Adenovirus Vector-mediated Upregulation Of Spermidine/Spermine N1-acetyltransferase Impairs Human Gastric Cancer Growth In Vitro And In Vivo

Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related death, with 700000 deaths annually. Now the first-line therapy is radical surgery with adjuvant chemotherapy. But overall 5 year survival rate for this disease is 20%～30%. Because of lacking targeting, these traditionary treatments may also damage the normal tissues and cells. Therefore, it is important to find a novel non-surgical targeting intervention for treating gastric cancer.Polyamines include spermidine, spermine and their diamine precursor, putrescine. Their positive charges enable polyamines to interact electrostatically with polyanionic macromolecules within the cell, such as DNA, RNA and proteins. They are critical for cell growth and differentiation.The level of polyamine is high in cancer cell and tissues, and rapid tumor growth has been associated with remarkable elevation of polyamine biosynthesis and accumulation. Therefore, polyamine pathway becomes an important target for preventing and treating cancer.The intracellular polyamine biosynthetic pathway is mainly regulated by the actions of two rate-limiting enzymes, Ornithine decarboxylase (ODC) and Sadenosylmethionine decarboxylase (AdoMetDC). Studies in our laboratory have demonstrated that ODC mRNA and ODC protein levels in colorectal cancer were higher than that were found in the surrounding normal tissues, the down regulation of ODC and AdoMetDC could inhibit colorectal cancer. Spermidine/spermine N1-acetyltransferase (SSAT) is the rate limiting step in polyamine catabolism,which is primarily responsible for regulation of intracellular polyamine concertrations in mammalian cells. SSAT catalyzes the transfers of the acetyl group from acetyl-CoA to the N1 positions of spermidine or spermine. SSAT is thought to prevent accumulation of higher polyamines from becoming toxic to the cell, and so maintains a balanced ration of intracellular polyamines according to cellular needs.SSAT activity is highly regulated and is induced rapidly in response to high intracellular levels of natural polyamine. Additionally, SSAT activity can be induced by a number of other stimuli, including hormones, physiological stimuli, drugs and toxic agents. In polyamine analogue-responsive cells, the super-induction of SSAT expression can result in a> 1000-fold increase in protein activity. Depletion of intracellular polyamine pools invariably inhibits cell growth. Although this is usually accomplished by inhibiting polyamine biosynthesis, Kristin Kee and Slavoljub Vujcicthis have found that this might be more effectively achieved by activating polyamine catabolism at the level of SSAT.Telomerase is an enzyme involved in the synthesis and maintenance of chromosome termini known as telomeres that conventional polymerases are unable to replicate fully. Telomerase activation is observed in approximately 90%of human cancers, irrespective of tumor type, while most normal tissues contain inactivated telomerase. Numerous studies have demonstrated that hTERT promoter expression is highly specific to cancer cells and tightly associated with telomerase activity, while the other subunits are constitutively expressed both in normal and cancer cells. Therefore, based on the specialty of hTERT promoter, we constructed an adenovirus which can express SSAT mediated by hTERT promoter, valued its inhibitory effects on gastric cancer. We also investigated the in vivo growth inhibitory effect using a SGC7901 xenograft model in nude mice.Part 1 Adenovirus vector-mediated upregulation of spermidine/spermine N1-acetyltransferase impairs human gastric cancer growth in vitroObjective:To construct a recombinant adenovirus that can expression human SSAT mediated by hTERT promoter, and study the inhibitory effects of Ad-SSAT on growth of SGC7901 and MGC803 cells. To construct the shRNA expression vector pGPU6/ GFP/Neo-shSSAT.Methods:1. Human SSAT cDNA was amplified by RT-PCR and was cloned into pGL3-hTERT plasmid. The pGL3-hTERT-SSAT construction was digested, and the small fragments were cloned into pAdTrack. Then the pAdTrack-hTERT-SSAT plasmids recombined with pAdEasy-1 vectors in AdEasy-1 cells. pAdeasy-hTERT-SSAT were linearized by Pac I, and transfected into the HEK293 to package. Green fluorescent protein expression and PCR technique was used