Tumor necrosis factor­related apoptosis­inducing ligand is a novel transcriptional target of runt­related transcription factor 1.

Runt­related transcription factor 1 (RUNX1), which is also known as acute myeloid leukemia 1 (AML1), has been frequently found with genomic aberrations in human leukemia. RUNX1 encodes a transcription factor that can regulate the expression of hematopoietic genes. In addition, tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) performs an important function for malignant tumors in immune surveillance. However, the regulatory mechanism of TRAIL expression remain to be fully elucidated. In the present study, tetradecanoylphorbol 13­acetate­treated megakaryocytic differentiated K562 cells was used to examine the effect of RUNX1 on TRAIL expression. Luciferase assay series of TRAIL promoters for the cells co­transfected with RUNX1 and core­binding factor ß (CBFß) expression vectors were performed to evaluate the nature of TRAIL transcriptional regulation. Electrophoresis mobility shift assay of the RUNX1 consensus sequence of the TRAIL promoter with recombinant RUNX1 and CBFß proteins was also performed. BloodSpot database analysis for TRAIL expression in patients with acute myeloid leukemia were performed. The expression of TRAIL, its receptor Death receptor 4 and 5 and RUNX1 in K562 cells transfected with the RUNX1 expression vector and RUNX1 siRNA were evaluated by reverse transcription­quantitative PCR (RT­qPCR). TRAIL and RUNX1­ETO expression was also measured in Kasumi­1 cells transfected with RUNX1­ETO siRNA and in KG­1 cells transfected with RUNX1­ETO expression plasmid, both by RT­qPCR. Cell counting, lactate dehydrogenase assay and cell cycle analysis by flow cytometry were performed on Kasumi­1, KG­1, SKNO­1 and K562 cells treated with TRAIL and HDAC inhibitors sodium butyrate or valproic acid. The present study demonstrated that RUNX1 is a transcriptional regulator of TRAIL. It was initially found that the induction of TRAIL expression following the megakaryocytic differentiation of human leukemia cells was RUNX1­dependent. Subsequently, overexpression of RUNX1 was found to increase TRAIL mRNA expression by activating its promoter activity. Additional analyses revealed that RUNX1 regulated the expression of TRAIL in an indirect manner, because RUNX1 retained its ability to activate this promoter following the mutation of all possible RUNX1 consensus sites. Furthermore, TRAIL expression was reduced in leukemia cells carrying the t(8;21) translocation, where the RUNX1­ETO chimeric protein interfere with normal RUNX1 function. Exogenous treatment of recombinant TRAIL proteins was found to induce leukemia cell death. To conclude, the present study provided a novel mechanism, whereby TRAIL is a target gene of RUNX1 and TRAIL expression was inhibited by RUNX1­ETO. These results suggest that TRAIL is a promising agent for the clinical treatment of t(8;21) AML.

Studies on the role of alpha 7 nicotinic acetylcholine receptors in K562 cell proliferation and signaling.

The results we obtained from this study gave information about the determination of alpha 7 nicotinic acetylcholine receptor (α7-nACh) expression in human erythroleukemia cells, as well as whether it has a role in calcium release and cell proliferation in the presence of nicotinic agonist, antagonists. Determining the roles of α7 nicotinic receptors in erythroleukemia cells will also contribute to leukemia-related signal transduction studies. This study is primarily to determine the role of nicotinic agonists and antagonists in cell proliferation, α7 nicotinic acetylcholine receptor expression, and calcium release. The aim of this study, which is a continuation and an important part of our previous studies on the cholinergic system, has contributed to the literature on the human erythroleukemia cell signaling mechanism. Cell viability was evaluated by the trypan blue exclusion test and Bromodeoxyuridine/5-Bromo-2'-deoxyuridine (BrdU) labeling. Acetylcholine, nicotinic alpha 7 receptor antagonist methyllycaconitine citrate, and cholinergic antagonist atropine were used to determine the role of α7-nACh in K562 cell proliferation. In our experiments, the fluorescence spectrophotometer was used in Ca2+ measurements. The expression of nicotinic alpha 7 receptor was evaluated by western blot. The stimulating effect of acetylcholine in K562 cell proliferation was reversed by both the α7 nicotinic antagonist methyllycaconitine citrate and the cholinergic antagonist, atropine. Methyllycaconitine citrate inhibited K562 cell proliferation partially explained the roles of nicotinic receptors in signal transduction. While ACh caused an increase in intracellular Ca2+, methyllycaconitine citrate decreased intracellular Ca2+ level in K562 cell. The effects of nicotinic agonists and/or antagonists on erythroleukemic cells on proliferation, calcium level contributed to the interaction of nicotinic receptors with different signaling pathways. Proliferation mechanisms in erythroleukemic cells are under the control of the α7 nicotinic acetylcholine receptor via calcium influx and different signalling pathway.

