The SH-SY5Y human neuroblastoma cell line was purchased from Peking Union Medical College (Beijing, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 µg/ml streptomycin and 100 U/ml penicillin (both Solarbio Science & Technology Co., Ltd., Beijing, China). The cells were cultured at 37°C in an humidified atmosphere of 95% air and 5% CO₂. Upon 70% confluency, isatin [in a 5 mM stock solution in 0.1% dimethly sulfoxide (DMSO); Sigma-Aldrich, St. Louis, MO, USA] was added with final concentrations of 100, 200 or 400 µM. Following incubation for 48 h, the cells were harvested and subjected to analysis.

The treated cells were harvested by centrifugation and washed three times with phosphate-buffered saline. The cells were fixed with ice-cold 75% ethanol for 18 h, stained with propidium iodide (Sigma-Aldrich) and then analyzed by flow cytometry (FACSCanto; BD Biosciences, Franklin Lakes, NJ, USA) to detect the cell cycle. A minimum of 10,000 events were analyzed in each experiment, and the results were analyzed using ModFit LT software, version 3.2 (Verity Software House, Inc., Topsham, ME, USA).

The invasive potential of SH-SY5Y cells was examined using Transwell inserts (Corning Inc., Corning, NY, USA). The membranes were coated with Matrigel (BD Biosciences) for 30 min. SH-SY5Y cells were trypsinized (Thermo Fisher Scientific, Inc.), re-suspended in serum-free medium and counted following serum starvation for 12 h. The bottom wells of the Transwell inserts were filled with DMEM containing 10% FBS. Cells (2×10⁵ in 200 µl serum-free medium) were added to the upper compartment of each Transwell insert and incubated for 24 h in the absence or presence of isatin (100 or 200 µM). Cells that failed to migrate through the filter following incubation were removed using a sterile cotton swab, while invaded cells on the lower side of the filter were fixed with methanol (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and stained with 0.1% crystal violet (Solarbio Science & Technology Co., Ltd.). The number of invaded cells in five random fields of the Transwell membrane was counted under a microscope (CKX41; Olympus Corporation, Tokyo, Japan).

An MTT assay was conducted in order to assess the survival of SH-SY5Y cells. Cells (10³ cells/well) were seeded into 96-well plates and isatin was added to a final concentration of 100 µM-400 µM 24 h later. Following incubation for 48 h, the cells were incubated with MTT (1 mg/ml; Sigma-Aldrich) for 3 h at 37°C, after which formazan crystals were dissolved in 100 µl DMSO. The absorbance was measured at 490 nm using a microplate reader (Synergy H1; BioTek Instruments, Inc., Winooski, VT, USA). The suppression rate was calculated using the following formula: Suppression rate = (1−A/C) × 100%, where A and C represent the number of cells treated with or without isatin, respectively. The MTT assay was performed six times.

Cells were seeded into individual wells of a six-well culture plate and grown to confluence. In order to suppress the contribution of cell proliferation, cells were grown in serum-free medium for 12 h; furthermore, cells were treated with mitomycin (10 µg/ml; Bio Basic Canada, Inc., Markham, ON, Canada) for 3 h prior to wounding. A sterile 10-µl pipette tip was then used to perform a longitudinal scratch in the confluent monolayer. The cell debris and medium were removed by aspiration and substituted with 2 ml fresh serum-free medium. Images were captured at 0, 12, 24, 36 and 48 h after wounding (corresponding to 12, 24, 36, 48 and 60 h post-treatment) using an inverted microscope (CKX41; Olympus Corporation). Ten randomly selected points along each wound, which were used to mark the horizontal distance between the initial wound and the migrated cells, was measured. All images were processed using Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).

Total RNA was extracted from SH-SY5Y cells cultured in the presence or absence of isatin using RNAiso Plus (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. Total RNA was converted into cDNA using the Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics, Basel, Switzerland), under the following conditions: 50°C for 1 h followed by 85°C for 5 min. cDNA was subjected to qPCR amplification in triplicate experiments using SYBR Green qPCR Master Mix (Takara Biotechnology Co., Ltd.) in a Real-Time PCR System (LightCycler^® 96; Roche Diagnostics). The qPCR amplification conditions were as follows: 95°C for 30 sec, followed by 45 cycles of 95°C for 5 sec, 60°C for 20 sec and 72°C for 30 sec. Quantification relative to GAPDH was performed using the 2^−ΔΔCq method (14). Real-time PCR was performed using the following primers (Shanghai Sunny Biotech Co., Ltd., Shanghai, China): GAPDH forward, 5′-AACAGCCTCAAGATCATCAGCAA-3′ and reverse, 5′-GACTGTGGTCATGAGTCCTTCCA-3′; MMP2 forward, 5′-TTCCCTCGCAAGCCCAAGTG-3′ and reverse, 5′-CTCCCAGCGGCCAAAGTTGA-3′; MMP9 forward, 5′-GCTGACTCGACGGTGATGGG-3′ and reverse, 5′-GCCCCACTTCTTGTCGCTGT-3′.

