title: Differential sensitivity of hepatocellular carcinoma cells to suppression of hepatocystin transcription under hypoxic conditions.
abstract: Mutations in the gene encoding hepatocystin/80 K-H (PRKCSH) cause autosomal dominant polycystic liver disease. Hepatocystin deficiency impairs glucosidase II activity, which is critical for processing and folding glycoproteins in the endoplasmic reticulum (ER). Hypoxia is known as a strong stimulus for generating survival signals in hepatocellular carcinoma (HCC) cells. However, hypoxia may induce cell apoptosis under conditions of severe ER stress. Thus, we hypothesized that suppression of hepatocystin transcription induces HCC cell death under hypoxic conditions due to excessive ER stress. A new human HCC cell line, SNU-3058, was established following primary culture of tumor cells harvested from a Korean patient with rapidly growing hypovascular HCC. In cell culture, human HCC cells (Huh-7, SNU-761, and SNU-3058) were treated with control siRNA or hepatocystin siRNA with or without doxorubicin under hypoxic conditions. Cell viability, ER stress, unfolded protein response (UPR), and apoptosis were assessed using the MTS assay, immunoblot assay, and RT-PCR. Suppression of hepatocystin transcription attenuated proliferation in Huh-7 and SNU-761 cells, while proliferation was amplified in SNU-3058 cells. Similar results were observed following treatment with doxorubicin. Hepatocystin siRNA transfection increased cell death in Huh-7 and decreased cell death in SNU-3058. In SNU-3058, hepatocystin siRNA amplified GRP78, known as a pro-survival and cyto-protective signal, and attenuated the pro-apoptotic signal CHOP. These findings suggest that suppression of hepatocystin transcription induce the UPR, which alleviates damage associated with ER stress in SNU-3058. UPR had a limited role in protecting SNU-761 cells, resulting in cell death through apoptosis. In addition, blocking of pro-survival UPR signal by bacitracin or GRP78 knockdown, attenuated hepatocystin siRNA-induced proliferation in SNU-3058 cells under hypoxia. In this study, we demonstrated that different sensitivities to hepatocystin siRNA among human HCC cell lines are dependent on appropriate UPRs to hypoxia-induced ER stress following hepatocystin siRNA transfection. Because UPR is the main evasive mechanism for apoptosis induced by suppression of hepatocystin, targeting hepatocystin via UPR suppression could be a strategy for treating HCC.
authors.affiliation: Department of Gastroenterology and Hepatology, Soonchunhyang University Bucheon Hospital, Gyeonggi do, Republic of Korea.
authors.forename: Jeong-Ju
authors.initials: JJ
authors.lastname: Yoo
authors.affiliation: Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
authors.forename: Dong Hyeon
authors.initials: DH
authors.lastname: Lee
authors.affiliation: Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Cho
authors.affiliation: Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
authors.forename: Eun Ju
authors.initials: EJ
authors.lastname: Cho
authors.affiliation: Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
authors.forename: Jeong-Hoon
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
authors.forename: Su Jong
authors.initials: SJ
authors.lastname: Yu
authors.affiliation: Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
authors.forename: Yoon Jun
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Republic of Korea.
authors.forename: Chung Yong
authors.initials: CY
authors.lastname: Kim
authors.affiliation: Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul National University Hospital, Seoul, Republic of Korea. yoonjh@snu.ac.kr.|Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul, 110-744, Republic of Korea. yoonjh@snu.ac.kr.
authors.forename: Jung-Hwan
authors.initials: JH
authors.lastname: Yoon
mesh_terms: D017209:Apoptosis; D002135:Calcium-Binding Proteins; D006528:Carcinoma, Hepatocellular / Q000473:pathology*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress; D015972:Gene Expression Regulation, Neoplastic; D005959:Glucosidases / Q000235:genetics*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D008113:Liver Neoplasms / Q000473:pathology*; D034741:RNA, Small Interfering; D014158:Transcription, Genetic; D056811:Unfolded Protein Response
chemical_list: D002135:Calcium-Binding Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D005959:Glucosidases; C070368:PRKCSH protein, human
keywords: Apoptosis; Hepatocellular carcinoma; Hepatocystin; Unfolded protein response