Human Chronic Leukemia Cells Research Articles

Hybrid Clustered Nanoparticles for Chemo-Antibacterial Combinatorial Cancer Therapy.

Background: A great number of therapeutic limitations, such as chemoresistance, high dosage, and long treatments, are still present in cancer therapy, and are often followed by side effects such as infections, which represent the primary cause of death among patients. Methods: We report pH- and enzymatic-responsive hybrid clustered nanoparticles (HC-NPs), composed of a PCL polymeric core loaded with an anticancer drug, such as Imatinib Mesylate (IM), and coated with biodegradable multilayers embedded with antibacterial and anticancer baby-ship silver NPs, as well as a monoclonal antibody for specific targeting of cancer cells conjugated on the surface. Results: The HC-NPs presented an onion-like structure that serially responded to endogenous stimuli. After internalization into targeted cancer cells, the clustered nanoparticles were able to break up, thanks to intracellular proteases which degraded the biodegradable multilayers and allowed the release of the baby-ship NPs and the IM loaded within the pH-sensible polymer present inside the mothership core. In vitro studies validated the efficiency of HC-NPs in human chronic leukemic cells. This cellular model allowed us to demonstrate specificity and molecular targeting sensitivity, achieved by using a combinatorial approach inside a single nano-platform, instead of free administrations. The combinatory effect of chemotherapic drug and AgNPs in one single nanosystem showed an improved cell death efficacy. In addition, HC-NPs showed a good antibacterial capacity on Gram-negative and Gram-positive bacteria. Conclusions: This study shows an important combinatorial anticancer and antimicrobial effect in vitro.

Molecular mechanism for apoptosis of leukemia cells induced by chromosome region maintenance 1 selective inhibitor KPT-330

Objective To investigate the induction effect and molecular mechanisms of chromosome region maintenance 1(CRM1) selective inhibitor KPT-330 on apoptosis of human leukemia cells. Methods Human acute promyelocytic leukemia cells(NB4), human chronic myelogenous leukemia cells (K562) and human promyelocytic leukemia cells(HL-60) were divided into a control group(without KPT-330 treatment) and KPT-330 treatment group(NB4, K562 and HL-60 were treated with 0.01, 0.10, 0.20, 0.50, 1.00, 5.00, 10.00 μmol/L KPT-330, respectively). Cell counting kit-8 assay was used to quantify the growth inhibition of cells after exposure to KPT-330.Fluorescence microscope was used to observe the morphological change in leukemia cells when treated with KPT-330.Annexin V/propidium iodide staining followed by flow cytometry was used to detect the apoptosis of leukemia cells.Then, Western blot was used to detect the expression level of CRM1 and the activation of apoptosis marker cysteinyl aspartate specific proteinase-9(Caspase-9), cysteinyl aspartate specific proteinase-3(Caspase-3) and poly ADP-ribose polymerase(PARP). Long non-coding RNA(lncRNA) microarray was used to analyze the differentially expressed genes differentially by the cluster analysis. Results The inhibition of cell proliferation of leukemia cells was in a dose-dependent manner with KPT-330 treatment.Expression of CRM1 was also down-regulated when leukemia cells were treated with KPT-330.Compared with the control group, abnormal cells were observed under fluorescence microscopy.Much more cells showed apoptotic feature after being treated with 0.20 μmol/L and 1.00 μmol/L KPT-330, the apoptosis rate of NB4, K562 and HL-60 were respectively(4.95±0.21)%, (14.05±0.07)%, (4.95±0.63)%(0.20 μmol/L 24 h); (6.10±0.56)%, (20.75±0.21)%, (5.85±0.07)% (0.20 μmol/L 48 h); (41.15±0.21)%, (35.45±0.35)%, (15.65±0.07)% (1.00 μmol/L 24 h); (52.45±0.35)%, (47.45±2.47)%, (16.80±1.98)% (1.00 μmol/L 48 h), respectively, and the differences were statistically significant compared to the control group[24 h NB4, K562, HL-60: (3.30±0.28)%, (9.50±0.56)%, (2.00±0.14)%; 48 h NB4, K562, HL-60: (3.70±0.14)%, (3.50±0.28)%, (2.15±0.21)%](all P<0.05). G2 phase cells obviously reduced and subG1 phase cells increased.The expression level of cleaved Caspase-3, cleaved Caspase-9 and cleaved PARP increased.Molecular function analysis showed that apoptosis induced by KPT-330 in leukemia cells was partially rela-ted to Glutathione metabolism and Toll-like receptor signaling pathway. Conclusions KPT-330 can effectively inhibit proliferation and induce apoptosis of leukemia cells.KPT-330-induced apoptosis in leukemia cells is partially related to Glutathione metabolism and Toll-like receptor signaling pathway. Key words: KPT-330; Chromosome region maintenance 1; Leukemia cell; Apoptosis; Long non-coding RNA

