoles detection, sphere-forming assay	Cell growth inhibition was significantly upregulated when Dox was used in combination with EGCG. EGCG reduced the Dox-induced pro-survival autophagy by decreasing SOX2OT variant 7. EGCG partially inactivated the Notch3/DLL3 signaling cascade, targeting LncRNA SOX2OT variant 7 to reduce the stemness and abate drug-resistance of osteosarcoma cells.	EGCG produced synergistic effects with Dox on osteosarcoma cell growth inhibition by targeting LncRNA SOX2OT variant 7. This study provides a basis for developing anti-tumor treatments targeting osteosarcoma stem cells.	[86]

Abbreviations: TRAIL, Tumor necrosis factor-Related Apoptosis inducing Ligand; NAC, N-acetylcysteine (ROS scavenger); HHV8, Human Herpesvirus 8; 3-MA, 3-methyladenine (autophagy inhibitor); HK, PFK and LDH, hexokinase, pyruvate kinase and lactate dehydrogenase; qRT-PCR, quantitative Real-Time Polymerase Chain Reaction; Nrf2, Nuclear factor-erythroid factor 2-related factor 2; MDC, monodansylcadaverine (marker for autophagic vacuoles); Dox, doxorubicin (chemotherapy drug); NSCLC, non-small cell lung cancer; Gef, gefitinib (EGFR-tyrosine kinase inhibitor); CRC, colorectal cancer; AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; FASN, fatty acid synthase.