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Antibody-drug conjugates (ADCs) are one of the most important classes of anticancer therapeutics. Human epidermal growth factor receptor-2 (HER2), which is highly expressed in many types of aggressive cancers including breast and ovarian cancer, has been approved as an ideal target for ADCs. Lidamycin (LDM), developed by Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, is an enediyne-containing antibiotic with potent anti-tumor activity. LDM is a promising payload for ADCs. In the present research, using a special site-directed conjugating technology, we made a novel ADC (607-LDM) with a drug-to-antibody ratio (DAR) of 2 and composed of the anti-HER2 antibody 607 and LDM. The new ADC exhibited potent antitumor activity against human ovarian cancer SKOV3 and breast cancer BT-474 cells. It also induced apoptosis and G2/M arrest. In nude mice with SKOV3 xenografts and a tumor volume of 150-200 mm3, a single intravenous injection 607-LDM at 1 mg·kg-1 induced tumor growth inhibition of 72.4%, which was significant compared to either LDM (50.6%) or antibody (30.2%) treatment alone, or both in combination (50.1%, P < 0.05). All animal experiments were performed in accord with National Regulations and approved by the Animal Experiments Ethical Committee of College of Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences. The novel ADC designed in this study, 607-LDM, is a promising candidate for the treatment of HER2-positive cancers.
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OBJECTIVE: To study the distribution, excretion and other pharmacokinetic characteristics of dual target ligand-based lidamycin (DTLL) in rats. METHODS: [125l] radioactivity isotope tracing method was used to determine the pharmacokinetic parameters of DTLL which was injected intravenously via the tail vein of healthy Wistar rats at a single dose of [125l] DTLL 2625 kBq · 0.05 mg-1· kg-1. The samples from rat plasma, tissue, feces, urine and bile were obtained to analyze the distributions of DTLL in rats after administration and to calculate the cumulative excretion scores by using a liquid scintillation counting analyzer with the WinNolin program. RESULTS: The area under the concentration-time curve (AUC0-∞) of DTLL was (184±33) h·μg·L-1, with half time (t1/2) and cleanrance (Cl) at (3.98±0.75) h and (278±41) mL·h-1· kg-1, respectively. DTLL was mainly distributed in whole body tissues without any accumulation observed. The excretion rate of DTLL was (82.5±2.6)% for urine, compared with (10.4±6.6)% in feces and (92.9±7.4)% in total. CONCLUSION: DTLL is distributed rapidly in the whole body without obvious accumulation. Most of the DTLL administered is excreted through feces and urine.
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Aim To investigate the effects of lidamycin (LDM) on cell cycle, apoptosis, autophagy and epithelial mesenchymal transformation (EMT) of human cervical cancer Hela cells as well as its underlying mechanism. Methods MTT assay was used to analyze the effect of LDM on Hela cell growth. Flow cytometry was used to detect the effect of LDM on cell cycle, apoptosis and mitochondrial membrane potential. Western blot was used to detect the expression levels of proteins. Transwell assay was used to analyze the effect of LDM on migration and invasion of Hela cells. QT-PCR was employed to analyze the effects of LDM on LSD1 (lysine specific demethylase 1) expression level in Hela cells. Results LDM could significantly inhibit Hela cell proliferation in a time-and concentration-dependent manner, with the IC50 values of 19. 24 ng . mL-1 (24 h), 6.678 ng . mL- 1(48 h) and 3.221 ng . mL-1 (72 h). LDM could induce G2/M cycle arrest and mitochondrial-mediated endogenous apoptosis in Hela cells. Low concentration of LDM(5 ng . mL-1) could induce autophagy, but high concentration of LDM(20 ng . mL-1 ) could inhibit autophagy in Hela cells. In addition, LDM could inhibit the migration and invasion of Hela cells, change the expression levels of EMT-related factors, but LSD1 was not involved in this process. Conclusions LDM can effectively inhibit proliferation, invasion and metastasis of Hela cervical cancer cells, and its mechanism may be closely related to cell cycle arrest, apoptosis, interfering autophagy and inhibition of EMT.
