Motexafin Gadolinium Induces Apoptosis in Lymphoid Cell Lines and Demonstrates Enhanced Biological Activity with Akt Kinase Inhibitors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3406-3406
Author(s):  
Louie Naumovski ◽  
Jason Ramos ◽  
Jun Chen ◽  
Mint Sirisawad ◽  
David Lucas ◽  
...  
Keyword(s):  
Cell Death ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Lines ◽  
Specific Inhibitor ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Jurkat Cells ◽  
Motexafin Gadolinium ◽  
Akt Phosphorylation ◽  
Induced Apoptosis

Abstract Motexafin gadolinium (MGd, Xcytrin®) is a tumor-selective redox mediator that catalytically oxidizes intracellular reducing metabolites and produces reactive oxygen species (ROS). In this report, we demonstrate that MGd induces apoptosis or growth inhibition in several hematopoietic tumor-derived cell lines and tumor cells from patients with chronic lymphocytic leukemia. Lymphoma (HF-1, Ramos, DHL-4, DB, Hut78 and Raji) and leukemia (Jurkat, HL-60) cell lines were cultured in RPMI 1640 media with 10% heat inactivated fetal bovine serum with or without 50 uM MGd. MGd inhibited the growth of 6 of the cell lines (HF-1, Ramos, HL-60, DHL-4, Jurkat and DB) and was cytotoxic to HF-1. ROS were implicated in MGd-induced cell death since their presence was detected by dichlorofluorescein diacetate staining and peroxiredoxin oxidation in MGd treated HF-1 cells that undergo apoptosis, but not in Jurkat cells that do not undergo MGd-induced apoptosis. MGd triggered an apoptotic pathway in HF-1 cells as demonstrated by loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspases, cleavage of PARP and annexin-V binding. MGd also induced cell death and activated caspases in vitro in primary chronic lymphocytic leukemia cells. Protein lysates from cultured cell lines (HF-1, Ramos) were subjected to immunoblot analysis to determine caspase cleavage patterns, and the phosphorylation status of Akt, a kinase that regulates survival pathways. In MGd treated HF-1, phospho-Akt protein levels initially increased 2–3 fold between 30 min and 1 hr (n=4) and then decreased to 40–50% of control levels by 24–48 hrs (n=4). The drop in phospho-Akt protein coincided with an increase in apoptotic cell death as indicated by morphology, staining with Annexin-V and activation of caspases. Addition of a specific inhibitor of Akt phosphorylation (SH-5) reduced Akt phosphorylation in MGd treated HF-1 cells by 90% and enhanced the cytotoxic effect of MGd. In Ramos cells, which do not undergo apoptosis when treated with MGd, co-treatment with MGd and SH-5 decreased phospho Akt levels by only 15% and did not result in cytotoxicity. These data point to a potential role for Akt in MGd-induced apoptosis and suggest that MGd activity may be enhanced by inhibition of Akt. These data show that the pro-apoptotic effects of MGd involve caspase activation and provide a rationale to evaluate MGd in the treatment of lymphoma and leukemia patients.

scholarly journals Inhibition of the Notch1 Pathway in Chronic Lymphocytic Leukemia Promotes Apoptosis and Inhibits Proliferation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1972-1972 ◽  
Author(s):  
Josephine L. Klitgaard ◽  
Reuma Magori-Cohen ◽  
Reina Improgo ◽  
Stacey M. Fernandes ◽  
Bethany Tesar ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Expression Profiles ◽  
Annexin V ◽  
Receptor Activation ◽  
Lymphocytic Leukemia ◽  
Gamma Secretase ◽  
Induced Apoptosis ◽  
Notch1 Mutations ◽  
Notch1 Receptor

