MOLT-4 Cell Lines Research Articles

Pharmacological Inhibition of MAP2K7 Induces Apoptosis and Cell Cycle Arrest in T-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and relapsed ALL is a leading cause of cancer-related death in children. While advances in frontline therapy have led to an 85% cure rate, relapsed ALL patients face a dismal prognosis necessitating identification of novel targets and development of alternative therapies. Amongst the two sub types of ALL, T-cell acute lymphoblastic leukemia (T-ALL) occurs less frequently but T-ALL patients have worse prognosis and higher rate of relapse. Our group discovered that the transcription factor KLF4 is heavily repressed via DNA methylation coinciding with aberrant activation of MAP2K7 and the MAP2K7 pathway (Shen et al. Leukemia, 2017). Additionally, we demonstrated, as a proof of principle, that pharmacological inhibition of JNK, the only downstream target of MAP2K7, possesses anti-leukemic properties against T-ALL but presented obstacles in terms of low potency and off-target effects. In order to overcome these limitations, we hypothesize that direct pharmacological inhibition of MAP2K7 improve specificity for targeted approach to T-ALL therapeutics. To examine this hypothesis, we explore the anti-leukemic effects of the MAP2K7 pathway inhibitor, 5Z-7-oxozeaenol, against T-ALL cells because it has been shown to covalently react with a unique cysteine-218 in the ATP binding pocket of MAP2K7. We found that T-ALL cell lines exhibit increased cytotoxic sensitivity to 5Z-7-oxozeaenol (IC50 ranging 0.2-1.1 µM) compared to a non-leukemic control (1.5 µM), and more potent than JNK inhibitor IC50 of 5µM. Additionally, 5Z-7-oxozeaenol reduces the amount of phospho-JNK, in a dose dependent manner, indicative of MAP2K7 pathway inhibition. We purified MAP2K7 protein to directly address specificity of kinase inhibition and found that 5Z-7-oxozeaenol inhibits enzymatic activity in vitro of MAP2K7. In addition, 5Z-7-oxozeaenol also inhibited TAK1, which is the MAP3K upstream of MAP2K7. In a panel of T-ALL cell lines, 5Z-7-oxozeaenol treatment induced apoptosis in MOLT3, Jurkat, and KOPTK1 T-ALL cell lines. Although P12-Ichikawa, RPMI-8402, DND-41, and ALL-SIL T-ALL cell lines underwent cell cycle arrest evidenced by a reduction in percentage of S/G2/M phase cells and increase in percentage of G0 phase cells, further increasing 5Z-7-oxozeaenol dosage proved sufficient for induction of apoptosis based on increase of caspase-3 and caspase-7 cleavage. Through reverse-phase protein array analysis we identified reduced expression of several cell cycle regulator proteins, including CDC25C, Cyclin B1, Cyclin D3, and CDC2 resulting from 5Z-7-oxozeaenol treatment. Conversely, we detected increased expression of cleaved caspase-3 and caspase-7, particularly in MOLT3 and Jurkat cell lines treated with MAP2K7 inhibitor. Immunoblot analysis revealed that 5Z-7-oxozeaenol inhibits the MAP2K7 signaling pathway and induces cell cycle arrest and apoptosis in T-ALL cells. Based on these findings, we demonstrated that 5Z-7-oxozeaenol induces cell cycle arrest and apoptosis in T-ALL cells through inhibition of the MAP2K7 pathway, suggesting that MAP2K7 represents a novel pharmacological target for the development of targeted therapy for high-risk patients with T-ALL. Disclosures No relevant conflicts of interest to declare.

CARMA1 Is Required for Notch1-Induced NF-κb Activation in T-Cell Acute Lymphoblastic Leukemia

