Resveratrol (Rsv) has been shown to extend the lifespan of diverse range of species to activate sirtuin (SIRT) family proteins, which belong to the class III NAD+ dependent histone de-acetylases (HDACs).The protein deacetylating enzyme SIRT1 has been implicated in the regulation of cellular senescence and aging processes in mammalian cells. However, higher concentrations of this natural compound cause cell death. Therefore, novel compounds that have reduced cellular toxicity will be required for anti-aging therapy, especially for dermatological treatments. In this study, the Luciferase (Luc) expression vector pGL4-SIRT1 containing 396-bp of the 5’-upstream region of the human SIRT1 gene was transfected into HeLa S3 cells and Luc assay was performed. The results showed that treatments with the natural compound, α-, β- and γ-thujaplicins increase the SIRT1 promoter activity more than that with Rsv. Moreover, we carried out multiple transfection of Luc reporter vectors containing 5’-upstream regions of various human telomere maintenance factor encoding genes, and observed that β−thujaplicin (hinokitiol) activates TERT, RTEL, TRF1, DKC1, RAP1 (TERF2IP) and TPP1(ACD) promoters. These results suggest that that the β−thujaplicin could be used as anti-aging drugs to delay cellular senescence through activating SIRT1 transcription along with strengthening stability of telomeres.
Keywords: Aging, Cellular senescence, Shelterins, Telomere, Telomerase, Thujaplicin, Resveratrol, SIRT
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)  are up-regulated by Rsv treatment , 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 . It has been reported to have a variety of biological effects, including induction of apoptosis  and differentiation , anti-inflammatory , antibacterial  and anti-fungal  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.
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) . Structures of these compounds are shown in (Figure 1).
Human cervical carcinoma (HeLa S3) cells  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 . 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 , 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) . 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 , 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 . 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 . 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 . Because these protein factors function as metabolism regulators, SIRT1 could be referred as a key regulator of healthspan of organisms .
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 . It has been shown that FOXO1, CREB, PPAR proteins and PARP2 play roles in regulation of the SIRT1 promoter . 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’ . 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 . 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 . The CREB element is located in the 5’-upstream regions of the RTEL  and TPP1  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 . On the other hand, SIRT1 deacetylates HIF-1α to suppress its activity . 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 . 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 . 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.