The synergistic effect of lidamycin and rituximab on human B cell lymphoma / 药学学报

This study aimed to investigate the synergistic effect of lidamycin (LDM) and rituximab on human B cell lymphoma Ramos cells. Cell proliferation was measured using MTS assay, cell apoptosis was analyzed by Annexin V-FITC/PI assay, the expression of apoptosis related proteins was analyzed by Western blotting, and the in vivo lymphoma inhibition was verified using BALB/c mice inoculated via tail vein using Ramos cells which stably expressed pEGFP-N1 plasmid. The results showed that, after the pretreatment with rituximab for 48 h, rituximab and LDM showed significantly synergistic effects on cell proliferation. Cells in combined treatment group had a higher apoptosis rate than that in LDM treatment group. Compared with the LDM treatment group, the expression of apoptosis-related proteins such as Cleaved caspase-3, Cleaved caspase-7, Cleaved caspase-9 and Cleaved PARP in combined treatment groups increased, and expression of cIAP-2 and Bcl-2 decreased. The result of in vivo experiment showed that, in the combined treatment group, the survival time of BALB/c mice was significantly longer than the mice in control group and LDM treatment group, and the degree of tumor accumulation and metastasis to lymph nodes and spleen was lower.

Optimization of the preparation process for fusion protein Fv-LDP that composes lidamycin apoprotein and single-chain Fv antibody directed against type IV collagenase / 药学学报

This study is to optimize the preparation process of fusion protein Fv-LDP which was expressed in the form of inclusion body and consisted of lidamycin apoprotein LDP and single-chain Fv antibody (scFv) directed against type IV collagenase. The preparation and the dissolution of inclusion body, the immobilized metal affinity chromatography of the target protein and the renaturization by stepwise dialysis were optimized by single-factor analysis or orthogonal design. In addition, the refolded fusion protein Fv-LDP was refined by Sephadex G-75 chromatography followed by fluorescence-activated cell sorter (FACS)-based saturation binding assay to measure its antigen-binding activity. After optimization of the process, the purity of fusion protein Fv-LDP existed in the inclusion body was 63.9% and the corresponding solubility was 95.7%; Under denaturing conditions, the purity of fusion protein Fv-LDP was more than 95% after the purification process. The percentage of monomeric fusion protein Fv-LDP was 60% after the refolding process, while it was further refined to 85% which was 5.6-fold higher than that of the initial refolding condition. The refined fusion protein Fv-LDP could bind to human lung adenocarcinoma PAa cells and human hepatoma BEL-7402 cells with the dissociation constants (Kd) of 0.176 micromol x L(-1) and 0.904 micromol x L(-1), respectively. The preparation process of fusion protein Fv-LDP has been successfully optimized, which provides the experimental basis for the production and future development of fusion protein Fv-LDP, and might serve as a relatively practical system for the preparation of other scFv-based proteins expressed in the form of inclusion body.

Optimization of the assembly efficiency for lidamycin chromophore bound to its apoprotein: a case study using orthogonal array / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Lidamycin (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.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>A 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.</p><p><b>CONCLUSION</b>The 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.</p>

Bioluminescence imaging evaluation of the inhibitory effect of lidamycin on lung metastasis of human fibrosarcoma in athymic mice / 药学学报

This study is to investigate the inhibitory effect of lidamycin (LDM) and its combination with methotrexate (MTX) on lung metastasis of fibrosarcoma by bioluminescence imaging in athymic mice. A stable luciferase transfected HT-1080 cell line was constructed and the capability to establish experimental lung metastasis in athymic mice was confirmed. The optical imaging system was applied to evaluate the formation of lung metastasis in vivo. In addition, metastatic nodules were counted for the evaluation of inhibition rates. As shown, the fluorescent intensity of luciferase-transfected HT-1080 cells was colinear with the cell population and the minimal detected cell population was 100 cells/well. Optical imaging showed that the fluorescent intensity of treated group was apparently lower than that of the control. The inhibition rates of lung metastasis by LDM alone at 0.025 mg x kg(-1) and 0.05 mg x kg(-1) were 53.9% and 75.9%, respectively, while that of MTX alone at 0.5 mg x kg(-1) was 70.2%. The combination of LDM at 0.025 mg x kg(-1) and MTX at 0.5 mg x kg(-1) showed an inhibition rate of 88.7%. The coefficient of drug interaction (CDI) was 0.82. The results herein demonstrated that LDM alone had strong anti-metastasis effect on human fibrosarcoma HT-1080 and the inhibition efficacy is strengthened when combined with MTX.

