Transcription Activator-Like Effector Nucleases (TALENs)-Mediated Deletion Of MIR17HG In Burkitt Lymphoma Cells Decreases mTOR Pathway Activity and Increases Chemosensitivity

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 243-243
Author(s):  
Faye Pais ◽  
Sanghoon Lee ◽  
Vladimir Rodic ◽  
Matthew J. Barth ◽  
Mitchell S. Cairo ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Caspase 3 ◽  
Raji Cell ◽  
Therapy Resistance ◽  
Taqman Assay ◽  
Raji Cells ◽  
Pathway Activation ◽  
Pten Mrna ◽  
Phosphoflow Cytometry

Abstract Background Burkitt lymphoma (BL) is the most common form of non-Hodgkin lymphoma in children. Although curable in most cases, those patients who relapse have a dismal prognosis with a 4-year survival of 16% (Cairo, Blood 2007). Identification of biomarkers to predict relapse/refractory disease is needed, and an understanding of the underlying mechanisms is vital to develop novel targeted therapies. We previously identified a recurrent gain of chromosome 13q31 that was associated with relapse and increased expression of miR-17-92 in pediatric BL (Schiffman/Miles, Br J Haematol, 2011). In the present study, we sought to determine the role of miR-17-92 expression in vitro using TALENs mediated deletion of the miR-17-92 locus (MIR17HG) in Raji BL cells that express high levels of miR-17-92 (Shiffman/Miles, 2011). Methods The modified restriction enzyme and ligation assembly method (Lee/Cairo, ASH 2012) was used to construct MIR17HG TALENs. Expression of miR-17 was assayed by TaqMan assay, and expression of the miR-17-92 target PTEN mRNA and protein were assayed by RT-PCR and western blot, respectively. Proliferation and caspase activity were measured at baseline and after treatment with cyclophosphamide. AKT and phospho (p)AKT were assayed by western blotting. To evaluate mTOR and MAPK pathway activation, pS6 and pERK were assayed by phosphoflow cytometry. In addition, caspase 3 activity was assayed by phosphoflow cytometry to assess induction of apoptosis. Cells were treated with chemotherapy (cyclophosphamide or 4-hydroperoxycyclophosphamide, 4HC), rapamycin, the PI3 kinase inhibitor PIK90, and the dual PI3K/mTOR inhibitor BEZ235 (Axon Medchem). To further validate the role of miR-17 in chemoresistance, miR-17 expression levels were measured by RT-PCR TaqMan assay in the previously developed rituximab-chemotherapy resistant Raji cell lines (RCRCL) Raji 2R and Raji 4RH (Barth et al. Br J Haematol, 2012). Results MIR17HG TALENs were used to generate two Raji cell lines with a hemizygous deletion of MIR17HG, #31(+/-) and #48(+/-). Relative to vector control Raji cells, cell lines with hemizygous MIR17HG deletion showed lower expression of miR-17: Raji = 1.0, #31= 0.80-fold, and #48= 0.71-fold. PTEN mRNA was higher in #31 and #48 cells than in the control (Raji = 1.0, #31 = 1.38-fold, and #48 = 2.24-fold), but there was no apparent difference in PTEN protein by western blot. Western blotting showed decreased pAKT with no difference in total AKT. Cyclophosphamide (10mM) induced a significant decrease in proliferation at 48 hours (90.5 +/- 2.9%, p=0.007) in #31 cells vs. control cells that was associated with a trend toward increased caspase 3/7 activity (148.3+/-8.9%, p=0.08). By phosphoflow cytometry, Raji #48 cells showed decreased pS6 compared to control cells and a modestly greater induction of caspase 3 in response to 4HC (3.4 uM) or PIK90 after 48 hours. The combination of 4HC and PIK90, however, significantly induced caspase 3 in Raji #48 cells relative to control Raji cells. Raji cells showed high basal levels of pERK and pS6. BEZ235 or PIK90 treatment decreased p-ERK. Rapamycin did not affect p-ERK but did decrease pS6. While BEZ235 and 4HC each modestly induced caspase alone in Raji cells, the combination caused a much greater increase in caspase 3. In RCRCLs Raji 2R and Raji 4RH, which show high pAKT levels by western blot and phosphoflow cytometry, miR-17 levels were increased (1.73- and 1.69-fold, respectively) relative to chemosensitive Raji cells (1.0) that exhibit lower pAKT levels. Conclusions Increased miR-17-92 expression in BL may contribute to therapy resistance, which is further supported by finding increased miR-17 expression in chemotherapy resistant Raji cell lines. Hemizygous deletion of the miR-17-92 locus in BL cells led to increased PTEN mRNA, decreased mTOR pathway activation (decreased pAKT and pS6), and increased sensitivity to chemotherapy. Resistance to PIK90 and 4HC in parental Raji cells that overexpresses miR-17-92 may be due in part to a compensatory upregulation of the MAPK and mTOR pathways, as demonstrated by increased pERK and pS6. Inhibiting this survival mechanism with the PI3K/mTOR inhibitor BEZ235 restored sensitivity to cytotoxic chemotherapy. These findings suggest potential mechanisms underlying BL therapy resistance as well as targeted therapies to overcome resistance. Disclosures: Cairo: Roche/Genentech: advisory board Other.

