Myelogenous Leukemia Cell Line K562 Research Articles

Ursolic Acid Induced Apoptosis Involving Upregulation of Expression of PTEN Gene and Inactivation of PI3K/Akt Pathway in K562 Cells

Abstract 4453 ObjectUrsolic acid(UA), a pentacyclic triterpenoid derived from many kinds of medicinal plants, exhibits potent anticancer activity in many kinds of cancer cells. However, the anticancer mechanism of UA is not clearly understood. MethodThe toxic action of UA in K562 cells was assessed using CCK-8. K562 cells trested by UA were incubated with FITC-conjugated Annexin V and counterstained with PI in order to allow exclusion of necrotic cells, then were subsequently analyzed using a flow cytometer. Pro-caspase-3, cleaved caspase-3, Procaspase-9, cleaved caspase-9, Akt and Akt phosphorylation were analyzed by Western Blotting. The expression of PTEN gene is analyzed by Quantitative real-time RT-PCR. ResultHere we showed that UA could cause growth inhibition, and induce apoptosis in human chronic myelogenous leukemia cell line K562 cells. The expression of PTEN gene is rejected in many kinds of cancers including leukemia, then the PI3K/Akt pathway is actived. So the PTEN gene and PI3K/Akt pathway become the central targer for cancer therapy. UA treatment can up-regulate the expression of PTEN gene, inhibite the activity of Akt kinase, change the mitochondrial transmembrane potential, then induce the reduce of cytochrome C and the active of caspase family. ConclusionThese results suggest that UA might exhibit its strong antitumor effects via the up-regulation of PTEN gene and the inhibited the activity of PI3K/Akt pathway. Disclosures:No relevant conflicts of interest to declare.

Proliferation-inhibiting and apoptosis-inducing effects of ursolic acid and oleanolic acid on multi-drug resistance cancer cells in vitro

To investigate the proliferation-inhibiting and apoptosis-inducing effects of ursolic acid (UA) and oleanolic acid (OA) on multi-drug resistance (MDR) cancer cells in vitro. UA and OA in different concentrations (0-100 μmol/L) were added separately to cultures of different cancer cell lines, including the human colon cancer cell lines SW480 and SW620, human acute myelocytic leukemia cancer cell lines HL60 and HL60/ADR, human chronic myelogenous leukemia cell lines K562 and K562/ADR, and the human breast cancer cell lines MCF-7 and MCF-7/ADR. Effects of UA and OA on cell proliferation were detected by 3-(4,5-dimethyl-2-thiazole)-2-5-biphenly-tetrazole bromide (MTT) method and effects on cell apoptosis were tested by flow cytometry (FCM) and Western blot at 24, 48, and 72 h after treatment. Both UA and OA showed significant inhibition on parent and MDR cell lines in a time- and concentration-dependent manner; the drug-resistant multiple of them on K562 and K562/ADR as well as on HL60 and HL60/ADR was 1; the effects of UA were better than those of OA in inhibiting cell growth of solid colonic cancer and breast cancer. After SW480 cells were treated by UA at the concentrations of 0-40 μmol/L for 48 h, FCM showed that annexin V (AV) positive cells and hypodiploid peak ratio increased along with the increase in the drug's concentrations; and Western blot found that expressions of Bcl-2, Bcl-xL and survivin decreased in a concentration-dependent manner. Both UA and OA have antitumor effects on cancer cells with MDR, and the optimal effect is shown by UA on colonic cancer cells. Also, UA shows cell apoptosis-inducing effect on SW480, possibly by way of down-regulating the expressions of apoptosis antagonistic proteins, Bcl-2, Bcl-xL, and survivin.

