Investigating Apoptotic Effect through Blocking miR-181b and miR-222 Using LNA-anti-miRNA in HL-60 Cell Line: Strategies to Improve Hematopoietic Stem Cell Transplantation

Flt3 Kinase Regulates Microvesicle Transfer of miRNA Between AML and Stromal Cells

Background: Phenanthroindolizidine alkaloids have been isolated from many species of Vincetoxicum. These alkaloids show promising anti-cancer activity and are a current topic of interest. Objectives: The inhibition of cell viability by different extracts of V. pumilum was measured in vitro in HL-60 and K562 human leukemic cancer cell lines and in freshly-isolated peripheral blood lymphocytes as a normal cell line. Materials and Methods: Using resazurin staining, cell viability was measured. In addition, the presence of apoptotic cells was determined using propidium iodide (PI) staining of DNA fragments (sub-G1 peak). Employing western blot analysis, levels of Bax, Bcl-2, and PARP cleavage were measured. Results: The CH2Cl2 fraction of V. pumilum showed a potent dose-related inhibitory effect on cell viability. The CH2Cl2 fraction showed IC50 values of 8.3 and 12.5 µg/mL in the K562 and HL-60 cell lines, respectively. A sub-G1 peak in the flow cytometry histograms of treated cells induced by the CH2Cl2 fraction suggested an induction of apoptosis. Further, both the parent methanol extract and its CH2Cl2 sub-fraction increased the expression of the pro-apoptotic protein Bax and of cleaved PARP in the cells. Conclusions: Collectively, the V. pumilum extracts showed remarkable cytotoxic activity and merit further investigation as anticancer agents.

Fingolimod Is Cytotoxic in Acute Myeloid Leukemia Independent of Additional Chemotherapeutic Agents

METABOLOMIC PROFILING OF THE POLYPHENOL QUERCETIN RESPONSE IN LEUKEMIA CELL LINES

: Objective: The aim of the present study is to prepare poloxamer based formulations of meloxicam to evaluate various parameters like pH stability, drug release and in vitro anticancer activities in cell lines with an intention to formulate injectable sustained biodegradable drug delivery system. Method: Various strengths of meloxicam formulations were prepared by using poloxamer 407. Prepared formulations were analyzed for drug content and pH stability by using HPLC. Drug release studies were tested by using USP dissolution testing apparatus. Further, we evaluated in vitro anticancer activity among these formulations by using sulphorhodamine-B (SRB) assay in two leukemia cell lines such as HL-60 and K-562 cell lines. Results: It showed that among all formulations, F1 formulation showed stability at pH 6.8, 7.0 and 7.4. It also showed 60% drug release and exhibited good anti cancer activity in HL-60 cell line with GI50<10 μg/ml as similar to adriamycin. Conclusion: Comparing these results, we concluded that F1 formulation showed good anticancer activity in cell lines, therefore further studies are necessary to confirm the mechanism of toxicity action studies. Thus these formulations has a potential to be a sustained release, passive targeted deliver system for meloxicam, with reduced side effects associated with the drug.Key words: Meloxicam, Poloxamer 407, Sulphorhodamine-B, Cyclooxygenase, HL-60 and K-562.

Effect of Second Mitochondria-Derived Activator of Caspases (Smac) Mimetic Compounds on Leukemic Cell Lines.

Two new C(30)-epimeric polycyclic polyprenylated acylphloroglucinols (PPAPs), named uralodins B and C (1 and 2, resp.), were isolated from the aerial parts of Hypericum henryi subsp. uraloides together with two new chromone glucosides, urachromones A and B (3 and 4, resp.), as well as 16 known compounds. Their structures were established by extensive NMR techniques and MS analysis. The epimers 1 and 2 always behaved like a single compound when examined by TLC, and were separated by HPLC. Their configuration was distinguished by comparative analysis of the NMR data with known analogues together with the ROESY experiment. All the isolated PPAPs were evaluated for their cytotoxic activities against HepG2, SGC7901, HL-60, and K562 cell lines. Compound 1 showed modest cytotoxic activities against SGC7901 and HL-60 cell lines, and 2 showed modest cytotoxic activities against HepG2, SGC7901, HL-60, and K562 cell lines.

