Transcriptome and Literature Mining Highlight the Differential Expression of ERLIN1 in Immune Cells during Sepsis

Sepsis results from the dysregulation of the host immune system. This highly variable disease affects 19 million people globally, and accounts for 5 million deaths annually. In transcriptomic datasets curated from public repositories, we observed a consistent upregulation (3.26–5.29 fold) of ERLIN1—a gene coding for an ER membrane prohibitin and a regulator of inositol 1, 4, 5-trisphosphate receptors and sterol regulatory element-binding proteins—under septic conditions in healthy neutrophils, monocytes, and whole blood. In vitro expression of the ERLIN1 gene and proteins was measured by stimulating the whole blood of healthy volunteers to a combination of lipopolysaccharide and peptidoglycan. Septic stimulation induced a significant increase in ERLIN1 expression; however, ERLIN1 was differentially expressed among the immune blood cell subsets. ERLIN1 was uniquely increased in whole blood neutrophils, and confirmed in the differentiated HL60 cell line. The scarcity of ERLIN1 in sepsis literature indicates a knowledge gap between the functions of ERLIN1, calcium homeostasis, and cholesterol and fatty acid biosynthesis, and sepsis. In combination with experimental data, we bring forth the hypothesis that ERLIN1 is variably modulated among immune cells in response to cellular perturbations, and has implications for ER functions and/or ER membrane protein components during sepsis.

Antileukemic Activity and Molecular Docking Study of a Polyphenolic Extract from Coriander Seeds

Leukemia is a group of hematological neoplastic disorders linked to high mortality rates worldwide, but increasing resistance has led to the therapeutic failure of conventional chemotherapy. This study aimed to evaluate in vitro the antileukemic activity and potential mechanism of action of a polyphenolic extract obtained from the seeds of Coriandrum sativum L. (CSP). A methylthiazoletetrazolium assay was performed to assess the CSP cytotoxicity on chronic (K562) and acute (HL60) myeloid leukemia cell lines and on normal Vero cell line. CSP toxicity was also evaluated in vivo using the OECD 423 acute toxicity model on Swiss albino mice. The results demonstrated a remarkable antitumoral activity against K562 and HL60 cell lines (IC50 = 16.86 µM and 11.75 µM, respectively) although no cytotoxicity was observed for the Vero cells or mice. A silico study was performed on the following receptors that are highly implicated in the development of leukemia: ABL kinase, ABL1, BCL2, and FLT3. The molecular docking demonstrated a high affinity interaction between the principal CSP components and the receptors. Our findings demonstrated that CSP extract has remarkable antileukemic activity, which is mainly mediated by the flavonoids, catechins, and rutin, all of which showed the highest binding affinity for the targeted receptors. This study revealed a promising active compound alternative research-oriented biopharmacists to explore.

Deformation of an Encapsulated Leukemia HL60 Cell through Sudden Contractions of a Microfluidic Channel

Migration of an encapsulated leukemia HL60 cell through sudden contractions in a capillary tube is investigated. An HL60 cell is initially encapsulated in a viscoelastic shell fluid. As the cell-laden droplet moves through the sudden contraction, shear stresses are experienced around the cell. These stresses along with the interfacial force and geometrical effects cause mechanical deformation which may result in cell death. A parametric study is done to investigate the effects of shell fluid relaxation time, encapsulating droplet size and contraction geometries on cell mechanical deformation. It is found that a large encapsulating droplet with a high relaxation time will undergo low cell mechanical deformation. In addition, the deformation is enhanced for capillary tubes with narrow and long contraction. This study can be useful to characterize cell deformation in constricted microcapillaries and to improve cell viability in bio-microfluidics.