A novel bisindolymaleimide derivative (WK234) inhibits proliferation and induces apoptosis through the protein kinase Cβ pathway, in chronic myelogenous leukemia K562 cells

WK234, a novel bisindolymaleimide derivative, was designed as a protein kinase Cβ (PKCβ) inhibitor. The objective of this study was to evaluate the anti-tumor activity of WK234 in the human chronic myelogenous leukemia (CML) K562 cell line and to investigate possible mechanisms of its action. The results show that WK234 inhibited K562 cell proliferation in a time- and dose-dependent manner. WK234 increased cytochrome C release and caspase-3 cleavage, which indicates that it induced apoptosis via mitochondria- and caspase-mediated pathways. Western blotting showed that PKCβ1, PKCβ2, and their phosphorylation levels were effectively decreased after 2–4 h of WK234 treatment. Meanwhile the phosphorylation status of PKCβ downstream proteins, glycogen synthase kinase 3α/β (GSK3α/β) and extracellular signal-regulated kinase (ERK), were inhibited. WK234 blocked phorbol myristate acetate (PMA)-induced Ser660 phosphorylation of PKCβ2 located at the cell membrane, and increased Ser660 PKCβ2 expression within the cytoplasm and the nucleus. These results indicate that WK234 inhibited cell proliferation and induced apoptosis through suppressing the PKCβ signal pathway. WK234 might be a promising candidate for the treatment of CML.

PTEN’s regulation of VEGF and VEGFR1 expression and its clinical significance in myeloid leukemia

Phosphatase and tensin homolog (PTEN) acts as a novel tumor suppressor gene. PTEN protein plays an important role in regulating proliferation, apoptosis, invasion, and migration of many cancer cells. PTEN also modulates angiogenesis mediated by vascular endothelial growth factor (VEGF) via down-regulating PI3K/Akt pathway in many solid tumors. However, the effects of PTEN on VEGF and VEGFR1 (FLT1)-mediated angiogenesis, migration, invasion of leukemia cells, and its clinical significance are still unknown in myeloid leukemia. Therefore, we investigated the effect of PTEN on PI3K/Akt and VEGF/FLT1 pathways by transfecting wild-type PTEN gene to induce high expression of wild-type PTEN gene and protein in chronic myelogenous leukemia cell line K562 cells. We also observed the correlation between the expression levels of PTEN and VEGF/FLT1 and its clinical significance in myeloid leukemia patients. We found that the expression reconstitution of wild-type PTEN had significant effect on inhibiting proliferation, migration, and invasion abilities of K562 cells via down-regulation of Akt phosphorylation and inhibition of VEGF/FLT1 expression. In myeloid leukemia patients, a negative correlation was found between the expression level of PTEN mRNA and that of VEGF and FLT1 mRNA. Down-regulation of PTEN expression accompanied by up-regulation of VEGF and FLT1 mRNA indicated a higher tendency of extramedullary disease in acute myeloid leukemia patients. In conclusion, PTEN could modulate the function of VEGF/VEGFR signaling pathway down-regulation of Akt phosphorylation and that PTEN would be a candidate target to be addressed for inhibiting angiogenesis along with the treatment of myeloid leukemia.

Effects of biomaterials for Lab-on-a-chip production on cell growth and expression of differentiated functions of leukemic cell lines

The rapid increase of the applications for Lab-on-a-chip devices has attracted the interest of researchers and engineers on standard process of the electronics industry for low production costs and large scale development, necessary for disposable applications. The printed circuit board technology could be used for this purpose, in particular for the wide range of materials available. In this paper, assays on biocompatibility of materials used for Lab-on-a-chip fabrication has been carried out using two tumor cell lines growing in suspension, the human chronic myelogenous leukemia K562 cell line, able to undergo erythroid differentiation when cultured with chemical inducers, and the lymphoblastoid cell line (LCL), extensively used for screening of cytotoxic T-lymphocytes (CTLs). We have demonstrated that some materials strongly inhibit cell proliferation of both the two cell lines to an extent higher that 70-75%, but only after a prolonged exposure of 3-6 days (Copper, Gold over Nickel, Aramid fiber filled epoxy uncured, b-stage epoxy die attach film, Tesa 4985 adhesive tape, Pyralux uncured, Copper + 1-octodecanethiol). However, when experiments were performed with short incubation time (1 h), only Aramid fiber filled epoxy uncured was cytotoxic. Variation of the results concerning the other materials was appreciable when the experiments performed on two cell lines were compared together. Furthermore, the effects of the materials on erythroid differentiation and CTL-mediated LCL lysis confirmed, in most of the cases, the data obtained in cytotoxic and antiproliferative tests.