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplasm originated from early T cell progenitors. Constitutive activation of the Notch1-signaling pathway plays an important role in T-ALL. After three shears, Notch1 generates intracellular fragment of Notch1 (Notch1-IC), which translocates to the necleus and exerts its biological functions. NF-κB pathway is an important regulator of cell survival and a major downstream target of Nothc1 in T-ALL. However, the molecular mechanism of Notch1 activating NF-κB pathway is still unknown. CARMA1 forms a complex with MALT1 and BCL10 (CBM complex), which plays a role in activation of NF-κB pathway in leukomonocyte. Therefore, we asked whether CARMA1 mediates Notch1-induced NF-κB pathway. Methods Microarray gene expression data were obtained from GEO database (accession number GSE26713) to assess expression of CARMA1, BCL10, MALT1 in T-ALL patients. In addition, we apply microarray dataset (GEO accession number GSE42328) to examine the relationship between gene expression and patients outcome. Western blot was used to analyze CRAMA1 expression in different cell lines. CCK-8 assay, EdU assay, cell cycle and colony formation experiments were performed in CARMA1-shRNA and control-shRNA T-ALL cell lines to investigate the effect of CARMA1 in T-ALL. We intravenously injected the Control-shRNA and CARMA1-shRNA MOLT-4 cells into NOD-SCID/IL2Rgnull (NSG) mice to evaluate the influence of CARMA1 knockdown on overall survival. Dual luciferase reporter assay was applied to detect transcriptional activity of NF-κB. Results We found that CARMA1 was highly expressed in T-ALL cell lines, but not observed in B-ALL and AML cell lines. The T-ALL patients with high CARMA1 expression had better outcome compared with the low expression group. However, we failed to observe any significant differences in the expression of BCL10 and MALT1 between primary normal and T-ALL cells or relationship of survival with MALT1 and BCL10 in T-ALL. A previous finding showed that Notch1 mutations are correlated with an improved long-term prognosis (J Clin Oncol 2013;31:4333). The above results suggested both high expression of CARMA1 and activating Notch1 mutaions relate to favorable prognosis, indicating the possible relevance between CARMA1 expression and Notch1 activation. To elucidate the effect of CARMA1 on cell proliferation, we performed CARMA1 knockdown experiment with shRNAs in T-ALL cells. CARMA1 knockdown in MOLT-4 cells significantly decrease the quantity and size of cell colonies. MOLT-4 and Jurkat cell lines showed significant reductions in cell viability through CARMA1 silencing compared with control-shRNA cells. The EdU proliferation assay demonstrated that knockdown of CARMA1 in MOLT-4 and Jurkat cell lines markedly reduced the number of EdU-positive cells. Interestingly, CARMA1 had no significant effect on cell proliferation in CCRF-CEM cells. This finding is consistent with a recent report, which demonstrated that CARMA1 knockdown showed no difference in proliferation of CCRF-CEM cells, but only one cell line was used (Leukemia. 2017;31:255). CCRF-CEM is a SIL-TAL1 fusion gene positive cell line and its survival depends on the activation of TAL1 transcriptional complex. Therefore, the proliferation of SIL-TAL1 fusion gene positive cell lines may be independent of CARMA1 expression. We next explored the significance of CARMA1 in cell cycle and in vivo tumorigenic ability. Compared to the control-shRNA cells, CARMA1 knockdown arrested T-ALL cell lines in G1 phase. CARMA1 knockdown remarkably led to the downregulation of the percentage of CD45 positive cells and prolonged overall survival of NSG mice. To further determine the role of CARMA1 in Notch1-induced NF-Κb activation, we transfected Notch1-IC into control-shRNA and CARMA1-shRNA cells, respectively. We found that Notch1-IC significantly upregulated the activity of NF-κB in control-shRNA cells. However, the Notch1-induced NF-κB was abolished in CARMA1 knockdown T-ALL cells. The results demonstrate that CARMA1 is involved in Notch1-mediated NF-κB activation. Conclusion Taken together, CARMA1 markedly contributes to cell survival in SIL-TAL1 negative T-ALL cells and may play a crucial role in Notch1-induced activation of NF-κB pathway. Furthermore, CRAMA1 might be a prognostic marker and a prospective therapeutic target for T-ALL. Disclosures No relevant conflicts of interest to declare.

