Aclacinomycin A Sensitizes K562 Chronic Myeloid Leukemia Cells to Imatinib through p38MAPK-Mediated Erythroid Differentiation

Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and apoptosis. These results provided a potential management by which ACM might have a crucial impact on increasing sensitivity of CML cells to imatinib in the differentiation therapeutic approaches.     

Alantolactone induces apoptosis in chronic myelogenous leukemia sensitive or resistant to imatinib through NF-κB inhibition and Bcr/Abl protein deletion

Alantolactone, an allergenic sesquiterpene lactone, has recently been found to have significant antitumor effects on malignant tumor cells. Here, we investigated the potential effect of alantolactone on Bcr/Abl⁺ imatinib-sensitive and -resistant cells. Alantolactone treatment resulted in obvious apoptosis in both imatinib-sensitive and -resistant K562 cells, as shown by the increase in Annexin V-positive cells, caspase-3 activation, poly(ADP-ribose) polymerase-1 (PARP-1) cleavage and mitochondrial membrane potential collapse. Alantolactone significantly inhibited NF-κB-dependent reporter gene activity, decreased the DNA-binding activity of NF-ОκB, and blocked TNF-α-induced IκBα phosphorylation. Of interest, the oncogenic Bcr/Abl fusion protein but not its mRNA levels were quickly reduced upon alantolactone exposure in imatinib-sensitive and -resistant K562 cells. Bcr/Abl knockdown enhanced the apoptosis driven by alantolactone. Bcr/Abl protein reduction could not be reversed by the addition of proteasome or caspase-3 inhibitors. The overexpression of p65 inhibited alantolactone-induced apoptosis, whereas p65 or Bcr/Abl silencing enhanced its apoptotic-inducing effect. Furthermore, Bcr/Abl-transfected 32D cells showed more sensitivity to alantolactone than vector-transfected control cells, and the Bcr/Abl protein was depleted, as observed in K562 cells. Finally, alantolactone-induced apoptosis was also observed in primary CD34⁺ CML leukemic cells. Collectively, these findings suggest that alantolactone is a promising potent agent to fight against CML cells via the inhibition of the NF-κB signaling pathway and depletion of the Bcr/Abl protein.     

Icaritin shows potent anti-leukemia activity on chronic myeloid leukemia in vitro and in vivo by regulating MAPK/ERK/JNK and JAK2/STAT3 /AKT signalings

Purpose

To explore the effects of Icaritin on chronic myeloid leukemia (CML) cells and underlying mechanisms.

Method

CML cells were incubated with various concentration of Icaritin for 48 hours, the cell proliferation was analyzed by MTT and the apoptosis was assessed with Annexin V and Hoechst 33258 staining. Cell hemoglobinization was determined. Western blotting was used to evaluate the expressions of MAPK/ERK/JNK signal pathway and Jak-2/Phorpho-Stat3/Phorsph-Akt network-related protein. NOD-SCID nude mice were applied to demonstrate the anti-leukemia effect of Icaritin in vivo.

Results

Icaritin potently inhibited proliferation of K562 cells (IC50 was 8 µM) and primary CML cells (IC50 was 13.4 µM for CML-CP and 18 µM for CML-BC), induced CML cells apoptosis and promoted the erythroid differentiation of K562 cells with time-dependent manner. Furthermore, Icaritin was able to suppress the growth of primary CD34+ leukemia cells (CML) and Imatinib-resistant cells, and to induce apoptosis. In mouse leukemia model, Icaritin could prolong lifespan of NOD-SCID nude mice inoculated with K562 cells as effective as Imatinib without suppression of bone marrow. Icaritin could up-regulate phospho-JNK or phospho-C-Jun and down-regulate phospho-ERK, phospho-P-38, Jak-2, phospho-Stat3 and phospho-Akt expression with dose- or time-dependent manner. Icaritin had no influence both on c-Abl and phospho-c-Abl protein expression and mRNA levels of Bcr/Abl.

