Human Leukemia K562 Cells Research Articles

DABCO-catalyzed sp3 C–H activation: rapid access to isoxazole or coumarin-fused 3-quaternary carbon oxindoles and isoxazole-fused pyrrolidinones

A facile and efficient methodology was developed for the synthesis of isoxazole or coumarin-fused 3-quaternary carbon oxindoles and isoxazole-fused pyrrolidinones via DABCO-catalyzed sp3 C–H activation of 5-methyl-isoxazole or 4-methylcoumarin. Furthermore, the biological activity of the isoxazole or coumarin-fused 3-quaternary carbon oxindoles 3 and 5 has been preliminarily demonstrated by in vitro evaluation against human prostate cancer cells PC-3 and human leukemia cells K562 by the MTT-based assays using the commercially available standard drug Cisplatin as a positive control. These results suggested that most of the compounds 3 and 5 showed considerable cytotoxicities to these two cell lines K562 and PC-3, and a methyl or an ethyl group of acrylates and an oxindole moiety located in the isoxazole-fused 3-quaternary carbon oxindoles 3 are beneficial for the activity. In addition, the activity of all the afforded isoxazole-fused pyrrolidinones 7 were also evaluated against Gram-positive bacteria Staphylococcus aureus (MTCC96) and Gram-negative bacteria Escherichia coli (ATCC25835) using Penicillin as a standard drug for Gram-positive organism or Streptomycin as standard drug for Gram-negative organism. The results demonstrated that most of the compounds 7 had comparable in vitro inhibitory activity against Gram-positive bacteria Staphylococcus aureus (MTCC96) with the positive control Penicillin. The results also indicated that isoxazole-fused pyrrolidinone analogs had significantly better inhibition ability against Gram-positive bacteria Staphylococcus aureus (MTCC96) than against Gram-negative bacteria Escherichia coli (ATCC25835).

A facile and efficient synthesis of polycyclic spiropyrrolidine oxindoles bearing mesityl oxide unit via a three-component 1,3-dipolar cycloaddition reaction

A facile and efficient methodology was developed for the synthesis of multifunctional polycyclic spiropyrrolidine oxindoles bearing mesityl oxide unit via a three-component 1,3-dipolar cycloaddition reaction of dienones 2 with azomethine ylides (thermally generated in situ from isatin derivatives and proline or thioproline). Products bearing adjacent four chiral carbon centers were smoothly obtained in high yields (up to 98% yield) with diastereoselectivities up to >20:1. Furthermore, the key structural characteristic of the products of such a reaction is the natural product ingredient turmerone motif fused at the 3-position of the spirooxindole core, with varying degrees of substitution around it. In addition, their biological activity has been preliminarily demonstrated by in vitro evaluation against human prostate cancer cells PC-3, human lung cancer cells A549 and human leukemia cells K562 by the MTT-based assays, using the commercially available standard drugs Cisplatin as a positive control. These results suggested there is a trend that lipophilicity groups improve the potency, and also suggested an mesityl oxide moiety located in the polycyclic spiropyrrolidine oxindoles is beneficial for the activity. The results also demonstrated that most of the compounds showed considerable cytotoxicities to these three cell lines K562, A549 and PC-3, and indicated that polycyclic spiropyrrolidine oxindole analogs bearing mesityl oxide unit may be potential leads for further antitumor activity screenings.

Matrine suppresses the growth of human chronic myeloid leukemia K562 cells via inhibiting bcr-abl-mediated MEK-ERK pathway