Targeting WNT10B-Driven Signalling through Induction of FZD6 By Porcupine Inhibition in T Cell Acute Lymphoblastic Leukemia

Background Wnt/Fzd signaling is known to play a pervasive influence in hematopoietic stem cell maintenance, T-cell development in the thymus and function as well as an important role in T-cell acute lymphoblastic leukemia (T-ALL) establishment. We have previously described a recurrent rearrangement involving the WNT10Blocus (WNT10BR) expressing a transcript variant (WNT10BIVS1) in acute myeloid leukemia. To determine the occurrence of this rearrangement in T-ALL we analyzed retrospectively an italian cohort of patients (n=20) and detected the WNT10BRrearrangement with a high prevalence (14/20). We also confirmed the relevance of these findings to human disease, detecting the molecular circuit triggered by the WNT10B over-expression using the MOLT-4 T-ALL cell model.In this report, we examined the expression of components of the Wnt signaling cascade mediated by WNT10B and the effects of specific gene silencing by short hairpin RNA (shRNA) and exposure to the potent PORCN inhibitor (LGK974), or the TGFbRI inhibitor (A83-01) on the WNT10B-mediated Wnt signaling activation. Methods We used the T-ALL model MOLT-4 cell line to assess the WNT10B/FZD signaling axis driven by WNT10BR. In order to identify interaction between WNT10B and FZD receptors we performed in situ proximity ligation assay (PLA) a method used to visualize protein-protein interactions.MOLT4 cells were infected with WNT10B/WNT10BIVS1-shRNA silencing lentiviral vectors versus empty vector control and treated with increased concentration of LGK974 or A83-01, subsequently the effects of pharmacological inhibition on the WNT10B/FZD interactions and on Wnt effector proteins were evaluated by PLA and expression analyses. Cell proliferation and cell death were measured by EdU assay and Annexin-V/Propidium Iodide (PI) analyses. Results We found that WNT10BRdrives Wnt signaling activity in T-ALL through interaction of WNT10B with FZD6 receptor. The effects of WNT10B/FZD6 interaction on Wnt-mediated signal in MOLT-4 were interfered by short hairpin RNAs (shRNAs)-mediated gene silencing and by small molecules-mediated disruption of Wnt-dependent signaling. We performed WNT10BIVS1knockdown or pharmacological inhibition of WNT10B release by the porcupine (PORCN) inhibitor LGK974 and these in turn progressively down-modulate WNT10B/FZD6 protein complex formation and significantly impairs intracellular effectors and leukemic expansion. Finally, we induced interference to the WNT10B/FZD6 protein complex formation by exposure to the TGFbRI inhibitor A83-01 via inhibiting FZD6 expression, confirming its role in the WNT10B-mediated signaling activation. Conclusion Our study describes the molecular circuit of WNT10BR-mediated activation and highlight a strategy for a major improvement in T-ALL treatment.By altering FZD6-WNT10B complex formation, may provide the basis for therapeutic strategies to eradicate leukemic stem cells in patients selectively deployed depending on the underlying genetics of disease. Disclosures No relevant conflicts of interest to declare.

PHYTOCHEMICAL SCREENING, GAS CHROMATOGRAPHY–MASS SPECTROMETRY PROFILING, IN VITRO CYTOTOXICITY, AND ANTHELMINTIC ACTIVITY OF HYDROALCOHOLIC EXTRACT OF DURANTA ERECTA LEAVES

Objective: The present study aims at the preliminary phytochemical evaluation, gas chromatography–mass spectrometry (GC–MS) analysis, in vitro cytotoxicity, and anthelmintic activity of the hydroalcoholic extract of D. erecta leaves.
 Methods: The hydroalcoholic extraction of the plant material is done with Soxhlet apparatus and the GC–MS profiling of the extract is performed. The in vitro cytotoxicity activity of the extract is done using the MOLT-3 cell lines. The anthelmintic activity is performed using Pheretima posthuma. 
 Results: The GC–MS analysis shows the presence of various secondary metabolites with proved pharmacological activity. The in vitro cytotoxicity and the anthelmintic activity of the extract show the beneficial effect of the plant.
 Conclusion: The flavonoids, phenolic compounds, and tannins present in the plant extract may support the cytotoxic and anthelmintic activity of the plant.

Activity of Fluorescent Samarium Complex Containing 1,10 Phenanthroline Ligand against Human T-Cell Acute Lymphoblastic Leukaemia Cell Line

This paper is the study of the structure effect of samarium-phenanthroline complex on DNA molecule binding for cancer treatment. So the water soluble complex has been synthesized and characterized by using chemical analysis. By positive cooperative interaction, Sm complex was connected with calf thymus DNA (ct-DNA) and this interaction was investigated by circular dichroism and fluorescence spectroscopy. The result indicates that samarium complex with phenanthroline ligands can be as a fluorescence probe and intercalate in DNA additionally groove binding. Also, the cytotoxicity of this complex was investigated against human T-cell acute lymphoblastic leukemia, MOLT-4 cell line. Finally, the interaction of complex was studied by molecular docking. The results showed during DNA-Sm complex formation, the complex is surrounded by DT8, DC9, DG10, DA18, DT19 and DT20 via hydrophobic forces.