scholarly journals Microrna (miR)-17-92 Contributes to Therapy Resistance in Burkitt Lymphoma Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2944-2944 ◽  
Author(s):  
Rodney R. Miles ◽  
Vladimir Rodic ◽  
Matthew J. Barth ◽  
Mitchell S. Cairo ◽  
Michelle L. Hermiston
Keyword(s):  
Cell Lines ◽  
Western Blotting ◽  
Burkitt Lymphoma ◽  
Genomic Dna ◽  
Caspase 3 ◽  
Therapy Resistance ◽  
Taqman Assay ◽  
Refractory Disease ◽  
Raji Cells ◽  
Dna Pcr

Abstract Background Burkitt lymphoma (BL) is curable, but patients with relapse or refractory disease have very poor outcomes. The underlying biological differences leading to relapse/refractory disease are unknown. We previously identified a potential association between microRNA (miR)-17-92 and relapse in patient samples. MiRs encoded by this gene have been implicated in therapy resistance, so we are seeking relationships between miRNA expression and therapy resistance in BL cells. Methods The miR-17-92 locus was targeted for deletion in Raji BL cells using custom CRISPR-Cas9 lentiviral vectors. Single cell-derived clones were established and locus deletion was determined by genomic DNA PCR. Inducible miR-17-92 expression in Raji cells was established by transduction of a cumate switch inducible lentivector system containing the miR-17-92 gene along with a CymR repressor expression vector (Raji/CymR/miR-17-92). MiR expression was assessed by TaqMan assay, and protein expression and caspase-3 cleavage were assessed by western blotting. Cells were treated with 4HC, the active metabolite of cyclophosphamide, or doxorubicin, and viability was assessed by AlamarBlue assay. Results Genomic DNA PCR verified hemizygous deletion of the miR-17-92 gene in several Raji BL clones; no homozygous deleted clones were identified. In two hemizygous clones R5 and R24, miR-17 expression was decreased to 0.24-fold and 0.37-fold, respectively, relative to the parental Raji line. The miR-17-92 target proteins PTEN and Bim were increased in the R5 and R24 clones by western blotting. Compared to Raji, R5 and R24 showed increased basal caspase-3 cleavage and much greater induction of caspase-3 cleavage with cytotoxic chemotherapy (10 µM 4HC). In addition, 0.5 and 1 µM doxorubicin showed a dose dependent induction of cleaved caspase in R5 and R24 cells that was much greater than in parental Raji cells. The IC50 for doxorubicin in R5 and R24 miR-17-92 hemizygous cells was markedly decreased (0.00025 and 0.00032 µM, respectively) compared to parental Raji cells (0.064 µM). Finally, miR-17 expression was inducible by treating cumate repressor vector transduced Raji/CymR/miR-17-92 cells with 30 µg/mL cumate (+2.8-fold at 3 days, +7.5-fold at 5 days). Conclusion We established miR-17-92 hemizygous-deleted cell lines that show increased PTEN and pro-apoptotic Bim, increased basal caspase-3 cleavage, and increased chemosensitivity. We have established Raji BL cell lines with inducible miR-17-92 expression to extend these experiments by determining the effect of increased miR-17-92 on chemosensitivity. Our findings show that high miR-17-92 expression in BL cells can contribute to therapy resistance, which may be important for understanding refractory BL in patients and could lead to more effective targeted therapies. Disclosures Cairo: Celgene: Research Funding.

scholarly journals Knockdown of NOLC1 Inhibits PI3K-AKT Pathway to Improve the Poor Prognosis of Esophageal Carcinoma

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Fanguo Kong ◽  
Yansheng Shang ◽  
Xingyuan Diao ◽  
Jiaguo Huang ◽  
Hui Liu
Keyword(s):  
Overall Survival ◽  
Western Blot ◽  
Esophageal Carcinoma ◽  
Cell Line ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Caspase 3 ◽  
Cyclin B1 ◽  
Akt Pathway ◽  
Pi3k Inhibitor Ly294002

