Cytoskeletal changes during epithelial-to-fibroblastoid conversion as a crucial mechanism of des-γ-carboxy prothrombin production in hepatocellular carcinoma

Abstract

Abstract. 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)-ß1. 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 clathrinmediated 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.

Hypoxia-induced des-γ-carboxy prothrombin production in hepatocellular carcinoma

“…Recently, we demonstrated the mechanism of DCP production in HCC, which demonstrated that impairment of vitamin K uptake through cytoskeletal changes during chemical induction of epithelial-to-fibroblastoid conversion (EFC) has a crucial role for DCP production (3,4). Importantly, our proposed mechanism is supported by several clinical observations.…”

“…Indeed, chronic chemotherapy induces EMT in colorectal cancer cells in vitro (16) and chemotherapy-resistance is associated with EMT in several cancer cell lines (17)(18)(19). We also demonstrated that E-cadherin repression, which is involved in the early recurrence of HCC after surgical treatment (10) or metastasis (11), is observed in DCP-producing HCC cells during EFC (3,4). Clinical evidence that high serum DCP in HCC patients is associated with a high risk of vascular invasion (7,8), intrahepatic metastasis (8) and tumor recurrence (9) may support our results because E-cadherin is a central component of cellcell adhesion junctions.…”

“…2B) cells, whereas linear E-cadherin expression around the cell boundary was observed clearly in both control cells. CoCl 2 -treated cells or cells cultured in 1% O 2 became scattered and adopted a fibroblast-like appearance, whereas control cells showed round-shaped and clustered forms, although it was not prominent as chemicals (3,4). In addition, hypoxia-stimulated cells with reduced E-cadherin expression produced DCP.…”

“…From the above accumulating evidence, we examined if hypoxia, which is a common feature during cancer development, could induce EFC or EMT, resulting in DCP production in HCC cells through the same mechanism as chemicals (3,4).…”

Enhanced Antitumor Effects of Novel Intracellular Delivery of an Active Form of Menaquinone-4, Menahydroquinone-4, into Hepatocellular Carcinoma

“…Recent studies have revealed that DCP functions as a growth and metastasis factor; it may play a significant role in cancer progression (19)(20)(21)(22)(23) and partly contributes to the poor prognosis of HCC (24). Previous reports demonstrated that tumor vitamin K content has a critical role in DCP formation (15,(25)(26)(27).…”