Adenovirus-mediated expression of SSAT inhibits colorectal cancer cell growth in vitro.
OBJECTIVE
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.


CONCLUSIONS
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.
Sun
Hui
H
Institute of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan 250012, China.
Liu
Bin
B
Yang
Ya-Pei
YP
Xu
Chun-Xiao
CX
Yan
Yun-Fei
YF
Wang
Wei
W
Liu
Xian-Xi
XX
D000123:Acetyltransferases / Q000235:genetics* / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000737:chemistry; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D007668:Kidney / Q000166:cytology; D008969:Molecular Sequence Data; D010957:Plasmids; D013095:Spermidine / Q000378:metabolism*; D013096:Spermine / Q000378:metabolism*; D014162:Transfection; D000099228:Diamine N-Acetyltransferase
D000123:Acetyltransferases; D018076:DNA, Complementary; D049452:Green Fluorescent Proteins; D013095:Spermidine; D013096:Spermine; D000099228:Diamine N-Acetyltransferase