HeLa cells were cultured in Minimal Essential Medium (MEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FBS (Biochrom AG, Berlin, Germany), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich) and maintained in 5% CO₂ at 37°C. EX527, AGK2 and hemin were purchased from Sigma-Aldrich. Fugene HD transfection reagent was from Promega (Madison, WI, USA). Polyclonal anti-HSF1, anti-HSP27, anti-caspase-3, and anti-PARP antibodies were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Monoclonal anti-Sirt1 and anti-Sirt2 antibodies were from Cell Signaling Technology (Danvers, MA, USA).

Total RNA of HeLa cells was extracted using TRIzol reagent (Life Technologies, Gaithersburg, MD, USA) according to the manufacturer's instructions. A total of 1 μg of total RNA was reverse-transcribed to cDNA in a reaction mixture of 20 μl with the Reverse Transcription system (Promega, Leiden, The Netherlands). Amplification was performed for 30 cycles in a DNA thermal cycler. GAPDH was used as a control for the PCR reaction. The PCR products were resolved on a 1.5% agarose gel and were stained with ethidium bromide.

Cell proliferation was assessed using a 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were seeded in a 96-well plate at a density of 1×10⁴ cells/well. The next day, the cells were washed twice with PBS, and 500 μg/ml MTT (Sigma-Aldrich) was added to the wells. The MTT solution was removed after 4 h of incubation at 37°C. A mixture of 0.01 M glycine and DMSO (Sigma-Aldrich) was added to each well. The absorbance was measured at 540 nm with a Benchmark microplate reader (Bio-Rad Laboratories, Philadelphia, PA, USA).

Total cell extracts were incubated with anti-HSF1 in NP-40 lysis buffer (0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 50 mM Tris HCl pH 7.4, 1 mM EDTA, 50 mM NaF, 1 mM B-glycerophosphate, 1 mM sodium orthovanadate, 0.5 μg/ml leupeptin, 1 μg/ml pepstatin, 0.2 mM PMSF). The extract mixtures were incubated at 4°C overnight with rotation before the addition of 20 μl of protein A/G beads (Life Technologies) for 3 h at 4°C. The beads were washed three times with the same buffer and suspended in 2X SDS sample buffer. The samples were resolved in SDS-polyacrylamide gels for western blot analysis with specific antibodies as indicated.

Cell lysates (50 μg) were placed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, and a protease inhibitor cocktail containing 1 μg/ml aprotinin and leupeptin) and separated by 12% SDS-PAGE. The resolved proteins were transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, UK) according to standard procedures. The membrane was blocked in 5% non-fat dry milk for 3 h and incubated with the primary antibodies for 3 h at room temperature (RT). After incubation with a specific peroxidase-coupled secondary antibody (Sigma-Aldrich) for 1 h, the blotted bands were detected using an enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech).

Cells were allowed to grow in a culture dish overnight, and a scratch ~3-mm wide was created in the monolayer using a pipette tip. After washing twice with PBS, the cells were treated with or without the Sirt1/Sirt2 inhibitors, and images were captured after 12 or 36 h. Cells were imaged in 5 random microscopic fields per well using an Olympus IX2-SLP inverted microscope (Japan) at a ×100 magnification.

The cells were harvested and fixed with 70% ethanol for 1 h at 4°C for cell cycle analysis. After washing with cold PBS, the cells were incubated with DNase-free RNase and propidium iodide (PI) at 37°C for 30 min. The specific binding of Annexin V-FITC/PI was performed by incubating the cells for 15 min at RT in a binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4) containing saturated concentrations of Annexin V-FITC and PI. After incubation, the cells were pelleted and analyzed in a FACScan analyzer (Beckman Coulter Inc., Fullerton, CA, USA).

The cell cycle distribution was analyzed by flow cytometry. Briefly, 1×10⁶ cells were harvested and washed in PBS, and then fixed in 70% alcohol for 30 min at 4°C. After washing three times in cold PBS, the cells were resuspended in 1 ml of PBS solution containing 50 μl of 1 mg/ml PI and 1 unit of DNase-free RNase for 30 min at 37°C. The samples were then analyzed for their DNA content by FACS (Beckman Coulter Inc.).

Briefly, the cells (8×10³ cells/well) were exposed to different concentrations of EX527 or AGK2 in 1 ml of 0.3% basal medium Eagle's agar containing 10% FBS. The cultures were maintained at 37°C in a 5% CO₂ incubator for 10–15 days, and the cell colonies were scored using an Olympus IX2-SLP inverted microscope (Japan).

All experiments were performed at least three times. The mean values for the experiments are expressed as the mean ± SE. Significant differences were assessed by analysis of variance. p<0.05 was considered to indicate a statistically significant difference.

To examine the effect of Sirt1/2 inhibitors, EX527 and AGK2, on the growth of HeLa cells, we performed MTT assays. The cells were exposed to increasing concentrations of nicotinamide (NAM), EX527 and AGK2 for 24 h, and the cell viability was monitored (Fig. 1). EX527 and AGK2 significantly inhibited cell proliferation in a dose-dependent manner. EX527 at concentrations of 50 and 100 μM resulted in cell growth inhibition of 68 and 54%, respectively, at 24 h (Fig. 1B). Similarly, AGK2 also significantly inhibited cell growth in a dose-dependent manner without inducing cytotoxicity at low doses (≥1 μM; Fig. 1C). However, these cells exhibited no significant decrease in cell proliferation after NAM treatment for 24 h (survival rate >93%; Fig. 1A).

