title: COX-2 inhibitors sensitize human hepatocellular carcinoma cells to TRAIL-induced apoptosis.
abstract: Cyclooxygenase (COX)-2 is upregulated in a variety of human cancers, including in hepatocellular carcinoma (HCC), whereas it is undetectable in most normal tissue. Evidence suggests that COX-2 is likely to be involved in hepatocarcinogenesis and, thus, COX-2 may be involved in an early process in carcinogenesis, dedifferentiation. To address this possibility, we investigated the effect of COX-2 inhibitors on TNF-related apoptosis, inducing ligand (TRAIL) sensitivity and its molecular mechanisms, with special attention to anti-apoptotic proteins. We used the highly selective COX-2 inhibitors, NS398 and CAY10404. We also used the MTT assay and cytological analysis of DAPI-stained DNA to assess viability and apoptosis in two HCC cells (SK-Hep1 and HLE). In order to ask what led to increased sensitivity to TRAIL in HCC cells, cell surface expression of TRAIL and TRAIL-receptors was investigated using flow cytometry analysis. Expression of survivin, X-chromosome-linked IAP (XIAP), Bcl-xL, AKT and phospho-AKT was also investigated using immunoblotting. COX-2 inhibitors resulted in a concentration-dependent decrease in cell viability in the two HCC cell lines tested. Subtoxic levels of COX-2 inhibitors did not significantly augment TNFalpha-induced apoptosis but did dramatically enhance TRAIL-induced apoptosis in both cell lines. TRAIL receptor 2/death receptor 5 (TRAIL-R2/DR5) expression was significantly up-regulated in SH-Hep1 and HLE cells. TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) expression was up-regulated only in SK-Hep1. Expression of survivin and Bcl-xL was down-regulated in SK-Hep1 and HLE cells in the presence of CAY10404 but XIAP was not affected. Expression of survivin, Bcl-xL and XIAP was down-regulated in SK-Hep1 cells in the presence of NS398. Survivin expression was also down-regulated in the presence of NS398 in HLE cells. Finally, NS398 also decreased phospho-AKT in SK-Hep1 cells. These results demonstrate that COX-2 inhibitors can induce apoptosis and augment TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of both survivin and AKT signaling.
authors.affiliation: First Department of Internal Medicine, Mie University School of Medicine, Tsu, Mie 514-8507, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Yamanaka
authors.forename: Katsuya
authors.initials: K
authors.lastname: Shiraki
authors.forename: Tomoko
authors.initials: T
authors.lastname: Inoue
authors.forename: Kazumi
authors.initials: K
authors.lastname: Miyashita
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Fuke
authors.forename: Yumi
authors.initials: Y
authors.lastname: Yamaguchi
authors.forename: Norihiko
authors.initials: N
authors.lastname: Yamamoto
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ito
authors.forename: Kazushi
authors.initials: K
authors.lastname: Sugimoto
authors.forename: Takeshi
authors.initials: T
authors.lastname: Nakano
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism / Q000494:pharmacology*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D051034:Inhibitor of Apoptosis Proteins; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D008562:Membrane Glycoproteins / Q000378:metabolism / Q000494:pharmacology*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D000077022:Survivin; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology*; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C494811:BCL2L1 protein, human; C107489:BIRC5 protein, human; D052246:Cyclooxygenase 2 Inhibitors; D051034:Inhibitor of Apoptosis Proteins; D008562:Membrane Glycoproteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D000077022:Survivin; D053221:TNF-Related Apoptosis-Inducing Ligand; C505639:TNFRSF10B protein, human; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D051020:bcl-X Protein