bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Cyclosporine A and Estrogen-Receptor Inhibitor on the Reversion of Multidrug Resistance of K562/A02 Line.
Authors: Chen Bao-An , Bao Wen , Gao Feng , Cheng Jian , Gao Chong , Ding Jia-Hua , Sun Yun-Yu , Wang Jun , Zhao Gang , Chen Ning-Na , Fei Fei 
Published: April 2020

Abstract
Objective: This paper was to study the reverse effect of cyclosporine A and the estrogen-receptor inhibitor, raloxifene, on multidrug resistance cell line K562/A02 and to investigate the reversal mechanism of this combination, and to provide theoretic evidence for the clinical application of them as resistance modifying agents.
Method: The IC50 (the concentration causing 50% inhibition of cell growth) of DNR were assayed by MTT method; mdr-1 mRNA was assayed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR).; intracellular DNR concentrations and the apoptosis and p-glycoprotein (p-gp) expression in K562 and K562/A02 were detected by fluorometry .
Results:
The IC50 of DNR for K562/A02 and K562 cells were 23.51mg/L and 0.29mg/L respectively. Pretreating K562/A02 cells with CsA(1mg/L) or raloxifene(2.5mg/L) for 48 hours partially restored the sensitivity of K562/A02 cells to DNR (IC50 were5.98mg/L and 8.15mg/L respectively) ,but had no effect on K562 cells;IC50 of combined cyclosporine A and raloxifene was 3.68mg/L The intracellular DNR concentrations( [DNR]i) in K562 cells were higher than that in K562/A02 cells,[DNR]i in K562/A02 cells was 12% of K562. Pretreating K562/A02 cells with cyclosporine A and raloxifene alone or combination for 48 hours induced the enhancement of [DNR]i in K562/A02 cells([DNR]i:CsA alone 33% of K562; raloxifene alone 12.78%of K562;CsA+raloxifene 45.29% of K562 Cyclosporine A and raloxifene alone or combination could decrease the expression of P-gp in K562/A02,the effect for 48 hours as follows: control(K562) 3.58,control(K562/A02) 104.96; CsA alone 78.29; raloxifene alone 85.02;CsA+raloxifene 59.89 K562/A02 showed apoptotic characteristics after treated with cyclosporine A for 48 hours and no apoptotic characteristics after treated with raloxifene for 48 hours Cyclosporine A and raloxifene alone could down regulate the expression of mdr-1 gene mRNA in K562/A02, cyclosporine A and raloxifene combination could markedly down regulate the expression of mdr-1 gene mRNA in K562/A02, the effect for 48 hours as follows(mdr1/β-action):control 1.313;CsA alone 1.232;raloxifene alone 1.052;CsA+raloxifene 0.449.
Data was analyzed by SPSS 11.0 software and expressed as mean ± SD.
Conclusions:
Activation of P-gp may be involved in the mechanism of MDR of K562/A02 cell line; Multidrug resistance (MDR) can be partially reversed by CsA or raloxifene, of which the combination shows a great synergistic reversal effect.
A Farnesyltransferase Inhibitor Manumycin A Induces Oxidative DNA Damage and Apoptosis Via Oxidative Stress in Leukemia Cells.
Authors: She Miaorong , Kunyuan Guo , Hu Haiyan , Niu Xinqing , Tu Sanfang , Lu Xiaoxun 
Published: April 2020

Abstract
Farnesyltransferase inhibitors (FTIs) are currently under investigation for the treatment of leukemia. The mechanism of action of FTIs has not been clarified .In this study, we investigated the mechanism of actions of FTI manumycin in acute myeloid leukemia (AML) cells. We found that manumycin A (Manu) had an antineoplastic and apoptotic effect on leukemia cells. Manu significantly increased 8-OhdG, a reliable marker for assessment of oxidative DNA damage, formation. Manu also rapidly increased the phophorylation of H2AX in HL-60 and U937 cells. Methoxyamine, an inhibitor of base excision repair, enhanced the phophorylation of H2AX induced by Manu. Furthermore, Manu could induce phosphated p38 MAPK in HL-60 and U937 cells, and inhibitor of p38 MAPK could rescue leukemia cells from Manu-induced cytotoxicity. Moreover, Quenching of ROS with NAC prevented DNA damage, p38 phosphorylation and apoptosis by Manu. These results suggest the hierarchical position of ROS in oxidative DNA damage and apoptosis pathway. In conclusion, FTI induced oxidative DNA damage by induction of ROS and initiated the DNA damage responses then trigger the apoptosis involved p38 MAPK activation.
