Antitumor effect of c-myc antisense phosphorothioate oligodeoxynucleotides on human melanoma cells in vitro and in mice

Background: Phosphorothioate oligodeoxynucleotides ([S]ODNs) contain a modified internucleoside phosphate backbone. Antisense [S]ODNs targeted to specific oncogenes have been used with some therapeutic success in animal models of human leukemia; however, the potential for antisense [S]ODN treatment of solid tumors has only recently been explored. Purpose: We evaluated the effects of antisense [S]ODNs targeted to the c-myc oncogene on the proliferation of human melanoma cells in vitro and on the growth of human melanoma xenografts in CD-1 nude (nu/nu) mice. Methods: The effects of 15-mer [S]ODNs containing c-myc sense, c-myc antisense, and two different scrambled sequences on the proliferation and viability of cultures of three established human melanoma cell lines (M14, JR8, and PLF2) were determined by measuring cell numbers and use of the trypan blue exclusion test. The induction of apoptosis in these cells following treatment with [S]ODNs was evaluated by fluorescence-activated cell sorter (FACS) analysis. FACS analysis was also used to determine the effects of [S]ODN treatment on the proliferation of primary cultures of a human melanoma explant (NG cells). The expression of c-Myc protein in cultured NG cells after treatment with [S]ODNs was examined by western blot analysis. The antitumor activity and the toxic effects of several [S]ODN treatment regimens were monitored by measuring differences in tumor weight (percent tumor weight inhibition), tumor growth rate (tumor growth inhibition), animal lifespan (percent increase in lifespan), the number of toxic deaths, and the median number of lung metastases in treated and control mice bearing NG xenografts, c-Myc protein expression in NG tumor cells following [S]ODN treatment was evaluated by FACS analysis, and the extent of apoptosis in these cells was determined by FACS analysis and morphologic examination. Results: Treatment with antisense [S]ODNs, but not the others, inhibited the growth of all tested melanoma cultures in vitro; FACS analysis revealed that growth inhibition was associated with the induction of apoptosis. Antisense [S]ODN treatment also led to reduced cellular levels of c-Myc protein. In vivo, [S]ODN antitumor activity and toxicity were dose and schedule dependent; however, only antisense [S]ODNs exhibited antitumor activity. Mice bearing NG xenografts treated with antisense [S]ODNs showed a marked inhibition of tumor growth, a reduction in the number of lung metastases, and an increase in lifespan. Reduced levels of c-Myc protein and increased levels of apoptosis were also observed in NG tumor cells following antisense [S]ODN treatment. Conclusions: Treatment of human melanoma cells and solid tumors with antisense [S]ODNs targeted to c-myc inhibits their growth and is associated with the induction of apoptosis.