Feiyue Xing

In vivo toxicological evaluation of Anisomycin

Anisomycin is a pyrrolidine antibiotic isolated from Streptomyces griseolus. Recent studies have shown that Anisomycin as a novel immunosuppressive agent is superior to Cyclosporine A (J. Immunother. 31, 858-870, 2008). In order to make toxicological evaluation of Anisomycin, acute and four-week continuously intravenous toxicity studies were performed in mice. IC(50) value tested on peripheral lymphocytes was 25.44 ng/ml. The calculated LD(50) for Anisomycin was 119.64 mg/kg. The mice were intravenously injected through mouse tail vein with a total dose of 5, 15, 30 and 60 mg/kg/mice of Anisomycin every other day for 4 weeks. Just in the high-dose mice, death of three mice happened and body weight of the mice was significantly decreased. Statistically significant changes in organ index included increases in ratios of the spleen, liver, lung and brain to the body weight, and decrease in ratio of the thymus to the body weight. Changes in clinical biochemistry parameters included increases in the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, and decreases in the glucose (GLU) activity. The distinct inflammation appeared in the lung, liver and kidney, and the number and size of megakaryocytes in the spleen were significantly increased. Anisomycin did not induce formation of the peripheral blood micronucleus, but increased the number of micronucleated polychromatic erythrocytes in bone marrow and sperm aberrations. However, the above aberrant changes occurred only in the mice treated with the high-dose Anisomycin. These results indicate that although Anisomycin has no significant side effects at effectively therapeutic doses, its over-dosage may lead to toxicity, particularly pulmo-, nephro- and hepato-toxicity.

Comparison of immature and mature bone marrow-derived dendritic cells by atomic force microscopy

A comparative study of immature and mature bone marrow-derived dendritic cells (BMDCs) was first performed through an atomic force microscope (AFM) to clarify differences of their nanostructure and adhesion force. AFM images revealed that the immature BMDCs treated by granulocyte macrophage-colony stimulating factor plus IL-4 mainly appeared round with smooth surface, whereas the mature BMDCs induced by lipopolysaccharide displayed an irregular shape with numerous pseudopodia or lamellapodia and ruffles on the cell membrane besides becoming larger, flatter, and longer. AFM quantitative analysis further showed that the surface roughness of the mature BMDCs greatly increased and that the adhesion force of them was fourfold more than that of the immature BMDCs. The nano-features of the mature BMDCs were supported by a high level of IL-12 produced from the mature BMDCs and high expression of MHC-II on the surface of them. These findings provide a new insight into the nanostructure of the immature and mature BMDCs.

Anisomycin inhibits the behaviors of T cells and the allogeneic skin transplantation in mice.

There is still a lack of a high potent and low toxic immunosuppressive drug. We accidentally found that a quite low dose of anisomycin was sufficient to block proliferation of T cells. In this study, carboxy-fluorescein diacetate-succinimidyl ester staining showed that over 10.0 ng/mL of anisomycin markedly inhibited the proliferation of T cells induced by ConA. Propidium iodide staining revealed that anisomycin led to G0/G1 arrest and blocked S phase entry stimulated by ConA or phorbol 12, 13-dibutyrate plus ionomycin. Anisomycin down-regulated remarkably the CD69 and CD25 expression on the surface of T cells. The response of T cells was repressed by treatment of anisomycin, which was partly restored by adding exogenous interleukin-2, and there was no difference between anisomycin and dexamethasone, although the used dose of the latter was 100-fold of the former. The inhibition of cytotoxicity of T cells against 7919 cells by anisomycin was observed without the direct cytotoxicity to T cells or 7919 cells. The level of transforming growth factor-β1 fell by <80.0 ng/mL in vitro and 30.0 mg/kg of anisomycin in vivo and enhanced by more than the doses. The treatment of anisomycin prolonged the survival of the transplanted skin and depressed the delayed type hypersensitivity development and the T-cell response in the skin-transplanted mice. Moreover, the effect of its restraining allograft rejection might be superior to cyclosporine A, with relatively slight toxic signs. These results indicate anisomycin significantly inhibits the behaviors of T cells and the transplantation rejection, providing important evidence for anisomycin as a novel immunosuppressant.

Low-dose anisomycin is sufficient to alter the bio-behaviors of Jurkat T cells

Anisomycin is a pyrrolidine antibiotic isolated from Streptomyces griseolus. It has been found that a quite low dose of anisomycin is sufficient to block proliferation of primary T lymphocytes. The focus of this study is to explore the possibility of anisomycin to treat human acute leukemia Jurkat T cells in vitro. The results indicated that the low dose of anisomycin could significantly inhibit the colony formation of Jurkat T cells and elevate the inhibition rate of Jurkat T cell growth along with its increasing concentrations. Jurkat T cell cycle was blocked into S-phase by anisomycin. Consistent with the increased proportion of sub-G1 phase, anisomycin promoted Jurkat T cell apoptosis. The CD69 and CD25 expression on the surface of Jurkat T cells was also down-regulated prominently along with the enhancing concentrations of anisomycin, followed by the decreased production of IL-4, IL-10, IL-17, TGF-β and IFN-γ, and the down-regulated expression of phosphorylated-ERK1/2. The results suggest that the suppressive effect of anisomycin on Jurkat T cell growth may be related to inhibiting TGF-β production and ERK1/2 activation, arresting the cell cycle at S-phase and promoting the apoptosis of Jurkat T cells.

