Therapeutic Effects and Targeted Delivery of Eupenifeldin in Castration-Resistant Prostate Cancer

Castration-resistant prostate cancer (CRPC) poses a major therapeutic challenge, as it often fails to respond to standard treatments such as taxane-based chemotherapies, androgen receptor pathway inhibitors, and radioligand-based approaches1–4. Despite these treatments, CRPC remains incurable, and novel therapeutic strategies are urgently needed1,5. This thesis investigates Eupenifeldin, a bistropolone natural product, as a potential therapeutic agent for CRPC and explores targeted drug delivery strategies to enhance its efficacy and specificity6. Eupenifeldin demonstrated potent cytotoxicity in multiple CRPC cell lines, with nanomolar IC₅₀ values and significantly lower toxicity toward non-malignant prostate epithelial cells, indicating selectivity for cancer cells. Compared to docetaxel, a standard chemotherapy for CRPC, Eupenifeldin exhibited a more favorable selectivity profile. Further studies revealed that Eupenifeldin inhibits cell proliferation in a dose-dependent manner and induces apoptotic cell death through caspase-3/7 activation, suggesting that apoptotic pathways mediate its anticancer effects. Mechanistic studies revealed that Eupenifeldin caused a broad reduction in kinase phosphorylation, which correlated with a marked depletion in intracellular ATP levels. This metabolic disruption was attributed to Eupenifeldin’s ability to inhibit both glycolysis and oxidative phosphorylation (OXPHOS), key energy-producing pathways in CRPC cells. Metabolic flux analysis confirmed that Eupenifeldin significantly decreased extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), indicating glycolytic and mitochondrial metabolism suppression. Further analysis of metabolic enzyme expression demonstrated that Eupenifeldin downregulated key glycolytic regulators, including GLUT-1, HK2, and PKM2, which are critical for glucose uptake and metabolism. This metabolic stress was accompanied by a substantial increase in reactive oxygen species (ROS) production, further contributing to cellular damage and apoptosis. The addition of a ROS scavenger partially rescued cell viability, confirming the role of oxidative stress in Eupenifeldin’s mechanism of action. To enhance the therapeutic potential of Eupenifeldin, this study also focused on developing an optimized PSMA-targeted liposomal delivery system to improve drug accumulation in CRPC tumors while minimizing systemic toxicity. Given the overexpression of prostate-specific membrane antigen (PSMA) in advanced prostate cancer, Eupenifeldin-loaded liposomes functionalized with J-591, a monoclonal antibody targeting PSMA, were developed and evaluated. In vitro studies demonstrated that J-591-functionalized Eupenifeldin-loaded liposomes exhibited significantly greater cytotoxicity in PSMA-expressing CRPC cells compared to non-targeted liposomal Eupenifeldin or free drug, confirming the efficacy of active targeting. An in vivo xenograft study was conducted to further assess the therapeutic potential of targeted Eupenifeldin delivery. The rationale for this in vivo investigation was twofold: first, to evaluate the effectivity of PSMA-targeted liposomal Eupenifeldin in selectively reducing tumor burden in CRPC models, and second, to establish intravenous (IV) administration as a feasible route for delivering Eupenifeldin in a systemic anti-tumor setting. Given that previous studies on Eupenifeldin delivery were primarily limited to intraperitoneal administration, it was critical to determine the maximum tolerated dose (MTD) and safety profile for IV injection, which is a preferred route for clinical translation due to its potential for systemic drug distribution and controlled pharmacokinetics. A dose-escalation study established 0.025 mg/kg as the maximum tolerated dose (MTD) for IV Eupenifeldin administration, providing a foundation for subsequent efficacy studies7. In xenograft-bearing mice, IV administration of Eupenifeldin led to a significant reduction in tumor burden, with PSMA-targeted liposomal Eupenifeldin demonstrating the highest therapeutic effect. Notably, tumors in mice receiving J-591-functionalized Eupenifeldin-loaded liposomes exhibited significantly greater growth inhibition than those treated with non-targeted formulations or free drug, confirming the efficacy of antibody-mediated tumor targeting.