Molecular networking-based strategy for the discovery of polyacetylated 18-norspirostanol saponins from Trillium tschonoskii maxim.

Nine undescribed polyacetylated 18-norspirostanol saponins, trilliumosides A‒J, were obtained after a guidance based on a molecular networking strategy from the rhizomes of Trillium tschonoskii. Their structures were established by analysis of comprehensive spectroscopic data and chemical methods after their isolation in pure form. All isolated saponins were evaluated for their cytotoxicities against five selected human cancer cell lines (Huh7,A549,MCF-7,HepG2, and MOLT-4) and anti-inflammatory effects on a lipopolysaccharide (LPS)-stimulated NO production model in RAW264.7 macrophages. Trilliumoside D showed significant cytotoxicity against MOLT-4 cell lines with an IC50 value of 1.0 ± 0.1 μM, whereas trilliumosides H and I displayed remarkable anti-inflammatory effects on NO production with inhibitory rates of 56.3 ± 1.5 and 56.2 ± 2.2% at the concentration of 1.0 μM, respectively.

Stereospecific Cross-Coupling Reactions Provide Conformationally-Biased Arylalkanes with Anti-Leukemia Activity.

A focused small library of carbamates and alcohols was prepared employing stereospecific Kumada-ring opening reactions of tetrahydropyrans. The core framework of the library members is acyclic and incorporates 1,3-substituents, to provide a conformational bias in avoiding syn-pentane interactions. A new compound with micromolar activity against MOLT-4, CCRF-CEM, and HL-60(TB) leukemia cell lines was identified from this series.

Combination of ERK2 inhibitor VX-11e and voreloxin synergistically enhances anti-proliferative and pro-apoptotic effects in leukemia cells

ERK1/2 inhibitors are new promising anticancer drugs. The aim of this study was to investigate the effect of the combination of ERK2 inhibitor VX-11e and voreloxin on MOLM-14, K562, REH and MOLT-4 leukemia cell lines. We found that VX-11e alone and in combination with voreloxin significantly decreased ERK activation in all cell lines tested. To evaluate the interactions of the drugs, cells were treated for 24 h with VX-11e or voreloxin alone and in combination at fixed ratios based on IC50 values. The combinatorial effects of both drugs were synergistic over a wide range of concentrations in MOLM-14, REH and MOLT-4 cell lines. In K562 cells, three effects were found to be additive, one antagonistic and only one synergistic. The results showed that incubation with both VX-11e and voreloxin inhibited the growth of leukemia cells, affected cell cycle and induced apoptosis. Furthermore, the molecular mechanism of these effects might be attributed to an increased expression of p21 and a decreased expression of survivin and NF-κB in all cell lines tested except from K562 cells. In conclusion, combination of VX-11e and voreloxin can exert a synergistic anticancer effect in leukemia cells.

Structural tuning of acridones for developing anticancer agents targeting dihydrofolate reductase.

Targeting dihydrofolate reductase, here, we report the tumor growth inhibitory activity of substituted acridones. The screening of the molecules over 60 cell line panel of human cancer cells identified (S)-oxiran-2-ylmethyl 9-oxo-9,10-dihydroacridine-4-carboxylate (19) with average GI50 0.3 μM. The specificity of the compound to CCRF-CEM, MOLT-4 and SR cell lines of leukemia and SW-620, SF268, LOXIMVI, ACHN and MCF7 cancerous cells exhibiting GI50 in the nM range was observed. C6 Glioma cells treated with compound 19 showed differentiated cell morphology and cell cycle arrest in G2/M phase. The interactions of the compound with dihydrofolate reductase were ascertained with the help of enzyme immunoassays, molecular docking and molecular dynamic studies.