Phase 2 Clinical Trial of Infusing Haploidentical K562-mb15-41BBL-Activated and Expanded Natural Killer Cells as Consolidation Therapy for Pediatric Acute Myeloblastic Leukemia.

BACKGROUND: Acute myeloid leukemia (AML) accounts for approximately 20% of pediatric leukemia cases; 30% of these patients experience relapse. The antileukemia properties of natural killer (NK) cells and their safety profile have been reported in AML therapy. We proposed a phase 2, open, prospective, multicenter, nonrandomized clinical trial for the adoptive infusion of haploidentical K562-mb15-41BBL-activated and expanded NK (NKAE) cells as a consolidation strategy for children with favorable and intermediate risk AML in first complete remission after chemotherapy (NCT02763475). PATIENTS AND METHODS: Before the NKAE cell infusion, patients underwent a lymphodepleting regimen. After the NKAE cell infusion, patients were administered low doses (1 × 106/IU/m2) of subcutaneous interleukin-2. The primary study endpoint was AML relapse-free survival. We needed to include 35 patients to demonstrate a 50% reduction in relapses. RESULTS: Seven patients (median age, 7.4 years; range, 0.78-15.98 years) were administered 13 infusions of NKAE cells, with a median of 36.44 × 106 cells/kg (range, 6.92 × 106 to 193.2 × 106 cells/kg). We observed chimerism in 4 patients (median chimerism, 0.065%; range, 0.05-0.27%). After a median follow-up of 33 months, the disease of 6 patients (85.7%) remained in complete remission. The 3-year overall survival was 83.3% (95% confidence interval, 68.1-98.5), and the cumulative 3-year relapse rate was 28.6% (95% confidence interval, 11.5-45.7). The study was terminated early because of low patient recruitment. CONCLUSION: This study emphasizes the difficulties in recruiting patients for cell therapy trials, though NKAE cell infusion is safe and feasible. However, we cannot draw any conclusions regarding efficacy because of the small number of included patients and insufficient biological markers.

Identification and functional characterization of a Siglec-7 counter-receptor on K562 cells.

Sialic acid (Sia)-binding immunoglobulin-like lectin 7 (Siglec-7) is an inhibitory receptor primarily expressed on natural killer (NK) cells and monocytes. Siglec-7 is known to negatively regulate the innate immune system through Sia binding to distinguish self and nonself; however, a counter-receptor bearing its natural ligand remains largely unclear. Here, we identified a counter-receptor of Siglec-7 using K562 hematopoietic carcinoma cells presenting cell surface ligands for Siglec-7. We affinity-purified the ligands using Fc-ligated recombinant Siglec-7 and diSia-dextran polymer, a strong inhibitor for Siglec-7. We then confirmed the counter-receptor for Siglec-7 as leukosialin (CD43) through mass spectrometry, immunoprecipitation, and proximity labeling. Additionally, we demonstrated that the cytotoxicity of NK cells toward K562 cells was suppressed by overexpression of leukosialin in a Siglec-7-dependent manner. Taken together, our data suggest that leukosialin on K562 is a counter-receptor for Siglec-7 on NK cells and that a cluster of the Sia-containing glycan epitope on leukosialin is key as trans-ligand for unmasking the cis-ligand.

Abrogation of histone deacetylases (HDACs) decreases survival of chronic myeloid leukemia cells: New insight into attenuating effects of the PI3K/c-Myc axis on panobinostat cytotoxicity.