Lidamycin metabolism in vitro / 药学学报

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.

Lidamycin inhibits the proliferation of HERG K+ channel highly expressing cancer cells and shows synergy with anticancer drugs / 药学学报

This study is to investigate inhibitory effects of lidamycin (LDM) on the proliferation of HERG K+ channel highly expressing cancer cells and its synergy with anticancer drugs. MTT assay was used to examine the inhibitory effects of lidamycin combined with various anticancer drugs on the proliferation of human lung cancer A549 cells, human colon cancer HT-29 cells and herg-stably-transfected A549 cells. Using the xenograft model of subcutaneously transplanted HT-29 in nude mice, inhibitory effect was appraised in vivo. The coefficient of drug interaction (CDI) was used to evaluate the synergistic effect of drug combination. LDM significantly inhibited the proliferation ofA549 cells and HT-29 cells with IC50 values of 2.14 and 4.64 ng mL(-1), respectively. The efficacy in HT-29 cells with high HERG potassium expression level is less potent than that in A549 cells with low expression level. In terms of IC50 values, LDM suppressed the growth of herg-stably-transfected A549 cells less potently than pCDNA3.1-stably-transfected A549 cells. There existed synergistic effects in the combinations of fluorouracil (5-FU) and LDM, doxorubicin (DOX) and LDM, or hydroxycamptothecine (HCPT) and LDM. CDI values of the combinations of 5-FU and LDM were more than 0.75. CDI values of LDM and DOX were more than 0.70, but some CDI values of LDM and HCPT were less than 0.70. As for the CDI values, synergistic effects of the combination of LDM and HCPT were the most potent of the three groups. There is no relationship between the inhibitory effect of the growth of cancer cells by 5-FU and HERG potassium expression level. HERG expression level negatively correlated with inhibitory effect on the proliferation of cancer cells by DOX. HERG expression levels and chemosensitivity were positively correlated for HCPT. In the model of subcutaneously xenograft transplanted HT-29 in vivo, LDM and/or HCPT effectively inhibited the growth of HT-29 in nude mice, and the optimum CDI of the combination of LDM and HCPT was less than 1. HERG expression level negatively correlates the chemosensitivity of cancer cells to LDM. There exist synergistic effects in vitro and in vivo in the combination of LDM and HCPT, which inhibitory effects of the proliferation of cancer cells positively modulated by HERG potassium expression level. HERG K+ channel may become a target of combined therapy for choosing anticancer drugs.

Inhibitory effects of the immunoconjugate composed of anti-type IV collagenase antibody Fab' fragment and lidamycin on tumor invasion and metastasis / 药学学报

This study is to investigate the tumor invasion and metastasis inhibition effects of the immunoconjugate composed of lidamycin and anti-type IV collagenase monoclonal antibody Fab' fragment. Boyden chamber assay was used to evaluate the influence of Fab'-LDM on HT-1080 cells invasion ability, gelatinase spectrum was used to measure the change of invasion factor MMP-2 and MMP-9's secretion, and RT-PCR was adopted to determine TIMP-1 mRNA expression level. The immunoconjugate inhibition of tumor in situ metastasis was also tested in nude mice. The Fab'-LDM conjugates had dose-dependent inhibition effect on HT-1080 cells' invasion. At the concentrations of 5 and 10 nmol L(-1), the Fab'-LDM inhibited the invasion by (60 +/- 12) % and (79 +/- 11) % respectively. At the concentration of 5 and 10 nmol L(-1), the Fab'-LDM inhibited the secretion of MMP-2 by (42 +/- 8) % and (54 +/- 6) % and that of MMP-9 by (57 +/- 3) % and (87 +/- 1) %, respectively. RT-PCR indicated that conjugates increased the anti-invasion factor TIMP-1 level. The in vivo experiment showed that, compared with the control group, the tumor inhibition rate in Fab', Fab'-LDM, and LDM group equaled to (30 +/- 13) %, (86 +/- 26) %, (74 +/- 22) % respectively. In conclusion, Fab'-LDM could inhibit the invasion and metastasis of tumor and it might be a new tumor biotherapy agent.