SH-SY5Y cells were harvested and treated with isatin for 48 h. The cells were lysed in radioimmunoprecipitation assay buffer (Solarbio Science & Technology Co., Ltd.) for 20 min on ice. The homogenate was centrifuged for 5 min at 13,200 × g and the protein concentration in the supernatant was quantified using the bicinchoninic acid protein assay (Beyotime Institute of Biotechnology, Inc., Shanghai, China). Following storage at −80°C, 30 µg protein from each sample was separated by 0.1% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sigma-Aldrich) and electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were individually blocked with 5% bovine serum albumin (Amresco, LLC, Solon, OH, USA) and then incubated with mouse anti-β-actin monoclonal antibody (1:1,000; TA-09; Zhongshan Golden Bridge Biotechnology, Beijing, China), rabbit anti-cyclin D1 monoclonal antibody (1:1,000; 2978; Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-matrix metalloproteinase (MMP)2 polyclonal antibody (1:1,000; 4022; Cell Signaling Technology), rabbit anti-MMP9 monoclonal antibody (1:3,000; ab76003; Abcam, Cambridge, UK) and rabbit anti-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) monoclonal antibody (1:2,000; Tyr705; Cell Signaling Technology) for 2 h at room temperature, followed by further incubation at 4°C overnight. The blots were washed three times for 10 min each in Tris-buffered saline (Sangon Biotech Co., Ltd., Shanghai, China) containing 0.1% Tween 20 (TBST; Bio Basic Canada, Inc.) and then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit (1:2,000; BA1054; Boster Biological Technology, Ltd., Wuhan, China) and goat anti-mouse (1:5,000; ZB-2305; Zhongshan Golden Bridge Biotechnology) monoclonal antibodies for 1 h at room temperature. Following three washes in TBST for 10 min each, proteins were detected using an Enhanced Chemiluminescence Plus kit (Cyanagen, Bologna, Italy) by a chemiluminescence system (Fusion FX7; Vilber Lourmat, Collégien, France). Densitometric analysis was performed with Quantity One software, version 4.6.2 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Each experiment was performed at least three times. Values are expressed as the mean ± standard deviation. Statistical analysis included one-way analysis of variance, which was performed using SPSS software, version 20.0 (IBM SPSS, Armonk, NY, USA). When the differences between average levels among several groups were statistically significant, the Bonferroni multiple-comparisons test was performed. P<0.05 was considered to indicate as statistically significant difference.

Following administration of isatin for 48 h, cell cycle analysis was performed by flow cytometry. The results revealed that isatin significantly increased the proportion of cells in G₁ phase (P<0.01) and significantly decreased the proportion of cells in S phase (P<0.01), while the proportion of cells in the G₂/M phase was not altered (Table I). Compared with the 100 and 200 µM groups, treatment with 400 µM isatin resulted in a significantly higher increase in G₁-phase and decrease in S-phase populations (P<0.01). These results suggested that isatin significantly caused G₁-phase arrest in SH-SY5Y cells.

As cyclin D1 activation regulates the transcription of genes associated with cell proliferation (15), the present study investigated the impact of isatin on the expression of cyclin D1. As shown in Fig. 1, isatin significantly reduced the protein expression of cyclin D1 compared with that in the control group (P<0.01). Furthermore, cyclin D1 expresion in the 400 µM isatin group was significantly decreased compared with that in the 100 and 200 µM groups (P<0.01).

The impact of isatin on the invasion and migration of neuroblastoma cells was assessed using in vitro Transwell and wound-healing assays. As illustrated in Fig. 2A and B, 200 µM isatin significantly restrained the invasiveness of SH-SY5Y cells (P<0.01). Furthermore 200 µM isatin reduced the migratory ability of these cells after 36 h (P=0.046) and 48 h of incubation (P=0.035) (Fig. 2C and D). These results suggested that isatin reduces the invasion and migration of the SH-SY5Y human neuroblastoma cell line.

The effect of isatin on the proliferation of SH-SY5Y cells was investigated using an MTT assay. As is shown in Table II, isatin significantly inhibited the proliferation of SH-SY5Y cells (P<0.01), as compared with the control. In addition, the OD₄₉₀ and suppression rate of SH-SY5Y cells were significantly decreased (P<0.05). Notably, the 400 µM isatin group resulted in a more significant decrease, as compared with the 100 µM group (P<0.05).

As it is known that MMP-2 and MMP-9 expression is relevant to metastasis and progression of neuroblastoma (16), the present study assessed the impact of isatin on MMP2 and MMP9 mRNA and protein expression in SH-SY5Y cells. As shown in Fig. 3A and B, 100 µM isatin significantly reduced the mRNA expression of MMP2 (P=0.010) and MMP9 (P=0.040); however, at this concentration, isatin did not significantly affect the protein expression of these MMPs (P=0.520 and P=0.661) (Fig. 3C–E). Of note, at 200 and 400 µM, isatin significantly inhibited the mRNA and protein expression of MMP2 and MMP9 compared with that in the control (P<0.01).

As pSTAT3 has been suggested to be the active form of STAT3 (17), the present study determined the protein levels of pSTAT3 (Tyr705) by western blot analysis. As shown in Fig. 4, the phosphorylation of STAT3 in the 100-µM group was not markedly affected, while 200 µM isatin significantly inhibited the phosphorylation of STAT3 (P<0.05), and 400 µM isatin further diminished the activation of STAT3 (P<0.01).