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OBJECTIVE: To investigate the inhibitory effects of bispecific antibody-drug conjugate Fv-LDM-NGR targeting EGFR and CD13 on human tumor cells and endothelial cells,and possible mechanisms. METHODS: Human breast cancer cells MCF-7, human lung adenocarcinoma cells A549 and human microvascular endothelial cells HMEC-1, were studied. MTT assay was applied to measure proliferative activity of tumor cells. The influence of Fv-LDM-NGR on tube formation of HMEC-1 was observed. Transwell assay was applied to measure migration and invasion capacity in tumor cells. Western blot was applied for analyzing intracellular signaling transduction pathways. Flow cytometry, Hochest stain and Annexin -FITC/PI were used to detect cell cycle and apoptosis. RESULTS: Fv-LDM-NGR could inhibit the proliferation of tumor cells and microvascular endothelial cells with IC50 values of 10-14-10-12mol•L-1. Fv-LDM-NGR prevented tube formation in microvascular endothelial cells, and suppressed migration and invasion in tumor cells. Fv-LDM-NGR interfered with the intracellular signaling transduction pathways, then caused G2/M and S phase arrest and induced apoptosis. CONCLUSION :Bispecific antibody-drug conjugate Fv-LDM-NGR could prevent cell invasion in tumor cells and tube formation in microvascular endothelial cells through blocking activity of CD13. And it could down-regulate the expression and the phosphorylation of EGFR, interfere with cellular signal pathways, induce cell cycle arrest and cell apoptosis, and inhibit cell proliferation and migration.
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Objective:To investigate effects of Lidamycin (LDM,C-1027) on the proliferation and immunogenic transform of human Caski cervical cancer cells and to provide the basic experiment data and theoretical supports for establishment of the new immu-notherapy method mediated by LDM. Methods:MTT was used for the analysis of cell proliferation;apoptosis rate was analyzed by flow cytometry;Western blot was used to analyze the effect of LDM on the expression of Bax and Bcl-2 in Caski cells;the Flow cytometry was used to detect the expression of Calreticulin ( CRT ) on the cell surface. Results: Lidamycin inhibited proliferation of Caski cells significantly in the time-and dose-dependent manners;The apoptotic cell ratio induced by 5 μg/L Lidamycin was 11. 5% ,Comparing with the control group, Lidamycin treatment increased Bax but decreased Bcl-2 contents significantly within Caski cells, it also significantly increased the expression of CRT on the cell surface of Caski cells from 2. 31% to 67. 2%. Conclusion: Lidamycin has pharmacological activity in inhibiting proliferation of the human cervical Caski cells and the underlying mechanism is related with inducing the intrinsic mitochondrial pathway of apoptosis. In the same time,Lidamycin can increase the expression of CRT on the cell surface,so it may have the ability to promote the immunogenic apoptosis of tumor cells.
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OBJECTIVE: To construct a novel fusion protein contained insulin-like growth factor 1 (IGF-1) and lidamycin (LDM) and evaluate its antitumor activity on non-small cell lung cancer (NSCLC). METHODS: DNA fragment coding for fusion protein (ldp-igf) was synthesized by linking apoprotein of lidamycin (ldp) with igf-1, and then was cloned into the plasmid pET30a. Fusion protein LDP-IGF was expressed in E.coli as inclusion bodies and was purified by Ni2+ affinity chromatography. Binding affinity of LDP-IGF to NSCLC cells was evaluated by immunofluorescence assay and flow cytometry-based binding assay. MTT assay was used to measure the in vitrocytotoxicity of LDP-IGF and its enediyne-energized analogue LDP-IGF-AE. PI staining assay and Annexin V-FITC/PI staining assay were used to analyze the cell cycle arrest and cell apoptosis after treatment with LDP-IGF-AE, respectively. RESULTS: Active soluble LDP-IGF protein was prepared by isolation, purification, denaturation and refolding, and the production of LDP-IGF was 12 mg per liter fermentation broth. Both of immunofluorescence assay and flow cytometry-based binding assay showed that LDP-IGF has strong binding activity to NSCLC cells. Enediyne-energized fusion protein LDP-IGF-AE exhibited potent cytotoxicity to NSCLC cells in vitro, and it is more potent than that of LDM. Furthermore, fusion protein LDP-IGF without active enediyne was also cytotoxic to A549 cells at high concentrations (50 and 100 μg·mL-1). LDP-IGF-AE could cause significant G2-M arrest in A549 and H460 cells, and it also induced the apoptosis in NSCLC cells in a concentration-dependent manner. CONCLUSION: Fusion protein LDP-IGF-AE shows potent antitumor efficacy in vitro on NSCLC, suggesting it could be a promising candidate for targeted therapy.
