Human Leukemia K562 Cells Research Articles

Structural characterization and cytotoxicity studies of different forms of a combretastatin A4 analogue

It is well known that combretastatin A4 (CA-4), which is a natural stilbene isolated from Combretum caffrum, is used to inhibit angiogenesis. However, depending on the dose administered to the patient, it can cause some side-effects. Herein, we present the synthesis and structural characterization of a novel N-acylhydrazone derivative – LASSBio-1735 – a CA-4 analogue. LASSBio-1735 has displayed in vitro antiproliferative activity against HL-60 (human leukemia), SF-295 (human glioblastoma), MDA-MB435 (melanoma) and HCT-8 (ileocecal adenocarcinoma) tumor cells. We found different hydration levels in two batches of the as-synthesized compound. As a consequence, we could successfully determine the crystal structures – by using X-ray powder diffraction data and a simulated annealing procedure – of the anhydrous and hydrated forms. The effects on cell viability of anhydrous and hydrated forms of LASSBio-1735 were comparatively evaluated in different tumor cell lines, and the hydrated form exhibited higher cytotoxicity in human leukemia K562 cells. These findings lead us to perform a quantitative phase analysis on one of the samples and may shed some light on the search for possible new solvates and/or hydrates.

Reversal of ABCB1 mediated efflux by imatinib and nilotinib in cells expressing various transporter levels

Recently, it has been suggested that imatinib (IM) and nilotinib (NIL) could be studied beyond their original application, as inhibitors of the drug efflux pump ABCB1 (P-glycoprotein, MDR1). Since the reversal of ABCB1-mediated resistance has never been successfully demonstrated in the clinic, we addressed the question of whether IM and NIL may actually serve as efficient inhibitors of ABCB1. Here we define an efficient inhibitor as a compound that achieves full (90–100%) reversal of drug efflux at a concentration that does not exhibit significant off-target toxicity in vitro. In this study, human leukemia K562 cells expressing various levels of ABCB1 were used. We observed that cells expressing higher ABCB1 levels required higher concentrations of IM and NIL to achieve full reversal of drug efflux. Among the well-known ABCB1 inhibitors, a similar effect was found for cyclosporin A (CsA) but not for zosuquidar. IM was efficient only in cells with the low and moderate ABCB1 expression at high concentrations that were cytotoxic in the absence of Bcr-Abl. In contrast, NIL was as efficient an inhibitor of ABCB1 as CsA. Low and moderate expression levels of ABCB1 could be efficiently inhibited by NIL concentrations without cytotoxic effects in the absence of Bcr-Abl. However, high expression levels of ABCB1 required higher NIL concentrations with off-target cytotoxic effects. In conclusion, application of NIL, but not of IM, in clinics is promising, however, only in cells with low ABCB1 expression levels. We hypothesize that some patients may benefit from an inhibitor exhibiting an ABCB1 expression-dependent effect.

Splicing factor SF3B1K700E mutant dysregulates erythroid differentiation via aberrant alternative splicing of transcription factor TAL1.

More than 60% of myeloid dysplasia syndrome (MDS) contains mutations in genes encoding for splicing factors such as SF3B1, U2AF, SRSF2 and ZRSR2. Mutations in SF3B1 are associated with 80% cases of refractory anemia with ring sideroblast (RARS), a subtype of MDS. SF3B1K700E is the most frequently mutated site among mutations on SF3B1. Yet the molecular mechanisms on how mutations of splicing factors lead to defective erythropoiesis are not clear. SF3B1K700E mutant binds to an RNA binding protein, RBM15, stronger than the wild type SF3B1 protein in co-immunoprecipitation assays. In addition, K700E mutant alters the RNA splicing of transcription factors TAL1 and GATA1. Via alternative RNA splicing, a novel short TAL1 transcript variant (TAL1s) is generated. Enhanced interaction between SF3B1 and RBM15 promotes the production of full-length TAL1 (TAL1fl) mRNA, while reduction of RBM15 protein level via PRMT1-mediated degradation pathway changes TAL1s/TAL1fl ratio in favor of TAL1s. TAL1s contains the helix-loop-helix DNA binding domain but not the N terminal region upstream of the DNA binding domain. The TAL1s protein loses its interaction with ETO2, which represses early erythropoiesis. In this vein, overexpression of TAL1s stimulates the transcription of β-hemoglobin in human leukemia K562 cells and promotes erythroid differentiation of human cord blood CD34+ cells cultured in erythropoietin-containing medium. Therefore, mutations of SF3B1 may block erythropoiesis via dysregulation of alternative RNA splicing of transcription factor TAL1, and targeting PRMT1 may alleviate the anemic symptoms in MDS patients.

