Synergistic in vitro anticancer activity of doxorubicin-betulinic acid against neuroblastoma (SH-SY5Y) and lung carcinoma (A549) supported by in silico targeting of ALK

Neuroblastoma, a heterogeneous pediatric malignancy of neural crest origin, continues to exhibit poor prognosis in high-risk cases despite advances in multimodal therapy. Likewise, lung cancer, particularly non–small-cell lung carcinoma (NSCLC), frequently develops resistance to conventional chemotherapeutic agents. These challenges highlight the urgent need for novel therapeutic strategies with improved safety, selectivity, and the ability to target key molecular drivers of tumor progression. Combination approaches involving standard chemotherapeutics and bioactive phytochemicals represent a promising avenue for enhancing anticancer efficacy. In this study, betulinic acid (BA) was isolated for the first time from the ethyl acetate extract of Erythrophleum suaveolens (Guill. & Perr.) Brenan using column chromatography and thin-layer chromatography. Structural characterization was achieved through spectroscopic analyses, including 1 H-2D NMR and EI-MS. The cytotoxic activity of BA was evaluated using cell viability assays against SH-SY5Y human neuroblastoma and A549 human lung carcinoma cell lines, with doxorubicin (DOX) employed as a reference drug. Furthermore, molecular docking studies were conducted to investigate the interaction of BA with human anaplastic lymphoma kinase (ALK) in complex with crizotinib (PDB ID: 2XP2 ). Betulinic acid (3β-hydroxy-lup-20(29)-en-28-oic acid) exhibited significant cytotoxic activity against both cancer cell lines. In SH-SY5Y cells, BA demonstrated an IC₅₀ value of 61.65 ± 2.74 µM, markedly lower than that of doxorubicin (212.9 ± 32.63 µM), indicating superior potency. In A549 cells, BA showed an IC₅₀ of 26.86 ± 1.41 µM, comparable to doxorubicin (22.02 ± 3.14 µM). Notably, BA significantly potentiated DOX-induced cytotoxicity, particularly in SH-SY5Y cells, suggesting a strong chemosensitizing effect. Molecular docking revealed that BA binds within the ALK active site with a moderate binding energy (−5.149 kcal/mol), compared with crizotinib (−9.784 kcal/mol) and doxorubicin (−7.119 kcal/mol) supporting ALK as a potential molecular target. Overall, these findings highlight betulinic acid as a promising natural-product scaffold with chemosensitizing potential and warrant further mechanistic and in vivo investigations toward its development for pediatric and adult oncology.