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INTRODUCTION vasodilator-stimulated phosphoprotein (VASP), and is
[13]
localized both at cell-matrix and cell-cell adhesions. [14]
Extracellular matrix (ECM), focal adhesions, and their
interaction with actin cytoskeleton are fundamental for In the present study, we tested the expression and
a number of vital cellular processes such as cell survival, molecular mechanism of the action of Fascin-1 in two
differentiation, development, and tissue homeostasis. This is liver cell lines that differ in terms of aggressiveness; the
particularly true for the organ liver as the main parenchymal hepatoma cell line PLC/PRF/5 (Alexander) and the highly
cells of the liver, hepatocytes, interact to a great extent with invasive HCC cell line HepG2.
ECM, and express a number of focal adhesions and actin-
related proteins. Moreover, the fact that ECM and ECM- METHODS
related proteins are fundamental for liver biology is evident
by a number of studies showing how they affect hepatocyte Liver cell lines
differentiation, survival, and normal liver function. More Two liver cell lines of different invasive capacity were used in
[3,4]
[1]
[2]
specifically, matrix overlay on primary hepatocytes cultured in the present study; the hepatoma cell line PLC/PRF/5 (Alexander)
vitro inhibits the dedifferentiation that normally occurs due to and HCC cell line HepG2. Both cell lines were purchased from
culturing and maintains hepatocytes in a fully differentiated American Type Culture Collection.
[5]
state. Along the same line, loss of a cell adhesion protein
related to integrins (namely, integrin-linked kinase) from Transfection with small interfering RNAs
mouse hepatocytes in vitro and in vivo leads to increased Both Alexander and HepG2 cells were treated for 48 h with
apoptosis and hepatitis, showing clearly that cell-ECM 100 nmol/L small interfering RNA (siRNA), non-specific control
[2]
interactions are critical for hepatocyte survival. Finally, liver- (NSC) siRNA, or siRNA against Fascin-1 using the Lipofectamine
specific ablation of integrin-linked kinase leads to abnormal 2,000 transfection reagent (Invitrogen, Carlsbad, CA, USA)
liver histology and increased liver regeneration capacity, according to the company’s guidelines. The siRNA sequence
[4]
[3]
indicating the crucial role played by cell-ECM adhesion used to silence Fascin was as purchased from Santa Cruz,
proteins in liver structure and function. while the sequence 5’AAA CUC UAU CUG CAC GCU GAC3’ was
used as NSC. Silencing efficiency prior to every experiment
Moreover, ECM plays central role during carcinogenesis in performed was tested by western blot.
the liver, as hepatocellular carcinoma (HCC) is known to
result from a series of events which include fibrosis, cirrhosis, Antibodies
generation of phenotypically-altered hepatocytes, and Anti-β-actin antibody (Sigma-Aldrich) was used as loading
[6]
dysplasia ultimately leading to HCC. Thus, cell adhesion control. Antibodies VASP and Fascin-1 were purchased from
proteins that connect cells to the ECM and/or to neighboring Cell Signaling. The monoclonal antibody against migfilin
cells are evidently fundamental for HCC development and was kindly provided by Dr. Chuanyue Wu (Professor at the
progression. Moreover, cell adhesion proteins are often University of Pittsburgh Medical School, Pittsburgh, PA, USA).
directly or indirectly connected to actin cytoskeleton creating
a network of interacting proteins that is crucial for tissue Protein extraction and western blot analysis
homeostasis. Interestingly, most of these proteins are found Total cell lysates were obtained using 1% sodium dodecyl
to be deregulated in cancer and cancer metastasis. This sulfate in radioimmunoprecipitation assay buffer (20 mmol/L
[7,8]
deregulation destabilizes cell attachment to the surrounding Tris/Cl pH 7.5, 150 mmol/L NaCl, 0.5% NP-40, 1% TX-100,
ECM and neighboring cells, thus facilitating cancer cells 0.25% sodium deoxycholate, 0.6-2 μg/mL aprotinin,10 μmol/L
dissociation from the original tumor mass and invasion of leupeptin, and 1 μmol/L pepstatin). Protein concentrations in
surrounding tissues, and ultimately leads to metastases the samples were determined by the BCA protein assay kit
formation. (Pierce) using bovine serum albumin as standard. An equal amount
of protein was loaded on each lane of a 10-12% acrylamide gel and
Fascin-1 is an actin-bundling protein that is found in membrane transferred to a PVDF membrane (Millipore) using the Bio-Rad
ruffles and stress fibers, and its expression has been shown Semi-dry transfer system (Bio-Rad, Hercules, CA, USA). Signals
to be greatly increased in many transformed cells and a were detected using suitable secondary immunoglobulin
[9]
spectrum of cancers such as breast cancer, colon cancer, G, conjugated with horseradish peroxidase (Invitrogen).
pancreatic cancer and prostate cancer. In HCC in particular, Antibody detection was performed using super-signal ECL
Fascin-1 has been correlated with poor prognosis. [10] detection system (Pierce).
Moreover, Fascin-1 has been suggested as a novel marker of Cell adhesion assay
HCC progression and a significant indicator of poor prognosis Cell adhesion assay was performed as described previously.
[13]
for HCC patients. However, little is known regarding the Briefly, cells were transfected with a control NSC siRNA or
[11]
molecular mechanism of its action. In a recent work, we have siRNA against Fascin-1. Forty-eight hours post-transfection,
shown that Fascin-1 is negatively regulated by migfilin, a 10 cells/ well were seeded in 6 wells of a 96-well plate pre-
4
[12]
novel cell-matrix adhesion protein known to interact with coated with 0.1% gelatin. After a 60-min incubation at 37 °C,
Hepatoma Research | Volume 2 | Issue 2 | February 29, 2016 43