The differential pro-oxidant effects of the tea (Camellia sinensis) catechin, (-)-epigallocatechin-3-gallate (EGCG), in the context of oral cancer.

摘要

In the United States, an estimated 45,780 new cases and 8,650 deaths from cancers of the oral cavity and pharynx (throat) are expected in 2015. Dietary intervention is one potential strategy to reduce cancer burden. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea. Although widely considered as an antioxidant, recently a growing number of evidence shows that EGCG may exert pro-oxidant effects. EGCG has been found to undergo auto-oxidation upon the exposure to oxygen and transition metals to form reactive oxygen species (ROS). However, the role of EGCGinduced ROS and the differential effects of EGCG-induced ROS in cancer versus normal cells are poorly understood. Therefore, this thesis aims to provide insight into EGCG's pro-oxidant activities, with a focus on the potential differential effects in oral cancer and normal cells.;To explore the question, I treated the oral cancer cells with EGCG and detected intracellular ROS. First, I found that in oral cancer cells EGCG initially caused ROS formation localized in mitochondria, which resulted in mitochondrial dysfunction including the loss of mitochondrial membrane potential and the opening of the mitochondrial membrane transition pore. The accumulated mitochondrial ROS (mtROS) and mitochondrial dysfunction led to a systemic ROS burst and ultimately cell death. Interestingly, EGCG did not induce significant cell cytotoxicity and apoptosis in normal cells. In contrast to the ROS burst in oral cancer cells, no significant increase of ROS was observed in normal cells as well. PCR array analysis showed that EGCG down-regulated several antioxidant related genes in cancer cells (superoxide dismutase 2/3 and thioredoxin reductase 2), but up-regulated them in normal cells. The data suggest that EGCG exerts differential pro-oxidant effects in oral cancer and normal cells, in part through modulating antioxidant signaling.;In order to better understand the differential effects of EGCG, I examined the impact of EGCG on sirtuin3 (SIRT3). SIRT3 is a deacetylase localized in mitochondria that regulates the redox balance in that organelle. I found that EGCG suppressed the gene and protein expression as well as the activity of SIRT3 in cancer cells. By contrast, EGCG activated SIRT3 in normal cells, however, no significant changes in the mRNA and protein level of SIRT3 were observed. Further, I observed that estrogen-related receptor alpha (ERRalpha), a transcriptional factor of SIRT3, was selectively down-regulated by EGCG in cancer cells, indicating that EGCG may differentially modulate the mRNA expression of SIRT3 through ERRalpha. EGCG also differentially modulated the mRNA levels of SIRT3-associated downstream genes including glutathione peroxidase 1 and superoxide dismutase 2 in normal and oral cancer cells.;To determine the necessity of SIRT3 for EGCG's differential pro-oxidant effects, I knocked down SIRT3 through siRNA technology. Interestingly, siRNA-mediated knock-down of SIRT3 in cancer cells (SCC-25) resulted in decreased ROS, rendering a cell resistance to EGCG-induced growth inhibition. Accordingly, the mRNA levels of MT1G, MT1X and metal regulatory transcription factor 1 (MTF1), a transcriptional factor of MTs were significantly increased. MTs belong to a group of cysteine-rich, low molecular weight proteins. They are localized in cytoplasm and function as antioxidant through oxidation of thiol groups. Therefore, the up-regulated MTs and MTF1 expressions in si-SIRT3 cells suggest that MT may be an alternative modulator of intracellular antioxidant signaling. Further, I found EGCG down-regulated most MT isoforms in oral cancer cells, while up-regulating several MTs in normal cells, particularly MT1G (+20,900%) and MT1X (+350%). On the other hand, knocking down MTF1 in SCC-25 cells significantly decreased mRNA expression of MTs, SIRT3, NFE2L1 and NFE2L2. This indicates a crosstalk between SIRT3 and MT signaling that may be mediated by MTF1. Furthermore, EGCG decreased the transcriptional activity of MTF1 in cancer cells but slightly increased the activity in normal cells.;In conclusion, EGCG induced oxidative stress and apoptosis in oral cancer cells, but triggered antioxidant responses in normal cells. SIRT3 and MT appear to be molecular mediators of EGCG-induced differential pro-oxidant effects in oral cells. Moreover, a potential crosstalk between SIRT3 and MT signaling was identified and may be mediated by MTF1. Taken together, the current study provides novel mechanisms under which EGCG exerts differential pro-oxidant effects in oral cancer versus normal cells. The mechanistic study will provide helpful information for future work with oral cancer animal model.