AIMTo compare two methods, the total radioactivity assay (RA method) and the radioactivity assay after separation with high performance liquid chromatography (HPLC-RA method).

METHODS125I-Lidamycin was prepared by Iodogen method and separated by size exclusive high performance liquid chromatography. The pharmacokinetic parameters of lidamycin were assayed by two methods after intravenous injection to mice at the dose of 100 microg x kg(-1), and compared by statistical analysis.

RESULTSThe pharmacokinetic parameters (Vd, T1/2alpha, T1/2beta, K21, K10, K12, AUC and CL) showed significant difference between the two methods (P < 0.05).

CONCLUSIONThe HPLC-RA method was better than the RA method to determine unchanged 125I-lidamycin.

Aminoglycosides ; blood ; pharmacokinetics ; Animals ; Antibiotics, Antineoplastic ; blood ; pharmacokinetics ; Area Under Curve ; Chromatography, High Pressure Liquid ; methods ; Enediynes ; Female ; Iodine Radioisotopes ; Isotope Labeling ; Male ; Mice ; Sensitivity and Specificity

AIMTo investigate the synergetic effect and the mechanism of antitumor action of the antibiotic lidamycin in combination with cisplatin in vitro.

METHODSCytotoxicity of the drugs was measured by clonogenic assay. Chromatin condensation was observed by co-staining with fluorescent dyes, Hoechst 33342 and propidium iodide. Apoptotic sub-G1 was detected by flow cytometry and DNA ladder was observed using agarose gel electrophoresis. Bcl-2 protein level was detected by Western blot assay.

RESULTSBy using clonogenic assay, lidamycin in combination with cisplatin was found to have synergetic effects on the proliferation of human hepatoma BEL-7402 cells. The data showed that BEL-7402 cells treated with cisplatin and lidamycin in combination produced internucleosomal DNA fragmentation analysed by agarose gel electrophoresis. The results of flow cytometry showed that cisplatin and lidamycin administrated in combination showed no obvious change in G1 phase distribution compared with single treatment. However, this combination reduced the S phase arrest and reversed the reduction of G2/M phase induced by single treatment. The results also showed that there was 11.3% or 9.37% of cells undergoing apoptosis in BEL-7402 cells treated with cisplatin or lidamycin, respectively, while it showed 32.4% of apoptotic cells in combination treatment. Cisplatin, lidamycin and combination of cisplatin and lidamycin was shown to induce typical chromatin condensation in BEL-7402 cells. The study showed that 0.5 mumol.L-1 cisplatin or 1 x 10(-4) mumol.L-1 lidamycin alone decreased Bcl-2 protein level, while lidamycin in combination with cisplatin strongly inhibited expression of Bcl-2 proteins in BEL-7402 cells.

CONCLUSIONThe results suggest that lidamycin enhancement of cisplatin-induced apoptosis associates with decrease of Bcl-2 protein expression, which may be useful for cancer chemotherapy.

Aminoglycosides ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Carcinoma, Hepatocellular ; pathology ; Cisplatin ; pharmacology ; Drug Synergism ; Enediynes ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; S Phase ; drug effects ; Tumor Cells, Cultured

This paper is to report the study of the metabolism of lidamycin in vitro including in plasma and microsomes to guide clinical therapy. Lidamycin was quantified by detecting its active ingredient using HPLC-MS/MS. The metabolic stability of lidamycin in rat, Beagle dog, monkey and human plasma and liver microsomes, and its inhibition to cytochrome P450 isoforms in human liver microsomes were studied. Results showed that lidamycin was metabolized in the four species of plasma, and the sequence of metabolic rates in plasma were in rat > in dog > in human > in monkey. But among the four species of liver microsomes, lidamycin was metabolized only in monkey liver microsomes. There was almost no inhibition to cytochrome P450 isoforms at the concentrations of between 0.0005 and 10 ng x mL(-1). Therefore, the property of lidamycin metabolism in human is similar with that in dog, and metabolism of other drugs would not be decreased by cytochrome P450 as used along with lidamycin in clinic.
Aminoglycosides ; blood ; metabolism ; Animals ; Antibiotics, Antineoplastic ; blood ; metabolism ; Chromatography, High Pressure Liquid ; Cytochrome P-450 CYP1A2 ; metabolism ; Cytochrome P-450 Enzyme System ; metabolism ; Dogs ; Enediynes ; blood ; metabolism ; Enzyme Activation ; Humans ; Macaca ; Microsomes, Liver ; metabolism ; Rats ; Tandem Mass Spectrometry

AIMTo investigate the chemosensitivity to lidamycin (C-1027) in mdr1 gene overexpressing cancer cell lines established by drug induction and by gene-transfection.