Genome-wide TOP2A DNA cleavage is biased toward translocated and highly transcribed loci.

Type II topoisomerases orchestrate proper DNA topology, and they are the targets of anti-cancer drugs that cause treatment-related leukemias with balanced translocations. Here, we develop a high-throughput sequencing technology to define TOP2 cleavage sites at single-base precision, and use the technology to characterize TOP2A cleavage genome-wide in the human K562 leukemia cell line. We find that TOP2A cleavage has functionally conserved local sequence preferences, occurs in cleavage cluster regions (CCRs), and is enriched in introns and lincRNA loci. TOP2A CCRs are biased toward the distal regions of gene bodies, and TOP2 poisons cause a proximal shift in their distribution. We find high TOP2A cleavage levels in genes involved in translocations in TOP2 poison–related leukemia. In addition, we find that a large proportion of genes involved in oncogenic translocations overall contain TOP2A CCRs. The TOP2A cleavage of coding and lincRNA genes is independently associated with both length and transcript abundance. Comparisons to ENCODE data reveal distinct TOP2A CCR clusters that overlap with marks of transcription, open chromatin, and enhancers. Our findings implicate TOP2A cleavage as a broad DNA damage mechanism in oncogenic translocations as well as a functional role of TOP2A cleavage in regulating transcription elongation and gene activation.

Anthranilamide-based 2-phenylcyclopropane-1-carboxamides, 1,1'-biphenyl-4-carboxamides and 1,1'-biphenyl-2-carboxamides: Synthesis biological evaluation and mechanism of action

Several anthranilamide-based 2-phenylcyclopropane-1-carboxamides 13a-f, 1,1’-biphenyl-4-carboxamides 14a-f and 1,1’-biphenyl-2-carboxamides 17a-f were obtained by a multistep procedure starting from the (1S,2S)-2-phenylcyclopropane-1-carbonyl chloride 11, the 1,1'-biphenyl-4-carbonyl chloride 12 or the 1,1'-biphenyl-2-carbonyl chloride 16 with the appropriate anthranilamide derivative 10a-f. Derivatives 13a-f, 14a-f and 17a-f showed antiproliferative activity against human leukemia K562 cells. Among these derivatives 13b, 14b and 17b exerted a particular cytotoxic effect on tumor cells. Derivative 17b showed a better antitumoral effect on K562 cells than 13b and 14b. Analyses performed to explore 17b mode of action revealed that it induced an arrest in G2/M phase of cell cycle which was consequent to DNA lesions as demonstrated by the increase in phospho-ATM and γH2AX, two known markers of DNA repair response system. The effect of 17b was also related to ROS generation, activation of JNK and induction of caspase-3 dependent apoptosis.

Phytochemical Analysis and Cytotoxicity Evaluation of Kelussia odoratissima Mozaff.

