Preclinical Assessment: Fenbendazole as a Repurposed Therapeutic for Ovarian Cancer
Analysis confirms that FBZ initiates the intrinsic apoptotic machinery.
1. Clinical Context and Therapeutic Rationale
Ovarian cancer remains the most lethal malignancy of the female reproductive system, ranking as the sixth leading cause of cancer-related death among women in 2024. Despite aggressive surgical cytoreduction and the advent of poly (ADP-ribose) polymerase (PARP) inhibitors, five-year survival rates have stagnated between 30% and 40% for decades. This clinical plateau, driven largely by the inevitable emergence of chemoresistance, calls for a strategic interrogation of drug repurposing. By leveraging compounds with established safety profiles, we may bypass the traditional drug-development attrition rate.
Fenbendazole (FBZ) is a benzimidazole compound historically utilized as a broad-spectrum anthelmintic in veterinary medicine. Structurally related to other microtubule-disrupting agents such as Albendazole and Flubendazole, FBZ is emerging as a promising candidate for oncological repurposing. This assessment evaluates the therapeutic efficacy of FBZ against human epithelial ovarian cancer (EOC), focusing on its ability to trigger apoptosis and mitotic catastrophe. Mechanistic validation was conducted in high-grade EOC cell lines and systemic xenograft models to assess the compound’s viability for clinical translation.
2. In Vitro Efficacy: Proliferation Inhibition and Dose-Response Dynamics
In drug repurposing, the first requirement is establishing a robust, dose-dependent relationship at clinically relevant concentrations. This study used two distinct human EOC models—A2780 and the more chemoresistant SKOV3—to assess FBZ viability across varied histological profiles.
Mechanistic validation through Cell Counting Kit-8 (CCK-8) and colony formation assays established that FBZ exerts significant inhibitory pressure on EOC proliferation. The half-maximal inhibitory concentration (IC50) values revealed a clear temporal and dose-dependent progression of potency:
A2780 Cell Line: Demonstrated high sensitivity with IC50 values of 1.30 µM at 24 hours, which dropped to 0.44 µM at 48 hours and 0.38 µM at 72 hours.
SKOV3 Cell Line: Showed comparable inhibitory dynamics with IC50 values of 2.83 µM at 24 hours, 1.05 µM at 48 hours, and 0.89 µM at 72 hours.
Reproductive Integrity: Colony formation assays confirmed that FBZ suppressed long-term cell survival, with higher concentrations essentially eliminating the capacity for clonal expansion.
The transition from growth inhibition to a lethal effect at micromolar concentrations warrants deeper interrogation of specific cytotoxic thresholds and the activation of programmed cell death pathways.
3. Mechanistic Pathways I: Caspase-Dependent Apoptosis
A critical strategic differentiator for any proposed therapeutic is its ability to induce definitive cytotoxicity rather than mere cytostasis. Our analysis confirms that FBZ initiates the intrinsic apoptotic machinery. This is evidenced by a significant increase in the BAX/BCL-2 protein ratio, which signals the transition toward mitochondrial outer membrane permeabilization. Subsequent western blot analysis revealed the robust activation of cleaved caspase-3, the executioner protease of the apoptotic cascade.
To rigorously confirm the dependency of this effect on the caspase pathway, a “reversal” experiment was conducted using the pancaspase inhibitor Z-VAD-FMK. The results provided definitive pharmacological proof:
In SKOV3 models, FBZ treatment alone yielded an apoptosis rate of 54.28%.
Adding Z-VAD-FMK caused the apoptosis rate to plummet to 8.11%, partially restoring cell viability.
While these findings establish caspase-dependent apoptosis as a primary driver of efficacy, transcriptomic profiling suggests that FBZ’s impact extends beyond classical apoptosis into more profound structural dysregulation of the cell cycle.