Abstract In chronic lymphocytic leukemia (CLL), mutations in the NOTCH1 receptor occur in 4-10% of newly diagnosed patients and 15–20% of multiply relapsed patients. Using next-generation sequencing, our group previously reported NOTCH1 p.P2514fs mutations in 15 CLL patients (9.4%) in an initial cohort of 160 CLL patients in which NOTCH1 mutations were associated with IGHV unmutated (UM) CLL (p=0.0001). Further analysis using a three-group comparison (NOTCH1 mut, IGHV UM vs. NOTCH1 wild-type [wt] IGHV UM vs. NOTCH1 wt IGHV mut) showed that NOTCH1 mutations associated with both trisomy 12 (p=0.049) and 17p deletion (p=0.0008) and poor overall survival (HR 2.99, p=0.008). Given that targeting activating mutations has proven an effective therapeutic strategy in many cancers, we explored the therapeutic potential of a Notch1 inhibitor, PF-03084014, in CLL. Previous studies in T-cell acute lymphoblastic leukemia cells harboring NOTCH1 mutations have shown that gamma secretase inhibitors can induce apoptosis by blocking Notch1 receptor activation. When we tested the gamma secretase inhibitor (GSI) PF-03084014 in 18 CLL samples with NOTCH1 mutations, it consistently induced apoptosis of all CLLs after 48 hours in culture across all cytogenetic groups tested (13.3-47.2% death with 5 μM GSI, p<0.0001)(Figure A). The induction of apoptosis was similar (GSI vs. ibrutinib, p=ns) to that of ibrutinib (n=11,11.9-74.4% death with 5 μM ibrutinib, p<0.0001). In contrast, GSI treatment only induced apoptosis in some (n=10), but not all NOTCH1 wt CLLs (n=6) (p=0.0137). We next tested the effect of GSI PF-03084014 in the context of a stromal environment. Co culture of CLL cells with CD40L-expressing fibroblasts partially mimics the lymph node and bone marrow microenvironments, which are known sites of drug-resistance and proliferation of CLL in vivo. We tested whether the interaction with stromal cells protects CLL cells from Notch1 inhibitor-induced apoptosis, as seen with other drugs including ibrutinib. We found that co culture with CD40L-expressing 3T3s decreased GSI-induced apoptosis in NOTCH1 mutant CLLs (p=0.0006) and in the majority of the NOTCH1 wt CLLs that responded to the GSI (Figure A). Since NOTCH1 mutations have been reported to be an independent marker of aggressive disease in CLL, we tested whether CLL cells with NOTCH1 mutations were more proliferative in vitro compared to NOTCH1 wt CLL cells. We showed that CLL cells upregulate Ki67 expression in co culture with 3T3-CD40L cells and in a cohort of 10 NOTCH1 mutants and 11 NOTCH1 wt, we found the NOTCH1 mutants to be more proliferative than the NOTCH1 wt (median of 7.6% vs. 2.3%, p=0.015). To then address whether blocking of the Notch1 pathway decreases proliferation, we treated CLL cells in co culture with 3T3-CD40L cells with 5 μM GSI for 7 days. GSI treatment decreased the percentage of Ki67+ CLL cells in all but one NOTCH1 mutant (median decrease 28.3%, p=0.044) as wells as in the majority of NOTCH1 wt samples (median decrease 38.7%, p=0.037). Having established that inhibition of Notch1 can reduce proliferation and induce apoptosis in CLL cells in vitro, we were interested in determining the downstream genes that may be the effectors of this activity. We therefore compared the gene expression profiles (GEP) of NOTCH1 mut vs. NOTCH1 wt CLLs, and found upregulation of genes involved in the Notch1 pathway, in apoptosis and in chemokine signaling in the NOTCH1 mutants. Furthermore, comparing GEP of high Ki67 vs. low Ki67 expressing CLL cells revealed higher expression of a range of both upstream and downstream Notch1 pathway genes in high Ki67 expressing CLL cells. In conclusion, we show that PF-03084014 induces apoptosis and decreases proliferation in both NOTCH1 mutant and wt CLL cells. We find NOTCH1 mutant CLL cells to be more proliferative than NOTCH1 wt and show upregulation of Notch1 pathway genes in NOTCH1 mutants compared to wt CLL cells and in high Ki67 expressing compared to low Ki67 expressing CLL cells. Taken together, these results emphasize the important role of Notch1 signaling in CLL in general, perhaps particularly in proliferative compartments like lymph nodes, and demonstrate that Notch1 pathway inhibitors are worthy of therapeutic investigation in CLL. Figure A. GSI-induced apoptosis in NOTCH1 mut vs. wt CLL cells at 48 h. CLL cells naked or in co culture with 3T3-CD40L cells +/- 5 µM PF-03084014. Survival was assessed by CD19 and Annexin V/PI staining. Figure A. GSI-induced apoptosis in NOTCH1 mut vs. wt CLL cells at 48 h. CLL cells naked or in co culture with 3T3-CD40L cells +/- 5 µM PF-03084014. Survival was assessed by CD19 and Annexin V/PI staining. Disclosures No relevant conflicts of interest to declare.

