Effects of interferon β 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 β (IFN-β) 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-β, 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 ID₅₀s [the dose that inhibits growth by 50%] as low as 2 μM) than normal cell lines (with ID₅₀s of 85-135 μM). Single-agent NO-Cbl and IFN-β treatment of NIH-OVCAR-3 xenografts induced tumor regression, whereas combination treatment induced tumor eradication. IFN-β treatment increased TC II-R expression in vitro and uptake of [⁵⁷Co]cobalamin in vivo. Compared with NIH-OVCAR-3 cells treated with NO-Cbl, cells treated with NO-Cbl and IFN-β 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. Conclusion: NO-Cbl inhibited tumor growth in vivo by activating the extrinsic apoptotic pathway. The increased expression of TC II-R induced by IFN-β resulted in enhanced antitumor effects with NO-Cbl both in vitro and in vivo.