Synthesis, cytotoxic activity, and thermal studies of novel N‐[(1,3‐diphenylpyrazol‐4‐yl)methyl] α‐amino acids

Abstract magnified image New N‐[(1,3‐diphenylpyrazol‐4‐yl)methyl]α‐amino acids (1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i) have been synthesized and tested in vitro for their antiproliferative activity against human myelogenous leukemia K562, colon adenocarcinoma HT‐29, cervix carcinoma HeLa, and normal fetal lung fibroblasts, MRC‐5. Compounds derived from both phenylalanine enantiomer precursors appeared to be the most active against myelogenous leukemia K562 cell lines with a high cytotoxic potential. J. Heterocyclic Chem., (2010).

Effects of STAT3 silencing on fate of chronic myelogenous leukemia K562 cells

Signal transducer and activator of transcription 3 (STAT3), a transcription factor, is constitutively activated in various types of cancers. Previous investigations have demonstrated that this overexpression of STAT3 in human malignancies plays important roles in maintaining the characteristics of malignant tumors by having an effect on proliferation, differentiation, and/or immortalization. Thus, inhibition of STAT3 expression could be a potent therapeutic approach in cancer treatment. In this study, we introduced STAT3 siRNA into the human chronic myelogenous leukemia (CML) K562 cell line, which has constitutive activation of STAT3, to elucidate the role of STAT3 in CML. The cells were transducted with STAT3 siRNA using lentivirus. FACS, real-time PCR, and Western blot were used to study changes in STAT3 expression levels in transducted cells by comparing with negative control siRNA lentivirus transduction. Knockdown of STAT3 by STAT3 siRNA caused a decrease in STAT3 protein level, inhibition of growth and proliferation, cell cycle blockade, visible morphologic changes, and induction of apoptosis in K562 cells. These findings demonstrate that STAT3 does indeed play a critical role in the survival of K562 cells, which may have potential application in designing molecular therapies for CML treatment.

Liquid crystal-related compound-induced cell growth suppression and apoptosis in the chronic myelogenous leukemia K562 cell line

Liquid crystals are the state of matter existing between the liquid and the crystalline phase, and there is a recent surging interest in its biological effects. Our previous study showed that liquid crystal-related compounds (LCRCs), which are precursors of the liquid crystal, enhanced hematopoietic differentiation at a relatively low concentration (Biol Pharm Bull, 32, 2009). However, biological potentials of LCRCs on tumor cells are unclear. In this study, the biological activity of 16 LCRCs to a chronic myelogenous leukemia cell line, K562, was evaluated. As a result, two compounds, 2-(4-butoxyphenyl)-5-(4-hydroxyphenyl)pyrimidine (compound 7) and 2-{4-(4-hexyloxyphenyl)phenyl}-5-hydroxypyrimidine (compound 9) showed marked growth suppression of K562 cells at μM range. These compounds are similar in structure with a core of three aromatic rings including a pyrimidine ring and residues of one alkyl chain and one hydroxide on either side. In addition, only compound 7 induced the activation of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, and apoptosis of K562 cells. The contrasting results between compounds 7 and 9 indicate different mechanisms to suppress the cell proliferation between the two compounds. These results suggest the possibility of LCRCs for application as new antitumor drugs.

