The effects of specific expression of apoptin under control of pses and psa promoter on apoptosis of lncap cells
Vida Mohammadi,
1,* Mohammad ali takhshid,
2 Behzad-behbahani abbas ,
3 Hosseini seyed younes,
4 Rafiei dehbidi golamreza,
5
1. Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
2. Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
3. Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
4. Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
5. Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
Abstract
Introduction
Apoptotic effects of viral protein apoptin has been demonstrated in numerous studies. however, its tumor specificity has been questioned by some reports. the aim of present study was to more specify the expression of apoptin in prostate tumor cells under the control of a chimeric enhancer (pses) composing of prostate-specific membrane antigen (psma) and prostate-specific antigen (psa) regulatory elements.
Methods
The apoptin gene was amplified from cav genome with pcr, subsequently digested using xbai, and then ligated to psa promoter previously digested by xbai. the psa-apoptin fragment was amplified by pcr, digested using pvui and sali and replaced with cmv promoter region of the adeno shuttle vector of pad-cmv5. the presence of the cloned fragment was confirmed by colony pcr, restriction digestion and dna sequencing. lncap cell lines were then transfected with the final construct, along with lx-2 cell lines as control. the expression of apoptin was confirmed by real time-pcr and western blot analysis. in the end, the effects of apoptin expression on cell survival and apoptosis were evaluated using mtt assay and annexin v/7-aad flow cytometry assay, respectively. apoptin expression under the control of cmv promoter, was constructed similarly as control plasmid.
Results
Both western blot and real time pcr analysis confirmed the specific apoptin expression under the control of psa promoter in the lncap cells, while cmv promoter exhibited apoptin expression in tumor as well as normal cells. specific apoptin expression significantly increased the cell death and apoptosis in tumor cells when compared to normal cells (p<0.001).
Conclusion
These results suggest that new pses/psa sequence promoter effectively exhibit specialized expression of apoptin and consequently cell death to prostate related tumor cells.
Keywords
Apoptin, pses/psa, prostate cancer, apoptosis
