Apoptotic effect of Bulbine Natalensis and Chlorophytum Comosum in myelogenous Leukemia K562 cell line / Efeito apoptótico da natalensia bulbina e do clorofito comoso na linhagem celular mielogênea da leucemia K562

Abstract Bulbine natalensis and Chorophytum comosum are potential medicinal source for the treatment of cancers. Chronic myeloid leukaemia is a hematopoietic stem cells disorder treated by tyrosine kinase inhibitors but often cause recurrence of the leukaemia after cessation of therapy, hence require alternative treatment. This study determines the anti-cancer effect of leaf, root and bulb methanolic and aqueous extracts of B. natalensis and C. comosum in chronic human myelogenous leukaemia (K562) cell line by MTT, Hoechst bis-benzimide nuclear and annexin V stain assays. The root methanolic extract of B. natalensis and C. comosum showed a high cytotoxicity of 8.6% and 16.7% respectively on the K562 cell line at 1,000 μg/ml concentration. Morphological loss of cell membrane integrity causing degradation of the cell and fragmentation were observed in the root methanolic extract of both plants. A high apoptosis (p < 0.0001) was induced in the K562 cells by both leaf and root extracts of the C. comosum compared to the B. natalensis. This study shows both plants possess apoptotic effect against in vitro myelogenous leukaemia which contributes to the overall anti-cancer properties of B. natalensis and C. comosum to justify future therapeutic applications against chronic myelogenous leukaemia blood cancer.

Resumo Bulbine natalensis Baker e Chorophytum comosum (Thunb.) Jacques são potenciais fontes medicinais para o tratamento de cânceres. A Leucemia Mieloide Crônica (LMC) é um distúrbio das células-tronco hematopoiéticas que é tratado com inibidores da tirosina quinase, mas frequentemente, causa recorrência da leucemia após a interrupção da terapia, portanto, requer um tratamento alternativo. Este estudo determinou o efeito anticancerígeno de extratos metanólicos e aquosos de folha, raiz e bulbo de B. natalensis e C. comosum na linhagem celular de leucemia mieloide humana crônica (K562) por ensaios de MTT, Hoechst bis-benzimida nuclear e anexina V. O extrato metanólico da raiz de B. natalensis e C. comosum apresentou alta citotoxidade de 8,6% e 16,7% respectivamente, na linhagem celular K562 com a concentração de 1,000 μg / ml. Perda morfológica da integridade da membrana celular causando degradação dos núcleos, citoplasma e encolhimento celular foi observada no extrato metanólico da raiz de ambas as plantas. Uma alta apoptose (p <0,0001) foi induzida nas células K562 por extratos de folhas e raízes de C. comosum em comparação com B. natalensis. Este estudo mostrou que ambas as plantas possuem efeito apoptótico contra leucemia mieloide in vitro que contribui para as propriedades anticâncer gerais de B. natalensis e C. comosum para justificar futuras aplicações terapêuticas contra câncer de sangue de LMC.

Apoptotic effect of Bulbine Natalensis and Chlorophytum Comosum in myelogenous Leukemia K562 cell line

Abstract Bulbine natalensis and Chorophytum comosum are potential medicinal source for the treatment of cancers. Chronic myeloid leukaemia is a hematopoietic stem cells disorder treated by tyrosine kinase inhibitors but often cause recurrence of the leukaemia after cessation of therapy, hence require alternative treatment. This study determines the anti-cancer effect of leaf, root and bulb methanolic and aqueous extracts of B. natalensis and C. comosum in chronic human myelogenous leukaemia (K562) cell line by MTT, Hoechst bis-benzimide nuclear and annexin V stain assays. The root methanolic extract of B. natalensis and C. comosum showed a high cytotoxicity of 8.6% and 16.7% respectively on the K562 cell line at 1,000 g/ml concentration. Morphological loss of cell membrane integrity causing degradation of the cell and fragmentation were observed in the root methanolic extract of both plants. A high apoptosis (p 0.0001) was induced in the K562 cells by both leaf and root extracts of the C. comosum compared to the B. natalensis. This study shows both plants possess apoptotic effect against in vitro myelogenous leukaemia which contributes to the overall anti-cancer properties of B. natalensis and C. comosum to justify future therapeutic applications against chronic myelogenous leukaemia blood cancer.

