Anti-Cancer Effect of Thiacremonone through Down Regulation of Peroxiredoxin 6

et al. (2014) Anti-Cancer Effect of Thiacremonone through Down Regulation of Peroxiredoxin 6. PLoS ONE 9(3): e91508. doi:10.1371/journal.pone.0091508 Anti-Cancer Effect of Thiacremonone through Down Regulation of Peroxiredoxin 6 Miran Jo 0 Hyung-Mun Yun 0 Kyung-Ran Park 0 Mi Hee Park 0 Dong Hun Lee 0 Seung Hee Cho 0 Hwan-Soo Yoo 0 Yong-Moon Lee 0 Heon Sang Jeong 0 Youngsoo Kim 0 Jae Kyung Jung 0 Bang Yeon Hwang 0 Mi Kyeong Lee 0 Nam Doo Kim 0 Sang Bae Han 0 Jin Tae Hong 0 Peiwen Fei, University of Hawaii Cancer Center, United States of America 0 1 College of Pharmacy and Medical Research Center, Chungbuk National University , Chungbuk , Korea , 2 College of Agriculture, Life and Environments Science, Chungbuk National University , Chungbuk , Korea , 3 New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation , Daegu , Korea Thiacremonone (2, 4-dihydroxy-2, 5-dimethyl-thiophene-3-one) is an antioxidant substance as a novel sulfur compound generated from High-Temperature-High-Pressure-treated garlic. Peroxiredoxin 6 (PRDX6) is a member of peroxidases, and has glutathione peroxidase and calcium-independent phospholipase A2 (iPLA2) activities. Several studies have demonstrated that PRDX6 stimulates lung cancer cell growth via an increase of glutathione peroxidase activity. A docking model study and pull down assay showed that thiacremonone completely fits on the active site (cys-47) of glutathione peroxidase of PRDX6 and interacts with PRDX6. Thus, we investigated whether thiacremonone inhibits cell growth by blocking glutathione peroxidase of PRDX6 in the human lung cancer cells, A549 and NCI-H460. Thiacremonone (0-50 mg/ ml) inhibited lung cancer cell growth in a concentration dependent manner through induction of apoptotic cell death accompanied by induction of cleaved caspase-3, -8, -9, Bax, p21 and p53, but decrease of xIAP, cIAP and Bcl2 expression. Thiacremonone further inhibited glutathione peroxidase activity in lung cancer cells. However, the cell growth inhibitory effect of thiacremonone was not observed in the lung cancer cells transfected with mutant PRDX6 (C47S) and in the presence of dithiothreitol and glutathione. In an allograft in vivo model, thiacremonone (30 mg/kg) also inhibited tumor growth accompanied with the reduction of PRDX6 expression and glutathione peroxidase activity, but increased expression of cleaved caspase-3, -8, -9, Bax, p21 and p53. These data indicate that thiacremonone inhibits tumor growth via inhibition of glutathione peroxidase activity of PRDX6 through interaction. These data suggest that thiacremonone may have potentially beneficial effects in lung cancer. - Funding: This work was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Korean Government (MSIP) (No. MRC, 20080062275). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Peroxiredoxins (PRDXs) are a family of peroxidases as antioxidant enzymes [12]. The PRDX family includes six members. They are divided into two classes [3]. The 2-Cys group includes PRDX1-5, whereas PRDX6 is only a member of the 1-Cys group. PRDXs are a family of peroxidases that destroy peroxides using conserved cysteine residues in the catalytic center [4]. Among the six mammalian members of this family, PRDX6 is the only member that has glutathione peroxidase and calciumindependent phospholipase A2 (iPLA2) activities [5]. Whereas other PRDXs utilize thioredoxin as a physiological reductant, PRDX6 utilizes glutathione [6]. PRDX6 protects cells from membrane, DNA, protein damages, and lipid peroxidation [7]. The antioxidant response element (ARE) in the prdx6 promoter region, a cis-acting regulator element, is activated by oxidative stress [8]. Transcription of the PRDX6 gene is regulated by nuclear factor erythroid 2-related factors 1, 2, and 3 (Nrf1, Nrf2, and Nrf3) as transcription factors via binding to the ARE [9]. Among the Nrfs, Nrf2 positively regulates transcription of the PRDX6 gene [10]. As PRDXs are antioxidants, they support survival and tumor maintenance by protecting cells from oxidative stress-induced apoptosis [11]. In a recent study, over expression of PRDX 6 attenuates cisplatin-induced apoptosis in human ovarian cancer cells [12]. In contrast, reduction of PRDX6 expression increased peroxide-induced cell death in liver cancer cells [13]. The invasion and metastasis promoting actions of PRDX6 has been found in lung cancer cells through activation of Akt via activation of phosphoinositiede 3-kinase (PI3K) and p38 kinase [4,14]. The activity of PRDX6 contributes to the metastatic ability of lung cancer cells by stimulating invasion components including PI3K, Akt, and uPA [4]. It was also reported that PRDX6 expression in lung cancer cells was significantly associated with tumor progression [15]. Garlic has been used in traditional medicine as a food component to prevent the development of cancer [16]. Thiacremonone (2,4-dihydroxy-2,5-dimethyl-thiophene-3-one) is an antioxidant substance, as a novel sulfur compound, generated from High-Temperature-High-Pressure (HTHP)-treated garlic [17]. In the present study, we investigated the anti-cancer effect of thiacremonone through the inhibition of glutathione peroxidase activity via interaction in lung cancer cells. 6 hr, the cells were treated with thiacremonone. Luciferase activity was measured by using the luciferase assay kit (Promega, Wisconsin, USA) according to the manufacturers instructions (WinGlow, Bad Wildbad, Germany). Materials and Methods Extraction and characterization of thiacremonone The structure of a sulfur compound isolated from garlic (named thiacremonone) is shown in results (Fig. 1A). Garlic (Allium sativum L) was heated at temperatures of 130uC for 2 hrs. The heated samples were juiced and then filtered on a Buchner funnel under a vacuum. Heated garlic juice was partitioned consecutively in a separating funnel using ethyl acetate. Isolation of the compounds from the ethyl acetate layer of heated garlic juice was subjected to column chromatography on silica gel. This fraction containing thiacremonone was purified by preparative RP-HPLC on a Younglin SP930D Instrument [17]. Thiacremonone was resolved in 0.01% dimethyl sulfoxide, and treated at concentrations of 10, 20, and 50 mg/ml in culture cells. Cell Culture The A549 and NCI-H460 lung cancer cell lines were purchased from the American Type Culture Collection (Manassas, VA). The CCD-18Co colon normal cell line and LL24 lung normal cell line were purchased from the Korean cell line bank (Seoul, Korea). NCI-H460 and LL24 normal cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin (100 U/ml). A549 and CCD-18Co normal cells were cultured in a (...truncated)