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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.
Insights
Engineered toad BAX protein reactivates apoptosis in colorectal cancer (CRC) cells. Exosome delivery of optimized BAX shows potent anti-tumor effects and safety in preclinical models, offering a novel therapeutic strategy.
Area of Science:
- Oncology
- Molecular Biology
- Biotechnology
Background:
- Colorectal cancer (CRC) often evades apoptosis via anti-apoptotic BCL-2 proteins, limiting treatment efficacy.
- Current BCL-2 homology 3 (BH3) mimetics are less effective in solid tumors compared to hematologic malignancies.
- Targeting BAX, a pro-apoptotic effector, presents an alternative strategy for CRC treatment.
Purpose of the Study:
- To evaluate the therapeutic potential of Xenopus laevis (African clawed frog) BAX in colorectal cancer.
- To engineer and deliver optimized BAX using exosomes for enhanced anti-tumor activity.
- To establish a preclinical foundation for protein-based therapeutics against apoptosis-evasive solid tumors.
Main Methods:
- Assessed functional activity of wild-type and mutant toad BAX in human CRC cell lines and mouse models.
- Quantified direct binding of toad BAX to human BCL-2 using microscale thermophoresis.
- Utilized structure-guided mutagenesis to create an optimized triple mutant BAX.
- Engineered exosomes for targeted delivery of optimized BAX and evaluated anti-tumor efficacy and safety in xenograft and chemically induced CRC models.
Main Results:
- Toad BAX directly bound human BCL-2, induced mitochondrial outer membrane permeabilization (MOMP), and triggered apoptosis in CRC cells.
- The rationally designed triple mutant BAX demonstrated enhanced BCL-2 affinity and superior in vivo anti-tumor activity.
- Exosome-mediated delivery of optimized BAX effectively targeted CRC cells, inhibited tumor growth, and improved survival in preclinical models without significant toxicity.
Conclusions:
- Structurally optimized, cross-species BAX delivered via engineered exosomes is a potent and safe strategy to reactivate mitochondrial apoptosis in CRC.
- This approach provides a mechanistically distinct alternative to conventional BH3 mimetics for treating apoptosis-evasive solid tumors.
- The study lays the groundwork for developing novel protein-based therapeutics for challenging cancers.
