Human Chronic Myeloid Leukemia Cells Research Articles

Delayed Expansion of Murine BCR-ABL1+ Leukemia By Genetic and Pharmacological Inhibition of NOX2

Reactive oxygen species (ROS), produced from reduction of oxygen by the myeloid NADPH oxidase (NOX2), are pivotal effector molecules in the innate immune defense against microbes, but imbalance between the generation and detoxification of ROS may be injurious to cell components. The malignant cells of patients with BCR-ABL1+ chronic myeloid leukemia (CML) show elevated ROS production, which has been implicated in the genetic instability BCR-ABL1+ cells. However, further studies are required to define the role of ROS in leukemogenesis. We have utilized genetically modified murine hematopoietic cells to determine the influence of NOX2-derived ROS for the in vivo expansion of transplanted BCR-ABL1+ cells. Irradiated C57BL/6J mice received transplants of wild-type (WT) or Nox2 knockout (Nox2 -KO) hematopoietic cells that were transduced to express GFP-labeled human BCR-ABL1 by use of the p210 construct cloned into a MSCV vector. In vitro experiments using the NOX2 inducer D-peptide confirmed that Nox2 -KO BCR-ABL1+ cells were devoid of ROS production via NOX2. Peripheral blood was drawn biweekly from mice receiving WT and Nox2 -KO transplants for fluorimetric quantification of BCR-ABL1+ cells. We observed prolonged survival for mice receiving Nox2 -KO vs. WT BCR-ABL1 transplants (n=43, p=0.017, logrank test) along with a trend towards reduced expansion of leukemic cells in vivo . To determine effects of pharmacological inhibition of NOX2 on leukemic expansion, the NOX2 inhibitor histamine dihydrochloride (HDC) was administered to mice carrying WT or Nox2 -KO BCR-ABL1+ transplants. In vitro experiments confirmed that HDC reduced NOX2-dependent induction of ROS formation in WT BCR-ABL1+ cells. Treatment of age-matched mice with HDC (1.5 mg three times per week i.p.) delayed the expansion of WT BCR-ABL1+ cells at four weeks after transplantation (n=18, p<0.05, t-test) but did not reduce the burden of leukemia in mice receiving Nox2 -KO BCR-ABL1+ transplants. Initial experiments using primary human CML cells revealed differential expression of redox-related genes in CD34+CD38- leukemic stem cells vs. cord blood stem cells. Our results implicate NOX2 as a conceivable therapeutic target in BCR-ABL1+ leukemia. DisclosuresGrauers Wiktorin:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Thoren:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer. Hellstrand:Immune Pharmaceuticals: Patents & Royalties: Filed or pending patents protecting the use of NOX2 inhibitors in cancer. Martner:Immune Pharmaceuticals: Patents & Royalties: Pending patent protecting the use of NOX2 inhibitors in cancer.

RNA Binding Protein RBM47 Inhibits the K562 Cell Proliferation by Regulating HMGA2 mRNA Expression

To investigate the regulatory effects of RBM47 on HMGA2 and the function of RBM47 in human chronic myeloid leukemia cell K562. K562 cells were transduction by the overexpressed and knockdown RBM47 lentiviral vector. CCK-8 assay was used to detect the effect of RBM47 on the proliferation of K562 cells. Flow cytometry assay was used to detect the effect of RBM47 on the cell cycle progression of K562 cells. RNA immunoprecipitation assay was used to detect the association between RBM47 and HMGA2 mRNA. RT-qPCR was used to detect the effects of RBM47 on the stability of HMGA2 mRNA. Western blot was used to evaluate the effect of RBM47 on HMGA2 protein expression. The overexpressed RBM47 could inhibit the proliferation and cell cycle progression of K562 cells. However, the inhibitation of RBM47 could improve the proliferation and cell cycle progression of K562 cells. RBM47 combined with HMGA2 mRNA could promote the degradation of HMGA2 mRNA. Thus, the overexpressed RBM47 could decrease the expression of HMGA2 protein in K562 cells. RNA binding protein RBM47 can inhibit the proliferation of K562 cells by regulating HMGA2 expression.

