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Updated: Jul 5, 2026
A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer
Published on: July 6, 2017
Adenovirus-mediated expression of SSAT inhibits colorectal cancer cell growth in vitro
Hui Sun1, Bin Liu, Ya-Pei Yang
1Institute of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan 250012, China.
Aim:
To construct a recombinant adenovirus that can express human spermidine/ spermine N1-acetyltransferase (SSAT) and detect its inhibitory effect on colorectal cancer cell growth in vitro.
Methods:
A 516 bp cDNA of SSAT was amplified and cloned into a pGL3-hTERT plasmid. The pGL3-hTERT-SSAT recombinant was digested, and the small fragment was cloned into the shuttle vector pAdTrack. The pAdTrack-hTERT-SSAT plasmids were recombined with pAdEasy-1 vectors in AdEasy-1 cells. Positive clones were selected and transfected into the HEK293 packaging cells (transformed human embryonic kidney cells) after they were linearized by PacI. The process of adenovirus packaging and amplification was monitored by green fluorescent protein (GFP) expression. The SSAT protein levels were determined by Western blotting, and the intracellular polyamine content was detected by reverse-phase high performance liquid chromatography. The MTS (3-(4, 5-dimethylthiaol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(-4- sulfophenyl)-2H-tetrazolium, inner salt) and colony-forming assays were used to analyze the gene transduction efficiency and effect on the growth of HT-29 and LoVo cells. A viable cell count was used to determine the cell growth with or without exogenous polyamines.
Results:
The GFP expression in 293 cells during virus packing and amplification was observed by fluorescence microscopy. Western blotting results demonstrated that Ad-hTERT-SSAT could increase the expression of SSAT, and consequently, spermidine and spermine were reduced to low levels. The MTS and colony-forming assay results showed that HT-29 and LoVo cell growth were significantly inhibited, and the inhibitory effect could be partially reversed by exogenous spermidine and spermine.
Conclusion:
The successfully constructed recombinant adenovirus Ad-hTERT-SSAT could accelerate polyamine catabolism and inhibit the colorectal cell growth in vitro. It also has therapeutic potential in the treatment of colorectal cancer.
Insights
A novel recombinant adenovirus, Ad-hTERT-SSAT, was created to express human spermidine/spermine N1-acetyltransferase (SSAT). This therapy effectively inhibits colorectal cancer cell growth by reducing polyamine levels.
Area of Science:
- Molecular biology
- Oncology
- Gene therapy
Background:
- Colorectal cancer (CRC) is a leading cause of cancer-related mortality worldwide.
- Polyamines are essential for cell growth and proliferation, and their dysregulation is implicated in CRC development.
- Targeting polyamine metabolism presents a potential therapeutic strategy for CRC.
Purpose of the Study:
- To construct a recombinant adenovirus (Ad-hTERT-SSAT) expressing human spermidine/spermine N1-acetyltransferase (SSAT).
- To evaluate the efficacy of Ad-hTERT-SSAT in inhibiting colorectal cancer cell growth in vitro.
- To investigate the impact of SSAT expression on intracellular polyamine levels.
Main Methods:
- Amplification and cloning of SSAT cDNA into adenoviral vectors.
- Adenovirus packaging and amplification in HEK293 cells, monitored by GFP expression.
- Assessment of SSAT protein levels via Western blotting and polyamine quantification using HPLC.
- Evaluation of cell viability and growth inhibition using MTS, colony-forming, and cell count assays.
Main Results:
- Successful construction and packaging of the Ad-hTERT-SSAT recombinant adenovirus.
- Ad-hTERT-SSAT expression led to increased SSAT levels and significantly reduced spermidine and spermine concentrations.
- Significant inhibition of HT-29 and LoVo colorectal cancer cell growth was observed.
- Exogenous polyamine supplementation partially reversed the inhibitory effect of Ad-hTERT-SSAT.
Conclusions:
- The Ad-hTERT-SSAT recombinant adenovirus effectively accelerates polyamine catabolism.
- This approach demonstrates significant inhibition of colorectal cancer cell growth in vitro.
- Ad-hTERT-SSAT holds potential as a therapeutic agent for colorectal cancer treatment.
