Green Tea Extract EGCG Induces Apoptosis in CLL Cells and Overcomes the Supportive Effect of Primary Bone Marrow Stromal Cells Through the Regulation of PI3K/Akt Cascade and Proteasome Activity

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3916-3916 ◽  
Author(s):  
Elena Ponath ◽  
Susanne Schnabl ◽  
Martin Hilgarth ◽  
Dita Demirtas ◽  
Marlies Reiter ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Viability ◽  
Green Tea ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Cell Killing ◽  
Marrow Stromal Cells ◽  
Proteasome Activity ◽  
Leukemic Cells ◽  
Tea Extract

Abstract Abstract 3916 There is accumulating evidence that green tea extract EGCG [(-)-epigallocatechin-3-gallate] may exert a preventive or a direct anti-tumor effect in several tumor types including chronic lymphocytic leukemia (CLL) and clinical trials with EGCG are already on-going. However, EGCG has a broad spectrum of activities and downstream targets. Therefore, it would be necessary to precisely characterize the key targets of this compound and identify the CLL patients who would most likely profit from EGCG. Therefore, the aim of this study was to evaluate the effect of EGCG on the viability of CLL cells in a well characterized cohort of patients and to get insight into its mechanism of action in CLL. Peripheral blood mononuclear cells (PBMC) of 27 CLL patients were included in this study. Patients were characterized according to the Rai/Binet stage, IgVH mutation status and cytogenetics (13q-del, 11q-del, 17p-del, trisomy-12). The percentage of the leukemic cells (CD19+/CD5+) ranged between 60–98%. CLL cells were exposed to a wide range of concentrations of EGCG (0.1 – 200μM) and cell viability was evaluated by cell titer blue (CTB) assays and FACS analysis after 4 hours, 1, 2 and 3 days. Treatment with EGCG was performed in suspension cultures and under co-culture with primary human bone marrow stromal cells (BMSC). Cell viability assays demonstrated a dose and time dependent decrease in the cell viability after the exposure to EGCG with an IC50 ranging between 50–80μM (25–50μg/ml). A moderate variation in the response to EGCG was observed between patients demonstrating the heterogeneity of the disease. No clear correlation between the in vitro response to EGCG and the clinical background and prognostic markers could be observed in this cohort of patients. Annexin V/propidium iodide (Anx/PI) staining showed that EGCG increased the percentage of early apoptotic (Anx+/PI-) and late apoptotic/necrotic cells (Anx+/PI+). These data suggest that EGCG exerts a pro-apoptotic effect and activates other cell killing mechanisms in CLL cells. The leukemic cells (CD19/CD5) were relatively more sensitive to the compound compared to T cells and monocytes. Co-culture experiments showed that EGCG effectively overcomes the supportive effect of BMSC and induces apoptosis/cell killing in CLL cells. BMSC were less sensitive to the compound and a toxic effect was observed at a concentration of 200 μM or higher. RT-PCR showed a downregulation of the catalytic domain p110a and the regulatory domain p85 of phosphoinositide 3-kinases (PI3K) as well as Bcl-2 and Mcl-1 mRNA expression after exposure to EGCG. Western blotting analysis demonstrated a decrease in the phosphorylation of Akt particularly at pThr308 residue and de-phosphorylation of the tumor suppressor PTEN at pSer380 residue in parallel to the induction of PARP cleavage. In addition, EGCG induced a decrease in the protein expression of the activation marker CD23 and the adhesion molecule CD44. Furthermore, proteasome assays showed that EGCG inhibits the chymotrypsin-like activity within 4 hours of incubation in parallel to induction of early apoptosis. This effect was more remarkable after 24 hours. However, EGCG was less effective in proteasome inhibition compared to Bortezomib. In conclusion, these data demonstrate that EGCG induces cell death in CLL cells through a complex mechanism which may involve the inactivation of PI3K/Akt signaling cascade and inhibition of proteasome activity. The results also point to a potential therapeutic effect of EGCG in CLL which warrants further evaluation. Disclosures: No relevant conflicts of interest to declare.