Effect of PKF118-310 on apoptosis of leukemia K562 cells and its mechanism

Objective To investigate the effect of small molecule antagonists PKF118-310 on apoptosis of human chronic myeloid leukemia cell line K562 and its mechanism. Methods After the treatment of PKF118-310 at different concentration, the proliferation inhibition in K562 cells was detected by MTT, the β-catenin/TCF transcription complex in the nucleus was observed by immunofluorescence, the apoptosis was detected by flow cytometry, and the expressions of caspase-3, caspase-8, XIAP, bcl-2, β-catenin, TCF, c-myc and cyclin D1 were detected by Western blot. Results PKF118-310 inhibited the proliferation of K562 cells. The median inhibitory concentration (IC50) of PKF118-310-treated K562 cells for 24 h, 48 h and 72 h were 5.388, 3.290, 1.566 μmol/L, respectively. The β-catenin/TCF transcription complex in the nucleus was observed. After the treatment with 1.6 and 3.2 μmol/L PKF118-310 for 48 h, the apoptosis rates of K562 cells were (48.0±0.9) % and (80.2±1.2) %, respectively, and were higher than that in the control group [(1.2±0.6) %] (both P < 0.05). After the treatment of K562 cells with PKF118-310 at different concentration for 72 h, the expression levels of caspase-3 and caspase-8 were increased (both P < 0.05), and the expression levels of XIAP, bcl-2, β-catenin, TCF, c-myc and cyclin D1 were significantly decreased compared with control group (all P< 0.05). Conclusion PKF118-310 can inhibit K562 cells'proliferation, and induce the apoptosis. Key words: Leukemia, myeloid, chronic; K562 cells; Wnt/β-catenin signaling pathway; PKF118-310; Apoptosis

Effect of DMSA Modified Fe3O4 and Multidrug Resistance Modulator HZ08 on the Reversal of Multidrug Resistance in K562/A02 Cell Line

The objective of the present study was to investigate the reversible effect of HZ08 and daunorubicin(DNR) combined with dimercaptosuccinic acid modified iron oxide (DMSA-Fe3O4) magnetic nanoparticles (MNPs) in human chronic leukemia cell line K562/A02, and the mechanism potentially involved. The growth inhibition rate of K562/A02 cells was determined by Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis and intracellular concentration of DNR were detected by flow cytometry (FCM). DAPI staining was used to view apoptotic cellular morphology. Subsequently, transcription levels of MDR1 mRNA and expression levels of P-glycoprotein (P-gp) and caspase-3 were determined by real time polymerase chain reaction (real-time PCR) and Western blotting analysis, respectively. group clearly exhibited more morphological changes (severe structural alterations) than other groups. In addition, transcription of MDR1 gene and protein expression of P-gp and caspase-3 of K562/A02 cells were regulated at the most remarkable extent in DNR-HZ08-MNPs group when compared with other groups. These findings suggest that the remarkable effects of the novel DNR-HZ08-MNPs on multidrug resistant K562/A02 leukemia cells would be a promising strategy for overcoming multidrug resistance. DisclosuresNo relevant conflicts of interest to declare.

