Effects of interferon beta on transcobalamin II-receptor expression and antitumor activity of nitrosylcobalamin.
BACKGROUND
The ubiquitous plasma membrane transcobalamin II receptor (TC II-R) mediates uptake of cobalamin (Cbl; vitamin B12), an essential micronutrient. Tumors often require more Cbl than normal tissue, and increased Cbl uptake may result from increased TC II-R expression. To examine whether Cbl could therefore be used as a carrier molecule to target a chemotherapy drug, we tested an analogue of Cbl with nitric oxide as a ligand, nitrosylcobalamin (NO-Cbl). Because interferon beta (IFN-beta) has antitumor effects and increases expression of some membrane receptors, we examined whether it may enhance the effects of NO-Cbl.


METHODS
Antiproliferative effects of NO-Cbl were assessed in 24 normal and cancer cell lines. Xenograft tumors of human ovarian cancer NIH-OVCAR-3 cells were established in athymic nude mice, and tumor growth was monitored after treatment with NO-Cbl and IFN-beta, both individually and concomitantly. TC II-R expression and apoptosis was monitored in vitro and in vivo. RNA protection assays and mitochondrial membrane potential assays were used to distinguish the extrinsic and intrinsic apoptotic pathways, respectively.


RESULTS
Cancer cell lines were more sensitive to NO-Cbl (with ID(50)s [the dose that inhibits growth by 50%] as low as 2 microM) than normal cell lines (with ID(50)s of 85-135 microM). Single-agent NO-Cbl and IFN-beta treatment of NIH-OVCAR-3 xenografts induced tumor regression, whereas combination treatment induced tumor eradication. IFN-beta treatment increased TC II-R expression in vitro and uptake of [(57)Co]cobalamin in vivo. Compared with NIH-OVCAR-3 cells treated with NO-Cbl, cells treated with NO-Cbl and IFN-beta were more apoptotic and expressed higher mRNA levels of various apoptosis-associated genes. No changes in mitochondrial membrane potential were observed in cells treated with NO-Cbl.


CONCLUSIONS
NO-Cbl inhibited tumor growth in vivo by activating the extrinsic apoptotic pathway. The increased expression of TC II-R induced by IFN-beta resulted in enhanced antitumor effects with NO-Cbl both in vitro and in vivo.
Bauer
Joseph A
JA
Taussig Cancer Center, Center for Cancer Drug Discovery and Development, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Morrison
Bei H
BH
Grane
Ronald W
RW
Jacobs
Barbara S
BS
Dabney
Sally
S
Gamero
Ana M
AM
Carnevale
Kevin A
KA
Smith
Daniel J
DJ
Drazba
Judith
J
Seetharam
Bellur
B
Lindner
Daniel J
DJ
D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000627:therapeutic use; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D003131:Combined Modality Therapy; D004357:Drug Synergism; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D016899:Interferon-beta / Q000627:therapeutic use*; D008297:Male; D008545:Melanoma / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D008564:Membrane Potentials; D051379:Mice; D008819:Mice, Nude; D008928:Mitochondria / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D009603:Nitroso Compounds / Q000494:pharmacology*; D010051:Ovarian Neoplasms / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012235:Rhodamines; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014805:Vitamin B 12 / Q000031:analogs & derivatives* / Q000494:pharmacology*
D017304:Annexin A5; D000970:Antineoplastic Agents; D009603:Nitroso Compounds; D011956:Receptors, Cell Surface; D012235:Rhodamines; C113298:nitrosylcobalamin; C028176:transcobalamin receptor; C022027:lissamine rhodamine B; D009569:Nitric Oxide; D016899:Interferon-beta; D012259:Ribonuclease, Pancreatic; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D014805:Vitamin B 12