The aim of this study was to investigate the expression of prostaglandin E(2) receptors (EP(1-4)), cyclooxygenase-1 (COX-1), and COX-2 in nontumor and tumor human liver tissues, and also to evaluate the antitumor activity of selective EP(1) receptor antagonist used alone or in combination with COX-1 and COX-2 selective inhibitors. Semiquantitative PCR analyses revealed that EP(1-4), COX-1, and COX-2 mRNA expression was detected in nearly all the tissue samples assayed, although with a high variability between nontumor and tumor tissues. In vitro EP(1) receptor antagonist inhibited anchorage-independent cell growth and reduced the viability of hepatocellular carcinoma (HCC) cells in a dose-dependent manner. Moreover, treatment with the combination of EP(1) receptor antagonist and COX inhibitors produced a significantly greater cell growth inhibition than the single agent alone. These findings suggest that the EP(1) receptor may represent an important target for HCC treatment, and in addition they could provide preclinical support for a combined chemotherapeutic approach with EP(1) antagonists and COX inhibitors in the treatment of liver cancer.

The molecular mechanisms behind the anti-neoplastic effects of non-steroidal anti-inflammatory drugs (NSAIDs) are not completely understood and cannot be explained by the inhibition of the cyclooxygenase (COX) enzymes COX-1 and COX-2 alone. We previously reported that both the selective COX-1 inhibitor SC-560 and the selective COX-2 inhibitor CAY10404 exhibit anti-tumor effects in human hepatoma cells. NSAID inhibitors have many COX-independent actions and, among others, the mitogen-activated protein kinase (MAPK) pathways are targets for NSAIDs. Here, we examined the role of MEK/ERK1/2 signaling in the anti-neoplastic effects of both selective COX-1 and COX-2 inhibitors in two human hepatoma cell lines. Treatment of hepatoma cells with the selective COX-1 inhibitor SC-560, as well as with the selective COX-2 inhibitor CAY10404, was associated with activation of ERK1/2 in a time- and dose-dependent manner. Treatment with COX-1 and COX-2 inhibitors in the presence of the selective MEK1/2 inhibitor U0126 effectively suppressed ERK1/2 activation and combinations of either SC-560 or CAY10404 with U0126 resulted in synergistic effects on cell growth inhibition and induction of apoptosis. In HuH-6 hepatoma cells the combination-induced apoptosis was associated with caspase-9 and -3 activation, PARP cleavage, release of cytochrome c from the mitochondria into the cytosol and down-regulation of survivin and β-catenin levels. In conclusion, our study showed that growth inhibitory concentrations of selective COX-1 and COX-2 inhibitors increased ERK1/2 phosphorylation in hepatoma cells, and that inhibition of the MEK/ERK signaling pathway potentiates the antitumor activity of both types of inhibitors. Therefore, our results provide preclinical support for a combined chemotherapeutic approach with selective NSAIDs and MEK inhibitors for the treatment of hepatocellular carcinoma.

Two isoforms of cyclooxygenase (COX) are known, and to date most studies have implicated COX-2 in the development and progression of various human cancers. Increasing evidence suggests that COX-1 may also play a similar role. Indeed, we have recently observed that the dual COX-1/COX-2 inhibitor indomethacin induces apoptosis in human hepatocellular carcinoma (HCC) cell lines more effectively than the selective COX-2 inhibitors, possibly implicating COX-1 in HCC. In this study we investigated the expression of COX-1 in non-tumor and malignant human liver tissues, as well as the effects of the highly selective COX-1 inhibitor SC-560 on cell growth and apoptosis in human HCC cell lines. Expression of COX-1 was detected in nearly all the samples assayed, although with a high variability between non-tumoral (NT) and malignant tissues. The percentage of COX-1 positive cells was significantly higher in the NT tissues than in the tumors (p<0.0001). In well-differentiated HCC COX-1 expression was significantly higher than in the poorly-differentiated tissues (p<0.05). SC-560 showed a dose- and time-dependent inhibitory effect on HCC cell growth. The combination of the COX-1 inhibitor with nimesulide and CAY10404, two selective COX-2 inhibitors, resulted in additive effects on cell growth inhibition. SC-560 also inhibited colony formation in soft agar and induced apoptosis in HCC cells in a dose-dependent manner. Moreover, SC-560 decreased the levels of the anti-apoptotic proteins survivin and XIAP and activated caspase-3 and -7 in a dose- and time-dependent fashion. In conclusion, we report for the first time that the selective COX-1 inhibitor SC-560 exhibits anti-tumor and apoptotic effects in human HCC cells. Overall, our previous and present results suggest that both COX-1 and COX-2 inhibitors may have potential therapeutic implications in HCC patients.

