Glucosamine sulfate–induced apoptosis in chronic myelogenous leukemia K562 cells is associated with translocation of cathepsin D and downregulation of Bcl-xL

Cathepsin D (cat D) reportedly plays an important role in certain apoptotic processes, the downstream pathways of which involve release of cytochrome c (cyt c) from mitochondria and activation of the caspase cascade. Previous studies revealed that the B-cell lymphoma 2 (Bcl-2) family members Bax or Bid play important roles in apoptotic signal transduction between cat D and mitochondria. Here, we show that glucosamine sulfate (GS) inhibits the proliferation and induces apoptosis of human chronic myelogenous leukemia K562 cells in vitro. GS interfered with the maturation of cat D. Activation of caspase-3, cleavage of poly-(ADP-ribose)-polymerase, release of cyt c, and downregulation of Bcl-xL accompanied GS-induced apoptosis, and these processes were inhibited by the cat D inhibitor pepstatin A. However, we did not detect any altered gene expression of Bcl-2, Bax, or Bid during apoptosis. Translocation of cat D from the lysosome to the cytosol was observed in GS-treated K562 cells. These findings suggest that GS-induced K562 cell apoptosis involves the translocation of cat D from the lysosome to the cytosol. Furthermore, our findings suggest that downregulation of Bcl-xL (but not Bcl-2, Bax, or Bid) connects cat D and the mitochondrial pathway, which causes the release of cyt c and activation of the caspase cascade during GS-induced apoptosis of K562 cells.     

Ethyl acetate extract and its major constituent, isorhamnetin 3-O-rutinoside, from Nitraria retusa leaves, promote apoptosis of human myelogenous erythroleukaemia cells

Objective: Fractionation of ethyl acetate extract (EA) obtained from Nitraria retusa leaves was assessed using different methods of chromatography, and isorhamnetin3‐O‐rutinoside (I3‐O‐R) was isolated from this extract. Its structure was determined using data obtained from ¹H and ¹³C NMR spectra, as well as by various correlation experiments (COSY, HMQC and HMBC). Both EA extract and I3‐O‐R were investigated for their ability to induce apoptosis in human chronic myelogenous erythroleukaemia cells (K562). Materials and methods: Apoptosis of cells from the K562 line was detected by DNA fragmentation, PARP cleavage and by evaluating activities of caspases 3 and 8. Results: Apoptosis, revealed by DNA fragmentation and PARP cleavage, was observed after 48‐h incubation of these human myelogenous erythroleukaemia cells (K562), with the tested products. Likewise, caspase 3 and caspase 8 activities were induced in the presence of the EA extract and I3‐O‐R after 48 h of incubation. Conclusion: Our results strongly suggest the involvement of the extrinsic pathway of apoptosis in cells treated by both the original EA extract and its major component, I3‐O‐R.     

Induction of apoptosis in human lymphoblastoid cells by kaempferol 3-O-β-isorhamninoside and rhamnocitrin 3-O-β-isorhamninoside from Rhamnus alaternus L. (Rhamnaceae)

Objective: Kaempferol 3‐O‐β‐isorhamninoside (K3O‐ir) and rhamnocitrin 3‐O‐β‐isorhamninoside (R3O‐ir) from Rhamnus alaternus L leaves are investigated for their ability to induce apoptosis in human lymphoblastoid cells. We have attempted to characterize apoptotic pathway activated by these two flavonoids. Material and methods: Apoptosis of the human TK6 lymphoblastoid cell line was detected by DNA fragmentation, PARP cleavage and by evaluating caspase activity. Results: Apoptosis was observed after 24‐ and 48‐h incubation of the cells with the tested compounds. DNA fragmentation was observed after treatment with flavonoids; this was confirmed by demonstration of PARP cleavage. Caspase‐3 and caspase‐8 activities were induced by both K3O‐ir and R3O‐ir flavonoids showing highest activity with compound concentration of 400 μg/ml. Conclusion: We have demonstrated that K3O‐ir and R3O‐ir induce apoptosis in human lymphoblastoid cells by the extrinsic pathway of apoptosis.     

