Melittin induces human gastric cancer cell apoptosis via activation of mitochondrial pathway.

AIM: To investigate the apoptotic effects of melittin on SGC-7901 cells via activation of the mitochondrial signaling pathway in vitro. METHODS: SGC-7901 cells were stimulated by melittin, and its effect on proliferation and apoptosis of was investigated by methyl thiazolyl tetrazolium assay, morphologic structure with transmission electron microscopy, annexin-V/propidium iodide double-staining assay, measuring mitochondrial membrane potential (MMP) levels, and analyzing reactive oxygen species (ROS) concentrations were analyzed by flow cytometry. Cytochrome C (Cyt C), apoptosis-inducing factor (AIF), endonuclease G (Endo G), second mitochondria-derived activator of caspases (Smac)/direct IAP binding protein with low isoelectric point (Diablo), and FAS were analyzed by western blot. The expression of caspase-3 and caspase-8 was measured using activity assay kits. RESULTS: Melittin was incubated at 1.0, 2.0, 4.0, or 6.0 µg/mL for 1, 2, 4, 6, or 8 h and showed a time- and concentration-dependent inhibition of SGC-7901 cell growth. Melittin induced SGC-7901 cell apoptosis, which was confirmed by typical morphological changes. Treatment with 4 µg/mL melittin induced early apoptosis of SGC-7901 cells, and the early apoptosis rates were 39.97% ± 3.19%, 59.27% ± 3.94%, and 71.50% ± 2.87% vs 32.63% ± 2.75% for 1, 2, and 4 h vs 0 h (n = 3, P < 0.05); the ROS levels were 616.53% ± 79.78%, 974.81% ± 102.40%, and 1330.94% ± 93.09% vs 603.74% ± 71.99% (n = 3, P < 0.05); the MMP values were 2.07 ± 0.05, 1.78 ± 0.29, and 1.16 ± 0.25 vs 2.55 ± 0.42 (n = 3, P < 0.05); caspase-3 activity was significantly higher compared to the control (5492.3 ± 321.1, 6562.0 ± 381.3, and 8695.7 ± 449.1 vs 2330.0 ± 121.9), but the caspase activity of the non-tumor cell line L-O2 was not different from that of the control. With the addition of the caspase-3 inhibitor (Ac-DEVD-CHO), caspase-3 activity was significantly decreased compared to the control group (1067.0 ± 132.5 U/g vs 8695.7 ± 449.1 U/g). The expression of the Cyt C, Endo G, and AIF proteins in SGC-7901 cells was significantly higher than those in the control (P < 0.05), while the expression of the Smac/Diablo protein was significantly lower than the control group after melittin exposure (P < 0.01). Ac-DEVD-CHO did not, however, have any effect on the expression of caspase-8 and FAS in the SGC-7901 cells. CONCLUSION: Melittin can induce apoptosis of human gastric cancer (GC) cells through the mitochondria pathways, and it may be a potent agent in the treatment of human GC.

[Monoclonal antibody preparation and identification of vascular endothelial growth factor 165 expressed in vitro].

AIM: To prepare a monoclonal antibody against human vascular endothelial growth factor (VEGF165), for further study the VEGF165 in the tumorigenesis, tumor cell migration and the tumor cells escape from the immune response. METHODS: VEGF165 gene was cloned from the human umbilical vein endothelial cells (HUVEC) by RT-PCR, and then cloned into the pGEX-6P1, constructed the prokaryotic expression of pGEX-6P1-VEGF165. The fusion -protein of VEGF165 was expressed in E.coli (BL21) induced by the 1.0 mmol/L IPTG at 37DegreesCelsius after 4 h. The fusion-protein was purified by the MicroSpin GST purification kit for immunized the BALB/c mouse. The monoclonal antibodies (mAbs) against the VEGF165 were prepared by hybridoma technique, and ELISA and Western blot identified their immunoglobulin subclass and specificity. And we used the inhibition the embryo angiogenesis assay, inhibition the HUVEC migration assay and inhibition the HUVEC tubule information assay to study the bioactivity of the mAbs of VEGF165. RESULTS: The sequence of the VEGF165 is agreed to the GenBank, and we obtained five species VEGF165 mAbs, and the titer of the antibody is high, and we named, they are 5A6, 3F5, 6H3, 7D10 and 7A10. Our study showed that the 5A6, 3F5, 6H3, 7D10 were classified to IgG2a, 7A10 was classified to IgG2b, and the light chain is k.Meanwhile the purified mAbs inhibited formation of chicken embryo blood vessels, and inhibited tubule formation of the HUVEC and inhibited migration of the HUVEC. CONCLUSION: mAbs against human VEGF165 have the effective bioactivity, which would play a significant role for further study the mechanism of VEGF165.