Protection of ALL Cells by Bone Marrow Stromal Cells and the Underlying Molecular Mechanism.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2587-2587
Author(s):  
Yang Yang ◽  
Baohua Sun ◽  
Saradhi Mallampati ◽  
Zhen Cai ◽  
Xiaoping Sun
Keyword(s):  
Bone Marrow ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Stromal Cells ◽  
Cell Apoptosis ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Leukemic Cells ◽  
New Therapies ◽  
Induced Apoptosis

Abstract Abstract 2587 Acute lymphoblastic leukemia (ALL) is one of the fastest-growing hematological malignancies affecting patients with all ages, particularly children. Significant advances have been made in recent years in our understanding of the disease and the development of new therapies, which have led to a greatly improved outcome. Nevertheless, in a significant number of patients with ALL, the disease relapse and become resistant to treatment, causing death of the patients. Increasing evidence suggests that relapse of the disease and resistant to treatment are largely attributed to the protection of the leukemic cells by various components in the microenvironment, such as bone marrow stromal cells. However, the cross-talk between leukemic cells and their microenvironment remains poorly understood. Therefore, better understanding the mechanisms underlying the protection of ALL cells by the microenvironment is of ultimate importance in developing new therapies targeting such protection and eventually eradicating all the leukemic cells to cure the disease. In this study, we used a coculture system with leukemic cells and bone marrow stromal cells (MSC) to mimic the in vivo interaction between the two cell types to explore the molecular events that might be responsible for the protection of ALL cells from Ara-C induced apoptosis. We cocultured human primary ALL cells with hTERT-immortalized normal human MSC and evaluated ALL cell apoptosis by FACS after staining with Annexin V and propidium iodide. In all 8 cases, the MSC provided significant protection of ALL cells from both spontaneous and Ara-C induced apoptosis. For example, the mean Ara-C induced apoptosis of ALL cells cultured without MCS was 42.7% (range, 27–54%), whereas it was 19.1% (range, 8–27%) with MSC. Similar results were obtained with human leukemia cell lines Reh, SEMK2 and RS4.11. We also found that the murine MSC line M210B4 could provide similar protection to ALL cells, whether the ALL cells are primary or cell lines. The reduced apoptosis in the coculture were confirmed by Western blot which showed that MSC could protect ALL cells from Caspase-3 and PARP cleavage. Furthermore, our results showed no significant Ara-C induced reduction in S phase when cocultured with MSC. This phenomenon was associated with decreased cyclinA and CDK2 expression. In addition, we found that cocultured with MSC resulted in phosphorylation of AKT in ALL cells and PI3K inhibitor LY294002 specifically inhibited MSC-induced activation of AKT and promoted ALL cell apoptosis. In addition, beta-catenin and c-myc had increased expression in ALL cells cocultured with MSC, suggesting that Wnt pathway could play a role in MSC-mediated protection. To identify candidate molecules potentially involved in the protection of ALL cells by MSC, we performed gene expression microarray analyses with ALL cells exposed to Ara-C in presence or absence of MSC. Our data indicated that several signaling pathways might be involved in this process, including apoptosis signaling and cell cycle checkpoint control, which confirmed above findings. The top expressed genes identified in the microarray studies were confirmed by RT-PCR. Collectively, our results demonstrated that MSC can protect ALL cells from Ara-C induced apoptosis by multiple signaling pathways, such as those involving PI3K/AKT and Wnt signaling. Hence, targeting these pathways may become potential novel therapeutic strategies to disrupt the support of the microenvironment to ALL cells and to eventually eradicate leukemic cells. Disclosures: No relevant conflicts of interest to declare.

