The effects of specific expression of apoptin under the control of PSES and PSA promoter on cell death and apoptosis of LNCaP cells

Document Type: Original Article


1 Department of Medical Biotechnology, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

2 Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran

3 Department of Bacteriology and Virology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran


Objective(s): Apoptotic effect of apoptin has been demonstrated in numerous studies. However, its tumor specificity has been questioned by some reports. The aim of this study was to confine the expression of apoptin in the prostate tumor cells by inducing its gene expression under the control of a chimeric enhancer composing of prostate-specific membrane antigen (PSMA) and prostate-specific antigen (PSA) regulatory elements (PSES). Furthermore, we investigated the effects of apoptin expression on LNCaP prostate carcinoma cell survival and apoptosis using MTT assay and annexinV/7-AAD flow cytometry assay.
Materials and Methods: Recombinant plasmids containing apoptin gene under the control of PSES/PSA promoter or Cytomegalovirus (CMV) promoter were constructed. Tumor cell lines including LNCaP cells and HeLa cells, and LX-2 cells (as a normal control) were transfected with the plasmids and the expression of apoptin was evaluated by real time-PCR and western blot analyses. The effects of apoptin expression on cell survival and apoptosis were then investigated using MTT and annexinV/7-AAD flow cytometry assay, respectively.
Results: Western blot and real time PCR analyses confirmed the specific expression of apoptin under the control of PSES/PSA regulatory element in the LNCaP cells, while CMV promoter caused apoptin expression in both tumor and normal cell lines. Apoptin expression significantly increased cell death and apoptosis in tumor cells when compared with the normal cells (P<0.001).
Conclusion: These results suggest that PSES/PSA regulatory element may be considered as an efficient approach for specific expression of apoptin gene in prostate tumor cells and treatment of prostate cancer.


1. Hassanipour-Azgomi S, Mohammadian-Hafshejani A, Ghoncheh M, Towhidi F, Jamehshorani S, Salehiniya H. Incidence and mortality of prostate cancer and their relationship with the Human Development Index worldwide. Prostate Int 2016;4:118-124.

2. Merseburger AS, Alcaraz A, von Klot CA. Androgen deprivation therapy as backbone therapy in the management of prostate cancer. Onco Targets Ther 2016;9:7263-7274.

3. Heidenreich A, Bastian PJ, Bellmunt J, Bolla M, Joniau S, van der Kwast T, et al. EAU guidelines on prostate cancer. Part II: Treatment of advanced, relapsing, and castration-resistant prostate cancer. Eur Urol 2014; 65:467-479.

4. Marech I, Vacca A, Ranieri G, Gnoni A, Dammacco F. Novel strategies in the treatment of castration-resistant prostate cancer (Review). Int J Oncol 2012;40: 1313-1320.

5. Wong RS. Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res 2011 26;30:87-101

6. Javan B, Shahbazi M. Hypoxia-inducible tumour-specific promoters as a dual-targeting transcriptional regulation system for cancer gene therapy. Ecancermedicalscience. 2017;11:751.

7. Zhong X, Zhao H, Liang S, Zhou D, Zhang W, Yuan L. Gene delivery of apoptin-derived peptide using an adeno-associated virus vector inhibits glioma and prolongs animal survival. Biochem Biophys Res Commun 2017 15;482: 506-513.

8. Shen Ni L, Allaudin ZN, Mohd Lila MA, Othman AM, Othman FB. Selective apoptosis induction in MCF-7 cell line by truncated minimal functional region of Apoptin. BMC cancer. 2013; 13:488.

9. Li X, Liu Y, Wen Z, Li C, Lu H, Tian M, et al. Potent anti-tumor effects of a dual specific oncolytic adenovirus expressing apoptin in vitro and in vivo. Mol Cancer. 2010; 9:10.

10. Jangamreddy JR, Panigrahi S, Lotfi K, Yadav M, Maddika S, Tripathi AK, et al. Mapping of apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy. Oncotarget 2014; 5 :7198-7211.

11. Nastasie MS, Thissen H, Jans DA, Wagstaff KM. Enhanced tumour cell nuclear targeting in a tumour progression model. BMC cancer 2015; 15:76-87.

12. Backendorf C, Noteborn MH. Apoptin towards safe and efficient anticancer therapies. Adv Exp Med Biol 2014;818:39-59.

13. Li J, Wang H, Ma Z, Fan W, Li Y, Han B, et al. TAT-Apoptin induces apoptosis in the human bladder cancer EJ cell line and regulates Bax, Bcl-2, caspase-3 and survivin expression. Exp Ther Med 2012;31033-1038.

14. Zhou S, Zhang M, Zhang J, Shen H, Tangsakar E, Wang J. Mechanisms of apoptin-induced cell death. Med oncol 2012;29:2985-2991.

15. He X, Zhang Q, Liu Y, He P. Apoptin induces chromatin condensation in normal cells. Virus genes 2005;3149-3155.

16. Guelen L, Paterson H, Gaken J, Meyers M, Farzaneh F, Tavassoli M. TAT-apoptin is efficiently delivered and induces apoptosis in cancer cells. Oncogene 2004; 23: 1153-1165.

17. Lee SJ, Kim HS, Yu R, Lee K, Gardner TA, Jung C, et al. Novel prostate-specific promoter derived from PSA and PSMA enhancers. Mole Ther  2002; 6:415-421.

18. Zarei MA, Takhshid M, Behbahani AB, Hosseini S, Okhovat M, Dehbidi GRR, et al. Synergistic effects of NDRG2 overexpression and radiotherapy on cell death of human prostate LNCaP cells. J Biomed Physics Eng 2016;7:257-264.

19. Kashkin KN, Chernov IP, Didych DA, Sverdlov ED. Construction of a combinatorial library of chimeric tumor-specific promoters. Biotechniques 2017;63:107-116.

20. Qi Y, Guo H, Hu N, He D, Zhang S, Chu Y, et al. Preclinical pharmacology and toxicology study of Ad-hTERT-E1a-Apoptin, a novel dual cancer-specific oncolytic adenovirus. Toxicol appl pharmacol 2014 15;280:362-369.

21. Leliveld SR, Dame RT, Rohn JL, Noteborn MH, Abrahams JP. Apoptin's functional N- and C-termini independently bind DNA. FEBS lett 2004 16;557:155-158.

22. Kucharski TJ, Gamache I, Gjoerup O, Teodoro JG. DNA damage response signaling triggers nuclear localization of the chicken anemia virus protein apoptin. J virol 2011; 85:12638-12649.

23. Chaabane W, Ghavami S, Malecki A, Los MJ. Human gyrovirus-Gpoptin interferes with the cell cycle and induces G2/M arrest prior to apoptosis. Arch immuno ther exp 2017:464-468.