Resumo Bulbine natalensis Baker e Chorophytum comosum (Thunb.) Jacques são potenciais fontes medicinais para o tratamento de cânceres. A Leucemia Mieloide Crônica (LMC) é um distúrbio das células-tronco hematopoiéticas que é tratado com inibidores da tirosina quinase, mas frequentemente, causa recorrência da leucemia após a interrupção da terapia, portanto, requer um tratamento alternativo. Este estudo determinou o efeito anticancerígeno de extratos metanólicos e aquosos de folha, raiz e bulbo de B. natalensis e C. comosum na linhagem celular de leucemia mieloide humana crônica (K562) por ensaios de MTT, Hoechst bis-benzimida nuclear e anexina V. O extrato metanólico da raiz de B. natalensis e C. comosum apresentou alta citotoxidade de 8,6% e 16,7% respectivamente, na linhagem celular K562 com a concentração de 1,000 g / ml. Perda morfológica da integridade da membrana celular causando degradação dos núcleos, citoplasma e encolhimento celular foi observada no extrato metanólico da raiz de ambas as plantas. Uma alta apoptose (p 0,0001) foi induzida nas células K562 por extratos de folhas e raízes de C. comosum em comparação com B. natalensis. Este estudo mostrou que ambas as plantas possuem efeito apoptótico contra leucemia mieloide in vitro que contribui para as propriedades anticâncer gerais de B. natalensis e C. comosum para justificar futuras aplicações terapêuticas contra câncer de sangue de LMC.

Role of Calcium-Activated Potassium Channels in Proliferation, Migration and Invasion of Human Chronic Myeloid Leukemia K562 Cells.

Calcium-activated potassium channels (KCa) are important participants in calcium signaling pathways due to their ability to be activated by an increase in intracellular free calcium concentration. KCa channels are involved in the regulation of cellular processes in both normal and pathophysiological conditions, including oncotransformation. Previously, using patch-clamp, we registered the KCa currents in the plasma membrane of human chronic myeloid leukemia K562 cells, whose activity was controlled by local Ca2+ entry via mechanosensitive calcium-permeable channels. Here, we performed the molecular and functional identification of KCa channels and have uncovered their role in the proliferation, migration and invasion of K562 cells. Using a combined approach, we identified the functional activity of SK2, SK3 and IK channels in the plasma membrane of the cells. Selective SK and IK channel inhibitors, apamin and TRAM-34, respectively, reduced the proliferative, migratory and invasive capabilities of human myeloid leukemia cells. At the same time, the viability of K562 cells was not affected by KCa channel inhibitors. Ca2+ imaging showed that both SK and IK channel inhibitors affect Ca2+ entry and this could underlie the observed suppression of pathophysiological reactions of K562 cells. Our data imply that SK/IK channel inhibitors could be used to slow down the proliferation and spreading of chronic myeloid leukemia K562 cells that express functionally active KCa channels in the plasma membrane.

Antitumor Activity and Mechanism of Action of the Antimicrobial Peptide AMP-17 on Human Leukemia K562 Cells.

Cancer is one of the most common malignant diseases in the world. Hence, there is an urgent need to search for novel drugs with antitumor activity against cancer cells. AMP-17, a natural antimicrobial peptide derived from Musca domestica, has antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. However, its antitumor activity and potential mechanism of action in cancer cells remain unclear. In this study, we focused on evaluating the in vitro antitumor activity and mechanism of AMP-17 on leukemic K562 cells. The results showed that AMP-17 exhibited anti-proliferative activity on K562 cells with an IC50 value of 58.91 ± 3.57 µg/mL. The membrane integrity of K562 was disrupted and membrane permeability was increased after AMP-17 action. Further observation using SEM and TEM images showed that the cell structure of AMP-17-treated cells was disrupted, with depressions and pore-like breaks on the cell surface, and vacuolated vesicles in the cytoplasm. Furthermore, further mechanistic studies indicated that AMP-17 induced excessive production of reactive oxygen species and calcium ions release in K562 cells, which led to disturbance of mitochondrial membrane potential and blocked ATP synthesis, followed by activation of Caspase-3 to induce apoptosis. In conclusion, these results suggest that the antitumor activity of AMP-17 may be achieved by disrupting cell structure and inducing apoptosis. Therefore, AMP-17 is expected to be a novel potential agent candidate for leukemia treatment.