Stroma-Activated Integrin-Linked Kinase (ILK) Supports Survival of Leukemic Cells Via Stimulation of Notch-Hes Signaling: New Therapeutic Opportunities.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2466-2466
Author(s):  
Yoko Tabe ◽  
Linhua Jin ◽  
Nobuko Tanaka ◽  
Michael Andreeff ◽  
Marina Konopleva
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Survival ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Leukemic Cells ◽  
Nb4 Cells ◽  
Hes1 Expression ◽  
Integrin Linked Kinase

Abstract We have previously demonstrated that the BM microenvironment plays a crucial role in the pathogenesis of AML by influencing tumor growth, survival, and drug resistance. Integrin-linked kinase (ILK) has been shown to directly interact with β integrins and phosphorylate AKT in a PI3-kinase (PI3K)-dependent manner to promote cell survival and proliferation. HES-1 encodes a basic helix-loop-helix transcription factor downstream of the Notch receptor, and functions as a positive regulator of hematopoietic and neuronal stem cell self-renewal. Direct co-culture of human mesenchymal stem cell (MSC) and leukemic NB4 cells results in activation of PI3K/ILK/AKT signaling (elevated phospho(p)-Akt, p-GSK3β and nuclear-localized β-catenin), increased expression of Notch1 and Hes1 proteins and upregulation of p-STAT3 detected by Western blot and confocal microscopic analyses. Both, PI3K inhibitor LY294002 (20 μM) and ILK inhibitor QLT0254 (10 μM) specifically inhibited stroma-induced activation of AKT and Stat-3 signaling, suppressed GSK phosphorylation and decreased Notch 1 and HES1 expression. This resulted in massive induction of apoptosis which was not abrogated by stromal co-culture (AnnexinV positivity %, MSC(-) vs MSC(+); control 33.8±2.5 vs 27.3±1.9 p=0.02, QLT 51.4±2.5 vs 55.8±3.5 p=0.26, LY 47.0±8.1 vs 47.9±6.1 p=0.85, 48hrs). In contrast, GSK3b inhibitor BIO (0.1 μM) prevented the serum-withdrawal-induced apoptosis of NB4 cells (AnnexinV positivity %, control 38.1±4.0 vs BIO 25.9±3.4 p=0.003, 48hrs) with marked increase in Notch1 and Hes1 expression detected by confocal microscopy. These observations indicate that Notch signaling is involved in leukemic cell survival stimulated by BM stromal interactions via activation of the ILK-AKT-GSK3β pathway. We have next investigated the effects of leukemic cells on stroma cells. Coculture with NB4 cells caused significant increase in Hes1 and Bcl2 proteins in MSC along with phosphorylation of STAT3 and Akt, which were all abrogated by the treatment with QLT0254 or LY294002. In summary, these results demonstrate that interactions of leukemic and bone marrow stromal cells result in activation of PI3K/ILK/AKT and Notch-Hes signaling in both, leukemic and stromal cells. Disruption of these interactions by specific ILK inhibitors represents a novel therapeutic approach to eradicate leukemia in the bone marrow microenvironment via direct effects on leukemic cells and by targeting activated bone marrow stromal cells.

Bone marrow stromal cells regulate caspase 3 activity in leukemic cells during chemotherapy

2001 ◽  
Vol 25 (10) ◽  
pp. 901-907 ◽  
Author(s):  
James E Fortney ◽  
Wei Zhao ◽  
Sharon L Wenger ◽  
Laura F Gibson
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Caspase 3 ◽  
Marrow Stromal Cells ◽  
Leukemic Cells

scholarly journals Exosomes derived from bone marrow stromal cells decrease the sensitivity of leukemic cells to etoposide

2017 ◽  
Vol 14 (3) ◽  
pp. 3082-3088 ◽  
Author(s):  
Jianling Wang ◽  
Dong Li ◽  
Yong Zhuang ◽  
Jinqiu Fu ◽  
Xue Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Leukemic Cells

Bone marrow stromal cells attenuate mitoxantrone cytotoxicity against HL-60 leukemic cells

1992 ◽  
Vol 3 (6) ◽  
pp. 647-650 ◽  
Author(s):  
Atsushi Takahashi ◽  
Kohkichi Yamamoto ◽  
Minoru Okuma ◽  
Masataka Sasada
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Leukemic Cells

