Res was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and dissolved in DMSO as a stock solution of 100 mmol/L. The pan-caspase inhibitor Z-VAD-fmk was obtained from Promega. RPMI-1640 was obtained from Gibco (Rockville, MD, USA) and fetal bovine serum was purchased from Hangzhou Sijiqing Biological Engineering Materials (Hangzhou, China).

Human glioma U251 cells were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum and 0.03% L-glutamine (Sigma, St Louis, MO, USA) and incubated in a humidified 5% CO₂ incubator at 37°C. The cells in the mid-log phase were used in the experiments.

Cell viability was assessed by MTT assay as described previously 28. Cells were plated in 96-well plates at the density of 10,000 cells in 100 μL medium per well one day before the experiment. MTT solution was added to the culture medium (500 μg/ml final concentration) 4 h before the end of treatments and the reaction was stopped by addition of 10% acidified SDS (100 μl) to the cell culture. The absorbance value (A) at 570 nm was read using an automatic multiwell spectrophotometer (Bio-Rad, Richmond CA, USA). The percentage of cell death (growth inhibition) was calculated as follows: cell death(%) = (1-A of experiment well/A of positive control well) ×100%.

To measure the mitochondrial membrane potential (ΔΨm), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), a sensitive fluorescent probe for ΔΨm was used 29. The U251 cells were cultured in 24-well plates for 24 h, and then exposed to the Res (150 μM) for various times. Cells were then rinsed with PBS twice, stained with 1 mL 10% RPMI-1640 medium containing 5 μmol/L JC-1 (Molecular Probes, USA) for 30 min at 37°C. Cells were rinsed with ice-cold PBS twice, resuspended in 1 mL ice-cooled PBS, and instantly assessed for red and green fluorescence with flow cytometry (EPICS-XL, Beckman, USA) 3031. A 488 nm filter was used for the excitation of JC-1. Emission filters of 535 and 595 nm were used to quantify the population of mitochondria with green (JC-1 monomers) and red(JC-1 aggregates) fluorescence, respectively 30. Frequency plots were prepared for FL1 (green) and FL2 (red) to determine the percentage of the mitochondria stained green (low membrane potential) and red (normal membrane potential).

PI staining was used to analyze DNA content. U251 cells were plated in 10-cm culture dishes at density determined to yield 60–70% confluence within 24 h. Cells were then treated, with or without Res 150 μM for 24–48 h, were trypsinized, stained with PI by the Cellular DNA Flow Cytometric Analysis Reagent Set (Boehringer Mannheim, Indianapolis, IN, USA), and analyzed for DNA content using the FACScan (Becton Dickinson, San Jose, CA, USA) as previously described 32. Data were analyzed by Cell Quest software (Becton Dickinson). All experiments were performed in duplicate and yielded similar results.

To detect apoptosis morphologically, we treated U251 cells with Res for 72 h, fixed them with 4% paraformaldehyde, and stained them with Hoechst 33258(0.5 μg/ml) for 15 min.

The autofluorescent agent MDC (Sigma) was recently introduced as a specific autophagolysosome marker to analyze the autophagic process 33. U251 cells were treated with Res 150 μM for 3–24 h. Autophagic vacuoles were labeled with MDC by incubating cells with 0.05 mM MDC in RPMI1640 at 37°C for 10 min at various times after Res treatment. After incubation, cells were washed three times with PBS and immediately analyzed with a fluorescence microscope (Nikon Eclipse TE 300, Japan) equipped with a filter system (V-2A excitation filter:380/420 nm, barrier filter:450 nm) 34. Images were captured with a CCD camera and imported into Adobe Photoshop.

LC3, a mammalian homologue of Apg8p/Aut7p essential for amino acid starvation-induced autophagy in yeast 35, was recruited to the autophagosome membranes in the Apg5-dependent manner 36. Therefore, autophagosome membrane association with LC3 is a specific marker for autophagy 33. After being incubated with Res 150 μM for 3–24 h, cells were washed with PBS and then fixed with paraformaldehyde (4% w:v). After rinsing in PBS, the cells were blocked with 0.1% Triton X-100 containing 1% bovine serum albumin in PBS for 1 h. This was followed by incubation in goat polyclonal antibody against microtubule-associated protein 1 light chain 3 (LC3; 1:100, sc-16756; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 24 h at 4°C in a humidified chamber. After 3 washes in PBS, the cells were incubated in donkey anti-goat immunoglobulin G-fluoresceinisothiocyanate (1:400, sc-2024; Santa Cruz) for 1 h at 4°C. Finally, cells were rinsed in PBS, coverslipped and examined with a confocal microscope (C1 si, Nikon, Japan).

Whole cell lysates were prepared from treated cells for Western blot analysis as described previously 37. Before immunoblotting, the protein concentrations were determined with a BCA detection kit (Pierce, USA) and adjusted to equal concentrations across different samples. The proteins were separated by 10–12% SDS-PAGE gel and transferred to nitrocellulose membranes. The membrane was subjected to immunoblotting using enhanced chemiluminescence (ECL kit; Amersham Pharmacia Biotech, Piscataway, NJ, USA) and visualized by autoradiography. Protein β-actin (1:5000; A5441; Sigma) was used as the loading controls.

All of experiments were repeated at least three times. The data were presented as means ± SD. Statistical analysis was carried out by ANOVA followed by a Dunnett t-test, considering *P < 0.05 as significance. Statistical significance was assessed using GraphPad Prism 4 software.