Anti-chronic myeloid leukemia activity and quantitative structure-activity relationship of novel thiazole aminobenzamide derivatives

The anti-chronic myeloid leukemia activity of thiazole aminobenzamide derivatives in vitro was tested by a methanethiosulfonate (MTS)-based viability assay method, and the result showed that some compounds exhibited good inhibitory activities against human chronic myeloid leukemia cell line K562, imatinib-resistant strain K562/R and T135I mutant cell line BaF3-ABL-BCR-T315I. Comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods were used to analyze the relationship between the structure of thiazole aminobenzamide derivatives and the inhibition of K562/R cell activity. In CoMFA, Q2 was 0.899 and R2 was 0.963; in CoMSIA, Q2 and R2 were 0.840 and 0.903, respectively. These data indicated that the selected test set showed suitable external predictive ability. Combined with the contour map results, we further analyzed the three-dimensional quantitative structure (3D-QSAR) model. The results demonstrated that in the backbone of the thiazole aminobenzamide derivative, the substitution of a small group at R1 position, or the introduction of a hydrophilic group at R2 position, or the introduction of a large-volume amino acid at R3 position may be beneficial to improve the anti-CML activity of the compound.

Biological activity survey of Pereskia aculeata Mill. and Pereskia grandifolia Haw. (Cactaceae)

Background: Pereskia aculeata and P. grandifolia are non-traditional Brazilian vegetables with high nutritional value used in traditional medicine. The antioxidant, anticholinesterase, molluscicidal, cytotoxic, and antiproliferative properties of hydroethanolic extracts of P. aculeata and P. grandifolia leaves (PAL, PGL) and fruits (PAF, PGF) are investigated in this study. Methods: All extracts were prepared by maceration with ethanol 70%. Their antioxidant properties were assessed through DPPH, ABTS, FRAP, and β-carotene bleaching inhibition assays. A TLC bioautography method was employed to evaluate the inhibiting capacity of the acetylcholinesterase enzyme. The molluscicidal activity was tested against the snail Biomphalaria glabrata, which serves as an intermediate host for Schistosoma mansoni. The cytotoxic activity was assessed by a Artemia salina lethality test and the antiproliferative properties against seven human carcinoma cell lines. Results: Compounds with anticholinesterase activity were found in all extracts. Polar compounds present in PAF and PGL extracts were the most active (IC50 &lt; 25 μg of dry mass) and had an adequate inhibition capacity of the AChE. PGF and PGL were classified as moderate (LC50 = 19.2 μg/ml) and modest molluscicidal agents (LC50 = 66.6 μg/ml), respectively. All extracts exhibited selective antiproliferative activity against human chronic myeloid leukemia cell lines (K562). PAL, PGL, and PGF presented potent antiproliferative activity (TGI ≤ 5 μg/ml). Conclusion: Both species exhibited anticholinesterase, cytotoxic and antiproliferative properties. This research supports the potential of these species as sustainable sources of nutraceutical compounds.

Pharmacokinetics and tissue distribution study of hupehenenine in rats: A novel isosteroid alkaloid isolated from Bulbus Hupehensis Fritillariae.

Hupehenenine is a novel isosteroid alkaloid that was first isolated from Bulbus Hupehensis Fritillariae. The inhibitory proliferation effect of hupehenenine and its three related alkaloid derivatives, including o-caproyl-hupehenenine, o-(2-furanoyl)-hupehenenine, and Δ5(6) -isopeimine on human lung cancer cell line, human chronic myeloid leukemia cell line, and human thyroid duct cancer cell line in vitro, has been identified. This study first developed a sensitive HPLC-MS/MS method for the simultaneous quantification of hupehenenine and three alkaloid derivatives in rat plasma and tissues. The developed method was validated, and it was linear over the concentration range of 1-800 ng/mL for all analytes with R2 ≥ 0.9939 and 0.9972, respectively, in rat plasma and rat liver homogenate. The lower limit of quantitation was 1ng/mL for all analytes. The intra-day and inter-day precision and accuracy were satisfactory. This validated method was successfully applied to investigate the pharmacokinetics and tissue distribution of hupehenenine in rats. In pharmacokinetic study, the maximum plasma concentration of rats exists gender difference. Tissue distribution study showed that hupehenenine has good affinity for multiple tissues but is unable to cross the blood-brain barrier. These results may provide a useful reference for further research of hupehenenine and its three related alkaloid derivatives.

