Interleukin-21-Induced Apoptosis and Cell Death of Diffuse Large B-Cell Lymphoma (DLBCL) Cell Lines and Primary Tumors Are Associated with an Induction of Bim.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2503-2503
Author(s):  
Kristopher A. Sarosiek ◽  
Jun Chen ◽  
Dien G. Pham ◽  
Hovav Nechushtan ◽  
E. Avisar ◽  
...  
Keyword(s):  
Cell Death ◽  
Animal Models ◽  
Cell Line ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Lymphoma Cell ◽  
Primary Tumors ◽  
Protein Levels ◽  
Induced Apoptosis ◽  
Anti Tumor Activity

Abstract IL-21, a member of the IL-2 cytokine family, is reported to have an immune-mediated anti-tumor activity against renal cell carcinoma, malignant melanoma and Non-Hodgkin’s Lymphoma (NHL) cell lines in xenograph animal models. Whether IL-21 exhibits direct anti-tumor activity against NHL cell lines and primary tumors is presently unknown. We analyzed seven DLBCL and two Burkitt’s lymphoma cell lines for IL-21 receptor (IL-21R) expression. IL-21R was expressed at high levels in the two Burkitt’s lymphoma cell lines (RAJI and RAMOS). Out of the seven DLBCL lines, four expressed high levels of the receptor (OCI-LY-3, OCI-LY-7, OCI-LY-19, and RCK-8) while three had low receptor expression levels (OCI-LY-10, SU-DHL-4, and SU-DHL-6). IL-21 stimulation induced tyrosine phosphorylation of STAT-1, -3, and -5 as early as fifteen minutes post treatment in DLBCL lines expressing either high (OCI-LY-3 and RCK-8) or low (SU-DHL-6 and OCI-LY-10) levels of IL-21R. In six of the seven DLBCL lines tested, IL-21 dramatically inhibited or completely abolished cellular proliferation at 25 ng/mL and 100 ng/mL concentrations, respectively. In contrast, one DLBCL cell line (OCI-LY-3) exhibited a threefold increase in cellular proliferation after stimulation with 25 ng/mL IL-21, but proliferation was effectively inhibited by 100 ng/mL IL-21. Marked apoptosis and cell death were observed by flow cytometry at 72 hours post IL-21 exposure in all nine NHL cell lines tested with the exception of OCI-LY-3 which exhibited no significant change in cell viability. The IL-21-induced apoptosis was associated with an activation of caspases 3/7, 8, and 9, detected as early as 12 hours post IL-21 exposure. IL-21 also induced an increase in the levels of the pro-apoptotic protein Bim in all the cell lines exhibiting marked apoptosis. In contrast, Bim protein levels decreased in response to the IL-21 stimulation in the resistant OCI-LY-3 DLBCL cell line. IL-21 treatment led to a decrease in the protein levels of Bcl-2 in all the analyzed cell lines except OCI-LY-3. An increase in expression of Bcl-6 protein in three of the four tested DLBCL cell lines was observed in response to IL-21 exposure. In primary tumors, IL-21 induced apoptosis in two of two DLBCLs, two of three follicular lymphomas, and two of six chronic lymphocytic leukemias. No apoptosis or cell death was induced in normal peripheral B-lymphocytes or in the HeLa or 293T control cells. These results suggest that IL-21 exhibits a direct anti-tumor effect on the DLBCL cell lines and primary tumors and point to a potential applicability of IL-21 in anti-DLBCL therapy. Further work interrogating the molecular mechanism of the IL-21-induced apoptosis of DLBCL cells in vitro and in animal models is in progress.

scholarly journals Dissociation between p93B-myb and p75c-myb expression during the proliferation and differentiation of human myeloid cell lines

Blood ◽  
1994 ◽  
Vol 83 (7) ◽  
pp. 1778-1790 ◽  
Author(s):  
M Arsura ◽  
MM Luchetti ◽  
E Erba ◽  
J Golay ◽  
A Rambaldi ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Myeloid Cells ◽  
Indirect Evidence ◽  
Granulocytic Differentiation ◽  
Gm Csf ◽  
Protein Levels ◽  
Proliferation And Differentiation ◽  
Myb Protein