Hydroxylation of Diosgenin by Absidia coerulea

Microbial transformation of diosgenin (1) using Absidia coerulea yielded five new polar metabolites, which were identified as (25R)-spirost-5-en-3 beta,7 beta,12 beta,25 alpha-tetrol (2), (25S)-spirost-5-en-3 beta,7 alpha,12 beta,25 beta-tetrol (3), (25S)-spirost-5-en-3 beta,7 beta,12 beta,25 beta-tetrol (4), (25R)-spirost-5-en-3 beta,7 alpha,12 beta,25 alpha-tetrol (5), and (25R)-spirost-5-en-3 beta,7 beta,12 beta,24 beta-tetrol (6). Their structures were established on the basis of mass spectrometry and multi-dimensional NMR spectroscopy. The characteristic transformations observed were C-7 alpha, C-7 beta, C-12 beta, C-24 beta, C-25 alpha, and C-25 beta hydroxylation. The cytotoxicity of compounds 1-6 was evaluated against the human myelogenous leukemia K562 cell line and squamous cell carcinoma KB parental cell lines. Compounds 2-6 exhibited weak cytotoxicity against K562 and KB cells and were less potent than the parent compound 1.

Design, Synthesis, and Biological Evaluation of a New Palladium(II) Complex: β-Lactoglobulin and K562 as Targets

A water-soluble Pd(II) complex (2,2'-bipyridinglycinato Pd(II) nitrate) has been synthesized and characterized. The effect of synthesized complex on the carrier model protein structure and cell proliferation was investigated. Whey carrier protein beta-lactoglobulin-B (BLG-B) and chronic myelogenous leukemia cell line K562 were the targets. Fluorescence and CD instruments were used to assess effect of the complex on the protein structure at different temperatures. Growth inhibitory and apoptotic effect of the Pd(II) complex toward the cancer cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. This complex exhibited potent cytotoxic properties against chronic myelogenous leukemia cell line K562. The cells showed different sensitivity to complex. Cytotoxic studies shown that Pd(II) complex induced apoptosis of K562 cells in a concentration and time dependent manner. Then, it might be concluded that Pd(II) complex is a promising antiproliferative agent and should execute its biological effects by inducing apoptosis. Results of fluorescence studies revealed that Pd(II) complex can quench the intrinsic fluorescence emission of the protein at different temperatures. The far- and near-UV CD studies displayed that the Pd(II) complex induces changes in the secondary and tertiary structures of BLG-B at different temperatures. The biological significance of this work is evident since BLG serves as a carrier molecule for several antitumor compounds. Therefore, the interaction of the Pd(II) complex (with antitumor activity) can provide useful information to better design metal anticancer complexes with fewer side effects.

Receptor-binding cancer antigen expressed on SiSo cells induces apoptosis via ectodomain shedding

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a secreted antigen that induces apoptosis in putative receptor-expressing cells, including peripheral lymphocytes and natural killer (NK) cells. RCAS1 expression is associated with aggressive characteristics and poor overall survival for 15 different human malignancies. The putative RCAS1 receptor has not been isolated and the mechanism of RCAS1 apoptosis induction remains unclear. This study explores how RCAS1 is involved in apoptosis initiation. The cell lines SiSo and MCF-7, human uterine carcinoma and breast adenocarcinoma, respectively, both express RCAS1, but RCAS1 secretion is undetectable in MCF-7 cells. SiSo and MCF-7 cells were stimulated to induce RCAS1 ectodomain shedding followed by assessment of RCAS1 expression and secretion. Additionally, the RCAS1 putative receptor-expressing human chronic myelogenous leukemia cell line K562 was co-cultured with SiSo, MCF-7, or soluble RCAS1 to follow RCAS1 secretion in apoptosis initiation. RCAS1 secretion was strongly suppressed by inhibitors of metalloproteases, protein kinase C (PKC)-δ, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase kinase (MEK), epidermal growth factor (EGF), and G-protein-coupled receptor (GPCR). K562 apoptosis could be induced only by co-culturing with SiSo or soluble RCAS1. RCAS1 is thus secreted by ectodomain shedding, which may represent a pivotal step in RCAS1-induced apoptosis initiation.