Analysis of the erythroid differentiation effect of flavonoid apigenin on K562 human chronic leukemia cells

The erythroid differentiation-inducing effect of apigenin and its derivatives on human chronic myeloid leukemia K562 has been reported but the functional group in its structure responsible for the effect has not yet been elucidated. Here, we determined the moiety responsible for the erythroid differentiation induction effect of apigenin by using different flavonoids to represent the functional groups in its structure. In addition, we compared apigenin and apigetrin, a flavonoid similar in structure to apigenin except for the glycoside in its structure. Morphological changes as well as expressions of specific markers in K562 cells treated with apigenin were compared with those treated with apigetrin, flavone, 7-hydroxyflavone, chrysin, luteolin, or naringenin. The anti-proliferative and erythroid differentiation-inducing effect of apigenin and the five flavonoids were then investigated and their effects on the α, β, and γ globin genes expressions were compared using real-time PCR. Results of the comparison between apigenin and apigetrin revealed that the glycoside part of apigetrin does not have a role in the induction of cell differentiation. Based on glycophorin A expression, the potency of the other flavonoids for induction of differentiation, was: apigenin>chrysin>flavone/7-hydroxyflavone>luteolin/naringenin. Results of the analysis of the relationship between the structure and function of the flavonoids suggest that the apigenin-induced K562 cell differentiation was due to the 2–3 double bond and hydroxyl groups in its structure. This is the first study that identified the specific functional group in apigenin that impact the erythroid differentiation effect in K562 cells.

Hypoxia-inducible factor 1α regulates the expression of CD18 and ninjurin-1

Objective To investigate the effects of hypoxia-inducible factor 1α (HIF-1α) small interfere RNA construct pSUPERH1-siHIF1α on the expression of CD18 and ninjurin 1 by K562 (human chronic myelogenous leukemia cell line) cells cultured with serums from patients with early stage of diabetic retinopathy.Methods K562 cells were cultured in 4 groups as control group (group A),diabetic group (group B),diabetes and pSUPERH1-siHIF1α transfect group (group C) and diabetes and pSUPER-retro transfect group (group D).The cells in group A were cultured in human serum from age-matched healthy control,and in group B,C and D,the cells were cultured in serum from the subjects of early stage of diabetic retinopathy.Twenty-four hours before the cells were cultured by the serum from the subjects of early stage of diabetic retinopathy,the HIF-1α specific siRNA expression vector pSUPERH1-siHIF1α and empty vector pSUPER-retro were transfected into the cells of group C and D,respectively.The percentages of CD18 and ninjurin-1 positive cell on the surface of K562 cells were measured by Flow Cytometry.The adherent rate between K562 and RF/6A was measured by the rose Bengal staining test.Results The percentages of CD18 positive cell in the group A,B,C and D were significantly different (F=14.33,P=0.01).The percentage of group B was significantly higher than that in group A (P=0.001) ; the percentage of group C was significantly lower than that in group B (P=0.001) and group D (P=0.02) ; the difference between group C and A was not significant (95％CI=-14.89-2.13,P=0.12).The differences of the percentage of ninjurin-1 positive cell among the group A,B,C and D were significant (F=39.38,P=0.001).The percentage of group B was significantly higher than that in group A (P=0.00); the difference of the percentage between group C and B was not significant (P =0.06),that was also not significant between group C and D (P=0.49).The differences of the adherent rate between K562 and RF/6A (rhesus monkey retinal choroid blood vessel endothelial cell line) among the group A,B,C and D were significant (F=20.62,P=0.00).The adherent rate of group B was significantly higher than that in group A (P=0.00),the adherent rate in group C was significantly lower than that in group B (P=0.01),but it was still significantly higher than that in group A (P=0.002),the difference of adherent rate between group B and D was not significant (P=0.68).Conclusion Under the early stage of diabetic retinopathy,HIF-1α small interfere RNA pSUPERH1-siHIF1α may significantly suppress the expression of CD18 on the surface of K562 cells,but it may not significantly influence the expression of ninjurin-1 on the surface of K562 cells. Key words: Diabetic retinopathy/pathophsiology; Hypoxia-inducible factor 1, alpha subunit; RNA, small interfering

