Glioblastoma multiforme (GBM) is an aggressive brain tumor with limited treatment options and poor survival outcomes. This study evaluated the anticancer potential of seco-duocarmycin SA (seco-DSA), a potent DNA-alkylating agent, alone and in combination with proton radiation in human GBM cell lines. Human glioblastoma cell lines T98G and LN18 were treated with varying concentrations of seco-DSA, proton radiation doses (2, 4, or 8 Gy), or both. Proton irradiation was delivered with a 250-MeV beam. Clonogenic survival, cell proliferation, and cell cycle distribution were analyzed using colony formation and flow cytometry assays. Proteomic analysis of LN18 cells was performed by LC-MS/MS followed by bioinformatic pathway analysis. Statistical significance was determined using a two-tailed unpaired t-test (p ≤ 0.05), and Bliss synergy scores were calculated to assess treatment interactions. Combination therapy produced additive and synergistic inhibition of colony formation and enhanced G2/M phase arrest compared with either treatment alone. Apoptosis and necrosis increased modestly but did not fully account for observed cytotoxicity. Proteomic profiling revealed differential expression of proteins involved in DNA repair, apoptosis, and senescence, indicating that seco-DSA broadened radiation-induced stress responses. Seco-DSA potentiates the cytotoxic effects of proton radiation in GBM cells through enhanced clonogenic inhibition and modulation of cell cycle and DNA repair pathways. These findings support seco-DSA as a promising radiosensitizer for further preclinical evaluation.

Glioblastoma multiforme (GBM) is among the most lethal primary brain tumors and is characterized by significant cellular heterogeneity and resistance to conventional therapies. This study investigates the efficacy of seco-duocarmycin SA (seco-DSA), a novel DNA alkylating agent. Initial investigations using a colony formation assay revealed that seco-DSA exhibits remarkable potential with IC₅₀ values lower than its natural DSA counterpart. Cell viability assay indicated that LN18 cells showed a markedly greater sensitivity to DSA than T98G cells. Furthermore, seco-DSA achieved its full cytotoxic effect within 8 h of drug incubation in GBM cell lines. Although seco-DSA induced a concentration-dependent increase in apoptotic cell death, the extent of apoptosis did not fully account for the observed decrease in cell viability. Instead, seco-DSA treatment resulted in significant cell cycle arrest in S and G2/M phases. These findings suggest that seco-DSA’s cytotoxicity in GBM cells is primarily due to its ability to disrupt cell cycle progression, though the precise mechanisms of action remain to be fully established, and further research is needed. Proteomic analysis of treated cells also indicates dysregulation of proteins involved in senescence, apoptosis, and DNA repair, alluding to seco-DSA-induced arrest as a major mechanism of GBM disruption. Data are available via ProteomeXchange with the dataset identifier “PXD061023”. Our reports promote the future exploration of seco-DSA’s therapeutic potential, representing a critical step toward developing a more targeted and effective treatment for GBM.