THERAPY OF CANCER METASTASIS BY ACTIVATION OF THE INDUCIBLE NITRIC-OXIDE SYNTHASE

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; only a few survive to produce metastases. Th is survival is not random. Metastases are clonal in origin and are pro duced by specialized subpopulations of cells that preexist in a hetero geneous primary tumor. Experimental studies concluded that metastatic cells survive in the circulation whereas nonmetastatic cells do not. I n part, this difference is due to an inverse correlation between expre ssion of endogenous inducible nitric oxide synthase (iNOS) and product ion of nitric oxide (NO) and metastatic potential. Direct evidence for the role of iNOS in metastasis has been provided by our data on trans fection of highly metastatic murine K-1735 clone 4 (C4.P) cells which express low levels of iNOS, with a functional iNOS (C4.L8), inactive m utated iNOS (C4.S2), or neomycin resistance (C4.Neo) genes in medium c ontaining 3 mM of the specific iNOS inhibitor NG-L-methyl arginine (NM A). C4.P, C4.Neo, and C4.S2 cells were highly metastatic, whereas C4.L 8 cells were not. Moreover, C4.L8 cells produced slow-growing subcutan eous tumors in nude mice, whereas the other 3 cell lines produced fast -growing tumors. In vitro studies indicated that the expression of iNO S in C4.L8.5 cells was associated with either cytostasis or cytolysis via apoptosis, depending upon NO output. The tumor cells producing hig h levels of NO underwent autocytolysis and produced cytolysis of bysta nder cells under both in vitro and in vivo conditions. Multiple i.v. i njections of liposomes containing a synthetic lipopeptide upregulated iNOS expression in murine M5076 reticulum sarcoma cells growing as hep atic metastases. The induction of iNOS was associated with the complet e regression of the lesions. Transfection of interferon-beta suppresse d tumor formation and eradicated metastases, which was apparently link ed to iNOS expression and NO production in host cells such as macropha ge. Besides mediating cell death, NO produced tumor suppression by reg ulating expression of genes related to metastasis, e.g., survival, inv asion, and angiogenesis. Suppression of metastasis can be achieved thr ough use of immunomodulators that induce iNOS expression in tumor lesi ons or by the direct delivery of the iNOS gene to tumor cells or host cells through liposome and/or viral vectors.