This study shows for the first time that scirrhous gastric carcinoma cells are impacted in their ability to metastasise in a mouse model by the dietary administration of (n-6)-PUFA. This is true both when tumour cells are injected into the i.p. space as well as when tumours are grafted onto the stomach wall. The levels of LA in the diets used (in terms of energy from LA) are higher than in most human diets (Arab, 2003
). However, the relative abundance of LA measured in circulating mouse plasma in the VHLA group of our study are very similar to those measured in humans on certain high-fat diets (Skeaff et al, 2006
), suggesting that the studies presented here are relevant to potential consequences in human disease. In this experimental metastasis study, the incidence of peritoneal metastasis, as well as the number of tumour nodules and total tumour volume, increased when LA was included at high levels in the diet. Although this model excludes the early steps of metastasis, gastric tumours are known to metastasise to the peritoneal cavity, and the presence of gastric tumour cells in the peritoneal cavity is a well-described marker for identifying patients who are at increased risk of peritoneal recurrence (Burke et al, 1998
; Hayes et al, 1999
). To better simulate the full metastatic cascade, we used an orthotopic graft model in which tumour sections, derived from tumours grown subcutaneously in a different mouse, were implanted on the stomach wall. This procedure led not only to efficient peritoneal metastasis but also to liver invasion and lymph node metastasis. Although the incidence of lymph node metastasis was not altered between LLA and HLA groups, liver invasion and peritoneal metastasis were higher in VHLA group. This result suggested that fatty acid intake may influence organ-specific metastasis of these cells, perhaps by altering the environment within host target sites and facilitating specific steps in the process of metastasis.
To address this question, we investigated the effect of LA on invasion, a critical step in the metastatic cascade. Linoleic acid increased OCUM-2MD3 cell invasion through Matrigel in an in vitro
invasion assay. These results are consistent with reports indicating increased invasion of breast carcinoma cells treated with LA (Rose et al, 1994
). Interestingly, LA did not affect the proliferation, apoptosis or adhesion of OCUM-2MD3 cells to Matrigel, indicating that increased invasion by LA was not caused by the enhancement of growth or adhesion of this cell line. To confirm that the response to LA is common in gastric carcinoma cells, we next sought to address whether other scirrhous gastric carcinoma cell lines had the similar response to LA. Interestingly, three cell lines, OCUM-12, NUGC3 and MKN-45, treated with LA showed a significant enhancement of invasive ability, indicating that LA effect might be common in scirrhous-type gastric cancer. These results contrast with previous reports in other types of tumour cells. For example, both our group and Johanning and Lin (1995)
previously demonstrated that (n-6)-PUFA promoted adhesion of breast carcinoma cell lines to extracellular matrix. These differences may be the result of cell-specific signalling pathways. Nie et al (2000)
described that eicosanoids regulated prostate cancer progression both positively and negatively, depending on the expression of enzymes such as COX, LOX and P450.
One mechanism by which fatty acids may enhance invasion is the secretion or activation of metalloproteases (Rose et al, 1994
). We did not detect a change in the production or secretion of several metalloproteinases in OCUM-2MD3 cells stimulated with LA based on immunoblotting and gelatin zymography (data not shown). We also examined the expression of urinary plasminogen activator (UPA), an important factor known to induce proteolytic activity. Linoleic acid treatment did not induce UPA expression in OCUM-2MD3 cells, as measured by immunoblotting (data not shown).
We did find that the ERK inhibitor, PD98059, clearly reduced the LA-enhanced invasion of OCUM-2MD3 cells, suggesting that this signalling pathway has an important role in LA-stimulated gastric carcinoma cell invasion. Linoleic acid did not consistently activate p38 or SAPK/JNK, and the increased invasion was not sensitive to the p38 inhibitor SB203580, suggesting that these pathways are not necessary for LA-enhanced invasion in these cells. Other studies have shown that LA activates p38 and SAPK/JNK in stromal fibroblasts (Westermarck et al, 2000
) and endothelial (Shin et al, 2001
) cells. Therefore, it is likely that there is cell-type specificity with respect to LA activation of p38 and JNK pathways. Moreover, we found that pre-treatment of OCUM-2MD3 cells with indomethacin inhibited ERK phosphorylation in vitro
, suggesting that the observed ERK activation is downstream of COX activity.
Our finding that a COX inhibitor, but not a LOX inhibitor, impaired LA-induced invasion of gastric carcinoma cells led us to investigate the effect of a COX inhibitor on peritoneal metastasis in vivo. Administration of indomethacin in drinking water inhibited the frequency and extent of peritoneal implantation, thus, serving as a proof of principle experiment for reducing tumour metastasis with inhibitors of fatty acid metabolism.
These results led us to examine the expression pattern of COX in OCUM-2MD3 cells. The COX-1, but not COX-2, was present in both cultured cells and metastatic nodules. Thus, it appears that COX-1 is primarily responsible for the enhanced metastatic effects of LA. As reviewed by Tsujii et al (1998)
, COX-1 expression regulates angiogenesis in endothelial cells. Therefore, it is conceivable that the ability of COX-1 to increase metastatic tumour formation in vivo
may be through enhanced angiogenesis. Moreover, the unaltered expression of COX-1 in the presence of LA either in OCUM-2D3 cells or metastatic nodules in vivo
suggests that any increase in LA metabolism may primarily be due to increased activity of COX-1, related to a higher substrate availability. Although our study has focused on COX expression in cultured cells and metastatic nodules, a complete understanding of the metastatic process will require measurement of the whole spectrum of LA metabolites, as a variety of biological effects, including dissemination and metastasis, may be exerted by several different eicosanoid metabolites (Mathias and Dupont, 1985
A number of studies have suggested the importance of dietary fatty acids and their metabolism for human cancer development, either through animal models (Connolly et al, 1996
), epidemiology of human populations (Armstrong and Doll, 1975
) or cell culture systems (Horia and Watkins, 2007
). Examples now exist in melanoma (Xia et al, 2006
), hepatocellular (Rohr-Udilova et al, 2008
), breast (Rose, 1997a
), prostate (Angelucci et al, 2008
) and pancreatic (Funahashi et al, 2008
) cancers in which tumour cells are effected by (n-6)-PUFA. In some studies, (n-3)-PUFA appear to antagonise the effects of the (n-6) fatty acids (Funahashi et al, 2008
), or have growth or metastasis inhibitory effects by themselves (Xia et al, 2006
). Our work now adds gastric carcinoma to the list, and, when considered with the other models, suggests that specific components in the diet may influence a wide variety of cancers, making dietary modulation an attractive candidate for altering the aggressiveness of pre-existing cancers.
We (Palmantier et al, 1996
; Nony et al, 2005
) and others (Liu et al, 1991
) have previously shown that metabolites of LA and AA can activate signalling pathways that regulate tumour cell behaviour. Our work with breast carcinoma cells showed that activation of TAK-1, a transforming growth factor receptor-associated kinase, is a key component of one of these pathways (Nony et al, 2005
). It is interesting that others have now found the TGF-β
pathway to be important for spreading and invasion of gastric carcinoma cells (Lee et al, 2005
). This raises the question of whether metabolites of LA are working through the TGF-β
pathway in gastric carcinoma cells.
Our results suggest that dietary LA promotes invasion and peritoneal metastasis of gastric carcinoma cells through an ERK-dependent pathway. Indomethacin treatment may contribute to impaired invasion and COX-1 activity. Results from our studies may provide information to suggest novel treatment and prevention options, as well as information useful in guiding the formulation of diets to enhance the protection of humans treated for cancer.