Down-regulation of AP-4 inhibits proliferation, induces cell cycle arrest and promotes apoptosis in human gastric cancer cells

Abstract

Background: AP-4 belongs to the basic helix-loop-helix leucine-zipper subgroup; it controls target gene expression, regulates growth, development and cell apoptosis and has been implicated in tumorigenesis. Our previous studies indicated that AP-4 was frequently overexpressed in gastric cancers and may be associated with the poor prognosis. The purpose of this study is to examine whether silencing of AP-4 can alter biological characteristics of gastric cancer cells.

Methods: Two specific siRNAs targeting AP-4 were designed, synthesized, and transfected into gastric cancer cell lines and human normal mucosa cells. AP-4 expression was measured with real-time quantitative PCR and Western blot. Cell proliferation and chemo-sensitivity were detected by CCK-8 assay. Cell cycle assay and apoptosis assay were performed by flow cytometer, and relative expression of cell cycle regulators were detected by real-time quantitative PCR and Western blot, expression of the factors involved in the apoptosis pathway were examined in mRNA and protein level.

Results: The expression of AP-4 was silenced by the siRNAs transfection and the effects of AP-4 knockdown lasted 24 to 96 hrs. The siRNA-mediated silencing of AP-4 suppressed the cellular proliferation, induced apoptosis and sensitized cancer cells to anticancer drugs. In addition, the expression level of p21, p53 and Caspase-9 were increased when AP-4 was knockdown, but the expression of cyclin D1, Bcl-2 and Bcl-x(L) was inhibited. It didn't induce cell cycle arrest when AP-4 was knockdown in p53 defect gastric cancer cell line Kato-III.

Conclusions: These results illustrated that gene silencing of AP-4 can efficiently inhibited cell proliferation, triggered apoptosis and sensitized cancer cells to anticancer drugs in vitro, suggesting that AP-4 siRNAs mediated silencing has a potential value in the treatment of human gastric cancer.

Figure 1. AP-4 specific-siRNAs suppressed the AP-4 expression in gastric cancer cells and human normal mucosa cells.

The different siRNAs were transfected into the cells for 24 h, 48 h, 72 h, and 96 h. The mRNA and protein expression were examined by real-time quantitative PCR and Western blot. AP-4 specific-siRNAs could effectively inhibit the gene expression. The induced suppression of AP-4 expression started at 24 hours, and the inhibition ratio decreased after 72 hours. The most efficient time point were 48 h and 72 h, the relative levels of mRNA transcripts significantly decreased by nearly 90%. There was statistical significance between AP-4 siRNAs groups and control groups. (* p<0.05; ** p<0.01).

Figure 2. Down-regulation of AP-4 expression inhibits the proliferation of gastric cancer cells, and enhances the chemo-sensitivity.

Forty-eight hours post-transfection, the cell proliferation and inhibitory effects of different concentration of 5-FU, ADR or Cis-plantinum were evaluated by CCK-8 assay. The results indicated that AP-4 specific-siRNAs could inhibit the proliferation of gastric cancer cells but not the normal mucosa cells (p<0.01) and enhance the chemo-sensitivities of gastric cancer cells to 5-FU, ADR or Cis-plantinum (p<0.0001). There was statistical significance between AP-4 siRNAs groups and control groups (* p<0.05; * p<0.01).

Figure 3. The effect of AP-4 on gastric cancer cell cycle was investigated by flow cytometry.

Forty-eight hours post-transfection, the cells were harvested and stained with propidium iodide, and proportion of cells in each phase of cell cycling was assayed. In the graph, the proportion of cells at G0/G1 phases in the cell transfected with AP-4 siRNAs was significantly higher than that of the mock cells or control siRNA. (* p<0.05; ** p<0.01).

Figure 4. The cell cycle regulators were evaluated with Real time PCR and Western blot.

Inhibition of AP-4 expression could up-regulate the expression of p53 and p21 mRNA and protein, but down-regulate the cyclin D1. (* p<0.05; ** p<0.01).

Figure 5. Effect of AP-4 specific-siRNAs on the induction of apoptosis in gastric cancer cells.

Forty-eight hours post-transfection, the cells were harvested and double stained with Annexin-V and PI. The apoptosis rate of cells transfected with AP-4 siRNAs was higher than the control siRNA (p<0.05) and mock cells (p<0.05). (* p<0.05; ** p<0.01).

Figure 6. The expressions of the factors involved in the apoptosis were examined with real time PCR and Western blot.

Inhibition of AP-4 expression could up-regulate the transcription of Caspase-9 but down-regulate the Bcl-2 and Bcl-x_L expression in human gastric cancer But Caspase-8 and Bax expression were difference in different cell lines after transfection. Forty eight hours after transfection, Caspase-8 and Bax were over expression in AGS cells. In SGC7901 cells, Bax was over expression, Caspase-8 expression was also up-regulated in siRNA-1 group, but in siRNA-2 group, Caspase-8 expression was not significant affected. (* p<0.05; ** p<0.01).

Figure 7. Silencing AP-4 regulated cell cycle and apoptosis in both p53-dependent and independent-manners.

In Kato-III cell, the AP-4 expression was obviously down-regulated at 48 hours post-transfection. However, the induction of cell cycle arrest and apoptosis were not observed in Kato-III cells. The proportions of cells at G0/G1, G2/M and S phases were not significant difference between AP-4 siRNAs groups and control or mock groups. The apoptosis rate of Kato-III cells transfected with AP-4 siRNAs was higher than the control siRNA (p<0.05) and mock cells (p<0.05). However, the apoptosis rate in Kato-III cells with AP-4 silencing was lower than AGS and SGC7901 cells with AP-4 silencing (p<0.05).