The omega-3 polyunsaturated fatty acid eicosapentaenoic acid inhibits mouse MC-26 colorectal cancer cell liver metastasis via inhibition of PGE2-dependent cell motility.

BACKGROUND AND PURPOSE: The omega-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) has antineoplastic activity at early stages of colorectal carcinogenesis, relevant to chemoprevention of colorectal cancer (CRC). We tested the hypothesis that EPA also has anti-CRC activity at later stages of colorectal carcinogenesis, relevant to treatment of metastatic CRC, via modulation of E-type PG synthesis. EXPERIMENTAL APPROACH: A BALB/c mouse model, in which intrasplenic injection of syngeneic MC-26 mouse CRC cells leads to development of liver metastases, was used. Dietary EPA was administered in the free fatty acid (FFA) form for 2 weeks before and after ultrasound-guided intrasplenic injection of 1 × 10(6) MC-26 cells (n= 16 each group). KEY RESULTS: Treatment with 5% (w w(-1)) EPA-FFA was associated with a reduced MC-26 mouse CRC cell liver tumour burden compared with control animals (median liver weight 1.03 g vs. 1.62 g; P < 0.034). Administration of 5% EPA-FFA was also linked to a significant increase in tumour EPA incorporation and lower intratumoural PGE(2) levels (with concomitant increased production of PGE(3)). Liver tumours from 5% EPA-FFA- treated mice demonstrated decreased 5-bromo-2-deoxyuridine-positive CRC cell proliferation and reduced phosphorylated ERK 1/2 expression at the invasive edge of tumours. A concentration-dependent reduction in MC-26 CRC cell Transwell® migration following EPA-FFA treatment (50-200 µM) in vitro was rescued by exogenous PGE(2) (10 µM) and PGE(1)-alcohol (1 µM). CONCLUSIONS AND IMPLICATIONS: EPA-FFA inhibits MC-26 CRC cell liver metastasis. EPA incorporation is associated with a 'PGE(2) to PGE(3) switch' in liver tumours. Inhibition of PGE(2)-EP(4) receptor-dependent CRC cell motility probably contributes to the antineoplastic activity of EPA.

Prostaglandin E2-EP4 receptor signalling promotes tumorigenic behaviour of HT-29 human colorectal cancer cells.

The predominant product of cyclooxygenase (COX) activity in the colon, prostaglandin (PG) E2 promotes intestinal tumorigenesis. Expression of the PGE2 receptor EP4 is upregulated during colorectal carcinogenesis. Therefore, we investigated the role of elevated PGE2-EP4 receptor signalling in the protumorigenic activity of PGE2 by increasing EP4 receptor expression in HT-29 human colorectal cancer (CRC) cells (HT-29-EP4) by stable transfection. Elevated PGE2-induced EP4 receptor activity in HT-29 cells increased resistance to spontaneous apoptosis and promoted anchorage-independent growth, but had no effect on proliferation of HT-29-EP4 cells. EP4 receptor activation by PGE2 in HT-29-EP4 cells also led to development of fluid-filled cysts, which was associated with increased tight junction protein (occludin and zonula occludens-1) expression. Overexpression of the EP4 receptor in HT-29 cells led to basal EP4 receptor signalling in the absence of exogenous PGE2, which was explained by autocrine activity of endogenous, COX-2-derived PGE2 and constitutive, ligand-independent EP4 receptor activity. The predominant signalling pathway mediating antiapoptotic activity downstream of PGE2-EP4 receptor activation in HT-29-EP4 cells was elevation of cyclic adenosine monophosphate (cAMP) levels, which was associated with phosphorylation of cAMP-response element binding protein. EP4 receptor activation led to a small increase in phosphorylated extracellular signal-regulated kinase (ERK) 2 protein levels but inhibition of ERK phosphorylation did not abrogate the antiapoptotic activity of PGE2. However, PGE2-EP4 receptor signalling did not lead to trans-activation of the epidermal growth factor receptor in HT-29 cells. Inhibition of protumorigenic PGE2-EP4 receptor signalling represents a potential strategy for anti-CRC therapy that may avoid the toxicity associated with systemic COX inhibition.

