Original Scientific Article
Suppression effects of excessively expressed gene bcl-2 in cell lines of prostate cancer
Igor Esmerov * ,
Branko Atanasov ,
Aleksandra Angeleska ,
Radmila Crceva ,
Ljupco Mickov ,
Ljupco Angelovski ,
Slavica Josifovska ,
Nikolaj Markov ,
Nikola Adamov

Mac Vet Rev 2022; 45 (2): 187 - 199


Received: 01 July 2022

Received in revised form: 14 September 2022

Accepted: 19 September 2022

Available Online First: 28 September 2022

Published on: 15 October 2022

Correspondence: Igor Esmerov, iesmerov@gmail.com


The aim of this study was to construct two plasmid-specific shRNA transcripts of the bcl-2 gene in order to prepare for reverse of cell apoptosis. The plasmid was designed according to a previously published sequence of interfering RNA following an appropriate reference, using appropriate software. By annulling complementary oligonucleotides, doublestranded inserts were formed. Recombinant shRNA-encoding plasmids were constructed by digestion of psiRNAx7SKGFPzeo plasmid (psiRNA-x7SKGFPzeo, with restrictive endonuclease BbsI electrophoresis in ultra-pure agarose with low melting point (LMP-Agarose). For each of the constructs, a suitable double-stranded insert downstream of x7SK (strong RNA III promoter) with T4 DNA ligase was cloned. The control plasmid psiRNAScr was used directly for transformation. The PC-3 cell lines were transfected with 2 plasmids, psiRNA-Bcl-2 and psiRNAScr to suppress the bcl-2 gene construct. The results have shown that the lowest level of bcl-2 genes was 48 h, and even lower 72 h after the transfer, and the mRNA levels returned to normal in 120 h. An increase in the percentage of cells with spontaneous apoptosis has been observed with successful inhibition of the bcl-2 gene. The induction of apoptosis in transfected cells increased the percentage of necrotic cells proportionally. The percentage of apoptotic cells transfected with psiRNA-bcl-2 plasmid increased proportionally to the increase of hydrogen peroxide concentration. The transfection of the PC-3 cell line from prostate cancer with constructed shRNA plasmid has induced suppression of bcl-2 gene expression versus control Scr plasmid. Suppression of bcl-2 gene expression significantly increased cell sensitivity to apoptosis induction.

