Human neuroblastoma cell line LA-N-1 was purchased from RIKEN BioResource Center Cell Bank (Japan), while SH-SY5Y, SK-N-DZ, human embryonic kidney HEK-293 and human hepatocyte-like WRL-68 cell lines were purchased from the American Type Culture Collection (ATCC, USA). LA-N-1 cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin sulfate and 0.25 μg/ml amphotericin B in 0.85% saline). SH-SY5Y cells were grown in DMEM/F12 medium (Gibco) supplemented with 15% FBS, 1% antibiotics, 1 mM sodium pyruvate and 1 mM MEM with non-essential amino acids (NEAA) solution. SK-N-DZ and HEK 293 cells were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics. WRL-68 cells were cultured in MEM medium (Gibco) supplemented with 10% FBS and 1% antibiotics. The primary embryonic cortical neurons from SD rats were kindly provided by Professor K.F. Lau, School of Life Sciences, The Chinese University of Hong Kong. The primary embryonic cortical neurons were cultured in MEM medium supplemented with 5% FBS, glucose (18 mM), L-glutamine (2 mM), insulin (5 μg/ml), progesterone (0.02 μM), putrescine (100 μM), selenium (30 pM), β-mercaptoethanol (25 μM) and 1% antibiotics. All cell lines were cultured at 37°C in a humidified incubator supplied with 5% CO₂. I3M was purchased from Sigma-Aldrich (USA). Stock solution of I3M (10 mM) was prepared by dissolving the powdered form of I3M (with a molecular weight of 277.28 and >98% purity) in 100% dimethyl sulfoxide (DMSO; Sigma, USA) and stored in the dark at −20°C until use. All other chemicals were purchased from Sigma unless otherwise stated.

Cell growth was measured using both the colorimetric MTT assay and fluorometric cell proliferation assay. Cells were seeded in 96-well plates, cultured overnight and then treated with different concentrations of I3M. Following treatment for the indicated periods of time, cell growth was measured using the MTT assay and recorded by a Benchmark microplate reader (Bio-Rad Laboratories, USA) as previously described (19). In addition, cell proliferation was measured using the CyQUANT^® NF Cell Proliferation Assay kit (Molecular Probes, Invitrogen Corp., USA) and recorded by a fluorescence plate reader (Tecan Polarion, USA) (20).

Cell colony-forming ability was determined as previously described (21). Briefly, the 6-well tissue culture plates were pretreated with poly-D-lysine hydrobromide for 4 h and washed once with deionized water. Then, 400 cells were added into each well of a 6-well plate and allowed to settle overnight. Cells were treated with I3M for 24 h. On the following day, the medium was replaced with completed RPMI medium. The medium was changed every 3 days. After 6 days, colonies were fixed with methanol and stained with Hemacolor staining solutions. Colonies were counted and the percentage of colonies relative to solvent-treated control was calculated.

The Cell Death Detection ELISA^PLUS kit (Roche Applied Science, Switzerland) was used to quantify the enrichment of cytoplasmic nucleosomes which reflects the induction of apoptosis in the cells. LA-N-1 cells were seeded in 96-well plates at a density of 1.5×10⁴ cells per well. On the following day, cells were treated with solvent control (0.1% DMSO) or different concentrations of I3M for 48 h. Doxorubicin (0.2 μM) was used as positive control. Procedures were performed according to the manufacturer’s instructions and absorbance was measured at 405 nm with reference to 490 nm by a Benchmark microtiter plate reader (Bio-Rad Laboratories) after color development, which was proportional to the amount of nucleosomes present in the samples. The results were expressed as enrichment factor which represents the relative levels of mono- and oligo-nucleosomes compared with the solvent control.

LA-N-1 cells were seeded in 60-mm dishes at a density of 9×10⁵ cells per dish and incubated overnight. After synchronizing in plain RPMI medium for 24 h, cells were treated with different concentrations of I3M for 48 h. Following treatment, cells were fixed with 70% ethanol, treated with 50 μg/ml RNase A (Sigma), stained with 40 μg/ml propidium iodide (Sigma) and analyzed by the FACSCanto flow cytometer (BD Biosciences, USA) for DNA synthesis and cell cycle status. The percentages of cells in the G0/G1, S and G2/M cell cycle phases were calculated by the ModFit 3.0 program (BD Biosciences).

Total RNA from LA-N-1 cells was extracted by TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. The first-strand cDNA was generated with random primers (Invitrogen) by M-MLV reverse transcription kits (Invitrogen). Quantitative real-time PCR analysis was performed with SYBR premix Ex Taq kit (Takara, China) using ABI-7500 Real-Time PCR System (Applied Biosystems, USA). Relative gene expression was normalized to β-actin level. The sequences of primers used are listed in Table I.

