HepG2.2.15 and HepG2 cells were cultured in DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA), in 5% CO₂ at 37°C. A final concentration of 380 mg/L G418 (Invitrogen) was added into the medium for the maintenance of HepG2.2.15 cells.

To detect the expression and replication of HBV in HepG2.2.15 cells, hepatitis B surface antigen (HBsAg) and hepatitis B envelope antigen (HBeAg) levels in the medium at 24 h, 48 h and 72 h were determined semi-quantitatively using enzyme-linked immunosorbent assay (ELISA) kits (Sino-American Biotechnology Company, Shanghai, China) according to the manufacturer’s instructions. All experiments were performed in triplicate.

HepG2.2.15 and HepG2 cells were collected and fixed in 2.5% glutaraldehyde (GA) overnight, dehydrated in a graded series of ethanol and embedded in Quetol-812. The ultrathin sections were cut and stained with lead citrate. The girds were examined under a JEM-1220 electron microscope.

HepG2.2.15 and HepG2 cells were seeded in triplicate into 96-well plates at 4 × 10³ cells per well. Twenty microliters 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/mL) was added to the medium and cultured for another 4 h. DMSO (150 μL) was added and the absorbance of each well was read using a Bio-Rad model 550 microplate reader at a wavelength of 490 nm. It was tested for 5 d and the data were expressed as mean ± SD.

Cell cycle assay was monitored using propidium iodide (PI) staining of the nuclei. The cells were fixed in 75% cold alcohol overnight, resuspended in 300 μL PBS and stained with 500 μL PI (250 μg/mL) for 30 min in the dark. Annexin V/PI double staining was used for apoptosis assay. Annexin V and PI were added for incubation for 15 min at 4°C. The cells were analyzed by flow cytometry (BD BioSciences, San Jose, CA, USA).

The invasive abilities of HepG2.2.15 and HepG2 cells were determined using matrigel (BD) coated 24-well trans-well chambers (Corning Costar, NY, USA) as described previously[10]. In brief, trans-well was coated with 10 μL matrigel and dried in the air; and 5 × 10⁴ cells in serum-free DMEM were seeded into the upper chamber, with the lower chamber supplemented with DMEM containing 10% FBS. The trans-well was incubated at 37°C in 5% CO₂. Incubation time was different due to different invasive abilities of the two cell lines. The cells that had penetrated through the pores were fixed, stained with hematoxylin and eosin (HE) and photographed under light microscope. The experiments were conducted in triplicate.

Total protein extracts of cultured cells were performed routinely. Twelve-microgram samples were size fractionated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. The membranes were incubated with F-actin or Ezrin antibodies (Bioss, Beijing, China), and detected using Western blue (Promega, Madison, WI, USA). GAPDH (Calbiochem, Gibbstown, NJ, USA) was used as internal control.

Four-week-old female BALB/c nude mice were maintained in the laboratory for animal experiments under specific pathogen-free conditions. The experiments were conducted in accordance with the Guideline for Animal Experiments of the National Cancer Center of China.

HepG2.2.15 and HepG2 cells were harvested and resuspended to 2 × 10⁷/mL with PBS, and 300 μL was injected subcutaneously into the left flank of each of eight nude mice. The tumor volume was calculated according to the formula: V = mean diameter³×π/6[11] and measured and recorded every 2 d.

Once the subcutaneous tumor reached 1 cm in diameter, it was removed and cut into 1 mm × 1 mm × 1 mm cubes freshly at 4°C. Another group of mice were anesthetized with an intra-peritoneal injection of pentobarbital at a dose of 60 mg/kg. Tumor cubes were implanted into the liver as described previously[12]. The mice were killed 60 d after implantation to harvest the liver and lung. The volume of tumor was determined according to the method described by Janik et al[13]. The liver and lung were removed and fixed in 4% formalin for standard pathological studies.

HepG2.2.15 and HepG2 cells (1.5 × 10⁶ cells/150 μL) were prepared in PBS at 4°C for injection. The mice were anesthetized and the liver was exposed as mentioned above. The cells were injected into the left lobe of liver of ten nude mice. The mice were observed for 60 d. Liver and lung were sampled for standard pathological studies as described above.

