Date of Award
2025
Degree Type
Dissertation
Degree Name
Doctor of Philosophy in Pharmaceutical Sciences
Department
Biomedical and Pharmaceutical Sciences
First Advisor
David Rowley
Abstract
Natural products have long been an invaluable source of therapeutic agents. Their structural diversity, biological activity, and relatively favorable safety profiles have led to wide applications across drug discovery, dietary supplements, skin care products, and functional foods. Many phytochemicals have demonstrated beneficial effects in skin protection, particularly through their antioxidant and anti-inflammatory properties. Among them, compounds derived from Cannabis sativa have shown promising activity in promoting skin health.
Despite increasing attention on natural compounds for skin health, their potential roles in targeting critical cancer-related pathways, including ferroptosis and immune checkpoint regulation, are still not well understood. Melanoma remains one of the most aggressive and treatment-resistant forms of skin cancer, with limited long-term response to current therapies. Although immunotherapy and targeted drugs have shown clinical success, challenges such as drug resistance, relapse, systemic toxicity, and cost persist. There is a critical need for safer, more effective, and affordable therapies.
The investigations detailed in this dissertation examined two distinct approaches for melanoma treatment. Section 1 describes how cannabinoids modulate ferroptosis to protect healthy skin cells while inducing ferroptotic cell death in melanoma. Section 2 focuses on the discovery of PD-1/PD-L1 immune checkpoint inhibitors by establishing a novel surface plasmon resonance (SPR)-based platform, which was further applied to screen a library of FDA-approved drugs and validate their effects through cell-based assays.
Section 1 (Manuscripts 1-3) investigated the effects of cannabis-derived phytochemicals on ferroptosis pathways and melanoma cell viability.
Manuscript 1 (published in Nutrients; 2023, 15(21)) evaluated the protective effects of cannflavin A and cannflavin B against ferroptosis and carbonyl stress in human skin keratinocytes, using a combined model of reactive carbonyl species and erastin-induced cell death and reduced lipid peroxidation. Proteomic analysis revealed the combined model in oxidative stress’ and glycation related pathways. These findings indicate that cannflavins modulate multiple stress-related mechanisms and may offer protective benefits against oxidative and carbonyl damage in skin cells, highlighting their potential relevance for aging- and stress-related skin disorders.
Manuscript 2 (published in Journal of Natural Products; 2024, 87(5)) evaluated the anti-ferroptotic activity of cannabidiol (CBD) in human skin keratinocytes, employing a data-independent acquisition (DIA)-based proteomics approach. CBD treatment significantly improved cell viability in erastin induced ferroptosis and reduced key markers of lipid peroxidation and iron level. Proteomic analysis identified six biomarkers that may be associated with ferroptosis regulation. These findings suggest that CBD modulates multiple ferroptosis-related mechanisms and may offer protective effects against oxidative stress in skin cells, providing insight into its potential application in skin-related disorder therapy.
Manuscript 3 (published in Antioxidants; 2024, 13(4)) evaluated the antiproliferative activity of phytocannabinoids and synthetic CBD derivatives against melanoma cells. CBD demonstrated the strongest cytotoxicity among natural cannabinoids in both murine (B16F10) and human (A375) melanoma cell lines. Two bipiperidinyl derivatives of CBD exhibited significantly enhanced cytotoxicity, with lower IC10 values than CBD. Mechanistic studies using apoptosis, ferroptosis, and pyroptosis assays revealed that the enhanced efficacy of these derivatives may involve multiple programmed cell death pathways. These findings suggest that structural modification of CBD can enhance its anticancer potential and may serve as promising lead compounds for melanoma treatment.
Section 2 (Manuscripts 4-6) focuses on natural products and small molecules targeting the PD-1/PD-L1 immune checkpoint axis.
Manuscript 4 (published in Frontiers in Oncology; 2022, 12) evaluated the blockade effects and binding affinities of a panel of natural compounds targeting the PD-1/PD-L1 pathway using two complementary methods: surface plasmon resonance (SPR) and enzyme-linked immunosorbent assay (ELISA). Based on their ability to inhibit PD-1/PD-L1 interaction and/or bind to PD-1 or PD-L1 proteins, compounds were categorized into four functional groups. This study demonstrated that combining functional (ELISA) and biophysical (SPR) assays improves the accuracy of identifying true PD-1/PD-L1 inhibitors and reduces false positives or negatives.
Manuscript 5 (published in Bio Protocol; 2023, 13(15)) developed and validated a novel surface plasmon resonance (SPR)-based platform to assess the blockade effect of small molecules on the PD-1/PD-L1 immune checkpoint interaction. This real-time, label-free assay immobilizes human PD-1 on a sensor chip and measures the interaction kinetics with PD-L1 in the presence of test compounds. Known inhibitors (BMS-1166 and BMS-202) and a negative control (Losartan A) were used to verify assay performance. Compared to conventional ELISA or AlphaLISA methods, the SPR assay offers advantages such as higher sensitivity, reduced sample consumption, and faster throughput. This platform enables accurate determination of binding affinities and blockade percentages, supporting its application in the discovery and screening of small-molecule immune checkpoint inhibitors for cancer immunotherapy.
Manuscript 6 (unpublished) describes the utilization of a validated SPR-based high-throughput screening platform to evaluate a library of FDA-approved drugs for PD-1/PD-L1 inhibitory activity. Three hit compounds, including verteporfin, methylene blue, and rose Bengal, were identified and further validated using SPR and cell-based assays. These findings support the potential for using an SPR-based approach to screening a drug repurposing library for the identification of novel small-molecule immune checkpoint modulators.
In total, this dissertation explores the protective and therapeutic potential of cannabis-derived phytochemicals and innovative biosensing technologies in combating ferroptosis and immune checkpoint interactions. Phytochemicals from cannabis: cannflavins A and B and cannabidiol (CBD), were shown to protect human skin keratinocytes from erastin-induced ferroptosis. Proteomic analyses revealed modulation of ferroptosis-associated markers by CBD, while bipiperidinyl derivatives of CBD exhibited enhanced antiproliferative effects against melanoma cells. In parallel, a surface plasmon resonance (SPR)-based biosensor platform was developed and validated for real-time assessment of PD-1/PD-L1 interactions. This platform was employed to evaluate FDA-approved small molecules, identifying verteporfin, methylene blue, and rose bangle with modest PD-1/PD-L1 inhibitory activity. These research contributions highlight the dual promise of cannabis-derived compounds and advanced biosensing strategies as complementary approaches for mitigating ferroptosis and modulating immune checkpoint pathways in skin and cancer therapeutics.
Recommended Citation
Li, Huifang, "EVALUATIONS OF THE MODULATORY EFFECTS OF NATURAL PRODUCTS ON FERROPTOSIS AND CHECKPOINT PATHWAYS IN CANCER THERAPY" (2025). Open Access Dissertations. Paper 4526.
https://digitalcommons.uri.edu/oa_diss/4526