Laminarin induces apoptosis of human colon cancer LOVO cells through a mitochondrial pathway

Abstract

Many scientific studies have shown that laminarin has anti-tumor effects, but the anti-tumor mechanism was unclear. The purpose of this study was to investigate the effect of laminarin on the induction of apoptosis in human colon cancer LOVO cells and the molecular mechanism involved. LOVO cells were treated with different concentrations of laminarin at different times. Morphology observations were performed to determine the effects of laminarin on apoptosis of LOVO cells. Flow cytometry (FCM) was used to detect the level of intracellular reactive oxygen species (ROS) and pH. Laser scanning confocal microscope (LSCM) was used to analyze intracellular calcium ion concentration, mitochondrion permeability transition pore (MPTP) and mitochondrial membrane potential (MMP). Western blotd were performed to analyze the expressions of Cyt-C, Caspase-9 and -3. The results showed the apoptosis morphology, which showed cell protuberance, concentrated cytoplasm and apoptotic bodies, was obvious after 72 h treatment. Laminarin treatment for 24 h increased the intracellular level of ROS and Ca²⁺; decreased pH value; activated intracellular MPTP and decreased MMP in dose-dependent manners. It also induced the release of Cyt-C and the activation of Caspase-9 and -3. In conclusion, laminarin induces LOVO cell apoptosis through a mitochondrial pathway, suggesting that it could be a potent agent for cancer prevention and treatment.

Figure 1

Structural formula of laminarin.

Figure 2

Effect of laminarin on LOVO cells morphology. (A) Cells were treated as control group; (B) Cells were treated with 400 µg·mL⁻¹ laminarin; (C) Cells were treated with 800 µg·mL⁻¹ laminarin; (D) Cells were treated with 1,600 µg·mL⁻¹ laminarin; (E) Cells were treated with 5 µg·mL⁻¹ HCPT.

Figure 3

Effect of laminarin on ROS of LOVO cells by FCM analysis. (A) Cells were treated as control group; (B) Cells were treated with 400 µg·mL⁻¹ laminarin; (C) Cells were treated with 800 µg·mL⁻¹ laminarin; (D) Cells were treated with 1,600 µg·mL⁻¹ laminarin; (E) Cells were treated with 5 µg·mL⁻¹ HCPT.

Figure 4

Effects of laminarin on intracellular fluorescent intensity of [Ca²⁺] in LOVO. (A) Cells were treated as control group; (B) Cells were treated with 400 µg·mL⁻¹ laminarin; (C) Cells were treated with 800 µg·mL⁻¹ laminarin; (D) Cells were treated with 1,600 µg·mL⁻¹ laminarin; (E) Cells were treated with 5 µg·mL⁻¹ HCPT.

Figure 5

Effect of laminarin on pH of LOVO cells by FCM analysis. (A) Cells were treated as control group; (B) Cells were treated with 400 µg·mL⁻¹ laminarin; (C) Cells were treated with 800 µg·mL⁻¹ laminarin; (D) Cells were treated with 1,600 µg·mL⁻¹ laminarin; (E) Cells were treated with 5 µg·mL⁻¹ HCPT.

Figure 6

Effects of laminarin on intracellular fluorescent intensity of MPTP in LOVO. (A) Cells were treated as control group; (B) Cells were treated with 400 µg·mL⁻¹ laminarin; (C) Cells were treated with 800 µg·mL⁻¹ laminarin; (D) Cells were treated with 1,600 µg·mL⁻¹ laminarin; (E) Cells were treated with 5 µg·mL⁻¹ HCPT.

Figure 6

Effects of laminarin on intracellular fluorescent intensity of MPTP in LOVO. (A) Cells were treated as control group; (B) Cells were treated with 400 µg·mL⁻¹ laminarin; (C) Cells were treated with 800 µg·mL⁻¹ laminarin; (D) Cells were treated with 1,600 µg·mL⁻¹ laminarin; (E) Cells were treated with 5 µg·mL⁻¹ HCPT.

Figure 7

Effects of laminarin on intracellular fluorescent intensity of ΔΨm in LOVO. (A) Cells were treated as control group; (B) Cells were treated with 400 µg·mL⁻¹ laminarin; (C) Cells were treated with 800 µg·mL⁻¹ laminarin; (D) Cells were treated with 1,600 µg·mL⁻¹ laminarin; (E) Cells were treated with 5 µg·mL⁻¹ HCPT.

Figure 8

Effect of laminarin on Cyt-C, Caspase-9 and Caspase-3 expression in LOVO. (A) Cells were treated as control group; (B) Cells were treated with 400 µg·mL⁻¹ laminarin; (C) Cells were treated with 800 µg·mL⁻¹ laminarin; (D) Cells were treated with 1,600 µg·mL⁻¹ laminarin; (E) Cells were treated with 5 µg·mL⁻¹ HCPT. * p < 0.05, ** p < 0.01 vs. control group.