Objective. Esophageal carcinoma (ESCA) is a common malignant gastrointestinal tumor. The abnormal expression of NOLC1 is involved in the tumorigenesis of various human tumors, whereas the function and mechanism of NOLC1 in ESCA remain unclear. In this study, we explored the relationship between NOLC1 and poor prognosis of ESCA, and its role and mechanism in the occurrence of ESCA. Methods. The NOLC1 expression in ESCA tissues and cell lines was determined by qRT-PCR, immunohistochemistry, or western blot. The Kaplan–Meier method was conducted to estimate the overall survival. Cox regression analysis was carried out to examine the association between patient characteristics and prognosis. A recombined lentiviral vector containing NOLC1 was applied for transfecting ESCA cells (Eca109 and TE-13) and established a stable cell line with low NOLC1 expression or high NOLC1 expression, in the absence or presence of PI3K inhibitor (LY294002) treatment. Cell proliferation, apoptosis rate, invasion ability, migration ability, and PI3K/AKT pathway were detected by CCK8 assay, flow cytometry, Transwell assay, wound-healing assay, and western blot. Results. NOLC1 overexpression was observed in ESCA tissues and ESCA cell lines (EC9706, Eca109, TE-13, Kyse170, T.TN) compared with adjacent normal tissues and normal esophageal cell line HEEC. NOLC1 overexpression was markedly associated with bigger tumor size, lymph node metastasis, and advanced TNM stage. Patients with NOLC1 overexpression have shorter overall survival than that of those with low NOLC1 expression. NOLC1 overexpression was considered to be an independent poor prognostic factor affecting overall survival. NOLC1 knockdown inhibited proliferation, migration, invasion, and cyclin B1 expression and promoted the apoptosis and cleaved-caspase-3 expression of Eca109 and TE-13 cells. NOLC1 overexpression accelerated proliferation, migration, invasion, and cyclin B1 expression and inhibited the apoptosis and cleaved-caspase-3 expression of ESCA cells via activating PI3K/AKT pathway. Rescue experiments showed that PI3K inhibitor (LY294002) could reverse the phenomenon caused by NOLC1 overexpression. Conclusion. NOLC1 may be a marker for poor prognosis. It can participate in the occurrence and development of ESCA via the PI3K/AKT pathway.

scholarly journals ERβ1 Expression Patterns Have Different Effects On EGFR TKIs Treatment Response in EGFR Mutant Lung Adenocarcinomas.

2020 ◽  
Author(s):  
Lijuan Zhang ◽  
Meng Tian ◽  
Jiamao Lin ◽  
Jianbo Zhang ◽  
Haiyong Wang ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Expression Patterns ◽  
Estrogen Therapy ◽  
Growth Factor Receptor ◽  
Treatment Resistance ◽  
Progression Free Survival ◽  
Pathway Activation ◽  
Egfr Tkis

Abstract Background: Estrogen receptor β (ERβ) can regulate cellular signaling through non-genomic mechanisms, potentially promoting resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, the mechanisms underlying the ERβ-mediated resistance to EGFR TKIs remain poorly understood. Methods: qRT-PCR was performed to investigate ERβ1 and ERβ5 expression levels in cell lines. The localization of ERβ and ERβ1 within cells was assessed using immunocytochemistry and immunofluorescence. The effect of estradiol and/or gefitinib on EGFR signaling pathways was determined by western blot. Cell viability and colony formation assays were used to assess gefitinib response for different cell lines. The apoptosis was verified by tunel and western blot. Immunohistochemistry was used to assess the expression of ERβ1 in lung adenocarcinoma tissues. Patient survival was estimated using the Kaplan-Meier method, and comparisons between groups were conducted using log-rank tests. Results: PC9 cell lines stably overexpressing ERβ1 or ERβ1/ERβ5 were established successfully. Immunofluorescence revealed that ERβ5 overexpression partly retained ERβ1 in the cytoplasm. Immunoblotting analyses revealed that EGFR pathway activation levels were higher in PC9/ERβ1/5 cells than those in PC9/ERβ1 or control PC9 cells. In the presence of estradiol, PI3K/AKT/mTOR pathway activation levels were higher in ERβ1/5-expressing cells than those in ERβ1-expressing cells. Additionally, PC9/ERβ1/5 cells were less prone to the cytotoxic and pro-apoptotic effects of gefitinib compared with PC9/ERβ1 or control PC9 cells. Conclusion: Cytoplasmic ERβ1 was associated with poor progression-free survival in lung cancer patients treated with EGFR TKIs. These results suggest that anti-estrogen therapy might reverse EGFR TKI treatment resistance to some extent in selected patients.

scholarly journals Combining RANK/RANKL and ERBB-2 targeting as a novel strategy in ERBB-2-positive breast carcinomas

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Ilianna Zoi ◽  
Michalis V. Karamouzis ◽  
Evangelia Xingi ◽  
Panagiotis Sarantis ◽  
Dimitra Thomaidou ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Lines ◽  
Family Members ◽  
Metastatic Potential ◽  
Nuclear Factor Κb ◽  
Proximity Ligation Assay ◽  
Breast Cancers ◽  
Tissue Samples ◽  
Erbb Family ◽  
Pathway Activation

