Proliferation Of KG-1 Cells Research Articles

Anti-invasive and antiproliferative effects of Pleurotus ostreatus extract on acute leukemia cell lines.

Currently, mushrooms have been used in traditional and folk medicines for their therapeutic activities, such as antibiotic, antitumor, anti-inflammatory, anticancer, antileukemic and immunomodulatory actions. This investigation evaluates the anti-invasive, antiproliferative and cytotoxic effects of Pleurotus ostreatus (Pleurotaceae) on leukemia cell lines. The proliferation of KG-1 cells was measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after treatment with gradient dilutions of P. ostreatus extract. Then, the minimum inhibitory concentration (MIC) of the extract was determined. Moreover, the proliferation of Jurkat cells and bone marrow mesenchymal stem cells (BMSCs), a cancerous cell line and normal body cells, respectively, was considered. The apoptotic morphology of treated KG-1 cells was evaluated with Giemsa staining. The invasion and migration of cells were evaluated using transwell invasion assay. Thereafter, the rates of apoptosis and necrosis were measured by using flow cytometry, and BAX and MMP-9 gene expression were evaluated using quantitative reverse transcription-polymerase chain reaction as apoptotic and metastatic genes, respectively. The MIC of the extract was determined to be 1 mg/mL after 48 h. According to the results, the extract decreased the proliferation of leukemia cell lines (KG-1 and Jurkat cells) but had no antiproliferative effects on BMSCs. Moreover, KG-1 cell migration and MMP-9 gene expression decreased after the treatment, and the rate of apoptosis and BAX gene expression increased significantly. According to the efficient therapeutic properties of P. ostreatus on leukemia cell lines, this mushroom could be introduced as a natural medicine to cure leukemic patients who suffer from the harmful side effects and enormous costs of chemotherapy.

Effect of reactive nitrogen metabolite scavengers on sensitivity of human leukemia cells to immunotherapy

Objective To investigate the effect of Tiopronin (TIP) on interleukin (IL)-2 immunotherapy of human leukemia KG-1 cells and its possible mechanism. Methods KG-1 cells in logarithmic growth phase were randomly divided into KG-1+ IL-2 group and KG-1+ IL-2+ TIP group. Methyl thiazolyl tetrazolium (MTT) assay and colony formation assay were used to detect the sensitivity and proliferation of KG-1 cells. The changes of reactive nitric metabolites (RNM) were detected with nitrate reductase method. The production of tumor necrosis factor (TNF)-β and interferon (IFN)-γ was detected with enzyme linked immunosorbent assay (ELISA). The expression of CD3ζ was detected with Western blot and real time polymerase chain reaction (RT-PCR). Results IL-2 and IL-2+ TIP could inhibit the growth of KG-1 cells. The inhibitory rate of KG-1+ IL-2+ TIP group was significantly higher than that of KG-1+ IL-2 group, and the sensitivity of KG-1 cells to IL-2 was 6.2 times higher. Both IL-2 and IL-2+ TIP group inhibited the colony formation of KG-1 cells. Compared to KG-1+ IL-2 group, KG-1+ IL-2+ TIP group inhibited the colony formation of KG-1 cells by 3.5 times. The RNM production of KG-1 + IL-2 group was (158.26±3.82) μmol/ml, which was significantly higher than (45.18±4.29) μmol/ml of KG-1 + IL-2 + TIP group (P<0.05). The levels of TNF-β and IFN-γ in KG-1+ IL-2+ TIP group were (253.28±7.84) pg/ml and (181.25±6.41) pg/ml, which was significantly higher than (98.45±6.43) pg/ml and (68.74±8.26) pg/ml of KG-1+ IL-2 group (P<0.05). The expression of CD3ζ in KG-1+ IL-2+ TIP group was significantly higher than that in KG-1+ IL-2 group. Conclusions Tiopronin can promote NK/T cell activity and increase the sensitivity of leukemia KG-1 cells to IL-2 by eliminating reactive nitrogen metabolites. Key words: Thiopronine/PD; Leukemia/TH; Immunotherapy

Soluble GM-CSF Receptor α Is Cytotoxic to Myeloid Cells in a Manner That Is Independent of GM-CSF Binding Inhibition.