BackgroundKelussia odoratissima Mozaff. (Apiaceae) is an edible, indigenous, and ethnomedicinal plant that grows only in Iran. Although antioxidant and anti-inflammatory properties of K. odoratissima have been reported, cytotoxic activity of this plant has not been investigated previously. ObjectiveThis study aims to evaluate the cytotoxicity of K. odoratissima leaf extract against a panel of human cancer cell lines. A secondary aim was to perform a phytochemical analysis of the plant’s leaf oil. MethodsTo extract the plant oil, dried leaves were subjected to hydrodistillation using a Clevenger-type apparatus for up to 3 hours. For the phytochemical analysis, essential oil was subjected to gas chromatography/mass spectrometry. Plant extraction was performed by macerating leaf powder of K. odoratissima (50 g) in 70% methanol (500 mL) at room temperature (25–28°C) for 24 hours. To perform cytotoxicity assays, methanolic extract of K. odoratissima was tested against a panel of cell lines including MDA-MB468 (human breast cancer cell line), K562 (human leukemia cell line), SKOV3 (human ovarian cancer cell line), Y79 (human eye cancer cell line), A549 (lung cancer cell line), and HEK 293 (normal human embryonic kidney cell line). ResultsGas chromatography/mass spectrometry analysis revealed that sesquiterpens are dominant volatile components of the plant, followed by phthalides comprising 3-butyldine phthalide and 3-n-butyl phthalide, the latter compound being the major component of the leaf oil (25.1%). The leaf extract showed selective and dose-dependent cytotoxicity against MDA-MB468, K562, SKOV3, Y79, and A549 cancer cell lines with IC50 values (concentration that inhibits cell growth by 50%) of 85 μg/mL, 70 μg/mL, 120 μg/mL, 82 μg/mL, and145 μg/mL, respectively. ConclusionsThe present results suggest a direct cytotoxic activity of K. odoratissima leaf extract against human cancer cell lines. This activity of K. odoratissima may find application in combination with traditional herbal medicines to develop a new anticancer pharmacopuncture therapy.

Et2NH catalysis-enabled construction of convolutamydine A-fused morusignin L-scaffolds and their biological evaluation for anticancer activities

ABSTRACTDescribed herein is a facile and efficient methodology toward the synthesis of novel convolutamydine A-incorporated morusignin L scaffold 3 from an aldol addition event of carbonyl compounds to isatins through enamine catalysis. Products featuring a quaternary carbon center were smoothly obtained in good yields (up to 85% yield). This protocol also represents the first construction of flavonoid skeleton-fused oxindole molecules, thus leading to new knowledge in the fields of both molecular complexity and diversity synthesis and the lead compound discovery. Furthermore, their biological activities against human prostate cancer cells PC-3 and human leukemia cells K562 have been preliminarily demonstrated by in vitro assays. The results demonstrated that most of these compounds obtained by this protocol showed comparable or even much better activity than the positive control of cisplatin.

LncRNA MEG3 Regulates Imatinib Resistance in Chronic Myeloid Leukemia via Suppressing MicroRNA-21.

Imatinib resistance has become a major clinical problem for chronic myeloid leukemia. The aim of the present study was to investigate the involvement of MEG3, a lncRNA, in imatinib resistance and demonstrate its underlying mechanisms. RNAs were extracted from CML patients’ peripheral blood cells and human leukemic K562 cells, and the expression of MEG3 was measured by RT-qPCR. Cell proliferation and cell apoptosis were evaluated. Western blotting was used to measure the protein expression of several multidrug resistant transporters. Luciferase reporter assay was performed to determine the binding between MEG3 and miR-21. Our results showed that MEG3 was significantly decreased in imatinib-resistant CML patients and imatinib-resistant K562 cells. Overexpression of MEG3 in imatinib-resistant K562 cells markedly decreased cell proliferation, increased cell apoptosis, reversed imatinib resistance, and reduced the expression of MRP1, MDR1, and ABCG2. Interestingly, MEG3 binds to miR-21. MEG3 and miR-21 were negatively correlated in CML patients. In addition, miR-21 mimics reversed the phenotype of MEG3-overexpression in imatinib-resistant K562 cells. Taken together, MEG3 is involved in imatinib resistance in CML and possibly contributes to imatinib resistance through regulating miR-21, and subsequent cell proliferation, apoptosis and expression of multidrug resistant transporters.