In Vitro Characterization of Inhibitors for Lung A549 and Leukemia K562 Cell Lines from Fungal Transformation of Arecoline Supported by In Silico Docking to M3-mAChR and ADME Prediction.

The search for anticancer drugs is of continuous interest. Arecoline is an alkaloid with anticancer activity. Herein, the metabolism of arecoline through fungal transformation was investigated for the discovery of potential anticancer drugs with higher activity and selectivity. Compounds 1-5 were isolated, and their structures were fully elucidated using various spectroscopic analyses, including 1D and 2D NMR, ESIMS, and HRESIMS. This is the first report for the isolation of compounds 1 and 2. An MTT assay was performed to determine the cytotoxic activity of arecoline and its metabolites in vitro using non-small-cell lung cancer A549 and leukemia K562 cell lines compared to staurosporine and doxorubicin as positive controls. For the non-small-cell lung A549 cell line, arecoline hydrobromide, staurosporine, and doxorubicin resulted in IC50 values of 11.73 ± 0.71 µM, 10.47 ± 0.64 µM, and 5.05 ± 0.13 µM, respectively, while compounds 1, 3, and 5 exhibited IC50 values of 3.08 ± 0.19 µM, 7.33 ± 0.45 µM, and 3.29 ± 0.20 µM, respectively. For the leukemia K562 cell line, the IC50 values of arecoline hydrobromide, staurosporine, and doxorubicin were 15.3 ± 1.08 µM, 5.07 ± 0.36 µM, and 6.94 ± 0.21 µM, respectively, while the IC50 values of compounds 1, 3 and 5 were 1.56 ± 0.11 µM, 3.33 ± 0.24 µM, and 2.15 ± 0.15 µM, respectively. The selectivity index value of these compounds was higher than 3. These results indicated that compounds 1, 3, and 5 are very strong cytotoxic agents with higher activity than the positive controls and good selectivity toward the tested cancer cell lines. Cell cycle arrest was then studied by flow cytometry to investigate the apoptotic mechanism. Docking simulation revealed that most compounds possessed good binding poses and favorable protein-ligand interactions with muscarinic acetylcholine receptor M3-mAChR protein. In silico study of pharmacokinetics using SwissADME predicted compounds 1-5 to be drug-like with a high probability of good oral bioavailability.

Post-Translational Modifications of Extracellular Proteasome.

The ubiquitin-proteasome system (UPS) is one of the major protein degradation pathways in eukaryotic cells. Abnormal functioning of this system has been observed in cancer and neurological diseases. The 20S proteasomes, essential components of the UPS, are present not only within the cells but also in the extracellular space, and their concentration in blood plasma has been found to be elevated and dependent upon the disease state, being of prognostic significance in patients suffering from cancer, liver diseases, and autoimmune diseases. However, functions of extracellular proteasomes and mechanisms of their release by cells remain largely unknown. The main mechanism of proteasome activity regulation is provided by modulation of their composition and post-translational modifications (PTMs). Moreover, diverse PTMs of proteins are known to participate in the loading of specific elements into extracellular vesicles. Since previous studies have revealed that the transport of extracellular proteasomes may occur via extracellular vesicles, we have set out to explore the PTMs of extracellular proteasomes in comparison to cellular counterparts. In this work, cellular and extracellular proteasomes were affinity purified and separated by SDS-PAGE for subsequent trypsinization and matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) analysis. In total, we could identify 64 and 55 PTM sites in extracellular and cellular proteasomes, respectively, including phosphorylation, ubiquitination, acetylation, and succinylation. We observed novel sites of acetylation at K238 and K192 of the proteasome subunits ß2 and ß3, respectively, that are specific for extracellular proteasomes. Moreover, cellular proteasomes show specific acetylation at K227 of α2 and ubiquitination at K201 of ß3. Interestingly, succinylation of ß6 at the residue K228 seems not to be present exclusively in extracellular proteasomes, whereas both extracellular and cellular proteasomes may also be acetylated at this site. The same situation takes place at K201 of the ß3 subunit where ubiquitination is seemingly specific for cellular proteasomes. Moreover, crosstalk between acetylation, ubiquitination, and succinylation has been observed in the subunit α3 of both proteasome populations. These data will serve as a basis for further studies, aimed at dissection of the roles of extracellular proteasome-specific PTMs in terms of the function of these proteasomes and mechanism of their transport into extracellular space.