Direct and indirect evidence strongly indicates that the proto-oncogene c-myb plays an important role in the regulation of both the proliferation and differentiation of hematopoietic cells. In addition, recent data suggest that the structurally related B-myb gene is also necessary for the proliferation of these cells. To help understand the relationship between these two related gene products during proliferation and differentiation of myeloid cells, we have studied in parallel the regulated expression of c-myb and B-myb RNAs and proteins in human myeloid cells that were either growth-arrested or induced to differentiate along different pathways. For this purpose, we have produced a polyclonal antibody directed against a fragment of the recombinant B-myb protein. We have thus been able to detect the B-myb protein in human cell lines and have found it to be a 93-kD protein localized in the nucleus. We have chosen two models to study the expression of both c-myb and B-myb mRNAs and proteins during myeloid proliferation and differentiation. One of the models was the HL-60 cell line, which can be induced to differentiate towards the monocytic pathway with either phorbol ester (phorbol myristate acetate) or vitamin D3 and towards the granulocytic pathway with either dimethyl sulfoxide or retinoic acid. In addition, we have studied another recently established human leukemic cell line, called GF-D8, which is strictly dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) for proliferation. The results show that the expression of B- myb RNA and protein closely correlates with proliferation in all experimental setups studied, whereas the c-myb protein levels do not always do so. We observed that the c-myb protein levels decreased well before the decrease of B-myb protein and of proliferation itself during differentiation toward monocytes. Such a difference was not present during granulocytic differentiation, in which c-myb levels decreased, if anything, later than those of B-myb and proliferation. Most striking was the finding that high levels of c-myb RNA and protein, but not of B- myb, were present in the GF-D8 cell line, even after growth arrest by GM-CSF deprivation. These data suggest that B-myb may function solely in the regulation of cellular proliferation, whereas c-myb has additional functions, for example, in the maintenance of an undifferentiated state.

In Vitro Evaluation of Apoptosis and Cell Death of Neuroblastoma Cell Lines Exposed to Busulfan.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4459-4459
Author(s):  
Morris Kletzel ◽  
Sarah C. Tallman ◽  
Marie Olszewski ◽  
Wei Huang
Keyword(s):  
Cell Death ◽  
Cell Viability ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Annexin V ◽  
Resistant Cell ◽  
Primary Tumors ◽  
Induced Apoptosis ◽  
Neuroblastoma Cell Lines

Abstract Objective: While busulfan is a commonly used chemotherapeutic agent in the treatment of many hematological diseases, its effectiveness against neuroblastoma is still in question. This study aims to assess the degree of apoptosis and cell death in neuroblastoma cell lines and primary neuroblastoma tumors when exposed to varying doses of busulfan. Materials and Methods: Cultures from established cell lines SKN-SH, SKN-DOX-R, IMR-5, and NGP (n=4), as well as cultures from primary tumors (n=2) were seeded at 106 cells/ml in RPMI640 supplemented with 10% fetal bovine serum (FBS) and transferred to 24-well plates, where cells were exposed to 1ml of busulfan at 0, 0.001, 0.005, 0.01, 0.05, and 0.1mg/ml per well. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 for 72 hours. Wells were sacrificed after 0, 6, 24, 48 and 72 hours and tested with Annexin V and PI; 10,000 events were measured by flow cytometry. The percentage of apoptotic and dead cells was plotted in a graph and a t-test was performed against the untreated control. Results: After 24 hours, there was a significant decrease in cell viability of each dose when compared to the control untreated cells (p<0.005). 24 Hour % Cell Viability for Varying Doses of Busulfan (mg/ml) Dose 0 Dose 0.001 Dose 0.005 Dose 0.01 Dose 0.05 Dose 0.1 Mean 66.1 44.4 40.3 40.7 37.7 39 SEM 5.56 5.17 5.96 6.17 6.03 5.60 Median 65 33.5 38 39 37 31 Range 39 to 97 14 to 87 4 to 89 6 to 93 4 to 77 5 to 88 The overall mean decrease in cell viability when compared to the control was 25.7%. However, there were only modest differences in effectiveness when comparing the doses, with an average of only 5–7% difference between doses. Further, there was much variability between the different cell lines, some with changes in apoptosis and cell death of over 50%, while other lines showed no changes at all. Limited differences were seen after 6 hours, and after 72 hours any effect of busulfan was masked by cell death due to other factors, as seen through increased cell death in untreated cells. Conclusion: Busulfan induced apoptosis and cell death in vitro in neuroblastoma cell lines at a mean of 76.43% for non-resistant lines, 59.33% for primary tumors and 35% for resistant cell lines (at middle dose 0.01mg/ml). The resistance of certain cell lines confirms the difficulties of treating multi-drug resistant cells in often heterogeneous neuroblastoma tumors. That some cell lines were responsive shows the potential of using busulfan to treat neuroblastoma in the future.

