Through receptor-mediated uptake and nitric oxide release, nitrosylcobalamin (NO-Cbl) was designed to induce cell death in malignancies with high requirement for cobalamin. Cellular NO-Cbl uptake was dependent upon the transcobalamin II receptor (TC II-R), the specific plasma membrane receptor for vitamin B 12. NO (the active cytotoxic moiety) was released from the cobalamin (Cbl) carrier following receptor-mediated endocytosis in the acidic environment of the lysosome. NO-Cbl had potent antiproliferative activity against several human cancer cell lines in vitro, characterized by ID50s ranging from 1-75 p,M. NO-Cbl induced apoptosis via a death receptor/caspase pathway. Death was accompanied by induction of TRAIL/Apo2L, caspase-8, and caspase-7 mRNAs, and rapid induction of caspase-8 enzymatic activity. NO-Cbl, at doses sufficient to induce apoptosis, induced nitrosylation of cysteine residues of cellular proteins (S-nitrosylation), but did not alter mitochondrial trans-membrane potential (psi-delta m). Non-malignant cell lines were relatively resistant to NO-Cbl compared to malignant cell lines. Tumor regression was observed in established ovarian and breast carcinoma xenografts in a nude mouse model. NO-Cbl doses of 170 mg/kg/day for 60 days resulted in tumor apoptosis and necrosis, but no histopathologic changes in normal tissues. Interferon-beta (IFN-beta) up-regulated expression of TC II-R, resulting in enhanced cytotoxicity mediated by NO-Cbl (1). Co-treatment with human IFN-beta and NO-Cbl accelerated xenograft regression compared to single agent therapy. Yet, co-treatment with murine IFN-beta and NO-Cbl was not toxic to normal tissues. Hence, NO-Cbl appears to be an attractive chemotherapeutic agent, whose effectiveness can be enhanced by pretreatment with IFN-beta to render NO-Cbl-resistant tumors more sensitive. The exact mechanism by which NO induces cell death is still undetermined. Identification of NO-Cbl's cellular target(s) will provide a mechanistic basis and rationale for use of NO-Cbl in clinical trials. Understanding the NO-Cbl-induced death process will also facilitate design of improved NO donors. We postulate that S-nitrosylation of critical cellular proteins, and potentiated NO-Cbl uptake by IFNs, are responsible for death induction in malignant cells. To test these hypotheses, we will perform the following Specific Aims: 1. Characterize functional changes in two cellular targets that are S-nitrosylated by NO-Cbl a. Define role of TRAIL, DR4, and DR5 in NO-Cbl-induced apoptosis b. Determine whether S-nitrosylation of TRAIL receptors leads to enhanced apoptosis c. Determine whether S-nitrosylation of PRL phosphatases inhibits their enzymatic activity 2. Dissect mechanism of augmentation of NO-Cbl activity by IFN-beta a. Correlate upregulation of TCII-R with NO-Cbl / IFN-beta antiproliferative synergy b. Measure effects of IFN-beta upon iNOS promoter activity (promoter mutagenesis) c. Determine whether inhibition of TCII-R expression causes resistance to NO-Cbl d. Define effects of IFN-beta upon S-nitrosylation of TRAIL receptors and PRL phosphatase