Comparison of the antitumor activities of immunoconjugates composed of lidamycin and monoclonal antibody fab' fragment with different linkers / 药学学报

To investigate the antitumor activities of the immunoconjugates composed of anti-type IV collagenase monoclonal antibody Fab' fragment and lidamycin (LDM) prepared with different linkers. The immunoconjugates were prepared by linking Fab' to lysine-69 of LDM apoprotein by SPDP, LCSPDP, SMBS or SSMPB as the intermediate drug linkers. Immunoreactivities of the conjugates were determined by ELISA. The cytotoxicities of the conjugates were examined by clonogenic assay. In vivo antitumor effects of the conjugates were evaluated in nude mice bearing subcutaneously implanted HT-1080 tumor. ELISA assay showed that the conjugates retained part of the immunoreactivity of 3G11 against the antigen. The cytotoxicities of the Fab'-SMBS-LDM and Fab'-SSMPB-LDM to HT-1080 cells were significantly potent, compared with Fab'-SPDP-LDM, Fab'-LCSPDP-LDM and free LDM. In animal models at the same condition, free LDM, Fab'-SPDP-LDM and Fab'-LCSPDP-LDM inhibited the growth of HT-1080 tumor by 70.9%, 74.8% and 72.3%, while Fab'-SMBS-LDM and Fab'-SSMPB-LDM reached 78.0% and 87.7%, respectively. The median survival time of the mice treated with free LDM, Fab'-SPDP-LDM and Fab'-LCSPDP-LDM were prolonged by 71.9%, 82.2% and 107.5%, respectively, compared with that of untreated group. Whereas, the median survival time of Fab'-SMBS-LDM and Fab'-SSMPB-LDM were prolonged by 145.2% and 165.8%, respectively, indicating that Fab'-SSMPB-LDM was more effective than Fab'-SMBS-LDM in tumor suppression and life span prolongation. Fab'-SSMPB-LDM has more marked selective antitumor efficacy and lower toxicity, and might be a novel candidate for cancer therapy.

Binding capability of lidamycin apoprotein to human breast cancer detected by tissue microarrays / 药学学报

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.

Synergistic effect and its possible mechanisms of lidamycin in combination with TRAIL in NSCLC / 药学学报

This study is to investigate the effect and its possible mechanisms of lidamycin (LDM) combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human non-small cell lung cancer (NSCLC) cells. MTT assay was used to determine the growth inhibition of the two ingredients on H460 cells. Apoptosis was examined by Annexin V-FITC/PI staining, flow cytometry assay and DNA-specific dye Hoechst 33342 staining. The level of TRAIL receptor and apoptosis-associated protein expression was detected by Western blotting analysis. The results showed that the IC50 value of LDM and TRAIL for H460 cells was 4.603 x 10(-10) mol x L(-1) and 915.3 ng x mL(-1) respectively, but the IC50 value of LDM was 3.064 x 10(-11) mol x L(-1) and 1.611 x 10(-11) mol x L(-1) when different concentrations of LDM was combined with 50 and 100 ng x mL(-1) TRAIL respectively. And the CDI value was less than 1. The apoptosis ratios also increased in the combination group relative to the single-agent treatment and the untreated control. Furthermore, the induction of the cleavage of PARP and the activation of Caspase-3 and Caspase-8 by the combination were more effective than LDM or TRAIL alone. At last, the level of death receptor 5 (DR5) expressions increased in a dose-dependent manner and time-related pattern. The data indicate that LDM inhibits the growth of H460 cells in vitro. DR5 induction contributes to enhancement of TRAIL-induced apoptosis by LDM in human non-small lung cancer cells.

Lidamycin inhibits angiogenesis of zebrafish embryo via down-regulation of VEGF / 药学学报

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.