Abstract Background ERBB-2 is overexpressed in about 20% of breast cancers (BCs), indicating poor prognosis. The receptor activator of nuclear factor-κB (RANK) pathway is implicated in ERBB-2 (+) BC. The purpose of this study was to elucidate the underlying molecular mechanism of this interaction and the beneficial impact of dual targeting of RANK and ERBB-2 pathways. Methods We used SKBR3, MCF7, MDA-MB-453, and BT-474 human BC cell lines. We examined RANK and RANKL expression using RT-PCR, Western blot, and immunofluorescence. The evaluation of RANK expression in a cohort of BC patients was performed using immunohistochemistry. The interaction between RANK and ERBB family members was detected using proximity ligation assay (PLA), which enables the visualization of interacting proteins. We used inhibitors of both pathways [trastuzumab (T), pertuzumab (P), denosumab (D)]. NF-κB pathway activation was studied using Western blot. Cell growth and viability was evaluated using XTT, flow cytometry, and clonogenic assay. For cell migration evaluation, scratch assay was performed. Data were analyzed by one-way ANOVA. Results Cell lines express RANK and RANKL. RANK immunostaining was also detected in human BC tissue samples. RANK receptor dimerizes with ERBB family members. RANK/ERBB-2 dimer number seems to be associated with ERBB-2 expression (SKBR3, 5.4; BT-474, 8.2; MCF7, 0.7; MDA-MB-453, 0.3). RANK/ERBB-2 dimers were decreased in the presence of the inhibitors D, T, and P, while they were increased after RANKL (R) treatment in SKBR3 (m, 5.4; D, 1.2; T, 1.9; DT, 0.6; TP, 1; DTP, 0.4; R, 11.8) and BT-474 (m, 8.2; D, 3.1; T, 4.3; DT, 0.7; TP, 3.4; DTP, 3.2; R, 11.6). Combination targeting of SKBR3 further decreased NF-κB pathway activation compared to single targeting. In SKBR3, RANKL and ERBB-2 blockage resulted in reduced cell proliferation, increased apoptosis, and lower metastatic potential compared to mock cells (m) and reversed values in RANKL presence. The combination treatment of SKBR3 with D, T, and P had an advantage in functional traits compared to single targeting. Denosumab suppressed NF-κB signaling and diminished proliferation rate in MDA-MB-453 cells. MCF7 did not correspond to inhibitors. Conclusions The results indicate a novel physical and molecular association between ERBB-2 and RANK pathways that affects ERBB-2 (+) BC growth. We also present data suggesting that the combination of anti-ERBB-2 agents and RANKL inhibitors have a potential direct anti-tumor effect and should be further tested in certain BC patients.

Pan-BCL-2 Family Small Molecule Inhibitor GX015-070 (GeminX, Inc.) Exhibits Single Agent Cytotoxicity, Is Synergistic with Select Anti-Leukemia Cytotoxic Agents, and Induces Apoptosis in Cell Lines with t(4;11)(q21;q23) Translocations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3368-3368 ◽  
Author(s):  
Jessicca M. Rege ◽  
Blaine W. Robinson ◽  
Manish Gupta ◽  
Jeffrey S. Barrett ◽  
Peter C. Adamson ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Family Member ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Single Agent ◽  
Cytotoxic Agents ◽  
Response Surface Modeling