4. Mechanistic Pathways II: Transcriptome Profiling and Mitotic Catastrophe
RNA sequencing was used to uncover FBZ's systemic impact on the EOC transcriptome, identifying 1,747 differentially expressed genes (DEGs) in SKOV3 cells (803 upregulated; 944 downregulated). KEGG and Reactome enrichment analyses identified the cell cycle and M-phase checkpoints as the primary targets.
Pharmacologically, FBZ functions as a microtubule-destabilizing agent. This is critical, as standard-of-care taxanes (e.g., Paclitaxel) also target microtubules but are frequently compromised by resistance. FBZ may offer a distinct therapeutic advantage by inducing mitotic catastrophe, characterized by the following hallmarks:
Morphological Failure: DAPI and α-tubulin staining revealed multinucleated giant cells and micronuclei.
Spindle Disarray: Treatment led to severe spindle disorganization, preventing productive chromosome segregation.
Temporal Arrest: Flow cytometry confirmed a dose-dependent arrest at the G2/M phase, observed significantly at both 12-hour and 24-hour intervals.
The “So What?” of this failure is disruption of the Cyclin B1/CDK1 complex. While the source data indicates an upregulation of CDC25C (a phosphatase that normally activates CDK1 to promote mitosis), this appears to be a failed compensatory response. Despite higher CDC25C expression, there is a concurrent increase in p-CDK1 (the inactive, phosphorylated form) and a decrease in total CDK1 protein at 24 hours. The resulting accumulation of Cyclin B1 and the inactivation of its catalytic partner, CDK1, forces the cells into a state of mitotic failure that precedes the observed apoptosis.
5. Preclinical Proof-of-Concept: Xenograft Mouse Model Analysis
Bridging the gap between in vitro findings and clinical application requires evidence of systemic efficacy and a favorable safety profile. We used an SKOV3 xenograft study in female BALB/c nude mice to provide this translational proof of concept.
We administered 50 mg/kg/day of FBZ via oral gavage to tumor-bearing mice, suspended in 1% sodium carboxymethyl cellulose (vehicle). Over the 21-day regimen, FBZ demonstrated potent antitumor activity:
Efficacy: FBZ significantly reduced both tumor volume (P < 0.001) and total tumor weight (P = 0.0002) compared with the vehicle control.
Systemic Safety: Critically, the FBZ-treated group maintained stable body weight over the entire 21-day period, with no significant deviation from the control group.
In preclinical pharmacology, maintained body weight is a primary indicator of low systemic toxicity. This suggests that the oral dose required to achieve significant tumor suppression is well-tolerated, reinforcing FBZ’s potential as a long-term maintenance or chronic-dosing candidate.
6. Strategic Assessment of Therapeutic Viability
The synthesis of in vitro, transcriptomic, and in vivo data positions Fenbendazole as a robust candidate for repurposing in the ovarian cancer landscape. Its efficacy is rooted in a dual Mitotic Catastrophe-Apoptosis axis, where it disrupts microtubule dynamics to induce a failed G2/M transition via the inactivation of the Cyclin B1/CDK1 complex.
Core Evidence Points
High-Potency Cytotoxicity: Consistent IC50 values in the low micromolar range across multiple epithelial ovarian cancer (EOC) models.
Mechanistic Precision: Transcriptomic reprogramming of cell-cycle genes (e.g., CDC25C, TPX2) and the pharmacological confirmation of caspase-dependent death.
Translational Validation: Significant in vivo tumor suppression (50 mg/kg/day) with a demonstrated safety profile (stable body weight).
Given that the current standard of care often fails due to microtubule-related resistance, the next strategic phase should involve combination therapy studies. Evaluating FBZ in tandem with taxanes or PARP inhibitors could reveal synergistic potential to overcome chemoresistance. Fenbendazole addresses an urgent clinical need, offering a strategically viable path toward improving the stagnant survival outcomes in epithelial ovarian cancer.
Source:
Transcriptome analysis reveals the anticancer effects of fenbendazole on ovarian cancer: an in vitro and in vivo study
BMC Cancer 24, 1593 (2024). https://doi.org/10.1186/s12885-024-13361-9