[An EGFR antisense oligodeoxynucleotides and its phosphorothioate analogue inhibit the growth of human hepatocarcinoma BEL-7404 cells].
We have previously reported that the malignant phenotype of human liver carcinoma cell line BEL-7404 was reversed by antisense EGFR RNA. The aim of this paper is to explore the effects of an oligomer targeted to mRNA for EGFR and growth of BEL-7404 cells. A 21-mer oligodeoxynucleotides (ODNs) complementary to the 5' initiation region of mRNA for EGFR was synthesized and added to medium. The results showed that the growth of BEL-7404 cells was inhibited by ODNs at concentration of 3.2 mumol/L. Inhibition of DNA synthesis of BEL-7404 cells was dose-dependent and reached to 62.1% at 3.2 mumol/L as measured by 3H-thymidine incorporation test. The inhibition of EGFR gene transcription of the cells was up to 10.5% and 14.3% respectively after incubation with ODNs by 5 and 24 hours as measured by densitometric scanning of dot (RNA) blots of EGFR. The EGFR protein (P 170) expression was also found to be blocked by 4 days' antisense oligomer treatment up to 37.4% as measured by densitometric scanning of specific band of Western blot. The oligonucleotide phosphorothioate (S-ODNs) complementary to the same region of the gene was also synthesized and its growth inhibition effects on BEL-7404 cells were compared with those of unmodified oligomers. ODNs attained their highest effect within 30 hours. The proliferation inhibition rate of the cells didn't increase when cells were cultured in serum free medium. In contrast, the S-ODNs induced inhibition reached comparable level after 96 hours treatment as measured by 3H-thymidine uptake and the effect lasted longer, 1 mumol/L S-ODNs showed a little effect on BEL-7404 cells' proliferation. We concluded that the antisense oligomers directed to mRNA for EG-FR could inhibit the BEL-7404 cells growth by blocking the EGFR gene expression in some degree and the phosphorothioate analogues were more stable than the unmodified ODNs in vitro.