Pharmaceutica Analytica Acta

A natural polyphenolic compound Resveratrol (Rsv), which is known as a stimulator of NAD⁺-dependent deacetylases sirtuin (SIRT1) family, sirtuin, elongates lifespan of model animals [1-5]. Previously, we reported that Rsv moderately activates the human SIRT1 and TERT promoters inducing telomerase activity in HeLa-S3 cells [6,7]. Moreover, multiple transfection assays showed that promoter activities of the genes encoding human telomere maintenance factors (shelterin proteins) [8] are up-regulated by Rsv treatment [9], suggesting that natural polyphenol compounds, such as Rsv may affect chromosomal stabilities. Thus, Rsv and its related polyphenols are expected to become candidate drugs for anti-aging therapeutics. However, it should be noted that Rsv has cytotoxic effects by inducing apoptotic cell death, especially when it is used at higher doses [10-12]. Thus, in order to develop safe drugs with anti-aging effects, searching of alternative natural compounds that up-regulate SIRT1 and shelterin gene expression and their induction mechanisms should be investigated.

β-Thujaplicin, which is also known as hinokitiol, is a tropolone derivative found in the heartwood of cupressaceous plants [13]. It has been reported to have a variety of biological effects, including induction of apoptosis [14] and differentiation [15], anti-inflammatory [16], antibacterial [17] and anti-fungal [18] effects. In this study, we examined the effects of thujaplicins on the promoter activities of the human SIRT1 and shelterin-encoding genes by multiple transient transfection and Luc reporter assay. Here, we show the up-regulating effects of three types of thujaplicins (a, b and g) on the promoter activities of the human SIRT1 and shelterin-encoding genes by multiple transient transfection and Luc reporter assay. Here, we show that b-thujaplicin (hinokitiol) is able to up-regulate these promoter activities. Furthermore, we propose that b-thujaplicin could be used as one of lead-compounds for developing anti-aging drugs.

Materials

trans-Resveratrol was purchased from Cayman Chem. (Ann Arbor, MI) [6,7]. α−, β- and γ-Thujaplicins were purchased from Osaka Chemical Industry Ltd. (Osaka, Japan) [19]. Structures of these compounds are shown in (Figure 1).

Figure 1: The structures of trans-Resveratrol and thujaplicins.

Cell culture

Human cervical carcinoma (HeLa S3) cells [20] were grown in Dulbecco’s modified Eagle’s (DME) medium (WAKO-Pure Chemical, Tokyo, Japan), supplemented with 10% fetal bovine serum (FBS) (Sanko-Pure Chemical, Tokyo, Japan) and penicillin-streptomycin at 37°C in a humidified atmosphere with 5% CO₂.

Construction of Luc reporter plasmids

The Luc reporter plasmid pGL4-SIRT1 carrying 396-bp of the human SIRT1 promoter region was constructed as described previously [7]. Other Luc reporter plasmids, which contain 300 to 500-bp of 5’-upstream regions of the human PIF1, RTEL, TRF1, TRF2, TERT, TERC, TANK1, DKC1, TIN2, POT1, RAP1(TERF2IP) and TPP1(ACD) genes, were constructed as described previously [9,21].

Transient transfection and Luc assay

Plasmid DNAs were transfected into HeLa S3 cells by the DEAEdextran method [20-22]. The DNA-transfected cells were divided into at least four dishes. After 24h of transfection, Rsv or thujaplicins were added to the culture medium. After a further 24 h of incubation, cells were collected and lysed with 100 μL of 1 X cell culture lysis reagent, containing 25 mM Tris-phospate (pH 7.8), 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N’,N’,-tetraacetic acid, 10% glycerol and 1% Triton X-100, then mixed and centrifuged at 12,000 × g for 5 sec. The supernatant was stored at -80°C. The Luc assay was performed with a Luciferase assay system (Promega) and relative Luc activities were calculated as described previously [20-22]. Multiple transfection of human shelterin promoter-containing Luc reporter plasmids with 96-well culture plate was performed as described previously [8,21].