Crotamine-like from Southern Pacific rattlesnake (Crotalus oreganus helleri) Venom acts on human leukemia (K-562) cell lines and produces ultrastructural changes on mice adrenal gland.

Crotamine is a cationic, non-enzymatic, protein integrating a minor family of myotoxins, composed of 42 amino acid residues, described in Viperidae and Crotalidae snake's families that has been used in neuroscience research, drug progressing and molecular diversity reports. Crotamine-like protein (CLP) from C.o.helleri venom was isolated in fraction 5 from 7 peaks obtained by sulfopropyl waters protein pak cationic exchange column. In tricine-SDS-PAGE under non-reduced conditions this CLP showed a single band of ~8 kDa molecular weight. CLP induced toxicity of K-562 cells with a CC50 of 11.09 µM. In mice adrenal gland after 24 h of CLP injection, cortical cells exhibited swollen mitochondria with scarce tubular cristae, some elements of smooth and rough endoplasmic reticula, widened nuclear envelope, slightly osmiophilic lipid droplets, and autophagic vacuoles. In some areas cortical cells plasma membrane and endothelial walls disappeared, which indicated a necrosis process. In other areas, endothelial cell cytoplasm did not present the normal caveolae and pinocytotic vesicles. To our knowledge, this is the first report on mice adrenal gland damages, caused by the injection of CLP from rattlesnakes. Our results propose that adrenal cortex lesions may be significant in the envenoming etiopathogenesis by CLP.

Proteomic analysis of affinity-purified extracellular proteasomes reveals exclusively 20S complexes.

Proteasome-mediated proteolysis is important for many basic cellular processes. In addition to their functions in the cell, proteasomes have been found in physiological fluids of both healthy and diseased humans including cancer patients. Higher levels of these proteasomes are associated with higher cancer burden and stage. The etiology and functions of these proteasomes, referred to as circulating, plasmatic, or extracellular proteasomes (ex-PSs), are unclear. Here we show that human cancer cell lines, as well as human endometrium-derived mesenchymal stem cells (hMESCs), release proteasome complexes into culture medium (CM). To define ex-PS composition, we have affinity purified them from CM conditioned by human leukemia cell line K562. Using matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS), we have identified core 20S proteasome subunits and a set of 15 proteasome-interacting proteins (PIPs), all previously described as exosome cargo proteins. Three of them, PPIase A, aldolase A, and transferrin, have never been reported as PIPs. The study provides compelling arguments that ex-PSs do not contain 19S or PA200 regulatory particles and are represented exclusively by the 20S complex.

Induction of GD3/α1-adrenergic receptor/transglutaminase 2-mediated erythroid differentiation in chronic myelogenous leukemic K562 cells.

The disialic acid-containing glycosphingolipid GD3 recruited membrane transglutaminase 2 (TG2) as a signaling molecule for erythroid differentiation in human chronic myelogenous leukemia (CML) K562 cells. The α1-adrenergic receptor (α1-AR)/TG2-mediated signaling pathway regulated GD3 functions, including gene expression and production, to differentiate CML K562 cells into erythroid lineage cells. Epinephrine, an AR agonist, increased membrane recruitment as well as GTP-photoaffinity of TG2, inducing GD3 synthase gene expression. Epinephrine activated PI3K/Akt signaling and GTPase downstream of TG2 activated Akt. The coupling of TG2 and GD3 production was specifically suppressed by prazosin (α1-AR antagonist), but not by propranolol (ß-AR antagonist) or rauwolscine (α2-AR antagonist), indicating α1-AR specificity. Small interfering RNA (siRNA) experiment results indicated that the α1-AR/TG2-mediated signaling pathway activated PKCs α and δ to induce GD3 synthase gene expression. Transcription factors CREB, AP-1, and NF-κB regulated GD3 synthase gene expression during α1-AR-induced differentiation in CML K562 cells. In addition, GD3 synthase gene expression was upregulated in TG2-transfected cells via α1-AR with expression of erythroid lineage markers and benzidine-positive staining. α1-AR/TG2 signaling pathway-directed GD3 production is a crucial step in erythroid differentiation of K562 cells and GD3 interacts with α1-AR/TG2, inducing GD3/α1-AR/TG2-mediated erythroid differentiation. These results suggest that GD3, which acts as a membrane mediator of erythroid differentiation in CML cells, provides a therapeutic avenue for leukemia treatment.

