The role of sirtuin 3 in the differential pro-oxidant effects of (-)-epigallocatechin-3-gallate in oral cells Good morning, I am Ling Tao from Dr. Lambert’s lab. Today I am going to present my proposal about… Ling Tao Advisor: Joshua D. Lambert, Ph.D. Lab of Food and Disease Prevention Department of Food Science The Pennsylvania State University

We have no conflicts of interest to declare for this study.

Oral cancer has high morbidity and mortality Oral cancer or mouth cancer, a subtype of head and neck cancer, is any cancerous tissue growth located in the oral. It does not just cause disfiguration but also has high morbidity and mortality American cancer association reported that there were estimated… in 2014. In 2014, an estimated 42,440 new cases (2.6% increase from 2013) and 8390 deaths (6.3% increase from 2013) from cancer of the oral cavity and pharynx (throat) are expected in the United States [1, 2]. Oral squamous cell carcinoma accounts for greater than 90% of oral cancers and can develop from oral precancerous lesions including leukoplakia and erythroplakia [3]. Dietary interventions represent a potentially efficacious and cost-effective approach to reduce oral cancer burden. Both lining the cheek Left person: nice beautiful teeth Right person: color changes, white patches and lumps In 2010 36,540 new cases and 7,880 deaths from oral cavity and pharynx cancer Oral cancer starts in the mouth, also called the oral cavity. The oral cavity includes the lips, the inside lining of the lips and cheeks, the teeth, the gums, the front two-thirds of the tongue, the floor of the mouth below the tongue, and the bony roof of the mouth. www.cancer.org 42,440 new cases and 8,390 deaths expected Major risk factors: smoking & excessive alcohol American cancer society, 2014

Dietary intervention may be an important and effective approach to reduce oral cancer burden Most common oral cancer treatment is surgery and radiation therapy. However, they can cause severe tissue damage and irreparable facial disfigurement. Comparatively dietary intervention may be a simple and effective approach to reduce cancer risk.

EGCG is the most abundant polyphenol in green tea Green tea has cancer preventive effects Green tea is a rich source of catechins 1.25% (w/v) green tea has ~1.5 mM EGCG Green tea is a poplular drink worldwide and has been reported to have cancer preventive effects. Processed in a way that minimizes oxidation, green tea preserves the characteristic flavan-3-ols known as catechins. The major green tea catechins include: (–)-epicatechin (EC), (–)-epigallocatechin (EGC),(–)-epicatechingallate (ECG), and (–)-epigallocatechingallate (EGCG) EGCG is the most abundant polyphenol in green tea: 1.25% (w/v) green tea has ~1545 µM EGCG (1.5 mM) Numbers represent the amount of catechins contained in % dry weight Muto paper original from Graham HN. Green tea composition, consumption, and polyphenol chemistry. Preventive medicine 1992; 21: 334-50. Jankun et al, 1997; Muto et al., 2001; Yang et al., 2009

There is increasing evidence showing EGCG’s pro-oxidant activities under certain conditions EGCG produced reactive oxygen species (ROS) in cell culture medium and inside the cells EGCG induced dose-dependent oxidative stress, DNA damage, and apoptosis in xenograft lung tumor Under the exposure of O2 and transition metals, EGCG can be oxidized to form quinone radicals and produce hydrogen peroxide. Quinone radicals will further react with free EGCG to form reactive dimers which can turn oxygen into superoxide anions. The hydrogen peroxide and superoxide anions are reactivie oxygen species (ROS) that at high levels would cause tissue damage. EGCG has been reported to produce ROS in cell culture medium and inside the cells. Furthermore, EGCG could also cause oxidative stress in lung 100 µM EGCG caused chromosomal damage in B lymphocyte cells trace amount of transition metal in medium On the flip side, adding metal chelator would reduce oxidation High oxygen partial pressure (i.e. cell culture, oral cavity) Relatively high concentration pH (i.e. pH=3) Transition metals Will EGCG generate ROS in breast tissue or other organs with low oxygen pressure? Yang et al., 1998; Lambert and Elias, 2010; Li et al., 2010

