Analytica & l B y i tr o s c i h Song and Cho, Biochem Anal Biochem 2015, 4:4 m e m e h i Biochemistry & s c t o r i DOI: 10.4172/2161-1009.1000211 y B ISSN: 2161-1009 Analytical Biochemistry Research Article Open Access Phytol Induces Apoptosis and ROS-Mediated Protective Autophagy in Human Gastric Adenocarcinoma AGS Cells YeonWoo Song1 and Somi Kim Cho2* 1Faculty of Biotechnology, College of Applied Life Sciences, SARI, Jeju National University, Jeju 690-756, Republic of Korea 2Subtropical Horticulture Research Institute, Jeju National University, Jeju 690-756, Republic of Korea Abstract Phytol, a diterpene alcohol produced from chlorophyll, is used widely as a food additive and an aromatic ingredient. However, the molecular mechanisms behind the cytotoxic effects of phytol in cancer cells are not understood. The current study demonstrated that phytol induces apoptosis in human gastric adenocarcinoma AGS cells, as evidenced by an increased cell population in the sub-G1 phase, downregulation of Bcl-2, upregulation of Bax, the activation of caspase-9 and -3, PARP cleavage, and depolarization of the mitochondrial membrane. In addition, phytol induced autophagy, as evidenced by the induction of acidic vesicle accumulation, the conversion of microtubule-associated protein LC3-I to LC3-II, and the suppression of Akt, mTOR, and p70S6K phosphorylation. Most importantly, pretreatment with chloroquine, a lysosomal inhibitor, strongly augmented phytol-induced apoptosis in AGS cells, suggesting that phytol could induce protective autophagy. Furthermore, co-treatment with a reactive oxygen species (ROS) scavenger enhanced the phytol-induced cytotoxicity, which is accompanied by the decrease in the levels of p62 as well as the percentage of acidic vesicular organelles (AVOs) positive cells, suggesting that the cytoprotective Nrf2 pathway was induced by phytol. Taken together, these findings provide valuable insights into the mechanisms by which phytol induces cell death in AGS cells. Keywords: AGS human gastric adenocarcinoma cells; Apoptosis; Autophagy is an evolutionarily conserved cellular degradation Autophagy; Phytol; ROS pathway for long-lived proteins and organelles [17]. Induction of autophagy might maintain cancer survival under metabolic stress and Introduction mediate resistance to anticancer therapies [18]. Thus inhibition of autophagy enhances cancer cell death and potentiates various anticancer Phytol (3,7,11,15-tetramethyl-2-hexadecen-1-ol) is an acyclic therapies [19,20]. High levels of autophagy can induce a form of cell monounsaturated diterpene alcohol from chlorophyll. It is an aromatic death known as type II cell death or autophagic cell death [21]. From ingredient used in many fragrant compounds, and is also found a molecular point of view, several cellular signaling pathways play a in some cosmetic and non-cosmetic products [1]. Known for its medicinal value, phytol has antinociceptive and antioxidant activities role in regulating autophagy, including phosphatidylinositol 3-kinase [2], as well as anti-inflammatory [3] and immunostimulatory effects (PI3K)/Akt/mammalian target of rapamycin (mTOR). Previous [4]. Phytol also elicits antimicrobial activity against Mycobacterium studies have shown that inhibiting Akt and its downstream target tuberculosis [5,6] and Staphylococcus aureus [7]. Several reports have mTOR contribute to the initiation of autophagy [22]. During the initial assessed the anticancer activities of plant extracts that contain phytol stages of autophagy cellular proteins, organelles, and the cytoplasm are as a major component [8-10]. Similar to these findings, a recent reports sequestered and engulfed by intracellular double membrane-bound assessed the apoptosis induction in hepatocellular carcinoma cells [11] structures called autophagosomes. The microtubule-associated protein and the cytotoxic effects of phytol using MTT assays in several tumor 1 light chain 3 (LC3), a mammalian homolog of the yeast Atg8, a cytosolic cell lines in vitro [12]. However, the molecular mechanism by which form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine to phytol induced cell death remains unclear. In this study, we explored form LC3-II, which is recruited to autophagosomal membranes [23]. the molecular mechanisms by which phytol induces cell death in AGS These autophagosomes mature by fusing with lysosomes to form gastric cancer cells. the autolysosomes, in which the sequestered proteins and organelles are digested by lysosomal hydrolases and recycled to sustain cellular Cell death plays an important role in cancer growth and metabolism [24]. Through a comprehensive analysis of LC3-binding, progression, as well as the efficacy of