Abstract Background: Leukemias with MLL translocations, especially t(4;11), often are resistant to common chemotherapeutic agents, which may be due to abnormal apoptosis regulation. Pro- and anti-apoptotic BCL-2 family member interactions govern initiation of the intrinsic apoptosis pathway. GX015-070, which currently is in Phase I/IIA clinical trials, mimics the BH3 domain on pro-apoptotic BCL-2 family proteins and can bind the BH3 binding pocket of anti-apoptotic BCL-2 family members and modulate apoptosis. We performed comprehensive protein expression profiling of BCL-2 family member proteins and evaluated in vitro activity and mechanism of action of GX015-070 in cell lines with t(4;11). Methods: Baseline expression of BCL-2 family proteins was determined by Western blot analysis. Cytotoxicity was assessed by MTT after a 3 day exposure of RS4:11, SEM-K2 and MV4-11 cells in log phase growth to single agent GX015-070 at concentrations from 5 nM to 7.5 μM. Combined effects of fixed-concentration GX015-070 with cytotoxic agents over a range of concentrations were assayed by MTT, and the results were analyzed by pharmacostatistical response surface modeling. Disruption of specific pro- and anti-apoptotic BCL-2 family member interactions was investigated by co-immunoprecipitation/Western blot analysis. Flow cytometry and/or Western blot analysis of Caspase-3 activation, and a FACS TUNEL assay, were used to assess apoptosis in GX015-070 treated and untreated cells. Results: The three cell lines had similar baseline levels of expression of BCL-2 family proteins. BCL-2 and BAX were most abundant followed by PUMA, BAK, BCL-XL, BIM-EL, MCL-1, BIK and NOXA. Results of assays of GX015-070 activity and mechanism of action are in shown in the table. Conclusions: These data indicate that GX015-070 has potent cytotoxic activity in cell lines with t(4;11) as a single agent and that the cytotoxicity results from apoptosis. Response surface modeling in RS4:11 cells suggested ability to achieve effective doses with GX015-070 combined with cytosine arabinoside (Ara-C), dexamethasone (Dex) or doxorubicin (ADR) that are lower than projected from the single agents, but synergy was not suggested when GX015-070 was combined with etoposide, methotrexate or 6-thioguanine. The co-IP experiments give proof of principle that GX015-070 disrupts pro- and anti-apoptotic BCL-2 family protein interactions in cell lines with t(4;11). Additional pre-clinical experiments directed at overcoming drug resistance from abnormal cell death regulation in leukemias with t(4;11) using GX015-070 are in progress. These studies provide a framework to understand the cell death/survival machinery in primary leukemias with t(4;11) translocations more completely and manipulate that machinery to achieve better treatments. GX015-070 Activity and Mechanism Cell Line Single Agent Activity Synergy Inhibition Caspase-3 Activation TUNEL RS4:11 IC50=43.5 nM Ara-C, Dex, ADR Mcl1:Bak; Bcl2:Bak + + SEM-K2 IC50=156 nM In progress Mcl1:Bak; Bcl2:Bak + In Progress MV4-11 IC50=123 nM In progress Mcl1:Bak In progress +

The Oral PKC-β Inhibitor Enzastaurin (LY317615) Suppress Phosphorylation and Induces Apoptosis in Multiple Myeloma Cell Lines by Inhibition of AKT Pathway.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1371-1371
Author(s):  
Antonino Neri ◽  
Sandra Marmiroli ◽  
Pierfrancesco Tassone ◽  
Luigia Lombardi ◽  
Lucia Nobili ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Western Blot ◽  
Cell Lines ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Time Dependent ◽  
Parp Cleavage ◽  
Akt Phosphorylation ◽  
Downstream Target ◽  
Pkc Β

Abstract The PKC pathway has been shown to play a role in the regulation of cell proliferation in several hematologic malignancies. In this study we tested the oral PKC-β inhibitor, Enzastaurin (LY317615 - Eli Lilly) for its therapeutic efficacy in Multiple Myeloma (MM). We first analyzed PKC-β I and II expression by Western blot in a panel of 19 human MM cell lines, showing that 9 cell lines express either 1 or both isoforms. We next examined the growth inhibition effect of Enzastaurin in the same panel of MM cell lines using either WST-1 or MTT assay and cell viability assessment by Tripan Blue exclusion. Eighteen cell lines have IC50 value ranging from 1,2 μM to 12,5 μM. To examine molecular mechanisms whereby Enzastaurin induces cytotoxicity, we performed cell cycle profiling using PI and observed a significant increase of the percentage of cells in the sub G0–G1 fraction. To determine whether Enzastaurin-induced cell death is mediated by apoptosis, we studied by ELISA and Western blot caspase 3 and PARP cleavage. We observed induction of caspase 3 and PARP cleavage in a dose and time dependent fashion. Notably, the broad caspase (Z-VAD-FMK) inhibitor reduced Enzastaurin-induced cytotoxicity. We next determined whether Enzastaurin could inhibit AKT phosphorylation in MM cell lines with constitutive phosphorylation of AKT. Enzastaurin decreased AKT phosphorylation in a dose and time dependent fashion. Phosphorylation of GSK3β, a downstream target protein of AKT, was also markedly inhibited. Phosphorylation of PDK-1, a known upstream activator of AKT, was not affected by Enzastaurin. In conclusion, our results indicate that Enzastaurin-induced cytotoxicity is mediated via activation of caspase. This effect is associated with significant inhibition of AKT activity and its downstream target GSK3 β. Enzastaurin does not alter the phosphorylation of the upstream AKT activator PDK-1. These data suggest that Enzastaurin inhibit AKT signalling pathway and support its evaluation in a murine model of human MM.

