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, 22 (11), 3186-95

Melittin Induces Human Gastric Cancer Cell Apoptosis via Activation of Mitochondrial Pathway

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Melittin Induces Human Gastric Cancer Cell Apoptosis via Activation of Mitochondrial Pathway

Gui-Mei Kong et al. World J Gastroenterol.

Abstract

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.

Keywords: Apoptosis; Cytochrome C; Gastric cancer; Melittin; Mitochondrial.

Figures

Figure 1
Figure 1
Melittin alters the release of mitochondria proteins in SGC-7901 cells. A: Growth inhibition of a human gastric cancer cell line (SGC-7901) by melittin. Numbers 1-6 indicates the melittin concentration used (1-6 μg/mL) to inhibit the growth of the gastric cancer cells after 1, 2, 4, 6, or 8 h; B: The expression of Cytochrome C, apoptosis-inducing factor (AIF), endonuclease G (EndoG), second mitochondria-derived activator of caspases (Smac)/direct IAP binding protein with low isoelectric point, pI (Diablo) detected by western blot. (aP < 0.05 vs the control group; bP < 0.01 vs the control group); C: Ultra microstructure of mitochondria after incubation with 4 μg/mL melittin [a: SGC-7901 control group; b: SGC-7901 cells incubated with 4 μg/mL melittin for 4 h; (scale bar 0.5 μm, × 6600)]. The arrow is the mitochondria.
Figure 2
Figure 2
Melittin alters the mitochondrial membrane potential levels and caspase-3 expression of SGC-7901 cells. A: Mitochondrial membrane potential (MMP) (Δψm) levels of SGC-7901 cells after incubation with 4 μg/mL melittin for various times. [a: SGC-7901 cells control group; b: SGC-7901 cells incubated with 4 μg/mL melittin for 1 h; c: SGC-7901 cells incubated with 4 μg/mL melittin for 2 h; d: SGC-7901 cells incubated with 4 μg/mL melittin for 4 h (aP < 0.05 vs the control group); B: The effect of melittin on the release of caspase-3. L-O2 normal cells incubated with 4 μg/mL melittin for 4 h; SGC-7901 control cell; SGC-7901 cells incubated with 4 μg/mL melittin for 4 h, 2 h, and 1 h; (cP < 0.05 vs the control group); C: The release of caspase-3 in SGC-7901 cells after the addition of a caspase-3 blocker. SGC-7901 control group; SGC-7901 cells incubated with 4 μg/mL melittin for 4 h; SGC-7901 cells with caspase 3 blocker; SGC-7901 cells incubated with 4 μg/mL melittin for 4 h added with caspase-3 blocker; (eP < 0.05 vs the control group).
Figure 3
Figure 3
Melittin induced the apoptosis of SGC-7901 cells and increased the levels of reactive oxygen species. A: Melittin (4 μg/mL) induced the early apoptosis of SGC-7901 cells at different time points [a: SGC-7901 cells negative control group b: SGC-7901 cells incubated with 4 μg/mL melittin for 1 h; c: 4 SGC-7901 cells incubated with 4 μg/mL melittin for 2 h; d: SGC-7901 cells incubated with 4 μg/mL melittin for 4 h; (aP < 0.05 vs the control group)]; B: The ROS levels in SGC-7901 cells after incubation with 4 μg/mL melittin. NC: SGC-7901 cells negative control group; PC: Positive control group; 1 h: SGC-7901 cells incubated with 4 μg/mL melittin for 1 h; 2 h: SGC-7901 cells incubated with 4 μg/mL melittin for 2 h; 4 h: SGC-7901 cells incubated with 4 μg/mL melittin for 4 h (cP < 0.05 vs the control group).

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