Synthesis of copper and zinc 2-(pyridin-2-yl)imidazo[1,2-a]pyridine complexes and their potential anticancer activity.

A small library of novel copper and zinc imidazo[1,2-a]pyridine complexes have been synthesized. Their structures were confirmed by X-ray diffraction crystallography and a selection of these compounds was tested against five cancer cell lines originating from breast cancer (MCF-7 and MDA-MB-231), leukemia (K562 and HL-60) and colorectal cancer (HT-29). The imidazo[1,2-a]pyridines and their zinc complexes showed poor anticancer activity, while the copper complexes were active against the cancer cell lines with IC50 values comparable to and lower than camptothecin. For example, copper 6-bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate 21 had an IC50 value lower than 1 µM against the HT-29 cells. Fluorescence microscopy with acridine orange, Hoechst 33342 and ethidium bromide, used in a preliminary investigation to evaluate morphological changes showed that copper 6-bromo-N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amine acetate 21 caused both apoptosis, necrosis and paraptosis in the MCF-7 and HL-60 cells. A select group of copper N-cyclohexyl-2-(pyridin-2-yl)imidazo[1,2-a]pyridin-3-amines (26, 27, 29 and 31) induced apoptosis, paraptosis and deformed nuclei in MCF-7 cells.

The proteomics research of 4-amino-2-trifluoromethyl-phenyl retinate on human leukemia K562 cells / 中国药理学通报

Aim To explore the proteomics mechanism of the differentiation induction effect of 4-amino-2-trif-luoromethyl-phenyl retinate(ATPR)on human leukemi-a K562 cells. Methods Human leukemia K562 cells were incubated with the same concentration (1 × 10 - 6 mol·L - 1 ) of ATPR or ATRA for 48 hours. The total cell proteins were collected, purified and digested by trypsin, solid phase extraction, and the peptides were detected by ESI-LC-MS / MS. The difference of the pro-tein expression between the cells treated with ATPR and ATRA was compared by using the Discoverer Pro-teome 1. 2 software, and the molecular function, the biological process and other information of those pro-teins were analyzed based on the DAVID, KEGG, STRING databases. Results 120 specific proteins were identified only in the ATPR group, 143 only in the ATRA group, and 422 other proteins in both groups. Results of DAVID analysis showed that ATPR-induced specific proteins were mainly involved in 39 biological processes of proteins and macromolecules metabolism, protein transport and localization and so on. Results of KEGG analysis revealed that ATPR-in-duced proteins participated in signal pathways, mainly metabolic pathways, PI3K-Akt signal pathway, TGF-beta signal pathway and other pathways in cancer. String protein interaction network analysis displayed that ATPR-induced proteins, like EIF3A, EIF6, RPL3, RPL8, RPL13, RPL7A, RPL21, RPS3, RPS14, NACA, BTF3, NHP2L1, PPP2CA proteins had direct interactions with more than or equal to 10 associated proteins. Conclusion The differentiation induction effect of ATPR on K562 cells might be as-cribed to the ATPR-induced proteins interaction net-work and the specific central proteins it induced, which are involved in the regulation of cell prolifera-tion, differentiation and apoptosis.

The role of Beclin 1 in SDT-induced apoptosis and autophagy in human leukemia cells.