To Study the Proliferative Inhibition of Anticancer Drug in Two Kinds of Ph (+) Leukemia Cell Lines.

Abstract 4110 ObjectiveTo study the proliferative inhibition of imatinib, daunorubicin and bortezomib in two kinds of Ph(+) leukemia cell lines: chronic myelogenous leukemia cell line K562 expressing P210 protein and acute lymphoblastic leukemia cell line SUP-B15 expressing P190 protein. Methods(1) Cell proliferation with imatinib, daunorubicin and bortezomib for 72 hours was analyzed by the MTT assay and displayed by growth curve and IC50 value. (2) The change of bcr-abl gene mRNA levels after the 48 hours' intervention of imatinib (final concentration at 0μM, 0.35μM, 1 μM) was detected by reverse transcription polymerase chain reaction (RT-PCR). Results(1) The IC50 values of K562 and SUP-B15 cells inhibited by imatinib, daunorubicin and bortezomib for 72 hours was respectively 0.286±0.06 (μmol/L), 0.303±0.009 (μmol/L), 22.127±3.592 (nmol/L) and 1.387±0.180(μmol/L), 0.117±0.017 (μmol/L), 12.350±0.740 (nmol/L), which indicated that the K562 cell line was the more sensitive to imatinib than SUP-B15 cell line, whereas the SUP-B15 cell line had the more sensitivity to daunorubicin and bortezomib. (2) There was no change of bcr-abl gene expression after the 48 hours' intervention of imatinib in both cell lines. Conclusion(1) Imatinib, daunorubicin and bortezomib had good anti-cancer effect to Ph+ leukemia cells in vitro. What's more, the K562 cell was the more sensitive to imatinib and only imatinib will have good effect on chronic myelogenous leukemia. Whereas the SUP-B15 cell had the more sensitivity to daunorubicin and bortezomib and combining imatinib with daunorubicin or bortezomib, the effect will be better on Ph(+) acute lymphoblastic leukemia. (2) The short time intervention of imatinib had no effect on the bcr-abl gene expression and imatinib could need long time to show curative effect for the Ph+ leukemia. Disclosures:No relevant conflicts of interest to declare.

Second Mitochondria-Derived Activator of Caspases Mimetic Compounds (Smac) Enhance Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) Pro-Apoptotic Effect On Human Myeloid Leukemia Cell Lines.