Effects of thujaplicins on the human SIRT1 promoter

To examine whether the human SIRT1 promoter is affected by α−, β− ανδ γ−thujaplicins [19], transient transfection and Luc assays were carried out. Luc activities of pGL4-SIRT1 transfected cells were normalized to that of non-treated control cells. As shown in Figure 2A, the relative Luc activity of pGL4-SIRT1-transfected cells was prominently augmented by the addition of Rsv (10 μΜ) ορ α−, β− and γ-thujaplicins (10 μM) to the culture medium.

Figure 2: Effects of thujaplicins on the human SIRT1 promoter activity. (A) The Luc reporter plasmid, pGL4-SIRT1 [6,7], was transfected into HeLa S3 cells as described under Materials and Methods. After 24 h of transfection, cells were treated with Rsv (10 μΜ), then harvested after a further 24 h incubation. (B) A similar (experiment was performed as in (A) with 0 to 100 μM of β-thujaplicin. The results show relative Luc activities of the indicated Luc reporter plasmidtransfected cells relative to those of non-treated cells. The values are the mean + SD of four independent assays.

To examine the dose-dependent response to β-thujaplicin (hinokitiol), HeLa S3 cells were treated with 0 to 100 μM of β-thujaplicin after 24 h of transfection and collected after further 24 h incubation (Figure 2B). The half maximal effective concentration (EC₅₀) was estimated as 3.1 mM. These results indicate that 10 mM of m-thujaplicin is enough to induce SIRT1 promoter activity equal to Rsv (10 mM) treatment.

Effect of β−τhujaplicin on the 5’-upstream regions of human genes encoding telomere maintenance factors

Multiple transcription experiments were carried out with various Luc reporter plasmids containing 5’-flanking regions of the human shelterin encoding genes (Figure 3) [9]. By performing the multiple Luc assay, the effect of b-thujaplicin on these transcription-regulatory regions were examined. The results showed that b-thujaplicin (10 mM) could induce up-regulation of relative promoter activities of the RTEL, TRF1, TRF2, TERT, DKC1, TIN2, RAP1(TERF2IP) and TPP1(ACD) genes (Figure 3). Approximate 1.5 to 2-folds increases as compared with non-treated cells were observed in a similar manner as the 396-bp of the SIRT1 promoter (Figure 2A).

Figure 3: The effect of β-thujaplicin on the promoter activities of 5’-upstream regions of human shelterin encoding genes. Reporter plasmids (10 ng) and DEAE-dextran were spotted and dried onto each well of the 96-well culture plate. HeLa S3 cells (1 x 10⁵/well) were used for transfection and incubated for further 24 h, then treated with β-thujaplicin (10 μM) for 24 h. Results show relative Luc activities from various Luc reporter transfected cells compared with that of pGL4-PIF1 transfected cells..

It has been suggested that both cellular senescence and aging of organisms are accelerated by various factors, such as telomereshortening [23-25] and DNA damaging reactive oxygen species (ROS) that are mainly generated from mitochondria [26,27]. Another facet or aging is the demonstration that caloric restriction elongates lifespans of organisms [28], suggesting that metabolism regulatory systems controls lifespan. Genetic analyses of C.elegans showed that several genes encoding insulin/IGF1 receptor and transcription factor FoxO play important roles in controlling the lifespan [29]. Moreover, studies of budding yeast showed that Sir2, a member of the sirtuin proteins with an NAD+-dependent protein deacetylase activity, has silencing action on chronological aging of yeast cells [30]. Many proteins, including PGC-1α, p53, FOXO1, HIF1α, UCP2 and PPARγ, have been reported to be the targets of SIRT1, which is known as mammalian homologue of Sir2 [31]. Because these protein factors function as metabolism regulators, SIRT1 could be referred as a key regulator of healthspan of organisms [31].

