NS‐398, a selective cyclooxygenase 2 inhibitor, inhibited cell growth and induced cell cycle arrest in human hepatocellular carcinoma cell lines

Abstract

Cyclooxygenase 2 (COX-2) has been suggested to be associated with liver carcinogenesis. Several reports have shown that NSAIDs inhibit the growth of hepatocellular carcinoma cell lines. There is little evidence of how COX-2 inhibitors regulate the proliferation of hepatocellular carcinoma cells or the mechanism involved. In our study, we investigated the growth-inhibitory mechanism of a selective COX-2 inhibitor, NS-398, in 4 hepatocellular carcinoma cell lines by studying cell growth, COX-2 and proliferating cell nuclear antigen (PCNA) expression, cell cycle distribution and the evidence of apoptosis. NS-398 inhibited the growth of all 4 cell lines in a time-and dose-dependent manner and the inhibitory effects were independent of the level of COX-2 protein expression. PCNA expression was downregulated by NS-398 in a dose-independent manner. NS-398 caused cell cycle arrest in the S phase with a reduction in cell numbers and cell accumulation in the G0/G1 phase, for all 4 cell lines. No evidence of apoptosis was observed in our present study. Key words: selective COX-2 inhibitor; cell growth; cell cycle; hepatocellular carcinoma cellsCyclooxygenases (COX) are key rate-limiting enzymes involved in the conversion of arachidonic acid to prostaglandin H2, the precursor of various compounds including prostaglandins (PGs), prostacyclin and thromboxanes. There are at least 2 isoforms of COX: COX-1 and COX-2. COX-1 is expressed constitutively in a wide variety of tissues whereas COX-2 is highly inducible and is expressed in response to a variety of proinflammatory agents and cytokines. 1,2 Overexpression of COX-2 has been demonstrated in various tumor tissues such as colon cancer, pancreatic cancer and hepatocellular carcinoma. [3][4][5][6][7] Although several studies have shown the upregulation of COX-2 in cirrhotic tissues adjacent to hepatocellular carcinoma (HCC) and welldifferentiated HCC, the precise role of COX-2 in carcinogenesis remains unclear. 7,8 A few reports have shown that NSAIDs inhibited the growth of various cancer cell lines including those derived from hepatocellular carcinoma. 9 -11 The question arises whether COX-2 also is a target for the prevention or treatment of hepatocellular carcinoma, as it is for colon cancer. 12 There is little evidence, however, of how COX-2 inhibitors regulate the proliferation of hepatocellular carcinoma cells or the mechanism involved. In our study, we investigated the growth-inhibitory mechanism of a selective COX-2 inhibitor, NS-398, in hepatocellular carcinoma cell lines. MATERIAL AND METHODS Cell lines and cell cultureFour human hepatocellular carcinoma cell lines, PLC/PRF5, HepG2, Mahlavu and HuH-7, were obtained from the American Type Culture Collection. Cells (2 ϫ 10 5 cells) were grown for 24 hr in DMEM (PLC/PRF5 and HepG2) (ICN; Biomedicals Inc., Aurora, MI), minimum essential Eagle's medium (Mahlavu) (ICN) and RPMI 1640 (HuH-7) (ICN) supplemented with 10% FBS at 37°C under 5% CO 2 /95% air in 100 mm 2 cell culture dishes. Then, NS-398 (Cayman Chemical, Ann Ar...

Cyclooxygenase-2 selective inhibition with NS-398 suppresses proliferation and invasiveness and delays liver metastasis in colorectal cancer

“…Another large population-based survey reported that the use of NSAIDs significantly reduced the risk of both gastric and oesophageal carcinoma (Farrow et al, 1998). Although the precise mechanisms by which NSAIDs, and COX-2 selective inhibitors in particular, modulate tumour growth have not been elucidated, it appears that cell cycle arrest and apoptosis may play a critical role (Chang and Weng, 2001;Grosch et al, 2001;Cheng et al, 2002;Joe et al, 2002;Kundu et al, 2002;Toyoshima et al, 2002). Alternatively, Sawaoka et al (1998) and Tsujii et al (Tsujii and DuBois, 1995;Sawaoka et al, 1998;Tsujii et al, 1998) have suggested that a decrease in angiogenesis may account for the inhibitory properties of COX-2 selective NSAIDs on tumour growth.…”

Involvement of cell cycle regulatory proteins and MAP kinase signaling pathway in growth inhibition and cell cycle arrest by a selective cyclooxygenase 2 inhibitor, etodolac, in human hepatocellular carcinoma cell lines

“…Similar results have been reported for pancreatic and colorectal cancer and HCC cell lines. 4,9,30) Some mechanism different from COX-2 expression and PG production might be involved in growth inhibition by etodolac in HCC cells. To understand the mechanism of growth inhibition of HCC by COX-2 inhibition, we studied how etodolac regulates the expression of several key cell cycle regulatory proteins and growth factor signaling pathways.…”

“…5,8) A recent report of ours has also shown that NS-398, a selective COX-2 inhibitor, inhibited growth and induced cell cycle arrest in HCC cell lines. 9) However, the mechanism by which COX-2 inhibitors cause growth inhibition and cell cycle arrest in HCC cells is not known.…”

Endoplasmic Reticulum Stress Stimulates the Expression of Cyclooxygenase-2 through Activation of NF-κB and pp38 Mitogen-activated Protein Kinase

“…COX-2 inhibitor is a chemoprevention agent for colon cancer, and its use has been proposed recently (69,70) to decrease the incidence of other types of cancer including gastric and lung cancer. COX-2 inhibitor is used to treat hepatoma (71,72), but its role in prevention is not clearly defined (73). Recently a COX-2 inhibitor was shown to prevent hepatocarcinogenesis in an animal model (74).…”