The Diterpenoid Lactone Andrographolide (ANDRO) Stimulates Autophagy and Induces Reactive Oxygen Species (ROS)-Dependent Apoptosis in Lymphoma Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4991-4991
Author(s):  
Shuo Yang ◽  
Andrew M. Evens ◽  
Sheila Prachand ◽  
Leo I. Gordon
Keyword(s):  
Cell Proliferation ◽  
Cell Death ◽  
Cell Lines ◽  
Redox State ◽  
Annexin V ◽  
Lymphoma Cell ◽  
Time Dependent ◽  
Cellular Redox ◽  
Forkhead Transcription Factors ◽  
Induced Apoptosis

Abstract ANDRO is a diterpenoid lactone isolated from Andrographis paniculata (King of Bitters), an important herbal medicine used in China. It has been reported to have anti-inflammatory, anti-hypertensive, anti-viral and immunostimulant properties. It has also been shown to inhibit cancer cell proliferation and induce apoptosis in HL-60 (leukemia), PC-3 (prostatic adenocarcinoma), MDA-MB-231 (breast cancer), HepG2 (liver cancer), HeLa (cervical cancer) and HCT116 (colorectal cancer) cell lines. The diterpenoids have been found to generate ROS and may increase apoptosis by altering the cellular redox state. We hypothesized that ANDRO would lead to cell death in lymphoma cell lines and that the effect may be related to altered cellular redox state. We studied the Burkitt p53 mutated Ramos cell line, the mantle cell lymphoma line Granta and L428, a resistant EBV-negative Hodgkin lymphoma cell line. We found that after incubation with increasing concentrations of ANDRO, there was dose and time-dependent cell death as measured by MTT. The IC50 (concentration that achieved 50% cell proliferation inhibition) at 48h was 20μM for Ramos, 40μM for Granta, and 50μM for L428. ROS was measured by oxidation of 2’7’dichlorofluorescein diacetate (DCFDA) to dichlorofluorescein (DCF) and analyzed by fluorescence-activated cell sorting (FACS) following incubation at 1hour (h), 2h, 3h, 5h, 38h, and 48h with ANDRO (20–80μM). ANDRO increased ROS production in all lymphoma cell lines, which was abrogated by the antioxidant N-acetyl-L-cysteine (NAC). Maximum ROS generation with ANDRO was seen at 48h for Ramos (1.7 fold), 5h for Granta (1.6 fold), and 38h for L428 (2.4 fold). To determine the mechanism of cell death, we measured apoptosis by Annexin-V/propidium iodide (PI), and detected by flow cytometry (FACS). Cells were treated with ANDRO in the presence or absence of the reduced glutathione (GSH) depleting agent buthionine sulfoximine (BSO) (100μM) for 28h, 48h, and 72h. We found that the AC50 (concentration that achieved 50% apoptosis) was 40μM for Ramos at 72h, 40μM for Granta at 48h and &gt;80μM for L428 at 48h, while in the presence of BSO it was &lt;10μM for Ramos at 72h, between 30–40μM for Granta at 28h and between 30–40μM for L428 at 48h. Apoptosis was completely blocked, by NAC, both in the presence and absence of BSO. Further, ANDRO induced PARP cleavage and activation of caspases 3, 8, and 9 in Granta and Ramos. Next, we explored the relationship of ANDRO and Forkhead transcription factors. ANDRO caused dephosphorylation of FOXO3a or FOXO1, in a dose- and time-dependent manner, and this was reversible by NAC. Downstream proteins of FOXO3a, Bim, p27kip1 and the isoforms of the autophagy-related protein LC3B were upregulated, and this was reversed by NAC. The LC3B isoform-II, which is cleaved from LC3B-I, is a marker of autophagy activation. To determine the role of autophagy in cell death related to ANDRO, we inhibited autophagy with 3-methyladenine (1–2mM) and found significant enhancement of ANDRO-induced apoptosis in Granta and Ramos. Finally, ANDRO induced apoptosis (&gt;60% Annexin-V+/PI+) in malignant B-cells from a patient with chronic lymphocytic leukemia/small lymphocytic lymphoma (trisomy 12, peripheral blood absolute lymphocyte count 95.2 K/uL, bulky adenopathy) very low concentrations (5μM at 18h) in vitro, which was also reversible with NAC. We conclude that ANDRO induces ROS-dependent apoptosis in lymphoma cell lines and in a primary tumor sample, which is enhanced by depletion of GSH and inhibited by the antioxidant NAC. These effects appear to proceed through caspase activation and inhibition of autophagy, and are in part dependent on signaling through forkhead transcription factors and altered cellular redox pathways. Further studies of diterpenoids as single agents or in combination with other anti-lymphoma agents are warranted.

