Chlorogenic acid inhibits Bcr-Abl tyrosine kinase and triggers p38 mitogen-activated protein kinase–dependent apoptosis in chronic myelogenous leukemic cells

Blood ◽  
2004 ◽  
Vol 104 (8) ◽  
pp. 2514-2522 ◽  
Author(s):  
Gautam Bandyopadhyay ◽  
Tanusree Biswas ◽  
Keshab C. Roy ◽  
Swapan Mandal ◽  
Chhabinath Mandal ◽  
...  
Keyword(s):  
Chlorogenic Acid ◽  
Cell Lines ◽  
Salivary Gland Tumor ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Mitogen Activated Protein Kinase ◽  
Leukemic Cells ◽  
Abl Kinase ◽  
Mitogen Activated Protein

Abstract We report that chlorogenic acid (Chl) induces apoptosis of several Bcr-Abl–positive chronic myelogenous leukemia (CML) cell lines and primary cells from CML patients in vitro and destroys Bcr-Abl–positive K562 cells in vivo. In contrast, this compound has no effect on the growth and viability of Bcr-Abl–negative lymphocytic and myeloid cell lines and primary CML cells. Sodium chlorogenate (NaChl) exhibits 2-fold higher efficiency in killing K562 cells compared with Chl. NaChl also induces growth inhibition of squamous cell carcinoma (HSC-2) and salivary gland tumor cells (HSG), although at 50-fold higher concentration. NaChl inhibits autophosphorylation of p210Bcr-Abl fusion protein rapidly. We demonstrate that p38 phosphorylation is increased in Bcr-Abl–positive cells after treatment with NaChl and closely paralleled the inhibition of Bcr-Abl phosphorylation. NaChl did not increase phosphorylation of p38 in Bcr-Abl–negative cells including HSC-2 and HSG that are responsive to this compound, indicating that p38 activation by NaChl is dependent on Bcr-Abl kinase inhibition. Inhibition of p38 activity by SB203580 significantly reduced NaChl-induced apoptosis of K562 cells, whereas activation of p38 by anisomycin augmented the apoptosis. These findings indicate that inhibition of Bcr-Abl kinase leading to activation of p38 mitogen-activated protein (MAP) kinase may play an important role in the anti-CML activity of Chl.

scholarly journals Design, Synthesis and Biological Evaluation of Pentacyclic Triterpene Derivatives: Optimization of Anti-ABL Kinase Activity

Molecules ◽  
2019 ◽  
Vol 24 (19) ◽  
pp. 3535 ◽  
Author(s):  
Halil I. Ciftci ◽  
Mohamed O. Radwan ◽  
Safiye E. Ozturk ◽  
N. Gokce Ulusoy ◽  
Ece Sozer ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
K562 Cells ◽  
Biological Evaluation ◽  
Benzyl Ester ◽  
Myelogenous Leukemia ◽  
Pentacyclic Triterpene ◽  
Abl Kinase ◽  
Molecular Targeted Drug ◽  
Benzyl Esters

Imatinib, an Abelson (ABL) tyrosine kinase inhibitor, is a lead molecular-targeted drug against chronic myelogenous leukemia (CML). To overcome its resistance and adverse effects, new inhibitors of ABL kinase are needed. Our previous study showed that the benzyl ester of gypsogenin (1c), a pentacyclic triterpene, has anti-ABL kinase and a subsequent anti-CML activity. To optimize its activities, benzyl esters of carefully selected triterpenes (PT1–PT6), from different classes comprising oleanane, ursane and lupane, and new substituted benzyl esters of gypsogenin (GP1–GP5) were synthesized. All of the synthesized compounds were purified and charachterized by different spectroscopic methods. Cytotoxicity of the parent triterpenes and the synthesized compounds against CML cell line K562 was examined; revealing three promising compounds PT5, GP2 and GP5 (IC50 5.46, 4.78 and 3.19 μM, respectively). These compounds were shown to inhibit extracellular signal-regulated kinase (ERK) downstream signaling, and induce apoptosis in K562 cells. Among them, PT5 was identified to have in vitro activity (IC50 = 1.44 μM) against ABL1 kinase, about sixfold of 1c, which was justified by molecular docking. The in vitro activities of GP2 and GP5 are less than PT5, hence they were supposed to possess other more mechanisms of cytotoxicity. In general, our design and derivatizations resulted in enhancing the activity against ABL1 kinase and CML cells.