Rooting into the Soil, a Model for the Role of Sox4 in Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3794-3794
Author(s):  
Saradhi Mallampati ◽  
Baohua Sun ◽  
Yun Gong ◽  
Enze Wang ◽  
M. James You ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Leukemic Cell ◽  
Marrow Stromal Cells ◽  
Leukemic Cells ◽  
Cell Stage

Abstract Development and progression of leukemia requires interaction of leukemia-initiating cells with their bone marrow niches. The niches serve as the nursery and shelter for the leukemic cells, which can result in drug resistance, disease recurrence, and minimal residual disease, the most important causes for the death of patients with leukemia. Therefore, obliteration of the interaction between the leukemic cells and their niches is of utmost importance in eradicating leukemic cells during therapy to cure the disease. However, little is currently known of the molecular mechanisms underlying the interaction of the two types of cells. Sox4, a SRY-related HMG-box containing transcription factor that is vital during development, plays an important role in leukemia. Published mouse studies demonstrated that increased expression of Sox4 was associated with leukemogenesis. We determined the expression levels of Sox4 by real-time RT-PCR in 100 human leukemic samples and found high levels of expression in B- and T-ALL, but not in AML, CML, CLL, Sezary syndrome, or T cell prolymphocytic leukemia. In accordance, 7 of the 8 ALL cell lines (the exception was 697) we tested showed high expression levels of Sox4, but AML cell lines, normal mature B cells, T cells, and bone marrow CD34+ cells had low levels of expression. Since the majority of clinical B-ALL cases correspond to the pre-B cell stage, we investigated the role of Sox4 in a pre-B cell line (Nalm6) by lentivirus-mediated RNAi. Remarkably, knockdown of Sox4 in Nalm6 cells caused 70% reduction in the formation of leukemic cell clusters under the monolayer of co-cultured M2-10B4 bone marrow stromal cells, a phenomenon known as pseudo-emperipolesis. Similar results were obtained with ex vivo cultured bone marrow cells from conditional Sox4 knockout mice that displayed B cell developmental arrest at the transition from pro-B to pre-B cell stage and an absence of pre-B cells. These findings suggested that Sox4 is required for the interaction of the developing B cells or leukemic cells with bone marrow stromal cells, a component of the bone marrow niche. Since CXCR4/SDF1-mediated “homing” is known to be required for pseudo-emperipolesis, we tested the effect of Sox4 on Nalm6 cell migration toward SDF1 gradient and found that Sox4 did not affect the migration, suggesting that Sox4 is not acting through “homing”. Instead, our data indicated that the role of Sox4 in the interaction of leukemic cells with stromal cells is most likely mediated by its ability in enhancing the adhesion of the leukemic cells because we found that lentivirus-medicated overexpression of Sox4 in the 697 B cell line caused the suspension cells to display a spindle and adhesive morphology. In addition, 21% of the putative Sox4 downstream genes that we identified by multiple sets of gene expression microarray experiments are known to be involved in cell adhesion. Moreover, we found that the changes in gene expression profile of leukemic cells upon Sox4 knockdown or overexpression significantly overlap with the changes in response to the presence of bone marrow stromal cells in co-culture, indicating that Sox4 pathways are involved in leukemic cell response to stromal cell signaling. Based on these findings we hypothesize that deletion of Sox4 abolishes the interaction between the developing lymphocytes and their niches during lymphopoiesis. Conversely, overexpression of Sox4 may enforce these cells to over-interact with the niches so that they are overexposed to local growth factor stimuli. If superimposed with other genetic and/or epigenetic changes in the developing lymphocytes, such over-interaction may result in the development of leukemia. In case of established leukemia, such over-interaction may lead to the enhanced protection of leukemic cells by their niches. Therefore, the role of Sox4 in the interaction of developing lymphocytes or leukemic cells with their niches is like “rooting into the soil” of a growing tree, abbreviated as “rooting”.