Role of prosurvival molecules in the action of lidamycin toward human tumor cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Lidamycin, an enediyne antibiotic, leads to apoptosis and mitotic cell death of human tumor cells at high and low concentrations. The reason why tumor cells have distinct responses to lidamycin remains elusive. This study was to elucidate if cellular prosurvival molecules are involved in these responses.</p><p><b>METHODS</b>Cleavage of chromatin and DNA was observed by chromatin condensation and agarose gel electrophoresis. Accumulation of rhodamine 123 in lidamycin-treated cells was assayed by flow cytometry. Cell multinucleation was detected by staining with Hoechst 33342. Western blot and senescence-associated beta-galactosidase (SA-beta-gal) staining were used to analyze protein expression and senescence-like phenotype, respectively.</p><p><b>RESULTS</b>SIRT1 deacetylase remained unchanged in 0.5 nmol/L lidamycin whereas cleavage occurred when apoptosis was induced by lidamycin. Increased FOXO3a, SOD-1 and SOD-2 expression and transient phosphorylation of ERK were detected after exposure of human hepatoma BEL-7402 cells to 0.5 nmol/L lidamycin. High expressions of SIRT1 and Akt were found in colon carcinoma HCT116 p53 knock-out cells exposed to lidamycin. Degradation of PARP and p53 by lidamycin as a substitute for SIRT1 and Akt was confirmed with caspase inhibitor Q-VD-OPh and proteasome inhibitor MG132. Resistance to lidamycin-induced DNA cleavage was observed in breast cancer doxorubicin-resistant MCF-7 cells. This was not induced by P-glycoprotein as no accumulation of rhodamine 123 was detected in the resistant cells following exposure to lidamycin. In contrast to sensitive MCF-7 cells, a lower multinucleation rate for the resistant cells was measured following exposure to equal concentrations of lidamycin.</p><p><b>CONCLUSIONS</b>Cellular prosurvival molecules, such as SIRT1, Akt, SOD-1, SOD-2 and other unknown factors can influence the action of lidamycin on human tumor cells.</p>

Lidamycin induces apoptosis of human gastric carcinoma BGC823 cells and inhibits xenograft growth in nude mice / 药学学报

To investigate the effect of lidamycin (LDM) on human gastric carcinoma BGC823 cells and xenograft growth in nude mice, MTT (methyl thiazolyl tetrazolium) assay was used to determine the inhibition of BGC823 cell proliferation by LDM. Induction of apoptosis was studied by flow cytometry and TUNEL assay. The expression of VEGF was detected by Western blotting analysis. Athymic nude mice were used to determine in vivo antitumor activity. Proliferation inhibition and apoptosis induction were studied in lidamycin-treated cells. The expression of VEGF in BGC823 cells decreased in a dose-dependent manner. LDM at 0.02 mg x kg(-1) and 0.04 mg x kg(-1) suppressed the growth of BGC823 xenografts in nude mice by 57% and 72%, respectively. LDM potently induces apoptosis in human gastric carcinoma BGC823 cells and inhibits xenograft growth.

Effect of lidamycin on telomerase activity in human hepatoma BEL-7402 cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Chromatin 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.</p><p><b>RESULTS</b>Mitotic 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.</p><p><b>CONCLUSION</b>Mitotic 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.</p>

Antitumor activities of various immunoconjugates composed of lidamycin and anti-type IV collagenase monoclonal antibody / 药学学报

This study is to investigate the antitumor activities of the immunoconjugates composed of anti-type IV collagenase monoclonal antibody 3G11 and lidamycin (LDM) prepared by different methods. The immunoconjugates were prepared by linking 2-iminothiolane modified 3G11 to lysine-69 of LDM apoprotein by SPDP and SMBS as the intermediate drug linker. Immunoreactivity of the conjugates was determined by ELISA. The cytotoxicity of the conjugates was examined by clonogenic assay. Antitumor effects of the conjugates in vivo were evaluated in nude mice bearing subcutaneously implanted HT-1080 tumor. ELISA assay showed that the immunoconjugates retained the immunoreactivity of 3G11 against type IV collagenase. The cytotoxicity of the 3G11-SMBS-LDM to HT-1080 cells was significantly more potent than that of free LDM and 3G11-SPDP-LDM. In animal model at the same condition, free LDM inhibited the growth of HT-1080 tumor by 71.2%, while 3G11-SPDP-LDM and 3Gl1-SMBS-LDM reached 77.1% and 86.1%, respectively. The median survival time of the mice treated with free LDM was prolonged by 71.9% compared with that of untreated group. Whereas, the median survival time of 3G11-SPDP-LDM and 3G11-SMBS-LDM was prolonged by 125.3% and 163.7%, respectively, indicating that 3G11-SMBS-LDM was more effective than 3G11-SPDP-LDM in tumor suppression and life span prolongation. 3Gll-SMBS-LDM has more selective antitumor efficacy and lower toxicity, and might be a novel candidate for cancer therapy. LDM was more effective than 3G11-SPDP-LDM in tumor suppression and life span prolongation. 3Gll-SMBS-LDM has more selective antitumor efficacy and lower toxicity, and might be a novel candidate for cancer therapy.