New Anti-Leukemic Effect of Carvacrol and Thymol Combination through Synergistic Induction of Different Cell Death Pathways

Acute myeloid leukemia (AML) is a cancer of the myeloid lineage of blood cells, and treatment for AML is lengthy and can be very expensive. Medicinal plants and their bioactive molecules are potential candidates for improving human health. In this work, we studied the effect of Ptychotis verticillata (PV) essential oil and its derivatives, carvacrol and thymol, in AML cell lines. We demonstrated that a combination of carvacrol and thymol induced tumor cell death with low toxicity on normal cells. Mechanistically, we highlighted that different molecular pathways, including apoptosis, oxidative, reticular stress, autophagy, and necrosis, are implicated in this potential synergistic effect. Using quantitative RT-PCR, Western blotting, and apoptosis inhibitors, we showed that cell death induced by the carvacrol and thymol combination is caspase-dependent in the HL60 cell line and caspase-independent in the other cell lines tested. Further investigations should focus on improving the manufacturing of these compounds and understanding their anti-tumoral mechanisms of action. These efforts will lead to an increase in the efficiency of the oncotherapy strategy regarding AML.

Bis[Pyrrolyl Ru(II)] Triads: a New Class of Photosensitizers for Metal-Organic Photodynamic Therapy

A new family of ten dinuclear Ru(II) complexes based on the bis[pyrrolyl Ru(II)] triad scaffold, where two Ru(bpy)₂ centers are separated by a variety of organic linkers, was prepared to evaluate the influence of the organic chromophore on the spectroscopic and in vitro photodynamic therapy (PDT) properties of the compounds. The bis[pyrrolyl Ru(II)] triads absorbed strongly throughout the visible region, with several members having molar extinction coefficients (e) ≥10⁴ at 600–620 nm and longer. Phosphorescence quantum yields were generally less than 0.1% and in some cases undetectable. The singlet oxygen quantum yields ranged from 5% to 77% and generally correlated with their photocytotoxicities toward human leukemia (HL-60) cells regardless of the wavelength of light used. Dark cytotoxicities varied ten-fold, with EC₅₀ values in the range of 10–100 µM and phototherapeutic indices (PIs) as large as 5,400 and 260 with broadband visible (28 J cm^-2, 7.8 mW cm^-2) and 625-nm red (100 J cm^-2, 42 mW cm^-2) light, respectively. The bis[pyrrolyl Ru(II)] triad with a pyrenyl linker (5h) was especially potent, with an EC50 value of 1 nM and PI >27,000 with visible light and subnanomolar activity with 625-nm light (100 J cm^-2, 28 mW cm^-2). The lead compound 5h was also tested in a tumor spheroid assay using the HL60 cell line and exhibited greater photocytotoxcicity in this more resistant model (EC₅₀=60 nM and PI>1,200 with 625-nm light) despite a lower dark cytotoxicity. The in vitro PDT effects of 5h extended to bacteria, where submicromolar EC₅₀ values and PIs >300 against <i>S. mutans</i> and <i>S. aureus </i>were obtained with visible light. This activity was attenuated with 625-nm red light, but PIs were still near 50. The ligand-localized ³ππ* state contributed by the pyrenyl linker of 5h likely plays a key role in its phototoxic effects toward cancer cells and bacteria.<br><br>

Bis[Pyrrolyl Ru(II)] Triads: a New Class of Photosensitizers for Metal-Organic Photodynamic Therapy