Identification of the Epigenetic Reader BRD4 As a Novel Potential Target in Ph+ CML

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1571-1571
Author(s):  
Barbara Peter ◽  
Gregor Eisenwort ◽  
Gabriele Stefanzl ◽  
Daniela Berger ◽  
Wolfgang R Sperr ◽  
...  
Keyword(s):  
Cell Lines ◽  
Research Funding ◽  
Drug Target ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Thymidine Uptake ◽  
Dependent Manner ◽  
Potential Drug ◽  
Ku812 Cells

Abstract Chronic myelogenous leukemia (CML) is a bone marrow-derived hematopoietic neoplasm in which BCR/ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients with CML, leukemic cells can be kept under control by BCR/ABL1 tyrosine kinase inhibitors (TKI), including imatinib, nilotinib, dasatinib, bosutinib, and ponatinib. Nevertheless, resistance or intolerance against one or more of these TKI may occur. Therefore, current research is focusing on novel potential drug targets in CML. A promising class of targets may be epigenetic regulators of cell growth, such as members of the bromodomain and extra-terminal domain (BET) family. The epigenetic reader and BET family member BRD4 has recently been identified as a novel potential drug target in acute myeloid leukemia (AML). However, so far, little is known about the expression and function of BRD4 in CML cells. The aims of the present study were to determine the expression of BRD4 and its downstream target MYC in CML cells and to explore whether BRD4 can serve as a novel drug target in this disease. As determined by qPCR, primary CML cells (chronic phase patients, n=7) as well as the CML cell lines KU812 and K562 expressed BRD4 mRNA. In addition, both CML cell lines stained positive for BRD4 in our immunocytochemistry staining experiments. In one patient with accelerated phase CML, putative leukemic (CD34+/CD38-) stem cells were sorted to near homogeneity and found to express BRD4 mRNA by qPCR. In order to examine the functional role of BRD4 in CML cells, a BRD4-specific shRNA was applied. In these experiments, the shRNA-induced knockdown of BRD4 in KU812 cells and K562 resulted in reduced growth compared to a control shRNA. Furthermore, the BRD4-targeting drug JQ1 was found to inhibit 3H-thymidine uptake and thus proliferation in KU812 cells in a dose-dependent manner (IC50: 0.25-0.75 µM). In addition, we were able to show that JQ1 inhibits growth of primary CML cells with variable IC50 values (0.1-5 µM). However, no substantial growth-inhibitory effects of JQ1 were seen in K562 cells (IC50: >5 µM). As determined by Annexin V/PI staining, JQ1 induced apoptosis in KU812 cells whereas no apoptosis-inducing effect of JQ1 was observed in K562 cells. Nevertheless, we were able to show that both CML cell lines as well as primary CML cells express MYC mRNA, and treatment of KU812 cells or K562 cells with JQ1 resulted in a decreased expression of MYC mRNA and MYC protein. Next, we analyzed whether MYC expression in CML cells can be blocked by BCR/ABL1 TKI. We found that imatinib, nilotinib, dasatinib, and ponatinib decrease MYC mRNA- and MYC protein expression in KU812 and K562 cells. Finally, we found that JQ1 cooperates with imatinib, nilotinib, ponatinib and dasatinib in inhibiting the proliferation of KU812 and K562 cells. Together, our data show that BRD4 serves as a potential new target in CML cells, and that the BRD4 blocker JQ1 cooperates with BCR/ABL1 TKI in inducing growth-inhibition. Whether BRD4 inhibition is a pharmacologically meaningful approach in patients with TKI-resistant CML remains to be determined in clinical trials. Disclosures Sperr: Ariad: Consultancy; Celgene: Consultancy. Zuber:Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria.