EGCG selectively induced mitochondrial ROS and cell apoptosis in oral cancer cells SCC25: human oral squamous carcinoma cells HGF-1: human gingival fibroblast cells (B) (C) (D) The mechanism is not completely understood Tao et al., 2013; Tao and Lambert, 2014 (unpublished)

Sirtuin 3 is an important regulator of mitochondrial redox balance FOXO3a CYC GPX SOD2 CAT PRX3 mtROS Sirtuin 3 (SIRT3) is mainly localized in mitochondria and is an NAD+- dependent deacetylase SIRT3 mitigates mitochondrial ROS The transcription of SIRT3: ERRα: estrogen-related receptor α PGC-1α: peroxisome proliferator- activated receptor gamma, coactivator 1 alpha Sirtuin 3 (SIRT3) is an NAD+-dependent deacetylase and mainly localized in mitochondria. SIRT3 mitigates mitochondrial ROS by regulating the components in the electron transport chain and activating antioxidant defense signaling. It is an important mitochondrial redox balance regulator: Cytochrome c (CYC) is another important component of the electron transport chain which helps control ROS leakage [21]. SIRT3 activity is required for the mRNA expression of CYC [22]. , SIRT3 can modify the binding activity of the transcription factor forkhead box 3a (FOXO3a) and activate the expression of downstream antioxidant genes Directly associates with SOD2 Increased the expression of GPX1 Through deacetylation, it activates antioxidant response proteins. and plays an important role in maintaining mitochondrial redox balance [17]. The mitochondrial electron transport chain is the major site of ROS formation [18, 19]. SIRT3 physically interacts with at least one of the known subunits of Complex I (i.e. NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9) and enhances complex I activity by deacetylation. This prevents the formation of excess ROS in the mitochondria [20]. Cytochrome c (CYC) is another important component of the electron transport chain which helps control ROS leakage [21]. SIRT3 activity is required for the mRNA expression of CYC [22]. SIRT3 can also modulate the expression and activity of antioxidant response proteins. For example, SIRT3 can modify the binding activity of the transcription factor forkhead box 3a (FOXO3a) and activate the expression of downstream antioxidant genes [23, 24]. SIRT3 has also been shown to enhance the activity of superoxide dismutase 2 (SOD2) by lysine deacetylation, and to increase the expression of both glutathione peroxidase 1 (GPX1) and SOD2 [22, 25]. Bause and Haigis, 2013; Chen et al., 2014

Will SIRT3 play an important role in the differential pro- oxidant effects of EGCG?

EGCG induced differential changes of SIRT3 expression and activity in oral cells (B) (C)

EGCG regulates SIRT3 transcription in oral cancer cells through estrogen-related receptor α (ERRα) (B) suggest transcriptional activity (C)

EGCG differentially regulated SIRT3-modulated antioxidant defense genes Bigger graphs

In conclusion: Reduce the words In oral cancer cells, EGCG inhibited SIRT3, resulting in the induction of mtROS. The accumulation of mtROS will lead to mitochondrial dysfunction and ultimate cell death. In normal cells, however, EGCG activated SIRT3 and upregulated SIRT3-modulated downstream antioxidant genes to prevent the accumulation of mtROS, thereby protecting cells from oxidative damage. . The differential regulation of SIRT3 by EGCG indicates that SIRT3 may play an important role in EGCG-induced differential pro- oxidant effects in oral cells.

Acknowledgement People and facilities Dr. Joshua Lambert and lab members Dr. Jack (John) P. Vanden Heuvel Penn State Genomics Core Facility & Microscopy Facility Funding American Institute for Cancer Research grant (to JDL) Graduate Student Competitive Grant from the Pennsylvania State University College of Agricultural Sciences (to LT).