Opposite effects of spermidine and GC7 in cell culture are dictated by distinct molecular targets.

Spermidine (SPD) and related polyamines are small polycationic molecules typically elevated in cancer cells, where their depletion suppresses tumor growth both in vitro and in vivo. Paradoxically, SPD has also been proposed as a dietary supplement for its potential health benefits, including cancer prevention, prompting considerable interest in elucidating its mechanisms of action. In vitro studies using cultured cancer cell lines treated with exogenous SPD have yielded conflicting results, with reports of enhanced proliferation, cytotoxicity, or modulation of autophagy. To address these discrepancies, we used polyamine-depleted colorectal cancer (CRC) cells to systematically evaluate SPD's effects across a range of concentrations. Following depletion with difluoromethylornithine, SPD exhibited a biphasic response: at low concentrations (<20 µM), it promoted proliferation via deoxyhypusine synthase (DHPS)-dependent hypusination of eukaryotic initiation factor 5A, whereas high concentrations (>100 µM) induced DHPS-independent cytotoxicity mediated by bovine serum amine oxidase (BSAO) activity in fetal bovine serum. High SPD doses transiently inhibited the autophagic flux, while low doses did not display any effect at all time points tested. The DHPS inhibitor GC7 (N1-guanyl-1,7-diaminoheptane) suppressed SPD-induced proliferation at low concentrations and unexpectedly prevented cytotoxicity at high concentrations. Kinetic assays revealed that GC7 also inhibits BSAO in a non-competitive manner (Ki ≈ 300 nM), independent of DHPS. In silico docking analysis indicated that GC7 binds BSAO via non-covalent interactions, outside the topaquinone organic cofactor site. These findings clarify the concentration-dependent effects of SPD in CRC cells, reconcile conflicting in vitro data, and identify BSAO as a previously unrecognized target of GC7, providing new mechanistic insights into polyamine-driven cancer biology.