ROLE OF CYSTEINE REDOX STATE IN ALTERING THE EPITHELIAL-MESENCHYMAL TRANSITION IN LUNG CANCER

Author

Md Anisur Rahman, University of the PacificFollow

Date of Award

2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

John Livesey

First Committee Member

Roshanak Rahimian

Second Committee Member

David W. Thomas

Third Committee Member

Wade A. Russu

Fourth Committee Member

Lisa A. Wrischnik

Abstract

Epithelial-mesenchymal transition is a process by which cancer cells increase their capability for metastasis. EMT involves conversion of epithelial cells into a mesenchymal phenotype which makes cancer cells more invasive and motile. Lung cancer has the highest mortality rate among different types of cancers globally. Lung cancer exists in various types, the most prevalent of which is non-small cell lung cancer (NSCLC). Several factors, including oxidative stress, can induce EMT in cancer cells. However, the mechanism of how reactive oxygen species (ROS)-induced changes in cysteine redox state affect EMT remains elusive. Our studies aim to understand this mechanism by inducing cysteine redox state alteration with intracellular ROS generation, particularly inside A549 lung adenocarcinoma cells (a type of NSCLC). We also seek to determine how subcellular generation of peroxide influences EMT progression.We successfully generated hydrogen peroxide (H2O2) intracellularly using several methods including plasmid-based chemo-genetic approaches, by increasing H2O2 persistence with peroxidase enzyme inhibition, and by quinone metabolism. First, we used commercially available plasmids containing a peroxide generator. These plasmids deliver the gene coding for the enzyme D-amino acid oxidase (DAAO) which metabolizes D-amino acids and produces hydrogen peroxide which can be detected by the ‘HyPer’ fluorescent sensor, also a part of the fusion protein coded by the plasmid. By altering D-alanine concentrations, we were also able to regulate hydrogen peroxide production. We generated hydrogen peroxide at different subcellular locations (cytoplasm, nucleus, mitochondria). EMT progression was evaluated in each manner of H2O2 increase (whether by generation or persistence) by morphology observation, EMT-related marker protein expression change, as well as measurements of cell motility rate. Our studies found that H2O2 generation in the cytoplasm induces EMT in A549 cells, particularly when the peroxidase enzyme catalase is inhibited. This treatment resulted in production of the mesenchymal phenotype (elongated, loosely connected cells), downregulated the epithelial marker E-cadherin and upregulated the mesenchymal marker N-cadherin. The motility assay also showed a trend toward faster movement. Similar results were also obtained when catalase or catalase plus thioredoxin reductase was inhibited to increase H2O2 persistence. Quinone metabolism to generate H2O2 did not show EMT-related marker expression changes but showed an enhanced rate of motility. Investigation of the molecular targets of oxidation that promote EMT in the cytosol is in process. Βeta-catenin, SMAD2/3, SNAI1/2, and pan-AKT have been selected as the initial cytoplasmic targets. In initial results using a fluorescence-based microplate technique, peroxide persistence by catalase inhibition with 3-aminotriazole showed significantly higher oxidation of SNAI1/2 and pan-AKT, but the reproducibility of this observation must be verified. In summary, this research has shown that the cytoplasm is the probable subcellular location inside A549 cells where the molecular target of cysteine oxidation leading to EMT is present. Peroxide persistence by catalase inhibition also induces EMT and the probable molecular targets may be SNAI1/2 and pan-AKT.

Pages

210

Recommended Citation

Rahman, Md Anisur. (2023). ROLE OF CYSTEINE REDOX STATE IN ALTERING THE EPITHELIAL-MESENCHYMAL TRANSITION IN LUNG CANCER. University of the Pacific, Dissertation. https://scholarlycommons.pacific.edu/uop_etds/4176