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Objective To investigate the difference of effect between interventional treatments and intravenous therapy of lidamycin on VX2 rabbit liver cancer.Methods VX2 Carcinoma cells were surgically implanted into the left liver lobe of 12 New Zealand white rabbits to establish the VX2 rabbit liver tumor model.Tumor size was detected by type-B ultrasonic diagnostic instrument.The rabbits were randomly divided into two groups of six,respectively treated with the hepatic inter-ventional administration of lidamycin (LDM)(1 ml,0.05 mg/kg)under the guidance of digital subtraction angiography (DSA)(group A)and with the auricular intravenous administration of LDMat the same dose (group B).All the rabbits were sacrificed and anatomized on day 10 after treatment,whose liver tumor was fixed with 4% paraformaldehyde solution and embedded in paraffin.Proliferating cell nuclear antigen (PCNA)and CD34 expression in the sample sections of tumor tissue were assessed through immunohistochemical staining.The levels of alanine transaminase (ALT)and aspartate trans-aminase(AST)were detected by Cobas 8000.Finally,the inhibition of VX2 tumor was evaluated.Results The VX2 tumor volumes were all increased at 10 day after LDMtreatment.However,the tumors in group A were smaller than those of group B (P <0.05).The results of immunohistochemistry showed that the intervention therapy of LDM could further lower the expression of CD34 and PCNA compared to group B.Conclusion Hepatic interventional administration of LDM under the guidance of DSA produces a better effect on attenuating the tumor growth than the intravenous administration of LDM.
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OBJECTIVE: To construct a VEGFR1 and VEGFR2 bi-targeting oligopeptide-based fusion protein A7/G6-LDP and investigate its anti-angiogenic activity and the mechanism of action. METHODS: PCR and overlap PCR were used to construct the fusion protein A7/G6-LDP expression vector that consists of the gene encoding G6, A7, LDP, and the linker peptide. The product was purified by affinity chromatography and analyzed by SDS-PAGE and HPLC. The binding activity to endothelial cells was examined by ELISA and immunocytochemical staining. The inhibition of HMEC-1 proliferation was determined with CCK-8 assays. The phosphorylation of AKT and c-Raf was detected by Western blotting. HMEC-1 migration was determined using a wound healing assay and tube formation was measured after incubation on Matrigel. RESULTS: The data of DNA sequence confirmed that the A7/G6-LDP fusion protein was correctly constructed. The fusion protein was recovered in high yield (up to 20 mg·L-1) and high purity after His-tag purification. A7/G6-LDP bound to HMEC-1 and HUVEC, respectively; in addition, it inhibited endothelial cell proliferation more effectively than LDP alone when used higher concentration. Moreover, A7/G6-LDP disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. The mechanistic study showed that A7/G6-LDP decreased the phosphorylation of AKT in HMEC. CONCLUSION: The engineered VEGFR1 and VEGFR2 bi-targeting oligopeptide-based fusion protein A7/G6-LDP effectively inhibits anti-angiogenesis. It might serve as a drug delivery carrier in targeted cancer therapy.
