Growth arrest-specific gene 2 suppresses hepatocarcinogenesis by intervention of cell cycle and p53-dependent apoptosis

Abstract

Background: Growth arrest-specific gene 2 (GAS2) plays a role in modulating in reversible growth arrest cell cycle, apoptosis, and cell survival. GAS2 protein is universally expressed in most normal tissues, particularly in the liver, but is depleted in some tumor tissues. However, the functional mechanisms of GAS2 in hepatocellular carcinoma (HCC) are not fully defined.

Aim: To investigate the function and mechanism of GAS2 in HCC.

Methods: GAS2 expression in clinic liver and HCC specimens was analyzed by real-time PCR and western blotting. Cell proliferation was analyzed by counting, MTS, and colony formation assays. Cell cycle analysis was performed by flow cytometry. Cell apoptosis was investigated by Annexin V apoptosis assay and western blotting.

Results: GAS2 protein expression was lower in HCC than in normal tissues. Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type p53, while knockdown of GAS2 promoted the proliferation of hepatocytes (P < 0.05). Furthermore, GAS2 overexpression impeded the G1-to-S cell cycle transition and arrested more G1 cells, particularly the elevation of sub G1 (P < 0.01). Apoptosis induced by GAS2 was dependent on p53, which was increased by etoposide addition. The expression of p53 and apoptosis markers was further enhanced when GAS2 was upregulated, but became diminished upon downregulation of GAS2. In the clinic specimen, GAS2 was downregulated in more than 60% of HCCs. The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in paired non-tumor tissues (P < 0.05).

Conclusion: GAS2 plays a vital role in HCC cell proliferation and apoptosis, possibly by regulating the cell cycle and p53-dependent apoptosis pathway.

Keywords: Apoptosis; Cell cycle; Growth arrest-specific gene 2; Hepatocellular carcinoma; p53-dependent signaling pathway.

Figure 1

GAS2 exerts tumor-suppressive functions in HCC cells. A: Western blot analysis of GAS2 expression in liver and HCC cell lines. β-actin was used as the loading control; B: GAS2 transfected in SK-hep1 cells was identified by western blotting. β-actin was used as the loading control; C: Cell counting (^aP < 0.05 vs control); D: Cell viability (^aP < 0.05 vs control); E: Anchorage-dependent colony formation (^bP < 0.01 vs control). GAS2: Growth arrest-specific gene 2.

Figure 2

Effect of knockdown on endogenous GAS2 in MIHA cells. A: Western blot analysis of siGAS2, β-actin was used as the loading control; B: Cell counting (^aP < 0.05 vs control); C: Cell viability (^aP < 0.05 vs control); D: Anchorage-dependent colony formation (^bP < 0.01 vs control). GAS2: Growth arrest-specific gene 2.

Figure 3

Effect of GAS2 on cell cycle progression. SK-hep1 cells were transfected with pDEST40-GAS2 and pDEST40-CTRL plasmids followed by FACS analysis (FITC/PI). A: Cell populations in different fractions of the cell cycle phase were plotted (^aP < 0.05 vs control); B: The cell population in subG1 phase was determined by flow cytometry (^bP < 0.01 vs control). MIHA cells were transfected with siGAS2 and siCTRL followed by FACS analysis (FITC/PI); C: Cell populations in different fractions of the cell cycle phase were plotted (^cP < 0.05 vs control); D: Cell population in the subG1 phase was determined by flow cytometry. FACS: Fluorescence-activated cell sorting.

Figure 4

GAS2 inhibits HCC cell growth by increasing p53-mediated apoptosis. A: Effect of GAS2 overexpression on apoptosis was determined by FACS using the Annexin V-APC apoptosis assay. A: The effect of 100 μM etoposide (Eto) in SK-Hep1 cells transfected with pDEST40-CTRL or pDEST40-GAS2 (^aP < 0.05 vs control); (B) Effect of knocking down GAS2 in MIHA on apoptosis was determined by FACS using the Annexin V-APC apoptosis assay. The effect of 100 μM etoposide (Eto) in MIHA cells transfected with siCTRL or siGAS2 (^aP < 0.05 vs control); C: Expression of p53 in hepatocytes and HCC cell lines was identified by western blotting, β-actin was used as loading control; D: siRNA-mediated knockdown of p53 (sip53) and overexpression of GAS2 in SK-Hep1 cells as well as the apoptosis markers such as cleaved caspase-3 and cleaved PARP were confirmed by western blotting; E: Effect of knocking down p53 (siP53) and overexpression of GAS2 in SK-Hep1 cells on apoptosis was determined by FACS using the Annexin V-APC apoptosis assay (^bP < 0.01 vs control; mean values and SD from three replicate experiments); F: Cell apoptosis markers in the absence or presence of etoposide (Eto) in GAS2-overexpressing SK-Hep1 cells (left panel) or GAS2-ablated MIHA cells (right panel). FACS: Fluorescence-activated cell sorting; GAS2: growth arrest-specific gene 2.

Figure 5

Expression of GAS2 in HCC specimens as determined by qPCR and western blotting. A: Comparison of GAS2 mRNA expression in 54 paired tumor (T) and non-tumorous (N) tissues using PNN as an internal control. The bars (shown in log scale) illustrate the relative GAS2 mRNA level (T/N) in individual tissue pairs, of which negative and positive values respectively indicate downregulation and upregulation of GAS2 in HCC tumors. The differences of the T and N groups were statistically significant (P < 0.05 vs non-tumorous); B: Relative protein expression levels of GAS2 in 54 paired tumor (T) and non-tumorous (N) tissues using β-actin as an internal control. The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in the paired non-tumor tissues (^aP < 0.05 vs non-tumorous). GAS2: growth arrest-specific gene 2; HCC: Hepatocellular carcinoma.