Drug resistance of cancer cells is crucially affected by expression levels of ABC-transporters

Dasatinib (DAS), a second generation of tyrosine kinase inhibitor (TKI), represents excellent choice for the treatment of chronic myeloid leukemia resistant to imatinib. Unfortunately, recent laboratory studies suggested that antiproliferative effect of DAS might be significantly reduced due to the overexpression of the ATP-binding cassette (ABC) transporters, ABCB1 and ABCG2. However, whether these drug transporters might compromise therapeutic effect of DAS in clinic is unclear. We believe that the drug transporter expression level is a crucial factor that affects the results and its consideration may help to explain the existing controversy. In addition, clinically relevant concentrations of drug must be used. In our study, human leukemia K562 cells with high and low expression levels of ABCB1 or ABCG2 were used. DAS was applied at nanomolar concentrations. We observed that K562 cells expressing high levels of ABCB1 and ABCG2 contained significantly reduced intracellular levels of DAS and these cells exhibited significantly increased resistance to this drug. Importantly, cells with the low expression of ABCB1 or ABCG2 effluxed DAS less efficiently, however, still significantly. Accordingly, the observed resistance was lower but significant. Conclusions: The antiproliferative effects of DAS might be reduced by ABCB1 or ABCG2. However, the actual effect of these ABC transporters on DAS efficiency depends on their expression levels. The lower expression levels of ABC transporters mediate lower resistance. Considering the fact that expression levels of ABCB1 and ABCG2 transporters are usually low in clinical samples, their contribution to the overall resistance to DAS is probably low but significant.

Effects of shRNA Targeting mPGES-1 on Tumorigenicity of K562 Cells in Nude Mice In Vivo

To investigate the effects of shRNA targeting mPGES-1 on tumorigenicity of human acute leukemia K562 cells in nude mice in vivo and its mechanisms. For experiment 3 groups including KD group(expression of mPGES-1 in K562 cells was down-regulated by shRNA), CON (cells without any treatment) and NC group (cells treated with nonspecific-sequence shRNA) were set-up. Western blot was used to test the expression of β-catenin and cyclinD1 in cells. Then the cells of 3 groups were implanted into BALB/c nude mice subcutaneously to establish murine xenograft model. The growth state of the mice and the size of the xenograft tumor were recorded. HE staining was used to observe the morphology of xenograft tumor. Expressions of β-catenin and cyclinD1 in xenograft tumor were detected by immunohistochemical staining. In vitro the expression of β-catenin and cyclinD1 in KD group were lower than the CON group and NC group (P<0.05). In vivo the tumor volume and weight of KD group were significant smaller than the other two groups (P<0.01). HE staining showed that tissues in the KD group were relatively looser in arrangement with smaller cell nucleus and less cytoplasm. The expression of β-catenin and cyclinD1 in the KD group were remarkable weak as compared with that in CON group and NC group (P<0.05). Down-regulating the expression of mPGES-1 by shRNA may significantly inhibit the tumorigenicity of K562 cells in nude mice in vivo and its mechanism may be related with the inhibition of expression of β-catenin and cyclinD1.

Synthesis, characterization and in vitro biological activities of new water-soluble copper(II), zinc(II), and nickel(II) complexes with sulfonato-substituted Schiff base ligand

