| Objective: The purpose of the experiment is to examine whether melittin, one component isolated from bee venom, can inhibit hepatocarcinoma growth, proliferation and metastasis, to study its possible mechanisms, and to evaluate whether melittin can be an effective drug for clinical use in treating patients with hepatocarcinoma. Methods: H22 cells were cultured in the RPMI 1640 medium (Gibco, Glasgow, UK) supplemented with 10% new bovine serum. MTT reduction assay was conducted as follows:0.2ml of cell suspension containing 5×104 cells was added to each well of 96-well microplate and treated with melittin (2~128μg/ml) of different concentration,after being cultured 24h and 48h at 37℃, MTT solution (5mg/ml) 25μl/well was added, 4h later, the cells were collected by centrifugation at 120g for 5 min. The supernatant was discarded. The cells were dissolved in dimethyl sulfoxide(150μl/well) for 10 min, then were measured at miroplate reader with 490nm. The volume of PCNA expressed in H22 cells which were treated with melittin (2μg/ml, 6μg/ml ) for 24 and 48h, were analyzed with flow cytometer after being stained with immunocytochemistric methods.H22 cells mixed with N.S, were injected into axilla subcutaneous part with 0.2ml (2×106 cells/ml) each mouse. 24 hours later, 120 mice (male, 20±2 g) were divided into five groups randomly for experiments: negative control group (N.S), positive control group (cytoxan) and melittin-treated groups (4.5mg/kg/d, 3mg/kg/d, 1.5mg/kg/d). The mice were administrated by injection through caudal vein at the same time everyday. Five days later, the administration paused for two days, then the adminstration was undertaken in another five days. 12 mice of each group were killed to measure the neoplasm weight and volume after the administration stopped. The others were cultured to observe the survival period.Another 48 mice(male, 20±2 g) were injected through caudal vein with 0.2ml H22 cells(1×106/ml) to make the experimental lung metastasis models. 24 hours later, the mice were divided into normal control group and melittin-treated group (1.5mg/kg/d). The mice were administrated by injection through caudal vein at the same time everyday. Five days later, the administration paused fortwo days, then the administration was undertaken another five days. 12 mice of each group were killed to observe the metastasis tumor. The others were cultured to observe the survival period.Intercelluar adhesion molecule-1 (ICAM-1) were stained by monoclonal antibody immunofluorescence and determined with flow cytometry. Expression of ICAM-1mRNA were assayed by reverse transcription-polymerase chain reaction (RT-PCR) and analyzed by VDS image analyzing system. Results:1. Effect of melittin on growth of H22 cells and survival period of mice bearing H22 solid tumorThe cell growth was significantly inhibited by melittin (2-128μg/ml) of different concentration after being treated for 24h and 48h (p<0.01). There was no significant difference in inhibition of the different concentration melittin to H22 cells between 24h and 48h in statistics respectively (p>0.05). As is evident from this figure, the cell killing by melittin was dose-dependent (p<0.01). After treatment by melittin (2μg/ml, 6μg/ml for 24 or 48hours), the fluorescence density of PCNA decreased significantly compared with the control(p<0.01).Melittin showed significant and dose-dependent inhibition. The high-dosage had greater effect on H22 solid tumor (p<0.01). The cytoxan-treated and melittin-treated groups (4.5mg/kg/d, 3mg/kg/d) had lower mice neoplasm average weight than that of low-dosage melittin group (1.5mg/kg/d) and the control group (p<0.01). There was no significant difference between low-dosage melittin group and the negtive control group (p>0.05) in tumor weight. The survival period of the drug-treated mice were longer than that of the negtive control group (P<0.01), and there was no significant difference between cytoxan-treated group and 4.5mg/kg/d melittin-treated group (p>0.05).2. Eff...
|
PDF Full Text Request