METHODSDNA was cloned by RT-PCR and then eukaryotic expressing recombinant plasmid pcDNA3. 1/mdrl was constructed. Using Lipofectamine 2000, a strain of stably transfected human hepatoma cancer cells, HepG2/mdrl, was obtained. The mdr1 mRNA level, P-glycoprotein (P-gp) level and the activity of P-gp to extrude drugs in cancer cells were determined by RT-PCR, immunofluorescence analysis and rhodamine 123 efflux assay. The chemosensitivity of cancer cells with low or high mdr1 expression to lidamycin and other antitumor drugs was tested by MTT assay.

RESULTSThe mdr1 mRNA and P-gp levels in KBv200, MCF-7/ADR, and stably transfected HepG2/mdr1 cells were much higher than that in respective parent KB, MCF-7 and HepG2 cells. The IC50 values of lidamycin for KBv200, MCF-7/ADR and HepG2/mdrl cells were (0.24 +/- 0.20) nmol x L(-1), (0.028 +/- 0.011) nmol x L(-1), and (0.020 +/- 0.011) nmol x L(-1), respectively. Compared with parental cells, the values of resistant fold for KBv200, MCF-7/ADR and HepG2/mdr1 cells to lidamycin were 6.8, 1.6 and 1.3 fold; to adriamycin were 37.2, 181.3 and 8.8 fold; to taxol were 336.8, 49.2 and 40.3 fold, respectively.

CONCLUSIONLidamycin is highly active to multidrug resistant cancer cells. The chemosensitivity of those resistant cancer cells to lidamycin is approximately at the similar level as that of parent cancer cells.

ATP-Binding Cassette, Sub-Family B, Member 1 ; analysis ; genetics ; Aminoglycosides ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Cell Line, Tumor ; Drug Resistance, Multiple ; Drug Resistance, Neoplasm ; Enediynes ; pharmacology ; Genes, MDR ; Humans ; Neoplasms ; drug therapy ; pathology ; Transfection

AIMA bioassay method was established for the determination of active concentrations of lidamycin and studied its pharmacokinetics in mice and dogs.

METHODSCytotoxicity of lidamycin in vitro was used to determine drug serum concentrations in vivo.

RESULTSValidity of methodology met the requirements of pharmacokinetic study. The concentration-time profile in mice after iv lidamycin of 100, 50 and 10 microg x kg(-1) was best fitted with 2-compartmental model with T1/2alpha and T1/2beta of 0.77-1.8 min and 5.6-7.2 min, respectively. The AUC were 2851.3, 887.8 and 166.4 microg x min x L(-1), respectively and increased with dose nonlinearly. There were similar trends between AUC and the potency of tumor growth inhibition. After iv lidamycin of 12 microg x kg(-1) in dogs, the concentrations of lidamycin decreased rapidly and the AUC was 16 microg x min x L(-1), which were lower and quicker than those in mice. The levels in serum after second administration at day 15, were lower than those of the first.

CONCLUSIONActive concentrations and pharmacokinetics of lidamycin were obtained by bioassay method successfully. There are species differences and single and multi-dosing differences in the pharmacokinetics of lidamycin.

Aminoglycosides ; blood ; pharmacokinetics ; pharmacology ; Animals ; Antibiotics, Antineoplastic ; blood ; pharmacokinetics ; pharmacology ; Area Under Curve ; Biological Assay ; Dogs ; Enediynes ; Female ; Humans ; Injections, Intravenous ; KB Cells ; metabolism ; Liver Neoplasms ; pathology ; Male ; Mice ; Neoplasm Transplantation ; Sarcoma 180 ; pathology ; Species Specificity