Conclusion

Icaritin from Chinese herb medicine may be a potential anti-CML agent with low adverse effect. The mechanism of anti-leukemia for Icaritin is involved in the regulation of Bcr/Abl downstream signaling. Icaritin may be useful for an alternative therapeutic choice of Imatinib-resistant forms of CML.     

Bcr - Abl-mediated resistance to apoptosis is independent of PI 3-kinase activity

The Bcr - Abl tyrosine kinase is responsible for the oncogenic phenotype observed in Philadelphia chromosome-positive leukemia and induces resistance to apoptotic cell death in a variety of cell types. Recent evidence supports the hypothesis that these two properties of Bcr - Abl are derived from cooperative but distinct signaling pathways. Phosphatidylinositol 3-kinase (PI3K), which has been suggested to associate with and become activated by Bcr - Abl, has been shown to be required for Bcr - Abl-mediated cell growth. Also, PI3K has been implicated in resistance to apoptosis induced by some growth factors. We therefore examined the role of PI 3-kinase in the anti-apoptotic effect of Bcr - Abl. First, we confirmed that expression of p185(bcr - abl) in HL-60 cells, which renders these cells resistant to apoptosis, induces tyrosine phosphorylation of the p85 subunit of PI3K. Consistent with this result, we observed a 20-fold increase in PI3K activity upon immunoprecipitation of tyrosine-phosphorylated proteins from cells expressing Bcr - Abl versus control cells. Nevertheless, treatment of HL-60.p185(bcr - abl) cells with wortmannin, a potent inhibitor of PI3K, eliminated PI3K activity but did not interfere with the resistance of these cells to apoptosis. Similar results were obtained with the CML line K562 and with the BaF3.p185 (bcr - abl) line. We conclude that while PI3K participates in the anti-apoptotic response mediated by some growth factors and also seems to be important for the growth of Bcr-Abl-positive cells, it does not play any role in Bcr - Abl-mediated resistance to apoptosis.     

Study of the PTEN gene expression and FAK phosphorylation in human hepatocarcinoma tissues and cell lines

K562 cells contain a Bcr-Abl chimeric gene and differentiate into various lineages in response to different inducers. We studied the role of the mitogen-activated protein kinase (MAPK) kinase 1 (MEK1)/extracellular signal-regulated kinase (ERK) pathway during the erythroid differentiation of K562 cells induced by tyrosine kinase inhibitors (herbimycin A or STI571), using genetically modified cells (constitutively MEK1-activated K562: K562/MEK1, and inducible ERK-inactivated K562: K562/CL100). Basal expression of glycophorin A was markedly reduced in K562/MEK1 cells compared with that in parental cells, while it was augmented in K562/CL100 cells. Herbimycin A and STI571 differentiated K562 cells accompanying with the transient down-regulated ERK. Moreover, the erythroid differentiation was markedly suppressed in K562/MEK1 cells, and early down-regulation of ERK activity was not observed in these cells. In contrast, the induction of ERK-specific phosphatase in K562/CL100 cells potentiated erythroid differentiation. Once the phosphatase was induced, the initial ERK activity became repressed and its early down-regulation by the inhibition of Bcr-Abl was marked and prolonged. These results demonstrate that the erythroid differentiation of K562 cells induced by herbimycin A or STI571 requires the down-regulation of MEK1/ ERK pathway.     

c‐FLIP is involved in erythropoietin‐mediated protection of erythroid‐differentiated cells from TNF‐α‐induced apoptosis

