Epigallocatechin-3-Gallate Induces Cellular Differentiation and Reduces Leukemia Burden in PML/Rarα Mice By Increasing Reactive Oxygen Species and Reducing PIN1 Expression
Authors: Della Via Fernanda I , Shiraishi Rodrigo Naoto , Irene Santos , Ferro Karla P , Salazar-Terreros Myriam , Franchi Junior Gilberto C , Rego Eduardo M , Olalla Saad Sara T , Torello Cristiane Okuda 
Published: November 2019

Background: (-)-Epigallocatechin-3-gallate (EGCG) is a gallate ester obtained by the condensation of gallic acid with the (3R)-hydroxy group of (-)-epigallocatechin. This component, extracted from green tea, has multiple effects on signal transduction pathways and enzyme activities which could enhance apoptosis and suppress of cell proliferation, invasion, angiogenesis and metastasis in cancers. This study aims to evaluate the effect of EGCG in an experimental model of leukemia (PML-RARα mice).
Methods: NOD.CB17-Prkdcscid/J mice (12-16 weeks old) received 2Gy irradiation followed by transplantation of leukemia cells obtained from hCG-PML-RARα transgenic mice by i.v. injection in the caudal vein. Establishment of disease was confirmed at day 12 through presence of leukocytosis (>30x103/µL), and/or anemia (<10g/dL), and/or thrombocytopenia (<500x103/µL), associated to the presence of blasts in blood. At 12th day, mice (n=10/group) were randomly selected to receive EGCG (25mg/kg/day) (Cayman Chemical Co., Michigan, USA) or vehicle i.p. for five consecutive days. Mice were then sacrificed and peripheral blood, bone marrow and spleens were collected for flow cytometry and western blot analysis. All experiments were approved by the Ethical Committee for Animal Experimentation of Institution (nº3995-1/A).
Results: Hematological analysis revealed that EGCG treatment reversed leukocytosis (54.09±57.71 vs 11.45±16.08; p=0.0371), anemia (9.60±1.50 vs 11.32±1.36; p=0.0155) and thrombocytopenia (238.5±146.43 vs 475.8±247.91; p=0.0179) and prolonged survival of PML/RARα mice (13 vs 15 days; p=0.0017). Notably, EGCG reduced leukemia immature cells (CD45+CD34+) (8.04±2.49 vs 5.13±1.58; p=0.0060) and promyelocytes (CD45+CD117+) (73.54±12.85 vs 56.26±15.93; p=0.0157) in bone marrow of mice whereas increased mature myeloid cells (CD11b+Gr-1+) (6.15±3.00 vs 14.60±7.83; p=0.0051), possibly by inducing cellular differentiation. These results were corroborated by the reduction in promyelocytes (45.97±11.72 vs 30.29±11.01; p=0.0154), and the increase in neutrophils (CD45+Gr-1+) (38.20±14.34 vs 54.88±14.25; p=0.0178) and monocytes (CD45+CD11b+) (60.22±18.87 vs 76.79±15.59; p=0.0463) detected in peripheral blood. We then evaluated the effect of EGCG on cellular differentiation by studying degradation of PML/RARα oncoprotein. EGCG increased the percentage of cells with aggregated PML bodies stain in the bone marrow of PML-RARα mice, suggestive of higher degradation of oncoprotein, parallel to a reduction in PIN1 expression in bone marrow cells. Higher intracellular levels of reactive oxygen species (ROS) were also detected in leukemia immature cells (2101±1025 vs 3544±614; p=0.0051), promyelocytes (1765±1176 vs 3090±1282; p=0.0271) and neutrophils (1830±1093 vs 3532±1157; p=0.0033) of bone marrow. These results are consistent with literature data demonstrating that the ablation of PIN1 and/or induction of ROS could trigger PML/RARα degradation. EGCG has been reported to inhibit PIN1, a peptidyl isomerase overexpressed and/or over activated in human cancers, which is described as a key target in PML/RARα. Furthermore, apoptosis was detected in spleen cells of PML-RARα mice (5.97±4.19 vs 10.42±3.54; p=0.0197) in parallel to increased expression of BAX, reduced expression of BCL-2, and reduction of spleen weight (0.5587±0.05 vs 0.3949±0.10; p=0.0085).
Conclusion: Collectively, our results support further evaluation of EGCG in clinical trials for acute myeloid leukemia.

Disclosures
No relevant conflicts of interest to declare.