Objective To investigate the mechanism of matrine in inhibition of proliferation the proliferation of human chronic myeloid leukemia (CML) K562 cells via MEK-ERK signaling pathway. Methods Western blot was used to detect the expression of MEK1, ERK1/2, Shc and SHP2 (the signal effect molecules of MEK-ERK pathway) in K562 cells. The transcription and translation of bcr-abl and target protein (bcl-xL, Cyclin D1, c-myc and p27) were detected by RT-PCR and Western blot. Results Matrine was able to significantly inhibit the phosphorylation of MEK1, ERK1/2, Shc and SHP2 in K562 cells and suppress the protein and mRNA expression of bcr-abl. Moreover, the expressions of bcl-xL, Cyclin D1 and c-myc were down-regulated significantly, while the expression level of p27 (a negative regulator of cell cycle progression) was increased markedly after matrine treatment. Conclusions Suppression of the growth of human CML K562 cells is related to the inhibition of bcr-abl-mediated MEK-ERK pathway activity. The down-regulation of phosphorylated proteins or protein kinases activity in signaling pathways might be an important molecular mechanism in control the activity of MEK-ERK pathway. Key words: Matrine; Leukemia, myeloid, chronic; MEK-ERK signaling pathway; bcr-abl

Induction of Mitochondrial Dependent Apoptosis in Human Leukemia K562 Cells by Meconopsis integrifolia: A Species from Traditional Tibetan Medicine.

Objectives: Meconopsis integrifolia (M. integrifolia) is one of the most popular members in Traditional Tibetan Medicine. This study aimed to investigate the anticancer effect of M. integrifolia and to detect the underlying mechanisms of these effects. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and trypan blue assay were used to evaluate the cytotoxicity of M. integrifolia. Changes in cell nuclear morphology and reactive oxygen species (ROS) level were observed by fluorescent microscopy. Apoptosis ratio, DNA damage and mitochondrial membrane potential (MMP) loss were analyzed by flow cytometry. Western blotting assay was adopted to detect the proteins related to apoptosis. Immunofluorescence was used to observe the release of cytochrome C. Results: The obtained data revealed that M. integrifolia could significantly inhibit K562 cell viability, mainly by targeting apoptosis induction and cell cycle arrest in G2/M phase. Collapse in cell morphology, chromatin condensation, DNA damage and ROS accumulation were observed. Further mechanism detection revealed that mitochondrion might be a key factor in M. integrifolia-induced apoptosis. Conclusions: M. integrifolia could induce mitochondria mediated apoptosis and cell cycle arrest in G2/M phase with little damage to normal cells, suggesting that M. integrifolia might be a potential and efficient anticancer agent that deserves further investigation.

Loss of mitochondrial transmembrane potential and glutathione depletion are not sufficient to account for induction of apoptosis by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone in human leukemia K562 cells

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), an uncoupler of mitochondrial oxidative phosphorylation, inhibits cell proliferation and induces cell death with apoptotic features. It was reported that the cytotoxic effects of FCCP are preceded by a rapid glutathione (GSH) depletion with a subsequent loss of mitochondrial transmembrane potential (ΔΨ). The GSH depletion was suggested as the cause of apoptosis in FCCP treated cells. This conclusion was further supported by the finding that all adverse effects of FCCP including cell death can be prevented by N-acetylcysteine (NAC) a precursor of GSH synthesis (Han and Park, 2011).Here, we argue that neither loss of ΔΨ nor GSH depletion is sufficient to account for induction of apoptosis in FCCP treated leukemia K562 cells. Indeed, the lowest concentration of FCCP that brings about the permanent loss of ΔΨ and the extensive decrease in GSH level induces cell death in minor population of cells. Only much higher concentrations of FCCP, that exceed the range to achieve permanent collapse of ΔΨ, induce extensive apoptosis. The low proapoptotic activity of FCCP could be explained by hyperactivation of protein kinase B/Akt.A detailed LC/MS/MS analysis of cell extracts revealed extensive formation of FCCP adducts with GSH. This effect could explain the mechanism of GSH depletion, which is currently unknown. Although NAC induces an increase in the GSH pool, this effect is not crucial for abrogation of FCCP cytotoxicity. Indeed, the presence of NAC in the growth medium causes a rapid clearance of FCCP due to its quantitative conversion into the FCCP–NAC adduct, which is the real cause of abrogated FCCP cytotoxicity.

Induction of Apoptosis and Antitumor Activity of Eel Skin Mucus, Containing Lactose-Binding Molecules, on Human Leukemic K562 Cells.