scholarly journals Establishment of a Therapeutic Anti-Pan HLA-Class II Monoclonal Antibody That Directly Induces Lymphoma Cell Death Via Large Pore Formation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5488-5488
Author(s):  
Shuji Matsuoka ◽  
Yasuyuki Ishii ◽  
Atsuhito Nakao ◽  
Hiroshi Masutani ◽  
Satoshi Takahashi ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Class Ii ◽  
Hla Class Ii ◽  
Lymphoma Cell ◽  
Death Process

Abstract To develop a new therapeutic monoclonal Antibody (mAb) for Hodgkin lymphoma (HL), we immunized a BALB/c mouse with live HL cell lines, alternating between two HL cell lines. After hybridization, we screened the hybridoma clones by assessing direct cytotoxicity against a HL cell line not used for immunization. We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line. A newly established mouse anti-human mAb (4713) triggered cytoskeleton-dependent, but complement- and caspase-independent, cell death in HL cell lines, Burkitt lymphoma cell lines, and advanced adult T-cell leukemia cell lines. Intravenous injection of mAb 4713 in tumor-bearing SCID mice improved survival significantly. mAb 4713 was revealed to be a mouse anti-human pan-HLA class II mAb. Treatment with this mAb induced the formation of large pores on the surface of target lymphoma cells within 30 min. This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pores formation. This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma. Disclosures No relevant conflicts of interest to declare.

Synergy with combination of AKT inhibitor (MK-2206) and paclitaxel in head and neck squamous cell carcinoma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e13532-e13532 ◽  
Author(s):  
Omar Ahmed ◽  
Wen-Liang Kuo ◽  
Madhavi Nagilla ◽  
Jeannette S. Messer ◽  
David L. Boone ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Line ◽  
Cell Lines ◽  
Protein Localization ◽  
Competent Cell ◽  
Akt Inhibitor ◽  
Tissue Samples ◽  
Molecular Alterations ◽  
Protein Levels ◽  
Effective Combination

