benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Leukemia--Myelogenous--Chronic--BCR-ABL-Positive

Activated CDC42-associated kinase-1 (ACK1/TNK2) and epigenetic regulators of the histone deacetylase (HDAC) family regulate the proliferation and survival of leukemic cells. 18 HDACs fall into four classes (I-IV). We tested the impact of clinically relevant histone deacetylase inhibitors (HDACi) on ACK1 and if such drugs combine favorably with the therapeutically used ACK1 inhibitor Dasatinib.. We applied the broad-range HDACi Panobinostat/LBH589 and the class I HDAC-specific inhibitor Entinostat/MS-275 to various acute and chronic myeloid leukemia cells (AML/CML). We also used the replicative stress inducer Hydroxyurea (HU), a standard drug for leukemic patients, and the apoptosis inducer Staurosporine (STS). To assess cytotoxic effects of HDACi, we measured cell cycle profiles and DNA fragmentation by flow cytometry. Western blot was employed to analyze protein expression and phosphorylation.. LBH589 and MS-275 induce proteolysis of ACK1 in CML and AML cells. Panobinostat more strongly induces apoptosis than Entinostat, and this correlates with a significantly pronounced loss of ACK1. STS and HU also propel the degradation of ACK1 in leukemic cells. Moreover, the caspase inhibitor z-VAD-FMK reduces ACK1 degradation in the presence of HDACi. Concomitant with the attenuation of ACK1, we noticed decreased phosphorylation of STAT3. Direct inhibition of ACK1 with Dasatinib also suppresses STAT3 phosphorylation. Furthermore, Dasatinib and HDACi combinations are effective against CML cells.. HDACs sustain the ACK1-STAT3 signaling node and leukemic cell growth. Consistent with their different effects on ACK1 stability or auto-phosphorylation, Dasatinib and HDACi combinations produce beneficial antileukemic effects.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspases; Dasatinib; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Indoles; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Panobinostat; Phosphorylation; Protein-Tyrosine Kinases; STAT3 Transcription Factor

Quorum sensing is defined as the ability of microorganisms to sense their population density via the release of signaling molecules called autoinducers (AIs). Various types of AI analogs were prepared and their antitumor properties against chronic myeloid leukemia (CML) K562 cells were investigated. Two AI analogs induced progressive apoptosis with JNK activation and p21 induction. In addition, this induction of apoptosis is not related to bcr-abl kinase, which sustains CML proliferation. However, the progression of apoptosis was not inhibited by a caspase family inhibitor. These results suggested that AI analogs could induce caspase-independent apoptosis in CML K562.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Cell Cycle; Cell Proliferation; Cysteine Proteinase Inhibitors; Humans; JNK Mitogen-Activated Protein Kinases; K562 Cells; Lactones; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; p21-Activated Kinases; Quorum Sensing

Recently, plant lectins have attracted great interest due to their various biological activities such as anti-cancer, anti-fungal and anti-viral activities. We have reported earlier concerning anti-proliferation of human cancer cell lines by a galactose-binding lectin (AML), from a Chinese herb, ASTRAGALUS MEMBRANACEUS: In the present study, detailed investigations into the mechanism of such anti-proliferation properties have been carried out.. Mechanism of apoptosis initiation in K562 cells by AML was investigated by morphology, flow cytometry and western blot analysis.. AML induced apoptosis in a caspase-dependent manner in the chronic myeloid leukemia cell line, K562. Furthermore, we observed that cytotoxicity and apoptosis of K562 cells induced by AML were completely abolished in presence of lactose or galactose.. Our results suggest that AML could act as a potential anti-cancer drug.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Astragalus propinquus; Carbohydrates; Caspase 3; Cell Proliferation; Cysteine Proteinase Inhibitors; Drugs, Chinese Herbal; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Phytotherapy; Plant Lectins; Proto-Oncogene Proteins c-bcl-2

Tryptanthrin, a kind of indole quinazoline alkaloid, has been shown to exhibit anti-microbial, anti-inflammation and anti-tumor effects both in vivo and in vitro. However, its biological activity on human chronic myeloid leukemia cell line K562 is not fully understood. In the present study, we investigated the proliferation-attenuating and apoptosis-inducing effects of tryptanthrin on leukemia K562 cells in vitro and explored the underlying mechanisms. The results showed that tryptanthrin could significantly inhibit K562 cells proliferation in a time- and dose-dependent manner as evidenced by MTT assay and flow cytometry analysis. We also observed pyknosis, chromatin margination and the formation of apoptotic bodies in the presence of tryptanthrin under the electron microscope. Nuclei fragmentation and condensation by Hoechst 33258 staining were detected as well. The amount of apoptotic cells significantly increased whereas the mitochondrial membrane potential decreased dramatically after tryptanthrin exposure. K562 cells in the tryptanthrin treated group exhibited an increase in cytosol cyt-c, Bax and activated caspase-3 expression while a decrease in Bcl-2, mito cyt-c and pro-caspase-3 contents. However, the changes of pro-caspase-3 and activated caspase-3 could be abolished by a pan-caspase inhibitor ZVAD-FMK. These results suggest that tryptanthrin has proliferation-attenuating and apoptosis-inducing effects on K562 cells. The underlying mechanism is probably attributed to the reduction in mitochondria membrane potential, the release of mito cyt-c and pro-caspase-3 activation.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Proliferation; Cytochromes c; DNA Fragmentation; Enzyme Activation; Humans; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Quinazolines

