Roswell Park Memorial Institute medium-1640 (RPMI-1640), fetal bovine serum (FBS), and antibiotics (penicillin and streptomycin) were obtained from Gibco BRL Co. (Grand Island, NY, USA). Pemetrexed was purchased from Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada). Simvastatin, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), propidium iodide (PI), dimethyl sulfoxide (DMSO) and N-acetylcysteine (NAC) 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. Primary antibodies against the following targets: caspase-3, -8 and -9, poly(ADP-ribose) polymerase (PARP), Puma, Bim, Mcl-1, Bcl-XL, and XIAP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); antibodies against heme oxygenase-1 (HO-1), MnSOD, survivin, VDAC, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies to 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).

MSTO-211 cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and A549 human lung cancer cells were obtained from the Korean Cell Line Bank (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 three washes with phosphate-buffered saline (PBS; pH 7.4), the insoluble formazan product was dissolved in dimethyl sulfoxide (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 control cells was considered as 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-FITC and PI staining. Cells in the exponential growth phase (2.5×10⁵ cells) were seeded in 35-mm² dishes. Cells were left untreated or incubated with 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 (emission maximum at ~530 nM), whereas at higher concentrations it forms aggregates, known as J-aggregates, which emit orange-red fluorescence (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, Eugene, OR, USA) at 37°C for 30 min. Cells were then 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. Green fluorescence due to trapped DCF inside the cells was collected and plotted on a log scale. Data were acquired and analyzed with the CellQuest program. To measure mitochondria-derived ROS, the mitochondria-targeted, peroxide ion (O₂⁻) sensitive, hydroethidine analog probe MitoSOX (Invitrogen Life Technologies, M36008) was used to determine relative O₂⁻ levels. Cells were incubated with 5 μM MitoSOX for 10 min in RPMI-1640, washed twice with PBS, and analyzed with FACSCalibur flow cytometry.

Cells were harvested and lysed using radio-immunoprecipitation 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 method of Bradford (Bio-Rad protein assay). Equal amounts of protein were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and subsequently transferred to nitrocellulose membranes. 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, membranes were further incubated with 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 described previously (22) 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 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 cells.

Transcriptional expression of Bim was specifically suppressed by the introduction of 21-nucleotide duplex small interfering RNA (siRNA), which targets nucleotides of Bim mRNA coding sequence (23). Cells (10⁵ cells/well) were plated in 6-well plates and transiently transfected with 50 nM per well of Bim 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 were 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 statistically significant.

MSTO-211 and A549 cells were treated with different concentrations of simvastatin in the absence or presence of pemetrexed, and viability was measured by the MTT assay. As shown in Fig. 1, the combination of pemetrexed and simvastatin produced a synergistic inhibitory effect on the growth of both MSTO-211 and A549 cells. Simvastatin inhibited cell growth in a dose-dependent fashion in the presence of pemetrexed.

To examine whether the observed growth inhibition was due to enhanced apoptosis, the proportion of apoptotic cells was determined using Annexin V-PI staining. Annexin V staining showed that the combination of pemetrexed and simvastatin significantly enhanced apoptosis compared with either drug alone in MSTO-211 and A549 cells (Fig. 2A).

To further elucidate the mechanism of apoptosis induced by pemetrexed and simvastatin, cell lysates were evaluated by immunoblotting (Fig. 2B). Our results showed that the combination of pemetrexed and simvastatin enhanced the expression of the processed 85-kDa isoform of PARP, which is known to play a major role in circumventing the apoptosis process. Moreover, the combination of pemetrexed and simvastatin led to a marked increase in the expression of caspase-3, -8 and -9. These results indicate that pemetrexed and simvastatin enhanced caspase-dependent apoptosis in MSTO-211 and A549 cells.

We also investigated the upstream regulatory mechanisms leading to the induction of apoptosis by the combination of pemetrexed and simvastatin. Intracellular ROS generation was assessed by flow cytometry using the total ROS marker H₂DCFDA and the mitochondrial superoxide marker MitoSOX RED. The results demonstrate that ROS generation in MSTO-211 and A549 cells treated with both pemetrexed and simvastatin increased markedly compared to ROS generation in cells treated with pemetrexed or simvastatin alone (Fig. 3A). FACS analysis using MitoSOX revealed that intracellular O₂⁻ levels increased significantly, which correlated well with the onset of total ROS production. It is possible that the combination treatment increased cellular oxidative stress. We then investigated whether combined treatment with pemetrexed and simvastatin affected two markers for oxidative stress: inducible HO-1 and MnSOD (Fig. 3B). The combination of pemetrexed and simvastatin resulted in enhanced expression of HO-1 and MnSOD compared to MSTO-211 and A549 cells treated with either pemetrexed or simvastatin alone.

