Related Experiment Video
Updated: Jul 10, 2026
Preparation of Exosomes for siRNA Delivery to Cancer Cells
Published on: December 5, 2018
Engineered exosomes deliver structurally optimized toad BAX to reactivate mitochondrial apoptosis in colorectal
Xinqiang Xu1,2, Hongjie Wu2, Yixin Yan2
1Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing, China.
Introduction:
Mitochondrial apoptosis evasion, driven by overexpression of anti-apoptotic BCL-2 family proteins, remains a major obstacle in the effective treatment of colorectal cancer (CRC). While BCL-2 homology 3 (BH3) mimetics (such as venetoclax) have shown clinical efficacy in hematologic malignancies, their effectiveness in solid tumors is limited. Direct delivery of pro-apoptotic effectors, including BAX, offers an alternative strategy; yet, the therapeutic potential of non-human BAX orthologs and their delivery methods has not been explored.
Methods:
The functional activity of Xenopus laevis (African clawed frog) BAX was evaluated in human CRC cell lines (HCT116, LOVO) and in cell line-derived xenograft (CDX) and AOM/DSS-induced CRC mouse models. Direct binding to human BCL-2 was quantified by microscale thermophoresis. The mechanism of action was elucidated via JC-10 staining, subcellular fractionation and liposome permeabilization assays.Structure-guided mutagenesis based on the BCL-2-Beclin-1 BH3 complex (PDB: 5VAU) was employed to generate a triple mutant (I86T, A102S, R109M) . Optimized BAX was packaged into engineered exosomes for targeted delivery, and their anti-tumor efficacy and safety were assessed in CDX, patient-derived xenograft (PDX), and AOM/DSS models.
Results:
Toad BAX directly bound human BCL-2 (Kd = 12.7 ± 1.9 µM), triggered mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, and caspase-9/3 activation, thereby suppressing CRC cell proliferation and inducing apoptosis. The rationally designed triple mutant exhibited enhanced BCL-2 affinity and superior in vivo antitumor activity compared to wild-type toad BAX. Exosome-mediated delivery of the optimized BAX efficiently targeted CRC cells, inhibited tumor growth in PDX models, and extended overall survival in AOM/DSS-induced CRC without inducing overt toxicity.
Discussion:
This study establishes structurally optimized cross-species BAX, delivered via engineered exosomes, as a potent and safe strategy to reactivate mitochondrial apoptosis against CRC. It provides a preclinical foundation for protein-based therapeutics targeting apoptosis-evasive solid tumors, offering a mechanistically distinct alternative to conventional BH3 mimetics.