Autophagy and Apoptosis Are Induced Simultaneously in Leukemia Cells by Vitamin K2.
Authors: Miyazawa Keisuke , Yokoyama Tomohisa , Toyotake Juri , Gotoh Akihiko , Naito Munekazu , Ito Masahiro , Yuo Akira , Kondo Seiji , Ohyashiki Kazuma 
Published: April 2020

Abstract
Autophagy is a membrane trafficking process that leads to degradation of cytosolic proteins and organelles by lysosomes, and it is conserved throughout evolution. Autophagy is induced above basal levels in response to nutrient deprivation or trophic factor withdrawal, and it sustains metabolism through the targeted catabolism of long-lived proteins. Thus, autophagy acts as a self-limited survival mechanism. However, recent findings have suggested that autophagy is also as a potential contributor to programmed cell death.
We previously reported that vitamin K2 (VK2) selectively and potently induces the apoptosis of cultured acute myeloid leukemia cells (AML) and AML cell lines, but has almost no effect on normal hematopoietic cells. In addition, the clinical benefit of using VK2 for patients with AML and myelodysplastic syndrome has been reported. In the present study, we focused on whether autophagy was induced in AML cells by treatment with VK2.
HL-60bcl-2 cells with stable transfection of the bcl-2 gene showed 5-fold greater expression of BCL-2 compared with HL-60neo cells, and these cells were resistant to apoptosis induced by VK2, along with the occurrence of monocytic differentiation via G1 arrest. This suggested that VK2 had a differentiation-inducing effect on AML cells that were resistant to apoptosis. However, a cytocidal effect was still detectable in a caspase-independent manner. Treatment of HL-60bcl-2 cells with VK2 resulted in an increase of acid vesicular organelles (AVO) detected by acridine orange staining (for lysosomes).
Immunonoblotting with an antibody for microtubule-associated protein-1 light chain-3 (LC3) revealed the conversion of LC3-I into LC3-II a form that migrates more rapidly on SDS-PAGE and participates in the formation of autophagosomes (one of the hallmarks of autophagy). Electron microscopy revealed an increase of autophagosome formation after exposure to VK2. Finally, the induction of caspase-independent cell death by VK2 was suppressed in the presence of 3-methyladenine. These results suggest that autophagic death of HL-60bcl-2 cells was induced by VK2. Furthermore, detailed electron microscopic assessment of HL-60neo cells during exposure to VK2 revealed that autophagosome formation was more prominent compared with that occurring in H-60bcl-2 cells, although nuclear chromatin condensation was also observed at the same time. These findings indicated a mixture of the morphologic features of apoptosis and autophagy. The present results suggest that autophagy and apoptosis can both be induced simultaneously by VK2. However, autophagy only became detectable when cells were protected from rapid apoptotic death.
Polydeoxyribonucleotides Antagonize Nucleoside Analogues in Cancer Therapy.
Authors: Yang Ting , Steinmann Ingo , Tank Simone , Chen Zhizhe , Kolb Hans-Jochem , Buhmann Raymund 
Published: April 2020

Abstract
Background: In the past years, an increasing number of nucleic acid based drug candidates (e.g. antisense oligonucleotides, aptamers, ribozymes, RNA interference) have been identified and already tested in clinical trials. But until now, no information is available whether these treatment approaches might interfere with chemically related drugs, e.g. nucleoside analogues used in cancer or viral therapy.