We also investigated components upstream of caspase-3 in apoptotic signaling. Markers of mitochondrial dysfunction, including ΔΨ_m transition and cytosolic release of cytochrome c, were evaluated in cells treated with pemetrexed and simvastatin. JC-1 has been widely used for the detection of apoptosis by measuring mitochondrial depolarization. As shown in Fig. 4A, JC-1 monomer level was enhanced in MSTO-211 and A549 cells treated with the drug combination. Since the loss of ΔΨ_m results in cytochrome c release into the cytosol, cytochrome c levels were evaluated by western blotting in both mitochondrial and cytosolic fractions (Fig. 4B). Combination treatment with pemetrexed and simvastatin was associated with an increased level of cytochrome c in the cytosolic fraction over that seen with either agent alone and a corresponding decrease in levels in the mitochondrial fraction.

Members of the IAP and Bcl-2 families are important regulators of the mitochondrial apoptotic pathway. To identify the molecular mechanism underlying apoptosis induced by combined treatment with pemetrexed and simvastatin, we examined the expression levels of the IAP (XIAP and survivin), anti-apoptotic (Mcl-1 and Bcl-xL), and pro-apoptotic (Bim and Puma) Bcl-2 families, by immunoblot analysis in MSTO-211 and A549 cells treated with pemetrexed and/or simvastatin for 36 h. As shown in Fig. 4C, treatment of MSTO-211 and A549 cells with pemetrexed and simvastatin resulted in a significant decrease in XIAP and survivin levels relative to treatment with either drug alone. In addition, combination treatment with pemetrexed and simvastatin decreased the expression of the anti-apoptotic factors Mcl-1 and Bcl-xL, and increased the expression of pro-apoptotic factors Bim and Puma.

We next tested the effect of the free radical scavenger NAC in pemetrexed and simvastatin-treated MSTO-211 and A549 cells. Cells were pretreated with NAC, followed by the addition of pemetrexed and simvastatin for 24 h. As shown in Fig. 5A, the enhancement of ROS generation by combination treatment with pemetrexed and simvastatin was abrogated by NAC. Moreover, NAC markedly inhibited the effects of combination therapy on cell viability, as evaluated by the MTT assay (Fig. 5B).

Our results indicate that elevated ROS may be necessary for the potentiation of cell death in pemetrexed plus simvastatin-treated cells. To determine whether elevated ROS participated in the apoptosis induced by the combination of pemetrexed and simvastatin, the proportion of apoptotic cells was determined by Annexin V-PI staining (Fig. 5C). Annexin V-positive cells were increased in MSTO-211 and A549 cells treated with the drug combination. Pretreatment with NAC markedly reduced this increase.

We also observed that JC-1 monomers were increased in MSTO-211 and A549 cells treated with the drug combination, and the loss of ΔΨ_m was significantly reduced in cells pretreated with NAC (Fig. 5D). Western blot analysis of MSTO-211 and A549 cell lysates (Fig. 5E) showed that the combination of pemetrexed and simvastatin enhanced the expression of cleaved PARP, caspase proteins, Bim, and Puma, decreased the expression of XIAP, survivin, Mcl-1, and Bcl-xL. Pretreatment with NAC blocked these effects. Together, these findings indicate that ROS generation played a primary role in apoptosis induced by pemetrexed and simvastatin.

Previous studies revealed that the expression of Bim, a pro-apoptotic protein, was significantly induced by statins or gefitinib in lung cancer (24,25). To determine the role of Bim in apoptosis induced by pemetrexed and simvastatin, we decreased the level of Bim expression by introducing siRNA for Bim. We then examined the effect of Bim siRNA-transfected cells on apoptosis induced by pemetrexed and simvastatin. We spread an equal number of viable Bim siRNA-transfected and non-silencing siRNA-transfected cells at 48 h after siRNA transfection. After an additional 48-h incubation, the cells were treated with pemetrexed and simvastatin for 36 h, and the cell lysate was used to carry out western blotting. Both Bim siRNA and the pemetrexed-simvastatin combination decreased the expression of cleaved PARP, caspase-3, -8 and -9. Together, these data indicate that the induction of apoptosis by pemetrexed and simvastatin is due, at least in part, to the upregulation of Bim.