Albumin promotes proliferation of G1 arrested serum starved hepatocellular carcinoma cells

Abstract

Albumin is the most abundant plasma protein and functions as a transport molecule that continuously interacts with various cell types. Because of these properties, albumin has been exploited by the pharmaceutical industry to improve drug delivery into target cells. The immediate effects of albumin on cells, however, require further understanding. The cell interacting properties and pharmaceutical applications of albumin incentivises continual research into the immediate effects of albumin on cells. The HepG2/C3A hepatocellular carcinoma cell line is used as a model for studying cancer pathology as well as liver biosynthesis and cellular responses to drugs. Here we investigated the direct effect of purified albumin on HepG2/C3A cell proliferation in the absence of serum, growth factors and other serum originating albumin bound molecules. We observed that the reduced cell counts in serum starved HepG2/C3A cultures were increased by the inclusion of albumin. Cell cycle analysis demonstrated that the percentage of cells in G1 phase during serum starvation was reduced from 86.4 ± 2.3% to 78.3 ± 3.2% by the inclusion of albumin whereas the percentage of cells in S phase was increased from 6.5 ± 1.5% to 14.3 ± 3.6%. A significant reduction in the cell cycle inhibitor protein, P21, accompanied the changes in the proportions of cell cycle phases upon treatment with albumin. We have also observed that the levels of dead cells determined by DNA fragmentation and membrane permeabilization caused by serum starvation (TUNEL: 16.6 ± 7.2%, ethidium bromide: 13.8 ± 4.8%) were not significantly altered by the inclusion of albumin (11.6 ± 10.2%, ethidium bromide: 16.9 ± 8.9%). Therefore, the increase in cell number was mainly caused by albumin promoting proliferation rather than protection against cell death. These primary findings demonstrate that albumin has immediate effects on HepG2/C3A hepatocellular carcinoma cells. These effects should be taken into consideration when studying the effects of albumin bound drugs or pathological ligands bound to albumin on HepG2/C3A cells.

Keywords: Albumin; Cell cycle; Cyclin d1; Ethidium bromide staining; HepG2/C3A cells; Hepatocellular carcinoma; Proliferation; TUNEL assay; Western blot; p21.

Figure 1. Comparison of HepG2/C3A cell morphology and total cell counts after 72 h treatments.

Albumin alters morphology of serum starved HepG2/C3A cells demonstrated in micrographs (20×objective lens) of cells cultured for 72 h in (A) serum free media (serum starved control), (B) serum free media containing 5 mg/ml albumin and (C) serum free media containing 5 mg/ml dextran. Scale bar = 100 μm. (D) Albumin results in increased total cell counts after 72 h in culture. Values are mean ± SD (n = 4). ^∗p < 0.05, NS= not significant.

Figure 2. Cell cycle analysis of serum starved cells with or without albumin.

Flow cytometry histograms of propidium iodide stained cells treated for 72 h with (A) serum starved media or (B) serum starved media containing 5 mg/ml albumin. (C) Percentage of cell cycle stages calculated from the histogram using the Watson pragmatic algorithm shows a decrease in G1 phase and an increase in S phase in albumin containing media. Values are mean ± SD (n = 4). ^∗p < 0.05, ^∗∗p < 0.01, NS= not significant. Representative images of western blots demonstrating significantly reduced (D) P21 levels but no significant change in (E) cyclin D1 levels in albumin containing cultures compared to serum free controls.

Figure 3. TUNEL assay for the measurement of percentage of cells with fragmented DNA.

Percentages of apoptotic cells were not significantly different demonstrated in flowcytometry overlay of dot plots of TUNEL positive cells (red) and negative cells (black) of 72 h cultures of HepG2/C3A in (A) serum starved media or (B) serum starved media containing 5 mg/ml albumin. (C) Bar chart demonstrating percentage of TUNEL positive cells. Values are mean ± SD (n = 4). NS, not significant.

Figure 4. Live dead fluorescent micrographs of cells.

Percentages of dead cells were not significantly different demonstrated by fluorescence microscopy images of cells (10×  objective lens) cultured for 72 h in (A, B, C) serum starved media and (D, E, F) serum starved media containing 5 mg/ml albumin. Fluorochromes used were DAPI (nuclear), calcein AM (cytoplasmic) and ethidium bromide (nuclear). Scale bar = 100 μ m. (G) Chart demonstrating differences in the percentage of ethidium bromide stained cells between treatments. Values are mean ± SD (n = 4). NS, not significant.