e13532 Background: Though there have been many advances in the treatment of HNSCC, the 5-year survival rate remains at 50%. This is partly due to molecular alterations such as the activation of the PI3k-AKT pathway in HNSCC, which has been associated with treatment failure. We stained human tissue samples and saw increased expression of p-AKT and pS6 in HNSCC compared to normal and dysplasia. This led us to investigate the role of the AKT inhibitor MK-2206 in combination with an already established chemotherapeutic agent, paclitaxel. Methods: Cell viability and Combination index were used to determine synergy in 4 HNSCC cell lines. PI exclusion and Annexin/PI staining were used to determine cell death and apoptosis, respectively. Autophagy was measured through changes in LC3BII protein levels using western blot analysis and a stably transfected GFP-LC3BII cell line was used to detect changes in protein localization under confocal microscopy. Results: All cell lines were sensitive to paclitaxel while only some were sensitive to MK-2206. However, there was effective synergy to the combination treatment in all cell lines. Apoptotic synergy was observed in some cell lines indicating that the combination stimulates cell death in the appropriate context. In one autophagy competent cell line, MK-2206 induced robust autophagy while paclitaxel blocked a latter step in the pathway leading to an accumulation of autophagosomes prior to induction of apoptosis. Conclusions: Paclitaxel and MK-2206 appears to be an effective combination in HNSCC possibly through their effect on autophagy trafficking, leading to an accumulation of autophagosomes and eventual apoptosis. Clinical trials examining this combination are warranted.

Triptolide Sensitizes Leukemic Cells to TRAIL Induced Apoptosis Via Decrease of XIAP and p53-Mediated Increase of DR5.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 528-528
Author(s):  
Bing Z. Carter ◽  
Duncan H. Mak ◽  
Wendy D. Schober ◽  
Martin Dietrich ◽  
Clemencia Pinilla ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Chinese Herb ◽  
Leukemia Cell ◽  
Leukemic Cells ◽  
U937 Cells ◽  
Apoptosis Resistance ◽  
Protein Levels ◽  
P53 Signaling ◽  
Induced Apoptosis

Abstract Triptolide, a recently identified anticancer agent from a Chinese herb, has been shown to synergistically enhance TRAIL-induced cell death in various solid tumor cell lines. We have found that triptolide potently induces apoptosis in leukemic cell lines and blasts from AML patients at least in part by decreasing XIAP levels. XIAP is known to be a resistance factor in TRAIL-induced cell death. XIAP is highly expressed in AML blasts and primary AML cells generally are insensitive to TRAIL. We therefore hypothesize that triptolide will sensitize AML cells to TRAIL-induced apoptosis. We treated OCI-AML3, U937, and Jurkat cells with sub-optimal concentrations of triptolide, TRAIL, and their combination. At concentrations showing no or minimal effects by each agent alone, their combinations significantly promoted cell death with combination indices (CI) <1.0 for all three leukemia cell lines. To ensure that XIAP contributes to TRAIL resistance, we treated U937 cells overexpressing XIAP (U937XIAP) and the control cells (U937neo) with TRAIL. U937XIAP cells were 6-fold more resistant to TRAIL (IC50=455.8 ng/ml) than U937neo cells (IC50=74.3 ng/ml). We then treated these cells with TRAIL and 1396–11, a small molecule XIAP antagonist that binds to BIR2 of XIAP and promotes caspase-dependent apoptosis. At 3μM, 1396–11 had no significant effect on survival of either U937neo or U939XIAP cells. When combined with TRAIL, 1396–11 augmented TRAIL-induced cell death of both U937neo (IC50=45.0 ng/ml with 1396-11 vs. IC50=74.3 ng/ml without) and U939XIAP cells (IC50=318.9 ng/ml with 1396–11 vs. IC50=455.8 ng/ml without). Furthermore, we observed that triptolide decreased MDM2 and increased p53 protein levels in p53 wild type OCI-AML3 but not in p53 null U937 cells. TRAIL receptor DR5 has been shown to be regulated by p53. To elucidate the role of p53 in TRAIL-induced cell death sensitized by triptolide, we treated OCI-AML3 and U937 cells with triptolide and examined DR5 expression. We found that triptolide induced DR5 protein levels in OCI-AML3, but much less so in U937 cells. Treatment of OCI-AML3 cells with Nutlin3a, a MDM2 inhibitor that binds to MDM2 and stabilizes p53, increased DR5 protein levels and sensitized to TRAIL-induced cell death. Knockdown of p53 with retrovirus expressing p53siRNA in OCI-AML3 cells abolished cellular responses to nutlin3a and significantly decreased the sensitization to TRAIL by nutlin3a. Finally, we treated OCI-AML3 cells with 1396–11, nutlin3a, and TRAIL and our results showed that the triple combination (CI=0.045 at 24 hours, average of ED50, ED75, and ED90) was more effective in inducing cell death than either 1396–11 and TRAIL (CI=0.066) or nutlin3a and TRAIL (CI=0.190) combinations supporting our notion that triptolide sensitizes to TRAIL-induced cell death by modulating both XIAP expression and p53 signaling. Collectively, our studies suggest that inhibition of XIAP and induction of DR5 mediated by p53 activation both independently sensitize leukemic cells to TRAIL-induced apoptosis. Triptolide not only inhibits XIAP, which is overexpressed in AML, but also activates p53 signaling, which is intact in the majority of AMLs. Thus, combinations of triptolide and TRAIL may provide a novel strategy for treating AML patients by overcoming critical mechanisms of apoptosis resistance.