Cell pellets were collected at the indicated time points and total proteins were extracted by cell lysis buffer. Protein concentration was measured by the Bradford reagent (Sigma). Protein extracts were analyzed by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat dairy milk in Tris-buffered saline (20 mM Tris, 150 mM NaCl, pH 7.4) with 0.05% Tween-20 and incubated with mouse anti-human CDK2 antibody (Cell Signaling Technology, USA), rabbit anti-human-ERRα, -ERRγ, -PGC-1α and -PGC-1β antibodies (Cell Signaling Technology), rabbit anti-human cyclin E antibody (Santa Cruz Biotechnology, USA), or mouse anti-human β-actin antibody (Sigma) followed by HRP-conjugated secondary antibody (GE Healthcare UK Ltd., UK) and developed with the ECL reagent (Santa Cruz Biotechnology).

Mitotracker Green (Invitrogen) was added and quantified as described by Liao et al(22). Briefly, I3M-treated cells were incubated in serum-free medium (pre-warmed to 37°C) containing 150 nM Mitotracker Green FM for 20 min in the dark. After staining, the cells were washed twice with cold PBS and suspended in 500 μl PBS. Subsequently, cells were analyzed on a FACSCanto flow cytometer with excitation at 488 nm and emission at 516 nm. Data were processed using the CellQuest program (BD Biosciences).

ROS production in mitochondria was measured by a cell-permeable fluorogenic probe MitoSOX™ Red (Invitrogen) that is selectively targeted to the mitochondria, where it specifically reacts with superoxide anion as previously described (22). Briefly, I3M-treated cells were loaded with 5 μM MitoSOX Red for 10 min at 37°C, washed with PBS, and the fluorescence was detected with the FACSCanto flow cytometer with excitation at 488 nm and emission at 580 nm. Data were processed using the CellQuest program.

Mitochondrial membrane potential (ΔΨ_m) was analyzed by a fluorescent dye JC-1 (Invitrogen). JC-1 is capable of selectively entering mitochondria where it forms monomers and emits green fluorescence when ΔΨ_m is relatively low. At high ΔΨ_m, JC-1 aggregates and gives red fluorescence. Briefly, I3M-treated cells were incubated in serum-free medium (pre-warmed to 37°C) containing 4 μM JC-1 for 20 min in the dark. After staining, the cells were washed once with cold PBS and suspended in 500 μl PBS. Green (525 nm) and red (590 nm) fluorescence were detected on a FACSCanto flow cytometer. Data were processed by using the CellQuest program.

All assays were performed at least three times and the data are presented as the mean ± SD. The Student’s t-test was used to determine the significant difference between the drug-treated group and the control group. P<0.05 is regarded as statistically significant. Asterisks indicate significant differences: ^*P<0.05, ^**P<0.01, ^***P<0.001.

To examine the effect of I3M on the growth of human neuroblastoma cells, the MTT assay, CyQUANT NF cell proliferation assay and colony-forming assay were performed. Three human neuroblastoma cell lines LA-N-1, SH-SY5Y and SK-N-DZ, were exposed to increasing concentrations of I3M for 24, 48 and 72 h and the relative cell number was monitored by MTT assay. As shown in Fig. 2A, I3M dose- and time-dependently reduced the growth of the three cell lines. The 50% inhibitory concentration (IC₅₀) values of I3M on LA-N-1, SH-SY5Y and SK-N-DZ cells after 48-h treatment were 3.27±0.9, 18.15±1.91 and 5.4±0.28 μM respectively, indicating that I3M has the highest potency on LA-N-1 cells. I3M also inhibited the proliferation of LA-N-1, SH-SY5Y and SK-N-DZ cells in a dose-dependent manner after 48-h treatment (Fig. 2B). It has been shown that the colony-forming ability of drug-treated tumor cells is related to their tumorigenicity in vivo(23). I3M was found to decrease the colony-forming ability of LA-N-1 cells dose-dependently (Fig. 2C). A drug with a high therapeutic index indicates that it is potent in inhibiting the proliferation of cancer cells while exhibiting little or minimal cytotoxicity towards the normal cells. In this study, I3M was found to exhibit little, if any, direct cytotoxicity on the normal cells such as rat primary embryonic cortical neuronal cells, human embryonic kidney HEK-293 cells and human hepatocyte-like WRL-68 cells at or below 10 μM concentrations (Fig. 2D).