Synthesis of 4′-substituted 2′-deoxy-4′-thiocytidines and its evaluation for antineoplastic and antiviral activities

4′-Azido- (7), 4′-C-fluoromethyl- (8) 4′-C-ethynyl- (9) and 4′-C-cyano- (10) 2′-deoxy-4′-thiocytidines have been synthesized. In this study, it was found that the isolated yield of 4′-thiouracil nucleoside 13 in a Lewis acid-promoted Vorbrüggen-type glycosidation utilizing 12 was better than that of the electrophilic glycosidation reaction between silylated uracil and 11. This improved result prompted us to perform the glycosidation utilizing 36 and 43 for the synthesis of 37 and 44. Introduction of the azido group was carried out by nucleophilic substitution in the 4′-benzoyloxy derivative 22a. On the other hand, 9 and 10 were synthesized by way of the chemical manipulation of the hydroxymethyl group at the 4′-position of 46.Evaluation of the antineoplastic activity of 2 and 7–10 against human B-cell (CCRF-SB) and T-cell leukemia (Molt-4) cell lines revealed that 4′-azido- (7) and 4′-C-fluoromethyl- (8) derivatives exhibited cytotoxic activity whereas no cytotoxicity was observed in the 4′-C-ethynyl- (9) and 4′-C-cyano- (10) derivatives as well as the parent compound 2. Compound 7 was also found to possess promising antiviral activity against VZV and HSV-1 without any cytotoxity against HEL host cells. It is noteworthy that 7 exhibited potent inhibitory activities against the thymidine kinase-deficient (TK−) mutant of VZV and HSV-1.

Abstract 1741: Identification of microsatellite instability in hematologic malignant cell lines

Abstract Defective DNA repair machinery which is a consequence of chromosomal or molecular alterations is associated with variety of diseases including solid tumors. Cytogenetic analysis is an important tool in diagnosis and treatment of hematologic malignancies. Our aim is to understand the role of microsatellite instability in initiation and progression of blood cancers. Here we hypothesize that leukemogenic cells may carry defective repair mechanisms that contribute to other genomic instabilities. In order to explore the possible role of microsatellite instability (MSI) in leukemia and lymphoma, eight different DNA repair genes involved in mismatch repair (MMR) and direct reversal of damage (DRD) were analyzed in K562 (CML), Molt-4 (T-ALL) and Daudi (Burkitt’s lymphoma) cell lines. Fragment analysis was carried out using novel primers considering repeat motifs enriched with (A)n, (CA)n and (AT)n for instability. Almost all the cell lines showed moderate to high level of microsatellite instability and exhibited a shift in selected repeat regions indicating possible loss (66.7%) (MSH2, MSH4, MSH6, MLH1, MLH3, PMS1) or gain (25%) (MSH3, MGMT) of nucleotide repeats as compared to normal DNA. The K562, Philadelphia negative CML cell line appeared to be microsatellite stable (MSS) for genes MSH2 and MLH1. Treatment induced microsatellite instability is known in leukemic patient cells as also seen in Molt-4 cells. Our present and previous observations suggest that MSI is a non-random event in leukemia/lymphoma which needs confirmatory future studies in newly diagnosed untreated patient samples. Considering the fact that both MSI and MSS represents two distinct molecular pathways of carcinogenesis, assessment of MSI, if added as distinct diagnostic criteria, can possibly refine early detection and help in identifying novel targets in management of hematologic malignancies. Citation Format: Priyanjali Bhattacharya, Naveed Anjum Chikan, Arati Sharma, Dhimant Desai, Charyguly Annageldiyev, Pinaki Patel, Trupti N. Patel. Identification of microsatellite instability in hematologic malignant cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1741.

Initial human T-cell leukemia virus type 1 infection of the salivary gland epithelial cells requires a biofilm-like structure