scholarly journals Dissociation between p93B-myb and p75c-myb expression during the proliferation and differentiation of human myeloid cell lines

Blood ◽  
1994 ◽  
Vol 83 (7) ◽  
pp. 1778-1790 ◽  
Author(s):  
M Arsura ◽  
MM Luchetti ◽  
E Erba ◽  
J Golay ◽  
A Rambaldi ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Myeloid Cells ◽  
Indirect Evidence ◽  
Granulocytic Differentiation ◽  
Gm Csf ◽  
Protein Levels ◽  
Proliferation And Differentiation ◽  
Myb Protein

Abstract Direct and indirect evidence strongly indicates that the proto-oncogene c-myb plays an important role in the regulation of both the proliferation and differentiation of hematopoietic cells. In addition, recent data suggest that the structurally related B-myb gene is also necessary for the proliferation of these cells. To help understand the relationship between these two related gene products during proliferation and differentiation of myeloid cells, we have studied in parallel the regulated expression of c-myb and B-myb RNAs and proteins in human myeloid cells that were either growth-arrested or induced to differentiate along different pathways. For this purpose, we have produced a polyclonal antibody directed against a fragment of the recombinant B-myb protein. We have thus been able to detect the B-myb protein in human cell lines and have found it to be a 93-kD protein localized in the nucleus. We have chosen two models to study the expression of both c-myb and B-myb mRNAs and proteins during myeloid proliferation and differentiation. One of the models was the HL-60 cell line, which can be induced to differentiate towards the monocytic pathway with either phorbol ester (phorbol myristate acetate) or vitamin D3 and towards the granulocytic pathway with either dimethyl sulfoxide or retinoic acid. In addition, we have studied another recently established human leukemic cell line, called GF-D8, which is strictly dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) for proliferation. The results show that the expression of B- myb RNA and protein closely correlates with proliferation in all experimental setups studied, whereas the c-myb protein levels do not always do so. We observed that the c-myb protein levels decreased well before the decrease of B-myb protein and of proliferation itself during differentiation toward monocytes. Such a difference was not present during granulocytic differentiation, in which c-myb levels decreased, if anything, later than those of B-myb and proliferation. Most striking was the finding that high levels of c-myb RNA and protein, but not of B- myb, were present in the GF-D8 cell line, even after growth arrest by GM-CSF deprivation. These data suggest that B-myb may function solely in the regulation of cellular proliferation, whereas c-myb has additional functions, for example, in the maintenance of an undifferentiated state.

