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

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Forry, Suzanne L
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Cleveland Clinic Lerner
Other Basic Sciences
Schools of Medicine
United States
Zip Code
Tang, Zhuo; Bauer, Joseph A; Morrison, Bei et al. (2006) Nitrosylcobalamin promotes cell death via S nitrosylation of Apo2L/TRAIL receptor DR4. Mol Cell Biol 26:5588-94
Chawla-Sarkar, Mamta; Bauer, Joseph A; Lupica, Joseph A et al. (2003) Suppression of NF-kappa B survival signaling by nitrosylcobalamin sensitizes neoplasms to the anti-tumor effects of Apo2L/TRAIL. J Biol Chem 278:39461-9
Bauer, Joseph A; Morrison, Bei H; Grane, Ronald W et al. (2002) Effects of interferon beta on transcobalamin II-receptor expression and antitumor activity of nitrosylcobalamin. J Natl Cancer Inst 94:1010-9