SH-SY5Y neuroblastoma cells were purchased from American Type Culture Collection, and STR profiling was conducted. The cells cultured in high-glucose DMEM containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 µg/ml penicillin/streptomycin under 5% CO₂ at 37°C. The cells were passaged once they were 80-90% confluent, and logarithmic growth phase cultures were harvested for further analysis.

The lentiviral construct with the Luc (GV260) tag was synthesized by (Shanghai GenePharma Co., Ltd. After 48 h of cell transfection, the 70-80% confluent cells were infected with the virus at the MOI of 30. The stably transduced cells were screened 48 h later using puromycin, and tested for Luc tag using D-luciferin.

RNA was isolated from the isatin-treated cells using the TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions, and analyzed using spectro-photometry (BioTek Instruments, Inc.). Following reverse transcription (using a Prime-Script qPCR kit; TransGen Biotech Co., Ltd.), the cDNA was amplified using TransStart SYBR Probe qPCR SperMix (TransGen Biotech Co., Ltd.) on a Bio-Rad One-Step Plus system (Bio-Rad Laboratories, Inc.). The temperature protocol for reverse transcription was 42°C for 15min and 85°C for 5 sec. The thermocycling conditions for qPCR were 94°C for 30 sec, 94°C for 5 sec and 60°C for 30 sec, for 45 cycles. Primer sequences for GAPDH, lysine-specific histone demethylase (LSD)1 and p53 are provided in (Table SI). Relative gene expression levels were calculated using the 2^−∆∆Cq method (21).

Cells in the logarithmic growth phase were washed twice with PBS, harvested using trypsin, and seeded in a 96-well plate at the density of 3,000 cells/100 µl/well. After treating with different concentrations (0, 25, 50, 100, 200, 300, 400, 500 and 800 µmol/l) of isatin for 3 days at 37°C, the cells were washed and incubated at 37°C for 1 h with 10 µl CCK8 according to the manufacturer's protocols (Beijing Solarbio Science & Technology Co., Ltd.). Absorbance was measured at 450 nm using a microplate reader (Synergy H1; BioTek Instruments, Inc). Each experiment was performed three times.

The cells treated with isatin were seeded in a six-well plate at a density of 100 cells per well and cultured for 2 weeks. The ensuing colonies were fixed with 4% paraformaldehyde, stained with crystal violet (both at room temperature), air dried and counted manually under a light microscope at ×200.

Cells were seeded in six-well plates at the density of 3×10⁵ cells per well, and cultured until 80% confluent. The cells were harvested and stained using the FITC/PI Annexin V Apoptosis Detection kit I (BD Pharmingen; BD Biosciences) to detect the level of apoptosis using a Accuri C6 flow cytometer and analyzed with the corresponding software (BD Biosciences). The percentage of early apoptotic cells was analyzed using flow cytometry within 1 h of staining.

The cells treated with isatin were seeded in a 6-well plate and cultured until a uniform monolayer had formed. The layer was scratched with a sterile P200 pipette tip, and the wells were rinsed with the aforementioned medium to remove all cellular debris. Low-serum DMEM with mitomycin (2 µg/ml) was then added to inhibit cell proliferation. Images (Nikon, TI-SM) of the wound area were captured using a TI-SM light microscope (magnification, ×100 or ×200) at 0, 24 and 48 h post scratching, and the migration rate (%) was calculated as the percentage of area covered by migrated cells divided by the total wound area.

Precoating with Matrigel was conducted at 37°C for 4 h. The upper compartment of Transwell inserts were seeded with cells in serum-free medium, and the lower chambers were filled with 600 µl complete medium with 30% FBS. After 12 to 24 h of incubation, the inserts were removed, and the cells remaining on the filter surface were swabbed. The cells that had migrated/invaded to the other side the filter were fixed with paraformaldehyde for 5 min at room temperature), stained with crystal violet and images were captured using a light microscope (magnification, ×100).

The cells treated with isatin were lysed in RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with a protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA) on ice for 30 min, and centrifuged for 20 min at 4°C and 8,000 × g. The protein concentration in the cleared lysate was measured using a BCA assay (Beyotime Institute of Biotechnology). In total, 20 ng protein were loaded per lane onto a 10% gel, resolved using SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% BSA in TBST for 2 h at room temperature, the membranes were incubated overnight with the primary antibodies (all 1:1,000; Table I) at 4°C. The blots were washed thrice with TBST, incubated with HRP-conjugated secondary antibody (1:2,000; Wuhan Boster Biological Technology, Ltd.), and washed again with TBST. The protein bands were detected with an ECL detection system (Beijing Transgen Biotech Co., Ltd.). The Fusion FX7 luminescence imaging system (Vilber) was used to visualize and analyze protein bands.