For innate immune defense, lower animals such as fish and amphibian are covered with skin mucus, which acts as both a mechanical and biochemical barrier. Although several mucus sources have been isolated and studied for their biochemical and immunological functions, the precise mechanism(s) of action remains unknown. In the present study, we additionally found the eel skin mucus (ESM) to be a promising candidate for use in anti-tumor therapy. Our results showed that the viability of K562 cells was decreased in a dose-dependent manner by treatment with the isolated ESM. The cleaved forms of caspase-9, caspase-3 and poly adenosine diphosphate-ribose polymerase were increased by ESM. The levels of Bax expression and released cytochrome C were also increased after treatment with ESM. Furthermore, during the ESM mediated-apoptosis, phosphorylation levels of ERK1/2 and p38 but not JNK were increased and cell viabilities of the co-treated cells with ESM and inhibitors of ERK 1/2 or p38 were also increased. In addition, treatment with lactose rescued the ESM-mediated decrease in cell viability, indicating lactose-containing glycans in the leukemia cells acted as a counterpart of the ESM for interaction. Taken together, these results suggest that ESM could induce mitochondria-mediated apoptosis through membrane interaction of the K562 human leukemia cells. To the best of our knowledge, this is the first observation that ESM has anti-tumor activity in human cells.

Synergistic effect of HMGB1 knockdown and cordycepin in the K562 human chronic myeloid leukemia cell line.

The high-mobility group box 1 (HMGB1) protein is a DNA-binding nuclear protein, which is overexpressed in leukemia cells. Cordycepin is characterized by strong antileukemic properties and is regarded as an effective natural compound for leukemia therapy. The aim of the present study was to investigate the impact of HMGB1 knockdown and cordycepin treatment on proliferation, apoptosis, reactive oxygen species (ROS) levels and adhesion of K562 human chronic myeloid leukemia cells. The Cell Counting kit‑8 assay was used to determine the proliferation of K562 cells. The cell cycle and apoptosis of K562 cells was determined using flow cytometric analysis. In addition, a cell adhesion assay was performed. Western blotting was used to determine the protein expression of cyclooxygenase 2, Bax, receptor for advanced glycation end-products and Bcl‑2. The data collected demonstrated that HMGB1 knockdown combined with cordycepin treatment had significant anti‑proliferative and pro‑apoptotic effects. In addition, it increased the ROS levels and reduced the adhesion of K562 cells. It was also identified that HMGB1 knockdown had synergistic effects with cordycepin, which aided in accelerating apoptosis, and inhibiting proliferation and adhesion in chronic myeloid leukemia cells. These results indicated that HMGB1 may be used as a potential therapeutic target, with cordycepin having potential as an auxiliary drug. Therefore, it is suggested that HMGB1 knockdown and corycepin treatement may present a promising therapeutic strategy for leukemia.

A derivative of epigallocatechin-3-gallate induces apoptosis via SHP-1-mediated suppression of BCR-ABL and STAT3 signalling in chronic myelogenous leukaemia.

Epigallocatechin-3-gallate (EGCG) is a component of green tea known to have chemo-preventative effects on several cancers. However, EGCG has limited clinical application, which necessitates the development of a more effective EGCG prodrug as an anticancer agent. Derivatives of EGCG were evaluated for their stability and anti-tumour activity in human chronic myeloid leukaemia (CML) K562 and KBM5 cells. EGCG-mono-palmitate (EGCG-MP) showed most prolonged stability compared with other EGCG derivatives. EGCG-MP exerted greater cytotoxicity and apoptosis in K562 and KBM5 cells than the other EGCG derivatives. EGCG-MP induced Src-homology 2 domain-containing tyrosine phosphatase 1 (SHP-1) leading decreased oncogenic protein BCR-ABL and STAT3 phosphorylation in CML cells, compared with treatment with EGCG. Furthermore, EGCG-MP reduced phosphorylation of STAT3 and survival genes in K562 cells, compared with EGCG. Conversely, depletion of SHP-1 or application of the tyrosine phosphatase inhibitor pervanadate blocked the ability of EGCG-MP to suppress phosphorylation of BCR-ABL and STAT3, and the expression of survival genes downstream of STAT3. In addition, EGCG-MP treatment more effectively suppressed tumour growth in BALB/c athymic nude mice compared with untreated controls or EGCG treatment. Immunohistochemistry revealed increased caspase 3 and SHP-1 activity and decreased phosphorylation of BCR-ABL in the EGCG-MP-treated group relative to that in the EGCG-treated group. EGCG-MP induced SHP-1-mediated inhibition of BCR-ABL and STAT3 signalling in vitro and in vivo more effectively than EGCG. This derivative may be a potent chemotherapeutic agent for CML treatment.

