Mechanisms involved in Korean mistletoe lectin-induced apoptosis of cancer cells

Abstract

Aim: To investigate the anti-cancer mechanisms of Korean mistletoe lectin (Viscum album coloratum agglutinin, VCA) using a human colon cancer cell line (COLO).

Methods: Cytotoxic effects of VCA on COLO cells were determined by 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay in vitro and tumor-killing effects in vivo. To study the mechanisms involved, the expression of various pro-caspases, anti-apoptotic proteins, and death receptors was determined by western blot. To determine which death receptor is involved in VCA-induced apoptosis of COLO cells, cytotoxicity was examined by MTT assay after treatment with agonists or antagonists of death receptors.

Results: VCA killed COLO cells in a time- and dose-dependent manner and induced complete regression of tumors in nude mice transplanted with COLO cells. Treatment of COLO cells with VCA activated caspase-2, -3, -8, and -9 and decreased expression of anti-apoptotic molecules including receptor interacting protein, nuclear factor-kappaB, X-linked inhibitor of apoptosis protein, and Akt/protein kinase B. We then examined the involvement of death receptors in VCA-induced apoptosis. Only tumor necrosis factor receptor 1, among the death receptors examined, was involved in apoptosis of COLO cells, evidenced by inhibition of VCA-induced apoptosis and decreased activation of caspases, particularly caspase-8, by tumor necrosis factor receptor 1 antagonizing antibody.

Conclusion: VCA-induced apoptotic COLO cell death is due to the activation of caspases and inhibition of anti-apoptotic proteins, in part through the tumor necrosis factor receptor 1 signaling pathway.

Figure 1

Cytotoxic effects of VCA on COLO cells in vitro. A: Cells were treated with VCA for 24 h and cell viability was determined by MTT assay. Values are mean ± SE; n = 4/group; B: COLO cells were incubated with the indicated amounts of VCA for various times and cell viability was determined by MTT assay. Values are mean ± SE, n = 5/group. ^aP < 0.05 vs control group; C: COLO cells were treated with or without VCA (100 ng/mL) for 9 h. Cells were harvested, fixed in ethanol, and stained with propidium iodide in the presence of RNase A. The stained cells were analyzed by flow cytometry.

Figure 2

Tumor-killing effect of VCA in vivo. COLO cells (1 × 10⁷ cells/mouse) were injected into the neck of CD1 nude mice. Five weeks later, VCA was injected around the developed tumors every 2 d for 5 wk. (A) The tumors were measured at 0, 1, 3 and 5 wk after VCA treatment and (B) were photographed at the end of experiment. The size of the tumors decreased (a-c) at 5 wk of treatment and eventually disappeared at 2 mo after the termination of VCA treatment (d).

Figure 3

Activation of caspases in COLO cells treated with VCA. A: COLO and WI-38 cells were treated without (-) or with (+) VCA (100 ng/mL) for 12 h. Cell lysates were prepared and western blots were performed using antibodies specific for each pro-caspase (PCas). Immunoreactive bands were visualized by chemiluminescence. Representative western blots are shown (left panel). Each band was quantified and normalized against the internal control (®-actin). Normalized values were used to calculate the relative expression of each procaspase as a percentage of PBS-treated WI-38 cells. WP: WI-38 cells treated with PBS, WV: WI-38 cells treated with VCA, CP: COLO cells treated with PBS, CV: COLO cells treated with VCA. Values are mean±SE of three independent experiments (right panel). ^aP < 0.05 vs WP. ^cP < 0.05 vs CP; B: COLO cells were treated without (-) or with (+) VCA (100 ng/mL) for various times and western blots were performed and quantified as described for (A). Representative western blots of three independent experiments are shown (left panel). Each band was quantified and normalized against the internal control (®-actin). Normalized values were used to calculate the relative expression of each procaspase as a percentage of COLO cells without VCA treatment at each time point (right panel).

Figure 4

Decreased expression of anti-apoptotic molecules in COLO cells treated with VCA. COLO cells were treated without (-) or with (+) VCA (100 ng/mL) for the indicated times. Cell lysates were prepared and western blots were performed using specific antibodies. Immunoreactive bands were visualized by chemiluminescence. Representative western blots are shown (A). Each band was quantified and normalized against the internal control (®-actin). Normalized values were used to calculate the relative expression as a percentage of COLO cells without VCA treatment at each time point (B). p-Akt: phosphorylated Akt. Results are representative of three independent experiments.

Figure 5

Inhibition of VCA-induced cytotoxicity of COLO cells by antagonizing anti-TNFR1 antibody (A) COLO and WI-38 cells were treated without (-) or with (+) VCA (100 ng/mL) for 12 h, cell lysates were prepared, and western blots were performed using specific antibodies. Immunoreactive bands were visualized by chemiluminescence. Representative western blot is shown (left panel). Each band was quantified and normalized against the density of the internal control (®-actin). Normalized values were used to calculate the relative expression as a percentage of PBS-treated WI-38 cells. WP: WI-38 cells treated with PBS, WV: WI-38 cells treated with VCA, CP: COLO cells treated with PBS, CV: COLO cells treated with VCA. Values are mean±SE of three independent experiments (right panel). ^aP < 0.05 vs WP. ^cP < 0.05 vs CP. (B) COLO cells were treated without (Control) or with VCA (100 ng/mL) in the absence (PBS) or presence of the indicated amounts of antagonizing TNFR1 antibody (®-TNFR1) for 12 h. Cell viability was determined by MTT assay. Values are mean ± SE (n = 4). ^aP < 0.05 vs the PBS-treated control group. ^cP < 0.05 vs the PBS/VCA-treated group.

Figure 6

Inhibition of VCA-induced caspase activation and VCA-induced decrease of anti-apoptotic molecules in COLO cells treated with anti-TNFR1 antibody. COLO cells were treated without (-) or with (+) VCA (100 ng/mL) for 12 h in the presence (+) or absence (-) of anti-TNFR1 antibody (α-TNFR1, 10 μg/mL). Cell lysates were prepared and western blot was performed using specific antibodies for (A) various procaspases (PCas) and (B) anti-apoptotic molecules. Immunoreactive bands were visualized by chemiluminescence. Representative western blot is shown (left panels). Each band was quantified and normalized against the density of the internal control (actin). Normalized values were used to calculate the relative expression as a percentage of COLO cells without VCA or anti-TNFR1 antibody treatment. Values are mean ± SE of three independent experiments (right panels). ^aP < 0.05 vs VCA-treated cells without anti-TNFR1.