scholarly journals TR57, an Inhibitor of the Integrated Stress Response, Is Synergistic with Venetoclax Against CLL Cells, Independent of Their TP53 Status

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1735-1735
Author(s):  
Narjis Fatima ◽  
Yandong Shen ◽  
Kyle R Crassini ◽  
Edwin Iwanowicz ◽  
Richard Christopherson ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Stress Response ◽  
Stromal Cell ◽  
Single Agent ◽  
Significant Proportion ◽  
Integrated Stress Response ◽  
Significant Difference ◽  
Highly Effective ◽  
Ic50 Values

Introduction Despite recent advances in the treatment of chronic lymphocytic leukemia (CLL), a significant proportion of patients still relapse with drug-resistant disease. Patients with deletions and/or mutations in TP53 remain a poor prognostic sub-group. There remains a need for on-going research on identifying novel treatment strategies. We have investigated the therapeutic potential of TR57 (Madera Therapeutics, USA), as a single agent and in combination with the Bcl-2 inhibitor venetoclax in CLL. TR57 is an inhibitor of the integrated stress response (ISR) and has shown efficacy in pre-clinical studies of breast cancer. Methods TR57 and venetoclax, as single agents or in combination, were studied against primary CLL cells co-cultured with CD40L-expressing fibroblasts to mimic the tumour microenvironment (TME). The effects of the drugs were also assessed against an OSU-CLL TP53 knock-out cell line generated using CRISPr Cas-9. Cell viability was assessed using the mitochondrial membrane potential dye DiIC1(5), propidium iodide (PI) and flow cytometry. Cytotoxic synergy between TR57 and venetoclax was determined by calculating combination indices (CI) using the CompuSyn software, with CI values of <1 indicative of synergy. Primary CLL cells were stimulated into cycle primary using Dsp30 in combination with IL-2. Cell cycle distribution and proliferation were analysed by flow cytometry using PI and CFSE, respectively. The migratory and adhesive capacities of primary CLL cells was assessed using stroma-derived factor 1a and by assessing expression of CXCR4 and CD49d. The mechanisms of the synergy between TR57 and venetoclax were studied by immunoblotting. Statistical analyses were performed using the students t-test with P-values of < 0.05 considered significant. Results TR57 was cytotoxic towards primary CLL cells in a nanomolar range, with IC50 values of 38 ± 1.38 nM and 287 ± 50.45 nM against cells in medium and stromal cell co-culture, respectively. No significant difference was observed in the sensitivity of samples with ATM or TP53 aberrations (n=12). Synergy between TR57 and venetoclax against CLL cells in stromal cell co-culture was consistent with a significant (P < 0.001) decrease in the IC50 for both drugs (Figure 1A). A CI value of 0.13 was calculated at a fractional effect of 0.5. TR57 was cytotoxic towards OSU-CLL and OSU-CLLTP53ko cells, with IC50 values of 3.98 ± 1.03 nM and 90.7 ± 3.51 nM, respectively. Synergy between TR57 and venetoclax was evident in both lines with a CI = 0.1 at Fa 0.5. Dose-response analyses in the OSU-CLLTP53ko line are shown in Figure 1B. Following stimulation of primary CLL cells with Dsp30/IL-2 we observed a significant (P < 0.01) increase in the proportion of cells in S, G2 and M phases, which was consistent with an increase in cell proliferation. TR57 and venetoclax in combination had a greater effect than individual drug treatments, significantly reduced the proportion of cells in these cell cycle phases and the proliferative fraction of cells. TR57 and venetoclax in combination also had a significantly greater effect on the migratory capacity (P < 0.05) of CLL cells and on the expression of CXCR4 and CD49d (P < 0.001) than either drug as a single agent. Immunoblotting of primary CLL and OSU-CLL cells showed that treatment with TR57 decreased expression of Grp78, which supports the notion that this drug may function through inhibition of the ISR. We also observed that TR57, alone and in combination with venetoclax, down-regulated the expression of the pro-survival Mcl-1 and Bcl-2 proteins, up-regulated expression of the pro-apoptotic Noxa and Bax proteins and inhibited the phosphorylation of AKT and ERK1/2-MAPK. Conclusions The data presented demonstrate that TR57 is cytotoxic towards CLL cells under in vitro conditions that mimic the TME and cells with lesions of the TP53 pathway. The synergy observed between TR57 and venetoclax suggests that the drug combination may be highly effective at limiting the survival and proliferation of CLL cells as well as their ability to migrate to, and be retained within, the TME. The mechanisms of this synergy include a shift towards a pro-apoptotic balance in Bcl-2 family proteins and inhibition of signalling via the AKT and ERK1/2-MAPK pathways. Collectively, these data suggest that TR57 alone and in combination with venetoclax may be a highly effective treatment strategy for high risk CLL. Disclosures Iwanowicz: Madera Therapeutics, LLC: Other: President;Ownership.

