Anti-tumorigenic effect of a K-ras ribozyme against human lung cancer cellline heterotransplants in nude mice

Approximately 15-30% of human non-small cell lung cancers (NSCLC) carry K-r as mutations, among which point mutations at codon 12 are the most common. This study characterizes the anti-tumor effect of an anti-K-ras ribozyme ad enoviral vector (KRbz-ADV; replication-deficient, E1-deleted Ad5 backbone) against NSCLC lines that express the relevant mutation (K-ras codon 12 GGT --> GTT; H441 and H1725). KRbz-ADV significantly inhibited tumor cell growt h (38-94% reduction by H-3-thymidine uptake) in a time- and dose-dependent manner, but produced minimal growth inhibition on normal epithelial cells, or NSCLC H1650 cells that lack the relevant mutation. The in vivo anti-tumo ri-genic effect of KRbz-ADV treatment was characterized with cell line xeno grafts in nu/nu mice. Pre-treatment with KRbz-ADV (10 or 20 p.f u. per cell ) completely abrogated subcutaneous engraftment of H441 (n = 13) or H1725 c ells (n = 8), as compared with a 100% tumor take and progressive tumor grow th in animals that received untreated tumor cells, or control vector (lucif erase-adenovirus/Luc-ADV)-treated tumor cells. Pre-treatment with a mutant anti-K-ras ribozyme adenoviral vector (mutKRbz-ADV), which has the same spe cificity as KRbz but lacks ribozyme catalytic activity, did not produce an anti-tumorigenic effect. The in vivo effect of KRbz-ADV treatment was furth er examined by initiating injections (2 x 10(9) p.f. u.) at 7 days after tu mor induction. Preexisting tumor growth was reduced by 39% by a single intr atumoral injection. Repeat injections (three or five KRbz-ADV-intratumoral injections at 2 x 10(9) p.f.u. every other day) resulted in complete tumor regression in five of seven mice. In contrast, single or multiple injection s of control vector Luc-ADV did not significantly alter tumor xenograft out come. Ribozyme expression was confirmed in H441 cells that demonstrated red uced growth after KRbz-ADV treatment. Reduced growth corresponded to signif icantly lowered levels of K-ras mRNA, as defined by RT-PCR (51% of untreate d level, n = 3) and RNase protection assay (56% of untreated level, n = 4) analyses. Further 37.5% of KRbz-ADV-treated cells underwent apoptosis, as c ompared with 11.7%, and 19.0% in untreated and Luc-ADV-treated cultures, re spectively. A significantly higher proportion of KRbz-ADV-treated H441 cell s (58.2%) underwent apoptosis when maintained under anchor-independent cond itions that simulate in vivo tumorigenesis ('anoikis'). This is the first r eport that demonstrates that KRbz-ADV can effectively inhibit in vivo tumor i-genesis, and produces regression of pre-existing human lung tumor xenogra fts having the relevant K-ras mutation.