Key words melittin

   Melittin is the main component of bee venom, has a variety of biological activity, recent studies reveal its anti-tumor effect, aroused widespread concern, studies have shown that melittin BEL  7402, SMMC  7721, Hep  3B [1,2], T lymphoblastic leukemia 6T  CEM [3] and other tumor cells proliferation inhibition and apoptosis induced by melittin to induce tumor cell apoptosis mechanism is not very clear. This study was melittin on human osteosarcoma U2OS cell growth inhibition of proliferation and induction of apoptosis of mitochondrial membrane potential and apoptosis-related protein Bcl  2, Bax expression, to investigate the mechanism of the induction.

    1 Materials and methods

    1.1 Materials

    1.1.1 Chemical reagents and melittin MEM medium, fetal bovine serum purchased from Gibco, USA, trypsin and Rodamine  123 were purchased from Sigma (USA), mouse anti-human Bcl  2 monoclonal antibody, Bax and mouse anti-human polyclonal antibody purchased from Santa Cruz. Melittin from Changhai Hospital of Traditional Chinese Medicine, Laboratory of extraction, purification, 1mg/ml configured with saline -20 ℃ cryopreservation spare.

    1.1.2 cell lines and cell culture U2OS cells lines were purchased from the Shanghai Institute of Cell Institute, complete medium containing 10% fetal bovine serum in DMEM, 37 ° C, the volume fraction of 5% CO2 for fully saturated humidity conditions under conventional culture, the cells were digested with 0.25% trypsin and 0.02% EDTA 1:1 mixture of digestive juices, 2 ~ 3d subcultured take experiment logarithmic phase cells.

    1.2 Experimental Methods

    1.2.1 MTT assay and cell proliferation inhibition rate and median effective inhibitory concentration (IC50) U2OS cells in logarithmic growth phase, a density of 1 × 104 / well were seeded in 96-well plates, conventional culture culture system of 100 μl. 24h adherent cells after culture, the control group and the experimental group, each group of six wells in the experimental group were added to a final concentration of 1, 2, 4, 8, 16 and 32μg/ml melittin were 12, 24 , 36h. 4h before the end of the experiment, each hole by adding MTT 10μl (concentration of 5mg/ml), 96-well plates, and the supernatant was removed out of the termination of the experiment, filter paper floating liquid, each hole plus DMSO 150μl fully dissolved, the absorbance was measured on a markup A value measured wavelength of 492nm, and the growth inhibition rate was calculated as follows:

    Inhibition ratio = (control group A value – A value of experimental group) / control group A value × 100%

    Application of drugs that inhibit the concentration calculation software (Loggt method) to calculate the IC50 values.

    1.2.2 Transmission electron microscopy morphological observation after 12h the cells were collected, before fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, conventional electron microscopy taxidermy, H  600 transmission electron microscope observation cell ultrastructure.

    1.2.3 flow cytometry apoptosis rate after 12h, cells were collected and try hanging the Binding the Buffer (binding buffer), plus fluorescently labeled Annexin V and PI, dark at room temperature and incubated 15min, flow cytometry The rate of apoptosis.

    1.2.4 Flow cytometry determination of changes in mitochondrial membrane potential (Δψm) the 12h after digestion, the cells were collected. Washed twice with PBS, cells were resuspended in 0.5ml PBS added to Rh123 to give a final concentration of 10mg/ml. 37 ℃ for 30min, PBS wash once on flow cytometry.

    1.2.5 immunocytochemistry assay Bcl  2 and Bax protein expression in logarithmic growth phase U2OS cells, 2 × 105 / seeded pre-set to have a sterile coverslip tin plates and incubated overnight The latter were added to a final concentration of 2,4,8 μg / ml of melittin, another blank control group without drug treatment, cultured for 12h, remove the coverslip, Bcl  2 and Bax immunohistochemistry kit Operating Instructions staining, fixed. Bcl  2 and Bax signals are brownish yellow, located in the cytoplasm. The determination method for the light microscope brownish yellow cytoplasm of positive cells. IMS the computer cell image analysis system to the strength of positive expression quantitative analysis, light microscope (× 250) randomly selected three horizons, the percentage of positive area (Positive area rate), and the mean optical density value (Average by default on sampling procedures OD Value), the product of both immunocytochemistry positive index (Positive Index, PI).

    1.3 statistical methods

    The application SPSS 11.0 experimental data analysis, data results are used ± s, among groups ahead homogeneity of variance test, and homogeneity of variance single-factor analysis of variance, pairwise comparisons using Newman  Keuls test (q test); variance The arrhythmia those into a group of rank-sum test (Kruskal  Wallis method), the pairwise comparison law Nemenyi used.

    2 Results

    2.1 Melittin of U2OS cells growth inhibition

    Loggt method measured IC50 value 7.12μg/ml U2OS cells proliferation inhibition in the concentration range of 2 ~ 8μg/ml has a significant dose-and time-dependent manner, as shown in Figure 1.

    Figure 1 melittin U2OS cell growth inhibition

    Fig 1 The inhibitive effect of melittin on U2OS cell line

    2.2 Transmission electron microscopy morphology

    The control group osteosarcoma cells evenly distributed chromatin, the nucleus is irregular polygonal containing lipid droplets in the cytoplasm of individual cells. The melittin 2μg/ml treated cytoplasmic loose membrane looming, chromatin, typical apoptotic Early ultrastructural changes. Group 4μg/ml cytoplasm scattered chromatin condensation, apoptotic bodies, 8μg/ml apoptotic bodies, as shown in Figure 2.