Abstract 2756Poster Board II-732The Inhibitor of Apoptosis Proteins (IAP) are important regulators of programmed cell death. Among them, XIAP, which is characterized by 3 tandem BIR domains selectively blocking caspases 3, 7 and 9, is the most potent and is over-expressed in several hematological malignancies. Its activity is antagonized by Second Mitochondria-derived Activator of Caspases (Smac) and also by small molecules mimicking Smac able to induce apoptosis in tumor cells, alone or in combination with other drugs.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a pro-apoptotic cytokine, is capable of triggering programmed death in cancer cells, where it synergizes with chemotherapeutic agents and/or radiation by several malignant cell-type specific mechanisms, and it is currently under evaluation in clinical trials for its strong pro-apoptotic activity.Here we describe the pro-apoptotic effect of newly synthesized monovalent and bivalent Smac-mimetic compounds tested for cytotoxicity on a cohort of human leukemic cell lines over-expressing XIAP (HL60, K562 and Jurkat) as well as on normal CD34+ hematopoietic progenitor cells, alone or in combination with TRAIL. The Smac-mimetics, designed and produced by the University of Milan Center for biomolecular Interdisciplinary Studies and Industrial applications, were dissolved in dimethylsulfoxide (DMSO) to obtain a 10 mM solution and stored at −20°C. Drug stocks were diluted with phosphate buffer (PBS) prior to their use. The cells were treated with 0.1 nM – 50 μM doses for up to 72 hours. TRAIL was kindly provided by Prof. Henning Walczak (Imperial College, London, UK). In combined treatments, Smac-mimetics and TRAIL were used simultaneously. The cytotoxic effect was evaluated by a colorimetric assay for the quantification of cell proliferation and viability based on the cleavage of the WST-8 tetrazolium salt by mitochondrial dehydrogenases. The effect on fresh human CD34+ hematopoietic normal progenitor cells selected from healthy donors' peripheral blood stem cells (PBSC) was assayed by myeloid colony (CFU-GM) formation. Apoptosis was determined by flow cytometric analysis of DNA fragmentation and by Western blot analysis of caspases activation and PARP cleavage.The combination of some of our Smac-mimetics (both monomers and dimers) with TRAIL highly enhanced the inhibition of proliferation of the chronic myelogenous leukemia cell line K562, which was shown to be resistant to TRAIL single agent. In particular, a synergistic effect was observed in combined treatment with Smac012 10 μM and TRAIL 50 ng/ml (R Kern index = 6.2). Our Smac-mimetics, particularly the dimeric ones, were capable of inducing apoptosis, as demonstrated by DNA fragmentation and accumulation of cleaved PARP, caspase 8 and caspase 3, especially when administrated in combination with TRAIL. No cytotoxic effect was observed on normal CD34+ progenitor cells by Smac mimetics at doses ranging from 40 μM to 70 μM, even normal controls when were treated simultaneously with TRAIL.As many hematological malignancies are resistant to TRAIL alone, thus limiting its therapeutic effectiveness, the observed strong synergistic effect with Smac mimetic compounds not affecting the normal hematopoietic progenitor cell compartment might be of great consequence for the development of innovative potent pro-apoptotic drug combinations in myeloid leukemia treatment. Disclosures:No relevant conflicts of interest to declare.

Transcriptional Changes Induced by Imatinib and Nilotinib in the Chronic Myelogenous Leukemia (CML) Cell Line K562.

Abstract 4250Nilotinib is a selective bcr-abl tyrosine kinase inhibitor that is 30-fold more potent than Imatinib in vitro. To examine the molecular and functional effects of Nilotinib and Imatinib we performed gene expression and functional analyses in K562 cells following in vitro treatment with the two tyrosine kinase inhibitors. Affymetrix U133A 2.0 microarrays covering 21.722 probe sets were used to analyse the gene expression profile of 5×10 7 K562 cells after 24h in vitro treatment with Imatinib (0.5 μM) or Nilotinib (0.05 μM) (half maximal inhibitory concentration). Gene expression data of the treated cells were compared with data of untreated cells. In addition, proliferation (MTS Assay, Promega), apoptosis (Cell Death Detection ELISAPLUS, Roche) and cell cycle (FITC BrdU Flow Kit, BD Pharmingen) assays were performed. Protein levels of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2 were detected with Western blots. Particular emphasis was put on 303 genes which we found to be differentially expressed in primary CD34+ cells from patients with CML in vivo during treatment with Imatinib (Bruennert et al, Leukemia, 2008). In K562 cells, we found that Imatinib led to a significant differential expression of 45 of those genes. In general, the effect of Nilotinib with regard to the number of genes affected and degree of suppression was more pronounced resulting in a significant differential expression of 120 genes of the aforementioned genes. Of note, genes affected by Nilotinib included all genes altered by Imatinib. Downregulation of genes involved in cell cycle was observed in 17 genes following Nilotinib exposure, but only in the PIM-1 gene following Imatinib exposure. This effect of Nilotinib is in line with the results of cell cycle experiments showing that Nilotinib exposed cells had the lowest proportion of actively cycling cells. The proportion of apoptotic K562 cells was 5.5-fold greater following treatment with Nilotinib in comparison to treatment with DMSO, whereas Imatinib treated K562 cells had a 1.3-fold higher apoptosis rate compared to DMSO treated 562 cells after 24 hours. The superiority of Nilotinib is also reflected by the results of protein analysis of STAT5, pSTAT5(Thr694), PIM-1 and PIM-2: Both tyrosine kinase inhibitors completely inhibit the phosphorylation of STAT5, but Nilotinib inhibits PIM-1 and PIM-2 stronger than Imatinib (by 85% vs. 70%) in K562 cells. In summary, on a molecular level Nilotinib is apparently more potent than Imatinib with regard to the number of genes affected and the degree of their suppression. Among the 45 genes that were significant differentially expressed with both drugs, genes of Imatinib treated K562 cells were downregulated 1.68fold (mean) whereas genes of Nilotinib treated K562 cells were downregulated 2.41fold (mean). The genes altered are mainly associated with cell cycle regulation. In addition to this Nilotinib has a stronger inhibitory effect on PIM 1 and 2. Disclosures:Gattermann:Novartis: Honoraria, Participation in Advisory Boards on deferasirox clinical trials.

