Growth inhibition of human pancreatic cancer cell lines by anti-sense oligonucleotides specific to mutated K-ras genes.

Abstract

About 90% of human pancreatic cancers carry K-ras point mutation, which may play an important role in tumorigenesis. We investigated the inhibitory effects of anti-sense oligonucleotides targeting K-ras point mutation on the growth of cultured human pancreatic cancer cells. Eight human pancreatic cancer cell lines were screened for K-ras codon 12 point mutations by PCR-RFLP analysis and direct sequencing. Then, 3 cell lines with the major types of K-ras point mutation, i.e.,HuP-T1, HuP-T3 and PANC-1, and 1 without mutation, BxPC-3, were used for the experiments. Seventeen mer anti-sense oligonucleotides were designed, targeting the point mutation of K-ras codon 12, and transfected into the cells by the liposome-mediated method. Cell-growth activities were estimated by MTT assay. Levels of K-ras mRNA expression were determined using quantitative RT-PCR, and K-ras p21 protein synthesis was evaluated with Western blotting. Mutation-matched anti-sense oligonucleotides effectively inhibited the growth of these pancreatic cancer cell lines, except for BxPC-3, by suppressing K-ras mRNA expression and K-ras p21 protein synthesis. Moreover, mutation-matched anti-sense oligonucleotides showed stronger anti-proliferative effects than did mutation-mismatched ones. Our results suggest that anti-sense therapy specific to point mutations of K-ras mRNA is a practical approach to selective suppression of tumor growth, with little effect on normal cells.