The research team, led by Fengyi Li, Ke Zhang, and Miao Wang at the Affiliated Hospital of Inner Mongolia Medical University in Hohhot, built their investigation on prior work with Batri-7, a classic Mongolian medicinal formula prescribed for enteritis and other gastrointestinal disorders. The group had previously shown that Batri-7 markedly suppressed tumor growth in a mouse model of colitis-associated colorectal cancer, but the active constituents responsible for this effect had not been identified. The principal medicinal material in the formula derives from Aconitum species, whose major component, aconitine, possesses antitumor activity but severe cardiotoxicity that limits direct therapeutic use. Drug metabolism studies indicate that aconitine undergoes stepwise hydrolysis of the C8 acetyl group and the C14 benzoyl group, yielding benzoylaconine as the main secondary metabolite. Compared with aconitine, benzoylaconine shows markedly attenuated toxicity while retaining analgesic, anti-inflammatory, and cardioprotective properties, making it an ideal candidate for the toxicity reduction and efficacy retention paradigm of aconitine-type alkaloids.

To systematically characterize the impact of benzoylaconine on malignant phenotypes, the researchers used two human colorectal cancer cell lines, HCT116 and SW480, as model systems. MTT assays revealed that benzoylaconine inhibited proliferation in a dose- and time-dependent manner, with 24-hour IC50 values of 86 micromolar for HCT116 and 83.28 micromolar for SW480. EdU incorporation assays confirmed a marked reduction in the proportion of cells actively synthesizing DNA, and colony formation assays demonstrated a concentration-dependent decline in clonogenic capacity over 14 days of culture. Wound-healing experiments showed that benzoylaconine slowed wound closure, while Transwell invasion assays through Matrigel-coated membranes revealed reduced invasive ability, indicating impaired migration and invasion. Flow cytometric analysis using propidium iodide staining showed that treatment with 50 and 100 micromolar benzoylaconine for 24 hours markedly increased the G1 population and concomitantly reduced the S population in both cell lines, demonstrating effective arrest at the G1/S transition. Annexin V-FITC/PI dual staining further revealed a pronounced, dose-dependent increase in apoptotic cells, showing that benzoylaconine not only suppresses proliferation but also potently triggers programmed cell death.

To identify the molecular targets underlying these effects, the team applied network pharmacology, a computational approach that integrates compound target prediction with disease-associated target databases. Following retrieval from SwissTargetPrediction, GeneCards, OMIM, and DisGeNET, the analysis yielded 103 benzoylaconine-related targets and 1,727 colorectal cancer-associated targets, whose intersection produced 29 potential therapeutic targets. A protein-protein interaction network constructed from these shared targets and visualized in Cytoscape highlighted evident interactions among CDK2, MMP2, and CCNE1, identifying cyclin-dependent kinase 2 and matrix metalloproteinase 2 as core nodes. Gene Ontology enrichment indicated that the common targets were predominantly involved in cell cycle progression, protein phosphorylation, and positive regulation of cell migration, while KEGG pathway analysis showed significant enrichment in inflammation-associated signaling and the cell cycle pathway. These computational predictions aligned closely with the functional assays, suggesting that benzoylaconine may suppress colorectal cancer progression by interfering with cell cycle- and migration-related pathways.

The researchers then examined whether benzoylaconine directly binds the two core targets using molecular docking, molecular dynamics simulations, and surface plasmon resonance. Semi-flexible docking with AutoDock Vina gave binding free energies of -8.13 kilocalories per mole for CDK2 and -8.06 kilocalories per mole for MMP2, indicating favorable affinity. One hundred nanosecond molecular dynamics simulations in GROMACS under physiological conditions showed that root-mean-square deviation values remained below 0.3 nanometers throughout, reflecting stable binding of both the CDK2-benzoylaconine and MMP2-benzoylaconine complexes. The radius of gyration exhibited negligible fluctuation, supporting structural compactness, while solvent-accessible surface area analysis showed no marked changes after ligand binding. Root-mean-square fluctuation values were predominantly below 0.4 nanometers, indicative of low flexibility and high stability, and free energy landscape plots revealed thermodynamically favored low-energy conformations for both complexes. Surface plasmon resonance on a Biacore 1K system confirmed stable and concentration-dependent binding signals, yielding equilibrium dissociation constants of approximately 3.91 micromolar for CDK2 and 4.44 micromolar for MMP2, providing direct experimental evidence of specific binding.

