Antisense RNA of survivin gene inhibits the proliferation of leukemia cells and sensitizes leukemia cell line to taxol-induced apoptosis

Abstract

The effects of survivin antisense RNA on proliferation of leukemia cell line HL-60 and taxol-induced chemotherapy was explored. A cDNA fragment of survivin obtained by RT-PCR was inserted into a plamid vector named pcDNA3 in the reverse direction. The vector encoding antisense RNA of survivin was confirmed by restriction enzyme digestion and DNA sequencing. The recombinant plasmid was delivered into HL-60 cells by electroporation. Growth curves were plotted based on cell counting. Trypan blue dye exclusion assay and MTT assay were carried out after the cells were incubated with taxol. DNA gel electrophoresis and nuclear staining were performed for cell apoptosis assay. The correct construction of the recombinant plasmid has been identified by restriction enzyme digestion and DNA sequencing. A stable down-regulation has been achieved in HL-60 SVVas cells after G418 selection. Compared to HL-60 cells, the proliferation of HL-60 SVVas cells was significantly inhibited (P<0.05). Cytotoxicity assays indicated that IC₅₀ of HL-60 SVVas for taxol was relatively lower than controls (P<0.01). Apoptosis assays revealed that taxol-induced apoptosis was detected in HL-60 SVVas cells incubated with 50 ng/ml taxol for 12 h, while in HL-60 cells incubated with 100 ng/ml taxol for 72 h. It was suggested that Survivin antisense RNA could inhibit the proliferation of HL-60 cells and enhance taxol-induced apoptosis in HL-60 cells, which may lay an experimental foundation for further research on gene therapy in leukemia.

Keywords

survivin / leukemia / antisense RNA / taxol / apoptosis / proliferation

References

[1] AndersenM. H., SvaneI. M., BeckerJ. C., et al.. The universal character of the tumor-associated antigen surviving. Clin Cancer Res, 2007, 13(20): 5991-5994

[2] MarziaP., GennaroC., MarcoF., et al.. Ribozyme-mediated attenuation of survivin expression sensitizes human melanoma cells to cisplatin-induced apoptosis. J Clin Invest, 2002, 109: 285-286

[3] TsurusawaM., SoekiK., KatanoN., et al.. Bcl-2 expression and prognosis in childhood acute leukemia. Pediatr Haematol Oncol, 1998, 15: 143-155

[4] LiF., BrattainM. G.. Role of the Survivin gene in pathophysiology. Am J Pathol, 2006, 169(1): 1-11

[5] WangX. J., DaiG. Y., CaoL. M., et al.. Prognostic significance of expression of survivin in acute leukemia. Chin J Cancer Res (Chinese), 2002, 14(4): 265-269

[6] SelaB.. Survivin: anti-apoptosis protein and a prognostic marker for tumor progression and recurrence. Harefuah, 2002, 14: 103-107

[7] KamihiraS., YamadaY., HirakataY., et al.. Aberrant expression of caspase cascade regulatory genes in adult T-cell leukemia: survivin is an important determinant for prognosis. Bri J Haematol, 2001, 114: 63-69

[8] AdidaC., CrottyP. L., McGrathJ., et al.. Developmentally regulated expression of the novel cancer antiapoptosis gene survivin in human and mouse differentiation. Am J Pathol, 1998, 152: 43-49

[9] AmbrosiniG., AdidaC., SirugoG., et al.. Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J Biol Chem, 1998, 273(18): 11 177-11 182

[10] OlieR. A., Simes-WustA. P., BaumannB., et al.. A novel antisense oligonucleotide targeting survivin expression induces apoptosis and sensitizes lung cancer cells to chemotherapy. Cancer Res, 2000, 60: 2805-2809

[11] LiF., AltieriD. C.. The cancer antiapoptosis mouse survivin gene: characterization of locus and transcriptional requirements of basal and cell cycle-dependent expression. Cancer Res, 1999, 59(13): 3143-3151

[12] CasazzaA. M., FairchildC. R.. Paclitaxel (Taxol): mechanisms of resistance. Cancer Treat Res, 1996, 87: 149-171

[13] SchiblerM. J., CabralF.. Taxol-dependent mutants of Chinese hamster ovary cells with alterations in alpha-and beta-tubulin. J Cell Biol, 1986, 102(4): 1522-1531

[14] GiannakakouP., SackettD. L., KangY. K., et al.. Paclitaxel-resistant human ovarian cancer cells have mutant beta-tubulins that exhibit impaired paclitaxel-driven polymerization. J Biol Chem, 1997, 272(27): 17 118-17 125

[15] O’ConnorD. S., WallN. R., PorterA. C., et al.. A p34 (cdc2) survival checkpoint in cancer. Cancer Cell, 2002, 2(1): 43-54

[16] AnaC., MarC.. Survivin is required for stable checkpoint activation in taxol-treated Hela cells. J Cell Sci, 2003, 116: 2987-2998