Proteasome Inhibitors Can Induce Caspase-3 Activation and Apoptosis in Human CML Cell Lines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4681-4681
Author(s):  
Byung-Su Kim ◽  
Chang Up Kim ◽  
Young-Ju Kim ◽  
Eun Kyung Bae ◽  
Jinhee Kim ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Proteasome Inhibitor ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Expression Patterns ◽  
Proteasome Inhibitors ◽  
Acridine Orange Staining

Abstract The proteasome is a multi-enzyme complex that provides the ubiquitin-dependent degradation of many cytoplasmic and nuclear proteins involved in cell cycle progression and apoptosis. Inhibition of the proteasome represents a promising approach for the treatment of cancer because it can lead to cell cycle arrest and activation of caspases in tumor cells. There are several proteasome inhibitors that have been reported to induce apoptosis in various tumors. However, the effect of proteasome inhibition in human myeloid leukemia has not been reported so far. In this study, we tested two peptide-aldehyde proteasome inhibitors (MG115, MG132) on two human CML cell lines (K562, KCL22). At first, we treated both cell lines for 24, 48 and 72 hours with different doses of MG115 and MG132 and cell viability was tested by MTT assay. It showed substantial time and dose dependent cytotoxicity in both CML cell lines. Acridine orange staining also revealed DNA fragmentation. We then performed caspase-3 colorimetric assay after treating both cell lines for 6, 12 and 24 hours with 0.78μM of MG115, MG132. K562 showed the continuous rising of caspase-3 activity, while KCL22 exhibited the initial increase and subsequent mild decrease of caspase-3 activity. In addition, western blot analysis showed the reduction of procaspase-3 expression. The expression of Bcl-2 and Bcl-XL was reduced by western blot. p21 expression was slightly increased and that of cyclin D1 was decreased. Additionally, the treatment of proteasome inhibitor in CML cell lines initially induced phosphorylation of Jun kinase. We next examined the expression of heat shock proteins (Hsp70, Hsp90) after treating for 6, 12, 24 hours with the same proteasome inhibitors. Western blot analysis results indicated that expression patterns were different between MG115 and MG132. MG115 induced the slight increase of Hsp70 and Hsp90 in K562, but the reduction of both in KCL22. Meanwhile, MG132 produced the decrease of Hsp70 and Hsp90 in both K562, KCL22. In summary, our work supports that a proteasome inhibitor can induce apoptosis in human CML cell lines. We are currently focusing on the combined effect of proteasome inhibitor and Hsp90 inhibitor on CML. IC50 of Proteasome Inhibitors Cell line Proteasome Inhibitor 24hr 48hr 72hr K562 MG115 3.01 μM 1.14 μM 0.59 μM K562 MG132 μ 2.13 M 1.03 μM 0.57 μM KCL22 MG115 156.92 μM 1.36 μM 0.73 μM KCL22 MG132 1.56 μM 0.93 μM μ 0.75 M

Inhibition Of The Wnt Pathway Leads To Improved Chemosensitivity In Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1428-1428
Author(s):  
Smita C. Dandekar ◽  
Eleny Romanos-Sirakis ◽  
Faye Pais ◽  
Teena Bhatla ◽  
Courtney L Jones ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Wnt Pathway ◽  
Caspase 3 ◽  
Target Genes ◽  
Synergistic Effects ◽  
Lymphoblastic Leukemia ◽  
Facs Analysis ◽  
Protein Levels ◽  
Phosphoflow Cytometry