In Vitro and In Vivo Activity of the Src/Abl Kinase Inhibitor AP23464 and Analogs Against Activation Loop Mutants of KIT.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 793-793 ◽  
Author(s):  
Amie S. Corbin ◽  
Shadmehr Demehri ◽  
Ian J. Griswold ◽  
Chester A. Metcalf ◽  
William C. Shakespeare ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Activation Loop ◽  
Wild Type ◽  
Abl Kinase ◽  
Ic50 Values

Abstract Oncogenic mutations of the KIT receptor tyrosine kinase have been identified in several malignancies including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), seminomas/dysgerminomas and acute myelogenous leukemia (AML). Mutations in the regulatory juxtamembrane domain are common in GIST, while mutations in the activation loop of the kinase (most commonly D816V) occur predominantly in SM and at low frequency in AML. Several ATP-competitive kinase inhibitors, including imatinib, are effective against juxtamembrane KIT mutants, however, the D816V mutant is largely resistant to inhibition. We analyzed the sensitivities of cell lines expressing wild type KIT, juxtamembrane mutant KIT (V560G) and activation loop mutant KIT (D816V,F,Y and murine D814Y) to a potent Src/Abl kinase inhibitor, AP23464, and analogs. IC50 values for inhibition of cellular KIT phosphorylation by AP23464 were 5–11 nM for activation loop mutants, 70 nM for the juxtamembrane mutant and 85 nM for wild type KIT. Consistent with this, IC50 values in cell proliferation assays were 3–20 nM for activation loop mutants and 100 nM for wild type KIT and the juxtmembrane mutant. In activation loop mutant-expressing cell lines, AP23464, at concentrations ≤50 nM, induced apoptosis, arrested the cell cycle in G0/G1 and down-regulated phosphorylation of Akt and STAT3, signaling pathways critical for the transforming capacity of mutant KIT. In contrast, 500 nM AP23464 was required to induce equivalent effects in wild-type KIT and juxtamembrane mutant-expressing cell lines. These data demonstrate that activation loop KIT mutants are considerably more sensitive to inhibition by AP23464 than wild type or juxtamembrane mutant KIT. Non-specific toxicity in parental cells occurred only at concentrations above 2 μM. Additionally, at concentrations below 100 nM, AP23464 did not inhibit formation of granulocyte/macrophage and erythrocyte colonies from normal bone marrow, suggesting that therapeutic drug levels would not impact normal hematopoiesis. We also examined in vivo target inhibition in a mouse model. Mice were subcutaneously injected with D814Y-expressing (D816V homologous) murine mastocytoma cells. Once tumors were established, compound was administered three-times daily by oral gavage. One hour post treatment we observed >90% inhibition of KIT phosphorylation in tumor tissue. Following a three-day treatment regimen, there was a statistically significant difference in tumor size compared to controls. Thus, AP23464 analogs effectively target D816-mutant KIT both in vitro and in vivo and inhibit activation loop KIT mutants more potently than the wild type protein. These data provide evidence that this class of kinase inhibitors may have therapeutic potential for D816V-expressing malignancies such as SM or AML.

scholarly journals Human mitogen-activated protein kinase kinase kinase mediates the stress-induced activation of mitogen-activated protein kinase cascades

1998 ◽  
Vol 336 (3) ◽  
pp. 599-609 ◽  
Author(s):  
Po-Ying CHAN-HUI ◽  
Robert WEAVER
Keyword(s):  
Protein Kinase ◽  
Sequence Similarity ◽  
K562 Cells ◽  
Mitogen Activated Protein Kinase ◽  
Hek293 Cells ◽  
Mapk Cascades ◽  
Mitogen Activated Protein ◽  
Factor Α

