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Abstract(–)-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.

ARHGAP21 is a member of the RhoGAP family of proteins involved in cell growth, differentiation, and adhesion. We have previously shown that the heterozygous Arhgap21 knockout mouse model (Arhgap21+/−) presents several alterations in the hematopoietic compartment, including increased frequency of hematopoietic stem and progenitor cells (HSPC) with impaired adhesion in vitro, increased mobilization to peripheral blood, and decreased engraftment after bone marrow transplantation. Although these HSPC functions strongly depend on their interactions with the components of the bone marrow (BM) niche, the role of ARHGAP21 in the marrow microenvironment has not yet been explored. In this study, we investigated the composition and function of the BM microenvironment in Arhgap21+/− mice. The BM of Arhgap21+/− mice presented a significant increase in the frequency of phenotypic osteoblastic lineage cells, with no differences in the frequencies of multipotent stromal cells or endothelial cells when compared to the BM of wild type mice. Arhgap21+/− BM cells had increased capacity of generating osteogenic colony-forming units (CFU-OB) in vitro and higher levels of osteocalcin were detected in the Arhgap21+/− BM supernatant. Increased expression of Col1a1, Ocn and decreased expression of Trap1 were observed after osteogenic differentiation of Arhgap21+/− BM cells. In addition, Arhgap21+/− mice recipients of normal BM cells showed decreased leucocyte numbers during transplantation recovery. Our data suggest participation of ARHGAP21 in the balanced composition of the BM microenvironment through the regulation of osteogenic differentiation.

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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.

Beneficial effects of green tea (GT) consumption have been described, including the ability to reduce cancer development. Polyphenols are the main chemical constituents of GT extract and have been identified as the most effective substances that can inhibit tumorigenesis. Acute myeloid leukemia is an aggressive hematologic malignancy and there is no sufficient evidence that supports a protective role of tea intake on its development. In this concern, the aim of this study was to investigate GT effects in acute promyelocytic leukemia (APL) mice. A total of 1 × 106 leukemic cells obtained from hCG-PML-RARa transgenic mice were injected in the tail vein of 12- to 16-week-old NOD.CB17-Prkdcscid/J mice, after 4-6 h of sublethal cobalt irradiation with 2 Gy. The hematologic counts were monitored weekly, and the following criteria were used for the diagnosis of leukemia: presence of at least 1% of blast in peripheral blood associated with leukocytosis above 30 000 cells/L, hemoglobin levels below 10 g/dL, and thrombocytopenia below 500 × 103 cells/L (He et al, 1997). Twelve days after transplantation, mice were then submitted to daily oral treatment (gavage) with 250 mg/kg/day GT or vehicle only (water) for 5 consecutive days and were sacrificed; bone marrow (BM) and spleens were collected to the assays. Treatment with GT significantly increased the mean number of apoptotic cells in the BM (29.4 ± 5.2 vs untreated 21.0 ± 2.1 %, P < 0.05) and spleen (13.9 ± 3.1 vs untreated 9.2 ± 1.9 % P < 0.05) of mice, evaluated by Annexin V-FITC/PI. GT induced an increase in the median fluorescence intensity (MFI) of cleaved caspase-3 in the BM (83.9 ± 3.6 vs untreated 72.6 ± 4.7, P < 0.05) and in the spleen (75.5 ± 28.2 vs untreated 55.8 ± 7.3, P < 0.01); cleaved caspase-8 in the BM (117.3 ± 9.9 vs untreated 89.1 ± 12.3, P < 0.005) and in the spleen (118.0 ± 31.5 vs untreated 81.5 ± 14.8, P < 0.001); and cleaved caspase-9 in the BM (138.2 ± 52.4 vs untreated 85.8 ± 12.9, P < 0.001) and in the spleen (121.7 ± 49.2 vs untreated 76.5 ± 21.9, P < 0.001) of leukemic mice. Moreover, GT treatment reduced the percentage of CD34+ hematopoietic progenitor cells (32.4 ± 2.3 vs untreated 41.0 ± 0.5 %) as well as of CD117+ cells (33.4 ± 3.7 vs untreated 44.2 ± 1.8 %). We then evaluated the phenotype of cells infiltrated in the spleen. Interestingly, we found that GT induces a decrease in the percentage of CD117+ (40.7 ± 0.3 vs leukemic 44.6 ± 0.9 %) and Gr-1 cells (60.8 ± 0.2 vs untreated 65.6 ± 0.5 %) present in the spleen. We then analyzed the effects of GT in the production of intracellular ROS in the BM subpopulations of CD34+, CD117+ and Gr-1+ cells from leukemic mice. Significant increases in the median fluorescence intensity (MFI) of intracellular ROS production by Gr-1 cells of GT-treated mice were observed (670 ± 103 vs untreated 428.5 ± 5.2). Interestingly, GT induced a reduction of MFI of intracellular ROS production in the CD34+ (167.5 ± 27.1 vs untreated 405.5 ± 73.3) and CD117+ (360 ± 142 vs untreated 1635 ± 40.4) cells. We then studied the expression and localization of CXCR4 and HIF-1α proteins. Studies have shown that ROS increases expression of CXCR4 in cancer and immune cells (Li et al, 2009; Lin et al, 2011; Chetram et al, 2011; 2013) through nuclear translocation of HIF-1α (Lee et al, 2002; Salmeen et al, 2003). Our results showed that GT decreased the MFI of CXCR4 in the leukemic mice (9028 ± 1367 vs untreated 4196 ± 970). Reduction of the nuclear translocation of HIF-1α in GT-treated mice was also observed, using the ImageStream imaging flow. In conclusion, GT treatment in APL mice induces apoptosis of cells in the BM and spleen, confirmed by activation of caspase-3, -8 and -9, probably by modulating the production of intracellular ROS in the leukemic cells. Although GT and its polyphenols are well known as antioxidants, there is evidence that some of the effects of these compounds may be related to induction of oxidative stress in immune cells, which might be responsible for the induction of apoptosis of tumor cells. These pro-oxidant properties may also induce endogenous antioxidant systems in normal tissues that offer protection against cancer. On the other hand, it is possible that, in leukemic cells, which has excessive ROS, the antioxidant effect of GT become more evident. Several potential mechanisms have been proposed including both antioxidant and pro-oxidant effects to polyphenol compounds, but questions remain concerning the relevance of these mechanisms to cancer prevention. Disclosures Torello: Fundação de Amparo à Pesquisa do Estado de São Paulo - Fapesp: Research Funding; Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq: Research Funding. Shiraishi:University of Campinas: Employment.