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.

The Novel Organic Arsenical, Darinaparsin (ZIO-101), Induces Apoptosis through AKT and MEK/ERK Pathways In Hodgkin Lymphoma (HL) and T-Cell Lymphoma (TCL) Cell Lines

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2840-2840
Author(s):  
Savita Bhalla ◽  
Sheila Prachand ◽  
Leo I Gordon ◽  
Andrew M Evens
Keyword(s):  
Mass Spectrometry ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Annexin V ◽  
Inorganic Arsenic ◽  
Therapeutic Index ◽  
Mek Inhibitor ◽  
Jurkat Cells ◽  
Parp Activation ◽  
Induced Apoptosis

Abstract Abstract 2840 Background: The inorganic arsenic, arsenic trioxide (ATO), has a narrow therapeutic index, which has limited its clinical use in most malignancies. Darinaparsin (ZIO-101, S-dimethylarsino-glutathione), synthesized by conjugating dimethylarsenic to glutathione, is a novel organic arsenical that is under investigation as a novel agent for the treatment of cancer. Furthermore, early-phase clinical trials with darinaparsin have demonstrated low toxicity and encouraging clinical efficacy in relapsed/refractory hematologic malignancies. Methods: We treated several TCL cell lines (Jurkat, C10MJ, Hut-78, and MT2) and the resistant HL cell line, L428, with increasing concentrations of darinaparsin (0.5-5μM) +/− the MEK inhibitor, U0126, or ERK siRNA (Qiagen HiPerFect transfection). Cell survival and apoptosis were measured by MTT and Annexin-V/propidium iodide staining, respectively. Further, tumor intracellular darinaparsin and ATO concentrations were assessed with mass spectrometry, while transcription pathway intermediates were analyzed by Western blotting. Results: Darinaparsin inhibited cell growth and induced apoptosis in all cell lines at 1–3μM. At 2μm (48 hours), darinaparsin induced approximately 80% apoptosis in each of the four TCL lines, while 3μM resulted in 65% apoptosis in L428 cells. By comparison, >10μM of ATO (48 hours) was required to induce 40% apoptosis in TCL and 25% apoptosis in L428. At 1–3μM, darinaparsin induced significant increases in caspase 3 and PARP activation in TCL, while interestingly, minimal caspase or PARP was observed in L428. Notably, in L428 cells at 1 hour, mass spectrometry showed that intracellular accumulation of darinaparsin was >10-fold higher as compared with equivalent ATO concentrations (p<0.01). We also treated L428 cells with U0126 (5μM) or ERK2 siRNA, both combined with darinaparsin. Pre-incubation with U0126 or siRNA knock down of ERK2, followed by treatment with darinaparsin, significantly enhanced darinaparsin-induced apoptosis (p<0.05). To further investigate darinaparsin-induced signaling pathways, we analyzed phospho-AKT (p-AKT), and phospho-ERK (p-ERK) in Jurkat and L428. We found down-regulation of p-AKT in Jurkat as well as L428 cells, while total AKT remained unchanged. Additionally, an increase in p-ERK was observed in L428 cells with 2–3μM darinaparsin, while p-ERK was down-regulated in Jurkat cells. Conclusions: Darninaparsin induces significant cell death in HL and TCL cell lines that is mediated through AKT and MEK/ERK-based pathways. Additionally, markedly higher intracellular darinaparsin levels are achieved in lymphoma cells compared with equivalent concentrations of ATO. Continued pre-clinical and clinical trial investigation of darinaparsin in HL and TCL is warranted. Disclosures: No relevant conflicts of interest to declare.