The initial phase of the human T cell leukemia virus-1 (HTLV-1) infection of salivary gland epithelial cells (SGECs) was examined. SGECs of patients with Sjögren’s syndrome (SS) and non-SS subjects were co-cultured with the HTLV-1-infected cell line HCT-5 or MOLT-4, then immunofluorescence (IF), scanning and transmission electron microscopy (SEM/TEM) were employed.The extracellular matrix and linker proteins galectin-3, agrin, and tetherin were expressed on the surfaces of both HCT-5 and MOLT-4 cells. HTLV-1 Gag-positive spots were observed on adjacent SGECs after 1 h of co-culture with HCT-5. Both in subjects with and those without SS, agrin and tetherin were co-expressed with HTLV-1 Gag on SGECs after co-culture with HCT-5, although no polarization of HTLV-1 Gag and relevant molecules was observed. SEM showed HTLV-1 virions that were found on HCT-5 were observed in the interfaces between HCT-5 cells and SGECs. TEM imaging showed that HTLV-1 virions were transmitted to SGECs at the interface with thin film-like structure, while HTLV-1 virions were released from the surface of HCT-5 cells. No endogenous retroviruses were observed. These results showed that the initial phase of HTLV-1 infection toward SGECs of SS was mediated not by viral synapses, but by biofilm-like components.

Integrating microRNA and mRNA expression in rapamycin-treated T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) has a relatively improved remission rate, but the poor outcomes are primarily due to resistance and relapse. Moreover, organs infiltration trends to occur during remission. Rapamycin was applied to treat malignancies for decades. In this investigation, we aimed to explore the molecular mechanisms and pathway changes during the T-ALL therapeutic process. T-ALL cell line Molt-4 cells were treated with rapamycin and performed microarray analysis to identify the deregulated miRNAs and mRNAs (log2 fold change>2 or <-2). To obtain regulatory miRNA/mRNA network, miRNA target prediction softwares and Cytoscape were used to plot and modularize the rapamycin treatment-related network. Surprisingly, the enriched pathways were not involved in mediating either cell death or apoptosis but were responsible for angiogenesis, cell survival, and anti-apoptosis, which is consistent with the Gene Ontology analysis and PPI network based on all deregulated mRNAs, indicating that these elements likely play a role in promoting Molt-4 cell survival or escaping from rapamycin. The expression of 3 miRNAs (miR-149-3p, miR-361-3p, and miR-944) and their putative targets, which play central roles in their module, were validated by qRT-PCR. These results provide novel insight into potentially relevant biological pathways for T-ALL cells escaping from chemotherapy or developing central nervous system infiltration.

Costunolide and parthenolide from Champi Sirindhorn (Magnolia sirindhorniae) inhibit leukemic cell proliferation in K562 and molt-4 cell lines

The magnolia plant has been used in traditional medicine since ancient times. This study was designed to investigate the effects of active compounds from Thai Champi Sirindhorn (Magnolia sirindhorniae) on leukemic biomarker Wilms’ tumor 1 (WT1) protein expressions in K562 and Molt-4 cells. Costunolide (1) and parthenolide (2) were the major components used in this study, they were purified from ethyl acetate fractions. Costunolide (1) and parthenolide (2) had strong cytotoxic effects in K562 and Molt-4 cells measured with MTT assays. Their activities were compared to standard commercial costunolide (3) and parthenolide (4). Costunolide (1) and parthenolide (2) decreased WT1 protein levels and total cell numbers in K562 and Molt-4 cells. Both purified costunolide (1) and standard commercial costunolide (3) decreased WT1 protein levels in a time- and dose-dependent manner. Therefore, the active compounds from M. sirindhorniae were identified as promising sources for bioactive compounds for further applications in traditional medicine.

Structural characterization of polysaccharide-coated iron oxide nanoparticles produced by Staphylococcus warneri, isolated from a thermal spring.

The biocompatible-coated iron oxide nanoparticles (IONs) have attracted a great interest because of their various applications in biological science and medicine. In most cases, the toxic effect of naked iron oxide nanoparticles is completely cleared by adding a biocompatible coating, such as polysaccharides, polyethylene glycol (PEG), or biosynthesis of biocompatible-coated IONs using microorganisms such as bacteria. In the present study, polysaccharide-coated iron oxide nanoparticles were produced by a strain of Staphylococcus warneri isolated from a thermal spring. For identification of the isolated bacterium, 16S rRNA gene sequencing was done. Characterization of the nanoparticles was performed for the first time, using transmission electron microscopy (TEM), dynamic light scattering (DLS), thermogravimetric analysis (TGA), X-ray crystallography (XRD), Fourier-transform infrared (FTIR) spectroscopy, vibrating sample magnetometer (VSM), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results indicated that the spherical iron oxide nanoparticles were coated by a polysaccharide (13.6%), which provided a large negative charge of -91 mV and very low saturation magnetization of around 0.28 emu/g. The result of MTT assay on MOLT-4 cell lines showed that the percentage of viability was between 95.6% and 68.9% in the 10-100 µM of nanoparticle concentrations with a high IC 50 value, which makes it appropriate for biomedical applications such as cancer therapy.