Role of 5-aminolevulinic acid-conjugated gold nanoparticles for photodynamic therapy of cancer.

There are three possible mechanisms for 5-aminolevulinic acid (5-ALA) conjugated gold nanoparticles (GNPs) through electrostatic bonding for photodynamic therapy (PDT) of cancer: GNPs delivery function,singlet oxygen generation (SOG) by GNPs irradiated by light, and surface resonance enhancement (SRE) of SOG. Figuring out the exact mechanism is important for further clinical treatment. 5-ALA-GNPs and human chronic myeloid leukemia K562 cells were used to study delivery function and SOG by GNPs. The SRE of SOG enabled by GNPs was explored by protoporphyrin IX (PpIX)-GNPs conjugate through electrostatic bonding.Cell experiments show that the GNPs can improve the efficiency of PDT, which is due to the vehicle effect of GNPs. PpIX–GNPs conjugate experiments demonstrated that SOG can be improved about 2.5 times over PpIX alone. The experiments and theoretical results show that the local field enhancement (LFE) via localized surface plasmon resonance (LSPR) of GNPs is the major role; the LFE was dependent on the irradiation wavelength and the GNP's size. The LFE increased with an increase of the GNP size (2R ≤ 50 nm). However, the LSPR function of the GNPs was not found in cell experiments. Our study shows that in 5-ALA-conjugated GNPs PDT, the delivery function of GNPs is the major role.

Yessotoxin activates cell death pathways independent of Protein Kinase C in K-562 human leukemic cell line

Protein Kinase C (PKC) is a group of enzymes involved in pro-survival or pro-apoptotic events depending on the cellular model. Moreover, Yessotoxin (YTX) modulates its expression and activates different cell death pathways. In K-562 tumor cell line, YTX induces apoptosis and autophagy after 24 and 48h of incubation, respectively, and the toxin carries out its action through the phosphodiesterase 4A (PDE4A). Therefore, the levels of two subtypes of PKC, conventional (cPKC) and δ isotype of novel PKC (PKCδ) were studied at these times after YTX incubation. Also their involvement in the cell death activated by the toxin and their relationship with PDE4A was checked. The expression of cPKC and PKCδ in cytosol, plasma membrane and nucleus was studied in normal and PDE4A-silenced cells. Furthermore, cell viability of normal cells, as well as cPKC-, PKCδ- and PDE4A-silenced cells was tested by Lactate Dehydrogenase (LDH) assay. As a result, PKCδ showed a key role in K-562 cell survive, since without this protein, K-562 cell decreased their viability. Furthermore, modulation of PKCs by YTX treatment was observed, however, the changes in the expression of these proteins are independent of cell death activated by the toxin. In addition, the modulation of PKCs detected is PDE4A-dependent, since the silencing of this protein change PKC expression pattern.

