(–)-Epigallocatechin-3-gallate induces apoptosis and differentiation in leukaemia by targeting reactive oxygen species and PIN1

(–)-Epigallocatechin-3-gallate (EGCG), the major active polyphenol extracted from green tea, has been shown to induce apoptosis and inhibit cell proliferation, cell invasion, angiogenesis and metastasis. Herein, we evaluated the in vivo effects of EGCG in acute myeloid leukaemia (AML) using an acute promyelocytic leukaemia (APL) experimental model (PML/RARα). Haematological analysis revealed that EGCG treatment reversed leucocytosis, anaemia and thrombocytopenia, and prolonged survival of PML/RARα mice. Notably, EGCG reduced leukaemia immature cells and promyelocytes in the bone marrow while increasing mature myeloid cells, possibly due to apoptosis increase and cell differentiation. The reduction of promyelocytes and neutrophils/monocytes increase detected in the peripheral blood, in addition to the increased percentage of bone marrow cells with aggregated promyelocytic leukaemia (PML) bodies staining and decreased expression of PML-RAR oncoprotein corroborates our results. In addition, EGCG increased expression of neutrophil differentiation markers such as CD11b, CD14, CD15 and CD66 in NB4 cells; and the combination of all-trans retinoic acid (ATRA) plus EGCG yield higher increase the expression of CD15 marker. These findings could be explained by a decrease of peptidyl-prolyl isomerase NIMA-interacting 1 (PIN1) expression and reactive oxygen species (ROS) increase. EGCG also decreased expression of substrate oncoproteins for PIN1 (including cyclin D1, NF-κB p65, c-MYC, and AKT) and 67 kDa laminin receptor (67LR) in the bone marrow cells. Moreover, EGCG showed inhibition of ROS production in NB4 cells in the presence of N-acetyl-L-cysteine (NAC), as well as a partial blockage of neutrophil differentiation and apoptosis, indicating that EGCG-activities involve/or are in response of oxidative stress. Furthermore, apoptosis of spleen cells was supported by increasing expression of BAD and BAX, parallel to BCL-2 and c-MYC decrease. The reduction of spleen weights of PML/RARα mice, as well as apoptosis induced by EGCG in NB4 cells in a dose-dependent manner confirms this assumption. Our results support further evaluation of EGCG in clinical trials for AML, since EGCG could represent a promising option for AML patient ineligible for current mainstay treatments.

(-)-Epigallocatechin-3-gallate (EGCG) is a gallate ester obtained by the condensation of gallic acid with the (3R)-hydroxy group of the catechin (-)-epigallocatechin. EGCG is considered the most biologically active and abundant catechin found in green tea, accounting for at least 50% of the total amount of catechins [1][2][3] . Green tea (Camellia sinensis) represents 20% of the total tea produced and consumed worldwide 4,5 ; production involves steaming or pan-frying the freshly harvested leaves, in order to inactive enzymes such as polyphenols oxidase thus preserving the active chemical polyphenolic characteristics of catechins 3,5,6 . Epidemiologic studies have Results EGCG ameliorates the haematological parameters and prolonged survival of PML/RARα mice. EGCG treatment of PML/RARα mice decreased leukocytes number whereas increased platelets number and haemoglobin levels ( Fig. 1A-C). In addition, differential counts obtained from peripheral blood Leishman-Wright-Giemsa-stained smears showed a reduction in the percentage of blasts parallel to an increase of neutrophils ( Fig. 1D-F). Moreover, PML/RARα mice treated with EGCG showed a significant (P < 0.001) longer survival than vehicle-treated mice (Fig. 1G).