The TNF‐α (tumour necrosis factor) affects a wide range of biological activities, such as cell proliferation and apoptosis. Cell life or death responses to this cytokine might depend on cell conditions. This study focused on the modulation of factors that would affect the sensitivity of erythroid‐differentiated cells to TNF‐α. Hemin‐differentiated K562 cells showed higher sensitivity to TNF‐induced apoptosis than undifferentiated cells. At the same time, hemin‐induced erythroid differentiation reduced c‐FLIP (cellular FLICE‐inhibitory protein) expression. However, this negative effect was prevented by prior treatment with Epo (erythropoietin), which allowed the cell line to maintain c‐FLIP levels. On the other hand, erythroid‐differentiated UT‐7 cells – dependent on Epo for survival – showed resistance to TNF‐α pro‐apoptotic action. Only after the inhibition of PI3K (phosphatidylinositol‐3 kinase)‐mediated pathways, which was accompanied by negative c‐FLIP modulation and increased erythroid differentiation, were UT‐7 cells sensitive to TNF‐α‐triggered apoptosis. In summary, erythroid differentiation might deregulate the balance between growth promotion and death signals induced by TNF‐α, depending on cell type and environmental conditions. The role of c‐FLIP seemed to be critical in the protection of erythroid‐differentiated cells from apoptosis or in the determination of their sensitivity to TNF‐mediated programmed cell death. Epo, which for the first time was found to be involved in the prevention of c‐FLIP down‐regulation, proved to have an anti‐apoptotic effect against the pro‐inflammatory factor. The identification of signals related to cell life/death switching would have significant implications in the control of proliferative diseases and would contribute to the understanding of mechanisms underlying the anaemia associated with inflammatory processes.     

Imatinib mesylate (STI571) abrogates the resistance to doxorubicin in human K562 chronic myeloid leukemia cells by inhibition of BCR/ABL kinase-mediated DNA repair

Imatinib mesylate (STI571), a specific inhibitor of BCR/ABL tyrosine kinase, exhibits potent antileukemic effects in the treatment of chronic myelogenous leukemia (CML). However, the precise mechanism by which inhibition of BCR/ABL activity results in pharmacological responses remains unknown. BCR/ABL-positive human K562 CML cells resistant to doxorubicin (K562DoxR) and their sensitive counterparts (K562DoxS) were used to determine the mechanism by which the STI571 inhibitor may overcome drug resistance. K562 wild type cells and CCRF-CEM lymphoblastic leukemia cells without BCR/ABL were used as controls. The STI571 specificity was examined by use of murine pro-B lymphoid Baf3 cells with or without BCR/ABL kinase expression. We examined kinetics of DNA repair after cell treatment with doxorubicin in the presence or absence of STI571 by the alkaline comet assay. The MTT assay was used to estimate resistance against doxorubicin and Western blot analysis with Crk-L antibody was performed to evaluate BCR/ABL kinase inhibition by STI571. We provide evidence that treatment of CML-derived BCR/ABL-expressing leukemia K562 cells with STI571 results in the inhibition of DNA repair and abrogation of the resistance of these cells to doxorubicin. We found that doxorubicin-resistant K562DoxR cells exhibited accelerated kinetics of DNA repair compared with doxorubicin-sensitive K562DoxS cells. Inhibition of BCR/ABL kinase in K562DoxR cells with 1 microM STI571 decreased the kinetics of DNA repair and abrogated drug resistance. The results suggest that STI571-mediated inhibition of BCR/ABL kinase activity can affect the effectiveness of the DNA-repair pathways, which in turn may enhance drug sensitivity of leukemia cells.     

Comparative study of DNA damage, cell cycle and apoptosis in human K562 and CCRF-CEM leukemia cells: role of BCR/ABL in therapeutic resistance

The Philadelphia translocation t(9;22) resulting in the bcr/abl fusion gene is the pathogenic principle of almost 95% of human chronic myelogenous leukemia (CML). Imatinib mesylate (STI571) is a specific inhibitor of the BCR/ABL fusion tyrosine kinase that exhibits potent antileukemic effects in CML. BCR/ABL-positive K562 and -negative CCRF-CEM human leukemia cells were investigated. MTT survival assay and clonogenic test of the cell proliferation ability were used to estimate resistance against idarubicin. DNA damage after cell treatment with the drug at the concentrations from 0.001 to 3 microM with or without STI571 pre-treatment were examined by the alkaline comet assay. We found that the level of DNA damages was lower in K562 cells after STI571 pre-treatment. It is suggested that BCR/ABL activity may promote genomic instability, moreover K562 cells were found to be resistant to the drug treatment. Further, we provided evidence of apoptosis inhibition in BCR/ABL-positive cells using caspase-3 activity colorimetric assay and DAPI nuclear staining for chromatin condensation. We suggest that these processes associated with cell cycle arrest in G2/M checkpoint detected in K562 BCR/ABL-positive compared to CCRF-CEM cells without BCR/ABL expression might promote clone selection resistance to drug treatment.     