Gamma-linolenic acid induces apoptosis and lipid peroxidation in human chronic myelogenous leukemia K562 cells

Various polyunsaturated fatty acids, especially gamma-linolenic acid (GLA), inhibit the growth of a variety of tumor cells. Some evidence indicates that polyunsaturated fatty acid can kill cells by apoptosis. In the current study, we tested the apoptotic effect of GLA on human chronic myelogenous leukemia K562 cells. GLA induced K562 cell death in a dose-dependent manner. Typical apoptotic nuclei were shown by staining of K562 cells with DNA-binding fluorochrome Hoechst 33342, characterized by chromatin condensation and nuclear fragmentation. Flow cytometric analysis also demonstrated that GLA caused dose-dependent apoptosis of K562 cells. The apoptosis could be inhibited by a pancaspase inhibitor (z-VAD-fmk), suggesting the involvement of caspases. Further, release of cytochrome c, activation of caspase-3 and cleavage of PARP were found in GLA-induced apoptosis. GLA treatment could also elevate lipid peroxidation in K562 cells, and antioxidant α-tocopherol could reverse the cytotoxicity of GLA. The saturated fatty acid SA, which did not exhibit significant increase in lipid peroxidation, also did not induce cytotoxicity. Intracellular GSH was also determined, and there was no marked change of GSH levels in cells after incubation with GLA compared with the control. These results demonstrate that GLA could induce apoptosis in K562 cells. Apoptosis is mediated by release of cytochrome c, activation of caspase-3. Lipid peroxidation may play a role in GLA cytotoxicity.     

Discovery of a new RNA-containing nuclear structure in UVC-induced apoptotic cells by integrated laser electron microscopy

Background information. Treatment of cells with UVC radiation leads to the formation of DNA cross‐links which, if not repaired, can lead to apoptosis. γ‐H2AX and cleaved caspase 3 are proteins formed during UVC‐induced DNA damage and apoptosis respectively. The present study sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy, ILEM (integrated laser electron microscopy). Cleaved caspase 3 and γ‐H2AX were immunofluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analysed at high resolution with TEM (transmission electron microscopy). Results. Following the treatment of HUVECs (human umbilical vein endothelial cells) with UVC radiation, in the majority of the cells γ‐H2AX was formed, whereas only in a subset of cells caspase 3 was activated. In severely damaged cells with high levels of γ‐H2AX a round, electron‐dense nuclear structure was found, which was hitherto not identified in UV‐stressed cells. This structure exists only in nuclei of cells containing cleaved caspase 3 and is present during all stages of the apoptotic process. Energy‐loss imaging showed that the nuclear structure accumulates phosphorus, indicating that it is rich in nucleic acids. Because the nuclear structure did not label for DNA and was not affected by regressive EDTA treatment, it is suggested that the UV‐induced nuclear structure contains a high amount of RNA. Conclusions. Because the UV‐induced nuclear structure was only found in cells labelled for cleaved caspase 3 it is proposed as an electron microscopic marker for all stages of apoptosis. Such a marker will especially facilitate the screening for early apoptotic cells, which lack the well‐known hallmarks of apoptosis within a cell population. It also raises new questions on the mechanisms involved in the UV‐induced apoptotic pathway.     

Apoptotic effect of hesperidin through caspase3 activation in human colon cancer cells, SNU-C4.

Hesperidin, a known flavonoid constituent of citrus, reduces the proliferation of many cancer cells. The apoptotic effects of hesperidin on human colon cancer cells, SNU-C4, were determined at concentrations of 1-100 microM. At 100 microM, hesperidin reduced cell viability to 65.00+/-0.05% of control values in a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death induced by hesperidin showed apoptotic features in 4,6-diamidino-2-phenylindole (DAPI) staining and in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Examination of the expression of apoptosis-regulating genes indicated that hesperidin treatment decreased the expression of B-cell CLL/lymphoma 2 (BCL2) mRNA, and increased the expression of BCL2-associated X protein (BAX). The expression and activity of the major apoptotic factor caspase3 (CASP3) was increased significantly with hesperidin treatment. Hesperidin down-regulated the protein expression of pro-CASP3, and up-regulated the level of active CASP3. Thus, these results suggest that hesperidin could induce apoptosis in human colon cancer cells through CASP3 activation.     

Exosomes from mesenchymal stromal cells enhance imatinib-induced apoptosis in human leukemia cells via activation of caspase signaling pathway

Background aims

Imatinib (IM), a tyrosine kinase inhibitor targeting the BCR-ABL oncoprotein, remains a major therapeutic strategy for patients with chronic myelogenous leukemia (CML). However, IM resistance is still a challenge in the treatment of CML. Recently, it was reported that exosomes (Exo) were involved in drug resistance. Therefore, the present study investigated whether Exo secreted by human umbilical cord mesenchymal stromal cells (hUC-MSC-Exo) affected the sensitivity of K562 cells to IM.

