Cyclooxygenase 2 modulates killing of cytotoxic T lymphocytes by colon cancer cells

Abstract

Although anti-cancer effects of cyclooxygenase 2 (COX2) inhibitors have been reported, most studies focused on the direct effects of COX2 inhibiters on colon cancer cells. On the other hand, several types of cancers express Fas ligand (FasL) and/or TRAIL and mediate apoptosis of T cells in vitro. The "counter-attack" machinery may account for the mechanisms by which tumors evade host immune surveillance. In this study we determined if COX2 inhibitor could modulate effector molecules of cell death on colon cancer cells changing their effects on cytotoxic T lymphocytes. Colon adenocarcinoma cells, HCA7 and HCT116, the former COX2-positive and the latter COX2-negative, were pre-incubated with/without a COX2 inhibitor, NS398. Subsequently, the cells were co-cultured with Jurkat T cell leukemia cells and damage to Jurkat cells was determined. Treatment with NS398 resulted in reduction of expression of FasL and TRAIL in HCA7 cells, whereas NS398 did not affect the expression of FasL and TRAIL in HCT116 cells. The number of viable Jurkat cells was diminished when cells were co-cultured with naive, non-pretreated HCA7 or HCA116 cells. Preincubation of HCA7 cells with NS398 before co-culture blunted the HCA7 cell-induced cell toxicity on Jurkat cells. In contrast, pretreatment with NS398 failed to inhibit the HCT116-induced Jurkat cell killing. Our results suggest that COX2 regulates the expression of FasL and TRAIL on COX2-positive colon cancer cells thereby evoking a counter-attack against cytotoxic T cells, which may lead to compromised host immune responses.

Keywords: FasL; TRAIL; colon cancer cells; cyclooxygenase 2; cytotoxic T lymphocytes.

Fig. 1

Detection of COX-2 protein in human colonic adenocarcinoma cell lines. Colonic adenocarcinoma cells (HCA7and HCT116) were cultured in DMEM supplemented with 10% FCS. When cells became sub-confluent, cells were lysed as described in Materials and Methods. COX2 protein was detected in the cell lysates with Western blotting. Representative photograph of bands showing COX2 protein (72 kD).

Fig. 2

Effect of increasing concentrations of a COX2 selective inhibitor NS398 on viability of HCA7 cells. HCA7 cells were cultured for 48 h in the presence of 0–100 µM NS398. Subsequently, cell viability was determined using WST method. *p<0.005 vs 0 µM.

Fig. 3

Expression of proteins of the FasL and TRAIL systems on colon cancer cell lines, Jurkat and U937 cells. Upper panel: HCA7 and HCT116 (upper panel) , and Jurkat and U937 cells (lower panel) were cultured in the absent or presence of NS398 (10 µM). After an 18 h of incubation, whole cell lysate was extracted and the expression of apoptosis related proteins (FASL and TRAIL in HCA7 and HCT116 cells; Fas and DR4 in Jurkat and U937 cells) was examined by Western blotting. Representative photograph of bands showing FasL (38 kD), TRIAL (38 kD), Fas (48 kD) and DR4 (60 kD).

Fig. 4

Immunostaining analysis for FasL and TRAIL expression in HCA7 and HCT116 cells. After a 48 h of culture, HCA7 and HCT116 cells were fixed with paraformaldehyde and processed for immunostaining using a first antibody for TRAIL or FASL followed by visualization with an FITC-labeled secondary goat anti-rabbit antibody as described in Materials and Methods. Magnification (×400)

Fig. 5

Effects of NS398 on the cytotoxic ability of HCA7 and HCT116 cells against Jurkat cells. HCA7 and HCT116 cells pretreated with NS398 (0, 1, or 10 µM) for 18 h and used for co-culture with ³H thymidine labeled Jurkat cells for 18 h. Subsequently, cells were collected and radioactivity was counted by a scintillation counter. As a control, Jurkat cells were cultured for 18 h without colon cancer cells. *p<0.001, **p<0.05 vs appropriate control. NS; not significant.

Fig. 6

Effects of nerutralizing antibodies for FASL or/and TRAIL on the cytotoxic ability of HCA7 and HCT116 cells against Jurkat cells. Conditions as in Fig. 5. HCA7 and HCT116 cells pretreated with either anti-FasL or anti-TRAIL antibody, or with both (anit-Fas + anti-TRAIL) for 18 h. After co-culture with Jurkat cells, cytotoxicity of Jurkat cells was assayed as described in Fig. 5. *p<0.001 vs control.

Fig. 7

Working hypothesis. For details, refer to the text.