Apoptotic effect of Bulbine Natalensis and Chlorophytum Comosum in myelogenous Leukemia K562 cell line

Bulbine natalensis and Chorophytum comosum are potential medicinal source for the treatment of cancers. Chronic myeloid leukaemia is a hematopoietic stem cells disorder treated by tyrosine kinase inhibitors but often cause recurrence of the leukaemia after cessation of therapy, hence require alternative treatment. This study determines the anti-cancer effect of leaf, root and bulb methanolic and aqueous extracts of B. natalensis and C. comosum in chronic human myelogenous leukaemia (K562) cell line by MTT, Hoechst bis-benzimide nuclear and annexin V stain assays. The root methanolic extract of B. natalensis and C. comosum showed a high cytotoxicity of 8.6% and 16.7% respectively on the K562 cell line at 1,000 μg/ml concentration. Morphological loss of cell membrane integrity causing degradation of the cell and fragmentation were observed in the root methanolic extract of both plants. A high apoptosis (p < 0.0001) was induced in the K562 cells by both leaf and root extracts of the C. comosum compared to the B. natalensis. This study shows both plants possess apoptotic effect against in vitro myelogenous leukaemia which contributes to the overall anti-cancer properties of B. natalensis and C. comosum to justify future therapeutic applications against chronic myelogenous leukaemia blood cancer.

Metabolites From the Mangrove-Derived Fungus Cladosporium sp. HNWSW-1

Two new benzoic acids, cladoslide A (1) and cladoslide B (2); one new β-carboline derivative, cladospomine (3); and one new pyridin-2(1H)-one, cladoslide C (4), were isolated from the fermentation cultures of the mangrove-derived fungus Cladosporium sp. HNWSW-1, along with the previously reported N-acetyl-β-oxotryptamine (5), (4S,5S,11R)-iso-cladospolide B (6), (4S,5S,11S)-iso-cladospolide B (7), and (4R,5S,11R)-iso-cladospolide B (8). Their structures were elucidated by spectroscopic analysis, Rh2(OCOCF3)4-induced ECD experiments, and Marfey’s method. Compound 1 showed cytotoxicity against the K562 cell line with IC50 values of 13.10 ± 0.08 μM. Moreover, compounds 1 and 5 exhibited inhibitory activity against α-glycosidase with IC50 values of 0.32 ± 0.01 mM and 0.17 ± 0.01 mM, respectively.

HiCArch: A Deep Learning-based Hi-C Data Predictor

Hi-C sequencing analysis is one of the most popular methods to study three-dimensional (3D) genome structures, which affect the gene expression and other cellular activities by allowing distal regulations in spatial proximity. Hi-C sequencing analysis enhances understanding of chromatin functionality. However, due to the high cost of Hi-C sequencing, the publicly available Hi-C data of high resolutions (such as 10kb) are limited in only a few cell types. In this paper we present HiCArch, a light-weight deep neural network that predicts Hi-C contact matrices from 11 common 1D epigenomic features. HiCArch identifies topological associated domains (TADs) of 10kb resolution within the distance of 10Mb. HiCArch obtains train Pearson correlation score at 0.9123 and test Pearson correlation score at 0.9195 when trained on K562 cell line. which are significantly higher than previous approaches, such as HiC-Reg, Akita, DeepC, and Epiphany.

An Experimental and Theoretical Study of the 1,3-Dipolar Cycloaddition of Alloxan-Derived Azomethine Ylides to Cyclopropenes

A diastereoselective synthesis of biologically interesting spirobarbiturates has been achieved via [3 + 2] cycloaddition of alloxan-derived azomethine ylides to 3-R-1,2-diphenylcyclopropenes. With this approach, various spirobarbiturate-3-azabicyclo[3.1.0]hexanes and spirobarbiturate-cyclopropa[a]pyrrolizines were obtained in moderate to good yields with excellent diastereoselectivities. DFT calculations (M11 density functional theory) have been carried out to shed light on the molecular mechanism of 1,3-dipolar cycloaddition of alloxan-derived azomethine ylides to cyclopropenes. The cytotoxic activity of some obtained compounds against human erythroleukemia (K562) cell line was evaluated in vitro by MTS-assay.