Objective: In the current survey we investigated, whether randomly synthesized single-stranded polydeoxyribonucleotides of different length could interfere with the cytotoxic effects of nucleoside analogues (e.g. fludarabine, cytarabine) on lymphocytes or myeloid blasts.
Methods: For this purpose, T-lymphocytes or CD33 positive myeloid blasts (purity &gt;95%) were labelled with [1μM] carboxyfluorescein diacetate, succinimidyl ester (5(6)-CFDA-SE) and incubated with different concentrations of fludarabine or cytarabine (200μM, 20μM und 2μM). Cellular proliferation was induced by addition of CD3/CD28 Dynabeads for the T-cells, or a cytokine cocktail containing 50ng/mL of SCF, 25ng/mL of IL-3, 100ng/mL of GM-CSF, 100ng/mL of G-CSF, 2 U/mL of EPO, 0.47g/L of transferrin, and 5×10-5mmol/L of 2-ME for the myeloid blasts. Random polydeoxribonucleotides of different length (20–80b) and concentrations were added immediately or with a delay of 24, 48 or 72 hours to the assay. After 5 days, quantitative CFDA distribution was measured by flow cytometry to assess the cellular proliferation of the T cells and the myeloid blasts. Moreover, cell viability was measured by trypan blue exclusion. Each experiment was performed at least three times.
Results: Polydeoxyribonucleotides of different length and composition could antagonize the cytotoxic effects of nucleoside analogues (fludarabine, cytarabine) in both, T-cells and myeloid blasts. Thereby, the antagonistic effects of polydeoxyribonucleotides revealed to be concentration- and time dependent. Moreover interference with a equimolar concentrations of polydeoxyribonucletides could be detected even with a delay of 48 hours. Of importance, the concentration of nucleoside analogues used in these experiments, referred to concentrations applied in standard clinical treatment protocols.
Conclusion: Treatment approaches using polydeoxyribonucleotides, might be critical in context with nucleoside analogues, like fludarabine and cytarabine and diminish the efficacy of these drugs. Further experiments are necessary to elucidate the precise mechanism of these effects.
Hypoxia Induces Imatinib Resistance in CML Cell Lines.
Authors: Buglio Daniela , La Cava Piera , Stagno Fabio , Chiarenza Annalisa , Berretta Salvatore , Aloisi Alesandra , Palumbo Giuseppe A. , Vigneri Paolo , Di Raimondo Francesco 
Published: April 2020

Abstract
Background: Imatinib is a potent tyrosine kinase inhibitor that is highly effective in the treatment of chronic myeloid leukemia. However, some patients are resistant to imatinib. Some of the mechanisms leading to imatinib resistance include amplification of the BCR-ABL gene, determining overexpression of the protein, and mutations in the BCR-ABL protein with alteration of imatinib binding sites. Imatinib uptake is an active process mediated by a group of transporters that includes the organic cation transporters (hOCT) and it has been shown that different expression of OCT1 may play a critical role on intracellular drug levels and, hence, resistance to imatinib. Hypoxia is another important factor that may contribute to drug resistance. Hypoxia-Inducible Factor (HIF-1α) and its downstream target, Vascular endothelial Growth Factor (VEGF), have been shown to be overexpressed in leukemic bone marrow specimens compared to normal bone marrow. The purpose of this study is to determine if the hypoxic conditions and OCT1 inhibition affect imatinib sensitivity.
Methods: Chronic myeloid leukemic cell line K562 and LAMA84 were cultured in 10% serum RPMI medium under hypoxic (3% O2) or normoxic (21% O2) conditions. All samples were treated with imatinib 1μM ± prazosin 13 μM (OCT1 reversible inhibitor) for 24 hs.