scholarly journals Growth Effects of Some Platinum(II) Complexes with Sulfur-Containing Carrier Ligands on MCF7 Human Breast Cancer Cell Line upon Simultaneous Administration with Taxol

2002 ◽  
Vol 9 (1-2) ◽  
pp. 33-43 ◽  
Author(s):  
Gordana Bogdanović ◽  
Vesna Kojić ◽  
Tatjana Srdić ◽  
Dimitar Jakimov ◽  
Miloš I. Djuran ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Recovery Period ◽  
Cancer Cell Line ◽  
Inhibitory Effect ◽  
Mcf7 Cells ◽  
Cisplatin Analogues ◽  
Ic50 Values ◽  
G2m Phase ◽  
Induced Apoptosis

The platinum (II)complexes, cis-[PtCl2(CH3SCH2CH2SCH3)] (Pt1), cis-[PtCl2(dmso)2] (dmso is dimethylsulfoxide; Pt2) and cis-[PtCl2(NH3)2] (cisplatin), and taxol (T) have been tested at different equimolar concentrations. Cells were exposed to complexes for 2 h and left to recover in fresh medium for 24, 48 or 72 h. Growth inhibition was measured by tetrazolium WST1 assay Analyses of the cell cycle, and apoptosis were performed by flow cytometry, at the same exposure times. The IC50 value of each platinum(II) complex as well as combination index (CI; platinum(II) complex + taxol) for various cytotoxicity levels were determined by median effects analysis.MCF7 cells were found to be sensitive to both Pt1 and Pt2 complexe These cisplatin analogues influenced the cell growth more effectively as compared to cisplatin. Cytotoxic effect was concentration and time-dependent. Profound growth inhibitory effect was observed for Pt1 complex, across all its concentrations at all recovery periods. A plateau effect was achieved three days after treatment at Pt1 concentrations ≤ 1 μM. Pt2, however, decreased MCF7 cells survival only for the first 24 h ranging between 50-55%. Pt2 cytotoxicity sharply decreased thereafter, approaching 2 h - treatment cytotoxicity level. The median IC50 values for Pt1 and Pt2 were similar (0.337 and 0.3051 μM, respectively) but only for the first 24 h. The IC50 values for Pt1 strongly depend on the recovery period. On simultaneos exposure of cells to taxol and platinum(II) complexes no consistent effect was found. The Cls for combinations of taxol with Pt1 or Pt2 revealed cytotoxic effects that were in most Cases synergistic (Pt1) or less than addtiive (Pt2). Flow cytometry analysis has shown that each platinum(II) complex induced apoptosis in MCF7 cells. The level of apoptosis correlated with cytotoxicity level for the range concentrations. Both cisplatin analogues, at IC50 concentrations, increased the number of MCF7 cells in G0G1 phase of cell cycle. Pt2-treated cells remained arrested in G0G1 phase up to 72 h after treatment. Combination of Pt2 and taxol caused further arrest of cells in G0G1 phase (24 h) in parallel with strong decrement of G2M phase cells.

