TNFα-activated stromal COX-2 signalling promotes proliferative and invasive potential of colon cancer epithelial cells

Abstract

Objectives: Up to now it has been unclear whether stromal/epithelial interaction affects progression of colon cancer. This study was designed to examine effects of tumour necrosis factor alpha (TNFα)-activated stromal cyclooxygenase-2 (COX-2) signalling on proliferation and invasiveness of colon cancer epithelial cells.

Materials and methods: Cyclooxygenase-2 mRNA and protein were determined by real-time PCR and western blotting and prostaglandin E2 (PGE2 ) was assayed by radioimmunoassay. Cell proliferation and invasiveness were determined by transwell chamber assays and protein kinase C (PKC) was assayed by Biotrak(™) PKC Assay System.

Results: Our results indicated that TNFα, a powerful inflammatory cytokine, strongly promoted COX-2 expression and PGE2 production in colon cancer-associated fibroblasts. Using in vitro assays for estimating proliferative and invasive potential, we discovered that activation of stromal COX-2 signalling significantly promoted proliferation and invasiveness of colon cancer epithelial cells. In addition, selective COX-2 inhibitor N-[2-(Cyclohexyloxy)-4-nitrophenyl]methanesulfonamide, blocked such proliferative and invasive effects on the cancer epithelial cells. In this process, PKC was involved in activation of COX-2 signalling in the fibroblasts.

Conclusion: We conclude that activation of stromal COX-2 signalling by TNFα played a major role in promoting proliferation and invasiveness of colon cancer epithelial cells.

Figure 1

(a) Tumour necrosis factor alpha (TNFα) stimulated cyclooxygenase‐2 (COX‐2) mRNA in Normal fibroblast (NFs) and cancer‐associated fibroblasts (CAFs). NFs or CAFs were cultured in medium with or without TNFα (10 ng/ml). Stimulation by TNFα elicited 10‐fold upregulation of COX‐2 mRNA in NFs, compared to 7‐fold upregulation of COX‐2 mRNA in CAFs (n = 4, *P < 0.05 and **P < 0.01, respectively). Level of COX‐2 in resting CAFs was 6‐fold higher than that in resting NFs (n = 4, #P < 0.05), while level of COX‐2 in TNFα‐stimulated CAFs was 4‐fold higher than that in TNFα‐stimulated NFs (n = 4, #P < 0.05). (b) TNF α induced COX ‐2 protein expression in NF s and CAF s. NFs or CAFs were cultured in medium with or without TNFα (10 ng/ml). Stimulation by TNFα elicited 3‐fold upregulation of COX‐2 protein in NFs, but 2‐fold upregulation of COX‐2 protein in CAFs (n = 4, *P < 0.05). Level of COX‐2 protein in resting CAFs was 3‐fold greater that in resting NFs (n = 4, #P < 0.05), while level of COX‐2 protein in TNFα‐stimulated CAFs was 2‐fold greater than that in TNFα‐stimulated NFs (n = 4, #P < 0.05). (c) TNF α increased synthesis of PGE ₂ in NF s and CAF s. NFs or CAFs were cultured in medium with or without TNFα (10 ng/ml). Stimulation by TNFα elicited 13‐fold upregulation of PGE ₂ synthesis in NFs compared to 6‐fold upregulation of synthesis in CAFs (n = 4, *P < 0.05 and **P < 0.01 respectively). Level of COX‐2 protein in resting CAFs was 7‐fold greater than that in resting NFs (n = 4, #P < 0.05), while level of COX‐2 protein in TNFα‐stimulated CAFs was 4‐fold greater than that in TNFα‐stimulated NFs (n = 4, #P < 0.05).