Although the identification of tyrosine kinase inhibitors (TKIs) has changed the treatment paradigm of many cancer types including chronic myeloid leukemia (CML), still adjustment of neoplastic cells to cytotoxic effects of anticancer drugs is a serious challenge. In the area of drug resistance, epigenetic alterations are at the center of attention and the present study aimed to evaluate whether blockage of epigenetics mechanisms using a pan-histone deacetylase (HDAC) inhibitor induces cell death in CML-derived K562 cells. We found that the abrogation of HDACs using panobinostat resulted in a reduction in survival of the K562 cell line through p27-mediated cell cycle arrest. Noteworthy, the results of the synergistic experiments revealed that HDAC suppression could be recruited as a way to potentiate cytotoxicity of Imatinib and to enhance the therapeutic efficacy of CML. Here, we proposed for the first time that the inhibitory effect of panobinostat was overshadowed, at least partially, through the aberrant activation of the phosphoinositide 3-kinase (PI3K)/c-Myc axis. Meanwhile, we found that upon blockage of autophagy and the proteasome pathway, as the main axis involved in the activation of autophagy, the anti-leukemic property of the HDAC inhibitor was potentiated. Taken together, our study suggests the beneficial application of HDAC inhibition in the treatment strategies of CML; however, further in vivo studies are needed to determine the efficacy of this inhibitor, either as a single agent or in combination with small molecule inhibitors of PI3K and/or c-Myc in this malignancy.

Puerarin sensitized K562/ADR cells by inhibiting NF-κB pathway and inducing autophagy.

Puerarin is an isoflavone isolated from Pueraria lobata (Willd.) Ohwi. In the present study, reversal effect and underlying mechanisms of puerarin on multidrug resistance (MDR) were investigated in K562/ADR cells. K562/ADR cells exhibited adriamycin (ADR) resistance and higher levels of MDR1 expression compared with K562 cells. Puerarin enhanced the chemosensitivity of K562/ADR cells and increased the ADR accumulation in K562/ADR cells. The expression levels of MDR1 were down-regulated by puerarin in K562/ADR cells. Luciferase reporter assay further demonstrated the inhibitory effect of puerarin on TNF-α-induced NF-κB activation. The phosphorylation of IκB-α was significantly suppressed by puerarin. In silico docking analyses suggested that puerarin well matched with the active sites of IκB-α. Moreover, a large number of autophagosomes were found in the cytoplasm of K562/ADR cells after puerarin treatment. The significant increase in LC3-II and beclin-1 was also observed, indicating autophagy induction by puerarin in K562/ADR cells. Puerarin induced cell cycle arrest and apoptosis in K562/ADR cells. Finally, puerarin inhibited phosphorylation of Akt and JNK. In conclusion, puerarin-sensitized K562/ADR cells by downregulating MDR1 expression via inhibition of NF-κB pathway and autophagy induction via Akt inhibition.

Suppressive Effects of a Truncated Inhibitor K562 Protein-Derived Peptide on Two Proinflammatory Cytokines, IL-17 and TNF-α.

Inhibitor K562 (IK) protein was first isolated from the culture medium of K562 cells, a leukemia cell line, and is an inhibitory regulator of interferon-γ-induced major histocompatibility complex class II expression. Recently, exogenous truncated IK (tIK) protein showed potential as a therapeutic agent for inflammation-related diseases. In this study, we designed a novel putative anti-inflammatory peptide derived from tIK protein based on homology modeling of the human interleukin-10 (hIL-10) structure, and investigated whether the peptide exerted inhibitory effects against proinflammatory cytokines such as IL-17 and tumor necrosis factor-α (TNF-α). The peptide contains key residues involved in binding hIL-10 to the IL-10 receptor, and exerted strong inhibitory effects on IL- 17 (43.8%) and TNF-α (50.7%). In addition, we used circular dichroism spectroscopy to confirm that the peptide is usually present in a random coil configuration in aqueous solution. In terms of toxicity, the peptide was found to be biologically safe. The mechanisms by which the short peptide derived from human tIK protein exerts inhibitory effects against IL-17 and TNF-α should be explored further. We also evaluated the feasibility of using this novel peptide in skincare products.

