RPMI-1640, fetal bovine serum (FBS), and antibiotics were obtained from Gibco-BRL Co. (Grand Island, NY, USA). Pemetrexed was purchased from Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), propidium iodide (PI), dimethyl sulfoxide (DMSO), N-acetylcysteine (NAC) and resveratrol were purchased from Sigma-Aldrich (St. Louis, MO, USA). JC-1, a lipophilic fluorescent dye used to detect mitochondrial membrane depolarization, was obtained from Molecular Probes Co. (Eugene, OR, USA). Primary antibodies against the following targets, caspase-3, -8 and -9, and poly(ADP-ribose) polymerase (PARP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against SIRT1, voltage-dependent anion channel (VDAC) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against cytochrome c were obtained from Pharmingen (San Diego, CA, USA). Anti-rabbit IgG-conjugated horseradish peroxidase (HRP) antibodies and enhanced chemiluminescence (ECL) kits were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK).

The human mesothelioma cell line MSTO-211H was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA), and the human lung adenocarcinoma cancer cell line A549 was obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). These cell lines were grown in RPMI-1640 containing 100 U/ml penicillin, 0.1 mg/ml streptomycin and 10% FBS. The cells were incubated in a humidified atmosphere of 5% CO₂ in air at 37°C and maintained in log phase growth. Cell viability was determined by measuring the mitochondrial conversion of MTT to formazan, which was measured spectrophotometrically. After cells were treated with the specified study drugs, MTT was added to the cell suspension for 4 h. After 3 washes with phosphate-buffered saline (PBS; pH 7.4), the insoluble formazan product was dissolved in DMSO. The optical density (OD) of each well was measured using a microplate reader (Titertek Multiskan; Flow Laboratories, North Ryde, New South Wales, Australia) at 590 nm. The OD resulting from formazan production in the control cells was considered as representing 100% cell viability, and all other measurements were expressed as a percentage of the control cell value.

MSTO-211 and A549 cells undergoing early/late apoptosis were analyzed by Annexin V-fluorescein isothiocyanate (FITC) and PI staining. Cells in the log phase (2.5×10⁵ cells) were seeded in 35-mm² dishes. Cells were left untreated or were incubated with the specified drugs for the indicated times at 37°C. Both adherent and floating cells were collected and analyzed by the Annexin V assay according to the manufacturer’s instructions. Pelleted cells were briefly washed with PBS and resuspended in Annexin binding buffer. Cells were then incubated with Annexin V-FITC and PI for 15 min at room temperature. After incubation, the stained cells were analyzed using a fluorescence-activated cell sorting (FACS)Calibur system equipped with CellQuest software (Becton-Dickinson, San Jose, CA, USA). Cells with no drug treatment were used as controls.

MSTO-211 and A549 cells were harvested at the indicated treatment times, washed with PBS, and then stained with 10 μg/ml JC-1 at 37°C for 30 min. After a brief wash with PBS, cells were immediately analyzed using a FACSCalibur system equipped with CellQuest software. At low concentrations, JC-1 exists mainly in a monomeric form, emitting green fluorescence with an emission maximum at ~530 nM, whereas at higher concentrations it forms aggregates known as J-aggregates, which emit orange-red fluorescence with an emission maximum at ~590 nM.

To measure intracellular ROS, cells were incubated with 10 μmol/l 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA; Molecular Probes Co.) at 37°C for 30 min. Cells were washed, scraped gently, resuspended in PBS and kept on ice for immediate analysis by FACSCalibur flow cytometry using an argon laser (488 nm) for excitation (Becton-Dickinson). Green fluorescence due to DCF trapped inside the cells was measured and plotted on a log scale. Data were acquired and analyzed with the CellQuest program (Becton-Dickinson).

Cells were harvested and lysed using radioimmunoprecipitation assay buffer (50 mM Tris-Cl, pH 7.4, 1% NP40, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml each of aprotinin and leupeptin, and 1 mM Na₃VO₄). After centrifugation at 12,000 × g for 30 min, the supernatant was collected and the protein concentration was determined by the Bradford method (Bio-Rad Protein Assay; Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk in TBS-T (25 mM Tris, pH 7.6, 138 mM NaCl and 0.05% Tween-20) for 1 h and probed with primary antibodies (at 1:1,000–1:5,000). After a series of washes, the membranes were further incubated with a secondary antibody (at 1:2,000–1:10,000) conjugated with HRP. Detection of the immunoreactive signals was carried out using an ECL detection system.