To further assess the effect of EX527 and AGK2 on tumorigenicity, we compared the anchorage-independent growth rates. Twelve days after EX527 (50 μM) and AGK2 (5 μM) treatment, HeLa cells showed a significantly reduced colony forming ability in soft agar to ~95 and 46% of the control cells, respectively (Fig. 1D). Together, our observations showed that EX527 and AGK2 suppressed the malignant phenotype such as cell proliferation and colony formation.

It was hypothesized that EX527 or AGK2 induce alterations in cell cycle regulation. Using flow cytometry, we analyzed the effect of EX527 or AGK2 on cell cycle progression. As shown in Fig. 2A–C, EX527 and AGK2 induced cell cycle arrest at the G1 phase. Western blot analysis showed that the levels of CDK4 or CDK6 and cyclin D1 were decreased after EX527 or AGK2 treatment in a dose-dependent manner. In addition, EX527 and AGK2 inhibited the expression of p53 protein (Fig. 2D). These results suggest that the effects of EX527 and AGK2 on G1 cell cycle progression were associated with the inhibition of cell growth.

To determine whether EX527 and AGK2 induce death in HeLa cells, we quantified apoptosis by flow cytometry using the Annexin V-FITC/PI double staining assay. As shown in Fig. 2E–H, after 24 h of incubation, there was no significant decrease in living cells upon EX527 treatment; 93% of the control cells were alive, while only 3.3% of the EX527-treated cells underwent cell death. Similarly, apoptotic cells were not markedly observed after AGK2 treatment (Fig. 2G). Consistent with this observation, caspase-3 and PARP were not cleaved in cells treated with EX527 or AGK2 compared with the control (Fig. 2H). In addition, key autophagy proteins, LC3B and beclin-1, were not activated after treatment with EX527 or AGK2 (Fig. 2I).

A previous study suggested that Sirt1 deacetylates HSF1 and activates heat shock proteins (HSPs) under a heat stress condition (21). We investigated the possibility that Sirt1 and Sirt2 inhibition by EX527 and AGK2 may be responsible for HSF1 inactivation. Therefore, we subjected EX527- or AGK2-treated cells to heat shock (1 h at 42°C) and analyzed the expression of endogenous Sirt1, Sirt2 and HSF1, as well as HSPs such as Hsp27 and Hsp70. EX527 or AGK2 treatment decreased the expression of HSF1 and HSP27 in non-stress or heat stress condition (Fig. 3A and B), which indicated that Sirt1 and Sirt2 are required for the regulation of the HSF1 pathway. Furthermore, EX527 and AGK2 also decreased HSF1 phosphorylation after heat shock. However, NAM decreased HSF1 expression/phosphorylation and HSP27 expression under heat shock conditions (Fig. 3C).

To determine whether EX527 and AGK2 influence HSF1 transcription in HeLa cells, RT-PCR was performed using specific oligonucleotides against the HSF1 gene. As shown in Fig. 3D, the HSF1 mRNA level was not altered by EX527 or AGK2.

To verify the significance of the regulation of HSF1 by Sirt1, cells were transfected with or without Sirt1, exposed to a 42°C heat shock for 1 h, allowed to recover at 37°C for 24 h, and analyzed for HSF1 expression. As expected, the heat shock induced HSP27 expression, which increased with recovery time. Importantly, at the same time-points, we observed that the cells overexpressing Sirt1 showed increased HSF1 expression compared with the heat shock-treated cells (Fig. 3E).

To determine whether HSF1 deacetylation by Sirt1 is involved in HSF1 ubiquitination/stabilization, we examined the acetylation and ubiquitination status of HSF1 after EX527 and AGK2 treatment. HeLa cells were treated with or without EX527 and AGK2 and exposed to a 42°C heat shock for 1 h. Then, the samples were immunoprecipitated with an anti-HSF1 antibody and analyzed for HSF1 acetylation. Acetylated HSF1 was detected in the untreated cells but was decreased in the cells exposed to heat shock stress conditions. However, acetylated HSF1 levels were induced in the EX527- or AGK2-treated cells before heat shock (Fig. 4A). In addition, EX527 and AGK2 induced HSF1 ubiquitination (Fig. 4B).

To investigate whether proteasome-dependent protein degradation is involved in Sirt1 inhibitor-mediated downregulation of HSF1 protein, we treated cells with the proteasome inhibitor hemin and/or EX527. As shown in Fig. 4C and D, the HSF1 protein level in the hemin-treated cells was substantially higher than that in the EX526-treated cells. Our above data indicate that EX527 or AGK2 decreases the expression/phosphorylation of HSF1 protein. However, hemin inhibited the EX527-mediated reduction of the HSF1 protein. From the above data, we conclude that Sirt1 and/or Sirt2 inhibition results in a diminution of HSF1 protein stabilization.

To investigate whether Sirt1/HSF1 modulate cell motility, we treated HeLa cells with EX527 and AGK2 for 36 h and performed a wound-healing experiment. EX527- or AGK2-treated cells showed reduced migration ability when compared with the control or heat shock-treated cells (Fig. 5A).

To further assess the effect of the Sirt1/HSF1/HSP27 pathway on cell migration, we compared the migration capabilities of Sirt1 or HSF1 with or without siHSP27 transfection. First, the levels of the HSF1 protein increased in the HSF1-transfected cells compared to the control cells (Fig. 5B). As expected, HSF1-overexpression led to induced Sirt1 protein levels. A wound-healing assay showed that overexpression of HSF1 or Sirt1 led to increased cell migration. However, the increase in gap closure was delayed in cells transfected with siHSP27; the migration of siHSP27-transfected cells was similar to the control cells (Fig. 5C). These data indicate that Sirt1/Sirt2-mediated HSF1/HSP27 regulation is involved in cell migration.