Effects of the multidrug resistance modulator HZ08 on the apoptosis pathway in human chronic leukaemia cell line K562/A02

ōancer falls to respond to chemotherapy by acquiring multidrug resistance in over 90% of patients. A previous study revealed that multidrug resistance modulator HZ08 had great multidrug resistance reversal effect in vitro and in vivo. It could enhance adriamycin (doxorubicin) induced intrinsic apoptosis pathway and rectify cell cycle and some apoptosis related proteins in human breast resistant cancer MCF-7/ADM cells. This study detected Rh123 accumulation to assess the effect of HZ08 on P-glycoprotein function in human chronic leukaemia cell line K562/A02. Moreover, mitochondria membrane potential, cytochrome c release and caspase-3 activity were analyzed for HZ08 treatment with or without vincristine. Since pretreatment with HZ08 could also reverse the multidrug resistance to vincristine in K562/A02 cells, the individual influence of HZ08 was further detected on apoptotic regulator like Bcl-2, Bax, p53, cell cycle checkpoints and proliferation regulatory factors like survivin, hTERT, c-Myc, c-Fos, c-Jun. Finally, it revealed that HZ08 increased vincristine induced activation in intrinsic apoptosis pathway by inhibition of P-gp mediated efflux. In addition, the outstanding reversal effect of HZ08 should also attribute to its individual effect on apoptosis and proliferation related regulatory factors. It renders HZ08 possibility of application in pretreatment to reverse multidrug resistance while avoiding unexpected drug interactions and accumulative toxicity.

Bis(2‐methylbenzimidazolyl)amine‐Derived Copper Complexes and Their Antineoplastic Activity

AbstractCopper(II) complexes (1–4) of tri‐ or tetradentate bis(2‐methylbenzimidazolyl)amine ligands (L1–L4) have been prepared and characterized by spectroscopic methods in solution, as well as in the solid state by X‐ray crystallography. The ligands act as tridentate donors towards the cupric ions through one central amine and two benzimidazole N atoms in the solid state; a water ligand and a bridging perchlorate group define the distorted octahedral environments of 2 and 3. Complex 4 has square‐pyramidal coordination geometry, with an additional thioether donor attached to the central N atom in the axial position. Electrospray mass spectrometry characterized the complexes as monomeric in acetonitrile solution. Cyclic voltammetry studies established that amine N benzyl‐containing 3 has the highest half‐wave redox potential of all of these complexes at –0.08 V (vs. Fc+/Fc) for the Cu2+/Cu+ couple. The complexes display dose‐dependent cytotoxicity against one human and four murine cancer cell lines. Complexes 3 and 4 have good antiproliferative activity against the human chronic leukemia cell line K562. Moreover, for 3 the proliferation of all malignant cell lines decreases at concentrations lower than the IC50 for healthy bone marrow cells.

Inhibition of cell growth by Stellera chamaejasme extract is associated with induction of autophagy and differentiation in chronic leukemia K562 cells

Stellera chamaejasme has been used as a therapeutic plant for treatment of various inflammatory diseases and solid tumors. Our study was particularly interested in the differentiation inducing activity of the aerial parts of S.chamaejasme using human chronic leukemia cell line K562. The ethanol extract has been shown to be highly cytotoxic against K562 cells at >0.008% (v/v) doses. Cell cycle arrest at G(0)/G(1) phase, not detectable DNA fragmentation as well as positive staining with acridine orange and expression of beclin-1 protein in the treated cells led as to detect autophagic cell death during the plant extract treatment of K562 cells. Moreover, differentiation marker CD11b was also expressed in the treated cells.

A Small Molecule Inhibitor of D-Cyclin Transactivation Displays Preclinical Efficacy in Myeloma and Leukemia.