Figure 2

(a and b) Activation of cyclooxygenase‐2 ( COX ‐2) signalling by Tumour necrosis factor alpha ( TNF α) in Normal fibroblast ( NF s) and cancer‐associated fibroblasts ( CAF s) promoted proliferation of colon cancer epithelial cells. Colon cancer cell lines (HT29, and the others) and NFs or CAFs, respectively, were cultured in upper and lower compartments of Snapwell co‐culture chambers (0.4 μm pores). In selected experiments, some fibroblast cultures were stimulated with TNFα (10 ng/ml) for 24 h before co‐culture. Proliferation of colon cancer epithelial cells was measured by incorporation of 3[H]thymidine. Activation of COX‐2 signalling in CAFs (b), with or without treatment of TNFα, elicited quantitatively greater proliferative response in colon cancer epithelial cells than in NFs (a) (*P < 0.05 and **P < 0.01 compared to their corresponding −NFs control, n = 4; #P < 0.05 when compared to their corresponding +NFs group, n = 4). As a result, activation of COX‐2 signalling by TNFα in CAFs elicited quantitatively greater proliferative response in colon cancer epithelial cells when co‐cultured with CAFs than when co‐cultured with NFs (5‐ and 3‐fold increase in proliferation respectively). (c and d) Activation of COX ‐2 signalling by TNF α in NF s and CAF s promoted invasiveness of colon cancer epithelial cells. NFs or CAFs and the cancer epithelial cells were cultured in separate compartments of Matrigel chambers (Becton Dickinson, Bedford, MA, USA), separated by a filter with 0.8 μm pore size. Upper surface of the filter, on which epithelial cells were cultured, was coated with Matrigel; lower compartment contained no cells (control), or fibroblast cultures in unsupplemented medium or medium supplemented with TNFα (10 ng/ml) for 24 h before co‐culture. After co‐culture for 24 h, cells on the upper surface of filters were removed. Cells on the lower surface, which had invaded through the Matrigel layer and the filter pores, were counted (*P < 0.05, **P < 0.01 and ***P < 0.001, respectively when compared to the −NFs control, n = 4; #P < 0.05 when compared to the +NFs control, n = 4). As a result, activation of COX‐2 signalling by TNFα in CAFs elicited quantitatively greater invasiveness response in colon cancer epithelial cells when co‐cultured with CAFs than when co‐cultured with NFs (~120 versus 70 invasive cells per view).

Figure 3

(a) Inhibition of cyclooxygenase‐2 ( COX ‐2) signalling by NS 398 in Normal fibroblast ( NF s) and cancer‐associated fibroblasts ( CAF s) blocked proliferation of colon cancer epithelial cells. NS 398 (10 μm) was added with or without TNFα (10 ng/ml). NS 398, a specific COX‐2 inhibitor, blocked most paracrine effects induced by TNFα from NFs (a) and CAFs (b) on proliferation of colon cancer epithelial cells (*P < 0.05 and **P < 0.01 compared to their corresponding controls, n = 4). (B) Inhibition of COX ‐2 signalling by NS 398 in NF s and CAF s attenuated invasiveness of colon cancer epithelial cells. NS 398 (10 μm) was added with or without TNFα (10 ng/ml). As a result, NS 398 also blocked most effects of NFs and CAFs stimulated by TNFα on invasiveness of colon cancer epithelial cells (n = 4, *P < 0.05, **P < 0.01, ***P < 0.001 when compared to their corresponding controls, n = 4).

Figure 4

(a) Tumour necrosis factor alpha activated protein kinase C ( PKC ) activity in Normal fibroblast ( NF s) and cancer‐associated fibroblasts ( CAF s). NFs (a) and CAFs (b) were exposed to TNFα 10 ng/ml) for 1–10 min. Cell homogenates were pelleted, and particulate fractions were resolubilized. PKC activity of these samples was determined from transfer of [−32]ATP to a PKC‐specific peptide and were expressed as pmol/min/mg protein. Bars represent mean of results from 4 separated cultures ± SE. *P < 0.05 compared to their respective controls n = 4. (b) Effect of PKC on synthesis of PGE ₂ . NFs and CAFs were co‐incubated with TNFα 10 ng/ml) and PKC inhibitor, bisindoylmalemide (BIM, 10 μm) for 24 h. PGE ₂ levels in harvested media were determined by RIA. Bars represent mean of results from 4 separated cultures ± SE. *P < 0.05, **P < 0.01 compared to their respective controls, #P < 0.05 compared between NFs and CAFs.