Improved Therapy of Multiple Myeloma by Combining Milatuzumab (Anti-CD74 Humanized mAb) and Bortezomib: In Vitro and In Vivo Results.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1176-1176
Author(s):  
Rhona Stein ◽  
David M. Goldenberg
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Leukemic Cells ◽  
Tumor Type ◽  
Term Survival ◽  
Significant Difference ◽  
Highly Effective ◽  
Ic50 Values

Abstract Background: The humanized anti-CD74 monoclonal antibody, milatuzumab (hLL1, or IMMU-115; Immunomedics, Inc, Morris Plains, NJ), is in clinical evaluation for therapy of multiple myeloma (MM) after preclinical evidence of activity in this tumor type (Stein et al, Blood2004;104:3705). Here we examine the ability of milatuzumab to increase the efficacy of drugs in MM cell lines. Methods: MTT cytotoxicity assays were performed on a panel of MM cell lines, including CAG, KMS11, KMS12-PE, and MC/CAR, to examine the effects of bortezomib, doxorubicin (dox), and dexamethasone (dex) alone and combined with milatuzumab or milatuzumab + crosslinking 2nd Ab (goat anti-human IgG, GAH). In vivo studies used a CAG-SCID mouse model of disseminated disease. Results: Without drugs, crosslinked milatuzumab, but not milatuzumab alone, yielded significant anti-proliferative effects on the four MM cell lines. In combination studies, crosslinked milatuzumab produced significant reductions in the IC50 values of the anti-MM drugs. For example, in CAG, milatuzumab+GAH decreased the IC50 values 58%, 78%, and 98% for bortezomib, dox, and dex, respectively (P=0.0034, 0.0073, and 0.078, respectively). In vivo, milatuzumab at 100 μg/injection, 2x weekly for 4 weeks, starting 1 day after injection of CAG cells, more than doubled the median survival time (MST) from 42 days in untreated CAG-bearing SCID mice to 103 days. Combination therapy with milatuzumab and bortezomib or dox was compared to milatuzumab alone, with treatments initiated 5 days after injection of CAG cells. Bortezomib alone (1.0 mg/kg) increased MST from 33 to 44 days (P=0.0021 vs. untreated). Treatment with milatuzumab alone (100 μg/mouse) increased the MST to 73 days (P<0.0001 vs. untreated). When bortezomib and milatuzumab treatments were combined, the MST increased to 93 days (P=0.0441 vs. milatuzumab and P=0.0065 vs. bortezomib). Thus, the combination of milatuzumab and bortezomib increased survival significantly compared to either single treatment. Given alone, dox yielded little or no effect on survival compared with untreated animals, and there was no significant difference between milatuzumab monotherapy and milatuzumab plus doxorubicin in this model. In contrast, a milatuzumabdox immunoconjugate was found to be a highly effective therapeutic agent, with all mice achieving long-term survival. The inhibition of the NF-κB survival pathway of B-leukemic cells by milatuzumab supports its complementary effects when combined with drugs having different mechanisms of action, such as bortezomib. Conclusions: The therapeutic efficacies of bortezomib, dox, and dex are enhanced in vitro in MM cell lines when given in combination with milatuzumab. In vivo, milatuzumab alone or especially in combination with bortezomib is highly effective in MM. (Supported in part by USPHS grant P01CA103985 from the NCI, and grants from the Thomas and Agnes Carvel Foundation and the Walter and Louise Sutcliffe Foundation.)

Apoptosis Was Induced by Casticin in Acute Myelocytic Leukemia Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3991-3991
Author(s):  
Jie Jin ◽  
Jia-Kun Shen ◽  
Hua-ping Du ◽  
Min Yang ◽  
Yun-Gui Wang
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Death ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Acute Myelocytic Leukemia ◽  
Myelocytic Leukemia ◽  
Cycle Arrest ◽  
M Phase ◽  
Ic50 Values