2-phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells

Blood ◽  
2009 ◽  
Vol 114 (6) ◽  
pp. 1217-1225 ◽  
Author(s):  
Andrew J. Steele ◽  
Archibald G. Prentice ◽  
A. Victor Hoffbrand ◽  
Birunthini C. Yogashangary ◽  
Stephen M. Hart ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Annexin V ◽  
P53 Gene ◽  
Mitochondrial Pathway ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Cytochrome C Release ◽  
P53 Status ◽  
Induced Apoptosis ◽  
P53 Gene Deletion

Abstract We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 μM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.

scholarly journals PNU-74654 Suppresses TNFR1/IKB Alpha/p65 Signaling and Induces Cell Death in Testicular Cancer

2022 ◽  
Vol 44 (1) ◽  
pp. 222-232
Author(s):  
Wen-Jung Chen ◽  
Wen-Wei Sung ◽  
Chia-Ying Yu ◽  
Yu-Ze Luan ◽  
Ya-Chuan Chang ◽  
...  
Keyword(s):  
Cell Death ◽  
Testicular Cancer ◽  
Cell Viability ◽  
Cell Lines ◽  
Cancer Progression ◽  
Treatment Strategies ◽  
Annexin V ◽  
Western Blots ◽  
Execution Phase ◽  
Induced Apoptosis

Testicular cancer (TC) is a rare malignancy worldwide and is the most common malignancy in males aged 15–44 years. The Wnt/β-catenin signaling pathway mediates numerous essential cellular functions and has potentially important effects on tumorigenesis and cancer progression. The search for drugs to inhibit this pathway has identified a small molecule, PNU-74654, as an inhibitor of the β-catenin/TCF4 interaction. We evaluated the therapeutic role of PNU-74654 in two TC cell lines, NCCIT and NTERA2, by measuring cell viability, cell cycle transition and cell death. Potential pathways were evaluated by protein arrays and Western blots. PNU-74654 decreased cell viability and induced apoptosis of TC cells, with significant increases in the sub G1, Hoechst-stained, Annexin V-PI-positive rates. PNU-74654 treatment of both TC cell lines inhibited the TNFR1/IKB alpha/p65 pathway and the execution phase of apoptosis. Our findings demonstrate that PNU-74654 can induce apoptosis in TC cells through mechanisms involving the execution phase of apoptosis and inhibition of TNFR1/IKB alpha/p65 signaling. Therefore, small molecules such as PNU-74654 may identify potential new treatment strategies for TC.

Mechanism of staurosporine-induced apoptosis in murine hepatocytes

2002 ◽  
Vol 282 (5) ◽  
pp. G825-G834 ◽  
Author(s):  
Guoping Feng ◽  
Neil Kaplowitz
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Caspase 3 ◽  
Nuclear Translocation ◽  
Nuclear Factor Κb ◽  
Jurkat Cells ◽  
Primary Hepatocytes ◽  
Iκb Kinase ◽  
Apoptosis Protein ◽  
Induced Apoptosis

Staurosporine (STS) induces apoptosis in various cell lines. We report in this study that primary cultured mouse hepatocytes are less sensitive to STS compared with Jurkat cells and Huh-7 cells. In contrast to the cell lines, no apparent release of cytochrome c or loss of mitochondrial transmembrane potential was detected in primary hepatocytes undergoing STS-induced apoptosis. Caspase-3 was activated in primary hepatocytes by STS treatment, but caspase-9 and -12 were not activated, and caspase-3 activation is not dependent on caspase-8. These findings point to a novel pathway for caspase-3 activation by STS in primary hepatocytes. Pretreatment with caspase inhibitor converted STS-induced apoptosis of hepatocytes to necrotic cell death without significantly changing total cell death. Thus STS causes hepatocytes to commit to death upstream of the activation of caspases. We also demonstrated that STS dramatically sensitized primary hepatocytes to tumor necrosis factor-α-induced apoptosis. STS activated IκB kinase and nuclear factor-κB (NF-κB) nuclear translocation and DNA binding but inhibited transactivation of IκB-α, inducible nitric oxide synthase, and inhibitor of apoptosis protein-1 in hepatocytes and NF-κB reporter in transfected Huh-7 cells.

