Use of aspirin in the prevention of colorectal cancer through TIGIT-CD155 pathway

Abstract

Colorectal cancer (CRC) is one of the most widespread malignant cancers, with a high incidence and mortality all over the world. Aspirin (ASA) otherwise known as acetylsalicylic acid, is a non-steroidal anti-inflammatory drug that has shown promising results in the prevention of chronic diseases, including several cancers. In previous studies, aspirin has been shown to reduce the incidence of CRC. Immune checkpoint blockade of T cell Ig and ITIM domain receptor (TIGIT) alone or combined with other immune checkpoint blockades moleculars has gained impressive results in the treatment of the melanoma and glioblastoma. Here, we found that TIGIT and Poliovirus receptor (PVR, CD155) are expressed in tumour cells; the TIGIT and CD155 protein expression in cancer tissue has been found to be significantly higher than that in the precancerous tissue. T cell Ig and ITIM domain receptor and CD226 were expressed in the lymphocytes near the tumour tissue and the adjacent tissues. Aspirin has been found to inhibit cancer cell viability and promote CRC cell apoptosis.Similarly, aspirin has also been found to increase pro-apoptotic protein Bax's expression. We found that the expression of TIGIT decreased with an increase in the concentration of aspirin and that the suppression of TIGIT can affect the effect of aspirin on cell proliferation. In this paper, we found that aspirin attenuates cancer cell proliferation and induces CRC cells apoptosis by down-regulating the expression of TIGIT, which provides new evidence for the application of aspirin in cancer treatment.

Keywords: CD155; CD226; TIGIT; aspirin; cell proliferation; colorectal cancer.

Figure 1

TIGIT/CD226/CD155 expression in colorectal cancer and adjacent tissues. TIGIT and CD155 are expressed in malignant epithelial cells in colorectal carcinoma. CD226 is not expressed in colorectal cancer tissues. (A,A1) Colon adenocarcinoma shows immunoreactivity with polyclonal antibody TIGIT (X40, X400). (B,B1) Adjacent section stained with an irrelevant polyclonal antibody TIGIT (X40, X400). (C,C1) Colon adenocarcinoma shows immunoreactivity with polyclonal antibody CD226 (X40, X400). (D,D1) Adjacent section stained with an irrelevant polyclonal antibody CD226 (X40, X400). (E,E1) Colon adenocarcinoma shows immunoreactivity with polyclonal antibody CD155(X40, X400). (F,F1) Adjacent section stained with an irrelevant polyclonal antibody CD155(X40,X400). TIGIT, T cell Ig and ITIM domain receptor

Figure 2

TIGIT/CD226/CD155 expressions on lymphocyte in colorectal cancer and adjacent tissues. TIGIT and CD226 are expressed in lymphocyte cells in colorectal carcinoma. CD155 is not expressed in lymphocyte. (A,A1) Colon adenocarcinoma shows immunoreactivity with polyclonal antibody TIGIT (X40, X400). (B,B1) Adjacent section stained with an irrelevant polyclonal antibody TIGIT (X40, X400). (C,C1) Colon adenocarcinoma shows immunoreactivity with polyclonal antibody CD226 (X40, X400). (D,D1) Adjacent section stained with an irrelevant polyclonal antibody CD226 (X40, X400). (E,E1) Colon adenocarcinoma shows immunoreactivity with polyclonal antibody CD155(X40, X400). (F,F1) Adjacent section stained with an irrelevant polyclonal antibody CD155 (X40, X400). TIGIT, T cell Ig and ITIM domain receptor

Figure 3

Aspirin inhibits the viability of colorectal cancer cell. HT‐29 cells were treated with increasing concentrations of ASA (from 2 to 8 mmol/L). After 24, 48 and 72 h of incubation, cell viability was measured using CCK‐8 assay. (*Significantly different from control; *P < 0.05; **P < 0.01; ***P < 0.001; ^&Significantly different from control 0 h, ^& P < 0.05; ^&& P < 0.001; ^&&& P < 0.0001

Figure 4

Aspirin inhibits the migration of colorectal cancer cell. A, HT‐29 cells were treated with increasing concentrations of ASA (from 2 to 8 mmol/L). After 72 h of incubation, the distance was photographed. (B‐G) The expression of MMP‐9 and FAK proteins was deteced using western blotting in HT‐29 cells. Antibody against GAPDH was used as loading control.**P < 0.01, ***P < 0.001, compared to control group. All data are expressed as mean ± SD of three separate experiments

Figure 5

Aspirin induces the apoptotic of colorectal cancer cell. (A,B) HT‐29 cells were stained with annexin V‐FITC and PI following treatment with increasing concentrations of ASA (from 2 to 8 mmol/L) for 48 h. Data are indicative of three separate experiments.The statistical significance of the results was analysed using one way ANOVA followed by Least Significant Difference test. *P < 0.05, significantly different from control

Figure 6

Western blot for Bax, Bcl‐2 in HT‐29 cells after treatment with ASA. A, Effects of treatment of HT‐29 cells for 48 h with IC50 concentration of ASA or different concentrations ASA(2, 4, 8 mmol/L) on for Bax and Bcl‐2 level. B, Effects of treatment of HT‐29 cells with 8mM aspirin for 24, 48 and 72 h. Antibody against GAPDH was used as loading control. *P < 0.05, **P < 0.01, ***P < 0.001, compared to control group. All data are expressed as mean ± SD of three separate experiments

Figure 7

Western blot for TIGIT and CD155 in HT‐29cells after treatment with ASA. A, Effects of treatment of HT‐29 cells for 48 h with IC50 concentration of ASA or different concentrations ASA(2, 4, 8 mmol/L) on for Bax and Bcl‐2 level. B, Effects of treatment of HT‐29 cells with 8 mmol/L aspirin for 24, 48 and 72 h. Antibody against GAPDH was used as loading control. *P < 0.05, **P < 0.01, ***P < 0.001, compared to control group. All data are expressed as mean ± SD of three separate experiments. TIGIT, T cell Ig and ITIM domain receptor