U251 cells were treated with Res at concentrations ranging from 0 to 300 μM for various lengths of time. The cell viability of U251 cells was determined using the MTT assay. As shown in Figure. 1, Res reduced U251 cell viability in a time- and dose-dependent manner. After 48 h of treatment, the inhibitory rate of Res (150 μM) on viability of U251 cells had reached 51.29 ± 0.64%, and when the incubation time was prolonged to 72 h, the inhibitory rate increased to 62.56 ± 0.36%. While at the dose of 75 μM the inhibitory ratio was 41.03 ± 0.32% with 48 h of treatment. Based on this dose-effect relationship, Res concentration of 150 μM was used for subsequent studies.

To examine whether Res affects cell cycle and induces apoptosis, we performed the DNA flow cytometric analysis. As shown in Figure. 2, cells treated with Res (150 μM) for 48 h showed increased population of cells in sub-G1 phase, characteristic of apoptosis, from 4.04 ± 0.46% to 11.82 ± 1.30%.

To determine the incidence of apoptosis morphologically, we stained the nuclei of treated U251 cells with Hoechst 33258. As shown in Figure. 3, apoptotic morphological characteristics such as chromatin condensation and nuclear fragmentation were detected in U251 cells treated with Res.

It has been suggested that Res-induced apoptotic death may be mediated by mitochondria-dependent signaling pathways 22. Chemically promoted apoptosis mediated by the mitochondria/caspase-9 activation pathway is often, though not always, associated with the collapse of ΔΨm as a result of leakiness of the inner mitochondrial membrane 38. Therefore, to investigate whether mitochondrial membrane integrity is damaged by Res, mitochondrial membrane potential was measured using JC-1, a sensitive fluorescent probe for ΔΨm 29.

U251 cells were cultured in 24-well plates and treated with Res for various lengths of time. When ΔΨm was low, JC-1 existed mainly in a monomeric form, which emits green fluorescence. As shown in Figure. 4, an increase in the amount of mitochondria with collapsed membrane potential was detected as early as 6 h after Res treatment.

To make a quantitative analysis, we use a flow cytometer (EPICS-XL, Beckman, USA). As shown in the Figure. 5, the levels of FL1 (green) count were found to be increased 6 h after Res treatment. This alteration reached its peak 72 h after Res treatment. These results indicate that a dysfunction of ΔΨm is an early event in Res-induced cell death.

To confirm an apoptotic mechanism in Res-induced death of U251 cells, the effects of pretreatment with the pan-caspase inhibitor Z-VAD-fmk were investigated. U251 cells were pretreated with Z-VAD-fmk 1 h before Res treatment, cell viability was determined with MTT assay 48 h later. As shown in Figure. 6, Z-VAD-fmk significantly inhibited the cytotoxicity of Res.

As Res-treated U251 cells triggered a slow process of apoptosis, we next assessed whether Res induces autophagy in U251 cells. MDC accumulates in mature autophagic vacuoles, such as autophagolysosomes, but not in the early endosome compartment 33. MDC staining can be used to detect autophagic vacuoles. When cells are viewed with a fluorescence microscope, AVs stained by MDC appear as distinct dot-like structures distributed within the cytoplasm or localizing in the perinuclear regions. As shown in Figure. 7, there was an increase in the number of MDC-labeled vesicles at 24 h after Res treatment. The result indicates an induction of AV formation by Res.

LC3 is localized in autophagosome membranes during amino acid starvation-induced autophagy 3536. LC3 is considered a molecular marker of autophagosomes. To assess if LC3 is altered in Res-induced autophagy, we examined localization of LC3 immunoreactivity. Under a fluorescence microscope, punctate patterns of LC3 immunoreactivity in many cells were observed after Res (150 μM) treatment, representing increased formation of autophagic vacuoles (Figure. 8). In contrast, the control cells only showed diffuse distribution of LC3 immunoreactivity.

Recent investigations suggest that there are two forms of the LC3 proteins in various cells: LC3-I and LC3-II 36. LC3-I is the cytoplasmic form and is processed into LC3-II, which is autophagosome membrane bound. Therefore, the amount of LC3-II is correlated with the extent of autophagosome formation. Using Western Blot analysis with anti-LC3 antibody, we examined the expressions of LC3-I (18 kDa) and LC3-II (16 kDa) in U251 cells after treatment with Res (150 μM). As seen in Figure. 9, an apparent increase in the levels of LC3-II protein was detected in U251 cells 24 h after treatment with Res, with a peak effect occurred at 48 h.

Autophagy is controlled by a group of evolutionarily conserved genes (ATG genes) 3940. More than 30 ATG genes have been identified in yeast and at least 11 have orthologs in mammals, Atg6 is known as beclin1. Beclin 1/Atg6 is a part of type III PI3 kinase complex that is required for the formation of the autophagic vesicle and interference with beclin 1 can prevent autophagy induction. Therefore, beclin 1 plays considerable roles in the processes of autophagy. The Western blot analysis revealed that beclin 1 levels were markedly increased 3 h after Res treatment, but decreased dramatically 24 h later (Figure. 10).

3-MA, the specific class III PI3 kinase inhibitor, prevents autophagy at an early stage; while bafilomycin A1, a specific inhibitor of vacuolar H⁺-ATPase, prevents autophagy at a late stage by inhibiting fusion between autophagosomes and lysosomes. To investigate the effects of autophagy on Res-induced apoptosis, 3-MA was added 1 h before Res. Results showed that 3-MA alone had little effect on sub-G1 fraction, an indicator of apoptotic cell death; however, pretreatment of U251 cells with 3-MA significantly increased the sub-G1 fraction induced by Res (Figure. 11).

In addition, to explore inhibition of autophagy at a late stage, bafilomycin A1 was added before Res. As shown in Figure. 12, the specific inhibitor of vacuolar H⁺-ATPase bafilomycin A1 alone had no significant effect in U251 cells, however, Res-induced cytotoxicity was significantly potentiated by bafilomycin A1.