Efficacy of c-erbB-2 antisense oligonucleotide transf ection on uterine endometrial cancer HEC-1 A cell lines

Objective: Antigene therapy targeting only one oncogene has made much progress although it still has some limitations. To explore the potential for antigene therapy in uterine endometrial cancer, we examined the in vitro inhibitory effects of liposmal anti-sense phosphorothioate oligonucleotides targeting c-erbB-2 in the human uterine endometrial cancer HEC-1A cell line.

Methods: 1) To detect c-erbB-2 protein expression on HEC-1A cell membranes by immunohisto- chemistry. 2) To assay cellular growth inhibition by MTT after transfecting 0.1-0.6 microM ASODN. 3) To observe cellular and ultra-structural changes under transmission electron microscope and to assay the cellular apoptotic rate by flow cytometry and c-erbB-2 mRNA, and protein expression by RT-PCR and Western blot after transfecting 0.3 microM ASODN.

Results: 1) c-erbB-2 protein expression was positive on HEC-1A cell membranes. 2) With the increase of the transfecting ASODN concentration from 0.1-0.6 microM, HEC-1A cellular growth inhibition was also enhanced. The results of MTT showed that when the transfecting concentration of ASODN was 0.3 microM, the HEC-1A cellular growth inhibition rate was 50% while when the transfecting concentration of ASODN was 0.6 microM, the HEC-1A cell growth inhibition rate was 75%. 3) When the concentration of transfecting ASODNs was 0.3 microM, there were obvious vacuolar degenerations in the plasma of HEC-1A cells, disappearance of organelle and nuclear structure and obvious shrinkage of nuclei under transmission electron microscope. The cellular apoptotic rate was 62.80%, while c-erbB-2 mRNA and protein expression were 47.18% and 33.60%, respectively, compared with those of the normal control cells.

Conclusion: Transfecting c-erbB-2 ASODNs can obviously suppress the mRNA and protein expression in HEC-1A cells, cause cellular apoptosis and inhibit cell growth. It may be a more useful gene therapy for endometrial cancer.

Antisense; Oligodeoxynucleotides; Transfection; Uterine endometrial neoplasms

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