EGCG reduces leukaemia immature cells and promyelocytes in the bone marrow, spleen and peripheral blood. Total cells from the bone marrow, peripheral blood and spleen of PML/RARα mice were incubated with CD45, CD34 and CD117 markers. The percentage of CD45 positive cells were selected to exclude erythroid lineage, and subsets were then generated to evaluate the expression of CD34 (hematopoietic stem cells) and CD117 (promyelocytes) markers ( Fig. 2A). The CD34 marker represents leukaemia stem cells, typically observed in AML 29 ; the CD117 marker is the c-kit proto-oncogene encoding the receptor tyrosine kinase involved in the proliferation of leukaemia cells found in the stage one of myeloid differentiation 30 . EGCG treatment reduced the percentage of CD45 + CD34 + cells in the bone marrow (Fig. 2B) and spleen (Fig. 2C) of PML/ RARα mice, and also reduced the percentage of CD45 + CD117 + cells in the bone marrow (Fig. 2B) and spleen (Fig. 2C). In the peripheral blood, we observed a reduction of CD45 + CD117 + , with no changes in CD45 + CD34 + cells (Fig. 2D).
EGCG induces cellular differentiation. EGCG treatment also increased the percentage of myeloid cells (CD11b + Gr-1 + ) in the bone marrow of PML/RARα mice (Fig. 3A). Corroborating these findings, we further observed an increase of circulating granulocytes (CD45 + Gr-1 + cells) (Fig. 3B) and monocytes (CD45 + CD11b + cells) (Fig. 3C). To support these results, we studied EGCG effects on PML bodies of total bone marrow cells of PML/RARα mice by imaging flow cytometry. EGCG treatment decreased diffuse staining of PML bodies in the bone marrow cells of PML/RARα mice while increasing aggregated staining (Fig. 3D). In addition, decreased expression of PML/RAR oncoprotein was detected in the bone marrow cell lysates of PML/RARα mice by western blotting (Fig. 3E). Furthermore, in vitro assays performed in NB4 cells corroborate our in vivo results showing that EGCG treatment (12.5-20.0 µl), similarly to all-trans retinoic acid (ATRA; the drug approved for APL therapy), increased the expression of neutrophil differentiation markers such as CD11b, CD14, CD15 and CD66 in NB4 cells in a dose-dependent manner after 96 hours incubation (Fig. 3F). It is important to mention that EGCG is less potent than ATRA, however the combination ATRA plus EGCG yields a higher increase in the expression of CD15 marker (Fig. 3G).  4A), with no effects in c-Jun expression. In addition, since oxidative stress is important for APL treatment and EGCG displays both pro-oxidant and anti-oxidant effects, we investigated the role of EGCG in ROS levels of the bone marrow cells. EGCG increased mean fluorescence intensity (MFI) of ROS in CD45 + CD34 + cells (Fig. 4B), in CD45 + CD117 + cells (Fig. 4C), and in CD45 + Gr-1 + granulocytes (Fig. 4D). Supporting these findings, we observed an increase in ROS after 2 hours treatment of the NB4 cells with EGCG (12.5-50.0 μg/ml) (Fig. 4E). Furthermore, treatment of the NB4 cells with the antioxidant N-acetyl-L-cysteine (NAC), together with EGCG, induced inhibition of the intracellular ROS production (Fig. 4F), as well as led to a partial blockage of neutrophil differentiation ( Fig. 4G-J) and apoptosis (Fig. 4K), compared to NB4 cells treated with EGCG alone, indicating that EGCG-activities involve/or are in response of oxidative stress.

EGCG increases apoptotic cells in the spleen by modulating BAX, BAD, BCL-2 and c-MYC. As
previously reported, green tea extract reduced leucocytosis and immature cells in the bone marrow and spleen of PML/RARα mice by inducing apoptosis 23 . Thus, we investigated cell death by apoptosis in the bone marrow and spleen cells to verify whether isolated EGCG displays this ability. EGCG treatment induced a significant increase of apoptotic cells in the spleen of PML/RARα mice (Fig. 5A) and no difference was found in the bone marrow cells (Fig. 5B). Consistent with these findings, EGCG treatment also significantly reduced spleen weights of PML/RARα mice (Fig. 5C). To support these results, we next evaluated the expression of BAX, BAD, BCL-2 and c-MYC proteins in the spleen cells lysate. EGCG treatment increased expression of BAX and BAD while decreasing expression of BCL-2 and c-MYC (Fig. 5D). Moreover, the in vitro results further demonstrated that EGCG (12.5-50 µg/ml) induced cell death in NB4 cells by increasing the percentage of apoptotic cells in a dosedependent manner (Fig. 5E).