Interleukin-21 Potently Induces Direct and Indirect Cytotoxicity of Mantle Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1776-1776
Author(s):  
Shruti Bhatt ◽  
Kristopher Sarosiek ◽  
Salma Parvin ◽  
Julie Marie Matthews ◽  
Dekuang Zhao ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Tumor Regression ◽  
Resistant Cell ◽  
Primary Tumors ◽  
Mantle Cell ◽  
Direct Cytotoxicity ◽  
Anti Tumor Activity

Abstract Mantle cell lymphoma (MCL) is a distinct subtype of non-Hodgkin lymphomacharacterized by overexpression of cyclin D1 (CCND1) in 95% of patients due to the t(11;14)(q13;q32) chromosomal translocation. This incurable lymphoma is highly chemoresistant, with short response duration, frequent relapses and eventual death, even with the most aggressive chemotherapeutic regimens. Interleukin 21 (IL-21), a member of the IL-2 cytokine family, possesses potent anti-tumor activity against a variety of cancers not expressing IL-21 receptor (IL-21R) through activation of the immune system. Previously, we established that apart from its immuno-stimulatory effects, IL-21 exerts direct cytotoxicity on IL-21R-expressing diffuse large B cell lymphoma (DLBCL) cells (Sarosiek KA et al.Blood, 2010). Herein we carried out a comprehensive study to delineate the effects of IL-21 on MCL cell lines and primary tumors. Flow-cytometric analysis revealed that all MCL cell lines (Mino, HBL2, Jeko1, G519, IRM2, SP53, Z138, UPN1 and L128) as well as primary tumors expressed surface IL-21R at variable levels. Treatment of Mino, HBL2 and SP53 cells with IL-21 (100ng/mL) led to a marked time-dependent decrease in cell proliferation and increased cell death. In contrast, Jeko1, IRM2, L128, Z138, UPN1 and G519 cells exhibited resistance to IL-21 treatment. Similarly, primary MCL tumors treated with IL-21 in vitro exhibited significant cell death in 4 of 5 cases expressing IL-21R. To decipher the mechanism of IL-21-induced direct cytotoxicity, responsive and resistant cell lines as well as primary tumors were utilized. Similarly to our previous study in DLBCL, IL-21 stimulation resulted in dramatic phosphorylation of STAT1 and STAT3 in IL-21 responsive cell lines (Mino, HBL-2, SP53) and a primary tumor, while minimal STAT5 phosphorylation was observed only in Mino. We have previously demonstrated that IL-21-induced cell death in DLBCL is mediated by STAT3-induced upregulation of c-Myc expression. Correspondingly, IL-21 treatment led to c-Myc upregulation only in IL-21-sensitive MCL cell lines and primary tumors but not in the resistant cell lines and primary tumors, independent of the IL-21R expression levels. Knockdown of c-Myc prevented IL-21-induced Mino cell death, whereas c-Myc overexpression in resistant MCL cell lines facilitated IL-21-induced cytotoxicity. Furthermore, IL-21 resulted in upregulation of the pro-apoptotic protein Bax and downregulation of the anti-apoptotic proteins Bcl-XL and Bcl-2, as previously observed in DLBCL. Knockdown of STAT3 or Bax using specific siRNAs in Mino cells resulted in abrogation of the IL-21-induced cell death. In contrast to a previous report (Gelebart P et al. Leukemia, 2009), knockdown of STAT1 or overexpression of dominant negative STAT1 failed to prevent IL-21-induced Mino cell death. We also discovered that apart from its direct cytotoxic effects, IL-21 also leads to NK-cell dependent lysis of MCL cell lines resistant to direct cytotoxicity. In vivo treatment with IL-21 resulted in complete FC-muMCL1 tumor regression in syngeneic mice (p<0.0001). To understand the contribution of immune system components to the IL-21 induced anti-tumor activity, we carried out cell subset depletion studies. In vivo depletion of NK cells together with CD4+ T cells abrogated IL-21 induced tumor regression of the FC-muMCL1 in xenograft mice, suggesting that both NK and CD4+ cells contribute to the indirect immune mediated cytotoxicity of MCL. Collectively, our data indicate that IL-21 has potent anti-tumor activity against MCL cells via direct (IL-21R mediated) and indirect (immune cell mediated) effects, and should be evaluated in clinic for treatment of patients with MCL. Disclosures No relevant conflicts of interest to declare.