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The mechanism underlying quiescence and/or mobilization of hematopoietic stem cells and their bone marrow progenitors (HSPC) into circulation are tightly regulated for the continuous supply of peripheral blood cells; however, non-physiological or stress conditions, such as infections, can accelerate these mechanisms. Our results have shown that polyphenols modulate quiescence/mobilization of HSPC, but do not affect mature populations. Thrombin has been reported to induce the rapid HSPC mobilization through coagulation thrombin/PAR-1 axis, and quiescence is maintained across the APC/EPCR/PAR-1 axis (Nat. Med. 2015, 21:1307-17). Our objective was to investigate the effect of polyphenols on thrombin/PAR-1 and APC/EPCR/PAR-1 axis. C57BL/6J mice (6-8 weeks old) were treated with polyphenols from green tea extract (250 mg/kg body weight) orally (gavage) once every seven days and injected (i.p.) at day 7 with lipopolysaccharide (LPS) (100μg;Sigma) (n=6). The control group received vehicle and was injected with LPS (n=6). After 24h of LPS injection, mice were anesthetized for blood collection, and then sacrificed for bone marrow collection. PAR-1 and EPCR expression, nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) phosphorylation were evaluated in HSPC by flow cytometry. The functional ability of HSC was assessed by competitive repopulation assay. Vascular permeability was studied using Evans blue. After LPS injection, mice showed reduced expression of EPCR in bone marrow LSK parallel to an increase of PAR-1 expression in circulating immature and mature cells. Treatment of these mice with polyphenols partially prevented the reduced expression of EPCR in bone marrow LSK (13±3 vs 54±12; p<0.05), but did not affect the increased PAR-1 expression in circulating immature and mature cells. Evans blue assay revealed a reduction in the vascular permeability of the bone marrow of LPS-injected mice treated with polyphenols (3.9±0.5 vs 2.1±0.1; p<0.05). To assess whether polyphenols altered NO production, we measured NO levels and eNOS phosphorylation in immature LSK EPCRhigh (or LT-HSC) cells. NO production is activated by eNOS phosphorylation at Ser1177 and negatively regulated by eNOS phosphorylation at Thr495. LPS injection rapidly increased NO levels and eNOS phosphorylation at Ser1177 in bone marrow LSK of mice. Treatment of these mice with polyphenols reduced the percentage of bone marrow LSK EPCRhigh cells with higher intracellular NO (52±2.8 vs 28±5.6; p<0.01) and increased eNOS phosphorylation at Thr495 in immature LSK. In order to evaluate the action of polyphenols on the functional ability of HSC, a competitive bone marrow repopulation assay was performed. Donor mice (C57BL/6J) received or not polyphenols followed by LPS injection (treated group: Polyphenols+LPS; control group: LPS), and bone marrow cells were transplanted (1:1) together with bone marrow cells of competitors (B6.SJL-PtprcaPepcb/BoyJ) in lethally irradiated recipients (B6.SJL-PtprcaPepcb/BoyJ). Mice were followed for 16 weeks and hematological analysis revealed no difference in circulating leukocytes, platelets or hemoglobin levels. Transplanted mice (recipients) presented a higher percentage of CD45+ cells from Polyphenols+LPS donors (33.7±13 vs 78.6±0.9; p<0.05) in the peripheral blood, as well as increased number of T lymphocytes (6.7±4.5 vs 37.2±2.9; p<0.05) and myeloid cells (68.5±1.7 vs 82.5±3.5; p<0.05) from Polyphenols+LPS group. After 16 weeks, mice were euthanized and a higher percentage of LSK (or HSC) and LSK EPCRhigh (or LT-HSC) cells from Polyphenols+LPS donors were detected in the bone marrow, although only the percentage of LSK EPCRhigh was statistically different (0.0014±0.0001 vs 0.0032±0.001; p<0.05). Taken together, our results indicate that polyphenols increased the functional ability of HSC in LPS-injected mice showing increased percentage of bone marrow LSK EPCRhigh cells, which are the most quiescent stem cells with strong self-renewal ability. Polyphenols reduced EPCR expression and NO production in immature cells of LPS-injected mice, exhibiting an anti-inflammatory effect that leads to the maintenance of barrier integrity and quiescence of cells, which was corroborated by reducing vascular permeability in the bone marrow. Thus, polyphenols appear to modulate quiescence/mobilization of HSPC through APC/EPCR/PAR-1 axis. Disclosures No relevant conflicts of interest to declare.