Design, synthesis, and biological evaluation of novel bromo-pyrimidine analogues as tyrosine kinase inhibitors

In the present investigation we designed, synthesized and evaluated a novel series of bromo-pyrimidine analogues (6a-j,7a-e,9a-f,and10a-f) as anticancer agents. The compounds were characterized using spectroscopic studies and elemental analysis and screened for theirin vitrocytotoxic activity by MTT assay against four cancer cell lines including HCT116 (human colon cancer cell line), A549 (human lung cancer cell line), K562 (human chronic myeloid leukemia cell line), U937 (human acute monocytic myeloid leukemia cell line) as well as the normal human liver cell line, L02. Most of the compounds showed potent activity on K562 cells. Considering this, the compounds were evaluated for Bcr/Abl tyrosine kinase inhibitory activity by ADP-Glo assay. Dasatinib was used as standard drug for both cytotoxicity and tyrosine kinase inhibition studies. The compounds,6g,7d,9c,and10eemerged as potent Bcr/Abl kinase inhibitors. Hence, the potent compounds that arose out of this investigation are potential lead molecules to develop as an alternative to existing dasatinib therapy.

Overexpression of chromodomain helicase DNA binding protein 5 (CHD5) inhibits cell proliferation and induces cell cycle arrest and apoptosis in chronic myeloid leukemia.

BackgroundChromodomain helicase DNA binding protein 5 (CHD5) was reported to be a tumor suppressor and our previous work showed CHD5 was epigenetically inactivated in human chronic myeloid leukemia (CML). This study aimed to investigate the effect of its overexpression on CML tumorigenesis.MethodsQuantitative reverse-transcriptase PCR and Western blotting analysis were used to detect the expression of CHD5 in human CML cell lines. The endogenous CHD5 expression was activated in two CML cell lines by CRISPR/dCas9-SAM system. In vitro cell function experiments were performed including proliferation, colony formation, apoptosis, autophagy, senescence and differentiation assays. Furthermore, tumorigenicity was evaluated in vivo in nude mice xenograft model.ResultsCHD5 was down-regulated in CML cell lines compare to normal bone marrow mononuclear cells (MCs). Cell proliferation after activating CHD5 was significantly inhibited. Moreover, overexpression of CHD5 induced G2/M phase arrest and apoptosis in CML cells. In a tumor xenograft mouse model, CHD5 restoration was found to sharply repress tumor growth. Compared with the control group, overexpression of CHD5 enhanced the expression of p21 and cdc2 phosphorylation, whereas decreased the protein level of Cyclin B1. Furthermore, experiments revealed that up-regulation of CHD5 activated caspase-3, while anti-apoptosis protein Bcl-2 expression was reduced in CML cells.ConclusionsCHD5 plays a role of anti-tumorigenic effects involved in CML cell proliferation, cell cycle arrest and apoptosis.