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OBJECTIVE: To investigate the antitumor efficacy of anti-gelatinases dFv-LDP and its enediyne-energized fusion protein dFv-LDP-AE on human fibrosarcoma HT-1080 cancer cells. METHODS: Western blot was used to analyze the expression level of gelatinases in different cancer cell lines. The inhibitory effects of fusion protein dFv-LDP and its enediyne-energized fusion protein dFv-LDP-AE on HT-1080 were determined by MTT assay. The binding capability of fusion protein dFv-LDP with HT-1080 was detected by ELISA and immunofluorescence. FACS was used to analyze the cell cycle arrest by dFv-LDP, dFv-LDP-AE or their combination on HT-1080 cells. The anti-metastasis effects of dFv-LDP, dFv-LDP-AE or their combination on the experimental lung metastasis model established by HT-1080Luc via tail vein injection were also evaluated in this study. RESULTS: Expression level of gelatinases was higher in HT-1080 cells as compared to that of other cancer cell lines. The fusion protein dFv-LDP showed well binding capability with HT-1080 cells as determined by ELISA and immunofluorescence. The enediyne-energized fusion protein dFv-LDP-AE displayed extremely inhibitory effect on proliferation of HT-1080. Results of FACS indicated that the combination of dFv-LDP with dFv-LDP-AE could not further increase the G2/M proportion on cell cycle arrest. However, in vivo experiment as examined using the experimental lung metastasis model established via HT-1080Luc tail veil injection, the metastasis foci in group of fusion protein dFv-LDP (10 mg·g-1) was 55.8% compared to that of control group (P<0.01). The metastasis foci in group of dFv-LDP-AE at dosage of 0.4 and 0.6 mg·g-1 were 41.4% and 25.1% respectively compared to that of dFv-LDP 10 mg·g-1 group (P<0.01). The combination of dFv-LDP (10 mg·g-1) with dFv-LDP-AE (0.4 or 0.6 mg·g-1) showed an additive decrease of metastasis foci number in the lung of athymic mice, which were 20.3% (P<0.05, compared with dFv-LDP-AE at 0.4 mg·g-1) and 13.1% (P<0.05, compared with dFv-LDP-AE at 0.6 mg·g-1) respectively. CONCLUSION: The combination of dFv-LDP with its enediyne-energized fusion protein dFv-LDP-AE would intensify the anti-metastasis effect on experimental lung metastasis model as established via tail vein injection of HT-1080Luc cells.
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OBJECTIVE: To construct a novel fusion protein consisting of oligopeptides specific for human epidermal growth factor receptor 2(HER2) and lidamycin(LDM), and investigate its antitumor activity. METHODS: Coding sequences of oligopeptides from complementarity determining region 3(CDR3) of anti-HER2 antibody C6.5 heavy chain was fused to apoprotein of lidamycin to obtain the fusion gene ldp-Hr. Fusion protein LDP-Hr was expressed in E. coli and purified by affinity chromatography. The purity of LDP-Hr was analyzed by high HPLC. Immunofluorescence assay and flow cytometry-based binding assay were used to investigate the binding activity of LDP-Hr to HER2 overexpressed cancer cells. The energized fusion protein LDP-Hr-AE was prepared by integrating the active enediyne chromophore (AE) of lidamycin into the LDP-Hr protein. MTT assay was used to measure the in vitro cytotoxicity of LDP-Hr-AE and Annexin V-FITC/PI staining assay was used to analyze its apoptosis-inducing efficacy. RESULTS: Fusion protein LDP-Hr was constructed correctly and expressed in E. coli in a secretory manner. The production of LDP-Hr was 40 mg per liter fermentation broth, and the purity of fusion protein was 97.4% as analyzed by HPLC. LDP-Hr showed strong binding activity to cancer cells highly expressing HER2, such as SK-BR-3 and SK-OV-3 cells. The energized fusion protein LDP-Hr-AE exhibited more potent cytotoxicity to SK-BR-3 and SK-OV-3 cells than LDM as measured by MTT assay. The results from Annexin V-FITC/PI staining assay also revealed that LDP-Hr-AE significantly induced cell apoptosis even at very low concentrations. CONCLUSION: The novel energized fusion protein LDP-Hr-AE bounds to HER2 specifically, and shows potent cytotoxicity and apoptosis-inducing activity to cancer cells, which suggests that it would be a promising candidate for targeted cancer therapy.
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Aim To study the mechanism of inhibition of basic fibroblast growth factor (bFGF) related signal transduction by lidamycin in cancer cells. Methods 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. Results 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 1 000-fold more potent than that of ADR. LDM blocked the specific by anti-FGFR specific antibody, LDM inhibited the formation of bFGF-receptor immune complex. bFGF demonstrated that LDM inhibited the activity of PKC isoenzymes in cancer cells stimulated with bFGF.Conclusion The blocking of bFGF receptors in the signal transduction pathway may be involved in the effect of LDM on cancer cells.