VEGF receptor phosphorylation status and apoptosis is modulated by a green tea component, epigallocatechin-3-gallate (EGCG), in B-cell chronic lymphocytic leukemia

Blood ◽  
2004 ◽  
Vol 104 (3) ◽  
pp. 788-794 ◽  
Author(s):  
Yean K. Lee ◽  
Nancy D. Bone ◽  
Ann K. Strege ◽  
Tait D. Shanafelt ◽  
Diane F. Jelinek ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Annexin V ◽  
Lymphocytic Leukemia ◽  
Parp Cleavage ◽  
Vegf Receptor ◽  
Cell Leukemia ◽  
The Impact

AbstractWe recently reported that chronic lymphocytic leukemia (CLL) cells synthesize and release vascular endothelial growth factor (VEGF) under normoxic and hypoxic conditions. CLL B cells also express VEGF membrane receptors (VEGF-R1 and VEGF-R2), suggesting that they use VEGF as a survival factor. To assess the mechanism of apoptosis resistance related to VEGF, we determined the impact of VEGF on CLL B cells, and we studied the impact of epigallocatechin-3-gallate (EGCG), a known receptor tyrosine kinase (RTK) inhibitor, on VEGF receptor status and viability of CLL B cells. VEGF165 significantly increased apoptotic resistance of CLL B cells, and immunoblotting revealed that VEGF-R1 and VEGF-R2 are spontaneously phosphorylated on CLL B cells. EGCG significantly increased apoptosis/cell death in 8 of 10 CLL samples measured by annexin V/propidium iodide (PI) staining. The increase in annexin V/PI staining was accompanied by caspase-3 activation and poly–adenosine diphosphate ribose polymerase (PARP) cleavage at low concentrations of EGCG (3 μg/mL). Moreover, EGCG suppressed the proteins B-cell leukemia/lymphoma-2 protein (Bcl-2), X-linked inhibitor of apoptosis protein (XIAP), and myeloid cell leukemia-1 (Mcl-1) in CLL B cells. Finally, EGCG (3-25 μg/mL) suppressed VEGF-R1 and VEGF-R2 phosphorylation, albeit incompletely. Thus, these results suggest that VEGF signaling regulates survival signals in CLL cells and that interruption of this autocrine pathway results in caspase activation and subsequent leukemic cell death.

FTY720 (2-Amino-2-[2-(4-octylphenyl) ethyl] Propane 1, 3-diol hydrochloride), Mediates Cytotoxicity through Caspase Independent and Protein Phosphatase 2A Dependent Mechanisms in Chronic Lymphocytic Leukemia and Lymphoblastic Leukemia/Lymphoma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2095-2095
Author(s):  
Qing Liu ◽  
Xiaobin Zhao ◽  
Frank Frissora ◽  
Yihui Ma ◽  
Ramasamy Santhanam ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Protein Phosphatase ◽  
Lymphocytic Leukemia ◽  
Pp2a Activity ◽  
Phosphatase 2A ◽  
Induced Apoptosis ◽  
Pp2a Activation

Abstract FTY720 (2-Amino-2-[2-(4-octylphenyl) ethyl] propane 1, 3-diol hydrochloride) is a synthetic compound produced by modification of a natural immunosuppressant, ISP-1. It is an immunosuppressive agent that is being developed to prevent organ transplant rejection. Recent studies indicate additional role for FTY720 in inducing cell apoptosis. We demonstrate here a novel mechanism by which FTY720 mediates cytotoxic effects in cell lines representing different B cell malignancies and primary B cells from chronic lymphocytic leukemia (CLL) patients. FTY720 induced apoptosis as detected by annexin V/ propidium iodide staining in representative B cell lines and CLL patient derived CD19+ B cells in time and dose dependent manner (p<0.0001, untreated vs 10mM-treated CLL cells, n=15). In contrast to previous reports in T cell lines, FTY720 induced cytotoxicity in Raji cell line and primary CLL cells is independent of activation of caspase 3, 8 and 9 or poly-ADP ribose polymerase cleavage. Further, pan-caspase inhibitor Z-VAD-fmk rescued these cells from fludarabine but not FTY720 induced apoptosis (p=0.001 fludarabine vs fludarabine+z-VAD-fmk; p=0.99 FTY720 vs FTY720+z-VAD-fmk, n=5). Over-expression of Bcl-2 failed to protect transformed B-cells from FTY720 induced apoptosis suggesting Bcl-2 independent cytotoxic effect. Interestingly, FTY720 induced consistent increase in protein phosphatase 2a (PP2a) activity and concentrations of okadaic acid that inhibited the FTY720-induced PP2A activity also resulted in inhibition of FTY720-mediated cytotoxicity in B cell lines and primary CLL cells, indicating a role for PP2A activation in FTY720 induced cytotoxicity. Consistent with its activation of PP2A, FTY720 induced dephosphorylation of of Erk1/2 in CLL B cells. Further, FTY720 treatment resulted in significant in-vivo therapeutic efficacy associated with prolonged survival in a xenograft SCID mouse model of disseminated B cell lymphoma/leukemia (median survival time for FTY720 treated mice was 47 days (95 %CI 39–53) compared to 18 days in placebo controls (95% CI 17–19) P<0.0001-FTY720 vs placebo). These results provide first evidence for a PP2A dependent and caspase independent cytotoxicity of FTY720 in B cells. The novel caspase and Bcl-2 independent mechanism of cytotoxicity concurrent with identification of PP2a activation as a surrogate marker of cell killing provide further justification for clinical development of this agent in lymphoid leukemia.