Abstract Introduction While childhood acute lymphoblastic leukemia (ALL) is highly curable, up to 20% of children will relapse, with dismal prognosis, warranting the need for novel therapies. Previously, using an integrated genomic approach on matched diagnosis-relapse samples, we identified overactivation of the Wnt pathway as a mechanism of disease recurrence at relapse (Hogan et al, Blood 2011). Aberrant Wnt signaling has been linked to cancers of the liver, colon, breast, skin and more recently hematologic malignancies. To validate our findings and determine if Wnt inhibition could restore chemosensitivity in relapsed ALL, we sought to examine directly whether Wnt is activated at relapse in paired samples (examining expression of activated b-catenin and its downstream target Survivin (BIRC5) using multiparameter phosphoflow cytometry) and tested the efficacy of a recently developed small molecule Wnt inhibitor, iCRT14, that specifically interferes with the b-catenin-TCF interaction (Gonzalves et al, PNAS 2011), in ALL cell lines and patient samples. Methods B and T-ALL cell lines were treated with iCRT14 and the expression of target genes were determined by quantitative RT-PCR.10 paired diagnosis-relapse patient samples obtained from the Children’s Oncology Group were washed, fixed and stained simultaneously with caspase 3, CD10, activated b-catenin and survivin and the change in expression of activated b-catenin and survivin from diagnosis to relapse was measured by multiparameter phosphoflow cytometry in each patient by gating on the caspase 3 negative, CD10 positive leukemic blasts. To test the effect of Wnt inhibition on chemosensitivity, B-ALL cell lines were pretreated with iCRT14 for 48 hours prior to incubation with traditional chemotherapy for an additional 24 hours. The response to increasing doses of iCRT14 and chemo, alone and in combination, was assessed by cell viability (Cell Titer-Glo Luminescent Assay (Promega)) and apoptosis (FACS analysis with AnnexinV-PE/7AAD staining (BD Bioscience)). Protein levels of apoptotic markers were assessed. Also, 4 newly diagnosed and 4 relapsed patient samples were treated ex vivo with iCRT14 (20 and 30 uM) and prednisolone, alone and in combination. Drug combination results were analyzed using the Calcusyn program which calculates a Combination Index (CI): CI>1.1=antagonism, 0.9-1.1=additive and <0.9=synergy. Results Previously, we reported that treatment of ALL cell lines with iCRT14, downregulated the mRNA expression of the Wnt target genes BIRC5, axin 2, and c-myc (Romanos et al, ASPHO 2012 # 414). Comparison of Mean Fluorescent Intensity of activated b-catenin and survivin in the 10 pairs showed upregulation of activated b-catenin at relapse in 6 of 10 patients. Survivin expression was increased in 6 patients and in 4 patients the upregulation of activated b-catenin and survivin was concordant. iCRT14 pretreatment of cell lines followed by chemotherapy (prednisolone, etoposide, doxorubicin, cytarabine and 6TG) demonstrated additive to synergistic effects on viability. UOCB1 cells showed synergism with all 5 chemotherapy agents (CI=0.1-0.88). Nalm6 cells were very sensitive to iCRT, hence the combination with chemotherapy showed additive to synergistic effects (CI=0.05-1.I). In Reh cells, all agents besides cytarabine showed robust synergism (CI=0.03-0.55). FACS analysis revealed that iCRT14 alone contributed significantly to apoptosis and combination with chemotherapy further increased cell death with >80% apoptosis by hour 72 with the maximal chemotherapy dose in all cell lines. Change in the protein levels of cleaved PARP and cleaved caspase 3 was seen. The 4 diagnosis patients were very sensitive to prednisolone as expected, precluding synergism with iCRT14. The relapsed patient samples were much less sensitive to prednisolone alone (40% decrease in viability in relapsed patients vs 80% in new diagnoses). Interestingly, all the relapsed patients showed enhanced chemosensitivity with Wnt inhibition. 3 out of 4 relapsed patients showed strong synergism (CI=0.03-0.6) with both doses of iCRT14 and 1 patient showed additive to synergistic effects (CI=0.7 and 1). Conclusion Overactivation of the Wnt pathway may lead to chemoresistance in relapsed ALL. Wnt Inhibition restores chemosensitivity and induces apoptosis in ALL cell lines and primary patient samples making it a potential therapeutic approach. Disclosures: No relevant conflicts of interest to declare.

New Targets and Potential Strategy to Enhance the Anticancer Efficacy BKM-120 and BEZ235 in Lymphoma Cell Lines

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1773-1773
Author(s):  
Monica Civallero ◽  
Maria Cosenza ◽  
Stefano Sacchi
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Cell Cycle Arrest ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Caspase 3 ◽  
Lymphoma Cell ◽  
Tubulin Polymerization ◽  
Anticancer Efficacy ◽  
Cycle Arrest