ICBP90 mediates Notch signaling to facilitate human hepatocellular carcinoma growth

The Notch signaling pathway plays a key role in cell proliferation and development that is closely related to an inverted CCAAT box binding protein (ICBP90), but little is known about whether there is a correlation between Notch signaling and ICBP90. The aim of the current study was to elucidate this. MTT assay and flow cytometry were used to determine the proliferation, cell cycle and apoptosis of HepG2 or Hepa1-6 cells treated by N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a specific inhibitor of the Notch pathway. RT-PCR, Western Blot and in situ immunofluorescence staining were employed to examine expression of ICBP90 in the cells. DAPT caused inhibition of the activation of the Notch signaling pathway, followed by preventing the cells at the G0/G1 phases to enter S and G2/M phases. ICBP90 and Hes-1 proteins were highly expressed in the untreated cells. The reduced levels of Notch intracellular domain (NICD) protein were observed in the DAPT-treated cells, thereby bringing about the down-regulation of ICBP90 with the increment of the DAPT dose. Consistent with this, knockdown of the Hes-1 gene, which encodes a critical transcriptional factor in the Notch pathway, also led to the attenuation of ICBP90. On the contrary, Jagged-1, a Notch ligand, facilitated ICBP90 production. Adriamycin could result in the reduction of ICBP90, which was not accompanied with the alteration of Hes-1. ICBP90 was almost fully distributed within the nuclei, but Hes-1 was visible within both the cytoplasm and nuclei. Our novel findings strongly indicate that inactivation of the Notch signaling pathway impedes hepatocellular carcinoma progress via reduction of ICBP90.

Anisomycin represses in vitro and in vivo colon adenocarcinoma CT26 cell growth via activation of caspase-cascade with reduction of carcinoembryonic antigen

Aims: In vitro evidence indicates that anisomycin can induce cell apoptosis. This study was to evaluate potential of anisomycin to treat colon adenocarcinoma in vitro and in vivo. Methods: Cell viability was determined by methyl thiazolyltetrazolium bromide (MTT). Cell cycle and apoptosis were detected by flow cytometry. CT26 colon adenocarcinoma model was established. Cellular apoptosis and tumor necrosis were detected by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling), and H&E staining. Carcinoembryonic antigen (CEA) was detected by in situ immunofluorescence. Expressions of caspases were measured by Western Blot. Results: Our results showed that multipoint intratumoral administration of anisomycin significantly suppressed colon adenocarcinoma CT26 cell growth, and resulted in the survival of approximately 80% of CT26bearing mice 50 days after CT26 inoculation, superior dramatically to adriamycin. Anisomycin inhibited the proliferation of CT26 cells, and

Hes-1-targeting siRNA inhibits the maturation of murine myeloid-derived dendritic cells

Activation of Notch by Jagged-1 may plays a pivotal role in maturation of dendritic cells (DCs), but the mechanism has not been completely defined. In the present study, Hes-1 (Hairy/enhancer-of-split)-targeting siRNA was used to confirm a role of Jagged-1-Notch signaling pathway activation in maturation of murine bone marrow-derived DCs and to search for a target that plays a critical role. The results showed that compared with the control, lipopolysaccharide or Zymosan A groups, Jagged-1 (a soluble Jagged 1/Fc chimera protein) effectively increased expression of Hes-1 and Deltex-1 mRNA, which could be reversed by DAPT (2, 4-diamino-5-phenylthiazole), a specific inhibitor of the Notch signaling pathway. Hes-1-targeting siRNA could successfully down-regulate the endogenous Hes-1 expression in the DCs. Concurrently, a significant down-regulation of CD40, CD80, CD86 and MHC-II expressions on the surface of the DCs was found with the reduction of IL-12 yielded by the DCs. Our results demonstrate that Hes-1-targeting siRNA can inhibit the maturation of the DCs induced by Jagged-1, indicating Hes-1 may be an important target of Notch signaling mediating the maturation of DCs.

Nano-characterization of Jagged-1-educated dendritic cells

Jagged-1-educated dendritic cells (DCs) are of distinct phenotypes different from Lipopolysaccharide (LPS)-educated DCs. However, to date, little is known as to nano-features of Jagged-1-educated DCs. In this study, nanostructure of Jagged-1-educated DCs was observed through atomic force microscopy. Our results showed that the volume, surface area and width of Jagged-1-educated DCs, and the number of protrusion, pseudopodia and lamellapodia on the surface of Jagged-1-educated DCs were significantly more than those of GM-CSF-induced DCs, but less than those of LPS-educated DCs. Compared with GM-CSF-induced DCs, the roughness on the surface of Jagged-1-educated DCs was greatly increased, similar to LPS-educated DCs, but the particle size and number of them on the membrane were markedly less than the latter. The change of DC nanostructure caused by Jagged-1 was abrogated with N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester, a γ-secretase inhibitor of Notch signaling pathway. Compared to LPS-educated DCs, Jagged-1-educated DCs highly expressed NICD, Deltex-1 and Hes-1 proteins, moderately produced IL-12, but not IFN-γ, and the level of CD40 molecules on the surface of them was much lower, suggesting that Jagged-1-educated DCs are in a semi-mature status and may have unique functions distinguished from LPS-educated DCs.