485. Targeted Epigenome Editing by CRISPR/Cas9-Based Repressors for Silencing of Distal Regulatory Elements

The CRISPR/Cas9 genome engineering platform has emerged as a powerful tool for gene regulation. Catalytically inactivated, “dead” Cas9 (dCas9) can be fused to a variety of transcriptional modulators to activate or silence gene expression. The KRAB domain, a common heterochromatin-forming motif in naturally occurring zinc finger transcription factors, has been genetically linked to dCas9 to create an RNA-guided synthetic repressor. KRAB-based synthetic repressors can effectively silence the expression of single genes and have been used to repress oncogenes, inhibit viral replication, and treat dominant negative diseases. However, the specificity of silencing endogenous genes and the distance of heterochromatin formation catalyzed by dCas9-KRAB repressors have not been studied. Understanding the precision of dCas9-KRAB repressors is critical to their further therapeutic application. As a model system to investigate the epigenetic consequences of KRAB-based silencing, dCas9-KRAB was targeted to the HS2 enhancer of the globin locus in the K562 human erythroid leukemia cell line. Enhancers are regulatory elements characterized by open chromatin that can activate distal genes targets. Genetic variation in enhancer sequences has been linked to many disease states, including cancer and cardiovascular disease. The HS2 enhancer is a potent activator of the developmentally regulated globin genes, which are involved in sickle cell anemia and beta thalassemia. We demonstrated that dCas9-KRAB targeted to HS2 by a single guide RNA (sgRNA) can disrupt the expression of multiple downstream globin genes. These findings expand the capabilities of synthetic repressors by showing that dCas9-KRAB targeted to a distal regulatory element can silence expression of multiple genes located 10 to 50 kb away. Genome-wide analysis of DNA-binding and mRNA expression by ChIP-seq and RNA-seq, respectively, established that RNA-guided synthetic repressors were highly specific for targeting and silencing the globin genes. To further investigate the utility of dCas9-KRAB repressors as a targeted epigenetic modifier, we performed ChIP-seq for tri-methylation of H3K9, a mark of heterochromatin, and DNase I hypersensitivity-sequencing (DNase-seq), a genome-wide measure of chromatin accessibility. dCas9-KRAB repressors induced H3K9 methylation at the HS2 enhancer. Off-target H3K9 methylation events were minimal and generally depended on the sgRNA. Reduced DNase I hypersensitivity was also observed at the enhancer region and its promoter targets, indicating that dCas9-KRAB repressors can modulate the chromatin structure of active enhancers to affect the activity of downstream target genes. These studies establish that dCas9-KRAB repressors can interfere with distal enhancer activity by highly specific remodeling of the epigenetic state of targeted loci. Enhancers play critical roles in differentiation, development, and disease, and understanding the epigenetic implications of targeting engineered repressors to distal regulatory elements will inform further use of genome engineering technologies for therapeutic applications.

Long noncoding RNA, CCDC26, controls myeloid leukemia cell growth through regulation of KIT expression.

BackgroundAccumulating evidence suggests that some long noncoding RNAs (lncRNAs) are involved in certain diseases, such as cancer. The lncRNA, CCDC26, is related to childhood acute myeloid leukemia (AML) because its copy number is altered in AML patients.ResultsWe found that CCDC26 transcripts were abundant in the nuclear fraction of K562 human myeloid leukemia cells. To examine the function of CCDC26, gene knockdown (KD) was performed using short hairpin RNAs (shRNAs), and four KD clones, in which CCDC26 expression was suppressed to 1% of its normal level, were isolated. This down-regulation included suppression of CCDC26 intron-containing transcripts (the CCDC26 precursor mRNA), indicating that transcriptional gene suppression (TGS), not post-transcriptional suppression, was occurring. The shRNA targeting one of the two CCDC26 splice variants also suppressed the other splice variant, which is further evidence for TGS. Growth rates of KD clones were reduced compared with non-KD control cells in media containing normal or high serum concentrations. In contrast, enhanced growth rates in media containing much lower serum concentrations and increased survival periods after serum withdrawal were observed for KD clones. DNA microarray and quantitative polymerase chain reaction screening for differentially expressed genes between KD clones and non-KD control cells revealed significant up-regulation of the tyrosine kinase receptor, KIT, hyperactive mutations of which are often found in AML. Treatment of KD clones with ISCK03, a KIT-specific inhibitor, eliminated the increased survival of KD clones in the absence of serum.ConclusionsWe suggest that CCDC26 controls growth of myeloid leukemia cells through regulation of KIT expression. A KIT inhibitor might be an effective treatment against the forms of AML in which CCDC26 is altered.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0364-7) contains supplementary material, which is available to authorized users.

