Hepatoma Cells

Hepatoma cells, particularly HepG2 cells, are a type of cell line derived from liver cancer. They are characterized by their unlimited lifespan, stable phenotype, high availability, and ease of handling, which makes them an invaluable tool in biological and medical research. However, one of their limitations is their lower expression of some metabolic activities compared to normal hepatocytes, or liver cells. Despite this, HepG2 cells, which originate from a human hepatoma (a type of liver tumor), are most commonly used in studies related to drug metabolism and hepatotoxicity. These cells retain many characteristics of normal hepatocytes, and thus, they provide a useful model for studying liver diseases, the liver's response to drugs, and other related topics. They are widely used in labs around the world for this purpose.

Biological Properties of Hepatoma Cells

• Cell morphology and growth characteristics. Hepatoma cells often exhibit a more irregular and disorganized appearance, with a less uniform shape and size compared to the well-organized structure of healthy hepatocytes. This morphological transformation is believed to be a reflection of the underlying genetic and molecular alterations that drive the malignant transformation of these cells.
• Proliferation and cell division patterns. In terms of growth patterns, hepatoma cells demonstrate a significantly higher proliferation rate than normal liver cells. These cells are characterized by a more aggressive cell division pattern, with a shorter doubling time and a greater capacity for self-renewal.
• Metabolic and functional differences from normal liver cells. Unlike normal liver cells, which primarily rely on oxidative phosphorylation for energy production, hepatoma cells exhibit a pronounced shift towards aerobic glycolysis, a phenomenon known as the "Warburg effect." This metabolic reprogramming provides these cancer cells with a competitive advantage, as it allows them to generate ATP more quickly, even in the presence of oxygen. Furthermore, hepatoma cells often display dysregulation of various liver-specific functions, such as bile acid synthesis, xenobiotic metabolism, and the production of essential proteins.

Drug Testing and Toxicology Studies

Hepatoma cells exhibit many of the characteristic features of liver cells, including drug-metabolizing enzymes and transporters, making them a relevant system for assessing the potential liver-related adverse effects of new therapeutics. Researchers can utilize hepatoma cells to screen for hepatotoxicity, study the metabolism and pharmacokinetics of drugs, and investigate the underlying mechanisms of liver injury, helping to identify and mitigate potential liver-related toxicities early in the drug development process.

Liver Disease and Regeneration Studies

Hepatoma cells can provide insights into the pathogenesis of various liver diseases, as they often display functional and metabolic alterations that mimic those observed in diseased liver tissue. These cell lines can be used to study the impact of genetic, environmental, or pharmacological interventions on liver-specific processes, such as bile acid metabolism, xenobiotic detoxification, and protein synthesis. Furthermore, some hepatoma cells exhibit characteristics of hepatic progenitor cells, allowing researchers to explore the potential of these cells for liver regeneration and cell-based therapies.

Metabolic and Signaling Pathway Investigations

Hepatoma cells also serve as models for studying dysregulated signaling cascades, such as growth factor-mediated pathways and oncogenic signaling, that contribute to the malignant phenotype of liver cancer cells. By understanding these metabolic and signaling alterations, researchers can identify novel therapeutic targets and develop strategies to selectively target the vulnerabilities of hepatoma cells.

KIF11 Negatively Correlates With Senescence Biomarkers in HCC Tissues and Hepatoma Cells

KIF11, also known as Kinesin-5, is a necessary molecular motor protein during mitosis. KIF11 mediates centromeric separation and bipolar mitotic spindle formation, thereby promoting mitosis to support cell proliferation. KIF11 is highly expressed in various malignancies, including hepatocellular carcinoma (HCC). The role of KIF11 in modulating cellular senescence has not been reported. The TCGA database and GTEx database were re-analyzed and found that KIF11 was highly expressed in HCC tissues compared with their adjacent normal tissues (Fig. 1A). Likewise, KIF11 was highly expressed in cultured hepatoma cells (HCCLM3, Huh7, HepG2, and SNU398) compared with normal liver cells (HLSEC and THLE-3) (Fig. 1B, C). Furthermore, KIF11 is negatively correlated with p16 and p14 in cultured liver cells and hepatoma cells (Fig. 1D, E). These effects were also observed in ROS stress-induced cultured normal liver cells (THLE-3) and hepatoma cells (HepG2 and Huh7) (Fig. 1F, G).