Effect of nonsteroidal anti-inflammatory drugs on beta-catenin protein levels and catenin-related transcription in human colorectal cancer cells.

Elevated beta-catenin levels in human colorectal cancer (CRC) cells lead to increased trans-activation of 'protumorigenic' beta-catenin/T-cell factor (TCF) target genes such as cyclin D1. Therefore, possible targets for the anti-CRC activity of nonsteroidal anti-inflammatory drugs (NSAIDs) are beta-catenin and catenin-related transcription (CRT). We tested the antiproliferative activity and the effects on levels of beta-catenin and cyclin D1 protein, as well as CRT (measured using a synthetic beta-catenin/TCF-reporter gene [TOPflash]), of a panel of NSAIDs (indomethacin, diclofenac, sulindac sulphide and sulphone, rofecoxib; range 10-600 microM) on SW480 human CRC cells in vitro. Following NSAID treatment, there was no consistent relationship between reduced cell proliferation, induction of apoptosis and changes in beta-catenin protein levels or CRT. All the NSAIDs, except rofecoxib, decreased nuclear beta-catenin content and cyclin D1 protein levels in parallel with their antiproliferative activity. However, cyclin D1 downregulation occurred prior to a decrease in total beta-catenin protein levels and there was no correlation with changes in CRT, suggesting the existence of CRT-independent effects of NSAIDs on cyclin D1 expression. In summary, NSAIDs have differential effects on beta-catenin protein and CRT, which are unlikely to fully explain their effects on cyclin D1 and their antiproliferative activity on human CRC cells in vitro. British Journal of Cancer (2004) 91, 153-163. doi:10.1038/sj.bjc.6601901 www.bjcancer.com Published online 8 June 2004

Activity of the non-steroidal anti-inflammatory drug indomethacin against colorectal cancer.

A substantial body of evidence from rodent colon carcinogenesis models, in vitro experiments with human colorectal cancer cells and limited clinical observations in humans suggest that the non-steroidal anti-inflammatory drug indomethacin has anti-colorectal cancer activity. However, although many mechanisms of the anti-neoplastic activity of indomethacin have been suggested, e.g., cyclooxygenase inhibition and peroxisome proliferator-activated receptor gamma activation, the precise relevance of the majority of in vitro pharmacological observations to the in vivo anti-neoplastic activity of indomethacin remains unclear. Herein, we review the existing literature describing the chemopreventative and chemotherapeutic efficacy of indomethacin against colorectal cancer, and draw together the disparate literature describing potential mechanisms of action of indomethacin in human colorectal cancer cells in vitro. Although indomethacin itself has significant adverse effects, including serious upper gastrointestinal toxicity, the development of novel derivatives that may have an improved safety profile means that further investigation of the anti-colorectal cancer activity of indomethacin is warranted.

Activation of peroxisome proliferator-activated receptor gamma does not explain the antiproliferative activity of the nonsteroidal anti-inflammatory drug indomethacin on human colorectal cancer cells.

The mechanism of the anticolorectal cancer activity of the nonsteroidal anti-inflammatory drug indomethacin is poorly understood. Indomethacin inhibits both cyclooxygenase (COX) isoforms, but it may also act via COX-independent targets. Indomethacin can bind and activate the transcription factor peroxisome proliferator-activated receptor (PPAR) gamma. Moreover, natural and synthetic PPARgamma ligands can induce growth arrest and apoptosis of human colorectal cancer cells in vitro. Therefore, we tested the hypothesis that the antiproliferative activity of indomethacin on human colorectal cancer cells in vitro is explained by a PPARgamma-dependent mechanism of action. Human colorectal cancer cell lines SW480 and HCT116 both expressed functional PPARgamma. Indomethacin directly activated PPARgamma in both cell lines (HCT116 > SW480). A dominant-negative PPARgamma strategy was used to demonstrate that endogenous PPARgamma represses proliferation of HCT116 cells (compatible with tumor suppressor activity) but that the presence of functional PPARgamma is not necessary for the antiproliferative activity (or reduction in cyclin D1 protein) associated with indomethacin in vitro. In summary, indomethacin (>100 microM) directly activates PPARgamma in human colorectal cancer cells. However, PPARgamma activation does not underlie the antineoplastic activity of indomethacin on human colorectal cancer cells in vitro.