Transcriptome sequencing provided a genome-wide view of the molecular changes induced by benzoylaconine. Comparing control and benzoylaconine-treated HCT116 cells with three biological replicates per group, the researchers identified 1,111 differentially expressed genes, comprising 424 upregulated and 687 downregulated transcripts. Gene Ontology and KEGG enrichment analyses showed that these genes were predominantly associated with cell cycle regulation, and several key cell cycle regulators, including CDK2, CCNE1, CDK6, CCND1, and CDKN1A, displayed marked transcriptional changes. Validation at the protein level by Western blotting demonstrated a dose-dependent decrease in CDK2 and MMP2 protein levels in both HCT116 and SW480 cells after 48 hours of treatment with 50 and 100 micromolar benzoylaconine. Cyclin D1 and Cyclin E1, the upstream activators of CDK2, were also downregulated in a concentration-dependent manner, whereas the endogenous cyclin-dependent kinase inhibitor p21 was markedly upregulated. These coordinated protein changes support a mechanism whereby benzoylaconine exerts anti-cancer effects by modulating the CDK2/MMP2 axis, downregulating Cyclin E1 and Cyclin D1, and enhancing p21-mediated inhibition.

The choice of CDK2 and MMP2 as targets reflects their well-established roles in tumor biology. CDK2 is a pivotal kinase that drives the G1-to-S phase transition primarily through its interaction with Cyclin E, thereby triggering DNA replication. In various human malignancies, including colorectal cancer, CDK2 is frequently overexpressed or aberrantly activated, and its expression levels correlate closely with tumor proliferative activity, aggressiveness, and poor prognosis, making it an attractive therapeutic target. MMP2, a prominent member of the matrix metalloproteinase family, specifically degrades type IV collagen, a major structural component of the basement membrane and extracellular matrix, thereby facilitating tumor cell migration and dissemination. Elevated MMP2 expression in colorectal cancer tissues is positively correlated with depth of invasion, lymph node involvement, distant metastasis, and reduced overall survival. Natural small-molecule inhibitors capable of concurrently targeting both nodes are exceedingly rare, and the study suggests that benzoylaconine may not only exert independent inhibitory effects on proliferation and metastasis but also cooperatively disrupt the malignant crosstalk between these two pathogenic nodes, a hypothesis supported by prior reports that CDK6 facilitates recruitment of c-Jun and Sp1 to the MMP2 promoter.

To evaluate activity in a more clinically relevant model, the team established five patient-derived colorectal cancer organoid models from fresh surgical specimens. Immunohistochemical analysis confirmed that the organoids were consistent with the original tumor tissues in the expression of Ki-67, CK7, CK20, CDX2, and E-cadherin, verifying phenotypic concordance. Bright-field imaging showed that benzoylaconine treatment led to reduced proliferative activity, with morphological changes including reduced size and decreased budding. The CellTiter-Glo 2.0 viability assay revealed that benzoylaconine reduced organoid viability, with half-maximal inhibitory concentration values ranging from 300 to 500 micromolar across the five patient-derived lines. The researchers noted that such potency discrepancy between monolayer cells and three-dimensional organoids is commonly observed for natural-derived small-molecule agents, attributing it to the Matrigel matrix and compact glandular architecture impeding drug diffusion, inter-patient tumor heterogeneity, and a subset of quiescent non-proliferative cells within organoid spheroids exhibiting reduced susceptibility to this cell-cycle-targeting compound. Despite elevated IC50 values, the obvious morphological deterioration in treated organoids confirmed anti-proliferative activity in this clinically relevant model.

The study carries important implications for anticancer drug discovery rooted in natural products, which have long been a prolific source of therapeutic leads, with plant-derived compounds such as paclitaxel and camptothecin entering clinical practice as first-line agents. The findings provide experimental evidence that benzoylaconine represents a key active form underlying the pharmacological efficacy of aconitine and the Batri-7 formula while offering a relatively favorable safety profile. However, several limitations warrant further investigation. This work was primarily conducted at the in vitro level, and in vivo efficacy validation in whole-animal models is absent. The absorption, distribution, metabolism, and excretion properties of benzoylaconine remain poorly defined, and it is uncertain whether effective exposure concentrations in solid tumor tissues can reach levels comparable to those used in vitro. Systematic toxicological safety evaluation has not been performed, and cardiac safety is an indispensable consideration for Aconitum-derived compounds. Future studies should employ subcutaneous xenograft and tail-vein injection lung metastasis models in nude mice to evaluate in vivo antitumor efficacy and pharmacokinetic characteristics, explore whether benzoylaconine regulates the CDK2-Rb pathway, and investigate potential synergistic effects in combination with established agents such as irinotecan and regorafenib.

