Small Interfering RNA of cyclooxygenase-2 Induces Growth Inhibition and Apoptosis Independently of Bcl-2 in Human Myeloma RPMI8226 Cells

Acta Pharmacol Sin. 2007 Jul;28(7):1031-6. doi: 10.1111/j.1745-7254.2007.00550.x.

Abstract

Aim: To investigate the effects of small interfering RNA of cyclooxygenase-2 (COX-2) on the proliferation and apoptosis of human multiple myeloma RPMI8226 cells and its relation with the Bcl-2 family in vitro.

Methods: Transcription and expression of COX-2 in human myeloma RPMI8226 cells were checked by RT-PCR and Western blot analysis, respectively. The COX-2 siRNA fragment targeting exon 5 of COX-2 gene was transfected into the cells with the Amaxa nucleofection technique. The inhibition of cell growth was detected by 3-(4,5-dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was estimated by Annexin-V/ propidium iodide double-labeled cytometry and confirmed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. Bcl-2 and Bax expression was evaluated by Western blot analysis.

Results: The COX-2 siRNA fragment could be successfully transfected into RPMI8226 cells, which resulted in the significant inhibition of transcription and expression of COX-2 in the myeloma cells. Proliferation of the transfected cells was inhibited and apoptosis was induced (6.52%+/-0.32%, 12.53%+/-2.52%, 24.39%+/-3.51% and 36.48%+/-4.96% for 0, 12, 24, and 48 h, respectively) in a time-dependent manner (P<0.01). However, the expression of Bcl-2 and Bax in the RPMI8226 cells had no significant changes after nucleofection.

Conclusion: COX-2 siRNA transfection can suppress COX-2 expression in human myeloma RPMI8226 cells, which leads to growth inhibition and apoptosis independent of Bcl-2.

MeSH terms

  • Apoptosis / physiology*
  • Cell Line, Tumor
  • Cell Proliferation
  • Cyclooxygenase 2 / genetics*
  • Cyclooxygenase 2 / metabolism
  • Humans
  • Multiple Myeloma / metabolism*
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism*
  • bcl-2-Associated X Protein / genetics
  • bcl-2-Associated X Protein / metabolism

Substances

  • Proto-Oncogene Proteins c-bcl-2
  • RNA, Small Interfering
  • bcl-2-Associated X Protein
  • Cyclooxygenase 2
  • PTGS2 protein, human