Date of Award

Spring 2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Siqi Guo

Committee Member

Stephen J. Beebe

Committee Member

Lesley H. Greene

Committee Member

Chunqi Jiang

Committee Member

Lifang Yang

Abstract

Pancreatic ductal adenocarcinoma is a highly lethal malignancy characterized by poor prognosis and resistance to conventional therapies. Novel treatment strategies are needed to overcome this resistance. Non-thermal physical modalities, including nanosecond pulsed electric field (nsPEF) and cold atmospheric pressure plasma discharge (CAP), have emerged as potential anticancer approaches; however, the mechanisms underlying their combined effects remain unclear.

This study evaluated whether nsPEF enhances CAP-induced cytotoxicity and investigated the associated molecular mechanisms using the murine Pan02 pancreatic cancer cell line. Cell viability was assessed by WST-1 metabolic activity assays and electric cell–substrate impedance sensing (ECIS). Intracellular reactive oxygen species (ROS) were measured using H₂DCFDA and MitoSOX. Mitochondrial membrane potential (ΔΨm) was analyzed using TMRE, and apoptosis was examined using caspase-3/7 activity assays and Western blotting. Label-free quantitative proteomics (LC–MS/MS) was performed to analyze global protein expression changes. Sequential nsPEF–CAP/CAP-nsPEF treatments resulted in near-complete loss of cell viability compared with either treatment alone, indicating synergistic cytotoxicity. Antioxidant rescue experiments demonstrated that cell death was dependent on reactive oxygen species. Combination iii treatment significantly increased mitochondrial superoxide production and caspase-3/7 activation relative to CAP alone, while nsPEF alone primarily induced non-apoptotic cell death. Pyroptotic and necroptotic pathways were not significantly activated.

Proteomic analysis revealed distinct responses to individual treatments: nsPEF induced adaptive stress responses involving redox regulation and protein homeostasis, whereas CAP promoted oxidative stress–associated apoptotic signaling and translational suppression. In contrast, combined treatment led to widespread downregulation of metabolic, translational, and antioxidant pathways, along with disruption of stress-response mechanisms, including NRF2- and MAT2A associated processes.

These results indicate that nsPEF enhances CAP-induced cytotoxicity through amplification of oxidative stress, mitochondrial dysfunction, and apoptosis, accompanied by failure of cellular stress-adaptation mechanisms. Collectively, these findings provide important mechanistic insight and support further investigation of combined nsPEF–CAP therapy as a potential strategy to overcome therapeutic resistance in pancreatic cancer.

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ISBN

9798197809438

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