**5. Intracellular Consequences Associated with ABCC6 Transporter Activity in HepG2 Cells**

It is widely evident that changes in nuclear lamin expression are associated with cellular senescence and age-related diseases [36]. However, cells undergoing aging also adapt a phenomenon to proliferate inappropriately, migrate, and colonize, which are the hallmarks of cancer cells [37]. In both cases, changes in lamin expression traduce in morphological changes in nuclei, which are known as "nuclear atypia". Contrarily, induction of cellular senescence is recognized as an important tumor-suppressive mechanism [38].

In previous studies with *ABCC6* knockdown HepG2 cells, we have found decreased cell growth and lamin A/C expression [33]. Interestingly, in the recent study, pharmacological inhibition of ABCC6 by probenecid or its knockdown in HepG2 cells not only decreased the amount of extracellular ATP content but also decreased the expression of CD73 and lamin A/C proteins. Lamins are the most important structural component of the cytoskeleton and are required to maintain cells in a proper shape. In this context, we examined whether down-regulation of lamin expression affected the actin filaments, required for cytoskeleton rearrangement and cellular movement. In this study, a typical organization of actin filament in filopodia, which is a hallmark of moving cells, was absent in both probenecid-treated and *ABCC6* knockdown HepG2 cells (Figure 6). However, administration of adenosine or ATP restored the normal architecture of filopodia and migration rate (Figure 7). This finding indicates that ABCC6 can be a potential therapeutic target for anti-metastatic treatment and, with coordination of the purinergic system, also regulates the intracellular functions [32].

**Figure 6.** Representative confocal image of (**A**)—scrambled HepG2 cells; (**B**)—Abcc6-shRNA.HepG2 cells; (**C**)—Abcc6-shRNA HepG2 cells treated with 500 µM ATP; (**D**)—Abcc6-shRNA HepG2 cells treated with 100 µM adenosine. F-actin was stained with Texas Red-phalloidin. In the insets superposition of cytoskeleton (red) and EGFP (green) to monitor the infection efficiency. The scale bar in the inserts is 40 µm [32].

**Figure 7.** Effect of probenecid and ABCC6 silencing on the migration rate of HepG2 cells. Cells were treated with 250 µM probenecid for 48 h (gray plain bars, Probenecid+). DMSO-treated cells were used as the control (gray plain bars, Probenecid-). A total of 500 µM ATP was added to either the control cells (gray plain bars, Probenecid-, ATP+) or to probenecid-treated cells (gray plain bars, Probenecid+, ATP+). HepG2 cells were transduced with scrambled shRNA (grey-texturized bars, scr-shRNA) or with specific ABCC6-shRNA (black bars, ABCC6-shRNA). A sample of 500 µM ATP was added to either control cells (grey-texturized bars, scr-shRNA, ATP+) or to ABCC6 silenced cells (black bars, ABCC6-shRNA, ATP+). Data are expressed as mean ± standard error (SE) of three replicates from three independent experiments and were analyzed by one-way ANOVA followed by Dunnett's post hoc: \*\*\* *p* < 0.001 probenecid-treated cells vs. control cells in the absence of ATP and ABCC6-shRNA vs. scr-shRNA in the absence of ATP; ### *p* < 0.001 probenecid + ATP-treated cells vs. probenecid-treated cells and ABCC6-shRNA cells + ATP vs. ABCC6-shRNA cells without ATP. NS, not significant [32].