NT157, an IGF1R-IRS1/2 inhibitor, exhibits antineoplastic effects in pre-clinical models of chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is successfully treated with BCR-ABL1 tyrosine kinase inhibitors, but a significant percentage of patients develop resistance. Insulin receptor substrate 1 (IRS1) has been shown to constitutively associate with BCR-ABL1, and IRS1-specific silencing leads to antineoplastic effects in CML cell lines. Here, we characterized the efficacy of NT157, a pharmacological inhibitor of IGF1R-IRS1/2, in CML cells and observed significantly reduced cell viability and proliferation, accompanied by induction of apoptosis. In human K562 cells and in murine Ba/F3 cells, engineered to express either wild-type BCR-ABL1 or the imatinib-resistant BCR-ABL1T315I mutant, NT157 inhibited BCR-ABL1, IGF1R, IRS1/2, PI3K/AKT/mTOR, and STAT3/5 signaling, increased CDKN1A, FOS and JUN tumor suppressor gene expression, and reduced MYC and BCL2 oncogenes. NT157 significantly reduced colony formation of human primary CML cells with minimal effect on normal hematopoietic cells. Exposure of primary CML cells harboring BCR-ABL1T315I to NT157 resulted in increased apoptosis, reduced cell proliferation and decreased phospho-CRKL levels. In conclusion, NT157 has antineoplastic effects on BCR-ABL1 leukemogenesis, independent of T315I mutational status.

Tanshinone I inhibited growth of human chronic myeloid leukemia cells via JNK/ERK mediated apoptotic pathways.

Tanshinone I (Tan I) is one of the main bioactive ingredients derived from Salvia miltiorrhiza Bunge, which has exhibited antitumor activities toward various human cancer cells. However, its effects and underlying mechanisms on human chronic myeloid leukemia (CML) cells still require further investigation. This study determined the effects and mechanisms of anti-proliferative and apoptosis induction activity induced by Tan I against K562 cells. The cytotoxic effect of Tan I at varying concentrations on K562 cells was evaluated via MTT assay. Cell apoptosis was further investigated through DAPI staining and flow cytometry analysis. The expression levels of apoptosis-related proteins and activities of JNK/ATF2 and ERK signaling pathways were analyzed by western blot. Quantitative PCR was performed to further determine mRNA expression levels of JNK1/2 and ERK1/2 after Tan I treatment. The results indicated that Tan I significantly inhibited K562 cell growth and induced apoptosis in a concentration- and time-dependent manner. It induced significant cellular morphological changes and increased apoptosis rates in CML cells. Tan I promoted the cleavages of caspase-related proteins, as well as increased the expression levels of PUMA. Furthermore, Tan I significantly activated JNK and inhibited ATF-2 and ERK signaling pathways. The mRNA expression levels of JNK1/2 and ERK1/2 were up-regulated by Tan I, further confirming its regulatory effects on JNK/ERK signaling pathways. Overall, our results indicated that Tan I suppressed cell viability via JNK- and ERK-mediated apoptotic pathways in K562 cells, suggesting that it might be a promising candidate as a novel anti-leukemia drug.

8-Hydroxydaidzein, an Isoflavone from Fermented Soybean, Induces Autophagy, Apoptosis, Differentiation, and Degradation of Oncoprotein BCR-ABL in K562 Cells.

8-Hydroxydaidzein (8-OHD, 7,8,4′-trihydoxyisoflavone) is a hydroxylated derivative of daidzein isolated from fermented soybean products. The aim of this study is to investigate the anti-proliferative effects and the underlying mechanisms of 8-OHD in K562 human chronic myeloid leukemia (CML) cells. We found that 8-OHD induced reactive oxygen species (ROS) overproduction and cell cycle arrest at the S phase by upregulating p21Cip1 and downregulating cyclin D2 (CCND2) and cyclin-dependent kinase 6 (CDK6) expression. 8-OHD also induced autophagy, caspase-7-dependent apoptosis, and the degradation of BCR-ABL oncoprotein. 8-OHD promoted Early Growth Response 1 (EGR1)-mediated megakaryocytic differentiation as an increased expression of marker genes, CD61 and CD42b, and the formation of multi-lobulated nuclei in enlarged K562 cells. A microarray-based transcriptome analysis revealed a total of 3174 differentially expressed genes (DEGs) after 8-OHD (100 μM) treatment for 48 h. Bioinformatics analysis of DEGs showed that hemopoiesis, cell cycle regulation, nuclear factor-κB (NF-κB), and mitogen-activated protein kinase (MAPK) and Janus kinase/signal transducers and activators of transcription (JAK-STAT)-mediated apoptosis/anti-apoptosis networks were significantly regulated by 8-OHD. Western blot analysis confirmed that 8-OHD significantly induced the activation of MAPK and NF-κB signaling pathways, both of which may be responsible, at least in part, for the stimulation of apoptosis, autophagy, and differentiation in K562 cells. This is the first report on the anti-CML effects of 8-OHD and the combination of experimental and in silico analyses could provide a better understanding for the development of 8-OHD on CML therapy.