Lidamycin (LDM) is a potent antitumor antibiotic. Previous studies have shown that LDM could inhibit proliferation and migration in endothelial cells. In the present report, the effect of LDM on angiogenesis of zebrafish embryo was studied. The results showed that treatment of zebrafish embryos with LDM resulted in significant inhibition of angiogenesis. Morphological observation, quantitative endogenous alkaline phosphatase (EAP) assay, alkaline phosphatase staining, and transgenic zebrafish assay were performed to evaluate vascular development defects in zebrafish. The results indicated that after the zebrafish embryos were exposed to LDM, angiogenesis defects of zebrafish embryos were observed, including pericardial edema, reduced numbers of circulating red blood cells, suppression of zebrafish vessel growth, and absences of SIV (subintestinal vein). The expression of VEGF was detected by RT-PCR assay, quantitative reverse transcriptase real-time PCR (qRT-PCR) assay and Western blotting analysis. The results revealed that LDM could inhibit the expression of VEGF protein, while the expression of mRNA was not significantly affected. The study suggests that LDM could inhibit the zebrafish embryo angiogenesis by down-regulation ofVEGF expression.
Aminoglycosides ; pharmacology ; Animals ; Animals, Genetically Modified ; embryology ; genetics ; physiology ; Antibiotics, Antineoplastic ; pharmacology ; Down-Regulation ; Embryo, Nonmammalian ; drug effects ; Enediynes ; pharmacology ; Neovascularization, Physiologic ; drug effects ; genetics ; RNA, Messenger ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; Zebrafish ; embryology ; genetics ; physiology

This study is to investigate the binding capability of lidamycin apoprotein (LDP), an enediyne-associated apoprotein of the chromoprotein antitumor antibiotic family, to human breast cancer and normal tissues, the correlation of LDP binding capability to human breast cancer tissues and the expression of tumor therapeutic targets such as VEGF and HER2. In this study, the binding capability of LDP to human breast cancer tissues was detected with tissue microarray. The correlation study of LDP binding capability to human breast tumor tissues and relevant therapeutic targets was performed on breast cancer tissue microarrays. Immunocytochemical examination was used to detect the binding capability of LDP to human breast carcinoma MCF-7 cells. As a result, tissue microarray showed that LDP staining of 73.2% (30/41) of breast cancer tissues was positive, whereas that of 48.3% (15/31) of the adjacent normal breast specimens was positive. The difference between the tumor and normal samples was significant (Chi2 = 4.63, P < 0.05). LDP immunoreactivity in breast cancer correlated significantly with the overexpression of VEGF and HER2 (P < 0.001 and < 0.01, r = 0.389 and 0.287, respectively). Determined with confocal immunofluorescent analysis, LDP showed the binding capability to mammary carcinoma MCF-7 cells. It is demonstrated that LDP can bind to human breast cancer tissues and there is significant difference between the breast cancer tissues and the corresponding normal tissues. Notably, the binding reactivity shows positive correlation with the expression of VEGF and HER2 in breast carcinoma tissues. The results imply that LDP may have a potential use as targeting drug carrier in the research and development of new anticancer therapeutics. This study may provide reference for drug combination of LDM and other therapeutic agents.
Aminoglycosides ; metabolism ; Antibiotics, Antineoplastic ; metabolism ; Apoproteins ; metabolism ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Enediynes ; metabolism ; Female ; Humans ; Protein Binding ; Receptor, ErbB-2 ; metabolism ; Tissue Array Analysis ; methods ; Vascular Endothelial Growth Factor A ; metabolism

AIMTo study the mechanism of inhibition of basic fibroblast growth factor (bFGF) related signal transduction by lidamycin in cancer cells.

METHODSMTT assay was used to determine the growth inhibitory effect of lidamycin (LDM) and adriamycin (ADR) in several cancer cell lines. The inhibition of bFGF bound to its receptor by LDM was measured with [125I]-bFGF binding assay. Intracellular Ca2+ stimulated by bFGF was measured by Fura-3. The formation of bFGF-receptor immune complex and the inhibitory effect of LDM on the activity of PKC isoenzymes induced by bFGF in cancer cells were identified by Western blotting analysis.

RESULTSLDM displayed extremely potent growth inhibitory effect on several cancer cell lines in a dose-dependent manner. A comparison of the IC50 values showed that the effect of LDM was 1000-fold more potent than that of ADR. LDM blocked the specific binding of [125I]-bFGF to rat lung membranes with an IC50 value of 2.0 x 10(-4) nmol x L(-1). As detected by anti-FGFR specific antibody, LDM inhibited the formation of bFGF-receptor immune complex. bFGF induced cytosolic Ca2+ response was obstructed by pretreatment with 10 nmol x L(-1) LDM. Immunoblotting demonstrated that LDM inhibited the activity of PKC isoenzymes in cancer cells stimulated with bFGF.

CONCLUSIONThe blocking of bFGF receptors in the signal transduction pathway may be involved in the effect of LDM on cancer cells.