Caspase Activation Is an Early Event in Anthracycline-Induced Apoptosis and Allows Detection of Apoptotic Cells before They Are Ingested by Phagocytes

An increasing number of methods are being described to detect apoptotic cells. However, attempts to detect apoptotic cells in clinical samples are rarely successful. A hypothesis is that apoptotic cells are cleared from the circulation by phagocytosis before they become detectable by conventional morphological or cytometric methods. Using LR73 adhering cells as phagocytes in a model ofin vitrophagocytosis, we found that phagocytosis of daunorubicin (DNR)-treated U937, HL60, or K562 leukemia cell lines occurred prior to phosphatidylserine externalization, DNA hydrolysis, chromatin condensation, nuclear fragmentation, or mitochondrial potential alteration. Moreover DNR-treated K562 cells were eliminated by phagocytes while apoptosis was never observed by any of the above methods. By contrast, using a fluorometric batch analysis assay to detect caspase activity in ceramide- or DNR-treated cells (fluorogenic substrate for caspase), we found that caspase activity increased in apoptosis-committed cells before they were detected by flow cytometry or recognized by phagocytes. Similarly a caspase activity increase was detected in circulating mononuclear cells of leukemic patients 15 h after the beginning of anthracyclin treatment. We suggest that recent findings on enzymatic events (caspase activation) occurring in the early events of apoptosis must now allow the development of new markers for apoptosis, irrespective of the morphological features or internucleosomal fragmentation which are late events in apoptosis.     

Infection of epithelial cells with Chlamydia trachomatis inhibits TNF‐induced apoptosis at the level of receptor internalization while leaving non‐apoptotic TNF‐signalling intact

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of medical importance. C. trachomatis develops inside a membranous vacuole in the cytosol of epithelial cells but manipulates the host cell in numerous ways. One prominent effect of chlamydial infection is the inhibition of apoptosis in the host cell, but molecular aspects of this inhibition are unclear. Tumour necrosis factor (TNF) is a cytokine with important roles in immunity, which is produced by immune cells in chlamydial infection and which can have pro‐apoptotic and non‐apoptotic signalling activity. We here analysed the signalling through TNF in cells infected with C. trachomatis. The pro‐apoptotic signal of TNF involves the activation of caspase‐8 and is controlled by inhibitor of apoptosis proteins. We found that in C. trachomatis‐infected cells, TNF‐induced apoptosis was blocked upstream of caspase‐8 activation even when inhibitor of apoptosis proteins were inhibited or the inhibitor of caspase‐8 activation, cFLIP, was targeted by RNAi. However, when caspase‐8 was directly activated by experimental over‐expression of its upstream adapter Fas‐associated protein with death domain, C. trachomatis was unable to inhibit apoptosis. Non‐apoptotic TNF‐signalling, particularly the activation of NF‐κB, initiates at the plasma membrane, while the activation of caspase‐8 and pro‐apoptotic signalling occur subsequently to internalization of TNF receptor and the formation of a cytosolic signalling complex. In C. trachomatis‐infected cells, NF‐κB activation through TNF was unaffected, while the internalization of the TNF–TNF‐receptor complex was blocked, explaining the lack of caspase‐8 activation. These results identify a dichotomy of TNF signalling in C. trachomatis‐infected cells: Apoptosis is blocked at the internalization of the TNF receptor, but non‐apoptotic signalling through this receptor remains intact, permitting a response to this cytokine at sites of infection.     

Reprogramming of nuclear proteasomes under apoptosis induction in K562 cells I. Effect of glutathione-depleting agent diethylmaleate

In the present work, changes in the subunit composition, phosphorylation state, and enzymatic activities of 26S proteasomes undergoing programmed cell death were studied. Apoptosis in proerythroleukemic K562 cells was induced by the glutathione-depleting agent, diethylmaleate (DEM). We have shown for the first time that proteasomes isolated from the nuclei of control and apoptotic K562 cells differ in their subunit patterns, as well as in the phosphorylation state of subunits on threonine and tyrosine residues. As well, the trypsin-and chymotrypsin-like activities of nuclear proteasomes and the specificity of proteasomal nucleolysis of several individual messenger RNAs (c-fos and c-myc) were found to change under DEM action in K562 cells. DEM treatment of K562 cells led to a modification of proteasomal zeta/α5 and iota/α6 subunits associated with RNase activity. The obtained results argue in favor of changes of proteasomal subunit composition, phosphorylation state, and enzymatic activities, i.e., indicate the so-called reprogramming of the nuclear proteasome population during induced apoptosis in K562 cells.     

