Inhibitory effects of EGFR antisense oligodeox ynucleotide in human colorectal cancer cell line.

Abstract

Epidermal-growth-factor receptor[1] (EGFR) is a polypeptide with 1186 amino acids, which binds to EGF family growth factors. Two major natural ligands in the family interact with EGFR: one is EGF, the other is transforming growth factor-α (TGF-α)[2]. When EGF or TGF-α, binds to EGFR, tyrosine kinase activity is induced which in turn triggers a series of events regulating the cell growth[3-8]. The importance of EGFR in growth regulating pathways was confirmed by the fact that enhanced expression of this receptor was found in brain glioblastomas, breast, lung, ovarian, colorectal, and renal carcinomas[9,10]. Elevated EGFR levels correlated with poor prognosis in human tumors[11-17], for this reason, it seemed to be that EGFR would be a logical target for cancer therapy. Previous reports had shown that monoclonal antibodies to EGFR were effective in the treatment of many human carcinoma cells[18-20]. Other drug therapies which targeted the EGFR had also been successful. Kunkel had shown a drug that inhibited EGFR tyrosine kinase activity could inhibit the growth of A431 cells in nude mice[10]. Yoneda also reported that selective inhibitors of EGFR tyrosine kinase activity, such as tyrphostins, could inhibit the growth of squamous carcinoma in nude mice[21]. Antisense oligodeoxynucleotides inhibit gene expression on a highly selective and target sequence in a specific manner[22-25]. Specific oligonucleotides hybridize to complementary mRNA and decrease protein expression[26-29]. Antisense oligonucleotides against proto-oncogenes of growth factors had already been shown to be successful in cell lines[18,30]. For example, an antisense oligonucleotide to the erB2 gene product had been shown to inhibit protein production in a breast cancer cell line[31]. Akino reported inhibition of in vivo growth and metastases in malignant pituitary tumors with an antisense compound to the PTHrp (parathyroid hormone-related peptide)[32]. An oligonucleotide to the c-myc gene inhibited the growth of thyroid carcinoma cell lines[33]. Phosphorothioate antisense oligodeoxynucleotides targeted against human c-raf-1 kinase producing potent antiproliferative effects on cell culture and in vivo antitumor effects against a variety of tumor types[34]. The co-expression of EGFR along with TGF-α in human colon cancer cell lines, also in colon carcinoma tissue, had led to the suggestion that the autocrine stimulation of EGFR by its ligands could be a mechanism for tumor cells to escape from normal growth controls[35]. Previous studies in our laboratory confirmed over-expression of EGFR in HR8348 cells[36]. In this investigation, we hypothesized that growth and proliferation of HR8348 could be inhibited by EGFR ASODN. In this report 15-mer EGFR ASODN was synthesized and the effects of EGFR ASODN on cell proliferation and tumorigenic rate of HR8348 cells were observed.