Nitrosylcobalamin Selectively Targets Tumors via Cobalamin Uptake and Lysosomal Processing

Background: Nitrosylcobalamin (NO-Cbl) is a vitamin B12 analog designed to exploit the “Trojan horse” vulnerability created by the heightened need of cancer cells for cobalamin and one-carbon metabolism. Building on our recent biophysical studies confirming the affinity of NO-Cbl for intrinsic factor, this work aimed to investigate the mechanistic basis for the selective anticancer activity of NO-Cbl through the cobalamin transport axis and lysosomal processing. Materials and Methods: Human cancer cell lines (NIH-OVCAR-3, MCF-7, WM9, and DU145) were cultured and transfected to overexpress transcobalamin II (TCII). Cell proliferation and cytotoxicity were measured using the sulforhodamine B (SRB) assay. TCII-R (CD320) expression was quantified by flow cytometry. The impact of anti-CD320 antiserum and lysosomal alkalization (chloroquine) on NO-Cbl activity was assessed. Results: Antiserum inhibition of the TCII receptor resulted in dose-dependent inhibition of NIH-OVCAR-3 and MCF-7 cell proliferation. Lysosomal alkalinization by chloroquine pretreatment abrogated NO-Cbl-induced cytotoxicity in OVCAR-3 cells. Flow cytometric analysis demonstrated an inverse correlation between TCII-R (CD320) expression (MFI ratio) and NO-Cbl ID50. TCII overexpression significantly reduced NO-Cbl ID50 in NIH-OVCAR-3 cells. Conclusion: NO-Cbl utilizes tumor cell cobalamin transport and processing pathways to deliver nitric oxide selectively to cancer cells. These results, integrated with recent binding studies, validate NO-Cbl as a cobalamin-based targeted anticancer agent with efficacy in tumors expressing high levels of TCII and CD320.