Abstract Casticin, a component from Vitex rotundifolia wich was widely used as an anti-inflammatory agent in Chinese traditional medicine, was reported to have anti-tumor activities in lung cancer and breast cancer. There are yet no reports on roles against acute myelocytic leukemia (AML). This study aims to elucidate the anti-leukemic activity of casticin on AML cells. We investigated the efficient efficacy and the mechanisms by which casticin triggers cell death in AML cells by analyzing cell cycle perturbations, apoptosis-related marker expression. Cell viability was measured by MTT method; apoptosis and cell cycle arrest were determined by flow cytometry and AV-PI assay. Western blot was performed to measure the apoptosis-related marker. Concentration-dependant cell deaths were observed in AML cell lines including K562, U937 and THP-1, with IC50 values of 24h (hours) being 47.4μM, 67.8μM and 61.7μM, respectively. Time-dependant cell deaths were also observed. At the concentration of 20μM casticin, 45.7%, 76.1% and 80.9% of K562 cells were inhibited at 24h, 48h and 72h, respectively; 24.7%, 30% and 61% of U937 cells were inhibited at 24h, 48h and 72h, respectively; while for THP-1, 29%, 41.8% and 53.9% were inhibited at 24h, 48h and 72h, respectively. Apoptosis was found using AV-PI staining by flow cytometry analysis. We observed an obvious G2/M phase increase prolongation in casticin treated K562 cells. BThe distribitions of G2/M phase were 2.9%, 33.6%, 75.3%, 54.9%, 29.7% and 27.0% in K562 cells after treated by 20μM casticin for 0h, 6h, 12h, 24h, 36h and 48h, respectively. Furthermore, apoptosis-related proteins, PARP and caspase 3, were cleaved in casticin treated K562 cells. Taken together, these results demonstrated that casticin can induce leukemic cell death through apoptosis, suggesting that casticin could be a promising therapeutic agent against acute myeloid leukemia.

scholarly journals Evaluation of Anticancer Activity of Olmesartan and Ramipril On A549 Cell Line

2018 ◽  
Vol 11 (3) ◽  
pp. 1351-1357 ◽  
Author(s):  
E Gayathri ◽  
K. Punnagai ◽  
D. Darling Chellathai
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Anticancer Activity ◽  
Cell Line ◽  
A549 Cell ◽  
Enzyme Inhibitor ◽  
A549 Cell Line ◽  
Ic50 Values

Angiotensin Converting Enzyme Inhibitor (ACEI) and Angiotensin II type 1 receptor antagonist (ARBs) are the most efficient cardiovascular drugs and exhibited efficient cytostatic activity in vitro in many malignant and normal cells1.OBJECTIVE: This study aims to assess the anticancer activity of these two drugs in a dose dependant manner using A549 cell line through MTT assay and Cell cycle analysis.. MATERIALS AND METHODS: Ramipril and Olmesartan were added to A549 at various concentrations ranging from 10⁻⁶ to 10mM.The dot plot of the cytotoxicity results were used to extrapolate the IC50 values. The dot plot of flow cytometry results were used to extrapolate the DNA percentage in phases of cell cycle. The plates were read at 570 nm by using a PERCLIN ELMER (multimode reader). Measurements for concentration required for 50% inhibition was noted. RESULTS: Ramipril and Olmesartan were added to A549 at various concentrations ranging from 10⁻⁶ to 10mM.The dot plot of the cytotoxicity results were used to extrapolate the IC50 values. The dot plot of flow cytometry results were used to extrapolate the DNA percentage in phases of cell cycle.

scholarly journals Abemaciclib-Based Combinational Therapies to Overcome Therapeutic Resistance in Mantle Cell Lymphoma

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-34
Author(s):  
Yuxuan Che ◽  
Yang Liu ◽  
Lingzhi Li ◽  
Holly Hill ◽  
Joseph McIntosh ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Research Funding ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Therapeutic Resistance ◽  
Cycle Arrest ◽  
Mantle Cell ◽  
Ic50 Values