In Vitro Stromal Co-Culture Model For The Evaluation Of Reovirus Oncolytic Therapy For Refractory Pediatric Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5407-5407
Author(s):  
Gaya Narendran ◽  
Matthew F. Clarkson ◽  
Randal Johnston
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Leukemia Cell ◽  
Marrow Stromal Cells ◽  
Oncolytic Viruses ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Pediatric Leukemia

Abstract Introduction Acute lymphoblastic leukemia (ALL) is the most common malignant disease in children, constituting more than a quarter of all childhood cancers. The survival rate of standard/good risk ALL now exceeds 80%. In spite of the significant improvements in outcome, the emergence of resistant disease remains the most common cause of death. Escalation of the intensity of combination chemotherapy with the introduction of second-line drugs is accompanied by cumulative toxicity, with marginal incremental benefits. Hence novel therapeutic approaches are urgently needed to improve the outcome in these patients. In addition to the inherent molecular alterations, recent reports have suggested an active role for the bone marrow stromal microenvironment in promoting both leukemia cell viability and drug resistance. Previous studies have shown a pivotal role for the stromal chemokine CXCL12 (SDF-1) and its receptor CXCR4 in this process. In the multiple myeloma model, inhibition of CXCR4 by the targeted agent AMD3100 (Plerixafor) abrogates the chemotherapy protection conferred by stromal cells. Recent years have seen evidence for the effectiveness of oncolytic viruses as cytotoxic agents against refractory tumors. The strategy behind this approach is to develop viruses that can replicate and cause cell lysis specifically in cancer cells while leaving non-malignant cells unaffected. In this study we explore the utility of reovirus mediated anti-leukemic therapy in an experimental model of pediatric leukemia in the context of co-culture with bone marrow stromal cells. Methods A panel of leukemia cell lines (n=4) representing pediatric ALL and acute myeloid leukemia (AML) were evaluated when co-cultured in the presence of stromal conditioned medium, live bone marrow stromal cells, stromal cells fixed with 0.5% glutaraldehyde, or control human skin fibroblasts (1Br3 cells). After three days in culture, cell proliferation kinetics under each condition was measured by WST-1 assay. The influence of nonspecific leukemia cell attachment was evaluated by the culture of leukemia cells in poly-L-lysine coated plates. Next the leukemic cells were treated with oncolytic reovirus, at various concentrations with and without co-cultures conditions to evaluate the potential of leukemia cells to escape reovirus mediated cytolysis. Finally, the ability of stromal cells to modulate reovirus anti-leukemic activity was evaluated in the presence of various concentrations of AMD3100. Results and Discussion An average of a 2-fold increase in leukemia cell proliferation (in the absence of reovirus) was seen with live stromal co-cultures compared to conditioned media or fixed stromal cells, indicating the requirement of live cell-cell contact with stromal cells in this process. Poly-L lysine coated plates did not increase leukemia cell growth. Our data reveals that, as previously seen with chemotherapeutic drug exposure, the presence of stromal cells is able to decrease the cytolytic activity of reovirus on leukemic cells. Leukemia cells alone exhibit a survival of 10% following a three-day exposure to reovirus (50 MOI). However, in the presence of stromal the survival rate was increased to 40% (p < 0.05). Furthermore, the addition of AMD3100 provided partial restoration of the oncolytic activity (mean 15% survival, n=3 experiments). Stromal conditioned media also decreased reovirus infectivity, although to a lower extent (mean 17% cell survival). Co-culture of leukemia cells with control 1br3 human skin fibroblast cells provided no protection from reoviral cytolysis (mean 10% survival). In this proof-of-concept study, we provide evidence for the first time that the bone marrow niche may provide at least partial protection from oncolytic virotherapy. This indicates the importance of evaluating the efficacy of oncolytic viruses in the context of the tumor microenvironment in future studies. Furthermore, we provide evidence for the consideration of combination therapies with targeted agents such as AMD3100, which may disrupt the stromal protective activities, to increase the effectiveness virotherapy in the treatment of refractory leukemia. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document