Inhibition of the Nrf2-TrxR Axis Sensitizes the Drug-Resistant Chronic Myelogenous Leukemia Cell Line K562/G01 to Imatinib Treatments.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is involved in tumor drug resistance, but its role in imatinib resistance of chronic myeloid leukemia (CML) remains elusive. We aimed to investigate the effects of Nrf2 on drug sensitivity, thioredoxin reductase (TrxR) expression, reactive oxygen species (ROS) production, and apoptosis induction in imatinib-resistant CML K562/G01 cells and explored their potential mechanisms. Stable K562/G01 cells with knockdown of Nrf2 were established by infection of siRNA-expressing lentivirus. The mRNA and protein expression levels of Nrf2 and TrxR were determined by real-time quantitative polymerase chain reaction and western blot, respectively. ROS generation and apoptosis were assayed by flow cytometry, while drug sensitivity was measured by the Cell Counting Kit-8 assay. Imatinib-resistant K562/G01 cells had higher levels of Nrf2 expression than the parental K562 cells at both mRNA and protein levels. Expression levels of Nrf2 and TrxR were positively correlated in K562/G01 cells. Knockdown of Nrf2 in K562/G01 cells enhanced the intracellular ROS level, suppressed cell proliferation, and increased apoptosis in response to imatinib treatments. Nrf2 expression contributes to the imatinib resistance of K562/G01 cells and is positively correlated with TrxR expression. Targeted inhibition of the Nrf2-TrxR axis represents a potential therapeutic approach for imatinib-resistant CML.

Counteracting the effect of leukemia exosomes by antiangiogenic gold nanoparticles.

PURPOSE: Progression of chronic myeloid leukemia (CML) is frequently associated with increased angiogenesis at the bone marrow mediated by exosomes. The capability of gold nanoparticles (AuNPs) functionalized with antiangiogenic peptides to hinder the formation of new blood vessels has been demonstrated in a chorioallantoic membrane (CAM) model. METHODS: Exosomes of K562 CML cell line were isolated and their angiogenic effect assessed in a CAM model. AuNPs functionalized with antiangiogenic peptides were used to block the angiogenic effect of CML-derived exosomes, assessed by evaluation of expression levels of key modulators involved in angiogenic pathways - VEGFA, VEGFR1 (also known as FLT1) and IL8. RESULTS: Exosomes isolated from K562 cells promoted the doubling of newly formed vessels associated with the increase of VEGFR1 expression. This is a concentration and time-dependent effect. The AuNPs functionalized with antiangiogenic peptides were capable to block the angiogenic effect by modulating VEGFR1 associated pathway. CONCLUSION: Exosomes derived from blast cells are capable to trigger (neo)-angiogenesis, a key factor for the progression and spreading of cancer, in particular in CML. AuNPs functionalized with specific antiangiogenic peptides are capable to block the effect of the exosomes produced by malignant cells via modulation of the intrinsic VEGFR pathway. Together, these data highlight the potential of nanomedicine-based strategies against cancer proliferation.

High-level embryonic globin production with efficient erythroid differentiation from a K562 erythroleukemia cell line.

A reliable cell line capable of robust in vitro erythroid differentiation would be useful to investigate red blood cell (RBC) biology and genetic strategies for RBC diseases. K562 cells are widely utilized for erythroid differentiation; however, current differentiation methods are insufficient to analyze globin proteins. In this study, we sought to improve erythroid differentiation from K562 cells to enable protein-level globin analysis. K562 cells were exposed to a variety of reagents, including hemin, rapamycin, imatinib, and/or decitabine (known erythroid inducers), and cultured in a basic culture medium or erythropoietin-based differentiation medium. All single reagents induced observable erythroid differentiation with higher glycophorin A (GPA) expression but were insufficient to produce detectable globin proteins. We then evaluated various combinations of these reagents and developed a method incorporating imatinib preexposure and an erythropoietin-based differentiation culture containing both rapamycin and decitabine capable of efficient erythroid differentiation, high-level GPA expression (>90%), and high-level globin production at protein levels detectable by hemoglobin electrophoresis and high performance liquid chromatography. In addition, ß-globin gene transfer resulted in detectable adult hemoglobin. In summary, we developed an in vitro K562 erythroid differentiation model with high-level globin production. This model provides a practical evaluation tool for hemoglobin production in human erythroid cells.

Genetically re-engineered K562 cells significantly expand and functionally activate cord blood natural killer cells: Potential for adoptive cellular immunotherapy.

