Spermidine's Double-Edged Sword: Low Doses Boost Growth, High Doses Kill Cells

Spermidine has attracted significant interest as a dietary supplement with potential anti-aging and cancer-preventive properties. Yet in vitro studies using cancer cell lines have produced confusing results—sometimes showing cell growth stimulation, sometimes cytotoxicity, and sometimes effects on autophagy. This study from Sapienza University of Rome systematically investigated these discrepancies using human colorectal cancer (CRC) cell lines.

The researchers first depleted HCT116 and LoVo CRC cells of endogenous polyamines using difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine synthesis enzyme ODC. They then treated these polyamine-depleted cells with a wide range of exogenous SPD concentrations (10 nM to 1 mM) and measured cell proliferation, eIF5A hypusination, MYC protein levels, autophagy markers, and cell death indicators.

A clear biphasic dose-response emerged. At low concentrations (≤10–20 µM), SPD significantly rescued cell proliferation in a dose-dependent manner. This was accompanied by increased eIF5A hypusination and MYC protein expression, consistent with DHPS-dependent translational activation of the MYC oncogene. Genetic knockdown of either DHPS or eIF5A abolished this pro-proliferative effect, and blocking the polyamine transporter AMXT-1510 also prevented low-dose SPD from entering cells and promoting growth. At high concentrations (≥100 µM), however, SPD paradoxically inhibited and ultimately killed cells—an effect that persisted even when DHPS or eIF5A were knocked down, indicating a completely separate mechanism.

The authors traced the cytotoxic high-dose effect to bovine serum amine oxidase (BSAO), a copper-containing enzyme present in the fetal bovine serum (FBS) routinely used in cell culture. BSAO oxidatively deaminates polyamines like SPD, generating cytotoxic hydrogen peroxide and acrolein. This was confirmed using cell-free BSAO assays, catalase co-treatment, and serum-free conditions—all of which abolished or attenuated high-dose SPD toxicity.

A key unexpected discovery involved GC7 (N1-guanyl-1,7-diaminoheptane), a well-established DHPS inhibitor used as a research tool and explored therapeutically. When GC7 was added alongside high-dose SPD, it not only blocked the DHPS-mediated pro-proliferative effect of low-dose SPD (as expected), but also completely prevented the cytotoxicity of high-dose SPD—an effect independent of DHPS. Kinetic enzyme assays revealed that GC7 is a potent, non-competitive inhibitor of BSAO with a Ki of approximately 300 nM. Molecular docking simulations confirmed that GC7 binds BSAO at a site distinct from the enzyme's topaquinone catalytic cofactor, via non-covalent interactions. This dual inhibitory activity of GC7—targeting both DHPS and BSAO—explains why it paradoxically protected cells from high-dose SPD-mediated death.

High-dose SPD transiently inhibited autophagic flux (as measured by LC3-II accumulation and p62 levels), while low-dose SPD had no detectable effect on autophagy at any tested time point, clarifying another source of conflicting literature reports. Taken together, these findings establish a rigorous mechanistic framework for the concentration-dependent actions of SPD in CRC cell culture and identify BSAO as a previously unrecognized molecular target of GC7.