In this study, we have examined promoter activities of the 396-bp 5’-flanking region of the human SIRT1 gene to find out its response to the treatments with three types of thujaplicins (a, b and g) in HeLa-S3 cells. The 396-bp region has no apparent TATA-box but contains several well known transcription factor binding elements, including CREB, C/EBPβ, c-ETS, USF, SREBP1, Sp1, GATA and c-MYC binding motifs [7]. It has been shown that FOXO1, CREB, PPAR proteins and PARP2 play roles in regulation of the SIRT1 promoter [31]. However, at present, the Rsv or β-thujaplicin-responsive elements in the SIRT1 promoter region have not been precisely determined. Previously, it was indicated that the 5’-upstream regions of the WRN, BLM, TERT, p21 (CDKN1A) and HELB genes possess one or more Sp1/GC-box elements and that they positively respond to Rsv treatment in HeLa S3 cells [6,32]. The GC-box consensus sequence of the Sp1 transcription factor binding site is: 5’-(G/T)GGGCGG(G/A)(G/A)(C/T)-3’ or 5’- (G/T)(G/A)GGCG(G/T)(G/A)(G/A)(C/T)-3’ [33]. It has been shown that two GC-boxes, 5’-AGGGCGGGGG-3’ and 5’GGGGCGGGTC -3’ (-83 to -74 and -66 to -57, respectively), play important roles in the SIRT1 promoter activity [34]. As shown in Figure 3, the 5’-upstream regions of the RTEL, TRF1, TRF2, TERT, DKC1, TIN2, RAP1 and TPP1 genes positively responded to the treatment with β-thujaplicin. All of the 5’-upstream regions in the Luc-reporter vectors except pGL4-RTEL have at least one Sp1/GC-box. Although TF-search analysis did not find Sp1/GC-box, 5’-CGGGCGGGAC-3’, 5’-TTTCCGCCGG-3’ and 5’-TGCGCGCCTC-3’, namely GC-box like sequences are contained in the pGL4-RTEL. Taken together, the Sp1 binding motif is possibly one of the candidate elements that respond to β-thujaplicin. Moreover, Rsv is known to up-regulate cAMP level to activate CREB, which plays important roles in hormonal metabolism, including that of the insulin signaling system [35]. The CREB element is located in the 5’-upstream regions of the RTEL [21] and TPP1 [9] genes. This suggests that the CREB element in the human SIRT1 promoter region (-288 to -281) may respond to the b-thujaplicin treatment.

It should be noted that β−τhujaplicin (hinokitiol) stabilizes transcriptional active HIF-1α in HeLa and HepG2 cells to increase transcription of the VEGF gene [36]. On the other hand, SIRT1 deacetylates HIF-1α to suppress its activity [37]. Therefore, the induction of SIRT1 gene expression by β−thujaplicin might be required for reduction of over-stimulated HIF-1α to maintain cellular homeostasis. Moreover, it has been reported that β-thujaplicin induces G1 arrest via down-regulation of phosphorylated Rb and Skp2 ubiquitin ligase [38]. This cell cycle arrest is accompanied with an increase of p27 and p21 protein levels. Although the precise molecular mechanisms are not known, these biological properties of β-thujaplicin including transcriptional regulation and antiviral activity [16,36] might originate from its specific structure (Figure 1) that can act as a chelator of divalent metal ions [39]. In this study, we observed the up-regulation of the SIRT1 and shelterin-encoding gene promoter activities by the treatment of β−thujaplicin. Moreover, comparison of 5’-upstream regions of those genes suggested that transcription factors, including Sp1, may control lifespans of organisms responding to β-thujaplicin. The core structure of the thujaplicins could be applied to design lead compounds for novel anti-aging drugs, which could simultaneously activate the SIRT1 and shelterin-encoding gene promoter activities.

The authors are grateful to Tsutomu Iijima and Masanao Taniura for their outstanding technical assistance. This work was supported in part by a Research Fellowship grant from the Research Center for RNA Science and Drug Creation Frontier Research Center, RIST, Tokyo University of Science.