BCL2 and related prosurvival proteins require BAK1 and BAX to affect autophagy

It is widely thought that prosurvival BCL2 family members not only inhibit apoptosis, but also block autophagy by directly binding to BECN1/Beclin 1. To distinguish whether BCL2, BCL2L1/BCL-XL, or MCL1 influence autophagy directly, or indirectly, through their effects on apoptosis, we compared normal cells to those lacking BAX and BAK1. In cells able to undergo mitochondria-mediated apoptosis, inhibiting the endogenous prosurvival BCL2 family members induces both autophagy and cell death, but when BAX and BAK1 are deleted, neither inhibiting nor overexpressing BCL2, BCL2L1, or MCL1 causes any detectable effect on LC3B lipidation, LC3B turnover, or autolysosome formation. These results show that prosurvival BCL2 family members influence autophagy only indirectly, by inhibiting activation of BAX and BAK1.     

SfDronc,an initiator caspase involved in apoptosis in the fall armyworm Spodoptera frugiperda

Initiator caspases are the first caspases that are activated following an apoptotic stimulus, and are responsible for cleaving and activating downstream effector caspases, which directly cause apoptosis. We have cloned a cDNA encoding an ortholog of the initiator caspase Dronc in the lepidopteran insect Spodoptera frugiperda. The SfDronc cDNA encodes a predicted protein of 447 amino acids with a molecular weight of 51 kDa. Overexpression of SfDronc induced apoptosis in Sf9 cells, while partial silencing of SfDronc expression in Sf9 cells reduced apoptosis induced by baculovirus infection or by treatment with UV or actinomycin D. Recombinant SfDronc exhibited several expected biochemical characteristics of an apoptotic initiator caspase: 1) SfDronc efficiently cleaved synthetic initiator caspase substrates, but had very little activity against effector caspase substrates; 2) mutation of a predicted cleavage site at position D340 blocked autoprocessing of recombinant SfDronc and reduced enzyme activity by approximately 10-fold; 3) SfDronc cleaved the effector caspase Sf-caspase-1 at the expected cleavage site, resulting in Sf-caspase-1 activation; and 4) SfDronc was strongly inhibited by the baculovirus caspase inhibitor SpliP49, but not by the related protein AcP35. These results indicate that SfDronc is an initiator caspase involved in caspase-dependent apoptosis in S. frugiperda, and as such is likely to be responsible for the initiator caspase activity in S. frugiperda cells known as Sf-caspase-X.     

ZnPcS2P2-based photodynamic therapy induces mitochondria-dependent apoptosis in K562 cells

Mitochondria play a key role in the regulation of apoptosis induced by numerous antitumor chemotherapeutic and other toxic agents. Photodynamic therapy (PDT) exerts significant cellular killing efficacy through either an apoptotic or necrotic cell death pathway. This study investigated the mechanism underlying the killing effects of a novel amphipathic photosensitizer [di-sulfonated di-phthalimidomethy lphthalocyanine zinc (ZnPcS2P2)]-mediated photodynamic therapy (ZnPcS2P2-PDT) on K562 cells. Apoptosis was evident in the post-PDT cells through the TdT-mediated dUTP nick end labeling (TUNEL) method and DNA fragmentation assay. After ZnPcS2P2-PDT, K562 cells underwent mitochondria-dependent apoptosis as evidenced by the release of cytochrome c from mitochondria into cytosol, accompanied by mitochondrial membrane potential (A~m) reduction, indicating the opening of the mitochondrial permeability transition pore (PTP). The activities of protease from the caspase family and caspase-3 were also significantly elevated. Furthermore, ZnPcS2P2-PDT down-regulated the expression of chimaeric Bcr-Abl oncoprotein, which is the molecular hallmark of chronic myelogenous leukemia (CML).     

Galectin‐3 deficiency protects pancreatic islet cells from cytokine‐triggered apoptosis in vitro