Data were expressed as percentage, mean ± SD. Comparisons between two groups were analyzed by the χ² and Student t test. Mann-Whitney U-test was employed for analysis of subcutaneous tumor growth. P < 0.05 was considered statistically significant.

Ultrastructural analysis demonstrated that HepG2.2.15 cells had obviously decreased filopodia (Figure 1A) compared with HepG2 cells (Figure 1D). Plentiful filopodia formed around HepG2 cells and higher amplification showed microfilaments in the filopodia (Figure 1E). Moreover, viral inclusion bodies existed in the cytoplasm of HepG2.2.15 cells (Figure 1B), and many organelles, such as mitochondria, ribosome and endoplasmic reticulum, were found to be degenerated in HepG2.2.15 cells (Figure 1C). In contrast, HepG2 cells contained normal and abundant organelles including ribosome, glycogen, microfilament and microtubule (Figure 1F).

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Figure 1 Ultrastructure of HepG2. 2.15 and HepG2 cells. A: Filopodia disappearance in HepG2.2.15 cells (EM × 2500); B: Viral inclusion bodies in the cytoplasm of HepG2.2.15 cells. Arrows indicate the viral inclusion bodies (EM × 15 000); C: Arrows indicate degenerated mitochondria (EM × 25 000); D: Plentiful filopodia around HepG2 cells. Arrows indicate filopodia (EM × 2500); E: Microfilament appearance in filopodia in HepG2 cells in high power field. Arrows indicate microfilament (EM × 25 000); F: Abundant organelles in the cytoplasm of HepG2 cells (EM × 25 000).

HBsAg and HBeAg were detected in the culture supernatant of HepG2.2.15 cells by ELISA. While the HBsAg level increased in a time-dependent manner, HBeAg level peaked at around 24 h and remained largely unchanged until 72 h (Figure 2A). As shown in Figure 2B, HepG2 cells had a significantly higher proliferation rate than HepG2.2.15 cells from Day 2 (P < 0.01), especially on Day 4 and Day 5 (P < 0.001).

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Figure 2 Cell proliferation and apoptosis flow cytometry. A: The levels of hepatitis B surface antigen (HBsAg) and hepatitis B envelope antigen (HBeAg) in HepG2.2.15 cell supernatant. The supernatant was collected every 24 h and tested by enzyme-linked immunosorbent assay; B: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of cell proliferation. The absorbencies of test wells were read every 24 h and the data represent the mean ± SD (^bP < 0.001); C: Flow cytometry of cell cycle; D: Apoptosis percentages in B1, B2 and B4 areas. All experiments were repeated three times with similar results.

To further investigate the reduced proliferation of HepG2.2.15, we tested cell cycle and apoptosis by flow cytometry. The results indicated that the percentage of the G1 phase of HepG2 was significantly lower than that of HepG2.2.15 (P < 0.01), but the HepG2 cells in S phase were increased significantly (P < 0.001) (Figure 2C), indicating cell cycle arrest at the G1/S phase in HepG2.2.15 cells. The apoptosis analysis showed no significant difference in apoptosis between HepG2.2.15 and HepG2 cells (Figure 2D).

Trans-well analysis demonstrated that HepG2.2.15 and HepG2 cells were significantly different in invasion ability in vitro. HepG2 cells on the lower part of the membrane were detected as early as 2 h after incubation, with an increasing number of cells invading through the membrane at 4 h, 6 h and 12 h (Figure 3A). In contrast, the invasion of HepG2.2.15 cells into the lower chamber was detected at 24 h, 36 h, 48 h and 60 h, respectively (Figure 3B). These results suggested that HepG2.2.15 cells had lower invasion ability and took longer time to go through the membrane.

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Figure 3 Invasion assays of HepG2 and HepG2. 2.15 cells. The trans-well membranes were collected at different time points and the cells that went through the pores were stained with hematoxylin and eosin. The cell numbers were counted. A: HepG2 cells collected and stained at 2 h, 4 h, 6 h and 12 h after incubation; B: HepG2.2.15 cells collected and stained at 24 h, 36 h, 48 h and 60 h after incubation; C: Western blotting analysis of F-actin and Ezrin.

