Tetramethylpyrazine induces reactive oxygen species-based mitochondria-mediated apoptosis in colon cancer cells

Figure 1 N-acetyl-L-cysteine and Z-VAD-FMK inhibit tetramethylpyrazine-induced cell apoptosis. A: Cell morphology of SW480 and HCT116 cells treated with tetramethylpyrazine (TMP) with or without Z-VAD-FMK and N-acetyl-L-cysteine (NAC) observed by inverted light microscopy; B: Percentage of apoptotic cells in SW480 and HCT116 cells treated with TMP with or without Z-VAD-FMK and NAC determined by flow cytometry; C: Cell survival rate of SW480 and HCT116 cells treated with TMP with or without Z-VAD-FMK and NAC. ^aP < 0.05. TMP: Tetramethylpyrazine; NAC: N-acetyl-L-cysteine.

Figure 2 N-acetyl-L-cysteine and Z-VAD-FMK inhibit tetramethylpyrazine-induced accumulation of intracellular reactive oxygen species. A: Intracellular reactive oxygen species (ROS) of SW480 and HCT116 cells treated with tetramethylpyrazine with or without Z-VAD-FMK and N-acetyl-L-cysteine detected by flow cytometry; B: Bar chart of intracellular ROS level in four groups of SW480 and HCT116 cells treated as indicated. ^aP < 0.001. TMP: Tetramethylpyrazine; NAC: N-acetyl-L-cysteine.

Figure 3 Caspases 3 and 9 cleavage induced by tetramethylpyrazine is inhibited by N-acetyl-L-cysteine and Z-VAD-FMK. Western blot analysis showed increased cleaved caspase 3 and caspase 9 expression in SW480 and HCT116 cells treated with tetramethylpyrazine with or without Z-VAD-FMK and N-acetyl-L-cysteine. TMP: Tetramethylpyrazine; NAC: N-acetyl-L-cysteine.

Figure 4 Tetramethylpyrazine inhibits tumor growth in a xenograft mouse model. A: Tumor volumes were calculated in mice treated with different concentrations of tetramethylpyrazine (TMP; 0, 50, and 100 mg/kg body weight) for 30 days (n = 5 mice/group); B: Tumor weight was calculated in mice treated with different concentrations of TMP (0, 50, and 100 mg/kg body weight) for 30 days (n = 5 mice/group). ^aP < 0.05, ^bP < 0.01. TMP: Tetramethylpyrazine.

Figure 5 Tetramethylpyrazine increases reactive oxygen species accumulation and induces apoptosis in xenograft tumors. A: Malondialdehyde content in xenograft tumors increased with increasing tetramethylpyrazine (TMP); B: TMP treatment resulted in enhanced caspases 3 and 9 activities in xenograft tumors. ^aP < 0.01. TMP: Tetramethylpyrazine.