Spred2 Modulates the Erythroid Differentiation Induced by Imatinib in Chronic Myeloid Leukemia Cells

Differentiation induction is currently considered as an alternative strategy for treating chronic myelogenous leukemia (CML). Our previous work has demonstrated that Sprouty-related EVH1 domainprotein2 (Spred2) was involved in imatinib mediated cytotoxicity in CML cells. However, its roles in growth and lineage differentiation of CML cells remain unknown. In this study, we found that CML CD34⁺ cells expressed lower level of Spred2 compared with normal hematopoietic progenitor cells, and adenovirus mediated restoration of Spred2 promoted the erythroid differentiation of CML cells. Imatinib could induce Spred2 expression and enhance erythroid differentiation in K562 cells. However, the imatinib induced erythroid differentiation could be blocked by Spred2 silence using lentiviral vector PLKO.1-shSpred2. Spred2 interference activated phosphorylated-ERK (p-ERK) and inhibited erythroid differentiation, while ERK inhibitor, PD98059, could restore the erythroid differentiation, suggesting Spred2 regulated the erythroid differentiation partly through ERK signaling. Furthermore, Spred2 interference partly restored p-ERK level leading to inhibition of erythroid differentiation in imatinib treated K562 cells. In conclusion, Spred2 was involved in erythroid differentiation of CML cells and participated in imatinib induced erythroid differentiation partly through ERK signaling.     

Combined effects of doxorubicin and STI571 on growth, differentiation and apoptosis of CML cell line K562

STI571 (imatinib mesylate; Gleevec) is an inhibitor that targets the tyrosine kinase activity of Bcr-Abl present in chronic myelogenous leukemia (CML) cells. Some preclinical studies have demonstrated that the combination of STI571 with chemotherapeutic drugs results in enhanced toxicity in Bcr-Abl-positive leukemias. We investigated the potential benefit of using STI571 to down-regulate Bcr-Abl activity for the enhancement of doxorubicin anti-proliferative action in K562 cell line derived from blast crisis of CML. At low concentrations of both drugs (40 nM doxorubicin combined with STI571 in the range of 100-150 nM), the antiproliferative effects were mainly due to cellular differentiation as assessed by benzidine staining for hemoglobin synthesis level and real-time PCR for gamma-globin expression. Higher concentrations of STI571 used in combinations with doxorubicin caused mainly apoptosis as shown by DNA degradation and nuclear fragmentation visualized by fluorescence microscopy after DAPI staining, changes in cell morphology observed after Giemza-May Grünwald staining and cellular membrane organization estimated by flow cytometry after Annexin V staining. As compared with either drug alone, cotreatment with STI571 and DOX induced stronger cellular responses. A low concentration of STI571 in combination with a low concentration of DOX might be tested as an alternative approach to increasing the efficacy of chemotherapy against CML.     

Changes in cell adhesivity and cytoskeleton‐related proteins during imatinib‐induced apoptosis of leukemic JURL‐MK1 cells

The fusion protein Bcr–Abl, which is the molecular cause of chronic myelogenous leukemia (CML) interacts in multiple points with signaling pathways regulating the cellular adhesivity and cytoskeleton architecture and dynamics. We explored the effects of imatinib mesylate, an inhibitor of Bcr–Abl protein used in front‐line CML therapy, on the adhesivity of JURL‐MK1 cells to fibronectin and searched for underlying changes in the cell proteome. As imatinib induces apoptosis of JURL‐MK1 cells, we used three different caspase inhibitors to discriminate between direct consequences of Bcr–Abl inhibition and secondary changes related to the apoptosis. Imatinib treatment caused a transient increase in JURL‐MK1 cell adhesivity to fibronectin, possibly due to the switch off of Bcr–Abl activity. Subsequently, we observed a number of changes including a decrease in cell adhesivity, F‐actin decomposition, reduction of integrin β1, CD44, and paxillin expression levels and a marked increase in cofilin phophorylation at Ser3. These events were generally related to the proceeding apoptosis but they differed in their sensitivity to the individual caspase inhibitors. J. Cell. Biochem. 111: 1413–1425, 2010. © 2010 Wiley‐Liss, Inc.     

