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Role of Glypican-3 in the growth, migration and invasion of primary hepatocytes isolated from patients with hepatocellular carcinoma

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Abstract

Background

Recently, Glypican-3 (GPC3) has been identified as a potential hepatocellular carcinoma (HCC) diagnostic and/or therapeutic target. GPC3 has been found to be up-regulated in HCC and to be absent in normal and cirrhotic liver. As yet, however, the molecular characteristics of GPC3 and its role in HCC cell physiology and development are still undefined.

Methods

Human hepatocyte cultures were established from 10 HCC patients. Additional liver samples were obtained from 5 patients without cirrhosis and/or HCC. Soft agar colony formation, (co-)immunofluorescence and Western blot assays were used to characterize the hapatocyte cultures. The expression of GPC3 in the hepatocytes was silenced using siRNA, after which, apoptosis, scratch wound migration and transwell invasion assays were performed.

Results

We found that in HCC precursor hepatocytes GPC3 is increasingly expressed in different forms and at different locations, i.e., a non-cleaved form (70 kDa) was found to be localized in the cytoplasm while a N-terminal cleaved form (N-GPC3: 40 kDa) was fond to be localized in the cytoplasm and at the extracellular side of hepatocyte membranes. In addition, we found that the non-cleaved form of GPC3 co-localizes with Furin-Convertase in the Golgi apparatus. We also found that, similar to GPC3, Furin-Convertase is expressed in HCC precursor cells, suggesting a role in GPC3 processing. Subsequent siRNA-mediated GPC3 silencing resulted in a temporary inhibition of cell proliferation, migration and ivasion, while inducing apoptosis in transformed hepatocytes.

Conclusion

Our data reveal new aspects of the role of GPC3 in early hepatocyte transformation. In addition we conclude that GPC3 may serve as a new HCC immune-therapeutic target.

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Abbreviations

αSMA:

α-smooth muscle actin

BSA:

bovine serum albumin

CD:

cirrhotic distal tissue

CD-Hep:

hepatocytes from distal cirrhotic liver

CKI:

Cyclin Kinase Inhibitor

CP:

cirrhotic proximal tissue

CP-Hep:

hepatocytes from proximal liver tissue

ECM:

extracellular matrix

FBS:

fetal bovine serum

GPC3:

Glypican-3

HBV:

hepatitis B virus

HCC:

hepatocellular carcinoma

HCV:

hepatitis C virus

HSC:

hepatic stellate cell

HSPG:

heparan sulphate proteoglycan

IHC:

immunohystochemistry

LTX:

liver transplantation

NAFLD/NASH:

non-alcoholic fatty liver disease/non-alcoholic steatohepatitis

PHH:

primary human hepatocytes

GPI:

glycosylphosphatidylinositol

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Authors and Affiliations

  1. Department of Surgery, University of Texas Medical Branch, Galveston, TX, USA

    Mauro Montalbano, Cristiana Rastellini, Joshua T. McGuire, Janika Prajapati, Ali Shirafkan & Luca Cicalese

  2. Center of Biomedical Engineering, University of Texas Medical Branch, Galveston, TX, USA

    Mauro Montalbano

  3. Laboratory of Biochemistry, Department of Biological, Chemical and Pharmaceutical Sciences and Technologies, Polyclinic, University of Palermo, Palermo, Italy

    Renza Vento

  4. Institute for Cancer Research and Molecular Medicine and Center of Biotechnology, College of Science and Biotechnology, Temple University, Philadelphia, PA, USA

    Renza Vento

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  1. Mauro Montalbano
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Supplemental Figure 1

(A) Schematic representation of protocol used to isolate hepatocytes from human liver specimen. (B) Morphologic features of cultured cells obtained from HCC, CP, CD and NL tissues. Cells in primary culture at time 0, 1st and 8th passages. All primary cells proliferate in-vitro maintaining a shape hepatocytes-like in NL- and CD-Hep while showed a spindle-like shape in CP-Hep and HCC. At 8th passages were observed aggregates in CP-Hep and HCC cultures (white bar: 100 μm, magnification: 100X). (TIFF 1435 kb)

High resolution image (GIF 117 kb)

Supplemental Figure 2

(A) GPC-3 immunofluorescence in margin human CP-HCC tissue. (B) Co-immunofluorescence of N-GPC3 and C-GPC3 with β-Tubulin confirmed that co-localized, while C-GPC3 expressed in the central area of the cell has not interaction with microtubule system in CP-Hep. In HCC cells N- and C-terminal colocalize in the cell and showed an overlap with β-Tubulin (white bar: 200 μm, magnification: 100X). (TIFF 6829 kb)

High resolution image (GIF 137 kb)

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Montalbano, M., Rastellini, C., McGuire, J.T. et al. Role of Glypican-3 in the growth, migration and invasion of primary hepatocytes isolated from patients with hepatocellular carcinoma. Cell Oncol. 41, 169–184 (2018). https://doi.org/10.1007/s13402-017-0364-2

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