The mitogen-activated protein kinase (MAPK) cascades represent one of the important signalling mechanisms in response to environmental stimuli. We report the identification of a human MAPK kinase kinase, MAPKKK4, via sequence similarity with other MAPKKKs. When truncated MAPKKK4 (ΔMAPKKK4) was overexpressed in HEK293 cells, it was constitutively active and induced the activation of endogenous p38α, c-Jun N-terminal kinase (JNK)1/2 and extracellular signal-regulated kinase (ERK)2 in vivo. Kinase-inactive ΔMAPKKK4 partly inhibited the activation of p38α, JNK1/2 and ERK2 induced by stress, tumour necrosis factor α or epidermal growth factor, suggesting that MAPKKK4 might be physiologically involved in all three MAPK cascades. Co-expressed MAP kinase kinase (MKK)-1, MKK-4, MKK-3 and MKK-6 were activated in vivo by ΔMAPKKK4. All of the above MKKs purified from Escherichia coli were phosphorylated and activated by ΔMAPKKK4 immunoprecipitates in vitro. When expressed by lower plasmid doses, ΔMAPKKK4 preferentially activated MKK-3 and p38α in vivo. Overexpression of ΔMAPKKK4 did not activate the NF-κB pathway. Immunoprecipitation of endogenous MAPKKK4 by specific antibodies showed that MAPKKK4 was activated after the treatment of K562 cells with various stress conditions. As a broadly distributed kinase, MAPKKK4 might serve as a stress responder. MAPKKK4 is 91% identical with the recently described murine MEKK-4β and might be its human homologue. It is also identical with the recently cloned human MAP three kinase 1 except for the lack of an internal sequence homologous to the murine MEKK-4α isoform. Differences in the reported functional activities of the three kinases are discussed.

scholarly journals 9-cis-retinoic acid: effects on normal and leukemic hematopoiesis in vitro

Blood ◽  
1993 ◽  
Vol 81 (4) ◽  
pp. 1009-1016 ◽  
Author(s):  
A Sakashita ◽  
M Kizaki ◽  
S Pakkala ◽  
G Schiller ◽  
N Tsuruoka ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Lines ◽  
Clonal Growth ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Clonal Proliferation ◽  
Normal Individuals ◽  
Cellular Responsiveness ◽  
X Receptors

Retinoic acid exhibits effects on the proliferation and differentiation of many hematopoietic cells. Cellular responsiveness to retinoic acid (RA) is conferred through two distinct classes of nuclear receptors, the RA receptors (RARs) and the retinoid X receptors (RXRs). The RARs bind to both 9-cis- and all-trans-RAs, but 9-cis-RA alone directly binds and activates RXR. This suggested that 9-cis-RA could have expanded hematopoietic activities as compared with all-trans-RA. We compared the abilities of 9-cis- and all-trans-RAs to induce differentiation and inhibit proliferation of three acute myelogenous leukemia (AML) cell lines and fresh leukemic cells from 28 patients and found that: (1) 9-cis-RA in general was more potent than all-trans-RA in suppressing the clonal growth of two AML cell lines and 17 AML samples from patients, including four from individuals with acute promyelocytic leukemia (APL). Eleven leukemic samples, including three from patients with chronic myelogenous or chronic myelomonocytic leukemia, were relatively refractory to both retinoids. (2) The range of activities of both retinoids was similar except that the clonal growth of samples from three AML patients were inhibited by 9-cis-RA, but not by all-trans-RA. (3) Both retinoids inhibited the clonal proliferation of leukemia cells without necessarily inducing their differentiation; in fact, the only fresh AML cells that were able to undergo differentiation were from patients with APL and one individual with M2 AML. (4) Both retinoids enhanced myeloid and erythroid clonal growth from normal individuals, and 9-cis-RA showed slightly more stimulation of the myeloid clonal growth than did the all-trans-RA. Our study suggests that 9-cis-RA is worthy of further study for the treatment of selected individuals with AML.