Results: Cells treated with imatinib and cultured under hypoxic conditions demonstrated decreased apoptosis and increased cell viability compared to normoxic conditions (K562 Annexin + cells 62% vs 94%, p= 0.003, LAMA84 Annexin + cells 61% vs 92%, p=0.0028). The addition of prazosin almost abrogated imatinib efficacy in normoxic environment but did not modify the effect of imatinib under hypoxic conditions.
Conclusions: Our data confirm that OCT1 is the most important imatinib carrier. Exposure of CML cell lines to an hypoxic environment results in reduced sensitivity to imatinib and this effect wasn’t affected by OCT1 inhibition. Search for underlying mechanisms of these findings are in progress.
MDR-1 Gene and BCRP Are Involved in the Resistance to Imatinib in K-562 Cell Line.
Authors: Lardo Mabel , Gargallo Patricia , Bianchini Michele , Lucero Graciela , Koziner Benjamin , Larripa Irene , Lazarowski Alberto 
Published: April 2020

Abstract
Drug resistance to imatinib in chronic myeloid leukemia (CML) has been linked to BCR/ABL gene amplification or mutations in its tyrosine kinase domain of BCR/ABL gene (Phi). However the role of the ABC-transporters in the multidrug resistant phenotype still remains to be clarified.
Objective: to study the functional expression of multidrug resistance gene-1 (MDR-1) and breast cancer resistant protein (BCRP), in imatinib pharmacoresistant K562 Phi+ cells.
Materials and methods: K562 Phi+ cultured cells (ATCC catN°:CCL-2439) were immunophenotyped by flow cytometry, mRNA transcripts of BCR/ABL and MDR-1 were amplified by RT-PCR and P-glycoprotein (P-gp), the product of MDR-1 gene and BCRP, were investigated by inmunocytochemistry, using specific monoclonal antibodies (P-gp: clones C494 and C219; BCRP: clone BXP-21). Total efflux activity of Rhodamine-123 (Rho-123) and its inhibition by Cyclosporin A (CyA) or 4°C, were evaluated using flow cytometry. The apoptotic effect in K-562 cells after 24hs and 72hs of treatment with imatinib (2μmol.L−1) with or without CyA (3ugm.L−1), was evaluated by staining the cells with 1 mL of a mixture of acridine orange (100 mg.mL−1) and ethidium bromide (100 mg.mL−1) in PBS.
Results: The immunophenotype was: HLA-DR-,CD45++,CD11c−/+,CD13+,CD14−,CD15−/+,CD16−,CD33+,CD34−CD117−/+; CD56−, CD61−/+; CD2−, CD3−, CD4−, CD7−, CD8−, CD10+, CD19−, glyA+ . MDR-1 and BCR/ABL transcripts were highly expressed, and P-gp and BCRP immunostaining were also positive. Rho-123 efflux was inhibited by CyA (3ugm.L−1) or 4°C. Increased apoptotic effect (from 7, 21% to 20, 79%) at 24hs and (11% to 53%) at 72hs was observed with combined imatinib+CyA treatment compared with imatinib alone.(p&lt;0.01*)
Conclusions. Our results suggest that P-gp and BCRP might play an active role in the pharmacoresistance to imatinib treatment in K562Phi+ cells. Combined treatment with Imatinib+CyA (or other more specific inhibitors of P-gp and BCRP) could increase the apoptotic effect in BCR/ABL over-expressing refractory cells.
Figure Figure
ASK1 Is Activated by Treatment with Arsenic Trioxide through Accumulation of Reactive Oxygen Species and May Suppress Apoptosis through NF-kappaB in Leukemia Cells.