A water-soluble thiosemicarbazone Schiff base ligand, namely sodium 3-{[(aminocarbonothioyl)hydrazono]methyl}-4-hydroxybenzenesulfonate (NaH2L) was prepared by 1:1 condensation of sodium salicylaldehyde-5-sulfonate and thiosemicarbazide. The X-ray structure analysis of NaH2L shows that the organic fragments of the ligand are bridged by two different types of seven-coordinated sodium cations. The ligand was used in the synthesis of the following CuII, NiII, and ZnII complexes: [Cu2(HL)2(H2O)3] (1), Ni(HL)(H2O)1.5 (2), and Zn(HL)(H2O)2 (3). The metal complexes were characterized by physico-chemical and spectroscopic methods. The X-ray structure analysis of the dinuclear copper complex (1) shows that two metal centers are five coordinated having distorted square pyramidal geometries. There are significant π-π interactions between the phenyl rings of the two ligands in the complex with the centroid-centroid distance 3.8571(10)Å. Solubility in water at 25°C was measured 100g/L for NaH2L, 50g/L for 1, 100g/L for 2 and 0.1g/L for 3. The stability of NaH2L, 1 and 2 in water solution exceeded 4days. The compounds 1 and 2 showed low antimicrobial activity while the inhibition efficiency of NaH2L and 3 was negligible. The ligand and complexes were evaluated for scavenging activity of picrylhydrazyl radical (DPPH) and ferric reducing antioxidant power (FRAP), using l-ascorbic acid (Vitamin C) as a reference antioxidant. The strongest radical scavenging was found for NaH2L. Cytotoxicity of the compounds was investigated against the human chronic myeloid leukemia K562 cells by the MTT assay method. The strongest cytotoxicity was found for 1 with the IC50 value of 16±1.0μg/mL for 72h and therefore 1 was chosen for further studies. The compound 1 caused a noticeable decrease in viability of the K562 cells in a dose- and time-dependent manner and induced apoptosis in the K562 cell line.

Isolation and characterisation of cytotoxic compounds from Euphorbia clementei Boiss.

A new phorbol-type diterpene ester, 4,20-dideoxy-4α-phorbol-12β-acetate-13α-isobutyrate, in addition to 11 known compounds were isolated from the latex and roots of Euphorbia clementei Boiss. Structure elucidation was performed by comprehensive 1D and 2D NMR analyses (1H and 13C NMR, COSY, ROESY, HSQC and HMBC experiments), mass spectrometry (HR-ESI-MS) and by comparison with literature data. The inhibitory activity of all isolated compounds was evaluated against promyelocytic leukemia HL60 and human erythromyeloblastoid leukemia K562 cell lines and seven of these compounds exhibited a weak cytotoxicity with IC50 values ranging from 40 to 97 μM.

Synthesis of Functionalized 3-Spiro[cyclopropa[a]pyrrolizine]- and 3-Spiro[3-azabicyclo[3.1.0]hexane]oxindoles from Cyclopropenes and Azomethine Ylides via [3 + 2]-Cycloaddition.

3-Spiro[cyclopropa[a]pyrrolizine]- and 3-spiro[3-azabicyclo[3.1.0]hexane]oxindoles were prepared in moderate to high yields via one-pot three-component reactions using substituted isatins, α-amino acids, and cyclopropenes. The key step is an intramolecular [3 + 2]-cycloaddition reaction of an in situ generated azomethine ylide onto a cyclopropene. Both N-substituted and N-unsubstituted α-amino acids, dipeptide Gly-Gly, and also benzylamine were used as the amine component for the azomethine ylide generation. The anticancer activity of some of the obtained compounds against human leukemia K562 cell line was evaluated by flow cytometry in vitro.

Synthesis of New trans-Dehydrocrotonin Nitrogenated Derivatives and their Cytotoxic and DNA-Topoisomerase I Inhibitory Activities

A new series of 19-nor-clerodane diterpene derivatives was synthesized from the natural trans-dehydrocrotonin obtained from stem barks of Croton cajucara (Euphorbiaceae), a native medicinal plant of the Brazilian Amazon. The new derivatives were obtained by changes in the ketone moiety of trans-dehydrocrotonin leading to nitrogenated derivatives which are: three substituted hydrazine diterpenes, oxime, and methyloxime. The cytotoxic effect of the diterpene derivatives was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay against Ehrlich carcinoma and K562 human leukemia cells. The cytotoxic activity of the hydrazine and oxime semi-synthetic derivatives was better than the one of the natural product trans-dehydrocrotonin. Moreover, all diterpenes were tested for their DNA topoisomerase I inhibitory activity, and the most effective one, in general, was observed to the phenyl-hydrazone derivative. Results indicated that the topoisomerase I inhibitory effect is correlated with the cytotoxic activity.