Keywords: shRNA plasmid, Bcl-2, apoptosis, cytotoxicity, prostate cancer


  1. Siegel, R.M., Frederiksen, J.K., Zacharias, D.A., Chan, F.K., Johnson, M., Lynch, D., Tsien, R.Y., Lenardo, M.J. (2000). Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations. Science 288(5475): 2354 -2357. https://doi.org/10.1126/science.288.5475.2354 PMid:10875918
  2. Chan, F.K., Chun, H.J., Zheng, L., Siegel, R.M., Bui, K.L., Lenardo, M.J. (2000). A domain in TNF receptors that mediates ligand-independent recpetor assembly and signaling. Science 288(5475): 2351 -2354. https://doi.org/10.1126/science.288.5475.2351 PMid:10875917
  3. Cooperberg, M.R., Park, S., Carroll, P.R. (2004). Prostate cancer 2004: insights from national disease registries. Oncology (Williston Park) 18(10): 1239-1247.
  4. Sun, Y., Niu, J., Huang, J. (2009). Neuroendocrine differentiation in prostate cancer. Am J Transl Res. 1(2): 148-162.
  5. Vashchenko, N., Abrahamsson, P.A. (2005). Neuroendocrine differentiation in prostate cancer: Implications for new treatment modalities. Eur Urol. 47(2): 147-155. https://doi.org/10.1016/j.eururo.2004.09.007 PMid:15661408
  6. Bonkhoff, H. (2001). Neuroendocrine differentiation in human prostate cancer. Morphogenesis, proliferation and androgen receptor status. Ann Oncol. 12 (Suppl 2): S141-S144. https://doi.org/10.1093/annonc/12.suppl_2.S141 PMid:11762342
  7. Huang, J., Yao, J.L., di Sant’agnese, P.A., Yang, Q., Bourne, P.A., Na, Y. (2006). Immunohistochemical characterization of neuroendocrine cells in prostate cancer. Prostate 66(13): 1399-1406. https://doi.org/10.1002/pros.20434 PMid:16865726
  8. Sambrook, J., Fritsch, E.F., Maniatis, T. (1989). Molecular cloning: a laboratory manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press
  9. Panov, S. (2003). Basic methods in molecular biology. Faculty of Natural Sciences and Mathematics, Ss. Cyril and Methodius University in Skopje.
  10. Panov, S. (2005). The effects of the RNA interferencemediated silencing of bcl-2 and hTERT genes in lung cancer cell lines [dissertation]. [Skopje, Macedonia]: Faculty of Natural Sciences and Mathematics, 147 p.
  11. Wigler, M., Pellicer, A., Silverstein, S., Axel, R., Urlaub, G., Chasin, L. (1979). DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci USA. 76(3): 1373-1376. https://doi.org/10.1073/pnas.76.3.1373 PMid:286319 PMCid:PMC383253
  12. Floch, V., Le Bolc’h, G., Audrezet, M.P., Yaouanc, J.J., Clement, J.C., des Abbayes, H., Mercier, B., et al. (1997). Cationic phosphonolipids as non viral vectors for DNA transfection in hematopoietic cell lines and CD34+ cells. Blood Cells Mol Dis. 23(1): 69-87. https://doi.org/10.1006/bcmd.1997.0123 PMid:9215752
  13. Guillaume-Gable, C., Floch, V., Mercier, B., Audrezet, M.P., Gobin, E., Le Bolch, G., Yaouanc, J.J., et al. (1998). Cationic phosphonolipids as nonviral gene transfer agents in the lungs of mice. Hum Gene Ther. 9(16): 2309-2319. https://doi.org/10.1089/hum.1998.9.16-2309 PMid:9829530
  14. Izumi, M., Miyazawa, H., Kamakura, T., Yamaguchi, I., Endo, T., Hanaoka, F. (1991). Blasticidin S-resistance gene (bsr): a novel selectable marker for mammalian cells. Exp Cell Res. 197(2): 229-233. https://doi.org/10.1016/0014-4827(91)90427-V
  15. Lin, Y., Ma, W., Benchimol, S. (2000). Pidd, a new death-domain-containing protein, is induced by p53 and promotes apoptosis. Nat Genet. 26(1): 122 -127. https://doi.org/10.1038/79102 PMid:10973264
  16. Osmak, M., Kapitanovic, S., Vrhovec, I., Beketic-Oreskovic, L., Jernej, B., Eljuga, D., Skrk, J. (1997). Characterization of human breast adenocarcinoma SK-BR-3 cells resistant to doxorubicin. Neoplasma 44(3): 157-162.
  17. Osmak, M., Brozovic, A., Ambriovic-Ristov, A., Hadzija, M., Pivcevic, B., Smital, T. (1998). Inhibition of apoptosis is the cause of resistance to doxorubicin in human breast adenocarcinoma cells. Neoplasma 45(4): 223-230.
  18. Osmak, M., Bordukalo, T., Kosmrlj, J., Kvajo, M., Marijanovic, Z., Eljuga, D., Polanc, S. (1999). Diazenes: modificators of tumor cell resistance to cisplatin. Neoplasma. 46(4): 201-206.
  19. Bishop, J.M., Weinberg, R.A. (1996). Molecular oncology. New York: Scientific American, Inc. 20.
  20. Panasci, M., Ballard, B.W., Breck, S., Rodriguez, D., Densimore, D.L. III, Weester, D.B., Baker, J.R. (2011). Evaluation of fecal DNA preservation techniques and effects of sample age and diet on genotyping success. J Wildl Manage. 75(7): 1616-1624. https://doi.org/10.1002/jwmg.221
  21. Hanahan, D., Weinberg, R.A. (2000). The hallmarks of cancer. Cell 100(1): 57-70. https://doi.org/10.1016/S0092-8674(00)81683-9
  22. Kramer, G., Erdal, H., Mertens, H.J., Nap, M., Mauermann, J., Steiner, G., Marberger, M., et al. (2004). Differentiation between cell death modes using measurements of different soluble forms of extracellular cytokeratin 18”. Cancer Res 64(5): 1751-1756. https://doi.org/10.1158/0008-5472.CAN-03-2455 PMid:14996736
  23. Degterev, A., Lugovskoy. A., Cardone, M., Mulley, B., Wagner, G., Mitchison, T., Yuan, J. (2001). Identification of small molecule inhibitors of interaction between the BH3 domain and BCL-XL. Nat Cell Biol. 3(2): 173 -182. https://doi.org/10.1038/35055085 PMid:11175750
  24. Brummelkamp, T.R., Bernards, R., Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296(5567): 550-553. https://doi.org/10.1126/science.1068999 PMid:11910072
  25. Cao, W., Shiverick, K.T., Namiki, K., Sakai, Y., Porvasnik, S., Urbanek, C., Rosser, C.J. (2008). Docetaxel and bortezomib downregulate Bcl-2 and sensitize PC-3-Bcl-2 expressing prostate cancer cells to irradiation. World J Urol. 26(5): 509-516. https://doi.org/10.1007/s00345-008-0289-5 PMid:18594829
  26. Cheng, T.L., Teng, C.F., Tsai, W.H., Yeh, C.W., Wu, M.P., Hsu, H.C., Hung, C.F., Chang, W.T. (2009). Multitarget therapy of malignant cancers by the head-to-tail tandem array multiple shRNAs expression systemA multiple shRNAs-based gene therapy, Cancer Gene Ther. 16(6): 516-531. https://doi.org/10.1038/cgt.2008.102 PMid:19165234
  27. Pulukuri, S.M.K., Gondi, C.S., Lakka, S.S., Jutla, A., Estes, N., Gujrati, M., Rao, J.S. RNA Interference-directed knockdown of urokinase plasminogen activator and urokinase Plasminogen activator receptor inhibits prostatecancer cell invasion, survival and tumorogenesis in vivo. J Biol Chem. 280(43): 36529-36540. https://doi.org/10.1074/jbc.M503111200 PMid:16127174 PMCid:PMC1351057
  28. Jia, J., Zhang, W., Wang, B., Trinko, R., Jiang, J. (2003). The Drosophila Ste20 family kinase dMST functions as a tumor suppressor by restricting cell proliferation and promoting apoptosis. Genes Dev. 17(20): 2514-2519. https://doi.org/10.1101/gad.1134003 PMid:14561774 PMCid:PMC218145


© 2022 Esmerov I. This is an open-access article published under the terms of the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Conflict of Interest Statement

The authors have declared that no competing interests exist.

Citation Information

Macedonian Veterinary Review. Volume 45, Issue 2, Pages 187-199, e-ISSN 1857-7415, p-ISSN 1409-7621, DOI: 10.2478/macvetrev-2022-0028, 2022