Authors: Arai Ayako , Yan Weihua , Ichijo Hidenori , Miura Osamu 
Published: April 2020

Abstract
Arsenic trioxide (ATO), a remarkably effective reagent for treatment of relapsed acute promyelocytic leukemia (APL), is reported to induce differentiation of APL cells at low concentrations and apoptosis at high concentrations. Induction of granulocytic differentiation is considered to be a specific effect of ATO on APL cells. By contrast, ATO commonly induces apoptosis of various tumor cells of hematological malignancies, including chronic myeloid leukemia cells expressing the BCR/ABL fusion kinase, as well as those of solid tumors. It was reported that ATO treatment induced intracellular accumulation of reactive oxygen species (ROS) by inhibiting glutathione peroxidase activity. Accumulated ROS induced a decline in cellular mitochondrial membrane potential, followed by cytochrome c release, caspase 3 activation, and apoptosis of cells. On the other hand, it is well known that adequate dose of ROS is indispensable for proliferation and survival of a variety of cells, including hematopoietic cells. Thus, various intracellular signaling pathways are strictly regulated and activated downstream of ROS to promote or suppress apoptosis, and the signaling pathways activated by ROS accumulation induced by ATO need to be defined to understand the mechanisms for ATO-induced apoptosis.
Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase that activates the JNK and p38 signaling pathways and is involved in regulation of apoptosis. Here we find that ATO treatment of NB4 and K562 leukemia cells induces activation of ASK1. ATO induced accumulation of ROS, and the ASK1 activation was suppressed by cotreatment with an antioxidant, N-acetyl-l-cysteine. Although the Rho family GTPases Rac and cdc42 were activated by ATO, overexpression of their dominant-negative mutants did not suppress ATO-induced ASK1 activation. ASK1 activation was induced most significantly at low concentrations of ATO, where G2/M arrest but not apoptosis was induced. On the other hand, ASK1 activation induced by ATO was barely detectable at high concentrations, where apoptosis was induced significantly. By contrast, JNK and p38 were activated in dose-dependent manners by ATO. Murine embryonic fibroblasts (MEFs) derived from ASK1-deficient mice were more prone to ATO-induced apoptosis than control MEFs. Moreover, ASK1 was activated by ATO in a more sustained manner in ATO-resistant leukemia cell lines than in sensitive cell lines. Finally, a dominant-negative ASK1 mutant reduced ATO-induced NF-kappaB activation in leukemia cells. Together, the results indicate that ASK1 is activated by ATO through ROS generation independent of activation of Rac and cdc42 and may play a negative role in induction of apoptosis, possibly through activation of NF-kappaB.
Notch Signaling Regulates Hematopoietic Stem Cell Homeostasis in the Fetal Liver through a Non-Cell-Autonomous Mechanism.
Authors: Maillard Ivan , High Frances , Shestova Olga , Dedhia Priya , Epstein Jonathan A. , Pear Warren S. 
Published: April 2020

Abstract
Notch is essential for the generation of definitive hematopoietic stem cells (HSCs) during fetal life, but its effects on subsequent HSC self-renewal are controversial. We have shown recently that canonical Notch signaling is dispensable for the maintenance of stem cell function in adult HSCs (Maillard et al., submitted). We now investigate whether Notch regulates HSC homeostasis during fetal development. For this purpose, we have developed a system to track individual Notch-deprived cells through expression of the GFP-tagged pan-Notch inhibitor DNMAML (Maillard et al., Blood 2004). We have used this system to conditionally inactivate Notch signaling at defined time points during development. Specifically, we crossed mice carrying an ubiquitously expressed Tamoxifen(TAM)-inducible Cre recombinase transgene (CAGG-CreERTm) with mice harbouring a Cre-inducible DNMAML construct (DNMAMLf). We injected TAM i.p. at mid-gestation (E11.5), after generation of fetal HSCs. At E13.5, DNMAML-GFP protein expression was induced in 20–45% of hematopoietic progenitors in the Lin-Sca-1hic-Kithi (LSK) fraction. As compared to control littermates, TAM-induced CAGG-CreERTm x DNMAMLf (TICD) fetuses initially showed increased numbers and decreased quiescence of fetal liver LSK cells: at E13.5, TICD LSK progenitors had increased cell size, expressed elevated levels of Sca-1, and showed an increase in the fraction of cells in S-G2M phases of the cell cycle. Significantly, these abnormalities were observed both within the GFP- and GFP+ TICD LSK fractions, indicating non-cell-autonomous disruption of their homeostasis. At subsequent time points (E15.5), TICD fetuses showed a marked decrease in the number of LSK progenitors, most prominently within the CD150+CD48− subset highly enriched for long-term HSCs (LT-HSC), suggesting HSC exhaustion. Histological examination revealed multiple small areas of hemorrhage in TICD tissues consistent with underlying vascular defects. Fetal death was observed at E17.5–18.5. Altogether, our findings show that continuous Notch signaling is absolutely required for normal development during mid-gestation and suggest that Notch controls a key element of the fetal HSC niche that promotes LT-HSC self-renewal and quiescence. Because inactivation of Notch signaling causes phenotypic changes associated with vascular defects, we speculate that Notch regulates critical vascular elements of the stem cell niche in the fetal liver.