Tuberostemonine reverses multidrug resistance in chronic myelogenous leukemia cells K562/ADR.

Objective: To investigate the reversal effect of tuberostemonine on MDR in myelogenous leukemia cells K562/ADR.Methods: Human myelogenous leukemia cells K562 and their adriamycin-resistance cells K562/ADR were used. The growth curve of cells treated by tuberostemonine and the Non-toxic concentration of tuberostemonine were determined by MTT, Cell apoptosis was determined by MTT and flow cytometry. The expression of MDR1, Survivin and Livin was detected by RT-PCR. The activity of P-gp was detected by flow cytometry. Western blot was used to detect the expression of NF-κB and Survivin.Results: The effect of tuberostemonine on K562/ADR showed a dose-dependence, and 350μg/mL and 500μg/mL of tuberostemonine could inhibit the expression of MDR1 (P<0.05). While no function difference of P-gp was detected. With the increased concentration of tuberostemonine, the inhibitory effect were enhanced to the expression of NF-κB. Tuberostemonine combined with adriamycin could time-dependently inhibit the cell proliferation (P<0.05) and obviously promoted the cell apoptosis (P<0.05). Also the tuberostemonine could inhibit the expression of Survivin.Conclusion: There are no direct relations between tuberostemonine and P-gp, but tuberostemonine could reverse the multidrug resistance of K562/ADR via down-regulating the expression of Nf-κB and inhibiting th1e expression of Survivin.

Inhibition of Suicidal Erythrocyte Death by Volasertib

Background/Aims: The Polo-like kinase 1 (Plk1) inhibitor volasertib is used in the treatment of malignancy. Volasertib is partially effective by triggering suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal cell death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i). The present study explored, whether volasertib impacts on eryptosis. Methods: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca²⁺ ionophore ionomycin (1 µM for 60 min) in absence and presence of volasertib (0.5-1.5 µg/ml) and flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3 fluorescence, reactive oxygen species from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance utilizing antibodies. For comparison, annexin-V-binding and forward scatter were determined following a 48 hours exposure of human leukemic K562 cells in RPMI-1640 medium to volasertib. Results: Treatment with volasertib alone did not significantly modify annexin-V-binding or forward scatter in mature erythrocytes. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Volasertib significantly blunted the effect of energy depletion and hyperosmotic shock, but not of oxidative stress and ionomycin on annexin-V-binding. Volasertib did not significantly influence the effect of any maneuver on forward scatter. In K562 cells, volasertib enhanced annexin-V-binding and decreased the forward scatter. Conclusions: Volasertib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and hyperosmotic shock, effects contrasting the stimulation of K562 cell apoptosis.

