PANC-1 Cell Line as an Experimental Model for Characterizing PIVKA-II Production, Distribution, and Molecular Mechanisms Leading to Protein Release in PDAC

Abstract

Pancreatic ductal adenocarcinoma (PDAC) represents a highly aggressive malignancy with a lack of reliable diagnostic biomarkers. Protein induced by vitamin K absence (PIVKA-II) is a protein increased in various cancers (particularly in hepatocellular carcinoma), and it has recently exhibited superior diagnostic performance in PDAC detection compared to other biomarkers. The aim of our research was to identify an in vitro model to study PIVKA-II production, distribution, and release in PDAC. We examined the presence of PIVKA-II protein in a panel of stabilized pancreatic cancer cell lines by Western blot analysis and indirect immunofluorescence (IFA). After quantitative evaluation of PIVKA-II in PaCa 44, H-Paf II, Capan-1, and PANC-1, we adopted the latter as a reference model. Subsequently, we analyzed the effect of glucose addiction on PIVKA-II production in a PANC-1 cell line in vitro; PIVKA-II production seems to be directly related to an increase in glucose concentration in the culture medium. Finally, we evaluated if PIVKA-II released in the presence of increasing doses of glucose is concomitant with the expression of two well-acknowledged epithelial-mesenchymal transition (EMT) markers (Vimentin and Snail). According to our experimental model, we can speculate that PIVKA-II release by PANC-1 cells is glucose-dependent and occurs jointly with EMT activation.

Keywords: PDAC; PIVKA-II; epithelial to mesenchymal transition; glucose.

Figure 1

PIVKA-II protein expression in different cell lines. One representative experiment out of three is shown. (A) Western blot analysis of PIVKA-II protein in different pancreatic adenocarcinoma cell lines. (B) Densitometric evaluation of PIVKA-II protein in PDAC cell lines. Histograms represent the mean of the densitometric analysis of the ratio of PIVKA-II/β-actin. Densitometric analysis was performed with ImageJ software (1.47 version, NIH, Bethesda, MD, USA), which was downloaded from the NIH website (http://imagej.nih.gov, accessed on 1 August 2022) and plotted with GraphPad Prism 5.0 software.

Figure 2

PIVKA-II localization in PANC-1 cells. (A) IFA performed on PANC-1 (left panel) and HaCaT (right panel) cell lines, showing PIVKA-II (red) and nuclei (blue). Representative images out of three are shown. (B) Double IFA performed on PANC-1 (upper panel) and HaCaT (lower panel) cell lines, showing PIVKA-II (red) and nuclei (blue) and actin (green). Representative images out of three are shown.

Figure 3

Expression and release of PIVKA-II in the presence of increasing doses of glucose in PANC-1 cells. One representative experiment out of three is shown. (A,B) Western blotting analysis performed on PANC-1 cells treated in the presence of increasing doses of glucose (5 mM, 25 mM, 50 mM) for 48 h. Following the treatment, Super (A) and Pellet (B) were recovered, separated on 12% SDS-PAGE, and analyzed by Western blotting with the indicated antibodies. Asterisks (*) in Figure 3A indicates released PIVKA-II protein. Correct loading of the supernatants was checked by Ponceau staining of the membrane (pink panel). b-actin was used as the lysates’ loading control. (C) Densitometric evaluation of PIVKA-II protein in different cell lines. Histograms represent the mean of the densitometric analysis (performed with ImageJ) of the ratio of PIVKA-II–Vimentin–Snail vs. β-actin.