Design, synthesis, biological activity evaluation of 3-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole derivatives as potent JAK 2/3 and aurora A/B kinases multi-targeted inhibitors.

In this study, a series of 3-(4-phenyl-1H-imidazol-2-yl)-1H-pyrazole derivatives were designed, synthesized, and evaluated for their biological activities. Upon performing kinase assays, most of the compounds exhibited potent inhibition against JAK2/3 and Aurora A/B with the IC50 values ranging from 0.008 to 2.52μM. Among these derivatives, compound 10e expressed the most moderate inhibiting activities against all the four kinases with the IC50 values of 0.166μM (JAK2), 0.057μM (JAK3), 0.939μM (Aurora A), and 0.583μM (Aurora B), respectively. Moreover, most of the derived compounds exhibited potent cytotoxicity against human chronic myeloid leukemia cells K562 and human colon cancer cells HCT116, while compound 10e expressed antiproliferative activities against K562 (IC50=6.726μM). According to western blot analysis, compound 10e down-regulated the phosphorylation of STAT3, STAT5, Aurora A, and Aurora B in a dose-dependent manner in K562 and HCT116cells. Cell cycle analysis revealed that compound 10e inhibited the proliferation of cells by inducing cell cycle arrest in the G2 phase. The molecular modeling suggested that compound 10e could maintain a binding mode similar to the binding mode of AT9832, a common JAK 2/3 and Aurora A/B kinases multi-target kinase inhibitor. Therefore, compound 10e might be a potential agent for cancer therapy deserving further research.

Circadian clock gene Period2 suppresses human chronic myeloid leukemia cell proliferation.

Circadian clock genes (CCGs) are reported to serve pivotal roles in regulating the development of certain tumors, including lung cancer and colon cancer . However, their expression patterns and function in chronic myeloid leukemia (CML) remains poorly understood. The present study aimed to investigate the expression and function of circadian clock gene Period2 (Per2) in human CML. Per2 expression levels in neutrophils isolated from patients with CML and healthy donors were measured via reverse transcription-quantitative PCR. Subsequently, through lentivirus transduction, Per2 was stably overexpressed in human CML cell line KCL22 cells, which were injected into nude mice to investigate the in vivo role of Per2 by measuring CML tumor size and weight. Additionally, Per2 expression levels in patients with acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL) were analyzed by re-analyzing microarray data in the Gene Expression Omnibus database. Per2 expression was significantly lower in neutrophils isolated from patients with CML patients compared with healthy donors, and was negatively correlated with the expression level of c-Myc. Similarly, patients with AML or CLL displayed lower Per2 expression levels compared with healthy controls. Per2 overexpression inhibited KCL22 cell proliferation in nude mice and in vitro, and induced cell cycle arrest at the G1 phase. By contrast, the results also indicated that KCL22 cell apoptosis was not regulated by Per2. The present study identified Per2 as a potential tumor suppressor in human CML.

Transcriptomics-Based Characterization of the Toxicity of ZnO Nanoparticles Against Chronic Myeloid Leukemia Cells.