to confirm the process.2. The siRNA duplex was designed to SSAT (NM₀02970). The shRNA expression vector pGPU6/GFP/Neo-shSSAT was commercially obtained from GenePharma (Shanghai, China).3. MTS assay was used to analyze adenovirus-mediated gene transduction efficiency of different MOI in SGC7901 and MGC803 cells.4. The SSAT protein levels were determined by Western blot, and intracellular polyamine content was detected by reverse-phase high performance liquid chromatography.5. MTS assay and Colony-Forming assay were used to analyze the effect of the the growth of SGC7901 and MGC803 cells.6. Cell cycle progression was detected by flow cytometry analysis.Results:1. A 516bp cDNA was amplified by RT-PCR and the sequencing result showed it was the domain of SSAT gene. Several positive recombinant clones were identified after transformation of Adeasy-1 cells with linearized pAdTrack-hTERT-SSAT. Fluorescent microscope observation and PCR conformed that pAd-hTERT-SSAT (abbreviate as Ad-SSAT) could infect 293 cell and replicate in the cell.2. The shRNA expression vector pGPU6/GFP/Neo-shSSAT was successfully constructed. 3. MTS assay results showed that Ad-SSAT could significantly inhibit the growth of SGC7901 and MGC803 about 65%and 55%, respectively, by 50MOI and 25MOI without cell toxicity.4. Western blot results showed that Ad-SSAT could increase the expression of SSAT about 265%and 210%, respectively, in SGC7901 and MGC803; pGPU6/ GFP/Neo-shSSAT could lower the expression of SSAT to 15%and 27% respectively. Spermidine and spermine were changed correspondingly.5. MTS assay and Colony-Forming assay results showed that Ad-SSAT could significantly inhibit the growth of SGC7901 and MGC803 cells.While silence of SSAT by pGPU6/GFP/Neo-shSSAT could accelerate the growth of gastric cells.6. Cell flow cytometry analysis showed that upregulated SSAT arrests gastric cancer cells in S phase.Conclusions:The recombinant adenovirus Ad-SSAT and shRNA expression vector pGPU6/GFP/Neo-shSSAT were successfully constructed. Ad-SSAT has significant inhibitory effects on gastric cancer cells and induces S arrest in SGC7901 and MGC803 cells.Part 2 Molecular mechanism of Adenovirus-mediated expression of SSAT expression induces S arrestObjective:To investigate underlying regulatory responses of Adenovirus-mediated expression of SSAT induces S arrest in MGC803and SGC7901 cells.Methods:1.The effect of Ad-SSAT on protein levels of cell cycle regulated proteins was measured by Western blotting analysis.2.The effect of Ad-SSAT on mRNA levels of cell cycle regulated proteins was measured by RT-PCR.3.The effect of Ad-SSAT on expression level of E2F-1 was measured by Western blotting and RT-PCR.4.Observe the effect of Ad-SSAT on E2F promoter activity.Results: 1. Ad-SSAT could decrease protein expression of cyclin A about 80%and 60%in SGC7901 and MGC803 cells, but there were no obvious changes in Cdk2 protein level.2. The mRNA level of cyclin A decreased significantly in Ad-SSAT-treated cells, but no changes in Cdk2 mRNA level.3. Ad-hTERT-SSAT could decrease protein and mRNA expression level of nuclear factor E2F-1.4. The E2F promoter activity measured by luciferase reporter decreased about 80% and 60%by Ad-SSAT infection in SGC7901 and MGC803 cells.Conclusions:SSAT expression mediated by Ad-SSAT induces S arrest in MGC803 and SGC7901 cells and the arrest was associated with suppression of cyclin A expression and inhibition of nuclear factor E2F-1.Part 3 Adenovirus vector-mediated up-regulation of spermidine/spermine N1-acetyltransferase suppress the gastric tumor growth in vivo.Objective:To investigate the antitumorigenicity of Ad-SSAT in vivo.Methods:1. Tumor cells injection:SGC7901, SGC7901/GFP and SGC7901/Ad-SSAT cells were injected subcutaneously in 4 week-old nude mice for tumor implantation studies.2. Tumor growth observation:Tumor growth and animal weight were monitored every 4 days since the eighth day after injection. On day 33day after inoculation, all the mice were sacrificed, and the tumor masses were weighted.Results:1. Visible tumors in SGC7901 and SGC7901/GFP groups were detectable 8 days after implantation and grew rapidly in the next days, while the cell treated with Ad-SSAT cells remarkably delayed tumor growth.2. In tumors from SSAT-up-regulated groups, size and weight were significantly decreased by 68.3%.Conclusions:Up-regulation of SSAT has an in vivo growth-inhibitory effect.