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Published on: September 9, 2010
Adenovirus-mediated expression of both antisense ODC and AdoMetDC inhibited colorectal cancer cell growth in vitro
Bing Zhang1, Xian-xi Liu, Yan Zhang
1Institute of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan 250012, China.
Aim:
To construct a recombinant adenovirus that can simultaneously express both antisense ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase ( AdoMetDC ) and detect its inhibitory effect on the intracellular polyamine pool and colorectal cancer cell growth.
Methods:
A 205-bp cDNA of AdoMetDC was reverse-inserted into recombinant pAdTrack-ODCas vectors and recombined with pAdEasy-1 vectors in AdEasy-1 cells. Positive clones were selected and transfected into the packaging cell HEK293 after they were linearized by PacI. Green fluorescent protein expression was used to monitor the process of adenovirus packaging. The ODC and AdoMetDC protein levels were identified by western blotting, and intracellular polyamine content was detected by reverse-phase high performance liquid chromatography. A viable cell count was used to determine the growth of HT-29 cells with or without exogenous polyamine.
Results:
Sequencing confirmed that AdoMetDC cDNA was successfully ligated into the pAdTrack-ODCas vector. GFP expression in 293 cells during virus packing and amplification was observed by fluorescence microscopy. Western blotting demonstrated that both ODC and AdoMetDC were downregulated by Ad-ODC-AdoMetDCas, and consequently 3 kinds of polyamine (putrescine, spermidine and spermine) were reduced to very low levels. HT-29 cell growth was significantly inhibited as compared with control conditions, and growth arrest was not reversed by exogenous putrescine.
Conclusion:
The successfully constructed recombinant adenovirus, Ad-ODC-AdoMetDCas, blocked polyamine synthesis and has therapeutic potential for treating colorectal cancer in vitro.
Insights
A novel recombinant adenovirus, Ad-ODC-AdoMetDCas, was created to inhibit colorectal cancer cell growth by blocking polyamine synthesis. This approach shows promise for in vitro cancer therapy.
Area of Science:
- Molecular Biology
- Oncology
- Virology
Background:
- Polyamines are crucial for cell growth and proliferation.
- Dysregulation of polyamine metabolism is implicated in colorectal cancer development.
- Targeting polyamine synthesis pathways offers a potential therapeutic strategy for cancer.
Purpose of the Study:
- To construct a recombinant adenovirus for simultaneous expression of antisense ornithine decarboxylase (ODC) and adenosylmethionine decarboxylase (AdoMetDC).
- To evaluate the inhibitory effect of this adenovirus on intracellular polyamine levels and colorectal cancer cell growth.
Main Methods:
- Construction of a recombinant adenovirus (Ad-ODC-AdoMetDCas) expressing antisense ODC and AdoMetDC.
- Verification of gene insertion and protein expression via sequencing and western blotting.
- Quantification of intracellular polyamines using high-performance liquid chromatography.
- Assessment of colorectal cancer cell (HT-29) growth inhibition.
Main Results:
- Successful construction and packaging of the Ad-ODC-AdoMetDCas adenovirus.
- Significant downregulation of ODC and AdoMetDC protein levels.
- Marked reduction in intracellular putrescine, spermidine, and spermine levels.
- Substantial inhibition of HT-29 cell growth, independent of exogenous putrescine.
Conclusions:
- The Ad-ODC-AdoMetDCas effectively blocks polyamine synthesis.
- This recombinant adenovirus demonstrates therapeutic potential for in vitro colorectal cancer treatment.