Abstract Background. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays an important role in many physiological functions, including cell cycle progression, differentiation, survival, motility, apoptosis, protein synthesis and metabolism modification. The PI3K/AKT/mTOR signaling pathway also inhibits cell autophagy, a catabolic process involving the degradation of a cell’s own components through the lysosomal machinery. BKM120 and BEZ235 are synthetic small molecules belonging to the class of imidazo-quinolones that show preclinical activity against a range of solid and hematological malignancies. BKM-120 inhibits the catalytic subunit of class I PI3K by competitive binding to its ATP binding site, while BEZ235 is a dual class I PI3K/mTOR inhibitor. In this study we investigated the effects of BKM120 and BEZ235 on survival rate, apoptosis, signaling pathways expression, autophagy, metabolism modification, cell cycle arrest and tubulin polymerization kinetic in lymphoma cell lines. Method. Lymphoma cell lines (WSU-NHL, Jeko-1 and Karpas-299) were treated with different concentrations of BKM120 and BEZ235 (Novartis) for 24 and 48h and the IC50 values were evaluated using MTT assay. To assess for apoptosis, we used annexin V/PI staining kit/ flow cytometer analysis and Western Blot to evaluate caspase 3, 8, 9 and PARP expression. The cell cycle was performed applying PI incorporation and flow cytometer analysis. A flow cytometry based technique for the analysis of tubulin polymerization using a-tubulin staining was done to test whether the two drugs could cause a mitotic block. Western blot was utilized for phosphorylation status of protein kinases and for monitoring autophagy and metabolism. Results and conclusions. BKM120 and BEZ235 induced significant increase of apoptosis evidenced by annexin IV/PI staining and confirmed by the cleavage of caspases -3, -9 -8 and PARP. BKM120 and BEZ235 induced an up regulation of pro apoptotic protein Bim, Bax and Bad. Treatment for 24h with BKM120 and BEZ235 resulted in different effects on cell cycle. BKM120 induced an increase of G2-phase with down regulation of Cyclin D and E, and an up-regulation of Cyclin A, p21 and p27. The increase in G2-M caused by BKM120 treatment occurred in a dose dependent manner. BEZ235 induced an increase of G0/G1-phase with up regulation of Cyclin A, D, E and p21 and p27. Quantitative analysis of a-tubulins polymerization of the cell lines revealed that treatment with BKM120 induced an accumulation of mitotic cells. BKM120 and BEZ235 are inhibitors of intracellular pathways in targeting p-Akt, p- mTOR, pS6K, 4EBP1, MYC and STAT. BKM120 and BEZ235 increased the levels of type II LC3 and p62, hallmarks of autophagy, in addition to increasing caspase 3 cleavage and annexin positive cells, suggesting that the two drugs induced both apoptosis and autophagy. The combination of BKM120 and BEZ235 with chloroquine which are in clinical use, with the lysosomotropic autophagy inhibitor chloroquine demonstrating marked cooperates with inhibition of autophagy to elicit apoptosis through the intrinsic mitochondrial pathway (BAX, BIM and BAD). Treatment with BKM120 and BEZ235 induced an increase of GLUT1 and HIF-1 protein expression. GLUT1 plays a role in regulation of ROS levels in particular after BEZ235 treatment. We concluded that BKM120 and BEZ235 inhibit PI3K/AKT and mTOR signaling, induce autophagy-apoptosis, affect lymphoid cell metabolism and promote ER stress via ROS increase. Moreover, we observed that BKM120 can act as a microtubule destabilizer inducing cell cycle arrest. In these study, we highlight new targets of BKM120 and BEZ235 in addition to the known PI3K/AKT signaling pathway; these results can help to identify new potential strategies to enhance the anticancer efficacy BKM-120 and BEZ235 in lymphoma cell lines. Disclosures No relevant conflicts of interest to declare.

Disruption of the Crosstalk CXCL12/CXCR4 Chemotactic Pathway To Enhance Melphalan-Induced Apoptosis in Multiple Myeloma Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3445-3445
Author(s):  
Ayman A. Saad ◽  
James Fortney ◽  
Lin Wang ◽  
Heather O’Leary ◽  
Laura Gibson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Western Blot ◽  
Cell Lines ◽  
Caspase 3 ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Apoptotic Markers ◽  
Induced Apoptosis ◽  
B1 Cells

Abstract INTRODUCTION: Multiple myeloma cells display functional CXCR4 chemokine receptor that stimulates the migration of these cells toward their natural ligand, CXCL12 (stromal-derived factor, SDF-1a). CXCL12 is secreted by bone marrow stroma. Consistent with their CXCR4 expression, myeloma cells home to the marrow microenvironment, where adhesive interactions promote growth, survival, and confer cell adhesion-mediated drug resistance. METHODS U266-B1 cells (ATCC myeloma cell line) were pre-treated with recombinant CXCL12 for 30 minutes prior to the addition of melphalan for up to 72 hours. Both melphalan and CXCL12 were added at 24 hour intervals. Cell lines alone and cell lines with only melphalan or only CXCL12 were used as controls. We have also tested the influence of adding AMD3100, a reversible inhibitor of CXCR4, on myeloma cell survival. U266-B1 cells in media alone were pre-treated with AMD3100 for 24 hours prior to treatment with melphalan for 16 hours. Cell viability following treatment was quantified by flow cytometry assay using Annexin-V-FITC staining. Western blot analysis was used to quantify the apoptotic activity of the cell lines using 4 apoptotic markers: PARP (poly ADP-ribose polymerase), caspase-3, Bcl-2, and Mcl-1. RESULTS: Recombinant CXCL12 conferred a protective effect to myeloma cell lines during melphalan treatment. This effect was more pronounced at 72 hours of treatment. Western blot analysis showed diminished expression of the apoptotic markers, cleaved PARP (poly ADP-ribose polymerase) and active caspase-3 in the melphalan-treated cell lines with prior exposure to CXCL12. Additionally, pretreatment with AMD3100 resulted in enhanced apoptosis following melphalan treatment. CONCLUSION: Our data showed that CXCL12, a naturally occurring cytokine secreted by bone marrow stromal cells confers a protective effect on myeloma cells against apoptosis. Disruption of this effect by AMD3100 resulted in enhanced melphalan-induced apoptosis of myeloma cells. We are continuing to study this effect with the potential future utility of AMD3100 as a melphalan chemo-sensitizer in the treatment of multiple myeloma.

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