5-Aminolevrinic Acid Exhibits Dual Effects on Stemness in Human Sarcoma Cell Lines under Dark Conditions

5-aminolevulinic acid (ALA) is used for tumor-targeting phototherapy because it is converted to protoporphyrin IX (PPIX) upon excitation and induces phototoxicity. However, the effect of ALA on malignant cells under unexcited conditions is unclear. This information is essential when administering ALA systemically. We used sarcoma cell lines that usually arise deep in the body and are rarely exposed to light to examine the effects of ALA treatment under light (daylight lamp irradiation) and dark (dark room) conditions. ALA-treated human SW872 liposarcoma cells and human MG63 osteosarcoma cells cultured under light exhibited growth suppression and increased oxidative stress, while cells cultured in the dark showed no change. However, sphere-forming ability increased in the dark, and the expression of stem-cell-related genes was induced in dark, but not light, conditions. ALA administration increased heme oxygenase 1 (HO-1) expression in both cell types; when carbon monoxide (CO), a metabolite of HO-1, was administered to sarcoma cells via carbon-monoxide-releasing molecule 2 (CORM2), it enhanced sphere-forming ability. We also compared the concentration of biliverdin (BVD) (a co-product of HO-1 activity alongside CO) with sphere-forming ability when HO-1 activity was inhibited using ZnPPIX in the dark. Both cell types showed a peak in sphere-forming ability at 60-80 μM BVD. Furthermore, a cell death inhibitor assay revealed that the HO-1-induced suppression of sphere formation was rescued by apoptosis or ferroptosis inhibitors. These findings suggest that in the absence of excitation, ALA promotes HO-1 expression and enhances the stemness of sarcoma cells, although excessive HO-1 upregulation induces apoptosis and ferroptosis. Our data indicate that systemic ALA administration induces both enhanced stemness and cell death in malignant cells located in dark environments deep in the body and highlight the need to pay attention to drug delivery and ALA concentrations during phototherapy.