Abstract 2036Poster Board II-13D-cyclins are universally dysregulated in multiple myeloma and frequently over-expressed in acute leukemia. To better understand the regulation of D-cyclins and the effects of targeting their expression in myeloma and leukemia cells, we conducted a chemical screen of the 56,000 compound Maybridge chemical library for inhibitors of the human cyclin D2 promoter using NIH 3T3 cells stably expressing the Cyclin D2promoter-driving a luciferase reporter gene. From this screen, 11 compounds that reproducibly inhibited transactivation of the cyclin D2 promoter, but did not inhibit transactivation of an unrelated RSV promoter driving luciferase, and did not reduce the growth and viability of NIH3T3 in an MTS assay. Among these 11 compounds, the most active was 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14161).Given the identification of S14161 as a potential inhibitor of cyclin D2 transactivation, we evaluated its effects on D-cyclin expression in myeloma and leukemia cell lines. S14161 decreased D-cyclin protein and mRNA levels across a range of events that dysregulated D-cyclins including cyclin D1 translocation (KMS12), c-maf over-expression (RPMI-8226, KMS11, LP1, JJN3), and FGFR3 translocation (KMS11) at low micromolar concentrations. Consistent with its effects on D-cyclin expression, S14161 arrested cells in the G0/G1 phase of the cell cycle at concentrations associated with its ability to decrease D-cyclins.Next, we tested the effects of S14161 on the viability of myeloma and leukemia cells. S14161 induced cell death in 6/7 myeloma and 6/7 leukemia cell lines with an IC50 <10 μM by the MTS assay. Cell death and apoptosis were confirmed by Annexin V-FITC staining. S14161 reduced the growth and viability of 4/5 primary AML samples with an IC50 <10 μM. In contrast, it was less toxic to normal hematopoietic cells with an IC20 > 25 μM.Given the ability of S14161 to induce apoptosis and reduce D-cyclin expression, we evaluated its efficacy in vivo. SCID mice were injected subcutaneously with K562 human chronic leukemia cells. After injection, mice were treated with S14161 (100mg/kg/day) intraperitoneally or vehicle control. Treatment with S14161 delayed tumor growth by up to 90% compared to vehicle control without evidence of gross organ toxicity or weight loss. Similar results were obtained in a U937 xenograft model. Of note, in our acute toxicity experiment, no weight loss or other toxic changes were noted in mice treated with 500 mg/kg/daily for two weeks.S14161 is a novel chemical compound with an unknown mechanism of action. However, the compound contains a chromene motif that is similar to the chromone motif found in the PI3 kinase inhibitor, LY294002. Therefore, we tested whether S14161 could inhibit the PI3 kinase signalling pathway and whether this inhibition was functionally important for its effects on D-cyclin expression and cell viability. Pre-treatment with S14161 blocked the increase in phospho-Akt without altering levels of total Akt. Moreover, S14161 inhibited the activities of Class I PI3 kinase α, β, γ, and δ with similar efficacy. In contrast, S14161 was less effective in inhibiting the PI3 kinase-associated enzymes PDK1, mTOR and DNA-PKcs with less than 20% inhibition at a concentration of 300 μM. Likewise, S14161 did not inhibit Akt1, Akt2, or Akt3, at a concentration up to 300 μM. To assess the relationship between inhibition of PI3 kinase signalling, decreased D-cyclin expression and cell death, we evaluated a series of chromene-containing compounds structurally related to S14161. Similar to the effects of S14161, the highly structurally related compound 8-methoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene (S14147) inhibited accumulation of phospho-Akt after IGF1 stimulation, decreased D-cyclin expression and induced cell death in KMS11 myeloma cells. In contrast, 4 other related compounds did not decrease cyclin D expression or prevent Akt phosphorylation after IGF1 stimulation. These inactive analogues were also not toxic to myeloma cells.Thus, we identified a novel chemical compound that inhibits D-cyclin transactivation. Moreover, we demonstrated that PI3 kinase inhibition can overcome dysregulation of D-cyclins across a broad array of transforming events in myeloma and leukemia. Disclosures:No relevant conflicts of interest to declare.