CD14, one of the main differentiation markers on the surface of myeloid lineage cells, acts as a key role in activation of LPS-induced monocytes. LPS (lipopolysaccharide) binds to LPS-binding protein in plasma and are delivered to the cell surface receptor CD14. In this study, Various stimuli [Dimethyl Sulfoxide (DMSO), active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and LPS], either alone or in combination, have been recognized that have an effect on the level of CD14 expression in the human HL-60 and U937 promonocytic cell lines and therefore induce their terminal differentiation into monocytes or mature macrophages. U937 and HL-60 cells were cultured in RPMI 1640 supplemented with 10% FBS. For each cell line, 1Ã106 cells were seeded for 72 hours with DMSO, 14 days with LPS and 18 days with 1, 25-D3 in each well plate; then ELISA method was used to study their responses to the factors by means of anti-CD14. ELISA assay demonstrated that U937 and HL-60 cells were induced by both [1,25(OH)2D3] and DMSO to obtain characteristics of adherent cells and express CD14 protein; moreover, LPS at a low dose increased CD14 expression on surface of this cells. According to the our results, it is speculated that CD14 gene expression may be induced in human U937 and HL-60 cell lines by different factors including 1,25-D3, DMSO and LPS.

Compound banmao capsule (CBC) is a traditional Chinese medicinal formula composed of extracts from 11 organisms. The present study investigated the mechanism of CBC on the biological behavior of human leukemia cell lines using seropharmacological methods. CBC-containing rat serum was prepared by intragastrical administration of CBC to rats. The proliferation of human leukemia HL60 and K562 cell lines was assayed by measuring cell viability with the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium method, while cell cycle distribution and the rate of apoptosis were evaluated with flow cytometry. The mRNA expression of vascular endothelial growth factor A (VEGF-A) and chemotactic and inflammatory genes in human leukemia cell lines was examined using reverse transcription quantitative-polymerase chain reaction methods. It was revealed that the proliferation of K562 and HL60 cells was significantly inhibited by the CBC-containing rat serum at 72 h. The CBC-containing serum also promoted the apoptosis of K562 and HL60 cell lines. The CBC-containing serum altered the cell cycle progression of K562 and HL60, increasing the proportion of the cells in G1 phase and decreasing the proportion of the cells in S phase. Attenuated expression of VEGF-A and a decreasing trend in the expression of chemotactic and inflammatory genes were identified following treatment with CBC-containing serum in HL60 and K562 cells. In conclusion, CBC-containing serum exerted an inhibitory effect on the growth of K562 and HL60 cells by decreasing cellular proliferation, promoting apoptosis and cell cycle arrest, and decreasing the expression of VEGF-A, and chemotactic and inflammatory genes.

Anti-leukemic activity of Wattakaka volubilis leaf extract against human myeloid leukemia cell lines

Synthesis and induction of G0–G1 phase arrest with apoptosis of 3,5-dimethyl-6-phenyl-8-(trifluoromethyl)-5,6-dihydropyrazolo[3,4- f][1,2,3,5]tetrazepin-4(3 H)-one

Biochemical and functional aspects as well as features of cellular distribution of the differentiation groups CD11a and CD18 were studied in the course of a detailed characterization of two new monoclonal antibodies which recognize the alpha (GRS3) and beta (GRF1) chains of the LFA-1 antigen. Both MAbs inhibited homotypic aggregation of an EBV cell line. In contrast, only GRF1 (anti-beta chain) was able to inhibit granulocyte aggregation as well. Different myeloid-monocyte antigen modulation was noted in PMA induced macrophage differentiation of the U937 and HL60 cell lines. PMA treated HL60 cells showed increased expression of alpha M (CD11b) and alpha X (CD11c) antigens but no change in HLA-DR or CD14 antigen expression. No variation in the expression of LFA complex antigen (CD11a, b and c, or CD18) was observed on U937 cells, which on the other hand presented de novo expression of HLA-DR and CD14 antigens.