Dipeptidyl Peptidase 2 - A Pro-Survival Molecule in Chronic Lymphocytic Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4964-4964
Author(s):  
Alexey V. Danilov ◽  
Olga V. Danilova ◽  
Andreas K. Klein ◽  
Brigitte T. Huber
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Serine Protease ◽  
Specific Inhibitor ◽  
Dipeptidyl Peptidase ◽  
Chemotherapeutic Agents ◽  
Lymphocytic Leukemia ◽  
Quiescent State ◽  
Induced Apoptosis

Abstract Chronic lymphocytic leukemia (CLL) is a unique malignancy characterized by persistent accumulation of quiescent B-cells. Disruption of survival pathways leads to apoptosis and renders CLL cells sensitive to chemotherapeutic agents. As clinical outcomes in CLL are heterogeneous, it is important to better predict prognosis in each individual case of the disease. In this respect, detection of somatic mutations in the immunoglobulin variable heavy chain (IgVH) genes has become the gold standard. Overexpression of ZAP-70 (Zeta-chain associated protein kinase - 70 kDa) by CLL cells correlates with unmutated IgVH genes and predicts poor outcome in B-CLL. Dipeptidyl Peptidase 2 (DPP2) is a newly discovered serine protease which maintains lymphocytes in a quiescent state (G0). In this study we investigated whether DPP2 is involved in cell cycle control in B-CLL. 38 patients with B-CLL and 20 healthy controls were included in the study. Median age of CLL patients was 67 years. Median time from diagnosis to enrollment in the study was 102 months. 21 patients (55.3%) received treatment before enrollment in the study. Standard Ficoll-Hypaque techniques were used to isolate peripheral blood mononuclear cells (PBMC) from healthy donors and CLL patients. CLL cells were isolated to &gt;98% purity by means of a MoFlo using CD19−specific antibodies. Cells were treated with Val-boro-Pro, a pan-inhibitor of DPP, or with DPP2-specific inhibitor (DSI), incubated for 16 hours and stained with propidium iodide and Annexin V. Expression of CD38 was assessed by flow cytometry, DPP2 and ZAP-70 - by real-time reverse-transcription polymerase chain reaction, Bcl-2 and p27 - by western blot analysis. We report that CLL B-cells expressed higher levels of DPP2 mRNA than normal B-cells. Inhibition of DPP2 in PBMC with VbP or DSI resulted in caspase-dependent apoptosis. In individuals with CLL, death of B-lymphocytes (and rescue by caspase inhibitors) was observed in 22 cases (57.9%). In the remaining 16 cases (42.1%) malignant B-cells did not undergo apoptosis upon inhibition of DPP2 with either VbP or DSI. We found that CLL cells resistant to DPP2 inhibition-induced apoptosis expressed higher levels of ZAP-70. Meanwhile, protein levels of p27 (cell cycle inhibitor) were decreased in resistant CLL. These patients exhibited worse disease prognosis such as shorter treatment-free period (p&lt;0.001), frequent episodes of treatment failure and faster disease progression. Between the two groups, we identified no differences in expression of Bcl-2. CLL B-cells express higher levels of DPP2, a serine protease involved in maintenance of G0 which may serve as a survival factor in CLL B-cells. Resistance vs. susceptibility to DPP2 inhibition-induced apoptosis can be employed as a prognostic factor in CLL.

Sign in / Sign up

Export Citation Format

Share Document