Time dependent response of daunorubicin on cytotoxicity, cell cycle and DNA repair in acute lymphoblastic leukaemia

BackgroundDaunorubicin is commonly used in the treatment of acute lymphoblastic leukaemia (ALL). The aim of this study was to explore the kinetics of double strand break (DSB) formation of three ALL cell lines following exposure to daunorubicin and to investigate the effects of daunorubicin on the cell cycle and the protein kinases involved in specific checkpoints following DNA damage and recovery periods.MethodsThree ALL cell lines CCRF-CEM and MOLT-4 derived from T lymphocytes and SUP-B15 derived from B lymphocytes were examined following 4 h treatment with daunorubicin chemotherapy and 4, 12 and 24 h recovery periods. Cell viability was measured via MTT (3-(4,5-dimethylthiazol-2-yl)-2–5 diphenyltetrazolium bromide) assay, reactive oxygen species (ROS) production by flow cytometry, double stranded DNA breaks by detecting γH2AX levels while stages of the cell cycle were detected following propidium iodide staining and flow cytometry. Western blotting was used to detect specific proteins while RNA was extracted from all cell lines and converted to cDNA to sequence Ataxia–telangiectasia mutated (ATM).ResultsDaunorubicin induced different degrees of toxicity in all cell lines and consistently generated reactive oxygen species. Daunorubicin was more potent at inducing DSB in MOLT-4 and CCRF-CEM cell lines while SUP-B15 cells showed delays in DSB repair and significantly more resistance to daunorubicin compared to the other cell lines as measured by γH2AX assay. Daunorubicin also causes cell cycle arrest in all three cell lines at different checkpoints at different times. These effects were not due to mutations in ATM as sequencing revealed none in any of the three cell lines. However, p53 was phosphorylated at serine 15 only in CCRF-CEM and MOLT-4 but not in SUP-B15 cells. The lack of active p53 may be correlated to the increase of SOD2 in SUP-B15 cells.ConclusionsThe delay in DSB repair and lower sensitivity to daunorubicin seen in the B lymphocyte derived SUP-B15 cells could be due to loss of function of p53 that may be correlated to increased expression of SOD2 and lower ROS production.

Co-operation of ABT-199 and gemcitabine in impeding DNA damage repair and inducing cell apoptosis for synergistic therapy of T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype of acute lymphoblastic leukemia with limited therapeutic options available. Here, we evaluated the therapeutic potential of the combination of the Bcl-2 antagonist ABT-199 and cytotoxic agent gemcitabine in T-ALL cell lines. Our results showed that the combination of ABT-199 and gemcitabine exhibited synergistic cytotoxicity and induced significant apoptosis in human T-ALL cell lines (Jurkat and Molt4). The augmented apoptosis induced by combination treatment was accompanied by the greater extent of mitochondrial depolarization and enhanced DNA damage. Importantly, single agent induced DNA damage alone but did not inhibit RAD51/BRCA1-mediated repair for DNA double-strand breaks. In contrast, the combination of ABT-199 and gemcitabine disrupted RAD51/BRCA1-dependent DNA repair and remarkably activated caspase-3 and PARP to trigger apoptosis. Moreover, ABT-199 exerted an antagonistic action towards Bcl-2 and Bcl-xL, but to a certain extent moderately increased Mcl-1 level that could be compromised by gemcitabine. In conclusion, our study showed that the combination of ABT-199 and gemcitabine exhibited synergistic cytotoxicity in T-ALL cells by cooperatively targeting DNA damage repair pathway and Bcl-2 family proteins.