PURPOSE: To prove the occurrence of autophagy after treatment by protoporphyrin IX (PpIX)-mediated sonodynamic therapy (SDT) of human chronic myelogenous leukemia K562 cells as well as its relationship with apoptosis. MATERIALS AND METHODS: The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenylter-trazolium bromide tetrazolium (MTT) assay was adopted to examine cytotoxicity of different treatments. Nuclear morphology changes were observed under a fluorescence microscopy with 4'-6-Diamidino-2-Phenylindole (DAPI) staining. Western blotting was used to analyze the expression of caspase-3, Beclin 1 (BECN 1) and the conversion of LC3- phosphatidylethanolamine conjugate/a cytosolic form of LC3 (LC3 II/I). Fluorescence microscope was used to identify the formation of autophagic vacuoles (AVO) during autophagy. RESULTS: Under optimal conditions, SDT was shown to induce autophagy in K562 cells, which caused the up-regulation of Beclin-1 and the formation of AVO. In addition, pre-treatment of cancer cells with Beclin 1-targeted short hairpin RNA (Beclin 1 shRNA) was shown to reduce the level of LC3-II accumulation and staining with punctate spots of monodansylcadaverine (MDC) staining. Besides, the cytotoxic effect of SDT was significantly increased by Beclin 1 shRNA. Furthermore, studies showed a marked effect on the apoptosis of cells by Beclin 1 shRNA to sonodamage with increased DAPI staining and caspase-3 cleavage. CONCLUSIONS: These results demonstrated that SDT significantly induced autophagy of K562 cells, probably to protect the K562 cells from sonodamage.

Sonodynamic therapy induces the interplay between apoptosis and autophagy in K562 cells through ROS.

Sonodynamic therapy (SDT) is a relatively new approach in the treatment of various cancers including leukemia cells. The aim of this study is to investigate the occurrence of apoptosis and autophagy after treated by protoporphyrin IX (PpIX)-mediated SDT (PpIX-SDT) on human leukemia K562 cells as well as the relationship between them. Firstly, mitochondrial-dependent apoptosis was observed through morphological observation and biochemical analysis. Meanwhile, SDT was shown to induce autophagy in K562 cells, which caused an increase in EGFP-LC3 puncta cells, a conversion of LC3 II/I, formation of acidic vesicular organelles (AVOs) and co-localization between LC3 and LAMP2 (a lysosome marker). Besides, pretreatment with autophagy inhibitor 3-MA or bafilomycin A1 was shown to provide protection against autophagy and to enhance SDT-induced apoptosis and necrosis, while the apoptosis suppressor z-VAD-fmk failed to affect formation of autophagic vacuoles or partially prevented SDT-induced cytotoxicity, which suggested that SDT-induced autophagy functioned as a survival mechanism. Additionally, this study reported apparent apoptosis and autophagy with dependence on intracellular reactive oxygen species (ROS) production. Preliminary data showed that ROS scavenger N-acetylcysteine (NAC) effectively blocked the SDT induced accumulation of ROS, reversed sono-damage, cell apoptosis and autophagy. Taken together, these data indicate that autophagy may be cytoprotective in our experimental system, and the ROS caused by PpIX-SDT treatment may play an important role in initiating apoptosis and autophagy.

Protoporphyrin IX-mediated sonodynamic action induces apoptosis of K562 cells.

OBJECTIVES: The present study aims to investigate apoptosis of human leukemia K562 cells induced by protoporphyrin IX (PpIX)-mediated sonodynamic therapy (PpIX-SDT). METHODS: The uptakes of intracellular PpIX in K562 cells were detected by flow cytometry. The sub-cellular localization of PpIX was imaged by confocal microscope. The cytotoxic effect of PpIX-SDT was assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenylter-trazolium bromide tetrazolium) assay. Apoptosis was evaluated by chromatin condensation with DAPI (4'-6-diamidino-2-phenylindole) staining, decrease of mitochondria membrane potential (MMP), re-distribution of Bax, and the expression changes of the key apoptosis-associated protein (Caspase-3 and polypeptide poly (ADP-robose) polymerase). The possible mechanism of SDT-induced apoptosis was investigated by detecting by intracellular ROS (reactive oxygen species) generation and effect of ROS scavenger-NAC (N-acetylcysteine) on SDT induced apoptosis. RESULTS: The intracellular PpIX increased quickly within 2 h after PpIX administration and PpIX mainly localized in the mitochondria. Compared with PpIX alone and ultrasound alone groups, the synergistic cytotoxicity of PpIX plus ultrasound was significantly boosted. In addition, the ultrasound induced some extent of chromatin condensation and MMP loss was greatly enhanced by the presence of 2 µg/ml PpIX, where PpIX alone treatment showed no or only slight effect. Time-dependent Bax translocation, caspase-3 activation and PARP cleavage were detected in SDT treatment groups. Besides, intracellular ROS production was significantly enhanced after SDT, and the general ROS scavenger NAC could obviously alleviate the SDT-caused cell viability loss, MMP loss, Bax redistribution and nuclear changes. CONCLUSIONS: These results indicated that PpIX-mediated sonodynamic action could induce apoptosis on K562 cells, and the intracellular ROS was involved in the PpIX-SDT induced apoptosis.