To elucidate the mechanism accounting for the difference in cell invasion between HepG2.2.15 and HepG2 cells, we examined the expression of F-actin and Ezrin, which are both cytoskeleton proteins that play crucial roles in maintaining cell shape and promoting cell invasion. Western blotting analysis showed a 0.7-fold decrease of F-actin level and a 3.8-fold decrease of Ezrin level in HepG2.2.15 cells compared with HepG2 cells (Figure 3C).

To explore the biological features of HepG2.2.15 cells in vivo, we monitored subcutaneous tumor growth in vivo. Two days after injection of HepG2 cells, the nodules were visualized and the diameter reached 0.6 cm on Day 6, and 100% (8/8) mice formed tumors. In contrast, only 25% (2/8) mice injected with HepG2.2.15 cells formed tumors, significantly lower than that of HepG2 cells (P < 0.01), and tumor formation was slower than the mice injected with HepG2 cells (Figure 4A). Notably, 100% (10/10) mice injected with HepG2 cells formed tumor in the liver 60 d after tumor cubes implantation, and the mean volume was 1.7 ± 0.4 cm³. Furthermore, all the mice (10/10) developed tumor in the liver after the injection of HepG2 cells and the mean volume of tumor was as big as 3.1 ± 1.1 cm³. Nevertheless, the incidence of tumor formation in HepG2.2.15 group (40%, 4/10) was significantly lower than the HepG2 group (P < 0.05). The mean volume was 2.3 ± 0.3 cm³ (Figure 4B). Only one case formed tumor (10%, 1/10) with a volume of 2.1 cm³ (Figure 4B). The incidence of tumor formation in the liver was significantly higher in HepG2 implantation group (P < 0.05) and injection group (P < 0.001) when compared with HepG2.2.15 group. Taken together, these results indicated the low tumorigenicity of HepG2.2.15 cells in vivo, being consistent with their reduced cell proliferation and invasion in vitro.

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Figure 4 Tumor formation of HepG2 and HepG2. 2.15 cells in vivo. A: The volume of subcutaneous tumors were measured and recorded every 2 d. The difference of tumor growth rate between HepG2 and HepG2.2.15 groups was significant (P < 0.01, Mann-Whithey U test); B: Tumor development in four groups in vivo; C: Expansive growth of tumor. The boundary of tumor and normal tissue is clear [hematoxylin and eosin (HE) stain, × 120]. T: Tumor; H: Liver tissue; D: Fatty changes in liver tissue of HepG2.2.15 groups (HE stain, × 120); E: Lung metastasis in HepG2 injected group (HE stain, × 460) and low power field.

Lung metastasis was observed under light microscope with a highest percentage of 50% (Figure 4E) in HepG2 group. The growth pattern (Figure 4C), invasion and changes in tumor and surrounding normal tissues were also analyzed in all the groups (Table 1). Most non-tumor livers showed obvious fatty changes in HepG2.2.15 groups (Figure 4D) and the invasion to surrounding organs occurred more frequently in HepG2 groups.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death in the world, and the second in China. It is generally accepted that hepatitis B virus (HBV) plays a major causative role in the development of HCC. Although considerable studies have been conducted, the precise mechanism remains unclear.

HBV infection is considered a high risk for the development of HCC. To investigate the pathogenesis of HBV in HCC, several HBV expressing cell lines have been established by viral DNA transfection. HepG2.2.15 cell line is one of the most common models for HBV-associated disease, but the cellular and biological features associated with HBV in HepG2.2.15 cells is seldom considered. Little is known about the effects of HBV on the biological features of host cells.

In this study, the authors demonstrated that HepG2.2.15 cells had lower proliferation and invasion ability than their parental HepG2 cells. HBV-induced cell cycle arrest and cytoskeletal alteration might be implicated in the mechanism.

The findings in this study will help us better understand the cellular and biological features of HepG2.2.15 cells associated with HBV and select the most suitable cell lines for research. They also shed new light on the interaction between HBV and host cells.

HepG2.2.15 cells are derived from HepG2 cells transfected with a full-length HBV plasmid. It is characterized by having stable HBV expression and replication in the culture system.

The study is well conducted and the methodology is sound.