Mitotic cell death in BEL-7402 cells induced by enediyne antibiotic lidamycin is associated with centrosome overduplication

AIM: Mitotic cell death has been focused on in tumor therapy.However, the precise mechanisms underlying it remain unclear. We have reported previously that enediyne antibiotic lidamycin induces mitotic cell death at low concentrations in human epithelial tumor cells. The aim of this study was to investigate the possible link between centrosome dynamics and lidamycin-induced mitotic cell death in human hepatoma BEL-7402 cells.METHODS: Growth curve was established by Ml-l assay.Cell multinucleation was detected by staining with Hoechst 33342. Flow cytometry was used to analyze cell cycle.Aberrant centrosomes were detected by indirect immunofluorescence. Western blot and senescenceassociated β-galactosidase (SA-β-gal) staining were used to analyze protein expression and senescence-like phenotype,respectively.RESULTS: Exposure of BEL-7402 cells to a low concentration of lidamycin resulted in an increase in cells containing multiple centrosomes in association with the appearance of mitotic cell death and activation of SA-β-gal in some cells, accompanied by the changes of protein expression for the regulation of proliferation and apoptosis. The mitochondrial signaling pathway, one of the major apoptotic pathways, was not activated during mitotic cell death. The aberrant centrosomes contributed to the multipolar mitotic spindles formation, which might lead to an unbalanced division of chromosomes and mitotic cell death characterized by the manifestation of multi- or micronucleated giant cells.Cell cycle analysis revealed that the lidamycin treatment provoked the retardation at G2/M phase, which might be involved in the centrosome overduplication.CONCLUSION: Mitotic cell death and senescence can be induced by treatment of BEL-7402 cells with a low concentration of lidamycin. Centrosome dysregulation may play a critical role in mitotic failure and ultimate cell death following exposure to intermediate dose of lidamycin.

Antitumor efficacy of lidamycin on hepatoma and active moiety of its molecule

AIM: To study the in vitro and in vivo antitumor effect of lidamycin (LDM) on hepatoma and the active moiety of its molecule.METHODS: MTT assay was used to determine the growth inhibition of human hepatoma BEL-7402 cells, SMMC-7721cells and mouse hepatoma H22 cells. The in vivo therapeutic effects of lidamycin and mitomycin C were determined by transplantable hepatoma 22 (H22) in mice and human hepatoma BEL-7402 xenografts in athymic mice.RESULTS: In terms of IC50values, the cytotoxicity of LDM was 10 000-fold more potent than that of mitomycin C (MMC)and adriamycin (ADM) in human hepatoma BEL-7402 cells and SMMC-7721 cells. LDM molecule consists of two moieties,an aproprotein (LDP) and an enediyne chromophore (LDC). In terms of IC50 values, the potency of LDC was similar to LDM. However, LDP was 105-fold less potent than LDM and LDC to hepatoma cells. For mouse hepatoma H22 cells, the IC50value of LDM was 0.025 nmol/L. Given by single intravenous injection at doses of 0.1, 0.05 and 0.025 mg/kg, LDM markedly suppressed the growth of hepatoma 22 in mice by 84.7%, 71.6% and 61.8%,respectively. The therapeutic indexes (TI) of LDM and MMC were 15 and 2.5, respectively. By 2 iv. Injections in two experiments, the growth inhibition rates by LDM at doses of 0.1, 0.05, 0.025, 0.00625 and 0.0125 mg/kg were 88.8-89.5%, 81.1-82.5%, 71.2-74.9%, 52.3-59.575%,and 33.3-48.3%, respectively. In comparison, MMC at doses of 5, 2.5, and 1.25 mg/kg inhibited tumor growth by 69.7-73.6%, 54.0-56.5%, and 31.5-52.2%,respectively. Moreover, in human hepatoma BEL-7402 xenografts, the growth inhibition rates by LDM at doses of 0.05 mg/kg ×2 and 0.025 mg/kg ×2 were 68.7%and 27.2%, respectively. However, MMC at the dose of 1.25 mg/kg ×2 showed an inhibition rate of 34.5%. The inhibition rate of tumor growth by LDM was higher than that by MMC at the tolerated dose.CONCLUSION: Both LDM and its chromophore LDC display extremely potent cytotoxicity to hepatoma cells. LDM shows a remarkable therapeutic efficacy against murine and human hepatomas in vivo.