IntroductionZinc oxide nanoparticles (ZnO NPs) have recently attracted attention as potential anti-cancer agents. To the best of our knowledge, the toxicity of ZnO NPs against human chronic myeloid leukemia cells (K562 cell line) has not been studied using transcriptomics approach.ObjectiveThe goals of this study were to evaluate the capability of ZnO NPs to induce apoptosis in human chronic myeloid leukemia cells (K562 cells) and to investigate the putative mechanisms of action.MethodsWe used viability assay and flowcytometry coupled with Annexin V-FITC and propidium iodide to investigate the toxicity of ZnO NPs on K562 cells and normal peripheral blood mononuclear cells. Next we utilized a DNA microarray-based transcriptomics approach to characterize the ZnO NPs-induced changes in the transcriptome of K562 cells.ResultsZnO NPs exerted a selective toxicity (mainly by apoptosis) on the leukemic cells (p≤0.005) and altered their transcriptome; 429 differentially expressed genes (DEGs) with fold change (FC)≥4 and p≤0.008 with corrected p≤0.05 were identified in K562 cells post treatment with ZnO NPs. The over-expressed genes were implicated in “response to zinc”, “response to toxic substance” and “negative regulation of growth” (corrected p≤0.05). In contrast, the repressed genes positively regulated “cell proliferation”, “cell migration”, “cell adhesion”, “receptor signaling pathway via JAK-STAT” and “phosphatidylinositol 3-kinase signaling” (corrected p≤0.05). Lowering the FC to ≥1.5 with p≤0.05 and corrected p≤0.1 showed that ZnO NPs over-expressed the anti-oxidant defense system, drove K562 cells to undergo mitochondrial-dependent apoptosis, and targeted NF-κB pathway.ConclusionTaken together, our findings support the earlier studies that reported anti-cancer activity of ZnO NPs and revealed possible molecular mechanisms employed by ZnO NPs to induce apoptosis in K562 cells.

Abstract 5267: Utilizing stimulated Raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells

Abstract Ponatinib is a clinically approved tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML). Drug resistance is a widespread problem in CML treatment, where ponatinib resistant patients have very limited treatment options. In this study stimulated Raman scattering (SRS) microscopy was used to allow label-free imaging of intracellular ponatinib with high sensitivity and specificity in live human CML cell lines, in the context of ponatinib resistance. Ponatinib has an alkyne moiety in its structure that makes it inherently Raman active in the cellular silent region of the Raman spectrum. SRS microscopy represents a powerful imaging tool for visualizing label-free drug molecules within cells with high resolution, without the need for additional labels, or nanoparticle sensors as used in many other optical imaging technologies. It provides Raman imaging with minimal spectral distortion and a quantitative output, allowing the intracellular concentrations of drug molecules to be accurately determined. Intracellular visualization of ponatinib was achieved at biologically relevant, nanomolar concentrations for the first time using SRS. It was determined that ponatinib is sequestered into the lysosomes, with a higher lysosomal concentration found in drug resistant cells. This was associated with increased lysosome biogenesis. Target engagement studies showed that treatment with chloroquine reduced ponatinib accumulation in lysosomes, but did not re-sensitise cells to ponatinib, confirming BCR-ABL independent resistance mechanism in this CML cell model. In summary, we have visualized intracellular ponatinib localization for the first time using SRS, demonstrating that acquired drug resistance can influence drug uptake and localisation in CML, which in turn has an effect on target engagement. Citation Format: Kristel Sepp, Martin Lee, Marie T. Bluntzer, G. Vignir Helgason, Alison N. Hulme, Valerie G. Brunton. Utilizing stimulated Raman scattering microscopy to study intracellular distribution of label-free ponatinib in live cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5267.

Inhibitory Effect of S1PR2 Antagonist JTE-013 on Proliferation of Chronic Myeloid Leukemia Cells

