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

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA098536-02
Application #
6889219
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Forry, Suzanne L
Project Start
2004-04-26
Project End
2008-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
2
Fiscal Year
2005
Total Cost
$250,920
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195
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