Nonclassical actions of estradiol-17beta are not detectable in the alphaT3-1 and LbetaT2 immortalized gonadotrope cell lines†

2019 ◽  
Vol 101 (4) ◽  
pp. 791-799
Author(s):  
Tracy L Davis
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Cell Lines ◽  
Surface Protein ◽  
Mitogen Activated Protein Kinase ◽  
Cell Type ◽  
Mitogen Activated Protein ◽  
Homogeneous Cell

Abstract The immortalized mouse gonadotrope cell lines alphaT3-1 and LbetaT2 cells have been a substitute model for primary gonadotropes. These cell lines have provided a homogeneous cell population, as compared to the dissociated anterior pituitaries, which contain a heterogeneous population of cells potentially responsive to estradiol-17beta (E2). Nonclassical actions of E2 assumed to occur through the plasma membrane estrogen receptor 1 (ESR1, also known as ERalpha). These actions have included inhibition of gonadotropin-releasing hormone (GnRH)-induced increases in intracellular calcium concentrations and phosphorylation of p44/42 mitogen-activated protein kinase (ERK-1/2) in ovine pituitaries including primary gonadotropes in vitro. The objective of the present experiment was to determine if alphaT3-1 and LbetaT2 are cell models with limitations to examine the nonclassical actions of E2 occurring in gonadotropes. Experiments were conducted to determine if the cells have ESR1 at the plasma membrane using biotinylation cell and isolation of surface protein and staining with a fluorescently labeled E2 conjugate. The alphaT3-1 cells contain ESR1 associated with but not enriched within lipid rafts of the plasma membrane and do not translocate to lipid rafts upon binding of E2. In contrast, LbetaT2 cells lack ESR1 associated with the plasma membrane. Pretreatment with E2 did not cause inhibition of GnRH-stimulated increases in intracellular concentrations of calcium for either cell type. Phosphorylation of ERK-1/2 was not stimulated by E2 in either cell type. Although these cells lines have been used extensively to study GnRH signaling, in vitro or in vivo effects of nonclassical actions of E2 cannot be replicated in either cell line.

The NRP1 gene regulates proliferation, apoptosis, migration, and invasion in T24 and 5637 bladder cancer cells

2020 ◽  
Author(s):  
Conghui Han ◽  
Yang Dong ◽  
Lin Hao ◽  
Kun Pang ◽  
Xiaoying Zhang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Biological Function ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Migration And Invasion ◽  
Mapk Signalling Pathway ◽  
Neuropilin 1 ◽  
Mitogen Activated Protein

Abstract Background Bladder urothelial carcinoma (BC) is a fatal invasive malignancy and the most common malignancy of the urinary system. In the current study, we investigate the function and mechanisms of Neuropilin-1 (NRP1), the co-receptor for vascular endothelial growth factor, in BC pathogenesis and progression. Methods The expression of NRP1 was assessed in several BC cell lines. Additionally, the biological function of NRP1 in proliferation, apoptosis, angiogenesis, migration, and invasion of BC were validated in vitro by silencing NRP1. Moreover, gene expression profiling chip analysis was conducted, and the related signalling pathways were confirmed by Western blot to reveal the potential molecular mechanisms by which NRP1 promotes the malignant progression of BC. Results Overexpression of NRP1 was observed in several human BC cell lines. NRP1 knockdown inhibited cell proliferation, promoted apoptosis, and decreased angiogenesis, migration, and invasion in T24 and 5637 human BC cells. Microarray analysis results indicated that the expression of NRP1 was correlated with the levels of cyclin dependent kinase (CDK) 4, baculoviral IAP repeat containing 3, Cyclin E 2, CDK2, and AP-1 transcription factor subunit in BC. We also demonstrated that the biological function of NRP1was associated with activation of the mitogen-activated protein kinase (MAPK) signalling pathway. Conclusions Our findings provide evidence that NRP1, as a potential tumour promoter, contributes to the metastasis and invasion of BC, which is associated with the activation of the MAPK pathway. Targeting NRP1 has the potential to become a new therapeutic strategy to benefit more patients with BC or other cancers.

scholarly journals 9-cis-retinoic acid: effects on normal and leukemic hematopoiesis in vitro