Nevertheless, the convergence of computational target prediction, structural binding confirmation, transcriptomic profiling, protein-level validation, and patient-derived organoid testing makes this study a compelling example of modern multi-platform drug discovery applied to a traditional medicine-derived compound. By demonstrating that a detoxified metabolite of aconitine can simultaneously arrest the cell cycle, suppress extracellular matrix degradation, and promote apoptosis through the CDK2/MMP2 signaling axis, the researchers have opened a promising avenue for developing benzoylaconine as a novel anti-colorectal cancer therapeutic candidate. As metastasis and drug resistance continue to challenge clinical management of colorectal cancer, agents that act through coordinated multi-target mechanisms and offer favorable efficacy-toxicity profiles represent exactly the kind of innovation the field needs, and benzoylaconine may well prove to be a valuable lead in that search.

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Subject of Research: Benzoylaconine suppresses colorectal cancer progression by modulating the CDK2/MMP2 signaling axis

Article Title: Benzoylaconine suppresses colorectal cancer progression by modulating the CDK2/MMP2 signaling axis

Article References: Li, F., Zhang, K., Wang, M., Su, L., & Yang, L. (2026). Benzoylaconine suppresses colorectal cancer progression by modulating the CDK2/MMP2 signaling axis. Frontiers in Pharmacology, 17, Article 1902707. https://doi.org/10.3389/fphar.2026.1902707

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1902707

Keywords: Benzoylaconine, suppresses, colorectal, cancer, progression, modulating, CDK2, MMP2, signaling, axis, scientific research

The protein at the center of the new work is the retinoic acid receptor alpha, or RARα, which is highly active in colorectal tumors and drives both proliferation and metastatic invasion. Traditional RARα modulators, built on retinoic acid scaffolds, suffer from chemical instability, excessive lipophilicity, and poor oral bioavailability, and they can provoke toxicity in the liver, skin, and bones. To escape these limitations, the researchers turned to proteolysis-targeting chimera, or PROTAC, technology. PROTACs are bifunctional molecules that simultaneously grip a target protein and an E3 ubiquitin ligase, forming a ternary complex that tags the target for destruction by the cell’s own ubiquitin–proteasome system. Because each PROTAC molecule dissociates after every degradation event and can engage targets repeatedly, this catalytic mechanism holds the promise of overcoming the drug resistance that plagues conventional inhibitors.

The team began with AR7, an atypical RARα antagonist, and selected its optimized derivative CA77.1 as the target-binding ligand. Molecular docking against the RARα structure (PDB:5K13) revealed that CA77.1 binds with a calculated energy of −7.379 kcal/mol, anchored by a key hydrogen bond to Arg276 and an extensive hydrophobic network. Crucially, a terminal methyl group on CA77.1 protrudes into the solvent-exposed region outside the ligand-binding pocket, providing an ideal attachment point for a linker without disturbing the core binding interactions. By covalently joining CA77.1 to thalidomide, a ligand for the E3 ubiquitin ligase CRBN, the researchers created a degrader that remains active in both oxygen-rich and hypoxic tumor regions, overcoming the hypoxia-dependent activation that limited the parent compounds.

Through systematic synthesis of six candidate molecules with varying carbon-chain and piperazine linkers, the team identified Z1 as the standout. Z1 degraded RARα with a DC50 of 6.02 ± 1.05 μM and a maximum degradation of 85.4 percent, an activity roughly 43.6 times greater than that of CA77.1 itself. Western blotting confirmed that Z1 left the related receptors RARβ and RARγ untouched, demonstrating high subtype selectivity. Mechanistic experiments sealed the case: cycloheximide chase assays showed accelerated decay of pre-existing RARα protein, while pre-treatment with the competitive ligand ATRA, the E1 enzyme inhibitor MLN4924, the proteasome inhibitor MG132, or excess thalidomide all blocked degradation, confirming the classical PROTAC pathway of direct binding and ubiquitin-proteasome-mediated clearance.

Molecular dynamics simulations running 100 nanoseconds provided atomistic support for the design. Z1 formed stable hydrogen bonds with both RARα and CRBN, with calculated binding free energies of −139.394 kJ/mol and −111.138 kJ/mol respectively, and per-residue energy decomposition pinpointed Pro407, Val395, and Ser225 as the thermodynamic core of RARα recognition. Functionally, Z1-induced degradation suppressed phosphorylation of AKT at Ser473 in a concentration-dependent manner while leaving total AKT unchanged, and it markedly reduced expression of MMP2, an enzyme that tumors use to invade surrounding tissue. This AKT–MMP2 axis, the authors note, had not been systematically characterized in earlier RARα degradation studies and offers a fresh mechanistic window into how acute protein elimination restrains cancer progression.