Synthesis, characterization, electrochemical studies and antitumor activity of some new chalcone analogues containing ferrocenyl pyrazole moiety

A series of new α,β-unsaturated conjugated ketones containing ferrocenyl pyrazole unit were synthesized and fully characterized by IR and NMR spectroscopy. Electrochemical characterization of subject compounds was performed by means of cyclic voltametry. The in vitro cytotoxic activity of all the synthesized compounds was studied against cervix adenocarcinoma HeLa, melanoma Fem-x and myelogenous leukemia K562 cell lines by the MTT method. Derivative 1l containing 3-pyridyl moiety exhibited a better cytotoxic activity in the cell growth inhibition of K562 cell lines in comparison with cisplatin as a reference compound.

Synthesis, characterization and antitumor activity of novel N-substituted α-amino acids containing ferrocenyl pyrazole-moiety

A series of new N-[(3-ferrocenyl-1-phenylpyrazol-4-yl)methyl] α-amino acids were prepared and characterized by a range of spectroscopic techniques and cyclic voltammetry. The in vitro antitumor activity of all synthesized compounds was investigated against cervix adenocarcinoma HeLa, melanoma Fem-x and myelogenous leukemia K562 cell lines using the MTT method. Tryptophan derivative 1l exhibited the highest cytotoxic activity in the cell growth inhibition of all three types of cell lines.

Discovery of 8-methoxypyrazino[1,2-a]indole as a New Potent Antiproliferative Agent Against Human Leukemia K562 Cells. A Structure-Activity Relationship Study

Identification of novel and selective anticancer agents remains an important and challenging goal in pharmacological research. The indole nucleus, frequently encountered as a molecular fragment in natural products and pharmaceutically active compounds, was employed as the initial building block for the synthesis of a series of pyrazino[1,2-a]indoles 1a-k, variably substituted at the 6, 7, 8 and 9-positions. Compound 1e, bearing the methoxy group at the 8-position of the pyrazino[1,2-a]indole nucleus was identified as a novel potent antiproliferative agent against the human chronic myelogenous leukemia K562 cell line, but it was much less active against several other cancer cell lines. Comparison of positional isomers indicated that moving the methoxy group from the 8- to the 7- or 6-position, to furnish compounds 1f and 1g, respectively, yielded inactive compounds. The analysis of structure-activity relationships observed in the series of investigated compounds may represent the basis for the design of more active molecules.

