ACTIVATION OF NITRIC-OXIDE SYNTHASE GENE FOR INHIBITION OF CANCER METASTASIS

The process of cancer metastasis consists of multiple sequential and h ighly selective steps, The vast majority of tumor cells that enter the circulation die rapidly and only a few survive and proliferate to for m distant metastases. This survival is not random, Metastases are clon al in origin and are produced by specialized subpopulations of cells t hat preexist in a heterogeneous primary tumor, Metastatic cells of the murine K-1735 melanoma survive in the circulation to produce experime ntal lung metasteses, whereas nonmetastatic cells do not, After incuba tion with different cytokines or LPS, nonmetastatic cells exhibit a hi gh level of inducible nitric oxide synthase (iNOS) activity and nitric oxide (NO) production, whereas metastatic cells do not, To provide di rect evidence for the inverse correlation between the production of en dogenous NO and the ability of K-1735 cells to produce metastasis in s yngeneic mice, highly metastatic clone 4 cells (C4.P), which express l ow levels of iNOS, were transfected with a functional iNOS (C4.L8), in active mutated iNOS (C4.S2), or neomycin resistance (C4.Neo) genes in medium containing 3 mM NMA, C4.P, C4.Neo3, and C4.S2.3 cells were high ly metastatic, whereas C4.L8.5 cells were not, Moreover, C4.L8.5 cells produced slow-growing subcutaneous tumors in nude mice, whereas the o ther three cell lines produced fast-growing tumors. In vitro studies i ndicated that the expression of iNOS in C4.L8.5 cells was associated w ith apoptosis, Multiple intravenous injections of liposomes containing a synthetic lipopeptide up-regulated iNOS expression in murine M5076 reticulum sarcoma cells growing as hepatic metastases, The induction o f iNOS was associated with the complete regression of the lesions, Col lectively, these data demonstrate that the expression of iNOS in tumor cells is associated with apoptosis, suppression of tumorigenicity, ab rogation of metastasis, and regression of established hepatic metastas es.