To investigate the effect of sphingosine-1-phosphate receptor 2 (S1PR2) specific antagonist JTE-013 on the proliferation of human chronic myeloid leukemia (CML) cell line K562. K562 cells were treated with JTE-013 (0, 0.5, 1, 5, 10, 20 μmol/L) for 24 and 48 hours respectively, CCK8 assay was used to detect the cell viability. K562 cells were treated with JTE-013 (0, 5, 10, 20 μmol/L) for 24 hours, propidium iodide (PI) DNA staining was used to analyze the cell cycle, Western blot was used to determine the levels of P21 and Cyclin D1 protein expression. JTE-013 inhibited the proliferation of CML cell line K562 in a dose dependent manner (r=-0.971). The proliferation rate of CML cells showed that the activity of CML cells decreased gradually with the increase of JTE-013 concentration (r=-0.971). The detection demonstrated that JTE-013 suppressed tumor cell proliferation through cell cycle arrest in G0/G1 phase. Further detection of the protein expressions of G1 phase regulators showed that level of P21 increased, and expression of Cyclin D1 decreased. JTE-013, a S1PR2 antagonist, can inhibit the proliferation of human CML K562 cells, which may be achieved by arresting the cells in G0/G1 phase.

ABL Genomic Editing Sufficiently Abolishes Oncogenesis of Human Chronic Myeloid Leukemia Cells In Vitro and In Vivo.

Chronic myelogenous leukemia (CML) is the most common type of leukemia in adults, and more than 90% of CML patients harbor the abnormal Philadelphia chromosome (Ph) that encodes the BCR-ABL oncoprotein. Although the ABL kinase inhibitor (imatinib) has proven to be very effective in achieving high remission rates and improving prognosis, up to 33% of CML patients still cannot achieve an optimal response. Here, we used CRISPR/Cas9 to specifically target the BCR-ABL junction region in K562 cells, resulting in the inhibition of cancer cell growth and oncogenesis. Due to the variety of BCR-ABL junctions in CML patients, we utilized gene editing of the human ABL gene for clinical applications. Using the ABL gene-edited virus in K562 cells, we detected 41.2% indels in ABL sgRNA_2-infected cells. The ABL-edited cells reveled significant suppression of BCR-ABL protein expression and downstream signals, inhibiting cell growth and increasing cell apoptosis. Next, we introduced the ABL gene-edited virus into a systemic K562 leukemia xenograft mouse model, and bioluminescence imaging of the mice showed a significant reduction in the leukemia cell population in ABL-targeted mice, compared to the scramble sgRNA virus-injected mice. In CML cells from clinical samples, infection with the ABL gene-edited virus resulted in more than 30.9% indels and significant cancer cell death. Notably, no off-target effects or bone marrow cell suppression was found using the ABL gene-edited virus, ensuring both user safety and treatment efficacy. This study demonstrated the critical role of the ABL gene in maintaining CML cell survival and tumorigenicity in vitro and in vivo. ABL gene editing-based therapy might provide a potential strategy for imatinib-insensitive or resistant CML patients.

Production Enhancement of an Anticoagulant Trypsin Inhibitor from Oceanimonas sp. BPMS22 and Its Anti-cancer Activity

The significant role of proteases in the cancer progression exposed them as potential targets for drug discovery. Regulating protease activities using protease inhibitors is one of the major therapeutic strategies for the treatment of cancer. The proteolysis activities of serine proteases in different stages of metastasis can be regulated by serine protease inhibitors. In the present work, the application of a peptide trypsin inhibitor (TI) from Oceanimonas sp. BPMS22 in cancer treatment was exploited. Initially, the process parameters such as initial medium pH, glucose, yeast extract, and sodium chloride concentrations were optimized for the maximum inhibitor activity using Response surface methodology. The maximum specific trypsin inhibitor activity observed was 40.16 ± 1.33 IU/mg protein at predicted critical conditions, glucose, 19.51 g/L; yeast extract, 23.04 g/L; sodium chloride, 28.29 g/L; and pH 7.26. Further, the apoptotic cell death of purified trypsin inhibitor-treated human chronic myeloid leukemia (K562) cell lines was demonstrated by MTT assay, AO/EtBr staining, and DNA ladder assay. The cellular oxidative stress in K562 cells was confirmed by glutathione levels and fluorescence imaging of ROS generation.

