Date

6-8-2023

Department

School of Health Sciences

Degree

Doctor of Philosophy

Chair

Gregory Raner

Keywords

AHR, Antioxidant, Apoptosis, ARE, NRF2, HSP, Cytoprotective

Disciplines

Biochemistry, Biophysics, and Structural Biology

Abstract

Oxidative damage is a cellular threat that is responsible for many pathologic conditions such as Alzheimer’s disease, Parkinson’s disease, chronic obstructive pulmonary disease (COPD), cancer, and many more. The primary cytoprotective pathway within the cell that is responsible for mitigating the harmful effects of oxidative stress is the antioxidant system. The master regulator of the antioxidant system is the transcription factor, nuclear factor erythroid 2-related factor 2 (NRF2) which operates by binding to the antioxidant response element (ARE) and inducing the production of many antioxidant genes. It is known that the early response of NRF2 is regulated by electrophilic interaction with Kelch-like ECH-associated protein 1 (Keap1) and the delayed response is regulated by glycogen synthase kinase 3 beta (GSK-3β). There have been many antioxidant natural products reported, including, but not limited to, broccoli, Cinnamomum cassia oil, turmeric powder, sulforaphane, cinnamaldehyde, and curcumin. Many of these function through interactions with Keap1 in the early response. This study identified two novel inducers of ARE activity: sabinene and tetrafluorohydroquinone. Further, Kenpaullone, is another compound that has been identified to have effects on the delayed response, as it is a known inhibitor of GSK-3β. Many studies have investigated the effects of these compounds on the induction of NRF2, however, in this study, the effect of a dual-targeted treatment of HepG2 cells was investigated in attempts to regulate the NRF2 pathway through both the early and the delayed response. This study showed that across multiple combinations of early response inducers in combination with kenpaullone HepG2/NRF2 cells had an enhanced activity of the ARE that was much greater than any compound or natural product could induce individually. This suggests that enhanced activation of the ARE can occur through a dual-targeted treatment. Further, the antioxidant pathway has been shown to have correlations to other cytoprotective pathways such as xenobiotic metabolism and heat shock proteins. For example, since many reactive oxygen species (ROS) are produced in the process of xenobiotic metabolism, the antioxidant system and xenobiotic system are typically activated simultaneously. Likewise, oxidative stress cannot only induce the antioxidant pathway but it can cause damage to proteins as well, and therefore activate heat shock proteins. In light of the correlations between NRF2 and other cytoprotective pathways, it would be logical to assume that enhanced activation of the antioxidant pathway could lead to the activation of other protective mechanisms. Therefore, this study also investigated the activation of xenobiotic metabolism and heat shock proteins in response to the NRF2 activators mentioned previously. Interestingly, the results indicate a novel activity of KP as an inducer of the xenobiotic response, as indicated by cytochrome P450, family 1 subfamily a, polypeptide 1 (CYP1A1), a classic marker for the activation of the xenobiotic response element (XRE) by the aromatic hydrocarbon receptor (AHR). Not only did the results of this study indicate that enhanced activation of the ARE could be achieved through a dual-targeting treatment, but also that attenuation of the activation occurred at high concentrations, suggesting that a negative-feedback mechanism was responsible for the inactivation of the antioxidant pathway. It has been well reported that the over-activation of cytoprotective pathways can induce programmed cell death, therefore experimentation was done to evaluate the potential activation of apoptosis at high concentrations of dual-targeted treatment. However, using qPCR assessment the treatment concentrations used did not seem to indicate attenuation of the NADPH quinone oxidoreductase-1 (NQO1), a classical marker of ARE activation. Rather, p53 upregulated modulator of apoptosis (PUMA) expression seemed to be reduced, and therefore the activation of apoptosis was not observed. In light of elevated NQO1 expression, PUMA expression would not be expected to be elevated as these results are consistent with the literature and are consistent with a relationship between cytoprotective pathways and programmed cell death.

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