Kaempferol Improves TRAIL-Mediated Apoptosis in Leukemia MOLT-4 Cells by the Inhibition of Anti-apoptotic Proteins and Promotion of Death Receptors Expression.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or Apo2L) is a member of the Tumor Necrosis Factor (TNF) superfamily, which stimulates apoptosis in a wide range of cancer cells through binding to Death Receptors 4 and 5 (DR4/5). Nevertheless, TRAIL has noticeable anti-cancer abilities; some cancer cells acquire resistance to TRAIL, and consequently, its potential for inducing apoptosis in target cells is strongly diminished. Acute lymphoblastic leukemia MOLT-4 cell line is one of the most resistant cells to TRAIL that developed resistance to TRAIL through different pathways. TRAIL plus kaempferol was used to eliminate the resistance of the MOLT-4 cells to TRAIL. Firstly, IC50 for kaempferol (95μM) was determined by using the MTT assay. Secondly, the viability of the MOLT-4 cells was assayed by FACS after Annexin V/PI staining, following treatment with TRAIL (50 and 100nM) and kaempferol (95μM) alone and in combination. Finally, the expression levels of the candidate genes involved in resistance to TRAIL were assayed by real-time PCR technique. Kaempferol plus TRAIL induced apoptosis robustly in MOLT-4 cells at 12, 24 and 48 hours after treatment. Additionally, it was found that kaempferol could inhibit the expression of c-FLIP, X-IAP, cIAP1/2, FGF-8 and VEGF-beta, and conversely augment the expression of DR4/5 in MOLT-4 cells. It is suggested that co-treatment of MOLT-4 cells with TRAIL plus kaempferol is a practical and attractive approach to eliminate cancers' resistance to TRAIL by inhibition of the intracellular anti-apoptotic proteins, upregulation of DR4/5 and also by suppression of the VEGF-beta (VEGFB) and FGF-8 expressions.

Searching for new cytotoxic agents based on chromen-4-one and chromane-2,4-dione scaffolds.

Cancer is a major cause of death worldwide and novel anticancer agents for its better management are much needed. Benzopyrone-based compounds, such as chromones, possess several distinctive chemical and biological properties, of which the cytotoxicity against cancer cells seems to be prominent. In this study, two series of compounds based on chromen-4-one (3-10) and chromane-2,4-dione (11-18) scaffolds were synthesized in moderate/high yields and evaluated for cytotoxicity against HL-60, MOLT-4, and MCF-7 cancer cells using MTT assay. In general, the compounds exhibited moderate cytotoxic effects against the cancer cell lines, among which, a superior potency could be observed against MOLT-4 cells. Chroman-2,4-dione (11-18) derivatives had overall higher potencies compared to their chromen-4-one (3-10) counterparts. Compound 13 displayed the lowest IC50 values against HL-60 (IC50, 42.0 ± 2.7 μM) and MOLT-4 cell lines (IC50, 24.4 ± 2.6 μM), while derivative 11 showed the highest activity against MCF-7 cells (IC50, 68.4 ± 3.9 μM). In conclusion, this study provides important information on the cytotoxic effects of chromone derivatives. Benzochroman-2,4-dione has been identified as a promising scaffold, which its potency can be modulated by tailored synthesis with the aim of finding novel and dissimilar anticancer compounds.

Enhanced cytotoxicity of indenyl molybdenum(ii) compounds bearing a thiophene function.

A series of six indenyl molybdenum compounds bearing a thiophenyl function in the side chain were prepared and characterized by analytical and spectroscopic methods. The structures of [(η5-C9H6CH2C4H3S)(η3-C3H5)Mo(CO)2] and [(η5-C9H6CH2C4H3S)Mo(CO)2(bpy)][BF4] were determined by single-crystal X-ray diffraction. The compounds bearing N,N-chelating ligands exhibit increased cytotoxic activity against human leukemia cell lines MOLT-4; up to two orders of magnitude lower IC50 values were observed compared to analogues with unsubstituted indenyl, which clearly demonstrates the strong effect of the indenyl ligand modification on the biological activity of the molybdenum(ii) compounds. The highest cytostatic potential was observed for the complex bearing 4,7-diphenyl-1,10-phenanthtoline [(η5-C9H6CH2C4H3S)Mo(CO)2(Ph2phen)][BF4] with IC50 (MOLT-4) = 0.19 ± 0.02 μM. Detailed regulation of the molecular and cellular mechanism by this derivative was investigated on the lung carcinoma cell line A549 and compared with the lung fibroblast cell line MRC-5. Rather unusual differences in the effects on tumor and non-tumor cell lines provide a unique insight into the cytostatic action of molybdenum(ii) complexes.