Wogonin induces apoptosis and endoplasmic reticulum stress in HL-60 leukemia cells through inhibition of the PI3K-AKT signaling pathway.

Wogonin is a flavonoid isolated from Scutellaria baicalensis root and has multiple pharmacological effects, including anticancer effects. Recent studies have shown that wogonin induces cell cycle arrest and reverses multi-drug resistance in the human K562 leukemia cell line. However, its pharmacological function in the apoptosis of leukemia cells remains unknown. Therefore, we hypothesized that wogonin can induce apoptosis in the HL-60 leukemia cell line. In the present study, the HL-60 cells were treated with different doses of wogonin (0-150 µM). Wogonin inhibited the viability of HL-60 cells in a dose-dependent and time-dependent manner. Flow cytometry and analyses of caspase and PARP-1 activation and the Bax/Bcl-2 ratio, demonstrated that the cytotoxic effect of wogonin on HL-60 cells was mediated by caspase-dependent and mitochondrial-dependent apoptosis. Wogonin also induced the expression of certain members of the endoplasmic reticulum (ER) stress pathway (CHOP, GRP94 and GRP78) and the activation of multiple branches of ER stress transducers (IRE1α, PERK-eIF2α and ATF6) in the HL-60 cells. In addition, wogonin reduced the phosphorylation of PI3K and AKT in the HL-60 cells. Furthermore, constitutive activation of AKT induced by adenoviral vectors inhibited the pro-apoptotic effects and ER stress induced by wogonin in the HL-60 cells. In summary, our results indicated that wogonin induced apoptosis and ER stress in HL-60 cells, which was mediated by the inhibition of the PI3K-AKT signaling pathway.

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 4ʹ-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.

Identification of a novel E-box binding pyrrole-imidazole polyamide inhibiting MYC-driven cell proliferation.

The MYC transcription factor plays a crucial role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Due to its oncogenic activities and overexpression in a majority of human cancers, it is an interesting target for novel drug therapies. MYC binding to the E-box (5′-CACGTGT-3′) sequence at gene promoters contributes to more than 4000 MYC-dependent transcripts. Owing to its importance in MYC regulation, we designed a novel sequence-specific DNA-binding pyrrole–imidazole (PI) polyamide, Myc-5, that recognizes the E-box consensus sequence. Bioinformatics analysis revealed that the Myc-5 binding sequence appeared in 5′- MYC binding E-box sequences at the eIF4G1, CCND1, and CDK4 gene promoters. Furthermore, ChIP coupled with detection by quantitative PCR indicated that Myc-5 has the ability to inhibit MYC binding at the target gene promoters and thus cause downregulation at the mRNA level and protein expression of its target genes in human Burkitt's lymphoma model cell line, P493.6, carrying an inducible MYC repression system and the K562 (human chronic myelogenous leukemia) cell line. Single i.v. injection of Myc-5 at 7.5 mg/kg dose caused significant tumor growth inhibition in a MYC-dependent tumor xenograft model without evidence of toxicity. We report here a compelling rationale for the identification of a PI polyamide that inhibits a part of E-box-mediated MYC downstream gene expression and is a model for showing that phenotype-associated MYC downstream gene targets consequently inhibit MYC-dependent tumor growth.

A microfluidic chip with hydrodynamic traps for in vitro microscopic investigations of single cells

The results on making a microfluidic chip for in vitro microscopic investigations of single cells are presented. Numerical simulation of the motion trajectories of microparticles makes it possible to determine the geometry of hydrodynamic traps, their number, and the trap arrangement in a reaction chamber. According to the developed design, microfluidic chips were fabricated from a SU-8 photoresist by photolithography. The microfluidic chips have been tested to prove their operating capacity for isolating and holding K562 human myeloid leukemia cells from a sample flow and their subsequent investigation by confocal laser scanning microscopy.

Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression.