    2.3 cell apoptosis were detected by flow cytometry

    Melittin dose group apoptosis rate compared with the control group were increased, the difference was statistically significant (P <0.01), and as the dose increased apoptosis rate increased (P <0.01). Table 1. Table 1 Annexin V / PI double staining melittin-induced U2OS cell apoptosis rate different concentration of melittin U2OS cells, showing the average fluorescence intensity of each concentration group gradually reduced to 4,8 μg / ml the group with 0μg/ml group phase the difference was statistically significant (P <0.05), are shown in Table 2. Table the Melittin around the average fluorescence intensity change

    0.05),Bax蛋白表达在蜂毒素浓度为4、8μg/ml时显著">2,4,8 μg / ml Melittin U2OS cells after 12 hours, Bcl  2 protein expression 0μg/ml group no significant change (P> 0.05), Bax protein expression melittin concentration of 4,8 μg / ml significantly enhanced, compared with 0μg/ml the group and 2μg/ml group, the differences were statistically significant (P <0.05), as shown in Table 3.

    3 Discussion

    Bee bee venom toxin is the main active ingredient, has anti-bacterial, anti-viral, anti-inflammatory, anti-arthritic, anti-tumor, and other aspects of the role. With the the bee venom separation, extraction and purification technology continues to improve, the study of the function and mechanism of action of melittin increasingly widespread. About bee venom anti-tumor mechanism research in recent years has made new progress, studies have found that melittin antitumor biological activity by inducing tumor cell apoptosis, it can be formed by inserting the cell membrane pore caused Ca2 + influx intracellular Ca2 + concentration can cause cell lysis, leading to apoptosis. Hwang et al [4] studies have shown that bee venom can induce the human osteosarcoma cell line MG  63 cell apoptosis and inhibit COX  2 protein expression. Since then, Ahn et al [5] reported that melittin-induced apoptosis in lung cancer cells with increased Bax protein expression and activation of cysteine ​​proteases. But generally speaking, the current specific mechanism of tumor cell apoptosis induced by melittin is not yet clear.

    The mitochondrial connection with a number of apoptotic pathways, plays an important role in apoptosis. The loss of the mitochondrial membrane potential is one of the salient features of the mitochondria is activated. Decline in mitochondrial transmembrane potential is considered to be the earliest events that occur in the process of apoptosis cascade, mitochondrial membrane potential collapse then apoptosis will be irreversible [6]. Bcl  2 family proteins and genes in the mitochondria involved in apoptosis pathway plays an important regulatory role in the regulation of cell signaling. Bcl  protein 2 family members are divided into anti-apoptotic and pro-apoptotic two categories, including the anti-apoptotic factor Bcl  2 and Bcl  x; composition of the pro-apoptotic members Bax, Bad, Bak and Bid. Apoptosis occurs and the to prevent relative proportions depending on the family members and reflect dimerize state. Expression of Bcl  2 homodimer blocking apoptosis, the Bax homodimer produce the opposite effect, Bcl  2 and Bax heterodimer is not active. The relative proportions of the two proteins in the cell determines the nature of the reaction in the mitochondrial apoptotic pathway, Bax / Bcl  2 ratio changes affect mitochondrial membrane permeability ratio increased mitochondrial membrane permeability increase, causing the cytochrome C release, activation of caspase  9, the last activation of caspase  3, and apoptosis.

    Our test results show U2OS cells, the mitochondrial membrane potential in the treatment of 4,8 μg / ml melittin significantly decreased, indicating that melittin can affect mitochondrial function and affect cell physiological activity of melittin may U2OS cells mitochondrial membrane potential depolarization-mediated activation of the mitochondrial apoptosis pathway.

    0.05),Bax蛋白表达在蜂毒素浓度为4、8μg/ml时较对照组及2μg/ml组显著">Different concentrations of melittin role U2OS cells 12 hours after Bcl  protein expression was no significant change (P> 0.05) and Bax protein expression in a concentration of melittin 4,8 μg / ml compared with the control group, and 2μg/ml group significantly enhanced, the difference was statistically significant (P <0.05), and induced Bax protein expression increased in a dose dependency. Huang Xueqiang [7], experimental studies have shown that melittin-induced T cell leukemia cell apoptosis and bcl  2 gene expression decreased significantly related. Also studied abroad [8] bee venom proliferation and induced apoptosis in leukemia U937 cells in vitro inhibition, apoptosis-inducing effect may be related to the expression of Bcl-2 downregulation and caspase  3 activation is closely related to. The results of this study are not the same, considering the possibility of the biological characteristics of the differences between the different cell lines, different levels of gene expression, cause the reaction to external stimuli. Our results demonstrate that melittin increased Bax / Bcl  2 ratio in to infer its subsequent effects may promote Bax  Bax homodimer formation, which is a large number located in the mitochondrial membrane and induce mitochondrial membrane potential conversion opening and mitochondrial membrane potential dissipation, induced U2OS apoptosis.

    The process of apoptosis, however, there is a complex regulatory network, melittin-induced apoptosis of osteosarcoma cells still needs further in-depth study by the regulation of other genes as well as affect the signal transduction pathway.