Mitotic catastrophe (MC) is induced when stressed cells enter prematurely or inappropriately into mitosis and can be caused by ionizing radiation and anticancer drugs. Foretinib is a multikinase inhibitor whose mechanism of action is incompletely understood. We investigated here the effect of Foretinib on chronic myelogenous leukemia (CML) cell lines either sensitive (IM-S) or resistant (IM-R) to the tyrosine kinase inhibitor Imatinib. Foretinib decreased viability and clonogenic potential of IM-S and IM-R CML cells as well. Foretinib-treated cells exhibited increased size, spindle assembly checkpoint anomalies and enhanced ploidy that collectively evoked mitotic catastrophe (MC). Accordingly, Foretinib-stimulated CML cells displayed decreased expression of Cdk1, Cyclin B1 and Plk1. In addition, Foretinib triggered caspase-2 activation that precedes mitochondrial membrane permeabilization. Accordingly, z-VAD-fmk and a caspase-2 siRNA abolished Foretinib-mediated cell death but failed to affect MC, indicating that Foretinib-mediated apoptosis and MC are two independent events. Anisomycin, a JNK activator, impaired Foretinib-induced MC and inhibition or knockdown of JNK phenotyped its effect on MC. Moreover, we found that Foretinib acted as a potent inhibitor of JNK. Importantly, Foretinib exhibited no or very little effect on normal peripheral blood mononuclear cells, monocytes or melanocytes cells but efficiently inhibited the clonogenic potential of CD34+ cell from CML patients. Collectively, our data show that the multikinase inhibitor Foretinib induces MC in CML cells and other cell lines via JNK-dependent inhibition of Plk1 expression and triggered apoptosis by a caspase 2-mediated mechanism. This unusual mechanism of action may have important implications for the treatment of cancer.

Topics: Amino Acid Chloromethyl Ketones; Anilides; Anisomycin; Antigens, CD34; Antineoplastic Agents; Benzamides; Caspase 2; Caspase Inhibitors; CDC2 Protein Kinase; Cell Cycle Proteins; Cell Death; Cell Size; Cell Survival; Cyclin B1; Cysteine Endopeptidases; Enzyme Activation; Enzyme Assays; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukocytes, Mononuclear; M Phase Cell Cycle Checkpoints; MAP Kinase Signaling System; Melanocytes; Mitosis; Phenotype; Piperazines; Ploidies; Polo-Like Kinase 1; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Pyrimidines; Quinolines; RNA, Small Interfering; Transfection

Autophagy that is induced by starvation or cellular stress can enable cancer cell survival by sustaining energy homeostasis and eliminating damaged organelles and proteins. In response to stress, cancer cells have been reported to accumulate the protein p62/SQSTM1 (p62), but its role in the regulation of autophagy is controversial. Here, we report that the plant phytoalexin resveratrol (RSV) triggers autophagy in imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia (CML) cells via JNK-dependent accumulation of p62. JNK inhibition or p62 knockdown prevented RSV-mediated autophagy and antileukemic effects. RSV also stimulated AMPK, thereby inhibiting the mTOR pathway. AMPK knockdown or mTOR overexpression impaired RSV-induced autophagy but not JNK activation. Lastly, p62 expression and autophagy in CD34+ progenitors from patients with CML was induced by RSV, and disrupting autophagy protected CD34+ CML cells from RSV-mediated cell death. We concluded that RSV triggered autophagic cell death in CML cells via both JNK-mediated p62 overexpression and AMPK activation. Our findings show that the JNK and AMPK pathways can cooperate to eliminate CML cells via autophagy.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Chloromethyl Ketones; AMP-Activated Protein Kinases; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Autophagy; Benzamides; Blotting, Western; Caspase Inhibitors; Caspases; Cell Survival; Drug Resistance, Neoplasm; Humans; Imatinib Mesylate; JNK Mitogen-Activated Protein Kinases; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Macrolides; Microscopy, Confocal; Microscopy, Electron; Piperazines; Pyrimidines; Resveratrol; RNA Interference; Sequestosome-1 Protein; Stilbenes; Tumor Cells, Cultured; Vacuolar Proton-Translocating ATPases