Beta cell apoptosis is a hallmark of diabetes. Since we have previously shown that galectin‐3 deficient (LGALS3^?/?) mice are relatively resistant to diabetes induction, the aim of this study was to examine whether beta cell apoptosis depends on the presence of galectin‐3 and to delineate the underlying mechanism. Deficiency of galectin‐3, either hereditary or induced through application of chemical inhibitors, β‐lactose or TD139, supported survival and function of islet beta cells compromised by TNF‐α + IFN‐γ + IL‐1β stimulus. Similarly, inhibition of galectin‐3 by β‐lactose or TD139 reduced cytokine‐triggered apoptosis of beta cells, leading to conclusion that endogenous galectin‐3 propagates beta apoptosis in the presence of an inflammatory milieu. Exploring apoptosis‐related molecules expression in primary islet cells before and after treatment with cytokines we found that galectin‐3 ablation affected the expression of major components of mitochondrial apoptotic pathway, such as BAX, caspase‐9, Apaf, SMAC, caspase‐3, and AIF. In contrast, anti‐apoptotic molecules Bcl‐2 and Bcl‐XL were up‐regulated in LGALS3^?/? islet cells when compared to wild‐type (WT) counterparts (C57BL/6), resulting in increased ratio of anti‐apoptotic versus pro‐apoptotic molecules. However, Fas‐triggered apoptotic pathway as well as extracellular signal‐regulated kinase 1/2 (ERK1/2) was not influenced by LGALS‐3 deletion. All together, these results point to an important role of endogenous galectin‐3 in beta cell apoptosis in the inflammatory milieu that occurs during diabetes pathogenesis and implicates impairment of mitochondrial apoptotic pathway as a key event in protection from beta cell apoptosis in the absence of galectin‐3. J. Cell. Physiol. 228: 1568–1576, 2013. © 2012 Wiley Periodicals, Inc.     

Kaempferol induces apoptosis in two different cell lines via Akt inactivation,Bax and SIRT3 activation,and mitochondrial dysfunction

Kaempferol (3,4′,5,7‐tetrahydroxyflavone) is a flavonoid with anti‐ and pro‐oxidant activity present in various natural sources. Kaempferol has been shown to posses anticancer properties through the induction of the apoptotic program. Here we report that treatment of the chronic myelogenous leukemia cell line K562 and promyelocitic human leukemia U937 with 50 µM kaempferol resulted in an increase of the antioxidant enzymes Mn and Cu/Zn superoxide dismutase (SOD). Kaempferol treatment induced apoptosis by decreasing the expression of Bcl‐2 and increasing the expressions of Bax. There were also induction of mitochondrial release of cytochrome c into cytosol and significant activation of caspase‐3, and ‐9 with PARP cleavage. Kaempferol treatment increased the expression and the mitochondria localization of the NAD‐dependent deacetylase SIRT3. K562 cells stably overexpressing SIRT3 were more sensitive to kaempferol, whereas SIRT3 silencing did not increase the resistance of K562 cells to kaempferol. Inhibition of PI3K and de‐phosphorylation of Akt at Ser473 and Thr308 was also observed after treating both K562 and U937 cells with kaempferol. In conclusion our study shows that the oxidative stress induced by kaempferol in K562 and U937 cell lines causes the inactivation of Akt and the activation of the mitochondrial phase of the apoptotic program with an increase of Bax and SIRT3, decrease of Bcl‐2, release of cytochrome c, caspase‐3 activation, and cell death. J. Cell. Biochem. 106: 643–650, 2009. © 2009 Wiley‐Liss, Inc.     

Taurine and deferiprone against Al-linked apoptosis in rat hippocampus

BackgroundAluminium (Al) overload has toxic effects on multiple organ systems, especially the nervous system. Al accumulation in the brain, especially the hippocampus, is an important factor contributing to Alzheimer’s disease (AD). Deferiprone (DFP), a metal chelator, is used as a potential treatment for AD. In this study, we investigated the combined effect of taurine and DFP on Al chelation and hippocampal apoptosis in Al-exposed rats, as well as the underlying mechanisms of these effects to explore a possible therapy for AD.MethodsMale Wistar rats were divided into seven groups: negative control group (administered saline), Al-exposure group (administered AlCl₃ and saline), and five experimental groups (administered AlCl₃ and taurine, varying doses of DFP, or taurine with varying doses of DFP). After 8 weeks of treatment, the rats were sacrificed, and the terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin nick end labelling (TUNEL) assay was used to detect hippocampal apoptotic cells. Real-time quantitative PCR was used to assess the expression of the Bcl2 and Bax genes, and a western blotting assay was used to evaluate BCL2, BAX, and cleaved caspase-3 levels.ResultsCompared to the negative control group, the number of apoptotic cells in the hippocampus increased, Bcl2 expression significantly decreased, and BAX and cleaved caspase-3 levels increased in the Al-exposure group. The combination of taurine and DFP exerted a protective effect by inhibiting hippocampal cell apoptosis through the BCL2, BAX, and caspase-3 signalling pathways. Compared with the taurine-administered group, the group administered taurine with DFP showed a significantly increased Bcl2 and decreased Bax expression.ConclusionThe combination of taurine and DFP is a potential candidate for the treatment of AD induced by Al exposure.