ATR/CHK1/WEE1 Dependency in SRSF2-Mutated MDS/AML

Background: Mutations in splicing factor gene SRSF2 are recurrent drivers in 5-15 % of patients with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). A key property of SRSF2 mutations is that they occur early in the pathogenesis of MDS and are therefore present in all tumor cells in a patient. This property makes targetable vulnerabilities caused by SRSF2 mutations exceptionally important as they provide a way to inhibit the whole tumor. We previously demonstrated that splicing factor mutations induce R-loop-dependent activation of ATR, rendering cells sensitive to ATR inhibition. R-loops are transcription intermediates consisting of an RNA:DNA hybrid and a displaced single-stranded DNA. Accumulation of aberrant R-loops induces the ATR kinase, which activates the G2-M cell cycle checkpoint via CHK1- and WEE1-mediated signaling. In normal cells, activation of the G2-M checkpoint halts the cell cycle until R-loops have been resolved. When the ATR pathway is inhibited, checkpoint activation does not occur, causing cells with unresolved R-loops to proceed to mitosis, resulting in DNA damage and cell death. We, therefore, sought to assess primary human MDS/AML samples for sensitivity to perturbation of the ATR/CHK1/WEE1 pathway and identify mechanisms of resistance. Methods: Sensitivity of 147 AML patient samples to 515 oncology drugs was tested ex vivo. Bone marrow mononuclear cells were incubated with 5 concentrations of each drug for 72 h followed by measurement of cell viability by CTG assay. Somatic mutations were identified by exome sequencing of matched leukemic bone marrow and skin biopsy samples. Isogenic K562 cell line clones carrying SRSF2 P95H/L/R mutations were generated using CRISPR/Cas9 editing. The presence of SRSF2 mutation was confirmed by CRISPR-sequencing and expression by whole transcriptome RNA-sequencing. Drug sensitivity of the K562 clones with and without SRSF2 mutation was determined by incubating cells with 16 concentrations of prexasertib, SRA-737, adavosertib, or BAY-1895344, followed by determination of cell viability by the MTS assay. Results: Analysis of ex vivo drug sensitivities in AML patient samples identified vulnerability to CHK1 and WEE1 inhibition in SRSF2-mutated AML: SRSF2 mutation is associated with sensitivity to the CHK1 inhibitors prexasertib (p = 0.006) (Fig 1 A and B) and PF-00477736 (p = 0.002) and the WEE1 inhibitor adavosertib (p = 0.003). To establish whether the isogenic SRSF2-mutant K562 cell line models recapitulate known downstream aberrations associated with SRSF2 mutations in patients, we analyzed gene expression and splicing. SRSF2 contains an RNA binding domain with affinity to CCNG or GGNG exonic splicing enhancer sequences. Similar to what has been observed in patients, the K562 clones with SRSF2 mutation show reduced use of GGNG sequence motifs at skipped exons. These results demonstrate that isogenic K562 clones recapitulate known alterations caused by mutant SRSF2. To determine whether SRSF2 mutations induce sensitivity to inhibition of ATR, CHK1, and WEE1, we tested 10 isogenic SRSF2 mutant and 4 wild-type K562 clones. Cells with SRSF2 mutation show increased sensitivity to ATR/CHK1/WEE1 inhibition (Fig 1C). We found no significant difference in drug sensitivity between clones carrying SRSF2 P95H/L/R substitutions. Clones with higher SRSF2 mutant allele dosage are more sensitive (Fig 1D). We identified a subset of SRSF2 mutated AML samples that were resistant to CHK1 and WEE1 inhibition. All resistant AML have co-occurring RUNX1 mutations (Fig 1B). In AML, RUNX1 mutations are associated with therapy resistance, suggesting that these mutations contribute to drug resistance. To test whether RUNX1 mutations induce resistance to ATR/CHK1/WEE1 inhibition in SRSF2-mutant leukemia, we introduced RUNX1 loss-of-function mutations in isogenic K562 carrying SRSF2 mutations. Candidate resistance factors identified by ATAC and RNA-sequencing will be validated in functional assays. Conclusions: Our results indicate that SRSF2-mutated leukemia harbor a vulnerability to the inhibition of ATR, CHK1, and WEE1 kinases. Cell line models indicate that sensitivity is similar across mutant alleles and dependent on allelic copy number. Several ATR/CHK1 and WEE1 inhibitors are in development, and our results suggest that these compounds could be effective treatments for SRSF2-mutated MDS and AML. Figure 1 Figure 1. Disclosures Graubert: astrazeneca: Research Funding; Janssen: Research Funding; Calico: Research Funding.