Caspase-independent programmed cell death can exhibit either an apoptosis-like or a necrosis-like morphology. The ABL kinase inhibitor, imatinib mesylate, has been reported to induce apoptosis of BCR-ABL-positive cells in a caspase-dependent fashion. We investigated whether caspases alone were the mediators of imatinib mesylate-induced cell death. In contrast to previous reports, we found that a broad caspase inhibitor, zVAD-fmk, failed to prevent the death of imatinib mesylate-treated BCR-ABL-positive human leukemic cells. Moreover, zVAD-fmk-preincubated, imatinib mesylate-treated cells exhibited a necrosis-like morphology characterized by cellular pyknosis, cytoplasmic vacuolization, and the absence of nuclear signs of apoptosis. These cells manifested a loss of the mitochondrial transmembrane potential, indicating the mitochondrial involvement in this caspase-independent necrosis. We excluded the participation of several mitochondrial factors possibly involved in caspase-independent cell death such as apoptosis-inducing factor, endonuclease G, and reactive oxygen species. However, we observed the mitochondrial release of the serine protease Omi/HtrA2 into the cytosol of the cells treated with imatinib mesylate or zVAD-fmk plus imatinib mesylate. Furthermore, serine protease inhibitors prevented the caspase-independent necrosis. Taken together, our results suggest that imatinib mesylate induces a caspase-independent, necrosis-like programmed cell death mediated by the serine protease activity of Omi/HtrA2.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Benzamides; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Nucleus; Cysteine Proteinase Inhibitors; Flavoproteins; Fusion Proteins, bcr-abl; High-Temperature Requirement A Serine Peptidase 2; Humans; Imatinib Mesylate; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Potentials; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Necrosis; Nucleosomes; Piperazines; Pyrimidines; Reactive Oxygen Species; Serine Endopeptidases

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) have been implicated in antitumor immunity and therapy. In the present study, we investigated the sensitivity of Philadelphia chromosome (Ph1)-positive leukemia cell lines to TRAIL- or FasL-induced cell death to explore the possible contribution of these molecules to immunotherapy against Ph1-positive leukemias. TRAIL, but not FasL, effectively induced apoptotic cell death in most of 5 chronic myelogenous leukemia-derived and 7 acute leukemia-derived Ph1-positive cell lines. The sensitivity to TRAIL was correlated with cell-surface expression of death-inducing receptors DR4 and/or DR5. The TRAIL-induced cell death was caspase-dependent and enhanced by nuclear factor kappa B inhibitors. Moreover, primary leukemia cells from Ph1-positive acute lymphoblastic leukemia patients were also sensitive to TRAIL, but not to FasL, depending on DR4/DR5 expression. Fas-associated death domain protein (FADD) and caspase-8, components of death-inducing signaling complex (DISC), as well as FLIP (FLICE [Fas-associating protein with death domain-like interleukin-1-converting enzyme]/caspase-8 inhibitory protein), a negative regulator of caspase-8, were expressed ubiquitously in Ph1-positive leukemia cell lines irrespective of their differential sensitivities to TRAIL and FasL. Notably, TRAIL could induce cell death in the Ph1-positive leukemia cell lines that were refractory to a BCR-ABL-specific tyrosine kinase inhibitor imatinib mesylate (STI571; Novartis Pharma, Basel, Switzerland). These results suggested the potential utility of recombinant TRAIL as a novel therapeutic agent and the possible contribution of endogenously expressed TRAIL to immunotherapy against Ph1-positive leukemias.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; Arabidopsis Proteins; Benzamides; Carrier Proteins; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 1; Death Domain Receptor Signaling Adaptor Proteins; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Fas Ligand Protein; Fatty Acid Desaturases; Fusion Proteins, bcr-abl; Humans; Imatinib Mesylate; Intracellular Signaling Peptides and Proteins; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leupeptins; Membrane Glycoproteins; Neoplasm Proteins; Neoplastic Stem Cells; NF-kappa B; Peptides; Piperazines; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Pyrimidines; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

We have previously shown that Bax translocation was crucial in TNFalpha or etoposide-induced apoptosis. Overexpression of Bax sensitized chronic myeloid leukemic K562 cells to etoposide-induced apoptosis. Treatment with TNF-related apoptosis-inducing ligand (TRAIL) induces a loss of mitochondrial membrane potential (DeltaPsim), cytochrome c release from mitochondria, activation of caspases-8, -9, and -3, and cleavage of Bid in the K562 cell line. Bax failed to sensitize K562 cells to TRAIL-induced apoptosis. TRAIL did not induce Bax expression and/or translocation from cytosol to mitochondria in the K562 cell line. However, 100 microM Z-VAD.fmk, a pan caspase inhibitor, completely blocked TRAIL-initiated mitochondrial alterations and cleavages of caspases and Bid. We propose that TRAIL-induced apoptosis in K562 cells is via Type I apoptotic signal pathway. Bax translocation is not essential for TRAIL-induced cytochrome c release and DeltaPsim collapse in the Type I cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Carrier Proteins; Caspase 3; Caspase 8; Caspase 9; Caspases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Enzyme Precursors; Humans; Intracellular Membranes; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Membrane Glycoproteins; Membrane Potentials; Mitochondria; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Transfection; Tumor Necrosis Factor-alpha