Hybrid compounds as new Bcr/Abl inhibitors

A series of 2,4-disubstituted thiazole derivatives were designed and synthesized as new Bcr/Abl inhibitors by hybriding the structural moieties from FDA approved imatinib, nilotinib and dasatinib. The new inhibitors strongly suppressed the activity of Bcr/Abl kinase and potently inhibited the proliferation of K562 and KU812 leukemia cancer cells. Compound 4i displayed comparable potency with that of nilotinib in both biochemical kinase assay and cancer cell growth inhibition assay. These inhibitors might serve as lead compounds for further developing new anticancer drugs.     

Treatment of Chronic Myeloid Leukemia Cells with Imatinib (STI571) Impairs p53 Accumulation in Response to DNA Damage

Chronic myelogenous leukaemia (CML) is induced by the Bcr-Abl fusion protein. Inhibition of Bcr-Abl by STI571 is widely used to treat CML patients. Unlike in most cancer types, the frequency of p53 mutations in CML is low. Here, we investigated the effect of STI571 treatment of CML cells on p53 regulation. Exposure of CML cells, including established cell lines and freshly isolated cells from patients, to STI571 reduced p53 protein levels, and severely impaired its accumulation in response to DNA damage. This may be explained by the status of p53 serine 20 phosphorylation. In non-stressed CML cells, serine 20 of p53 is constitutively phosphorylated by Chk1, and is inhibited by STI571. In response to DNA damage, however, this phosphorylation is mediated by Chk1 and Chk2, and is only partially inhibited by STI571. CML cells expressing wild-type p53 are more resistant to treatment with STI571, but moderately more sensitive to DNA damage, than CML cells lacking p53. An enhanced induction of apoptosis by STI571 and DNA damage is observed in CML cells bearing wild-type p53, but not in cells lacking functional p53. This implies that the status of p53 may affect the response of CML cells to this combined treatment.     

Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia （CML） cells

This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia （CML） cells （e.g., K562, LAMA84）. Treatment of K562 cells with shikonin （e.g., 0.5 pM） resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species （ROS）, striking activation of c-Jun-N-terminal kinase （JNK） and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events （i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis） following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.     

Sphingomyelin synthase 1 activity is regulated by the BCR-ABL oncogene

Sphingomyelin synthase (SMS) produces sphingomyelin while consuming ceramide (a negative regulator of cell proliferation) and forming diacylglycerol (DAG) (a mitogenic factor). Therefore, enhanced SMS activity could favor cell proliferation. To examine if dysregulated SMS contributes to leukemogenesis, we measured SMS activity in several leukemic cell lines and found that it is highly elevated in K562 chronic myelogenous leukemia (CML) cells. The increased SMS in K562 cells was caused by the presence of Bcr-abl, a hallmark of CML; stable expression of Bcr-abl elevated SMS activity in HL-60 cells while inhibition of the tyrosine kinase activity of Bcr-abl with Imatinib mesylate decreased SMS activity in K562 cells. The increased SMS activity was the result of up-regulation of the Sms1 isoform. Inhibition of SMS activity with D609 (a pharmacological SMS inhibitor) or down-regulation of SMS1 expression by siRNA selectively inhibited the proliferation of Bcr-abl-positive cells. The inhibition was associated with an increased production of ceramide and a decreased production of DAG, conditions that antagonize cell proliferation. A similar change in lipid profile was also observed upon pharmacological inhibition of Bcr-abl (K526 cells) and siRNA-mediated down-regulation of BCR-ABL (HL-60/Bcr-abl cells). These findings indicate that Sms1 is a downstream target of Bcr-abl, involved in sustaining cell proliferation of Bcr-abl-positive cells.