In vitro assays in HCT116 colorectal cancer cells painted a striking picture of Z1’s antitumor potency. Flow cytometry showed concentration-dependent arrest of cells at the G2/M phase, with the arrested population rising 3.7-fold at 100 μM. At 50 μM, Z1 completely abolished colony formation, whereas the same concentration of CA77.1 allowed more than 300 colonies to grow. Annexin V staining revealed that Z1 drove 34.6 to 55.2 percent of cells into late apoptosis, well above the 24.6 percent seen with CA77.1. Scratch assays demonstrated up to 90.4 percent inhibition of cell migration, and Transwell experiments showed that 50 μM Z1 cut the invasion rate to 10.8 percent, an 81.2 percent reduction relative to the parent compound.

Yet like all PROTACs, Z1 carries the field’s chronic handicaps: high molecular weight, poor water solubility, and weak membrane permeability. The team’s solution came from an unexpected source, the roots of Polygonatum sibiricum, a plant long valued in traditional medicine. Using a juicing, filtration, and ultracentrifugation workflow, the researchers isolated exosome-like nanoparticles, or PsELNs, that measure roughly 116 nanometers across, carry a mildly negative zeta potential of −5.8 mV, and display the characteristic disk-like morphology of plant vesicles. Their cargo of triglycerides forms a hydrophobic core that stabilizes hydrophobic drugs, while their natural tropism for colonic tissue and their ability to accumulate in tumors through the enhanced permeability and retention effect make them unusually well suited to colorectal cancer applications.

Encapsulation of Z1 within PsELNs achieved a drug-loading efficiency of 43.1 percent, exceeding values reported for goji berry and ginger vesicles, and the loaded particles grew only modestly to 143 nm while retaining an intact bilayer. Stability testing was impressive: at acidic pH 5.5 mimicking the tumor microenvironment, 77.38 percent of Z1 remained intact after 24 hours versus just 40.65 percent for the free drug, and the formulation resisted thermal stress at 44 °C, stayed stable in serum for 72 hours, and showed minimal leakage over 60 days of storage. Confocal microscopy and flow cytometry confirmed that Z1/PsELNs entered HCT116 cells far more efficiently than free Z1, and the encapsulated drug degraded RARα with a DC50 of 2.95 μM, a 2.2-fold improvement that translated into stronger apoptosis induction and greater cytotoxicity in vitro.

The decisive test came in living animals. Near-infrared imaging of tumor-bearing nude mice showed that DiR-labeled PsELNs accumulated in tumors at 2.13 times the level of the free dye, peaking at 24 hours after injection, while hemolysis rates below 5 percent confirmed blood compatibility. Over 16 days of tail-vein treatment, the Z1/PsELNs group showed significantly stronger tumor growth inhibition than mice receiving free Z1, with in vivo antitumor efficacy increasing 1.8-fold. Histopathology revealed extensive tumor cell damage, Ki67 staining showed the fewest proliferating cells, and both immunohistochemistry and Western blotting confirmed the lowest RARα levels in the nanoparticle-treated tumors, all without body-weight loss or organ damage. The authors propose that the intestinal stability and colon-targeting behavior of PsELNs could eventually support an oral PROTAC formulation, a goal that would address both gastrointestinal degradation and site-specific accumulation. By fusing event-driven protein degradation chemistry with a biocompatible, naturally derived delivery vehicle, the study charts a credible translational path for PROTAC therapeutics in colorectal cancer and illustrates how plant nanotechnology may help the next generation of degrader drugs reach their targets intact.

Subject of Research: A RARα-targeting PROTAC delivered by Polygonatum sibiricum exosome-like nanoparticles for colorectal cancer therapy

Article Title: Synthesis of RARα–PROTAC and its delivery via Polygonatum sibiricum exosome-like nanoparticles for the treatment of colorectal cancer

Article References: Zhu, G., Zhao, Y., Chen, M., Lu, S., Xu, L., Chen, G., Zeng, L., & Chen, J. (2026). Synthesis of RARα–PROTAC and its delivery via Polygonatum sibiricum exosome-like nanoparticles for the treatment of colorectal cancer. Materials Today Bio, 40, Article 103651. https://doi.org/10.1016/j.mtbio.2026.103651

Image Credits: AI Generated

DOI: 10.1016/j.mtbio.2026.103651

Keywords: colorectal cancer, PROTAC, RARα, protein degradation, Polygonatum sibiricum, exosome-like nanoparticles, drug delivery, nanomedicine, ubiquitin-proteasome system, AKT signaling, MMP2, targeted therapy