Effect of lidamycin on mitochondria initiated apoptotic pathway in human cancer cells / 药学学报

Although enediyne antibiotic lidamycin ( LDM) is a potent inducer of apoptosis, the underlying mechanisms of its apoptotic functions remain to be explored. Here, we aim to elucidate its possible mechanisms in mitochondria initiated apoptotic pathway involved in human BEL-7402 and MCF-7 cells. Cytochrome c released from mitchondria to cytosol fraction was detected by Western blotting. p53 and Bax, Bcl-2 expressions were detected by Western blotting and RT-PCR. MTT assay was used to detect cytotoxicity of LDM with or without caspase inhibitor z-VAD-fmk. After the BEL-7402 cells were exposed to 0. 1 micromol x L(-1) LDM within 6 h, the increase of cytochrome c in the cytosol and decrease in the mitochondria were observed when compared with untreated cells. The expression of Bax, an important proapoptotic member of the Bcl-2 family, increased gradually in the BEL-7402 cells after exposure to LDM of 0. 1 micromol x L (-1) for 2, 6, and 9 h, separately, while Bcl-2 increased at 2 and 6 h, and decreased at 9 h after LDM treatment. Enhanced protein expressions were parallel with respective increased mRNA level for Bax only, but not p53. Caspase inhibitor may inhibit partially the killing effects induced by LDM. Therefore we conclude that the rapid activation of mitochondrial pathway induced by LDM in tumor cells might contribute to its highly potent cytotoxicities.

Chemosensitivity of mdr1 gene overexpressed multidrug resistant cancer cells to lidamycin / 药学学报

<p><b>AIM</b>To investigate the chemosensitivity to lidamycin (C-1027) in mdr1 gene overexpressing cancer cell lines established by drug induction and by gene-transfection.</p><p><b>METHODS</b>DNA 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>Lidamycin 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.</p>

Lidamycin inhibits the cancer cell PKC activity induced by basic fibroblast growth factor / 药学学报

<p><b>AIM</b>To study the mechanism of inhibition of basic fibroblast growth factor (bFGF) related signal transduction by lidamycin in cancer cells.</p><p><b>METHODS</b>MTT 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.</p><p><b>RESULTS</b>LDM 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.</p><p><b>CONCLUSION</b>The blocking of bFGF receptors in the signal transduction pathway may be involved in the effect of LDM on cancer cells.</p>

Comparison of the pharmacokinetics of lidamycin in mice determined by two methods / 药学学报

<p><b>AIM</b>To compare two methods, the total radioactivity assay (RA method) and the radioactivity assay after separation with high performance liquid chromatography (HPLC-RA method).</p><p><b>METHODS</b>125I-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.</p><p><b>RESULTS</b>The pharmacokinetic parameters (Vd, T1/2alpha, T1/2beta, K21, K10, K12, AUC and CL) showed significant difference between the two methods (P < 0.05).</p><p><b>CONCLUSION</b>The HPLC-RA method was better than the RA method to determine unchanged 125I-lidamycin.</p>

Studies on the pharmacokinetics of lidamycin in mice and dogs using bioassay / 药学学报

<p><b>AIM</b>A bioassay method was established for the determination of active concentrations of lidamycin and studied its pharmacokinetics in mice and dogs.</p><p><b>METHODS</b>Cytotoxicity of lidamycin in vitro was used to determine drug serum concentrations in vivo.</p><p><b>RESULTS</b>Validity 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.</p><p><b>CONCLUSION</b>Active 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.</p>