ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Animals , Female , Humans , Mice , Aminoglycosides , Antibiotics, Antineoplastic , Antimetabolites, Antineoplastic , Antineoplastic Combined Chemotherapy Protocols , Therapeutic Uses , Cell Line, Tumor , Drug Synergism , Enediynes , Fibrosarcoma , Pathology , Luminescent Measurements , Lung , Pathology , Lung Neoplasms , Drug Therapy , Pathology , Methotrexate , Mice, Inbred BALB C , Mice, Nude , Random Allocation , Transfection , Xenograft Model Antitumor AssaysABSTRACT
<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>
Subject(s)
Humans , Aminoglycosides , Chemistry , Pharmacology , Antibiotics, Antineoplastic , Chemistry , Pharmacology , Apoproteins , Chemistry , Cell Line, Tumor , Cell Survival , Chromatography, High Pressure Liquid , Drug Design , Enediynes , Chemistry , Pharmacology , Recombinant Fusion Proteins , Chemistry , Single-Chain Antibodies , ChemistryABSTRACT
The recombinant fusion protein staphylokinase-hirudin(rSFH) was purified from the high density-fermented engineered E.coli by means of ion-exchange chromatography (IEC) and gel filtration (GF). The purity of rSFH reached to more than 98% determined by RP-HPLC and SDS-PAGE, and the yield was up to 0.7g per liter of fermentation broth. The analysis of homologous dimmer of rSFH appeared during the purification and calculation of the surface hydrophobic area had been carried out by means of hydrophobic chromatography and MALD-TOF. The influence of sodium chloride and temperature on the behavior of rSFH reversible dimerization was analyzed by high performance sized- exclusive chromatography(HPSEC). It is concluded that the hydrophobic interaction played an important role in the reversible dimerization of rSFH.