Tumor necrosis factor-related apoptosis-inducing ligand inhibits the growth and aggressiveness of colon carcinoma via the exogenous apoptosis signaling pathway

Abstract

Colon cancer is one of the most common types of gastrointestinal tumor. Previous studies have demonstrated that tumor necrosis factor-(TNF)-related apoptosis-inducing ligand (TRAIL) reduces the aggressiveness of colon cancer tumors and promotes the apoptosis of colon carcinoma cells. In the present study, the inhibitory effects of TRAIL were investigated and the potential mechanism of TRAIL-mediated apoptosis was explored in colon cancer cells. Reverse transcription-quantitative polymerase chain reaction, western blotting, immunofluorescence, immunohistochemistry, TUNEL and flow cytometry assays were used to analyze the effects of TRAIL on the growth, migration, invasion and apoptosis of colon tumor cells. In vivo experiments were performed in mice to analyze the therapeutic effects of TRAIL. The results demonstrated that TRAIL significantly suppressed the growth of colorectal tumor cells in a dose-dependent manner (0.5-2.5 mg/ml) and also promoted colon tumor cell death. The migration and invasion of colon tumor cells were inhibited by the downregulation of fibronectin, Vimentin and E-cadherin. The apoptotic rate revealed that TRAIL (2.0 mg/ml) significantly promoted the apoptosis of colon tumor cells by regulating apoptosis-related gene expression. TRAIL administration promoted the apoptosis of colon tumor cells via the exogenous apoptosis signaling pathway due to the upregulation of caspase-3, caspase-8 and nuclear factor-κB protein expression. In vivo assays revealed that TRAIL administration significantly inhibited tumor growth and promoted apoptotic body and lymphocyte infiltration, which led to increased survival in tumor-bearing mice compared with the control group. Immunohistochemistry revealed that P53 and B-cell lymphoma-2 were downregulated in TRAIL-treated tumors. In conclusion, TRAIL treatment significantly inhibited the growth and aggressiveness of colon tumors by inducing apoptosis via the exogenous apoptosis pathway, which suggests that TRAIL may be a potential anticancer agent for colon carcinoma therapy.

Keywords: aggressiveness; apoptosis; colon cancer; exogenous apoptosis pathway; tumor necrosis factor-related apoptosis-inducing ligand.

Figure 1.

Effect of TRAIL on the growth and cells cycle progression of colon tumor cells. (A) Affinity of TRAIL with its receptor tumor necrosis factor-α in LoVo and HT-29 cells as determined by ELISA. (B) Immunofluorescence assay for the affinity of TRAIL with LoVo and HT-29 cells. TRAIL inhibits (C) LoVo and (D) HT-29 cell growth in a dose-dependent manner. TRAIL inhibits (E) LoVo and (F) HT-29 cell growth in time-dependent manner. TRAIL treatment induced cell cycle arrest in (G) LoVo and (H) HT-29 cells. **P<0.05 vs. control. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Figure 1.

Effect of TRAIL on the growth and cells cycle progression of colon tumor cells. (A) Affinity of TRAIL with its receptor tumor necrosis factor-α in LoVo and HT-29 cells as determined by ELISA. (B) Immunofluorescence assay for the affinity of TRAIL with LoVo and HT-29 cells. TRAIL inhibits (C) LoVo and (D) HT-29 cell growth in a dose-dependent manner. TRAIL inhibits (E) LoVo and (F) HT-29 cell growth in time-dependent manner. TRAIL treatment induced cell cycle arrest in (G) LoVo and (H) HT-29 cells. **P<0.05 vs. control. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand.

Figure 2.

Effects of 48 h TRAIL treatment on the migration and invasion of colon cancer cells. TRAIL administration (0.20 mg/ml) inhibits the migration of (A) LoVo and (B) HT-29 cells. TRAIL administration (0.20 mg/ml) inhibits the invasion of (C) LoVo and (D) HT-29 cells. Reverse transcription-quantitative polymerase chain reaction analysis of FN, Vimentin and E-cadherin expression in (E) LoVo and (F) HT-29 cells. Western blotting of FN, Vimentin and E-cadherin protein expression in (G) LoVo and (H) HT-29 cells. **P<0.05 vs. control. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; FN, fibronectin.