Cytosolic and mitochondrial fractions were prepared as previously described (15) with modifications. Cells were harvested, washed with ice-cold PBS, and then incubated with 500 μM buffer A [250 mM sucrose, 20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl₂, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF and 10 μg/ml each of leupeptin, aprotinin and pepstatin A] on ice for 30 min. Cells were then disrupted by 20 passages through a 26-gauge needle and centrifuged at 750 × g for 10 min. The supernatant was centrifuged at 10,000 × g for 25 min. After centrifugation, the cytosolic fraction was frozen at 70°C. The pellet containing the mitochondria was washed with ice-cold buffer A and then resuspended with cell lysis buffer. The resuspended pellet was incubated on ice for 30 min and then centrifuged at 10,000 × g for 25 min. The supernatant thus collected represented the mitochondrial fraction of the cells.

Transcriptional expression of SIRT1 was specifically suppressed by the introduction of a 21-nucleotide duplex small interfering RNA (siRNA) targeting the coding sequence of SIRT1 mRNA (16). Cells (10⁵ cells/well) were plated in 6-well plates and transiently transfected with 50 nM/well of SIRT1 siRNA (Cell Signaling Technology) mixed with the X-tremeGENE siRNA transfection reagent (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions. Silencer Negative Control siRNA (Roche Applied Science) was used as a negative control and introduced into the cells using the same protocol.

Each experiment was performed at least 3 times, and all values are expressed as the mean ± SD of triplicate samples. The Student’s t-test was used to determine the statistical significance of the results. Values of p<0.05 were considered to indicate statistically significant results.

MSTO-211 and A549 cells were treated with different concentrations of pemetrexed, and the viability was measured by the MTT assay. As shown in Fig. 1A, pemetrexed significantly inhibited the growth of MSTO-211 and A549 cells in a dose-dependent manner. To determine whether the observed growth inhibition was due to enhanced apoptosis, the proportion of apoptotic cells was measured using Annexin V-PI staining. Annexin V staining showed that pemetrexed significantly enhanced apoptosis in the MSTO-211 and A549 cells (Fig. 1B). To further elucidate the mechanism through which pemetrexed induced apoptosis, cell lysates were evaluated by immunoblotting (Fig. 1C). Pemetrexed enhanced the expression of the processed 85-kDa isoform of PARP, which is known to play a major role in the process of apoptosis. Moreover, pemetrexed led to a marked increase in the expression of caspase-3, -8 and -9. These results indicate that pemetrexed enhanced caspase-dependent apoptosis in the MSTO-211 and A549 cells.

We examined the role of signal transduction pathways in modulating pemetrexed-induced apoptosis. Intracellular ROS generation was assessed by flow cytometry using the total ROS marker H₂DCFDA. Elevated ROS levels in the MSTO-211 and A549 cells were detectable 24 h after treatment with pemetrexed (Fig. 2A). Next, markers of mitochondrial dysfunction were evaluated in the cells treated with pemetrexed, including mitochondrial membrane potential transition (MPT) and cytosolic release of cytochrome c. JC-1 has been used to detect apoptosis induced by mitochondrial depolarization. As shown in Fig. 2B, JC-1 monomer levels were increased in the MSTO-211 and A549 cells treated with pemetrexed. Since the loss of ΔΨm resulted in cytochrome c release into the cytosol, cytochrome c levels were evaluated by western blotting in the mitochondrial and cytosolic fractions (Fig. 2C). Pemetrexed was associated with increased cytosolic cytochrome c and decreased mitochondrial cytochrome c. We next examined the effects of pemetrexed on the expression of SIRT1 in the MSTO-211 and A549 cells. SIRT1 expression was decreased in a time- and dose-dependent manner after pemetrexed treatment as compared with the control cells (Fig. 2D).