Since I3M caused a significant inhibition of cell growth in the human neuroblastoma LA-N-1 cells, the underlying mechanisms were investigated. The possible mechanisms of I3M leading to its potent anti-proliferative effect is its ability to induce apoptosis or interrupt the normal cell cycle progression. Initially, the Cell Death Detection ELISA^PLUS kit was used to determine whether I3M could induce apoptosis in the neuroblastoma LA-N-1 cells. I3M failed to induce significant apoptosis of the LA-N-1 cells at concentrations that are inhibitory for the growth of the cells (Fig. 3). Next, we investigated whether the I3M-induced cell growth inhibition was due to an arrest at a specific point of the cell cycle. It was found that at concentrations >2.5 μM, I3M significantly induced cell cycle arrest at the G0/G1 phase, accompanied by a significant decrease in the percentage of cells in the S and G2/M phase (Fig. 4A). The effects of I3M on the expression of the G0/G1 phase regulatory proteins including cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors in LA-N-1 cells were then examined. As shown in Fig. 4B, the I3M-treated cells exhibited an increase in p27^Kip1, and a decrease in the protein levels of CDK2 and cyclin E compared to the corresponding untreated control. Using the technique of real-time polymerase chain reaction, it was shown that the mRNA levels of CDK2 and cyclin E decreased in a dose-dependent manner following treatment with I3M (Fig. 4C and D).

The roles of mitochondrial biogenesis and function in cell growth and proliferation have been investigated. Mitochondrial function is known to play a pivotal role in the maintenance of cellular homeostasis. Previous studies have shown that mitochondrial dysfunction causes cell cycle arrest in neuronal PC12 cells (24). In the present study, whether the I3M-mediated growth suppression of LA-N-1 cells was correlated with mitochondrial biogenesis and function was examined. The changes in mitochondrial mass were measured by staining the cells with a fluorescent MitoTracker Green dye that stains the mitochondria independent of its ΔΨ_m. It was found that I3M dose-dependently reduced the mitochondrial mass (Fig. 5A). Correspondingly, the expression levels of mitochondrial transcription factor A (Tfam) and ATP synthase (ATP5b) that are involved in regulating mitochondrial mass and ATP production, respectively, were significantly reduced by I3M (Fig. 5B and C). It would be of interest to examine if I3M altered mitochondrial function in addition to reducing the mitochondrial mass in the human neuroblastoma LA-N-1 cells. First, the effect of I3M on the ΔΨ_m of LA-N-1 cells was studied. A fluorescent dye JC-1, which gives a red fluorescence when ΔΨ_m is high and green fluorescence when ΔΨ_m is low, was used to determine the overall electron transport chain activity. Indeed, quantification measurements indicated that I3M dose-dependently decreased the red to green fluorescence ratio, indicating a decrease in ΔΨ_m (Fig. 6). Decreased ΔΨ_m inhibits ATP generation, results in oxidative stress and leads to mitochondrial dysfunction. In order to demonstrate that I3M could result in oxidative stress in LA-N-1 cells, the ability of I3M to induce the generation of ROS was examined. Using a fluorescent MitoSOX Red dye, which selectively targets the mitochondria and reacts with superoxide anion, it was found that I3M elevated mitochondrial ROS levels in LA-N-1 cells dose-dependently (Fig. 5D). An endogenous antioxidant system that protects cells against ROS is characterized by superoxide dismutases (SODs). We also checked the mRNA expression levels of SOD1 and SOD2 in I3M-treated cells and found that it selectively suppressed the expression of SOD2, which resides in the mitochondria, but not SOD1 that is found mainly in intracellular cytoplasmic spaces (25) (Fig. 5E and F).

Our previous results showed that I3M suppressed the mRNA expression levels of SOD2, Tfam and ATP5b in LA-N-1 cells. It has been reported that ERRα, ERRγ, PGC-1α, and PGC-1β are responsible for controlling the expression of these genes at the transcriptional level and regulate mitochondrial mass and oxidative phosphorylation (17). Therefore, whether I3M can alter the mRNA and protein levels of these transcriptional factors and co-activators was investigated. Using western blot analysis, the protein levels of ERRα and PGC-1α were not found to be significantly altered by I3M (Fig. 7A). On the contrary, the protein levels of ERRγ and PGC-1β were markedly reduced (Fig. 7A). To determine whether the suppression was at the transcriptional level, the mRNA levels of ERRγ and PGC-1β were determined by quantitative RT-PCR analysis. The mRNA expression levels of ERRγ and PGC-1β were significantly decreased after I3M treatment (Fig. 7B and C).