Adenovirus vector-mediated upregulation of spermidine /spermine N1-acetyltransferase impairs human gastric cancer growth in vitro and in vivo

Abstract

Spermidine/spermine N(1)-acetyltransferase (SSAT) is the rate-limiting step in polyamine catabolism. In a previous study, we constructed a recombinant adenovirus, Ad-SSAT, which can express human SSAT. In the present study, we investigated the effect of upregulated and downregulated SSAT on gastric cancer cells. We found that upregulated SSAT could inhibit the growth of MGC803 and SGC7901 cells, whereas adverse results were found with downregulated SSAT. We further analyzed cell cycle profiles and the expression levels of the major cell cycle regulatory proteins of S phase. The results showed that the growth inhibition was caused by S phase arrest. Ad-SSAT suppressed the expression of cyclin A and nuclear factor E2F1 in MGC803 and SGC7901 cells. We observed the E2F promoter activity caused by Ad-SSAT using a reporter gene assay. We also investigated the antitumorigenicity of upregulated SSAT by Ad-SSAT using a SGC7901 xenograft model in nude mice. Our results suggest that the upregulation of SSAT by Ad-SSAT infection inhibited the growth of gastric cancer in vitro and in vivo. Ad-SSAT arrested gastric cancer cells in S phase, which was mediated through downregulation of the cyclin A-E2F signaling pathway.

Figure 1

Western blot analysis for spermidine/spermine N ¹‐acetyltransferase (SSAT) expression in MGC803 and SGC7901 cells. MGC803 and SGC7901 cells were transfected with pGPU6/GFP/Neo‐shNC and pGPU6/GFP/Neo‐shSSAT or infected with adenovirus by 50 and 25 MOI. I, mock; II, pGPU6/GFP/Neo‐shNC‐transfected; III, pGPU6/GFP/Neo‐shSSAT‐transfected; IV, mock; V, Ad‐GFP‐infected; VI, Ad‐SSAT‐infected.

Figure 2

Effect of adenovirus (Ad)‐spermidine/spermine N ₁‐acetyltransferase (SSAT) and pGPU6/GFP/Neo‐shSSAT on the growth of MGC803 and SGC7901 cells. MGC803 and SGC7901 cells were infected with Ad‐GFP (), Ad‐SSAT(‐) at MOI 25 and 50, and transfected with pGPU6/GFP/Neo‐shNC (×), pGPU6/GFP/Neo‐shSSAT (), or treated with PBS (). After 24 h of incubation, absorbance was measured every day for 5 days. Each bar represents the mean ± SD of five experiments.

Figure 3

Effect of adenovirus (Ad)‐spermidine/spermine N ¹‐acetyltransferase (SSAT) on growth of MGC803 and SGC7901 cells. Cells were cultured in triplicate sets of six‐well tissue culture plates with RPMI‐1640+10% FBS for 14 days. Colonies were visualized by Giemsa staining. Colonies transfected with pGPU6/GFP/Neo‐shSSAT increased, whereas Ad‐SSAT‐infected cells were significantly decreased compared to their two controls (P < 0.05). (A) MGC803; (B) Ad‐GFP‐infected MGC803; (C) Ad‐SSAT‐infected MGC803; (D) pGPU6/GFP/Neo‐shNC transfected MGC803; (E) pGPU6/GFP/Neo‐shSSAT‐transfected MGC803; (a) SGC7901; (b) Ad‐GFP‐infected SGC7901; (c) Ad‐SSAT‐infected SGC7901; (d) pGPU6/GFP/Neo‐shNC‐transfected SGC7901; (e) pGPU6/GFP/Neo‐shSSAT‐transfected SGC7901.

Figure 4

Effects of adenovirus (Ad)‐spermidine/spermine N ¹‐acetyltransferase (SSAT) on the cell cycle of MGC803 and SGC7901 cells. Cells were treated with 50 or 25 MOI of Ad‐GFP, Ad‐SSAT, or PBS (mock), then collected and dyed with propidium iodide for cell cycle analysis. The data are representative of three separate experiments.

Figure 5

Western blot analysis of Cyclin A, Cdk2, and E2F1 gene expression in MGC803 and SGC7901 cells. Total protein was extracted 3 days after infection with adenovirus (Ad)‐spermidine/spermine N ¹‐acetyltransferase (SSAT) or the control vector. The blot was probed with either anti‐cyclin A, anti‐Cdk2, or anti‐E2F1 antibodies. Equal loading was verified using an anti‐β‐actin antibody.

Figure 6

E2F promoter activity in (a) MGC803 and (b) SGC7901 cells. pGL3‐E2F and pRL‐TK were cotransfected into MGC803 and SGC7901. At 24 h after transfection, cells were infected with adenovirus (Ad)‐GFP and Ad‐spermidine/spermine N ¹‐acetyltransferase (SSAT) for 72 h. E2F promoter activity was measured as described in Materials and methods. Each bar represents the mean ± SD of three experiments.

Figure 7

Suppression of tumor growth in vivo. Approximately 2 × 10⁶ tumor cells were implanted subcutaneously into the right flank area of each mouse. Tumor growth and animal weight were monitored every 4 days after the eighth day after injection (c) showed the time course of estimated mean tumor volume of either SGC/7901/SSAT (), SGC7901/GFP (), or control group (). On the 33rd day after inoculation, all of the mice were killed. The (a) mice and (b) tumor appearance were photographed, and (d) the tumor masses were weighed. *P < 0.01 versus the other groups.