Acute myelogenous leukemia (AML) is a disease resulting from the clonal expansion and accumulation of hematopoietic stem cells arrested at various stages of development. B-cell Acute lymphoblastic leukemia (B-ALL) is a form of leukemia characterized by lymphoblast abnormal increase. B-ALL is the most common form of cancer in childhood with a peak of incidence at 2-5 years of age, and another peak in the elderly. The overall cure rate in children is about 80%, and about 38% to 60% of adults have long-term disease-free survival. Although the prognosis of AML and ALL has improved in the last decades, the outcome of relapsed and chemoresistant AML and ALL is still poor, especially in adults, with only a 35-40% survival at 5 years. Therefore, major efforts are being made to develop rationally targeted therapies against alterated signaling cascades that sustain AML and ALL cell proliferation, survival, and drug-resistance. A signaling pathway that is frequently upregulated in AML and ALL is the PI3K/Akt/mTOR network. To explore whether this pathway could represent a potential pharmacological target in AML and ALL, we evaluated the effects of the mTOR inhibitor, RAD001, on the HL60 AML cell line and on the SEM B-ALL cell line.RAD001decreased viability in AML and ALL cell lines, as demonstrated by MTT experiments. The values of IC50 at 24 and 48 hours in HL60 cell line was 8μM, while in SEM cell line, that displays an hyperactivation of the PI3K/Akt/mTOR pathway, the IC50 range was from 4,7 to 6,8 μM, thus showing an higher sensitivity of this cell line to this targeted therapy. As documented by Western Blotting, RAD001 showed a concentration-dependent induction of apoptosis with a relevant increment of PARP, Caspase 3, 8 and 9 cleavage in both cell lines. The ALL cell line SEM, with hyperactivated PI3K/Akt/mTOR pathway, displayed a much higher Caspase and PARP cleavage dependent apoptosis.PI3K/Akt/mTOR downstream targets were also partially dephosphorylated by RAD001, in particular GSK3β and 4E-BP1. RAD001 induced no significant changes in Akt dephosphorylation in HL60 and SEM cell lines, and this is in agreement with several reports showing no relevant changes in the phosphorylation of Akt. The cells always expressed unchanged total Akt. This indicates that PI3K/Akt/mTOR inhibition could be an attractive target to develop innovative therapeutic strategies directed towards AML and ALL leukemia cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3736. doi:1538-7445.AM2012-3736

BackgroundHuman neutrophils are central players in innate immunity, a major component of inflammatory responses, and a leading model for cell motility and chemotaxis. However, primary neutrophils are short-lived, limiting their experimental usefulness in the laboratory. Thus, human myeloid cell lines have been characterized for their ability to undergo neutrophil-like differentiation in vitro. The HL-60 cell line and its PLB-985 sub-line are commonly used to model human neutrophil behavior, but how closely gene expression in differentiated cells resembles that of primary neutrophils has remained unclear.ResultsIn this study, we compared the effectiveness of differentiation protocols and used RNA sequencing (RNA-seq) to compare the transcriptomes of HL-60 and PLB-985 cells with published data for human and mouse primary neutrophils. Among commonly used differentiation protocols for neutrophil-like cell lines, addition of dimethyl sulfoxide (DMSO) gave the best combination of cell viability and expression of markers for differentiation. However, combining DMSO with the serum-free-supplement Nutridoma resulted in increased chemotactic response, phagocytic activity, oxidative burst and cell surface expression of the neutrophil markers FPR1 and CD11b without a cost in viability. RNA-seq analysis of HL-60 and PLB-985 cells before and after differentiation showed that differentiation broadly increases the similarity in gene expression between the cell lines and primary neutrophils. Furthermore, the gene expression pattern of the differentiated cell lines correlated slightly better with that of human neutrophils than the mouse neutrophil pattern did. Finally, we created a publicly available gene expression database that is searchable by gene name and protein domain content, where users can compare gene expression in HL-60, PLB-985 and primary human and mouse neutrophils.ConclusionsOur study verifies that a DMSO-based differentiation protocol for HL-60 and PLB-985 cell lines gives superior differentiation and cell viability relative to other common protocols, and indicates that addition of Nutridoma may be preferable for studies of chemotaxis, phagocytosis, or oxidative burst. Our neutrophil gene expression database will be a valuable tool to identify similarities and differences in gene expression between the cell lines and primary neutrophils, to compare expression levels for genes of interest, and to improve the design of tools for genetic perturbations.

AML sensitivity to YM155 is modulated through AKT and Mcl-1