A new cytotoxic isoflavane from Dalbergiae Odoriferae Lignum

Fourteen compounds were isolated from the ethanol extract of Dalbergiae Odoriferae Lignum by various chromatographic techniques, including column chromatographies on silica gel, Sephadex LH-20 and semi-preparative HPLC. Their structures were determined by spectroscopic techniques as S-3'-hydroxy-7,2',4'-trimethoxyisoxane(1), 2-(2',4'-dimethoxyphenyl)-6-hydroxybenzofuran(2), 2-(2'-hydroxy-4'-methoxyphenyl)-6-methoxybenzofuran(3), 7,2',4'-trimethoxydihydroisoflavone(4), sativanone(5), 3,9-dimethoxy-6H-benzofuro[3,2-c]chromen-6-one(6),(6 aS,11 aS)-homopterocarpin(7),(6 aS,11 aS)-8-hydroxy-3,9-dimethoxypterocarpan(8),(6 aS,11 aS)-3,8,9-trimethoxypterocarpan(9), isodalbergin(10), isoliquiritigenin(11), butein(12), butin(13) and 3,7,4'-trihydroxyflavone(14). Among them, compound 1 was a new compound, while 2 and 3 were new natural products, 6, 8, 9 and 14 were isolated for the first time from Dalbergiae Odoriferae Lignum. Compounds 1-14 were tested for their cytotoxic activity against human hepatoma cell line BEL-7402, human gastric cancer cell line SCG-7901, human lung cancer cell line A549, human chronic myeloid leukemia cell line K562 and HeLa human cervical cancer cellline by MTT method. Compound 1 exhibited significant cytotoxicity with IC_(50) values ranging from 2.85 to 11.62 μg·mL~(-1). In addition, 2, 11 and 12 showed weak cytotoxic activities.

HERG K+ Channels Promote Survival of Irradiated Leukemia Cells

Many tumor cells express highly elevated activities of voltage-gated K+ channels in the plasma membrane which are indispensable for tumor growth. To test for K+ channel function during DNA damage response, we subjected human chronic myeloid leukemia (CML) cells to sub-lethal doses of ionizing radiation (0–8 Gy, 6 MV photons) and determined K+ channel activity, K+ channel-dependent Ca2+ signaling, cell cycle progression, DNA repair, and clonogenic survival by whole-cell patch clamp recording, fura-2 Ca2+ imaging, Western blotting, flow cytometry, immunofluorescence microscopy, and pre-plating colony formation assay, respectively. As a result, the human erythroid CML cell line K562 and primary human CML cells functionally expressed hERG1. Irradiation stimulated in both cell types an increase in the activity of hERG1 K+ channels which became apparent 1–2 h post-irradiation. This increase in K+ channel activity was paralleled by an accumulation in S phase of cell cycle followed by a G2/M cell cycle arrest as analyzed between 8 and 72 h post-irradiation. Attenuating the K+ channel function by applying the hERG1 channel inhibitor E4031 modulated Ca2+ signaling, impaired inhibition of the mitosis promoting subunit cdc2, overrode cell cycle arrest, and decreased clonogenic survival of the irradiated cells but did not affect repair of DNA double strand breaks suggesting a critical role of the hERG1 K+ channels for the Ca2+ signaling and the cell cycle control during DNA damage response.

Base Induced Condensation of Malononitrile with Erlenmeyer Azlactones: An Unexpected Synthesis of Multi‐Substituted Δ2‐Pyrrolines and Their Cytotoxicity

An efficient, metal free approach to synthesize multi-substituted Δ2 -pyrroline derivatives by mild base catalyzed cyclocondensation of malononitrile with Erlenmeyer azlactones via 1,2 addition was developed. The modularity of this reaction was used to assemble a range of poly-substituted pyrrolines. Further, synthesized products were screened for cytotoxic properties on different cancer cell lines such as A549 (Human lung adenocarcinoma cells), HeLa (Human cervical adenocarcinoma cells), Jurkat (Human chronic myeloid leukemia cells) and K562 (Human leukemic T cell Lymphoblast cells). Among the synthesized library of compounds, 6f and 6q displayed potent cytotoxic activity.