Co-Delivery of Berberine Chloride and Tariquidar in Nanoliposomes Enhanced Intracellular Berberine Chloride in a Doxorubicin-Resistant K562 Cell Line Due to P-gp Overexpression

The MDR phenomenon has become a major obstacle in the treatment of cancers, and among the strategies to reverse it, the inhibition of P-gp function and expression is essential to increase for effective anticancer drugs. In the present paper, the co-delivery of berberine chloride and tariquidar loaded nanoliposomes was investigated with the aim of enhancing solubility and improving desired effects for the antineoplastic drug and the P-gp inhibitor. Developed nanoliposomes were loaded with the electron-dense enzyme horseradish peroxidase, and analyzed by TEM to investigate their ability to enter in both K562 and K562/DOXO cell lines. Receptor-mediated endocytosis was evidenced for both cell lines. Nanoliposomes were loaded with tariquidar, berberine chloride, or both, maintaining chemical and physical characteristics—i.e., size, homogeneity, and encapsulation efficiency—and high suitability for parenteral administration. Tariquidar was able to reverse the MDR in the K562/DOXO cell line. Tariquidar- and berberine chloride-loaded nanoliposomes showed a significant increase of berberine chloride accumulation in tumor cells, which could be correlated with resensitization of the resistant cells to the antitumor agent. These results suggest that the co-delivery of the P-gp inhibitor, tariquidar, and the cytotoxicity inducer, berberine chloride, looks like a promising approach to overcome the MDR.

Synthesis and Erythroid Induction Activity of New Thiourea Derivatives

Background: The use of medicinal agents to augment the fetal hemoglobin (HbF) accretion is an important approach for the treatment of sickle-cell anemia and β-thalassemia. HbF inducers have the potential to reduce the clinical symptoms and blood transfusion dependence in the patients of β- hemoglobinopathies. Objectives: The current study was aimed to examine the erythroid induction potential of newly synthesized thiourea derivatives. Methods: Thiourea derivatives 1-27 were synthesized by using environmentally friendly methods. Compounds 3, 10 and 22 were found to be new. The structures of synthesized derivatives were deduced by using various spectroscopic techniques. These derivatives were then evaluated for their erythroid induction using the human erythroleukemic K562 cell line, as a model. The benzidine-H2O2 assay was used to evaluate erythroid induction, while HbF expression was studied through immunocytochemistry using the Anti-HbF antibody. Cytotoxicity of compounds 1-27 was also evaluated on mouse fibroblast 3T3 cell line and cancer Hela cell line using MTT assay. Result: All the compounds (1-27) have not been reported for their erythroid induction activity previously. Compounds 1, 2, and 3 were found to be the potent erythroid inducing agents with % induction of 45± 6.9, 44± 5.9, and 41± 6.1, at 1.56, 0.78, and 0.78 μM concentrations, respectively, as compared to untreated control (12 ± 1 % induction). Furthermore, compound 1, 2, and 3 significantly induced fetal hemoglobin the expression up to 4.2-fold, 4.06-fold, and 3.52-fold, respectively, as compared to untreated control. Moreover, the compounds 1-4, 6-9, 11, 12, 15, 17, 19, 22, 23, and 25 were found to be non-cytotoxic against the 3T3 cell line. Conclusion: This study signifies that the compounds reported here may serve as the starting point for the designing and development of new fetal hemoglobin inducers for the treatment of β- hemoglobinopathies.