Effects of AGEs on Oxidation Stress and Antioxidation Abilities in Cultured Astrocytes

Objective To investigate whether two kinds of in vitro prepared advanced glycation end products (AGEs), Glu-BSA and Gal-BSA, could change oxidation stress and anti-oxidation abilities in astrocytes, and thus might contribute to brain injury. Methods Changes of GSH, MDA, SOD, MAO-B, nitric oxide were measured after AGEs treatment. Results Both 0.1 g/L Glu-BSA and Gal-BSA could slightly decrease GSH level, while 1 g/L of them significantly decreased GSH level by 35% and 43% respectively. The MDA levels of both 1 g/L AGEs treated groups (306±13 and 346±22) were higher than that of the normal group (189±18), which could be inhibited by free radical scavenger NAC. The SOD activities of both 1 g/L AGEs treated groups (67.0±5.2 and 74.0±11.0) were lower than that of the normal group (85.2±8.0). Both 0.1 g/L AGEs could slightly increase the activity of MAO-B, while 1 g/L of them could increase MAO-B activity by 1.5 and 1.7 folds respectively. Both AGEs stimulation could produce NO level by 1.7 and 2 folds respectively. Conclusion Enhanced levels of astrocytic oxidation stress and decrease of antioxidation abilities may contribute to, at least partially, the detrimental effects of AGEs in neuronal disorders and aging brain.

Study of a Novel Antiosteoporosis Screening Model Targeted on Cathepsin K

Objective To establish an effective assay to access the effects of natural products on cathepsin K for screening antiosteoporosis drugs. Methods To obtain the purified cathepsin K, we cloned the target fragment from the mRNA of human osteosacoma cell line MG63 and demonstrated its correctness through DNA sequencing. Cathepsin K was expressed in a high amount in E.coli after IPTG induction, and was purified to near homogenetity through resolution and column purification. The specificity of the protein was shown by Western blotting experiment. The biological activity of the components in the fermentation broth was assayed by their inhibitory effects on cathepsin K and its analog papain. Results With the inhibition of papain activity as a screen index, the fermentation samples of one thousand strains of fungi were tested and 9 strains among them showed strong inhibitory effects. The crude products of the fermentation broth were tested for their specific inhibitory effects on the purified human cathepsin K, the product of fungi 2358 shows the highest specificity against cathepsin K. Conclusions The compounds isolated from fungi 2358 show the highest biological activity and are worth further structure elucidation and function characterization.

ANTITUMOR EFFECTS OF MONOCLONAL ANTIBODY FAB’FRAGMENT—CONTAINING IMMUNOCONJUGATES

Objective.Using monoclonal antibody (mAb) Fab′ fragment to develop mAb immunoconjugates for cancer. Methods.Fab′ fragment of mAb 3A5 was prepared by digestion of the antibody with pepsin and then reduced by dithiothreitol (DTT),while Fab′ fragment of mAb 3D6 was obtained by digestion of the antibody with ficin and subsequently reduced by β mercaptoethanol.The conjugation between Fab′ fragment and pingyangmycin (PYM),an antitumor antibiotic,was mediated by dextran T 40.Immunoreactivity of Fab′ PYM conjugates with cancer cells was determined by ELISA,and the cytotoxicity of those conjugates to cancer cells was determined by clonogenic assay.Antitumor effects of the Fab′ PYM conjugates were evaluated by subcutaneously transplanted tumors in mice. Results.The molecular weight of Fab′ fragment was approximately 53 kD,while the average molecular weight of Fab′ PYM conjugate was 170 kD.The Fab′ PYM conjugates showed immunoreactivity with antigen relevant cancer cells and selective cytotoxicity against target cells.Administered intravenously,Fab′ PYM conjugates were more effective against the growth of tumors in mice than free PYM and PYM conjugated with intact mAb. Conclusion.Fab′ PYM conjugate may be capable of targeting cancer cells and effectively inhibiting tumor growth,suggesting its therapeutic potential in cancer treatment.