Pyrrolo[2,3-b]Pyridinic Derivatives Induce Cell Growth Inhibition of the Myeloblastic HL-60 Cell Line.
Authors: Decrop Maylis , Espanel Claire , Guillard Jerome , Boissier Elodie , Gallay Nathalie , Binet Christian , Viaud-Massuard Marie-Claude , Herault Olivier 
Published: April 2020

Abstract
Cytokins, N6-subsitued adenine derivatives play an important role in many different processes in plant development (Mok et al, Annu Rev Plant. Physiol Plant Mol Biol2001, 52:89–118), including the cell growth and division control, the vegetable cell differentiation with auxin and the storage of various metabolites as alkaloids (Yahia et al, Plant Science1998, 133:9–15). Furthermore, several studies suggest that cytokinins and their purin derivated are able to control mammalian cell apoptosis and differentiation of human leukemic cells (HL-60 myeloblastic cell line) into mature granulocytes (Ishii et al, Cell Growth Differ2002, 13: 19–26). All these compounds exert their biological activity via Cyclin-Dependant Kinase (CDK) inhibition and particulary CDK1 and CDK2. The aim of our study was the synthesis of new 7-azaindole derivates as cytokinin analogues and the evaluation of their biological effects on HL-60 cells.
Eight analogues of 7-azaindole were prepared by the condension of (N-methyl-)4-chloropyrrolo[2,3-b]pyridine with corresponding amines using palladium-catalyzed reaction (Hartwig et al, Angew Chem IN Ed1998, 37: 2046–2067). The four derivates from 1H-pyrrolo[2,3-b]pyridine were 4-phenylamino-7-azaindole, 4-benzylamino-7-azaindole, 4-phenethylamino-7-azaindole and 4-phenylpiperazylamino-7-azaindole. The four derivates from 1-methyl-pyrrolo[2,3-b]pyridine were 4-phenylamino-N-methyl-7-azaindole, 4-benzylamino-N-methyl-7-azaindole, 4-phenetylamino-N-methyl-7-azaindole and 4-phenylpiperazylamino-N-methyl-7-azaindole. HL-60 cells were exposed to three concentrations of these compounds (10–100–500 μM) during 72h at 37°C. The number of viable cells was determined by Trypan blue exclusion, and the cell cycle was assessed by propidium iodide staining followed by flow cytometric analysis. All these compounds decreased the number of viable cells. The compounds of the NH serie were more active than their methyl-analogues, especially 4-phenylamino-N-methyl-7-azaindole and 4-phenethylamino-7-azaindole which presented an estimated IC50 of 2 μM. Moreover, when used at 10 μM, 4-phenylamino-N-methyl-7-azaindole induced apoptosis whereas 4-phenethylamino-7-azaindole promoted inhibition of the cell cycle without pro-apoptotic effect.
These results suggest that cytokinin analogues derived from 1H-pyrrolo[2,3-b]pyridine may present interesting therapeutic potential as cytostatic agents. Further studies will clarify their biological effects on leukemic cells.