A new family of ten dinuclear Ru(II) complexes based on the bis[pyrrolyl Ru(II)] triad scaffold, where two Ru(bpy)₂ centers are separated by a variety of organic linkers, was prepared to evaluate the influence of the organic chromophore on the spectroscopic and in vitro photodynamic therapy (PDT) properties of the compounds. The bis[pyrrolyl Ru(II)] triads absorbed strongly throughout the visible region, with several members having molar extinction coefficients (e) ≥10⁴ at 600–620 nm and longer. Phosphorescence quantum yields were generally less than 0.1% and in some cases undetectable. The singlet oxygen quantum yields ranged from 5% to 77% and generally correlated with their photocytotoxicities toward human leukemia (HL-60) cells regardless of the wavelength of light used. Dark cytotoxicities varied ten-fold, with EC₅₀ values in the range of 10–100 µM and phototherapeutic indices (PIs) as large as 5,400 and 260 with broadband visible (28 J cm^-2, 7.8 mW cm^-2) and 625-nm red (100 J cm^-2, 42 mW cm^-2) light, respectively. The bis[pyrrolyl Ru(II)] triad with a pyrenyl linker (5h) was especially potent, with an EC50 value of 1 nM and PI >27,000 with visible light and subnanomolar activity with 625-nm light (100 J cm^-2, 28 mW cm^-2). The lead compound 5h was also tested in a tumor spheroid assay using the HL60 cell line and exhibited greater photocytotoxcicity in this more resistant model (EC₅₀=60 nM and PI>1,200 with 625-nm light) despite a lower dark cytotoxicity. The in vitro PDT effects of 5h extended to bacteria, where submicromolar EC₅₀ values and PIs >300 against <i>S. mutans</i> and <i>S. aureus </i>were obtained with visible light. This activity was attenuated with 625-nm red light, but PIs were still near 50. The ligand-localized ³ππ* state contributed by the pyrenyl linker of 5h likely plays a key role in its phototoxic effects toward cancer cells and bacteria.<br><br>

The Effect of Cumin on HL60 Cell Line

Cancer is one of the most debilitating and traumatic diseases of modern life, for which no curative approach is presently available. Even though the recent therapies used to treat patients with various types of cancer have not been completely effective, adjuvant therapies, including the use of medicinal plants, may have some effect in achieving cancer treatment goals. Cumin has also been widely used in traditional medicine to treat a variety of diseases, including hypolipidemia, cancer, and diabetes. We used cumin in different concentrations to observe effect of cumin on HL60 cell line. We used MTT cell viability test to investigate cytotoxic effect of cumin. We made experiment for 24, 48 and 72 h and we incubate our cumin exposed drug 37 °C in CO2 incubator. According to MTT results we found IC50 values for cumin 8.5 mg/mL for 72 h incubation. Generally, cancer cells show drug resistant to especially chemical drugs. Use of plant derived substances may reduce drug resistant on cancer cells. Especially if we use cumin combine with chemical drug, probably we will observe more toxic effect on cancer cell. Because combination effect will reduce drug resistant.

CRISPR/Cas9 Mediated ELANE Knock-out Restores Survival and Granulocytic Differentiation of HL60 Cells Expressing Mutant Neutrophil Elastase: Is Neutrophil Elastase a Dispensible Granulocyte Protease?