We next tested the effect of the free radical scavenger NAC in pemetrexed-treated MSTO-211 and A549 cells. Cells were pretreated with 10 mM NAC, followed by the addition of pemetrexed at 2 μM for 24 h. As shown in Fig. 3A, the enhancement of ROS generation by pemetrexed was abolished by NAC. To determine whether elevated ROS mediated the apoptosis induced by pemetrexed, the proportion of apoptotic cells was determined by Annexin V-PI staining (Fig. 3B). The proportions of Annexin V-positive MSTO-211 and A549 cells were increased by treatment with pemetrexed, whereas pretreatment with NAC markedly reduced this effect. Moreover, western blot analysis of MSTO-211 and A549 cell lysates (Fig. 3C) showed that pemetrexed decreased SIRT1 expression, enhanced the cleavage of PARP, and enhanced the expression of caspase-3, -8 and -9, and pretreatment with NAC blocked these effects.

We found that JC-1 monomers were increased in the MSTO-211 and A549 cells treated with pemetrexed, whereas the loss of ΔΨm was significantly reduced in the cells pretreated with NAC (Fig. 3D). To provide further evidence of mitochondrial dysfunction, cytosolic cytochrome c was measured by western blotting in the mitochondrial and cytosolic fractions (Fig. 3E). Cytosolic cytochrome c was increased by pemetrexed, and pretreatment with NAC reduced this effect. Together, these findings indicate that ROS generation plays a primary role in apoptosis induction by pemetrexed through mitochondrial dysfunction and this effect involves, at least in part, the expression of SIRT1.

To determine the role of SIRT1 in pemetrexed-induced apoptosis in MSTO-211 and A549 cells, we knocked down SIRT1 with siRNA. The introduction of SIRT1 siRNA attenuated SIRT1 protein expression 48 h after transfection. No reduction in SIRT1 protein was observed in cells transfected with the scrambled siRNA, which contained the same number of each nucleotide found in the SIRT1 siRNA. As shown in Fig. 4A, the number of Annexin V-positive cells was increased in the SIRT1 siRNA transfectants, and combined treatment with SIRT1 siRNA and pemetrexed resulted in a greater apoptosis rate than combined treatment with the control siRNA and pemetrexed.

We next examined whether SIRT1 siRNA affects ROS generation. ROS generation in the MSTO-211 and A549 cells treated with SIRT1 siRNA and pemetrexed was increased markedly compared to that of cells treated with SIRT1 siRNA or pemetrexed alone (Fig. 4B). JC-1 monomer levels were consistently enhanced in the MSTO-211 and A549 cells treated with SIRT1 siRNA and pemetrexed (Fig. 4C). Moreover, the combination of SIRT1 siRNA and pemetrexed enhanced the expression of the processed 85-kDa isoform of PARP, caspase-3, -8 and -9 (Fig. 4D). Together, these data indicate that downregulation of SIRT1 enhanced apoptosis induced by pemetrexed through mitochondrial dysfunction that partially involves the generation of ROS.

We next examined whether resveratrol, an activator of SIRT1, could confer protection against the apoptosis induced by pemetrexed. As shown in Fig. 5A, pemetrexed resulted in 22.5 and 26.8%, respectively, more apoptotic MSTO-211 and A549 cells than were observed in the untreated cells. However, resveratrol decreased the number of apoptotic cells after pemetrexed treatment by only 16.0 and 18.1%, respectively, in the MSTO-211 and A549 cells. We next examined whether SIRT1 upregulation had a role in ROS generation by pemetrexed. The results demonstrated that ROS generation in the MSTO-211 and A549 cells treated with resveratrol and pemetrexed was decreased compared to the ROS generation in cells treated with pemetrexed alone (Fig. 5B). Consistently, JC-1 monomer levels were decreased in the MSTO-211 and A549 cells treated with resveratrol and pemetrexed (Fig. 5C) as compared to those treated with pemetrexed alone. Western blot analyses showed that SIRT1 overexpression decreased the cleavage of PARP and the expression of caspase-3, -8 and -9, even in the presence of pemetrexed (Fig. 5D). These results suggest that the reduction of apoptosis by pemetrexed is due, at least in part, to SIRT1 upregulation.