scholarly journals The XIAP Inhibitor BMT-062789 Displays Anti-Tumor Activity in Cell Line Models of De Novo and Rituximab Relapse/Refractory Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5375-5375 ◽  
Author(s):  
Kyle L. Runckel ◽  
Cory Mavis ◽  
Juan J Gu ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
De Novo ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Lymphoma Cell ◽  
Lymphoma Cell Line ◽  
Raji Cells ◽  
Ic50 Values ◽  
Anti Tumor Activity

Abstract The addition of rituximab to front line therapy for aggressive lymphomas has improved clinical outcomes, but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for rituximab associated chemotherapy cross-resistance our group developed several rituximab resistance cell lines (Raji 4RH and RL 4RH), which also display significant resistance to a wide range of chemotherapy agents. These rituximab resistant cell lines (RRCL)s exhibit multiple deregulations in the BCL-2 and inhibitor of apoptosis (IAP) protein families, including loss of the pro-apoptotic proteins Bax and Bak. We previously demonstrated that the X linked inhibitor of apoptosis protein (XIAP) is critically required for chemotherapy resistance in the RRCLs, and that an shRNA knockdown of XIAP increased chemotherapy response in both in vitro and in vivo models of rituximab resistant lymphoma. BMT-062789 is a heterodimeric mimetic of the second mitochondrial activator of caspases (SMAC) developed by Bristol-Myers Squibb, which can inhibit both the caspase 9 and caspase 3/7 binding domains of XIAP. BMT-062789 demonstrated dose and time dependent single agent anti-tumor effect (as measured by the Cell TiterGlo luminescent viability assay) in a panel of lymphoma cell lines, with IC50 values of less than 5uM for all cell lines tested except the rituximab resistant cell line Raji 4RH. The Burkitt's lymphoma cell line Daudi and diffuse large B-cell lymphoma cell line U2932 were particularly sensitive to BMT-062789 with IC50 values of 0.91uM and 0.76uM respectively. To investigate if the observed anti-tumor effect of BMT-062789 was due to increased apoptosis we exposed rituximab sensitive (Raji, RL) and rituximab resistant (Raji 4RH, RL 4RH) cells to escalating doses of BMT-062789 with or without the addition of 20uM etoposide for 48 hours. The induction of apoptosis was measured by flow cytometry with an Annexin-V:PE-Cy7 conjugate and Sytox blue (a DNA stain). 2uM BMT-062789 alone triggered apoptosis in 75% of Raji cells and 65% of RL cells. It is also worth adding that 2uM BMT-062789 induced higher rates of apoptosis than 20uM etoposide alone in both cell lines. The combination of 2uM BMT-062789 and 20uM etoposide together triggered apoptosis in 80% of Raji cells and 85% of RL cells, indicating that BMT-062789 may be able to augment the anti-tumor activity of conventional chemotherapy. More importantly, the combination of BMT-062789 and etoposide was also able to induce apoptosis in the rituximab resistant cell line models Raji 4RH and RL 4RH. 3uM BMT-062789 in combination with 20uM etoposide triggered apoptosis in 90% of Raji 4RH cells and 55% of the RL 4RH cells, which is a substantial improvement compared to 15% apoptosis with 20uM etoposide alone in each cell line. Additional studies are in progress to evaluate the anti-tumor effect of BMT-062789 in ex vivo samples from lymphoma patients with de novo and relapse/refractory disease. In summary, the novel heterodimeric XIAP inhibitor BMT-062789 has anti-tumor effect at low micromolar concentrations in lymphoma cell line models, including models of rituximab resistant disease. These results support earlier studies by our group indicating that XIAP is critical for survival in models of rituximab resistant lymphoma, and establish that XIAP inhibitors may have potential clinical value for the treatment of both de novo, and rituximab relapse/refractory lymphomas. Disclosures No relevant conflicts of interest to declare.

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