Ginsenoside Rh₂ induces apoptosis and autophagy of K562 cells by activating p38

To study the effect of ginseng saponin Rh₂ in inducing apoptosis of human leukemia K562 cells, and explore its mechanism from the aspect of autophagy pathway. CCK-8 assay was used to examine the growth inhibition of human leukemia cell lines K562 treated with ginsenoside Rh₂; flow cytometry (FCM) was used to detect cell apoptosis; Hoechst staining was used to observe the changes of cell morphological apoptosis; Acridine and MDC staining were used to detect the effects of the Rh₂ on autophagy; Western blot and RT-PCR were used to detect the expression levels of the proteins closely associated with autophagy and apoptosis. In order to study the effect of autophagy in proliferation and apoptosis, we used the autophagy inhibitor (3-MA).CCK-8 indicated that Rh₂ at low concentration could effectively inhibit the proliferation of leukemia cellsin dose- and time-dependent manners in K562 cells; FCM indicated that Rh₂ induced apoptosis; Hoechest staining showed that K562 cells had typical apoptotic morphological changes by treated Rh₂; Acridine and MDC staining showed that Rh₂ enhanced the green fluorescence and a large number of acidic autophagy vesicles were present; Western blot and RT-PCR results showed that Rh₂ increased the expression levels of Beclin-1, LC3A, LC3B, activated Caspase-3 and p-p38 in K562 cells; application of autophagy inhibitors(3-MA) could weaken the inhibition effect of Rh₂ on proliferation and induction effect on apoptosis in K562 cells. Ginsenoside Rh₂ inhibited the proliferation and induced apoptosis probably through activating p-p38, and inducing cell autophagy signaling pathway in K562 cells.

Reactions of copper(II), nickel(II), and zinc(II) acetates with a new water-soluble 4-phenylthiosemicarbazone Schiff base ligand: Synthesis, characterization, unexpected cyclization, antimicrobial, antioxidant, and anticancer activities

A new Schiff base thiosemicarbazone ligand with aqueous solubility, namely, sodium 3-{[(anilinocarbonothioyl)hydrazono]methyl}-4-hydroxy benzene sulfonate (NaH2L) was synthesized by condensation of 4-phenylthiosemicarbazide and sodium salicylaldehyde-5-sulfonate (NaSalSO3) and characterized by elemental analysis, spectroscopic methods (UV–Vis, 1H NMR, FTIR) and electrospray ionization mass spectrometry. A co-crystal (Na(H2L)·Na(SalSO3)) of NaH2L and NaSalSO3 was prepared which confirmed the expected structure of the ligand. Reactions of NaH2L·0.5H2O with copper(II), nickel(II), and zinc(II) acetates were investigated under reflux conditions in methanol to produce following complexes [Cu2(Lcycl)2(H2O)2]·2H2O (CuLcycl), Ni(HL)(H2O)3 (NiL), and Zn(HL)(H2O)3 (ZnL). In the copper case, dinuclear copper complex [Cu2(Lcycl)2(H2O)2]·2H2O (where HLcycl− represents incorporating of 1,3-benzothiazole ligand, formed by in situ cyclization of H2L−) was isolated. In the dimer structure copper atoms adopt a five-coordinate near square pyramidal geometry. Two oxygens derived from two sulfonate groups occupy the apical positions on both the metal centers and bridge them. The compounds have remarkable solubility in water at 25°C: 50, 10, 50, and 50g/L for NaH2L·0.5H2O, CuLcycl, NiL, and ZnL, respectively. The stability of the compounds was checked in acidic, neutral, and basic aqueous solutions. Antimicrobial activity results indicated CuLcycl and NiL possess activity against gram-positive bacteria and fungus while the free ligand and ZnL are inactive. Antioxidant activity of the compounds was evaluated and the ligand indicated the strongest radical scavenging relative to the complexes and l-ascorbic acid as a water-soluble standard. It was observed that copper and nickel complexes decreased the viability of the human chronic myeloid leukemia K562 cells in a dose- and time-dependent manner and the ligand and zinc complex were not active. The IC50 values were 31±2.5 and 6±1.0μg/mL (72h) for copper and nickel complexes, respectively. Morphological and DNA fragmentation assay studies clearly indicated that the nickel complex as the strongest compound induce apoptosis in the K562 cell line.

