Cytoskeletal changes during epithelial-to-fibroblastoid conversion as a crucial mechanism of des-gamma-carboxy prothrombin production in hepatocellular carcinoma.
Des-gamma-carboxy prothrombin (DCP) is a well-established tumor marker for hepatocellular carcinoma (HCC), but the precise mechanism by which HCC cells produce DCP remains unknown. Our preliminary experiments demonstrated that HepG2 cells with chemical induction of epithelial-to-fibroblastoid conversion (EFC) produced DCP through impairment of vitamin K uptake via cytoskeletal rearrangement. Therefore, in this study we further examined this mechanism in vitro and using human HCC samples. Hepatoma cell lines (HepG2 and PLC/PRF/5) were induced EFC or epithelial-mesenchymal transition (EMT) by phorbol 12-myristate 13 acetate (TPA) or transforming growth factor (TGF)-beta1. We analyzed these cells by ELISA, Western blotting and immunofluorescent studies. We also examined DCP production and E-cadherin expression in human surgically resected HCC samples by immunohistochemical studies. Labeled low-density lipoprotein (LDL) uptake (as a surrogate for vitamin K) was significantly impaired in DCP-producing hepatoma cells following induction of EFC or EMT. Further, filamentous actin, which plays a critical role in clathrin-mediated endocytosis, was dissociated in DCP-producing cells. Latrunculin A, an actin depolymerizer, induced naïve hepatoma cells to produce DCP with impairment of labeled-LDL uptake. In addition, an E-cadherin neutralizing antibody did not induce DCP production. Finally, immunohistochemical studies demonstrated that DCP production was inversely correlated with the intensity of E-cadherin expression in human HCC cells. In conclusion, cytoskeletal changes during EFC or EMT plays a critical mechanistic role in DCP production via impairments in vitamin K uptake.
Center for Community Medicine, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan. atarum@jichi.ac.jp
Kazumoto
K
Murata
Hideto
H
Suzuki
Hiroshi
H
Okano
Takashi
T
Oyamada
Yoshikazu
Y
Yasuda
Atsushi
A
Sakamoto
D000328:Adult; D000368:Aged; D015415:Biomarkers; D014408:Biomarkers, Tumor / Q000032:analysis; D015153:Blotting, Western; D015820:Cadherins / Q000096:biosynthesis; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D008078:Cholesterol, LDL / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism / Q000473:pathology*; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000473:pathology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D008113:Liver Neoplasms / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008648:Mesoderm / Q000473:pathology; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D011498:Protein Precursors / Q000096:biosynthesis*; D011516:Prothrombin / Q000096:biosynthesis*; D014812:Vitamin K / Q000378:metabolism
D015415:Biomarkers; D014408:Biomarkers, Tumor; D015820:Cadherins; D008078:Cholesterol, LDL; D011498:Protein Precursors; D014812:Vitamin K; C008208:acarboxyprothrombin; D011516:Prothrombin