The correlation among the KIF11, p16, and p14 protein levels was analyzed in clinical HCC tissues. A total of 83 cases of HCC tissues were stained by IHC and scored according to staining intensity. As shown in the pie chart (Fig. 1H), 2 cases were KIF11^high p16^low, 15 cases were KIF11^high p14^low, 33 cases were KIF11^high p16^low p14^low. IHC pictures showed different expressions of KIF11, p16, and p14 in clinical HCC tissues (Fig. 1I). In total, in about 60% (50 in 83 cases) of HCC patients, the expression of KIF11 is higher, while p16 or (and) p14 is low. These results indicate that the high expression of KIF11 inhibits the cellular senescence in the liver of patients with HCC. Altogether, KIF11 negatively correlates with senescence in both clinical HCC tissues and cultured hepatoma cells.

Fig. 1 Expression level of KIF11 and CDKN2A (p16 and p14) in HCC. (Chen D, et al, 2023)

HRP-3 Protects the Hepatoma Cells From Glucose Deprivation-Induced Apoptosis

To estimate the long-term and stable effect of HRP-3 knockdown on HCC cells, lentivirus-based small hairpin RNA (shRNA) expression vectors (Sh1 and Sh3) were generated for two effective HRP-3-siRNA sequences (i1 and i3) and ShC as control vector for NS-siRNA. HRP-3 stable knockdown clones and control clones of SMMC-7721 (Sh1-3, Sh3-5, and ShC-7) and SK-Hep1 (Sh1-5, Sh3-10, and ShC-7), which are infected with lentivirus harboring i1, i3, and NS siRNA respectively, were constructed for further analysis. The efficiency of knockdown was detected by Western Blot (Fig. 2).

The adhesion and morphology of culture-adherent cells could give an intuitional index to cell viability and apoptosis. So the cell morphology analysis was conducted. All clones, counted for the same number and divided into two portions, were plated in 6 well plates of the same diameter with normal high-glucose DMEM overnight. One portion of each clone was changed for glucose-free DMEM the next day and the other portion was used as a control group maintained in high-glucose DMEM. After 9 hours, stable HRP-3 knockdown clones of SMMC-7721 (Sh1-5 and Sh3-10) became shrinking, round, and even suspended compared with the shC-7 control clone which had no obvious injury morphology. Similarly, results were observed between the stable HRP-3 knockdown clones of SK-Hep1 (Sh1-3 and Sh3-5) and the control clone of normal adhesion (ShC-7) after 7 hours cultured in a glucose-free medium. The morphology of all clones was captured by microscope for stochastic observation for 3 areas and the pictures were analyzed by software Image J for calculating spreading areas (Fig. 3A, B).

Then all of these clone cells were digested and submitted to flow cytometry analysis. Consistent with the morphology results, during the glucose deprivation, a progressive aggregation in the sub-G1 phase appeared in cells knocking down HRP-3 compared with in control cells (Sh1-3 and Sh3-5 Vs. ShC-7 of SMMC-7721; Sh1-5 and Sh3-10 Vs. ShC-7 of SK-Hep1), which indicates the influence of HRP-3 on cell apoptosis (Fig. 4A, B).

Fig. 2 Silence of HRP-3 inhibits the de-phosphorylation of S6K1 induced by glucose deprivation. (Cai H, et al., 2015)

Fig. 3 Stable Knockdown of HRP-3 in HCC cells sensitized cells to become shrunk morphology of apoptosis under the energy pressure. (Cai H, et al., 2015)

Fig. 4 HRP-3 inhibits energy deprivation-triggered apoptosis of HCC cells. (Cai H, et al., 2015)