Discussion
In the present study, we investigated the effect of EGCG on the well-established transgenic model of APL [23][24][25] . Treatment of PML-RARα mice with EGCG (25mg/kg/day) for five consecutive days prolonged survival of mice and reduced leucocytosis, anaemia, and thrombocytopenia. Earlier results of the same mice model treated with green tea extract showed a reduction of leucocytosis only, suggesting that EGCG alone could be more efficient in APL when compared to the whole extract from green tea 23 . In this sense, we evaluated EGCG effect on leukaemia immature cells of PML-RARα mice. Reductions of CD45 + CD34 + and/or CD45 + CD117 + cells were   23,25 . The CD34 represents the leukaemia stem cells, typically observed in acute myeloid leukaemia 29 ; the CD117 is the c-kit proto-oncogene encoding the receptor tyrosine kinase involved in the proliferation of leukaemia cells found in stage one of myeloid differentiation 30 . EGCG treatment also increased the number of mature cells (CD11b + /Gr-1 + ) in bone marrow, and the number of circulating neutrophils and monocytes, suggesting that EGCG induced cell differentiation. To support these findings, we demonstrated that EGCG treatment decreased the expression of the PML/RARα oncoprotein and decreased the diffuse stain of PML bodies in the bone marrow cells while increasing aggregated stain of PML bodies, indicating a higher degradation of PML/ RARα oncoprotein by EGCG. According to Grimwade 31 , normal cells have an aggregated stain of PML bodies whereas leukaemia cells have a diffuse staining. Moreover, our in vitro results showed increased expression of neutrophil differentiation markers such as CD11b, CD14, CD15 and CD66 in NB4 cells after treatment with EGCG; and the combination of ATRA plus EGCG yield higher increase the expression of CD15 marker. These results are consistent with previous data reporting the ability of EGCG to induce leukaemia cell differentiation. Studies in vitro have demonstrated that EGCG upregulated the expression of differentiation markers (CD11B At the end of treatment (16th day) with EGCG (25 mg/kg/day i.p.) or vehicle (Ctrl), total bone marrow and spleen cells of PML/RARα mice were incubated with CD45 to exclude erythroid lineage, and then CD34 and CD117 antibodies were used to detect hematopoietic stem cells and promyelocytes, respectively (A); EGCG induced a reduction of CD45 + CD34 + and CD45 + CD117 + percentage in bone marrow (B) and spleen (C), as well as decreased CD45 + CD117 + percentage in the peripheral blood, with no changes in CD45 + CD34 + cells (D). Statistical significance (Student t test) is indicated as follows: * P < 0.05; ** P < 0.01.  21 demonstrated in vivo that a lower dose of EGCG (12.5 mg/kg) than the one used in our study (25 mg/kg) did not increase mature cells despite effectively reducing PML/RARα protein expression in the bone marrow of animals in pre-leukaemia phase. A possible mechanism that could explain EGCG effect on differentiation is related to its action on PIN1. PIN1 is involved in controlling the activity and stability of proteins 34 . In cancer, PIN is overexpressed and/or over activated 21 , correlating with poor outcomes. PIN1 upregulates >50 oncogenes or proliferation-promoting factors while inhibits >20 tumour suppressors or proliferation-restraining factors 20 . In case of APL, PIN1 stabilize the PML/RARα oncoprotein and the genetic or chemical ablation of PIN1 leads to PML/RAR degradation, thus being effective in treating APL mice or patients 20,21 . There is evidence that EGCG is capable of binding to both functional domains of PIN1, WW and PPIase, leading to the suppression of tumour-promoting activity of  Supplementary Fig. S1. Statistical significance (Student t test) is indicated, as follows: * P < 0.05; ** P < 0.01; *** P < 0.0001.  