Figure 2.

Effects of 48 h TRAIL treatment on the migration and invasion of colon cancer cells. TRAIL administration (0.20 mg/ml) inhibits the migration of (A) LoVo and (B) HT-29 cells. TRAIL administration (0.20 mg/ml) inhibits the invasion of (C) LoVo and (D) HT-29 cells. Reverse transcription-quantitative polymerase chain reaction analysis of FN, Vimentin and E-cadherin expression in (E) LoVo and (F) HT-29 cells. Western blotting of FN, Vimentin and E-cadherin protein expression in (G) LoVo and (H) HT-29 cells. **P<0.05 vs. control. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; FN, fibronectin.

Figure 3.

Effect of TRAIL on the apoptosis of colon cancer cells. TRAIL promotes the apoptosis of (A) LoVo and (B) HT-29 cells after 48 h incubation as determined by flow cytometry. Western blotting revealed that TRAIL promotes the expression of caspase-8, caspase-3 and NF-κB in (C) LoVo and (D) HT-29 cells, as well as P53 and Bcl-2 protein in (E) LoVo and (F) HT-29 cells. Si-FasL ameliorated TRAIL-mediated apoptosis in (G) LoVo and (H) HT-29 cells. *P<0.05 and **P<0.01 vs. control. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; Bcl-2, B-cell lymphoma-2; NF, nuclear factor; Si-FasL, short interfering RNA-Fas ligand.

Figure 3.

Effect of TRAIL on the apoptosis of colon cancer cells. TRAIL promotes the apoptosis of (A) LoVo and (B) HT-29 cells after 48 h incubation as determined by flow cytometry. Western blotting revealed that TRAIL promotes the expression of caspase-8, caspase-3 and NF-κB in (C) LoVo and (D) HT-29 cells, as well as P53 and Bcl-2 protein in (E) LoVo and (F) HT-29 cells. Si-FasL ameliorated TRAIL-mediated apoptosis in (G) LoVo and (H) HT-29 cells. *P<0.05 and **P<0.01 vs. control. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; Bcl-2, B-cell lymphoma-2; NF, nuclear factor; Si-FasL, short interfering RNA-Fas ligand.

Figure 4.

Effects of TRAIL on tumor growth and survival in LoVo- and HT-29-bearing mice. TRAIL suppresses tumor growth in (A) LoVo-bearing and (B) HT-29-bearing mice compared with the PBS group. Effects of TRAIL on apoptotic body and lymphocyte infiltration in (C) LoVo-bearing and (D) HT-29-bearing mice compared with the control group. TRAIL suppresses P53 and Bcl-2 expression levels in (E) LoVo-bearing and (F) HT-29-bearing mice compared with the control group. TRAIL prolongs the survival of (G) LoVo-bearing and (H) HT-29-bearing mice during a 120-day observation compared with the control group. **P<0.05 vs. control. Magnification, ×100. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; Bcl-2, B-cell lymphoma-2.

Figure 4.

Effects of TRAIL on tumor growth and survival in LoVo- and HT-29-bearing mice. TRAIL suppresses tumor growth in (A) LoVo-bearing and (B) HT-29-bearing mice compared with the PBS group. Effects of TRAIL on apoptotic body and lymphocyte infiltration in (C) LoVo-bearing and (D) HT-29-bearing mice compared with the control group. TRAIL suppresses P53 and Bcl-2 expression levels in (E) LoVo-bearing and (F) HT-29-bearing mice compared with the control group. TRAIL prolongs the survival of (G) LoVo-bearing and (H) HT-29-bearing mice during a 120-day observation compared with the control group. **P<0.05 vs. control. Magnification, ×100. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; Bcl-2, B-cell lymphoma-2.