Blood ◽  
1993 ◽  
Vol 81 (4) ◽  
pp. 1009-1016 ◽  
Author(s):  
A Sakashita ◽  
M Kizaki ◽  
S Pakkala ◽  
G Schiller ◽  
N Tsuruoka ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Cell Lines ◽  
Clonal Growth ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Clonal Proliferation ◽  
Normal Individuals ◽  
Cellular Responsiveness ◽  
X Receptors

Abstract Retinoic acid exhibits effects on the proliferation and differentiation of many hematopoietic cells. Cellular responsiveness to retinoic acid (RA) is conferred through two distinct classes of nuclear receptors, the RA receptors (RARs) and the retinoid X receptors (RXRs). The RARs bind to both 9-cis- and all-trans-RAs, but 9-cis-RA alone directly binds and activates RXR. This suggested that 9-cis-RA could have expanded hematopoietic activities as compared with all-trans-RA. We compared the abilities of 9-cis- and all-trans-RAs to induce differentiation and inhibit proliferation of three acute myelogenous leukemia (AML) cell lines and fresh leukemic cells from 28 patients and found that: (1) 9-cis-RA in general was more potent than all-trans-RA in suppressing the clonal growth of two AML cell lines and 17 AML samples from patients, including four from individuals with acute promyelocytic leukemia (APL). Eleven leukemic samples, including three from patients with chronic myelogenous or chronic myelomonocytic leukemia, were relatively refractory to both retinoids. (2) The range of activities of both retinoids was similar except that the clonal growth of samples from three AML patients were inhibited by 9-cis-RA, but not by all-trans-RA. (3) Both retinoids inhibited the clonal proliferation of leukemia cells without necessarily inducing their differentiation; in fact, the only fresh AML cells that were able to undergo differentiation were from patients with APL and one individual with M2 AML. (4) Both retinoids enhanced myeloid and erythroid clonal growth from normal individuals, and 9-cis-RA showed slightly more stimulation of the myeloid clonal growth than did the all-trans-RA. Our study suggests that 9-cis-RA is worthy of further study for the treatment of selected individuals with AML.

In Vitro and In Vivo Study of Synergistic Effect of Imatinib and Simvastatin in Chronic Myelogenous Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2202-2202
Author(s):  
Bora Oh ◽  
Dong Soon Lee ◽  
Tae Young Kim ◽  
Hyun Jung Min ◽  
Yun Song Lee ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Annexin V ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Killing Effect ◽  
Rna Transfection ◽  
Imatinib Resistant

Abstract Abstract 2202 Poster Board II-179 Background: Statins (HMG-CoA reductase inhibitors) are known to show anti-proliferative effects and are anticipated as a potential drug in the treatment of malignancies. To investigate the effect simvastatin on chronic myelogenous leukemia cells, we treated simvastatin on 3 kinds of CML cell lines and CD34+ primary CML cells from patients: erythrocytic lineage (K-562), granulocytic lineage (KCL-22), erythroid-megakaryocytic (LAMA-84) cell line. Also, antiproliferative effect on imatinib-resistant CML cell lines was assessed. Mehtods: Anti-proliferative effect was assessed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI). Apoptosis was assessed by Annexin V and Western blot study. Killing effect was calculated by CalcuSyn does effect analysis software (Biosoft, Ferguson, MO). CD34+cells for patients with CML were purified using immunomagnetic bead column. Cell cycle analysis was done by flow cytometrc method. Si-RNA transfection study for p27 gene was performed for verification of killing mechanism. Change of intracellular location of BCR/ABL protein was observed by confocal microscopy. Cellular changes of proteins and tyrosin phosphorylation after treatment of simvastain was evaluated by 2 dimensional electrophoresis and MALDI-TOF/TOF mass spectrophotometer. In vivo effect of simvastain was evaluated in BALB/c-nude. Results: Simvastatin inhibited the proliferation of imatinib-sensitive and imatinib-resistant K562 cell line in a dose dependent manner. The IC50 values of simvastatin and imatinib in imatinib-sensitive K562 cells were 14.5 and 0.4 μM. Treatment of simvastain induced apoptosis both in capase-dependent and caspase-independent pathways in all 3 cell lines; apoptosis by Annexin V analysis and increased apoptotic proteins (cytochrome C, AIF, Smac/Diablo, caspase-3, and caspase-9) by western blot. Cell cycle analysis revealed the G1/S arrest on treatment of simvastatin and si-RNA transfection specific for p27 reversed the G1/S arrest, suggesting cell cycle arrest as one of anti-proliferative mechanism. Co-treatment of imatinib and simvastain showed synergistic killing interactions between simvastatin and imatinib in imatinib-resistant K562 cells (mean combination index values were 0.56, P< 0.001). Enhanced killing effect was observed in all 3 imatinib-resistant CML cell lines (K-562: 0.6, KCL-22: 0.42 , LAMA-84; 0.99). Co-treatment with imatinib and simvastatin decreased the amount of Bcr-Abl protein and stimulated the import of Abl protein in the nuclei in K562 cells. In CML cells, simvastatin inhibited tyrosine phosphorylation included protease, cytocrome-c reductase, DNA/RNA processing proteins, oxidoructase protein, chaperones, glycolysis protein, cytoskeleton proteins, microtubule protein. Treatment of simvastain reduced subcutaneous tumor mass in nude mice. Conclusion: We showed that simvastatin killed CML cells in vitro and in vivo animal model and killing effect occurred via the induction of apoptosis, cell cyle arrest via p27 and inhibited BCR/ABL tyrosine kinase (TK) activity. Simvastatin may be a potential candidate for the treatment of imatinib-resistant CML patients and the effective dose of imatinib could be reduced in a combined treatment with simvastatin. Disclosures: No relevant conflicts of interest to declare.