The Mechanism of Resistance of Pseudomonas Aeruginosa toβ-lactam Antibiotics and Clinical Significance

To study the resistant mechanism and clinical significance of pseudomonas aeruginosa toβ- lactam antibiotics,the outer mem brane permeability rate of30 P.aeruginosa strains to5 β- lactam antibiotics was m easured and their production ofβ- lactamase and theβ- lactam ase genes they carried detected. Furthermore,the relationship between the perm eability,β- lactam ase and the clinical effects ofβ- lactam antibiotics was observed. By using 1 4C- penicillin and liquid- scintillant isotope assay,the affinity of penicillin binding proteins(PBPs) was m easured and their roles in the resistant m echanism studied.Itwas revealed thatthe perm eability rate was higher in sensitive strains than in resistantones(P<0 .0 5 ) .All strains harbored1- 4 β- lactamase genes and produced β- lactam ase.Higher permeability rate and higher degree of stability toβ- lactamase indicated better clinical therapeutic effects. The affinity of PBPs changed little without regard to the perm eability andβ- lactam ase. These results suggested that the permeability of outer mem brane andβ- lacta- mase,but not PBPs,played im portant roles in the resistant mechanism of P. aeruginosa toβ- lac- tam antibiotics and affected the clinical therapeutic effectiveness of som e patients.

Role of insulin/insulin-like growth factor 1 signaling pathway in longevity

The insulin/insulin-like growth factor 1 (IGF-1) signaling pathway is evolutionary conserved in diverse speciesincluding C.elegans, saccharomyces cerevisiae, Drosophila melanogaster, rodents and humans, which is involved in many interrelated functions that are necessary for metabolism, growth and reproduction. Interestingly, more and more research has revealed that insulin/IGF-1 signaling pathway plays a pivotal role in the regulation of longevity. Generally, disruption of the power of this pathway will extend longevity in species ranging from C.elegansto humans. The role of insulin/IGF-1 in longevit yis probably related to stress resistance. Although the underlying mechanisms of longevity are not fully understood, the Insulin/IGF-1 signaling pathway has attracted substantial attention and it will be a novel target to prevent or postpone age-related diseases and extend life span. In this review, we mainly focus on the similar constitution and role of insulin/IGF-1 signaling pathway in C.elegans, saccharomyces cerevisiae, rodents and humans.

Synthesis of Quinolone Analogues: 7-[(2S,4R)-2-Aminomethyl-4- hydroxypyrrolidin-1-yl] Quinolones

New quinolone derivatives of 7-[(2S, 4R)-2-aminomethyl-4-hydroxypyrrolidin-1-yl] quinolone-3-carboxylic acids were synthesized by condensation of 7-halo substituted quinolone- 3-carboxylic acids with (2S, 4R)-2-aminomethyl-4-hydroxypyrrolidine. These compounds were characterized by FAB-MS and 1H NMR.

Identification of a New Peptide Deformylase Gene From Enterococcus faecium and Establishment of a New Screening Model Targeted on PDF for Novel Antibiotics

Objective To identify a new peptide deformylase (PDF) gene (Genebank Accession AY238515) from Enterococcus faecium and to establish a new screening model targeted on PDF. Methods A new PDF gene was identified by BLAST analysis and PCR and was subsequently over-expressed in the prokaryotic expression host E.coli Bl21(DE3). Over-expressed protein was purified for enzymatic assay by metal affinity chromatography and a new screening model was established for novel antibiotics. Result A new PDF gene of Enterococcus faecium was identified successfully. Ten positive samples were picked up from 8000 compound library and the microbial fermentation broth samples. Conclusion A new PDF of gene Enterococcus faecium was first identified and the model had a high efficacy. Positive samples screened may be antibacterial agents of broad spectrum.