AIM:To investigate the association of cyclooxygenase-2 (COX-2) expression with angiogenesis and the number and type of inflammatory cells (macrophages/Kupffer cells; mast cells) within primary hepatocellular carcinoma (HCC) tissues and adjacent non-tumorous (NT) tissues. METHODS:Immunohistochemistry for COX-2, CD34, CD68 and mast cell tryptase (MC T ) was performed on 14 well-characterized series of liver-cirrhosis-associated HCC patients.COX-2 expression and the number of inflammatory cells in tumor lesions and surrounding liver tissues of each specimen were compared.Moreover, COX-2, CD34 staining and the number of inflammatory cells in areas with different histological degrees within each tumor sample were comparatively analyzed. RESULTS:The percentage of COX-2 positive cells was significantly higher in NT tissues than in tumors.COX-2 expression was higher in well-differentiated HCC than in poorly-differentiated tissues.Few mast cells were observed within the tumor mass, whereas a higher number was observed in the surrounding tissue, especially in peri-portal spaces of NT tissues.Abundant macrophages/ Kupffer cells were observed in NT tissues, whereas the number of cells was significantly lower in the tumor mass.However, a higher cell number was observed in the welldifferentiated tumor and progressively decreased in relation to the differentiation grade.Within the tumor, a positive correlation was found between COX-2 expression and the number of macrophages/Kupffer cells and mast cells.Moreover, there was a positive correlation between CD34 and COX-2 expression in tumor tissues.Comparison between well-and poorly-differentiated HCC showed that the number of CD34-positive cells decreased with dedifferentiation.However, COX-2 was the only independent variable showing a positive correlation with CD34 in a multivariate analysis. CONCLUSION:The presence of inflammatory cells and COX-2 expression in liver tumor suggests a possible relationship with tumor angiogenesis.COX-2 expressing cells and the number of macrophages/Kupffer cells and mast cells decrease with progression of the disease.

Abstract: The aim of the present study was to examine the effects of nonselective (indomethacin) and selective cyclooxygenase‐2 (COX‐2) inhibitors (NS‐398, nimesulide, and CAY10404) on cell growth, cell cycle distribution, and apoptosis in three human hepatocellular carcinoma cell lines (HepG2, HuH‐6, and HA22T/VGH) with different characteristics of differentiation and biological behavior. The four COX inhibitors showed a dose‐dependent growth‐inhibitory effect in all the cell lines. No substantial arrests in the progression of the cells through the cell cycle were observed after treatment of HuH‐6 or HA22T/VGH for 48 h with the various inhibitors. On the other hand, there were significant increases in apoptosis, with the highest effect of cell kill being seen after treatment with indomethacin, especially in HuH‐6. Our findings support the suggestion that selective or, perhaps more efficiently, nonselective COX‐2 inhibitors may have potential therapeutic effects in hepatocellular carcinoma. Further studies must be carried out to better determine the possible mechanisms of these effects.

A bstract : The aim of the present study was to examine the effects of nonselective (indomethacin) and selective cyclooxygenase-2 (COX-2) inhibitors (NS-398, nimesulide, and CAY10404) on cell growth, cell cycle distribution, and apoptosis in three human hepatocellular carcinoma cell lines (HepG2, HuH-6, and HA22T/VGH) with different characteristics of differentiation and biological behavior. The four COX inhibitors showed a dose-dependent growth-inhibitory effect in all the cell lines. No substantial arrests in the progression of the cells through the cell cycle were observed after treatment of HuH-6 or HA22T/VGH for 48 h with the various inhibitors. On the other hand, there were significant increases in apoptosis, with the highest effect of cell kill being seen after treatment with indomethacin, especially in HuH-6. Our findings support the suggestion that selective or, perhaps more efficiently, nonselective COX-2 inhibitors may have potential therapeutic effects in hepatocellular carcinoma. Further studies must be carried out to better determine the possible mechanisms of these effects.