Introduction The past decades witnessed dramatic improvement of overall survival rate of mantle cell lymphoma (MCL) patients by constant efforts in developing novel therapeutic strategies that include ibrutinib and venetoclax. Nevertheless, resistance is still a major challenge in refractory/relapsed MCL patients. Chromosomal translocation t(11:14)(q13:q32) of the cyclin D1 (CCND1) gene is the hallmark of MCL, which leads to overexpression of cyclin D1. This overexpression promotes aberrant cell cycle progression by activating CDK4/6. Abemaciclib is a selective CDK4/6 inhibitor used as a clinical treatment of breast cancer and has been shown to be effective in preclinical human MCL xenograft models. It has also been used in a phase II clinical trial as a single agent among refractory/relapsed MCL patients with an objective response rate of 35.7%. In this preclinical study, we aim to evaluate the benefit of a combinational therapeutic strategy using abemaciclib with other molecular targeting agents among MCL patients with therapeutic resistance. Methods Cytotoxic efficacy of abemaciclib as a single agent and in combination with other drugs on different MCL cell lines and primary lymphoma cells from MCL patients with or without resistance was used as a key criterion for screening beneficial therapeutic strategies. Cell apoptosis and cell cycle arrest assays were conducted to further evaluate those effective combinations. Western blot was performed to investigate the mechanism of action of the combinations. Finally, the efficacy of abemaciclib alone or in combination were assessed in ibrutinib-resistant or venetoclax-resistant MCL PDX models in vivo. Results Our preliminary data showed that all MCL cell lines involved in this study were highly sensitive to abemaciclib treatment with IC50 values ranging from 50 nM to 1 µM. Further investigation of abemaciclib cytotoxicity on ibrutinib and/or venetoclax resistant MCL cell lines showed effective inhibition with a higher IC50 values ranging from 5 µM to 10 µM. More importantly, abemaciclib had potent efficacy on cells from primary MCL patients as well as from patients with acquired ibrutinib resistance. Our recent findings revealed that the addition of PI3K inhibitor TGR-1202 significantly enhanced cytotoxicity of abemaciclib in both sensitive and resistant MCL cell lines. Abemaciclib significantly inhibited phosphorylation of Rb1, the active form of the protein, in 4 different MCL cell lines. The active Rb1 maintains the cell in the G1 phase, preventing progression through the cell cycle and acting as a growth suppressor. The result suggests that CDK4/6 inhibition with abemaciclib disrupts CDK4/6 suppressive activity towards pRb-E2F and induce cell cycle arrest in the MCL cells. Interestingly, abemaciclib somehow interrupted phosphorylation of Chk1, which is continuously phosphorylated and hence activated in the MCL cell lines. Inhibiting activation of Chk1 by abemaciclib may induce cell death via unmonitored and accumulated DNA damage. The efficacy of abemaciclib in combination with Bcl-2 or BTK inhibitors in MCL cell lines and isolated cells from MCL patients are ongoing. These data suggest that abemaciclib in combination with other therapeutic drugs could be beneficial in targeting therapeutic resistant MCL cells. Conclusions Abemaciclib showed impressive therapeutic potency on both MCL cell lines and isolated primary cells from MCL patients, which is likely due to the predominant contribution of cyclin D1-CDK4/6 pathway to malignancy. Other agents, such as PI3K inhibitors, can sensitize abemaciclib in therapeutic resistant MCL cells. Thus, an abemaciclib based multi-drug combinational strategy may be a promising therapy for refractory/relapsed MCL patients in the near future. Disclosures Wang: Beijing Medical Award Foundation: Honoraria; Lu Daopei Medical Group: Honoraria; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Targeted Oncology: Honoraria; OMI: Honoraria, Other: Travel, accommodation, expenses; Nobel Insights: Consultancy; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; Verastem: Research Funding; Molecular Templates: Research Funding; OncLive: Honoraria; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Oncternal: Consultancy, Research Funding; Juno: Consultancy, Research Funding; BioInvent: Research Funding; VelosBio: Research Funding; Acerta Pharma: Research Funding; InnoCare: Consultancy; MoreHealth: Consultancy; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

scholarly journals ONC-212 (I-39), a Novel Inhibitor of the UPR, Is Cytotoxic and Cytostatic Against CLL Cells Under in Vitro Conditions That Mimic the Tumor Microenvironment

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3145-3145
Author(s):  
Narjis Rizwan ◽  
Yandong Shen ◽  
Edwin Iwanowicz ◽  
Stephen P. Mulligan ◽  
Kyle R Crassini ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Effective Treatment ◽  
Cell Line ◽  
Propidium Iodide ◽  
Cycle Arrest ◽  
Migratory Capacity ◽  
Ic50 Values ◽  
Cells Cultured