Gambogic acid induces G0/G1 arrest and apoptosis involving inhibition of SRC-3 and inactivation of Akt pathway in K562 leukemia cells

Gambogic acid (GA), a major active component of gamboge, exhibits potent anticancer activity in many kinds of cancer cells. However, the anticancer mechanism of GA is not clearly understood. Here we showed that GA could cause growth inhibition, induce the G0/G1 phase cell cycle arrest and apoptosis in human chronic myelogenous leukemia cell line K562 cells. Since steroid receptor coactivator-3 (SRC-3), overexpressed in many human malignancies including leukemia, is a central target for cancer therapy, we also explored the effects of GA on SRC-3 and SRC-3-regulated gene products in K562. GA treatment downregulated the expression of SRC-3 and then inhibited the activity of Akt kinase and its downstream targets p70 S6 kinase 1 (S6K1) and glycogen synthase kinase 3β (GSK3β) without changes in total protein levels of these three proteins, which thus influenced the expression of the apoptosis related gene Bcl-2 in K562 cells. These results suggest that GA might exhibit its strong antitumor effects via the interruption of SRC-3.

Inhibition of imatinib-mediated apoptosis by the caspase-cleaved form of the tyrosine kinase Lyn in chronic myelogenous leukemia cells

Once cleaved by caspases, the Lyn tyrosine kinase (LynDeltaN) is relocalized from the plasma membrane to the cytoplasm of apoptotic cells, but the function of such a cleavage is incompletely understood. We evaluated the effect of LynDeltaN overexpression on imatinib sensitivity of the chronic myelogenous leukemia (CML) cell line K562. Therefore, we generated stable cells that express plasmids encoding LynDeltaN or its catalytically inactive counterpart LynDeltaNKD. We established that Lyn is cleaved in imatinib-treated parental K562 cells in a caspase-dependent manner. Lyn cleavage also occurred following BCR-ABL silencing by specific short hairpin RNA (sh-RNA). Imatinib-induced apoptosis was abrogated in LynDeltaN-overexpressing cells, but not in cells overexpressing its inactive counterpart. Conversely, the overexpression of LynDeltaN failed to affect the differentiation of K562 cells. Importantly, the protective effect of LynDeltaN was suppressed by two inhibitors of Lyn activity. LynDeltaN also inhibits imatinib-mediated caspase-3 activation in the small proportion of nilotinib-resistant K562 cells overexpressing Lyn that can engage an apoptotic program upon imatinib stimulation. Finally, Lyn knockdown by sh-RNA altered neither imatinib-mediated apoptosis nor differentiation. Taken together, our data show that the caspase-cleaved form of Lyn exerts a negative feedback on imatinib-mediated CML cell apoptosis that is entirely dependent on its kinase activity and likely on the BCR-ABL pathway.

Molecular imaging of Bcr-Abl phosphokinase in a xenograft model

The purpose of this study was to determine whether the Bcr-Abl tyrosine kinase can be assessed by gamma-imaging using an 111In-labeled anti-phosphotyrosine (APT) antibody, and if the response to treatment with imatinib could be detected using this imaging technique. APT antibody was labeled with 111In using ethylenedicysteine (EC) as a chelator. To determine if 111In-EC-APT could assess a nonreceptor tyrosine kinase, xenografts of the human chronic myelogenous leukemia cell line K562 were used. gamma-Scintigraphy of the tumor-bearing mice, before and after imatinib treatment, was obtained 1, 24, and 48 h after they were given 111In-EC-APT (100 microCi/mouse i.v.). 111In-EC-APT is preferentially taken up by Bcr-Abl-bearing tumor cells when compared with 111In-EC-BSA or 111In-EC-IgG1 controls and comparable with the level of uptake of 111In-EC-Bcr-Abl. Imatinib treatment resulted in decreased expression of phospho-Bcr-Abl by Western blot analysis, which correlated with early (4 days after starting imatinib) kinase down-regulation as assessed by imaging using 111In-EC-APT. The optimal time to imaging was 24 and 48 h after injection of 111In-EC-APT. Although tumor regression was insignificant on day 4 after starting imatinib treatment, it was marked by day 14. 111In-EC-APT can assess intracellular phosphokinase activity, and down-regulation of phosphokinase activity predates tumor regression. This technique may therefore be useful in the clinic to detect the presence of phosphokinase activity and for early prediction of response.