Background : Mutations in ELANE are the most common cause of both cyclic and severe congenital neutropenia. ELANE encodes neutrophil elastase (NE), a tissue specific serine protease expressed primarily in neutrophils. Expression of the mutant protein impairs survival and maturation of myeloid precursors in bone marrow. More than 130 different ELANE mutations have been found in patients with cyclic and congenital neutropenia, and genotype-phenotype studies suggest that specific mutations cause more severe disease. (Curr Op Hematol. 2015;22:3-11) Mutant NE is also implicated as the primary cause of ELANE associated neutropenia by studies showing that cell permeable inhibitors of NE correct the defect in cell survival and maturation in cellular models. (Makaryan et al. J Leukoc Biol. 2017;102:1143). Hypothesis: If inhibition of NE can correct the cellular defect, deletion of ELANE will have similar effects and permit studies to see if NE is an essential neutrophil protease. Methods: We used CRISPR/Cas9 mediated gene editing to create a cellular model of congenital neutropenia in the commercially available human promyelocytic cell line, HL60. We used CRISPR/Cas9 editing technology to create engineered HL60 cell lines with knock-in (KI) ELANE P139L, C151Y and G214R single point heterozygous mutations. Using the same technology, we then performed ELANE gene complete knock out (KO) of ELANE in each mutant cell line. All engineered cell lines as well as wild-type HL60 cells were cultured for 5-7 days in complete RPMI supplemented with 2uM all-trans retinoic acid (ATRA) to trigger myeloid differentiation. Survival of these cell lines was investigated using Annexin V-PE staining and flow cytometric analysis. Granulocytic differentiation was evaluated using CD11b surface marker staining and flow cytometry and by performing manual differential cell counts. We measured the unfolded protein response (UPR) by western blotting using UPR specific antibodies, phagocytosis with E. coli particles using fluorescence detection, IL-8 stimulated chemotaxis in trans-well system and PMA activated respiratory burst by flow cytometry. Results: Cells expressing the P139L and C151Y mutant ELANE had increased Annexin V staining more than a 2-fold increase in apoptotic cells at 7 days in culture. Granulocytic differentiation, measured by surface CD11b expression, was significantly impaired. (p&lt;0.0001). Cytospins stained with Diff-Quik showed a typical block of myeloid differentiation and a significant deficiency of mature neutrophils in 7-day cultures. Western blot analysis using antibodies to GRP78/BiP and ATF6 showed a typical UPR signature in both ELANE mutant cell lines compared to wild type. Chemotaxis to the IL8 chemo-attractant was severely impaired in cells expressing mutant NE. Respiratory burst and phagocytosis was also altered in cells expressing mutant NE. ELANE KO corrected all these cellular and functional abnormalities, reverting these functions toward the wild phenotype. Studies are in progress for the third cell line expressing the G214R mutation. Conclusions: CRISPR/Cas9 engineered HL60 cell lines expressing mutant NE are a highly reproducible and reliable cellular model for investigating ELANE associated neutropenia. The presence of mutant NE severely impairs neutrophil functions, and its deletion through KO of ELANE corrects the cellular abnormalities and reverses the induced abnormalities. These results suggest that CRISPR/Cas9 mediated genetic knock down of NE is a novel therapeutic approach for treatment of this disorder. This data also suggests that total absence of NE does not alter basic functions of granulocytes. Disclosures Dale: Sanofi Aventis: Consultancy, Honoraria; Athelas: Equity Ownership; Amgen: Consultancy, Research Funding; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Prolong: Consultancy; Beheringer/Ingelheim: Consultancy; Coherus: Consultancy; x4pharma: Consultancy, Honoraria, Research Funding.

HDAC and HMT Inhibitors in Combination with Conventional Therapy: A Novel Treatment Option for Acute Promyelocytic Leukemia

Acute promyelocytic leukemia (APL) is characterized by PML-RARA translocation, which causes the blockage of promyelocyte differentiation. Conventional treatment with Retinoic acid and chemotherapeutics is quite satisfactory. However, there are still patients who relapse or develop resistance to conventional treatment. To propose new possibilities for acute leukemia treatment, we studied the potential of histone deacetylase (HDAC) inhibitor and histone methyl transferase (HMT) inhibitor to enhance conventional therapy in vitro and ex vivo. NB4 and HL60 cell lines were used as an in vitro model; APL patient bone marrow mononuclear cells were used as an ex vivo model. Cell samples were treated with Belinostat (HDAC inhibitor) and 3-Deazaneplanocin A (HMT inhibitor) in combination with conventional treatment (Retinoic acid and Idarubicin). We demonstrated that the combined treatment used in the study had slightly higher effect on cell proliferation inhibition than conventional treatment. Also, enhanced treatment showed stronger effect on induction of apoptosis and on suppression of metabolism. Moreover, the treatment accelerated granulocytic cell differentiation and caused chromatin remodelling (increased H3K14 and H4 acetylation levels). In vitro and ex vivo models showed similar response to the treatment with different combinations of 3-Deazaneplanocin A, Belinostat, Retinoic acid, and Idarubicin. In conclusion, we suggest that 3-Deazaneplanocin A and Belinostat enhanced conventional acute promyelocytic leukemia treatment and could be considered for further investigations for clinical use.