Natural killer (NK) cells play a significant role in reducing relapse in patients with hematological malignancies after allogeneic stem cell transplantation, but NK cell number and naturally occurring inhibitory signals limit their capability. Interleukin-15 (IL-15) and 4-1BBL are important modulators of NK expansion and functional activation. To overcome these limitations, cord blood mononuclear cells (CB MNCs) were ex vivo expanded for 7 days with genetically modified K562-mbIL15-41BBL (MODK562) or wild-type K562 (WTK562). NK cell expansion; expression of lysosome-associated membrane protein-1 (LAMP-1), granzyme B, and perforin; and in vitro and in vivo cytotoxicity against B-cell non-Hodgkin lymphoma (B-NHL) were evaluated. In vivo tumor growth in B-NHL-xenografted nonobese diabetic severe combined immune deficient (NOD-scid) gamma (NSG) mice was monitored by tumor volume, cell number, and survival. CB MNCs cultured with MODK562 compared with WTK562 demonstrated significantly increased NK expansion (thirty-fivefold, p < 0.05); LAMP-1 (p < 0.05), granzyme B, and perforin expression (p < 0.001); and in vitro cytotoxicity against B-NHL (p < 0.01). Xenografted mice treated with MODK562 CB experienced significantly decreased B-NHL tumor volume (p = 0.0086) and B-NHL cell numbers (p < 0.01) at 5 weeks and significantly increased survival (p < 0.001) at 10 weeks compared with WTK562. In summary, MODK562 significantly enhanced CB NK expansion and cytotoxicity, enhanced survival in a human Burkitt's lymphoma xenograft NSG model, and could be used in the future as adoptive cellular immunotherapy after umbilical CB transplantation. Future directions include expanding anti-CD20 chimeric receptor-modified CB NK cells to enhance B-NHL targeting in vitro and in vivo.

Original Research: Stable expression of miR-34a mediates fetal hemoglobin induction in K562 cells.

Sickle cell anemia is a common genetic disorder caused by a point mutation in the sixth codon of the ß-globin gene affecting people of African descent worldwide. A wide variety of clinical phenotypes ranging from mild to severe symptoms and complications occur due to hemoglobin S polymerization, red blood cell sickling, and vaso-occlusion. Research efforts are ongoing to develop strategies of fetal hemoglobin (HbF; α2γ2) induction to inhibit sickle hemoglobin polymerization and improve clinical outcomes. Insights have been gained from investigating mutations in the ß-globin locus or transcription factors involved in the mechanisms of hemoglobin switching. Recent efforts to expand molecular targets that modulate γ-globin expression involve microRNAs that work through posttranscriptional gene regulation. Therefore, the goal of our study was to identify novel microRNA genes involved in fetal hemoglobin expression. Using in silico analysis, we identified a miR-34a binding site in the γ-globin mRNA which was tested for functional relevance. Stable expression of the shMIMIC miR-34a lentivirus vector increased fetal hemoglobin levels in single cell K562 clones consistent with silencing of a γ-globin gene repressor. Furthermore, miR-34a promoted cell differentiation supported by increased expression of KLF1, glycophorin A, and the erythropoietin receptor. Western blot analysis of known negative regulators of γ-globin including YY1, histone deacetylase 1, and STAT3, which are regulated by miR-34a showed no change in YY1 and histone deacetylase 1 levels; however, total- and phosphorylated-STAT3 levels were decreased in single cell miR-34a K562 clones. These data support a mechanism of fetal hemoglobin activation by miR-34a involving STAT3 gene silencing.

Magnetic Resonance Imaging of Mitochondrial Dysfunction and Metabolic Activity, Accompanied by Overproduction of Superoxide.