Abstract Introduction Despite the revolution in the treatment of chronic lymphocytic leukemia (CLL) over the past decade with the introduction of novel inhibitors targeting the B-cell receptor (BCR) signaling pathway and the Bcl-2 family of proteins, relapse is still common. Recent studies suggest that imipridones, a novel class of small molecule agents that attenuate mitochondrial respiration and modulate an immune response against cancer cells, may be an effective treatment option for several difficult to treat cancers. We investigated the effects of the imipridone, ONC-212 (I-39, first published by Nanjing Gator Meditech), as a potential therapeutic strategy for CLL using the OSU-CLL cell line and a modified OSU-CLL line in which TP53 was stably knocked out and primary CLL cells cultured under conditions that mimic the tumour microenvironment (TME). Methodology Primary CLL cells were co-cultured with CD40L-expressing fibroblasts to mimic aspects of the TME. The cytotoxicity of ONC-212 was assessed using the mitochondrial dye DiIC1(5), propidium iodide and flow cytometry. The effects of the drug on the adhesive and migratory capacity of primary CLL cells were evaluated using antibodies against CD49d, CXCR4 and an in vitro migration assay using stroma-derived factor 1a (SDF1-α). Changes in protein expression were assessed by immuno-blotting. The effects of ONC-212 on the cell cycle and proliferation were assessed using the OSU-CLL cell line. OSU-CLL cells were modified using the CRISPr-Cas9 technology to be TP53 deficient (OSU-TP53ko). The proportion of cells in each cycle phase was determined using propidium iodide and flow cytometry. Cell proliferation rates were determined using carboxyfluorescein succinimidyl ester (CFSE) and flow cytometry. Results ONC-212 induced apoptosis in a dose-dependent manner in primary CLL cells cultured in medium alone or in contact with CD40L-fibroblasts (Figure 1); the IC50 values were 72.97 nm +/- 1.45 nM and 472 +/- 2.04 nM, respectively. OSU-CLL and OSU-TP53ko cells were also sensitive to ONC-212, although the TP53 deficient line was less sensitive than OSU-CLL(Figure 1). IC50 values for the cell lines were 22 +/- 1.37 nM (OSU-CLL) and 48 +/- 3.25 nM (OSU-TP53ko). ONC-212 induced cell cycle arrest of the OSU-CLL and OSU-TP53ko lines at the G1/S phase transition. This effect was concomitant with a significant reduction in the proliferation of both lines. ONC-212 significantly down-regulated expression of the adhesion molecule CD49d and the G-coupled protein receptor CXCR4 on primary CLL cells. Down-regulation of CXCR4 translated into a decrease in the migratory capacity of CLL cells along an SDF1-α gradient. Immunoblotting suggested the mechanisms of action of ONC-212 include inhibition of ERK1/2-MAPK, a decrease in the Bcl-2/Bax ratio and upregulation of the pro-apoptotic Puma and Bak proteins. Conclusions ONC-212 is highly effective against CLL cells at nanomolar concentrations, against cells cultured under conditions that mimic aspects of the TME and against TP53-deficient cells. ONC-212 has cytotoxic effects, induces cell cycle arrest, slows proliferation and inhibits the mechanisms by which CLL cells migrate to and are retained within the TME. ONC-212 inhibited signaling downstream of the BCR and induced a pro-apoptotic 'tipping' of the balance in expression of BCl-2 family proteins. These data suggest ONC-212 may represent an effective treatment for CLL, particularly for patients who have high risk, relapsed/refractory disease associated with loss or mutation of TP53. Disclosures No relevant conflicts of interest to declare.

Bortezomib Can Overcome Stroma-Mediated Chemotherapy Resistance in Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2475-2475
Author(s):  
Ibraheem H. Motabi ◽  
Julie Ritchey ◽  
Matthew Holt ◽  
John F. DiPersio
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
S Phase ◽  
Cxcr4 Expression ◽  
Leukemic Cells ◽  
Apoptosis Rate ◽  
G2 Cells