A New Series of Antileukemic Agents: Design, Synthesis, In Vitro and In Silico Evaluation of Thiazole-Based ABL1 Kinase Inhibitors

Background: After the milestone approval of imatinib, more than 25 antitumor agents targeting kinases have been approved, and several promising candidates are in various stages of clinical evaluation. Objectives : Due to the importance of thiazole scaffold in targeted anticancer drug discovery, the goal of this work is the design of new thiazolyl hydrazones as potent ABL1 kinase inhibitors for the management of chronic myeloid leukemia (CML). Methods: New thiazolyl hydrazones (2a-p) were synthesized and investigated for their cytotoxic effects on K562 CML cell line. Compounds 2h, 2j and 2l showed potent anticancer activity against K562 cell line. The cytotoxic effects of these compounds on other leukemia (HL-60, MT-2 and Jurkat) and HeLa human cervical carcinoma cell lines were also investigated. Furthermore, their cytotoxic effects on mitogen-activated peripheral blood mononuclear cells (MA-PBMCs) were evaluated to determine their selectivity. Due to its selective and potent anticancer activity, compound 2j was benchmarked for its apoptosis-inducing potential on K562 cell line and inhibitory effects on eight different tyrosine kinases (TKs) including ABL1 kinase. In order to investigate the binding mode of compound 2j into the ATP binding site of ABL1 kinase (PDB: 1IEP), molecular docking study was conducted using MOE 2018.01 program. The QikProp module of Schrödinger’s Molecular modelling package was used to predict the pharmacokinetic properties of compounds 2a-p. Results: 4-(4-(Methylsulfonyl)phenyl)-2-[2-((1,3-benzodioxol-4-yl)methylene)hydrazinyl]thiazole (2j) showed antiproliferative activity against K562 cell line with an IC50 value of 8.87±1.93 µM similar to imatinib (IC50= 6.84±1.11 µM). Compound 2j was found to be more effective than imatinib on HL-60, Jurkat and MT-2 cells. Compound 2j also showed cytotoxic activity against HeLa cell line similar to imatinib. The higher selectivity index value of compound 2j than imatinib indicated that its antiproliferative activity was selective. Compound 2j also induced apoptosis in K562 cell line more than imatinib. Among eight TKs, compound 2j showed the strongest inhibitory activity against ABL1 kinase enzyme (IC50= 5.37±1.17 µM). According to molecular docking studies, compound 2j exhibited high affinity to the ATP binding site of ABL1 kinase forming significant intermolecular interactions. On the basis of in silico studies, this compound did not violate Lipinski's rule of five and Jorgensen's rule of three. Conclusion: Compound 2j stands out as a potential orally bioavailable ABL1 kinase inhibitor for the treatment of CML.

Effect of Chicken Bone Extracts on Metabolic and Mitochondrial Functions of K562 Cell Line

Background: Tetracyclines’ use in intensive animal farming has raised some concerns regarding the biosafety for humans. Increasing evidences have revealed the presence of these drugs in processed animal by-products, such as bone, throughout the food chain. A potential off-target of tetracyclines is the bacterial-like mitochondrial translational machinery, thereby causing proteostatic alterations in mitochondrial DNA-encoded components of the oxidative phosphorylation system. Methods: The Seahorse methodology, confocal microscopy imaging of mitochondrial potential and reactive oxygen species, and q-RT-PCR analysis of the expression of genes involved in mitochondrial biogenesis and mitophagy were carried out on human lymphoblast derived K562 cell line challenged with bone powder derived from chicken treated with or without oxytetracycline and pure oxytetracycline. Results: A complex dose-dependent profile was attained with a low dosage of bone powder extracts causing a metabolic adaptation hallmarked by stimulation of the mitochondrial respiration and enhanced expression of mitochondriogenic factors in particular in cells challenged with oxytetracycline-free bone extract. Conversely, a higher dosage of bone powder extracts, regardless of their source, caused a progressive inhibition of mitochondrial respiration and glycolysis, ultimately leading to cell death. No significant effects of the pure oxytetracycline were observed. Conclusion: Bone powder, regardless of chicken treatment, contains and releases factors/chemicals responsible for the observed effects on energy metabolism. Quantitative differential effects appear to depend on biochemical alterations in the bone matrix caused by antibiotics rather than antibiotics themselves.