Study on mechanism of apoptosis by bortezomib and imatinib in K562 cells in vitro.

Objective To investigate the effect of bortezomib alone and in combination with imatinib on apoptosis of human chronic myeloid leukemia cells of the line K562 ,what is more, to explore the mechanism. Methods K562 was cultured and treated with bortezomib and (or) imatinib in different concentrations for 12, 48, 72 hours. Cell proliferation was analyzed by MTT assay, the apoptosis of K562 cells was observed by flow cytometry, and the expression of Livin mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR). Results MTT assay and flow cytometry showed that bortezomib could inhibit K562 cell proliferation and induce its apoptosis, and it can strengthen cells' lethal effect by imatinib. The expression of Livin mRNA was decreased by bortezomib or imatinib to some extent, and was downregulated significantly when combined treatment was given. Conclusion Bortezomib alone and in combination with imatinib can induce K562 cells apoptosis, in which decrease of the expression of Livin mRNA is the possible mechanism. Key words: Antineoplastic agents; K562 cells; Livin mRNA; Apoptosis

Continuous Low-Voltage dc Electroporation on a Microfluidic Chip with Polyelectrolytic Salt Bridges

A microfluidic electroporator operating under a continuous low dc voltage (7 to approximately 15 V) is reported. The proposed electroporation microchip exploits the ionic conductivity of polyelectrolytic gel electrodes to precisely control the electric field that is applied to cells without bubble generation in the microchannel. In this study, pDADMAC (poly diallyldimethylammonium chloride) was used to efficiently apply the electric potential difference to the cells in the microchannels. Impedance analysis showed that the pDADMAC plugs could work as ionic conductors with a conductivity of approximately 16 S m(-1). In accordance with the calculation using CFD-ACE, an input voltage of only 10 V could generate an electric field of 0.9 kV cm(-1) across the microchannel; this meets the requirements for electropermeation. The electropermeation of K562 human chronic leukemia cells was observed in the microchip from 7 V, and the efficiency increased up to 60% upon the application of an input voltage of 15 V with a viability of 80%. An amount of 10(5) cells could be transfected every minute under a constant potential difference. The transfection and expression of DNA plasmids were also successfully demonstrated in the suspension cell line.

A new inhibitor of protein kinase C, RK-1409B (4'-demethylamino-4'-hydroxy-3'-epistaurosporine).

RK-1409B, a new inhibitor of protein kinase C, was isolated from the culture broth of Streptomyces platensis subsp. malvinus RK-1409. The structure was elucidated on the basis of spectroscopic analyses. RK-1409B inhibited protein kinase C in vitro and the morphological change of a human chronic leukemia cell line, K-562, induced by phorbol 12,13-dibutyrate with IC50 value of 0.4 microM.

Bovine leukemia virus (BLV)-infected B-cells express a marker similar to the CD5 T cell marker

In the course of generating monoclonal antibodies to bovine thymus-dependent differentiation antigens, we were able to characterize an antibody, termed 8C11, that detects an antigen shared by a majority of thymocytes and peripheral T cells (in blood and thymus-dependent area of spleen and lymph-nodes), but undetectable on normal B cells. However, this antibody was reactive with B cells from cows infected with bovine leukemia virus (BLV). These BLV-infected B cells were found to express simultaneously high concentrations of both surface IgM and 8C11-detected antigen. The antigen recognized by this antibody was shown to be a 67.5 kDa molecule. Because similar findings have been made on mouse myelomas and on human chronic leukemia cells, where this antigen was considered to be the equivalent of mouse Ly-1 antigen and human Leu-1 or CD5 antigen, the T cell antigen detected on BLV-infected cells could be the bovine counterpart of the CD5 antigen. By another way, it has been found that the CD5 T cell antigen is also present on a minor subpopulation of B cells in the spleen but not in the blood. We suggest that in the bovine a similar B cell subpopulation should be the BLV target and expand as a consequence of viral insertion, leading to the persistent lymphocytosis observed on BLV-infected animals.