Infliximab enhances the therapeutic effects of 5-fluorouracil resulting in tumor regression in colon cancer

Abstract

Colon cancer (CC) is among the most common malignant diseases with a dismal survival. Tumor necrosis factor-alpha (TNF-α) has been identified as a therapeutic target in various cancers, and anti-TNF-α treatment has shown promising effects in different cancer models. However, if TNF-α can be targeted in CC, the therapeutic values of anti-TNF-α treatment in CC remain unknown. Our study indicated that TNF-α is highly expressed in CC cell lines and patient tumor samples. High expression of TNF-α is an independent adverse prognosticator of CC. Targeting the TNF-α by its antibody infliximab induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity and enhanced apoptosis leading to cell death. The combination of infliximab with 5-fluorouracil showed better responses in vitro and in vivo than 5-fluorouracil alone. In conclusion, this study identified TNF-α as a target of CC and anti-TNF-α treatment synergized with chemotherapy leading to a better outcome in preclinical models.

Keywords: chemotherapy; colon cancer; prognosis; tumor necrosis factor-alpha; tumor regression.

Figure 1

TNF-α is overexpressed in CC. Notes: TNF-α expression in CC cell lines HCT116 and HT29 was measured using FACS analysis (gray peak shows the TNF-α expression in CC cell lines and white peak shows the value of isotype). (A and C) TNF-α was highly expressed in the CC cell line HCT116. (B and D) TNF-α was highly expressed in the CC cell line HT29. (E) TNF-α expression in CC tumor tissue and tumor-adjacent tissues was measured using ELISA. Comparing with tumor adjacent tissues, CC tumor tissues had much higher TNF-α concentration. *P<0.05, **P<0.01. Abbreviations: TNF-α, tumor necrosis factor-alpha; CC, colon cancer; FACS, fluorescence-activated cell sorter; ELISA, enzyme-linked immunosorbent assay.

Figure 2

High expression of TNF-α predicts poor survival in CC patients. Notes: (A) TNF-α expression in the CC patient tissue was tested by IHC. Here we show the representative pictures of low, medium, and high expressions of TNF-α in CC (from left to right). The brown color indicates TNF-α expression. (B) ROC analysis determined medium expression as dichotomy cutoff point for survival analysis with 0.630 AUC (95% CI =0.514–0.747). (C) Survival analysis by the Kaplan–Meier method and log-rank test showed that high expression of TNF-α correlated with poor survival of CC patients (P=0.01). The blue curve shows the survival curve of CC patients with low TNF-α expression, and the green curve shows the survival curve of CC patients with high TNF-α expression. Abbreviations: TNF-α, tumor necrosis factor-alpha; CC, colon cancer; IHC, immunohistochemistry; ROC, receiver operating characteristic; AUC, area under the curve; CI, confidence interval.

Figure 3

Infliximab inhibits CC cells’ viability via ADCC and CDC effects. Notes: ADCC and CDC effects were reflected by the reduction in the cell viability of CC cell lines HCT116 and HT29 cultured with effector cells (macrophage) or active complement with infliximab treatment. (A and B) In the control group, with the increase in infliximab concentration, neither HCT116 cell line nor HT29 cell line had an apparent reduction in cell viability. This suggested that there was no direct cytotoxicity of infliximab on CC cell lines. However, with the presence of effector cells and complement, infliximab decreased CC cells’ viability significantly with the increasing dose. *P<0.05. Abbreviations: CC, colon cancer; ADCC, antibody-dependent cellular cytotoxicity; CDC, complement-dependent cytotoxicity.

Figure 4

Infliximab enhances 5-FU action in CC cell lines. Notes: (A and B) In CC cell lines HCT116 and HT29, infliximab promoted the therapeutic effects of 5-FU under the presence of active complement showing decreased survival rate at each concentration of 5-FU treatment. (C and D) Cleaved caspase-3 was measured by FACS analysis. The bar area indicates the peak of cleaved caspase-3, and the bar chart at the right side shows the quantitative data of the cleaved caspase-3 level. In CC cell lines HCT116 and HT29, administration of 5-FU significantly activated apoptosis reflected by increased cleaved caspase-3; addition of infliximab further enhanced apoptosis. *P<0.05, **P<0.01. Abbreviations: 5-FU, 5-fluorouracil; CC, colon cancer; FACS, fluorescence-activated cell sorter.

Figure 5

Infliximab and 5-FU combination treatment promotes apoptosis in the CC tissue of the xenograft mouse model. Notes: Cleaved PARP was evaluated by IHC in the CC tissue of the xenograft mouse model. (A) Representative pictures of IHC staining of cleaved PARP in the CC tissue of the xenograft mouse model. 5-FU treatment induced an increased level of cleaved PARP compared with the control group, while combination of infliximab and 5-FU further increased the level of cleaved PARP in the CC tissue. (B) The bar chart shows the quantitative results of IHC staining of cleaved PARP in the CC tissue of the xenograft mouse model. Compared with the control group, 5-FU and 5-FU + infliximab-treated groups have a significantly increased level of cleaved PARP. *P<0.05, **P<0.01. Abbreviations: 5-FU, 5-fluorouracil; CC, colon cancer; PARP, poly(ADP-ribose) polymerase; IHC, immunohistochemistry.

Figure 6

Infliximab enhances the therapeutic effects of 5-FU in the CC xenograft mouse model. Notes: (A) Tumor growth of the group treated by infliximab + 5-FU is apparently slower than that of the group treated by 5-FU alone. (B) The survival curves indicated that the survival time of infliximab + 5-FU-treated group was extended compared with that of the group treated by 5-FU alone. Abbreviations: 5-FU, 5-fluorouracil; CC, colon cancer.