[Effect of survivin antisense oligodeoxynucleotide on carcinoma of larynx in vivo and in vitro]

Abstract

Objective: To observe the effect of survivin antisense oligodeoxynucleotide (ASODN) on the apoptosis of human carcinoma of larynx cell line Hep2 and the inhibitory rate in nude mice model so as to discuss the selective blocking activity of antisense technique on gene expression seeking a new way for gene therapy of carcinoma of larynx.

Methods: Antisense oligodeoxynucleotides survivin were transformed into human carcinoma of larynx cell line Hep2 by liposome Lipofectamine 2000. Within 72 h after transfection, 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cellular proliferation. Forty eight hours after transfection, reverse transcription-polymerase chain reaction (RT-PCR) assay was used to observe the expression of survivin gene, Western Blot assay for the protein, and terminal deoxynucleotide mediated nick end labeling (TUNEL) and flow cytometer for cellular apoptosis.

Results: Cellular inhibition rate of 72 h went up to 52. 5% and 71.4% at 1.0 micromol/L and 2.0 micromol/L value in Lipo-ASODN groups which differed statistically remarkably (P = 0.046), higher than that in controls in MTT assay (P =0. 003 and 0. 0004). Forty eight hours after transfection survivin gene expression in Lipo-ASODN groups were less than that in control group in RT-PCR assay. Survivin protein expression decreased in Western blot. In TUNEL assay, nuclear positive staining was observed and the apoptosis peak was observed in flow cytometer test, which were absent in controls. In nude mice of carcinoma of larynx model, the inhibitory rate in Lipo-ASODN groups got up to 48.1% and 61.3% higher than that of controls (P < 0.004 and 0. 0006), which differed remarkably (P = 0.032) in a dose-dependently way.

Conclusions: The findings showed that the expression of survivin gene and protein induced cellular apoptosis in Hep2 cells after transfection of Lipo-ASODN and that the carcinoma of larynx in the nude mice model were inhibited by Lipo-ASODN which suggested that antisense technique can be an effective means in the gene therapy of carcinoma of larynx.