Abstract Abstract 2475 Background: In spite of excellent results of chemotherapy induction resulting in high rates of complete remission in patients with ALL, relapse remains a major problem. This is likely due to a subset of leukemic cells that are chemotherapy resistant. The interaction between bone marrow stromal cells and leukemia cells protects leukemic cells from the cytotoxic effects of chemotherapy. Bortezomib is a proteozome inhibitor used to treat lymphoid malignancies. It has been shown to mobilize stem cells in myeloma patients. We hypothesized that, in addition to its direct cytotoxic effect, bortezomib can also overcome the protective effect of stromal cells by disrupting CXCR4 signaling through inhibition of CXCR4 turnover normally regulated by proteosome degradation. Methods: We used the human B-cell ALL cell lines, G2 and BV-173, and two human BM stromal cell lines, HS-5 and HS-27a for our studies. G2 cells were incubated alone or co-cultured with HS-5 or HS-27a for 24 hours then treated with RPMI (control), cytarabine, doxorubicin, or bortezomib. After 48 hours, cells were harvested and stained with FITC-conjugated anti-human annexin V antibody and analyzed by flow cytometry. To test the effect of bortezomib on CXCR4 expression, G2 cells were incubated with or without stromal cells and treated with cytarabine or bortezomib for 18 hours or left untreated. Then cells were stained with PE-conjugated anti-human CXCR4 antibody (clone 1D9) and analyzed by flow cyometry. The CXCR4 expression was reported as relative mean fluorescent intensity (RMFI) compared to isotype control. We then performed cell cycle analysis of G2 cells treated with cytarabine or bortezomib for 18 hours. All cells were treated with BrdU 1 hour prior to harvest, then stained with APC-conjugated anti-BrdU antibody and analyzed by flow cytometry. Results: Incubation of G2 cells with HS-5 protected them from apoptosis induced by cytarabine or doxorubicin but not bortezomib. The apoptosis rate for cytarabine treated cells was 55±0.3% without HS-5 vs. 15%±0.8 with HS-5(p<0.0001). The apoptosis rate of doxorubicin treated cells was 23±0.6% without HS-5 vs. 4.2±0.8% with HS-5 (p<0.0001). In the case of bortezomib, the apoptosis rate was 67±0.6% without HS-5 vs. 71±1.2% with HS-5. Similar results were observed with the BV-173 ALL cell line and with the use of HS-27a stromal cell line. We noted down-regulation of CXCR4 expression when G2 cells were co-cultured with stromal cells or after bortezomib treatment. In the absence of stromal cells, the CXCR4 RMFI was 40±3 in untreated cells vs. 16±0.4 in bortezomib treated cells (P=0.0015). In the presence of stromal cells CXCR4 RMFI 15±0.7% in untreated cells vs. 5±0.2% in bortezomib treated cells (P=0.0002). In cell cycle analysis, we found only 1.03±0.2% of cells treated with cytarabine were in S phase compared to 10.05±0.7% when cells were treated with bortezomib (P=0.0003). Conclusion: Bortezomib can overcome the stromal cell-mediated protection of human G2 ALL cells. This effect may be, in part, mediated by in the decreased surface expression of CXCR4 shown by others to mediate leukemia-stroma interaction and chemoprotection. In contrast to cytarabine which selectively kills cells in S phase, bortezomib induced killing of ALL cells regardless of cell cycle status. Future studies will use of in vivo mouse models of ALL to test the effects of bortezomib alone or in combination with chemotherapy on survival. Disclosures: DiPersio: genzyme: Honoraria.

Inhibition of mitochondrial translation overcomes venetoclax resistance in AML through activation of the integrated stress response

2019 ◽  
Vol 11 (516) ◽  
pp. eaax2863 ◽  
Author(s):  
David Sharon ◽  
Severine Cathelin ◽  
Sara Mirali ◽  
Justin M. Di Trani ◽  
David J. Yanofsky ◽  
...  
Keyword(s):  
Stress Response ◽  
Single Agent ◽  
Mitochondrial Protein ◽  
B Cell Lymphoma ◽  
Atp Depletion ◽  
Integrated Stress Response ◽  
Mitochondrial Translation ◽  
A Genome

Venetoclax is a specific B cell lymphoma 2 (BCL-2) inhibitor with promising activity against acute myeloid leukemia (AML), but its clinical efficacy as a single agent or in combination with hypomethylating agents (HMAs), such as azacitidine, is hampered by intrinsic and acquired resistance. Here, we performed a genome-wide CRISPR knockout screen and found that inactivation of genes involved in mitochondrial translation restored sensitivity to venetoclax in resistant AML cells. Pharmacologic inhibition of mitochondrial protein synthesis with antibiotics that target the ribosome, including tedizolid and doxycycline, effectively overcame venetoclax resistance. Mechanistic studies showed that both tedizolid and venetoclax suppressed mitochondrial respiration, with the latter demonstrating inhibitory activity against complex I [nicotinamide adenine dinucleotide plus hydrogen (NADH) dehydrogenase] of the electron transport chain (ETC). The drugs cooperated to activate a heightened integrated stress response (ISR), which, in turn, suppressed glycolytic capacity, resulting in adenosine triphosphate (ATP) depletion and subsequent cell death. Combination treatment with tedizolid and venetoclax was superior to either agent alone in reducing leukemic burden in mice engrafted with treatment-resistant human AML. The addition of tedizolid to azacitidine and venetoclax further enhanced the killing of resistant AML cells in vitro and in vivo. Our findings demonstrate that inhibition of mitochondrial translation is an effective approach to overcoming venetoclax resistance and provide a rationale for combining tedizolid, azacitidine, and venetoclax as a triplet therapy for AML.