The effect of non-steroidal anti-inflammatory drugs on human colorectal cancer cells: evidence of different mechanisms of action.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit proliferation and induce apoptosis in human colorectal cancer cells in vitro. It remains unclear whether individual NSAIDs act by cyclooxygenase-2 (COX-2) inhibition and how NSAIDs exert their anti-proliferative effects. We investigated the effects of NS-398 (a selective COX-2 inhibitor), indomethacin (a non-selective COX inhibitor) and aspirin on four human colorectal cancer cell lines (HT29.Fu, HCA-7, SW480 and HCT116). NS-398 completely inhibited proliferation, induced G1 arrest and promoted apoptosis in COX-2-expressing cells (HT29.Fu and HCA-7). However, indomethacin had similar effects on all cells, regardless of COX-2 expression. NS-398 also had anti-proliferative activity on COX-2-negative cell lines (SW480 and HCT116). Aspirin inhibited proliferation of all cell lines but did not induce apoptosis. Indomethacin decreased beta-catenin protein expression in all cells (unlike NS-398 or aspirin). NSAIDs act on human colorectal cancer cells via different mechanisms. Decreased beta-catenin protein expression may mediate the anti-proliferative effects of indomethacin.

Localization of cyclooxygenase-2 in human sporadic colorectal adenomas.

A putative target for the anti-colorectal cancer action of nonsteroidal anti-inflammatory drugs is the inducible isoform of cyclooxygenase (COX), COX-2. COX-2 is expressed within intestinal adenomas in murine polyposis models, but expression has been poorly characterized in human colorectal neoplasms. Therefore, we investigated the localization of the COX-2 protein in human sporadic colorectal adenomas. Immunohistochemistry for COX-2 and CD68 (a tissue macrophage marker) was performed on formalin-fixed, paraffin-embedded (n = 52) and frozen, acetone-fixed (n = 6) sections of human sporadic colorectal adenomas. Forty of 52 (77%) formalin-fixed adenomas expressed immunoreactive COX-2. COX-2 was localized to superficial interstitial macrophages in 39 cases (75%) and to deep interstitial macrophages in 9 cases (17%). COX-2 staining of dysplastic epithelial cells was observed in 15 cases (29%). A logistic regression analysis identified the adenoma site (P = 0.012) and histological type (P = 0.001) as independent predictors of superficial macrophage COX-2 expression. There was no relationship between the number of macrophages within an adenoma and macrophage COX-2 expression. These results indicate that COX-2 is expressed predominantly by interstitial macrophages within human sporadic colorectal adenomas. If COX-2 does indeed play a role in the early stages of colorectal carcinogenesis in man, these data suggest COX-2-mediated paracrine signaling between the macrophages and epithelial cells within adenomas.

KLP38B: a mitotic kinesin-related protein that binds PP1.

We have identified a new member of the kinesin superfamily in Drosophila, KLP38B (kinesin-like protein at 38B). KLP38B was isolated through its two-hybrid interaction with the catalytic subunit of type 1 serine/threonine phosphoprotein phosphatase (PP1). We demonstrate that recombinant KLP38B and PP1 associate in vitro. This is the first demonstration of direct binding of a kinesin-related protein to a regulatory enzyme. Though most closely related to the Unc-104 subfamily of kinesin-related proteins, KLP38B is expressed only in proliferating cells. KLP38B mutants show cell proliferation defects in many tissues. KLP38B is required for normal chromatin condensation as embryos from KLP38B mutant mothers have undercondensed chromatin at metaphase and anaphase. This is the first time that a kinesin-related protein has been shown to have such a role. Incomplete lethality of a strong KLP38B allele suggests partial redundancy with one or more additional kinesin-related proteins.