PIN1 35 . PIN1 has the property of binding to, and catalysing the conversion of proline-directed serine/threonine phosphorylation 20 , common and central signalling mechanism in oncogenic pathways 21 . In this context, we observed a reduction of cyclin D1, NF-κB p65, c-Myc and AKT proteins in bone marrow cell lysates of PML/ RARα mice after EGCG treatment, those all considered oncogenic proteins for PIN1. The literature proposes a correlation between PIN1 and cyclin D1, in which PIN1 increases cyclin D1 transcription directly or by Jun N terminal Kinase and/or cytokine-nuclear factor (NF)-κB pathways, resulting in increased proliferation of cancer cells 20,36 . AKT (also called protein kinase B) is another oncogenic protein stabilized by PIN1. PIN1 isomerization of AKT is critical for activation of the AKT signalling cascade that in turn activates the transcription of genes encoding cyclin D1. In cancer cells, high levels of PIN1 amplify the activation of the AKT cascade thus enhancing tumour progression 37 . Another interesting finding was the reduction of c-Myc expression by EGCG, a transcription factor co-overexpressed with PIN1 in human cancer cells 38 . PIN1 can positively regulate c-Myc transcriptional activity and promoter binding, and this sustained activation of c-Myc can inhibit terminal differentiation 39 . Moreover, we demonstrated herein the decreased expression of 67LR after EGCG treatment, which corroborates with literature data showing decreased expression of these receptor (commonly overexpressed in leukaemia cells) with the ATRA-induced neutrophil differentiation, in NB4 and HL60 cells 1,40 . The 67LR receptor is a non-integrin cell surface receptor for laminin with high affinity 41 and the expression level of the protein correlates with the basement membrane invasion and metastasis of cancer cells 1,40 as well as the drug resistance 42 . Interesting, the 67LR has been identified as surface receptor for EGCG activities 2,41,42 .

Scientific
Another mechanism that could be related to EGCG action on differentiation is the pro-oxidant profile of EGCG. We found increased levels of ROS in immature cells and neutrophils of PML-RARα mice after EGCG treatment. This results are consistent with the literature data demonstrating that EGCG induces the generation of oxidative stress (ROS formation) in vitro using APL cells, B cells and H1299 cells, as well as in vivo, in xenograft mice 14,15,43 . In previous work, we demonstrated that 250 mg/kg/day of green tea extract also increased ROS in neutrophils while decreasing their levels in immature cells 23 . This difference could be attributed to some additional component found in green tea as the whole extract was used. In addition, EGCG has been well established to be auto-oxidized under cell culture conditions, leading to the formation of ROS molecules, such as superoxide radicals and hydrogen peroxide 44 . In this respect, various drugs, such as anthracyclines and arsenic trioxide, have been used for leukaemia therapy and their mechanism of action involve ROS generation 45 . In APL, the induction of ROS by arsenic trioxide is a critical regulator both for the biogenesis of PML nuclear bodies and PML/ RARα degradation 27 . Our in vitro results showed inhibition of ROS production by treatment of the NB4 cells with NAC plus EGCG, as well as a partial blockage of neutrophil differentiation and apoptosis, indicating that EGCG-activities involve/or are in response of oxidative stress. A down-regulation of ROS by antioxidants, such as NAC, blocked the differentiation of APL cell line, while an over-expression of ROS increased cell differentiation has been previously described 46 . Moreover, in the murine APL model, ROS-inducers lead to PML/RARα degradation, regression of the disease and/or longer survival 47 ; our prolonged survival of PML/RARα mice after EGCG treatment corroborates these assumption.