scholarly journals Dose-Dependent Effect of Cordycepin on Viability, Proliferation, Cell Cycle, and Migration in Dental Pulp Stem Cells

2021 ◽  
Vol 11 (8) ◽  
pp. 718
Author(s):  
Nezar Boreak ◽  
Ahmed Alkahtani ◽  
Khalid Alzahrani ◽  
Amani Hassan Kenani ◽  
Wafa Hussain Faqehi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Growth Curve ◽  
Dental Pulp ◽  
Phase Cell ◽  
Ros Generation ◽  
Dependent Manner ◽  
Significant Difference ◽  
Dose Dependent

Objective: To examine the effect of Cordycepin on the viability, proliferation, and migratory properties of dental pulp-derived mesenchymal stem cells. Materials and methods: The pulp was derived from human premolar teeth extracted for orthodontic purposes after obtaining informed consent. The samples were transferred to the laboratory for processing. DPSCs were expanded and characterized using flow cytometry and differentiation to the bone, adipose, and cartilage cells was examined. MTT Assay was performed using various concentrations of Cordycepin. The growth curve was plotted for 13 days. Cell cycle analysis was performed by flow cytometry. Migratory ability was assessed by wound healing assay. ROS generation was detected by flow cytometry. Gene expression was quantified by RT-qPCR. Statistical analysis was performed. p < 0.05 was considered as significant and p < 0.01 was considered as highly significant (* p < 0.05, and ** p < 0.01). Results: DPSCs expressed characteristic MSC-specific markers and trilineage differentiation. Cordycepin at lower concentrations did not affect the viability of DPSCs. The growth curve of cells showed a dose-dependent increase in cell numbers till the maximum dose. DPSCs treated with 2.5 µM Cordycepin was found to have a reduced G1 phase cell percentage. DPSCs treated with 2.5 µM and 5 µM Cordycepin showed a significant decrease in G2 phase cells. No significant difference was observed for S phase cells. Cordycepin treatment affected the migratory ability in DPSCs in a concentration-dependent manner. Conclusion: Cordycepin can be used at therapeutic doses to maintain stem cells.

scholarly journals The Study on Biocompatibility of Porous nHA/PLGA Composite Scaffolds for Tissue Engineering with Rabbit ChondrocytesIn Vitro

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Lei Chen ◽  
Wei-Min Zhu ◽  
Zhi-Qiang Fei ◽  
Jie-Lin Chen ◽  
Jian-Yi Xiong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tissue Engineering ◽  
Flow Cytometry ◽  
Mtt Assay ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Control Group ◽  
Dependent Manner ◽  
Plga Scaffold ◽  
Significant Difference

Objective. To examine the biocompatibility of a novel nanohydroxyapatite/poly[lactic-co-glycolic acid] (nHA/PLGA) composite and evaluate its feasibility as a scaffold for cartilage tissue engineering.Methods. Chondrocytes of fetal rabbit were cultured with nHA/PLGA scaffoldin vitroand the cell viability was assessed by MTT assay first. Cells adhering to nHA/PLGA scaffold were then observed by inverted microscope and scanning electron microscope (SEM). The cell cycle profile was analyzed by flow cytometry.Results. The viability of the chondrocytes on the scaffold was not affected by nHA/PLGA comparing with the control group as it was shown by MTT assay. Cells on the surface and in the pores of the scaffold increased in a time-dependent manner. Results obtained from flow cytometry showed that there was no significant difference in cell cycle profiles between the coculture group and control (P>0.05).Conclusion. The porous nHA/PLGA composite scaffold is a biocompatible and good kind of scaffold for cartilage tissue engineering.

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