Regulation of the Erk2-Elk1 signaling pathway and megakaryocytic differentiation of Bcr-Abl+ K562 leukemic cells by Gab2

Blood ◽  
2002 ◽  
Vol 99 (4) ◽  
pp. 1388-1397 ◽  
Author(s):  
Jay F. Dorsey ◽  
Jess M. Cunnick ◽  
Shrikant M. Mane ◽  
Jie Wu
Keyword(s):  
Map Kinase ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Transcriptional Activation ◽  
Blast Crisis ◽  
Terminal Differentiation ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Megakaryocytic Differentiation

In the blast crisis phase of chronic myelogenous leukemia (CML), Bcr-Abl+ myeloblasts fail to undergo terminal maturation. The extracellular signal–regulated kinase (Erk) mitogen-activated protein (MAP) kinase has been shown to mediate terminal differentiation of myeloid cells. Interestingly, Bcr-Abl+ CML cell lines established from blast crisis were found to have low Erk MAP kinase activity. In this study, we analyzed the role of the Gab2 docking protein in regulation of the Erk MAP kinase in Bcr-Abl+K562 human CML cells. Overexpression of Gab2 in K562 cells resulted in transcriptional activation of the c-fos serum response element (SRE) promoter, whereas overexpression of SHP2, Grb2, and CrkL had no effect. Activation of the c-fos SRE transcriptional activity by Gab2 required tyrosine 604, which is a SHP2 docking site on Gab2, and the SHP2 tyrosine phosphatase activity. Elk1, c-Jun, and CHOPtrans-reporting assays indicated that overexpression of Gab2 selectively activated the Erk2-Elk1 signaling pathway. To determine cellular consequences of elevating the Gab2 level in K562 cells, stable cell lines for doxycycline-inducible expression of the wild-type Gab2 (Gab2WT) and an SHP2-binding defective Gab2 (Gab2Tyr604Phe) were established. Analysis of these cell lines indicated that induction of Gab2WT expression, but not Gab2Tyr604Phe expression, led to Erk activation, growth arrest, cell spreading, and enlargement; expression of megakaryocyte/platelet lineage–specific integrins αIIb/β3 (CD41/CD61); and upregulation of RNA for megakaryocyte/platelet proteins. All of these changes are characteristics of megakaryocytic differentiation. Together, these results reveal Gab2 as a limiting signaling component for Erk MAP kinase activation and terminal differentiation of K562 CML cells.

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