A new disposable electrode for electrochemical study of leukemia K562 cells and anticancer drug sensitivity test.

Developing cost-effective and simple analysis tools is of vital importance for practical applications in bioanalysis. In this work, a new disposable electrochemical cell sensor with low cost and simple fabrication was proposed to study the electrochemical behavior of leukemia K562 cells and the effect of anticancer drugs on cell viability. The analytical device was integrated by using ITO glass as the substrate of working electrodes and paper as the electrolytic cell. The cyclic voltammetry of the K562 cells at the disposable electrode exhibited an irreversible anodic peak and the peak current is proportional to the cell number. This anodic peak is attributed to the oxidation of guanine in cells involving two protons per transfer of two electrons. For the drug sensitivity tests, arsenic trioxide and cyclophosphamide were added to cell culture media. As a result, the electrochemical responses of the K562 cells decreased significantly. The cytotoxicity curves and results obtained corresponded well with the results of CCK-8 assays. In comparison to conventional methods, the proposed method is simple, rapid and inexpensive. More importantly, the developed sensor is supposed to be a single-use disposable device and electrodes were prepared "as new" for each experiment. We think that such disposable electrodes with these characteristics are suitable for experimental study with cancer cells or other types of pathogens for disease diagnosis, drug selection and on-site monitoring.

Construction,Identification and Expression of Recombinant Eukaryotic Vector pCAG-IRES-SHIP-GFP on Porliferation of Leukemia Cell Line K562 / 中国生物工程杂志

The aim was to construct and identify the mammalian expression vector of pCAG-IRES-SHIP-GFP and to detect whether it could express in human acute leukemia cell line K562.The cDNA fragment of SHIP obtained by RT-PCR was inserted into pCAG-IRES-GFP.The recombinant plasmid was confirmed by restriction enzyme digesiton,PCR and DNA sequecing.pCAG-IRES-SHIP-GFP was transfected into K562 cells with lipofectamine 2000.The expression of SHIP was determined by GFP fluorescence and Western blot analysis.FQ-PCR was used to quantitate SHIP mRNA.The expression of p-Akt,Akt were determined by Western blot.PI were tested by flow cytometry and MTT to verify whether exogenous SHIP could inhibit proliferation of K562 cells.The results showed that the correct constrution of the recombinant plasmid pCAG-IRES-SHIP-GFP has been shown by restriction enzyme digestion,PCR and DNA sequencing.pCAG-IRES-SHIP-GFP could express SHIP protein in K562 cells.The K562 cells viability after transfected with SHIP gene droped down.Western blot analysis showed that phospha-Akt308 and Akt473 were reduced to 38.7% and 68% respectively.It was concluded that the vector of pCAG-IRES-SHIP-GFP has been successfully constructed and it can be expressed in K562 cells.The expression of exogenous SHIP gene can lead to apoptosis of K562 cells by down-regulating the p-Akt expression.What found here might be one of the mechanisms involved in the pathogenesis of leukemia.

Apoptosis of Human Leukemia K562 Cell in vitro Induced by Toxoplasma gondii / 中国寄生虫学与寄生虫病杂志

Objective To investigate whether the Toxoplasma gondii can inhibit proliferation of human leukemia K562 cells and/or induce apoptosis of the cells in vitro. Methods K562 cells (5?104/ml) were harvested at mid-ex-ponential phase and planted in 96 well plates with 100 ?l each and in 50 ml culture bottles, 1.5 ml each. The cells were treated for 48 hours with different concentration of Toxoplasma tachyzoites. Growth inhibition rate was measured with MTT method. Apoptosis was detected through following ways: fluorescence microscopy with Hoechst 33 258 staining was used for observing the change of cell morphology, agarose electrophoresis was used to detect the DNA changes and FCM was used to observe sub-diploid. Results Toxoplasma can inhibit proliferation of K562 cells. K562 cells treated with Toxoplasma presented an inhibition rate of 17%, 28%, 48%, 50% and 55% under the tachyzoite concentration of 1?104, 2?104, 4?104, 8?104 and 16?104/ml respectively, with a significant difference to the control (t=3.606, 5.918, P