chemotherapy. Apoptosis is p62 was identified as an LC3-interacting protein [25-27]. Because LC3- thought to be the principal mechanism by which anti-cancer drugs II present in the inner membrane of the autophagosome is degraded kill cells. Apoptosis is a highly conserved form of cell suicide that is together with other cellular constituents by lysosomal proteases, p62 regulated by programmed cellular signaling pathways that control tissue homeostasis and/or eliminate damaged or infected cells [13]. There are two major representative apoptotic pathways: the extrinsic pathway, which is mediated by death receptors (DRs), and the intrinsic *Corresponding author: Somi Kim Cho, Faculty of Biotechnology, Jeju National University, 66 Jejudaehakno, Jeju, 690-756, Korea; Tel: +82-64-754-3348; Fax: pathway, which is mediated by mitochondria. The death-receptor- +82-64-756-3351; E-mail: [email protected], [email protected] mediated pathway activates caspase-8 by forming the death-induced signaling complex (DISC) [14]. Activated caspase-8 then cleaves Received: June 23, 2015; Accepted: September 07, 2015; Published September 09, 2015 Bid into truncated Bid (tBid), which connects the death receptor pathway to the mitochondrial pathway by translocating from the Citation: Song Y, Cho SK (2015) Phytol Induces Apoptosis and ROS-Mediated cytosol to the mitochondria. The mitochondrial pathway is initiated Protective Autophagy in Human Gastric Adenocarcinoma AGS Cells. Biochem Anal Biochem 4: 211. doi:10.4172/2161-1009.1000211 by the loss of mitochondrial membrane potential (MMP, ΔΨm) and release of cytochrome c, which activates caspase-9 and its downstream Copyright: © 2015 Song Y, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted effector caspases that cleave different substrates to eventually lead to use, distribution, and reproduction in any medium, provided the original author and dismantling of the cell [15,16]. source are credited. Biochem Anal Biochem ISSN: 2161-1009 Biochem, an open access journal Volume 4 • Issue 4 • 1000211 Citation: Song Y, Cho SK (2015) Phytol Induces Apoptosis and ROS-Mediated Protective Autophagy in Human Gastric Adenocarcinoma AGS Cells. Biochem Anal Biochem 4: 211. doi:10.4172/2161-1009.1000211 Page 2 of 7 trapped by LC3 is transported selectively into the autophagosome. The (A) p62 protein localizes to sites of autophagosome formation and can associate with both the LC3 and ubiquitinated proteins [26]. Therefore, p62 is considered to act as a receptor for ubiquitinated proteins, which (B) it sequesters into the autophagosome [28] and the impaired autophagy is accompanied by accumulation of p62. Reactive oxygen species (ROS) is a term that describes the collective species formed by the incomplete reduction of oxygen, including superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (HO•) [29]. Intracellular redox homeostasis is a key determinant of cell fate: excessive ROS production usually results in cytotoxic effects and might lead to apoptotic cell death, whereas moderate levels of ROS function as second messengers to regulate Figure 1: (A) Chemical structure of phytol (3,7,11,15-tetramethylhexadec-2-en- 1-ol). (B) The viability of cells treated with various concentrations of phytol for 24 diverse cellular processes such as cell survival, proliferation, and h. The results shown are the means ± S.Ds. of three independent experiments metastasis [30,31]. The accumulation of ROS can induce autophagy, (**p<0.01 vs. control). which in turn facilitates the clearance of excessive ROS to protect cells from oxidative damage; this might reflect the balance of either Cell viability assays cell survival or death [32,33]. During oxidative stress, ROS-mediated KEAP1 inhibition results in the activation of Nuclear factor-erythroid The effects of the treatments on cell viability were determined using 2-related factor 2 (Nrf2) and the expression of its target genes p62, MTT assays, which are based on the conversion of MTT to formazan heme oxygenase-1 (HO-1) and NAD(P)H dehydrogenase, quinine crystals by mitochondrial dehydrogenases. Briefly, cells were plated in 1 (NQO1) [34]. Moreover, p62 can interact directly with KEAP1 to 96-well plates at an initial density of 6 × 103 cells/well. After a 24-h promote the non-canonical activation of Nrf2 and creating a positive incubation, the cells were treated with phytol alone or combination of feedback loop that further amplifies p62 expression [35]. Therefore, 60 μM chloriquine or 2 mM NAC and then incubated for an additional p62 stimulates autophagy and the Nrf2-mediated antioxidant response 24 h. At the indicated time points 20 μL of MTT solution (5 mg/mL) during oxidative stress. was added to each well, and the plates