The reduction of immature cells found in the spleen cells could be explained by the increase of apoptotic cells and regulation of BCL-2 family proteins (EGCG reduced BCL-2 expression, whereas increased BAX and BAD expression), and c-Myc protein. These findings are in accordance with literature data showing that green tea catechins has the anti-leukaemia activity mainly due to the induction of apoptosis in vitro 1,14-16 and in vivo, both in murine xenograft model 14,15,48,49 and in APL model 23 . EGCG could affect apoptosis by modulating the level of expression of anti-apoptotic BCL-2 or pro-apoptotic BAX and BAD proteins 50 . In addition, BCL-2 is an apoptotic target suppressed by c-Myc 51 , thus supporting apoptosis induced by EGCG. Moreover, these findings were corroborated by the reduction of spleen weights of PML/RARα mice after EGCG treatment, as well as the apoptosis induced by EGCG in NB4 cells in a dose-dependent manner.
Collectively, our results indicate that EGCG reduced leukaemia burden, induced apoptosis and differentiation thus resulting in a longer survival of PML/RARα mice. This was explained by EGCG ability to modulate oxidative stress activity inducing ROS production and bind to molecules leading to inhibition of enzymes activities (PIN1), modulation of signalling molecules (BCL-2, BAX, BAD, Cyclin D1, c-Myc, NF-κB p65, AKT) and modulation of receptors function (67LR), converging to the induction of apoptosis and differentiation in APL cells ( Supplementary Fig. S6), thus providing new insights to the mechanisms of EGCG in leukaemia. Hence, as EGCG clinical effects has been reported in hematologic malignancies such as chronic lymphocytic leukaemia 7-9 ,  CB17-Prkdc scid /J mice, 18-20 g (The Jackson Laboratory, USA). Twelve days after transplantation, animals were diagnosed with leukaemia, characterized by leucocytosis (leukocytes >30 × 10,000/µL) and/or anaemia (haemoglobin levels < 10 g/dL) and/or thrombocytopenia (platelets < 500 × 10,000/µL) plus the presence of at least 1% of peripheral blood blast [23][24][25] . Mice were then randomly selected (n = 10 per group) to receive i.p. administration of EGCG (25mg/kg/day) (Cayman Chemical Co.) or vehicle (saline) for five consecutive days. At the end of the treatment (16th day), peripheral blood was collected for analysis and mice were then deepening anaesthetized for the sacrifice. Bone marrow cells were obtained using PBS flushing, and splenic cells were obtained by mechanical disruption with PBS. For survival analysis, mice (n = 10 per group) were submitted to the same treatment until the date of death. All experiments were conducted according to National Institutes of Health guide for the care and use of Laboratory Animals and ARRIVE guidelines, and were approved by the Ethics Committee for Animals of the University of Campinas (number 3995-1/A). Imaging flow cytometry. Bone marrow cell suspensions were stained with the antibodies anti-CD117 APC, anti-CD34 APC, PML Alexa-488 and 7-AAD (nuclear stain), from BD biosciences (San Jose, CA, USA) and fixed with BD Cytofix/Cytoperm Kit. A total of 5,000 events were acquired on the ImageStreamX (Amnis/ EMD Millipore, Seattle, WA, USA) and the image analysis was performed using IDEASVR software 31 .
Cell culture and differentiation assay. Human APL cell line, the NB4, obtained from the Deutsche Sammlung von Mikroorganismen and Zellkulturen Gmbh (DSMZ, Braunschweig, Germany) were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), penicillin/streptomycin and amphotericin B, and maintained at 37 °C in a 95% humidified atmosphere (incubator), containing 5% CO 2 . 1 × 10 5 /ml cells were seeded into petri dishes and they were treated with ATRA (1 µM), or EGCG (12.5 and 20 µg/ml) in the presence or absence of NAC (10 mM), from Sigma Chemical Co. (St. Loius, MO, USA) for three hours. After 96h, the expression of the differentiation markers was determined by flow cytometric analysis; the antibodies employed were anti-CD11b FITC, CD14 PE, CD15 APC and CD66 PE. For apoptosis assays, the NB4 cells was seeded as described above and treated with EGCG (12.5-50 µg/ml) for 48h in the presence or absence of NAC (10 mM).