Abstract 3618Poster Board III-554The potential influence over cytokine activity by soluble receptors and the precise cytokine specificity they display suggests that soluble cytokine receptors may play a significant role in the response of cells to cytokine-mediated signaling. GM-CSF biology is mediated by binding to a heterodimeric receptor consisting of a major binding subunit (GMRα) and a major signaling subunit (β). GM-CSF Receptor can signal an astonishing variety of cellular functions, including protection from apoptosis, differentiation, maturation, activation and homing. GM-CSF Receptor α (GMRα) exists in both trans-membrane (tmGMRα) and soluble isoforms (sGMRα). sGMRα is believed to arise exclusively through alternative splicing of the GMRα gene product which removes the exon encoding the transmembrane domain. The splicing is such that the amino terminal 317 residues of sGMRα are homologous to the extracellular domain of tmGMRα, however, the deletion and subsequent frameshift replaces the transmembrane and cytoplasmic domains of tmGMRα with a unique 16 amino acid tail on sGMRα. It has been demonstrated that normal human monocytes constitutively secrete sGMRα, and sGMRα has been implicated in the biology of acute myeloid leukemias and the tumor-nerve interactions evoking bone cancer pain.sGMRα has been shown to bind directly to GM-CSF. We hypothesized that recombinant sGMRα(r-sGMRα) competitively inhibits the biological properties of GM-CSF. Using a baculoviral expression system, we produced N-terminal-His-tagged r-sGMRα. We exposed the GM-CSF and IL-3 dependent myeloid cell line, Mo7e, to varying concentrations of r-sGMRα and found that r-sGMRα directly inhibits GM-CSF dependent cell survival and proliferation in a dose-dependent manner. We found that r-sGMRα was toxic to Mo7e cells at concentrations as low as the Kd of r-sGMRα for GM-CSF (3nM). The finding that lower concentrations of GM-CSF resulted in lower Mo7e stimulation, but not toxicity, indicated that the mechanism of r-sGMRα toxicity was unlikely to simply be a reduction in effective GM-CSF concentration.To test whether the r-sGMRα toxicity seen in Mo7e cells was a result of GM-CSF neutralization or a result of signaling inhibition, we demonstrated that GM-CSF neutralizing antibody is cytotoxic during GM-CSF withdrawal, but not cytotoxic in the presence of IL-3, whereas r-sGMRα was cytotoxic under both conditions. Previous kinetic studies have demonstrated a time-dependent strengthening of the GMRα : β : GM-CSF complex; forming discrete “loose” and “tight” binding forms over minutes. Our studies indicate that neither pre-incubation of r-sGMRα with GM-CSF (6 hours) nor pre-incubation of cells with GM-CSF (prior to r-sGMRα addition) diminishes r-sGMRα's ability to inhibit GM-CSF-dependent cell survival and proliferation. We also determined that both r-sGMRα and GM-CSF degradation is independent of the other in vitro.To determine if r-sGMRα mediated cell death resulted from mechanisms other than GM-CSF signaling inhibition, we also tested additional myeloid cell lines that do not require exogenous cytokines for survival, including K562, (which does not express GMRα) as well as KG-1 and HL-60 cells (which both co-express GMRα and GMRb). While r-sGMRα does not inhibit survival or proliferation of KG-1 cells, r-sGMRα does inhibit proliferation of both HL-60 and K562 cells, but only at concentrations tenfold higher than the Kd (30nM). This inhibition was independent of GM-CSF's presence. These data indicate that soluble receptor may have biological effects beyond competition for ligand with the membrane bound receptor, and beyond the modification of GM-CSF half-life.The ability of r-sGMRα to inhibit IL-3 dependent survival in Mo7e cells, but not K562 cells, supports the notion that r-sGMRα may be directly inducing cytotoxicity by interacting with surface components that are likely part of the GM-CSF Receptor complex. Collectively, our data represent the first evidence demonstrating the modulation of GM-CSF signaling in human myeloid neoplasms by r-sGMRα and further illustrates the potentially significant role of soluble receptors in the response of cells to cytokine-mediated signaling. Disclosures:No relevant conflicts of interest to declare.

3-Hydrogenkwadaphnine, a novel diterpene ester from Dendrostellera lessertii, its role in differentiation and apoptosis of KG1 cells

3-Hydrogenkwadaphnin (3-HK) (Fig. 1) is a daphnane-type diterpene ester isolated from the leaves of Dendrostellera lessertii (Thymelaeaceae) with differentiation and apoptotic potency among several leukemic cells without any measurable adverse effects on normal cells [Moosavi, M.A., Yazdanparast, R., Sanati, M.H., Nejad, A.S., 2005a. 3-Hydrogenkwadaphnin targets inosine 5'-monophosphate dehydrogenase and triggers post-G1 arrest apoptosis in human leukemia cell lines. Int. J. Biochem. Cell Biol. 37, 2366-2379]. In this study, we evaluated differentiating and apoptotic efficiency of a second new anti-proliferating agent from the same plant relative to 3-HK using acute myeloid leukemia (AML) KG1 cell line. 3-HK at 5-30 nM inhibited proliferation of KG1 cells after 24-96 h of treatment. NBT reducing assay and expression of cell surface markers (CD11b and CD14) confirmed that the inhibition of proliferation is associated with differentiation toward macrophage-like morphology. Regarding the relatively weaker potency of 3-HK in the induction of differentiation compared to that of the crude extract, we looked for additional compound(s) with similar properties in the crude extract. This effort led to isolation of the second compound from the leaves' extract with higher differentiating potency. The new compound inhibited proliferation of KG1 cells by almost 48+/-3.1% after 72 h of treatment with a single dose of 1.5 microg/ml. The treated cells differentiated along the monocyte/macrophage lineage based on the morphological features apparent after Wright-Giemsa staining, phagocytic activity and expression of cell surface markers as analyzed by flow cytometry. On the other hand, the results indicated that exposure of KG1 cells to either 3-HK or the new compound for 3-4 days induced apoptosis as assayed qualitatively by acridine orange/ethidium bromide (Ao/EtBr) double staining, agarose gel electrophoresis and quantitatively by Annexin-V technique and sub-G1 DNA staining using flow cytometry. Based on the present data, these two active constituents of D. lessertii have the novelty of being further evaluated for pharmaceutical applications.