Syntheses and anti-cancer activities of glycosylated derivatives of diosgenin

In order to obtain better anti-cancer compounds, nine glycosylated derivatives were designed and synthe-sized using diosgenin as starting material. Their structures were confirmed by 1H NMR, 13C NMR and MS spectra. The anti-cancer activities of intermediate compounds 5a—5i and the target compounds 6a—6i were investigated against human leukemia HEL, K562, HL60 and melanoma WM9 cell lines via MTT method. The bioassay results show that these derivatives possess good inhibitory activities against the four cancer cell lines. Furthermore, these derivatives show better inhibitory activities against K562 than against other cell lines, and most derivatives show better inhibitory activities than the parental material. Moreover, compounds 6a—6i are more active than their inter-mediates 5a—5i when against these cells. The above results demonstrate the effects of glycosylation on 3-OH of di-osgenin and acetylation of the sugar moiety on their antitumor activities.

The Novel Sulfonamidebenzamide ML291 Activates Apoptotic UPR Signaling in Pediatric Leukemia

Background: Acute leukemias are the most common cancers in childhood. Despite multi-agent chemotherapy protocols and the introduction of novel molecularly targeted therapies which have resulted in improved survival over the last few decades, relapsed acute lymphoblastic leukemia remains the second most common pediatric cancer diagnosis. In addition, morbidities from current chemotherapy regimens are unacceptably high. Abundant evidence point to a major role for mediators of the unfolded protein response (UPR) in normal and leukemic white blood cell biology. We have demonstrated that activation of the UPR is a productive approach to inhibit the proliferation of solid tumor cell lines in vitro and to reducing xenograft burden in vivo. The UPR consists of genetically distinct mechanisms that serve to clear misfolded proteins from the endoplasmic reticulum (ER) and enhance protein folding, or induce apoptosis if the initiating stress is prolonged or robust. ML291 is a novel UPR-inducing sulfonamidebenzamide, identified through cell-based high throughput screening and iterative SAR-guided chemical synthesis, that overwhelms the adaptive capacity of the UPR and induces apoptosis in a variety of solid cancer models.Objective: To determine the ability of ML291 to activate the UPR and induce apoptosis in a panel of leukemia cell lines, and to use CHOP-null K562 cells to elucidate the relative contribution of the UPR. We hypothesized that ML291 might activate the PERK/eIF2a/CHOP (apoptotic) arm of the UPR and reduce leukemic cell burden in vitro and in vivo.Methods: MTT and luciferase-based proliferation assays, flow cytometry and RT-qPCR were used to evaluate cell growth, UPR activation and apoptosis in a panel of leukemia cell lines that included AML, ALL and CML in cells exposed to ML291. CRISPR-Cas9 genome editing was used to delete CHOP in K562 (human myeloid leukemia) cells. Deletion was validated by immunoblot analysis and these cells were subjected to the same proliferation and gene analyses described above. The in vivo response to ML291 therapy was evaluated in an established zebrafish xenograft assay (Corkery et al. BJH 2011) in which embryos were xenotransplanted with wild type or CHOP knockdown K562 cells and embryos bathed in ML291.Results: Immunoblot and RT-qPCR analysis revealed an accumulation of proteins and increased gene expression for downstream UPR genes, including CHOP, GRP78/BiP, GADD34 and XBP1 in leukemia cells following ML291 treatment, indicating the activation of the UPR. Increased expression of the apoptotic genes, NOXA, PUMA and DR5 was also observed post-treatment with ML291; and dose response proliferation assays performed after 24 hours revealed IC50 concentrations of 1 - 30µM across cell lines. CHOP deleted K562 cells were protected from cell death when cultured with increasing concentrations of ML291, and were significantly less able to translocate phosphatidylserine across the cell membrane and activate the caspase cascade. When zebrafish embryos xenotransplanted with K562-wild type or -CHOP-null cells were bathed in water containing 5mM ML291 for three days, there was a significant reduction in leukemia cell burden exclusively in theK562 wild type xenografts.Conclusion: Collectively these data indicate that intact PERK/eIF2a/CHOP signaling is required for efficient leukemic cell apoptosis in response to ML291 in vitro and in vivo, and support the hypothesis that small molecule enforcement of the UPR might be a productive therapeutic approach in leukemia. DisclosuresNo relevant conflicts of interest to declare.