This study shows that a mitochondria-penetrating nitroxide probe (mito-TEMPO) allows detection of superoxide and visualization of mitochondrial dysfunction in living cells due to the effect of T1 shortening in MRI. Mitochondrial dysfunction was induced by treatment of cells with rotenone and 2-methoxyestradiol (2-ME/Rot). The MRI measurements were performed on 7T MRI. The 2-ME/Rot-treated cells were characterized by overproduction of superoxide, which was confirmed by a conventional dihydroethidium test. In the presence of mito-TEMPO, the intensity of MRI signal in 2-ME/Rot-treated cells was ∼30-40% higher, in comparison with that in untreated cells or culture media. In model (cell-free) systems, we observed that superoxide, but not hydrogen peroxide, increased the intensity of T1-weighted MRI signal of mito-TEMPO. Moreover, the superoxide restores the T1-weighted MRI contrast of mito-TEMPOH, a noncontrast (diamagnetic) analogue of mito-TEMPO. This was also confirmed by using EPR spectroscopy. The results demonstrate that superoxide radical is involved in the enhancement of T1-weighted MRI contrast in living cells, in the absence and presence of mito-TEMPO. This report gives a direction for discovering new opportunities for functional MRI, for detection of metabolic activity, accompanied by overproduction of superoxide, as well as by disturbance of the balance between superoxide and hydrogen peroxide, a very important approach to clarify the fine molecular mechanisms in the regulation of many pathologies. The visualization of mitochondrial activity in real-time can be crucial to clarify the molecular mechanism of the functional MRI in its commonly accepted definition, as a method for detection of neurovascular coupling.

BME, a novel compound of anthraquinone, down regulated P-glycoprotein expression in doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells via generation of reactive oxygen species.

P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) in tumor cells is still a main obstacle for the chemotherapeutic treatment of cancers. Thus, development of effective MDR reversing agents is an important approach in the clinic. The present study revealed that BME, a novel compound of anthraquinone, elevated intracellular accumulation of the P-gp substrates and reduced concentration resulting in 50% inhibition of cell growth (IC50) values for doxorubicin (DOX) in doxorubicin-resistant human myelogenous leukemia (K562/DOX) cells. Further more, BME was also reported to down regulated P-gp expression accompanying with generation of nontoxic low level of intracellular reactive oxygen species (iROS) and activation of extracellular signal-regulated kinase (ERK)1/2 as well as c-JUN N-terminal kinase (JNK). However, treatment with N-acetyl-cysteine (NAC), U0216 and SP600125 almost abolished actions of the BME mentioned above. These results indicated that the effect of the BME on the P-gp may be involved in generation of nontoxic low level of iROS and activation of ERK1/2 or JNK, which suggested valuable clues to screen and develop P-gp reversing agents.

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.

Inhibition effects of lamellarin D on human leukemia K562 cell proliferation and underlying mechanisms.

Lamellarin D (LamD) is a marine alkaloid with a pronounced cytotoxicity against a large panel of cancer cells, affecting cell growth and inducing apoptosis. However, the molecular mechanisms of action of this compound are poorly understood. In this study, the anticancer efficacy of LamD was investigated in human leukemia K562 cells. The results showed suppressed cell proliferation and induction of G0/G1-phase arrest,while expression of CDK1, and activity of smad3 and smad5 were reduced, but that of p27, p53 and STGC3 was increased. LamD induced cell apoptosis through activation of caspases-8/-3, inhibition of survivin and Bcl-2, suggesting that this compound may also act through a caspase-independent pathway. Moreover, LamD inhibited the secretion of TGF-ß, IL-1ß, IL-6, IL-8 and other inflammatory cytokines and the transcriptional activity of transcription factor NF-κB in human leukemia K562 cells. Taken together, our results suggest that LamD-mediated inhibition of leukemia cell proliferation may be related to the induction of apoptosis and the regulation of cell cycle, tumor- related gene expression and cytokine expression, which may provide a new way of thinking for the treatment leukemia.

Short peptide motifs for long-lasting anchoring to the cell surface.

A rational design strategy has been developed for the construction of stable peptide-based anchors for the efficient modification of cell surfaces. Six types of peptide composed of five residues with divalent hydrophobic groups have been designed using this new strategy. Among them, a peptide with a sequence of NBD-Lys-Lys(X)-Lys-Lys-Lys(X)-NH2 (NBD: fluorophore, Lys(X): N-ε-palmitoyl-l-lysine) was found to show the highest modification efficacy and longevity in culture medium. The good performance of this peptide was attributed to (1) its high aqueous solubility, which allowed it to partition from the medium to the cell surface, and (2) the high binding affinity of the saturated palmitoyl groups to the cell membrane. We found that the distribution of the peptide was affected by recycling endosome, which enabled the representation of the peptide following its endocytotic disappearance from the cell membrane. Biotin was also presented on the cell surface using this peptide-based anchor to examine its recognition by streptavidin. The efficacy of the recognition process increased as the length of the oligoethylene glycol spacer increased, indicating that it was necessary for the biotin tag to move away from the membrane glycoproteins on the cell surface to facilitate its efficient recognition by streptavidin.

