Yingting Zhu

Suppression of a sialyltransferase by antisense DNA reduces invasiveness of human colon cancer cells in vitro.

Transfer of terminal alpha 2,6-linked sialic acids to N-glycans is catalyzed by beta-galactoside alpha 2,6-sialyltransferase (ST6Gal I). Expression of ST6Gal I and its products is reportedly increased in colon cancers. To investigate directly the functional effects of ST6Gal I expression, human colon cancer (HT29) cells were transfected with specific antisense DNA. ST6Gal I mRNA and protein were virtually undetectable in six strains of transfected HT29 cells. ST6Gal activity was reduced to 14% of control (P<0.005) in transfected cells. Expression of terminal alpha 2,6- and alpha 2,3-linked sialic acids, and unmasked N-acetyllactosamine oligosaccharides, respectively, was assessed using flow cytometry and fluoresceinated Sambucus nigra, Maackia amurensis and Erythrina cristagalli lectins. Results indicated a major reduction in expression of alpha 2,6-linked sialic acids and counterbalancing increase in unmasked N-acetyllactosamines in antisense DNA-transfected cells, without altered expression of alpha 2,3-linked sialic acids or ganglioside profiles. The ability of transfected cells to form colonies in soft agar and to invade extracellular matrix material (Matrigel), respectively, in vitro was reduced by approx. 98% (P<0.0001) and more than 3-fold (P<0.005) compared to parental HT29 cells. These results indicate that N-glycans bearing terminal alpha 2,6-linked sialic acids may enhance the invasive potential of colon cancer cells.

Cytokine-mediated PGE2 expression in human colonic fibroblasts.

We investigated prostanoid biogenesis in human colonic fibroblasts (CCD-18Co and 5 primary fibroblast cultures) and epithelial cell lines (NCM460, T84, HT-29, and LS 174T) and the effect of PGE2 on fibroblast morphology. Cytokine-stimulated PGE2production was measured. PGH synthase-1 and -2 (PGHS-1 and -2) protein and mRNA expression were evaluated. Basal PGE2 levels were low in all cell types (0.15-6.47 ng/mg protein). Treatment for 24 h with interleukin-1β (IL-1β; 10 ng/ml) or tumor necrosis factor-α (50 ng/ml), respectively, elicited maximal 25- and 6-fold inductions of PGE2 synthesis in CCD-18Co cultures and similar results in primary fibroblast cultures; maximal inductions with IL-1β in colonic epithelial cell lines were from zero to fivefold. Treatment of CCD-18Co fibroblasts with IL-1β caused maximal 21- and 53-fold increases, respectively, in PGHS-2 protein and mRNA levels without altering PGHS-1 expression. PGE2 (0.1 μmol/l) elicited a dramatic shape change in selected fibroblasts. Colonic fibroblasts are potentially important as cytokine targets and a source of and target for colonic prostanoids in vivo.

iNOS signaling interacts with COX-2 pathway in colonic fibroblasts.

COX-2 and iNOS are two major inflammatory mediators implicated in colorectal inflammation and cancer. Previously, the role of colorectal fibroblasts involved in regulation of COX-2 and iNOS expression was largely ignored. In addition, the combined interaction of COX-2 and iNOS signalings and their significance in the progression of colorectal inflammation and cancer within the fibroblasts have received little investigation. To address those issues, we investigated the role of colonic fibroblasts in the regulation of COX-2 and iNOS gene expression, and explored possible mechanisms of interaction between COX-2 and iNOS signalings using a colonic CCD-18Co fibroblast line and LPS, a potential stimulator of COX-2 and iNOS. Our results clearly demonstrated that LPS activated COX-2 gene expression and enhanced PGE(2) production, stimulated iNOS gene expression and promoted NO production in the fibroblasts. Interestingly, activation of COX-2 signaling by LPS was not involved in activation of iNOS signaling, while activation of iNOS signaling by LPS contributed in part to activation of COX-2 signaling. Further analysis indicated that PKC plays a major role in the activation and interaction of COX-2 and iNOS signalings induced by LPS in the fibroblasts.

IL1β-mediated Stromal COX-2 signaling mediates proliferation and invasiveness of colonic epithelial cancer cells.

COX-2 is a major inflammatory mediator implicated in colorectal inflammation and cancer. However, the exact origin and role of COX-2 on colorectal inflammation and carcinogenesis are still not well defined. Recently, we reported that COX-2 and iNOS signalings interact in colonic CCD18Co fibroblasts. In this article, we investigated whether activation of COX-2 signaling by IL1β in primary colonic fibroblasts obtained from normal and cancer patients play a critical role in regulation of proliferation and invasiveness of human colonic epithelial cancer cells. Our results demonstrated that COX-2 level was significantly higher in cancer associated fibroblasts than that in normal fibroblasts with or without stimulation of IL-1β, a powerful stimulator of COX-2. Using in vitro assays for estimating proliferative and invasive potential, we discovered that the proliferation and invasiveness of the epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts than with normal fibroblasts, with or without stimulation of IL1β. Further analysis indicated that the major COX-2 product, prostaglandin E(2), directly enhanced proliferation and invasiveness of the epithelial cancer cells in the absence of fibroblasts. Moreover, a selective COX-2 inhibitor, NS-398, blocked the proliferative and invasive effect of both normal and cancer associate fibroblasts on the epithelial cancer cells, with or without stimulation of IL-1β. Those results indicate that activation of COX-2 signaling in the fibroblasts plays a major role in promoting proliferation and invasiveness of the epithelial cancer cells. In this process, PKC is involved in the activation of COX-2 signaling induced by IL-1β in the fibroblasts.

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

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.

Stromal COX-2 signaling activated by deoxycholic acid mediates proliferation and invasiveness of colorectal epithelial cancer cells

COX-2 is a major regulator implicated in colonic cancer. However, how COX-2 signaling affects colonic carcinogenesis at cellular level is not clear. In this article, we investigated whether activation of COX-2 signaling by deoxycholic acid (DCA) in primary human normal and cancer associated fibroblasts play a significant role in regulation of proliferation and invasiveness of colonic epithelial cancer cells. Our results demonstrated while COX-2 signaling can be activated by DCA in both normal and cancer associated fibroblasts, the level of activation of COX-2 signaling is significantly greater in cancer associated fibroblasts than that in normal fibroblasts. In addition, we discovered that the proliferative and invasive potential of colonic epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts pre-treated with DCA than with normal fibroblasts pre-treated with DCA. Moreover, COX-2 siRNA attenuated the proliferative and invasive effect of both normal and cancer associate fibroblasts pre-treated with DCA on the colonic cancer cells. Further studies indicated that the activation of COX-2 signaling by DCA is through protein kinase C signaling. We speculate that activation of COX-2 signaling especially in cancer associated fibroblasts promotes progression of colonic cancer.