Aminoglycosides ; administration & dosage ; pharmacology ; Animals ; Antibiotics, Antineoplastic ; administration & dosage ; pharmacology ; Breast Neoplasms ; pathology ; Calcium ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Doxorubicin ; pharmacology ; Enediynes ; administration & dosage ; pharmacology ; Female ; Fibroblast Growth Factor 2 ; pharmacology ; HT29 Cells ; Humans ; Membrane Proteins ; metabolism ; Protein Binding ; Protein Kinase C ; metabolism ; Rats ; Receptors, Fibroblast Growth Factor ; metabolism ; Signal Transduction

OBJECTIVELidamycin (LDM) can be dissociated to an apoprotein (LDP) and an active enediyne chromophore (AE). The detached AE can reassemble with its LDP-containing fusion protein to endow the latter with potent antitumor activity. However, the reassembly of AE with LDP is affected by several factors. Our aim was to optimize the assembly efficiency of the AE with a LDP-containing fusion protein and investigate the influence of several factors on the assembly efficacy.

METHODSA method based on RP-HPLC was developed to analyze the assembly rate, and an orthogonal experimental design L(9) (3(4)) was used to investigate the effects of temperature, assembly time, pH and molecular ratio of LDP-containing fusion protein to AE on the assembly rate. Furthermore, the determined optimum conditions for the assembly rate of the LDP-containing fusion protein with AE were applied and evaluated.

RESULTSA calibration curve based on the LDM micromolar concentration against the peak-area of AE by HPLC was obtained. The order in which individual factors in the orthogonal experiment affected the assembly rate were temperature>time>pH>molar ratio of AE to protein and all were statistically significant (P<0.01). The optimal assembly conditions were temperature at 10°C, time of 12 h, pH 7.0, and the molar ratio of AE: protein of 5:1. The assembly rate of AE with a LDP-containing fusion protein was improved by 23% after condition optimization.

CONCLUSIONThe assembly rate of chromophore of lidamycin with its LDP-containing fusion protein was improved after condition optimization by orthogonal design, and the optimal conditions described herein should prove useful for the development of this type of LDP-containing fusion protein.

Aminoglycosides ; administration & dosage ; chemical synthesis ; chemistry ; pharmacology ; Antibiotics, Antineoplastic ; administration & dosage ; chemical synthesis ; chemistry ; pharmacology ; Apoproteins ; chemistry ; Cell Line, Tumor ; Cell Survival ; Chromatography, High Pressure Liquid ; Drug Design ; Enediynes ; administration & dosage ; chemical synthesis ; chemistry ; pharmacology ; Humans ; Recombinant Fusion Proteins ; chemistry ; Single-Chain Antibodies ; chemistry

OBJECTIVETo investigate the effect of lidamycin (LDM) on telomerase activity in human hepatoma BEL-7402 cells under the condition of LDM inducing mitotic cell death and senescence.

METHODSChromatin condensation was detected by co-staining with Hoechst 33342 and PI. Cell multinucleation was observed by Giemsa staining and genomic DNA was separated by agarose gel electrophoresis. Fluorescent intensity of Rho123 was determined for mitochondrial membrane potential. MTT assay and SA-beta-gal staining were employed to analyze the senescence-like phenotype. The expression of proteins was analyzed by Western blot. Telomerase activity was assayed by telomerase PCR-ELISA.

RESULTSMitotic cell death occurred in LDM-treated cells characterized by unique and atypical chromatin condensation, multinucleation and increased mitochondrial membrane potential. However, no apoptotic bodies or DNA ladders were found. In addition, apoptosis-related proteins remained nearly unaltered. Senescence-like phenotype was identified by increased and elongated size of cells, growth retardation, enhanced SA-beta-gal activity and the changes of senescence-related protein expression. Telomerase activity markedly decreased (P<0.01) in LDM-treated hepatoma BEL-7402 cells.

CONCLUSIONMitotic cell death and senescence could be triggered simultaneously or sequentially after exposure of hepatoma BEL-7402 cells to LDM. The decrease in telomerase activity may play a key role in the defective mitosis and aging morphology. Further investigation of detailed mechanism is needed.

Aminoglycosides ; pharmacology ; Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Azure Stains ; Benzimidazoles ; Carcinoma, Hepatocellular ; enzymology ; pathology ; Cell Nucleus ; drug effects ; metabolism ; Cellular Senescence ; drug effects ; Chromatin ; metabolism ; DNA, Neoplasm ; analysis ; Dose-Response Relationship, Drug ; Enediynes ; pharmacology ; Genome, Human ; genetics ; Humans ; Liver Neoplasms ; enzymology ; pathology ; Membrane Potential, Mitochondrial ; drug effects ; Mitosis ; drug effects ; Phenotype ; Propidium ; Telomerase ; metabolism ; Time Factors ; beta-Galactosidase ; metabolism