Human cell-based artificial antigen-presenting cells for cancer immunotherapy.

Adoptive T-cell therapy, where anti-tumor T cells are first prepared in vitro, is attractive since it facilitates the delivery of essential signals to selected subsets of anti-tumor T cells without unfavorable immunoregulatory issues that exist in tumor-bearing hosts. Recent clinical trials have demonstrated that anti-tumor adoptive T-cell therapy, i.e. infusion of tumor-specific T cells, can induce clinically relevant and sustained responses in patients with advanced cancer. The goal of adoptive cell therapy is to establish anti-tumor immunologic memory, which can result in life-long rejection of tumor cells in patients. To achieve this goal, during the process of in vitro expansion, T-cell grafts used in adoptive T-cell therapy must be appropriately educated and equipped with the capacity to accomplish multiple, essential tasks. Adoptively transferred T cells must be endowed, prior to infusion, with the ability to efficiently engraft, expand, persist, and traffic to tumor in vivo. As a strategy to consistently generate T-cell grafts with these capabilities, artificial antigen-presenting cells have been developed to deliver the proper signals necessary to T cells to enable optimal adoptive cell therapy.

The matricellular protein CCN3 regulates NOTCH1 signalling in chronic myeloid leukaemia.

Deregulated NOTCH1 has been reported in lymphoid leukaemia, although its role in chronic myeloid leukaemia (CML) is not well established. We previously reported BCR-ABL down-regulation of a novel haematopoietic regulator, CCN3, in CML; CCN3 is a non-canonical NOTCH1 ligand. This study characterizes the NOTCH1­CCN3 signalling axis in CML. In K562 cells, BCR-ABL silencing reduced full-length NOTCH1 (NOTCH1-FL) and inhibited the cleavage of NOTCH1 intracellular domain (NOTCH1-ICD), resulting in decreased expression of the NOTCH1 targets c-MYC and HES1. K562 cells stably overexpressing CCN3 (K562/CCN3) or treated with recombinant CCN3(rCCN3) showed a significant reduction in NOTCH1 signalling (> 50% reduction in NOTCH1-ICD, p < 0.05).Gamma secretase inhibitor (GSI), which blocks NOTCH1 signalling, reduced K562/CCN3 colony formation but increased that of K562/control cells. GSI combined with either rCCN3 or imatinib reduced K562 colony formation with enhanced reduction of NOTCH1 signalling observed with combination treatments. We demonstrate an oncogenic role for NOTCH1 in CML and suggest that BCR-ABL disruption of NOTCH1­CCN3 signalling contributes to the pathogenesis of CML.

Acquired resistance of leukemic cells to AraC is associated with the upregulation of aldehyde dehydrogenase 1 family member A2.

The elucidation of drug resistance mechanisms is important in the development of clinical therapies for the treatment of leukemia. To study the drug resistance mechanisms, protein expression profiles of 1-ß-D-arabinofuranosylcytosine (AraC)-sensitive K562 (K562S) cells and AraC-resistant K562 (K562AC) cells were compared using two-dimensional fluorescence difference gel electrophoresis. In a comparison of protein expression profiles, 2073 protein spots were found to be altered, and 15 proteins of them were remarkably altered. These proteins were identified by mass spectrometry. The most differently expressed proteins were aldehyde dehydrogenase 1 family member A2 (ALDH1A2) and vimentin. Both proteins were verified using reverse transcriptase polymerase chain reaction and Western blot analysis. ALDH1A2 protein was found to be effective in AraC resistance. ALDH1A2 knock-down induced sensitivity to AraC treatment in K562AC cells, and ALDH1A2 overexpressed K562S cells acquired the AraC resistance. Furthermore, the findings also suggest that ALDH1A2 expression is increased after the appearance of AraC resistance in clinical cases. These results will be helpful in understanding the mechanism of AraC resistance.