Molecular design, synthesis and biological research of novel pyridyl acridones as potent DNA-binding and apoptosis-inducing agents

A series of novel pyridyl acridone derivatives comprised of a pseudo-five-cyclic system to extend the π-conjugated acridone chromophore, were designed and synthesized as potent DNA binding antitumor compounds. Most synthesized compounds displayed good activity against human leukemia K562 cells in MTT tests, with compound 6d exhibiting the highest activity with IC50 value at 0.46 μM. Moreover, 6d showed potent activities against solid tumor cell lines (0.16–3.79 μM). Several experimental studies demonstrated that the antitumor mode of action of compound 6d involves DNA intercalation, topoisomerase I inhibition, and apoptosis induction through the mitochondrial pathway. In summary, compound 6d represents a novel and promising lead structure for the development of new potent anticancer DNA-binding agents.

Anti-CD20 antibody induces the improvement of cytokine-induced killer cell activity via the STAT and MAPK/ERK signaling pathways.

There is a current requirement for novel therapeutic strategies for the treatment of hematopoietic tumors. Residual tumor cells are the main origin of tumor relapse. The aim of this study was to eliminate the residual tumor cells of hematopoietic tumors. Cytokine-induced killer (CIK) cells are used in immunotherapy to deplete the residual cells. However, it is necessary to increase the antitumor activity and clinical applicability of CIK cells. The present study investigated the antitumor activity of CIK cells to the SU-DHL2 human B-cell lymphoma and K562 human chronic myelogenous leukemia cell lines. CD3+CD56+ cells from healthy donors were expanded in culture with cytokines and anti-CD20 monoclonal antibody (mAb; rituximab) to generate CIK cells. A preliminary investigation of their mechanism was then performed. The increase in the cytotoxicity of the CIK cells induced by the anti-CD20 mAb was associated with an increase in the expression of cytotoxic factors. The expression of components of the signal transducer and activator of transcription (STAT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways was found to increase. Upregulation of the expression of STAT1, STAT3 and STAT5 is important as these co-stimulatory molecules enhance T-cell proliferation. Activation of the MAPK signaling pathway is a possible mechanism for the anti-apoptosis effect on the proliferation of CIK cells. In conclusion, anti-CD20 mAb may play an important role in the improvement of CIK-mediated cytotoxicity to tumor cells. These observations may aid in the improvement of the effects of immunotherapy in depleting the residual cells of hematopoietic tumors. Thus, the use of CIK cells cultured with anti-CD20 mAb could be a novel therapeutic strategy for the depletion of chemotherapy-resistant or residual cells in anaplastic large and B-cell lymphoma.

Human Wharton's jelly mesenchymal stem cell secretome display antiproliferative effect on leukemia cell line and produce additive cytotoxic effect in combination with doxorubicin.

Mesenchymal stem cell (MSC) therapy moves toward clinic progressively. Recent evidences establish anticancer effect of mesenchymal stem cells. However multiple factors including type of cancer, MSC source, study design, and animal model play role in final outcome. Wharton's jelly - a newly approved source of MSCs - possesses superiorities to bone marrow as the conventional source; therefore investigation of its medical effects can produce beneficial results. In this survey we examined cytotoxic and proapoptotic effect of human Wharton's jelly MSC secretome on K562 human leukemia cells. MSCs were isolated from human Wharton's jelly of umbilical cord by explant culture method, then characterized according to ISCT criteria (morphology and plastic adherence, surface antigenicity and differentiation potential). MSC secretome was collected and its cytotoxic and proapoptotic effects on K562 cells in combination with doxorubicin were evaluated using BrdU cell proliferation assay and Annexin V-PI staining. Our results showed antiproliferative effect of mesenchymal stem cell secretome on K562 cancer cells, the effect was also added to cytotoxic effect of doxorubicin without induction of drug resistance. Human Wharton's jelly derived mesenchymal stem cells exerted cytotoxic effect on leukemia cells. Addition of that effect to anticancer effect of chemotherapeutic agents can leads to cytotoxic drug dose reduction and diminished side effects.