The cells treated with isatin were lysed (1 ml RIPA buffer) at 4°C for 30 min, and the lysates were incubated overnight with anti-LSD1 antibody (1:500; Abcam). After adding 40 µl A/G sepharose (CST, 70024S), the lysates were incubated for 2 h at 4°C with constant agitation. Cells were centrifuged at 2,000 × g for 5 min at 4°C and the supernatant was discarded. Cells were washed six times with 500 µl wash buffer (50 Mm Tris-HCl, 150 mM NaCl, 1% Triton and 1% PMSF), then centrifuged at 4,000 × g at 4°C for 5 min and the resulting supernatant was discarded. The immunoprecipitates were separated using SDS-PAGE as aforementioned after washing with the same buffer, and analyzed by immunoblotting with the suitable antibodies.

After cells were transfected with luc-labelled lentivirus for 48 h, cells in the logarithmic growth phase were plated into a 96-well plate with 2,000 cells/well. After 24 h, the original culture medium was removed and D-luciferin sodium working solution was added (150 µg/ml) with 100 µl per well. D-fluorescein sodium working solution was diluted to 1:200 using D-fluorescein sodium stock solution (30 mg/ml) and pre-warmed culture medium. Within 10-20 min, a microplate reader was used to detect luminescence.

In total, 20 4-week old female athymic nude mice (14-15 g) were purchased from the Vital River Laboratory Animal Technology Co. Ltd. The mice were anesthetized via inhalation of 2% isoflurane, and inoculated with 1×10⁶ tumor cells in 100 µl medium between the second and third ribs. The inoculated mice were placed in a 37°C incubator until they became fully awake. On day 3 post-injection, the mice were randomly divided into the (A) control, (B) cyclophosphamide (CTX), (C) isatin (ISA) and (D) combination (D) groups (n=5 each), and treated with 5 ml/kg 1.25% Gummi Tragacanthae, 40 mg/kg CTX, 200 mg/kg ISA and 20 mg/kg CTX + 200 mg/kg ISA, respectively. The drugs were administered daily via the intragastric route for 4 weeks. Tumor formation and metastasis were observed using a live imager. All experimental procedures were performed in compliance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (22), and were approved by The Animal Ethics Committee of Qingdao University [approval no. QDU20190506b0200610(031)].

Weights of the mice were checked every three days, and fur gloss and behavior were also examined. After 3 days of modeling, the drug was continuously administered for 4 weeks, and mice were imaged in vivo. According to the results, it was found that the tumor metastasis of the mice in the control group had reached the standard, so the mice were sacrificed 4 weeks after drug treatment, and the experiment ended. Animals were euthanized with an overdose of 2% pentobarbital sodium (100 mg/kg) followed by cervical dislocation.

The following ELISA kits were used to detect specific markers in mouse serum samples: Mouse Matrix metalloproteinase (MMP)2 (cat. no. ZN2705) mouse vascular endothelial growth factor (cat. no. ZN2803), mouse MMP 9 (cat. no. ZN2808), superoxide dismutase (SOD; cat. no. A001-1), glutathione peroxidase (GSH-PX; cat. no. A005), malondialdehyde (MDA; cat. no. A003-1), urea nitrogen (BUN; cat. no. C013-2), creatinine (CREA; cat. no. C011-1), bilirubin (cat. no. C019-1-1), alkaline phosphatase (AKP; cat. no. A059-1) and γ-GT (cat. no. C017) (all Nanjing Jiancheng Bioengineering Institute). All operations are performed according to the kit instructions.

Molecular docking was performed using Glide (Schrodinger Inc) with LSD1 crystal structure (PDB Entry: 4KUM) as the receptor. Suitable structural modifications were made, and the water molecules and the crystallized ligand in the protein structure were removed. The refined structure was simulated in an OPLS 2005 force field, and visualized using Maestro in Schrodinger (Schrodinger Suite 2009). The active site was defined as a cubic box containing residues around ligand FAD901 at a distance of 20 Å. Other parameters were set at default levels.

The data were obtained from at least three independent experiments and analyzed using SPSS 17.0 (SPSS, Inc) for Windows. All data are expressed as mean ± SD or SEM. ANOVA followed by Tukey's post hoc test was performed to comparing differences between means in multiple groups. P<0.05 was considered to indicate a statistically significant difference.

SH-SY5Y cells treated with 25, 50, 100, 200, 300, 400, 500 and 800 µM isatin for 48 and 72 h showed a marked decrease in proliferation rates (Fig. 1 and Table SII). Concentrations below 200 µM did not show any significant effect on cellular morphology, whereas higher doses resulted in aberrant morphological changes, obvious shrinkage and the number of viable cells. Consistent with this, isatin also inhibited clonal expansion of the SH-SY5Y cells in a concentration-dependent manner (Fig. 2). Compared with the untreated control, the number of colonies decreased significantly following treatment with isatin (P<0.01) (Fig. 2). Similarly, apoptosis rates raised obviously following treatment with 100 and 200 µM isatin (P<0.01). Taken together, these data demonstrated that isatin inhibited the proliferation of neuroblastoma cells.

The effect of isatin on tumor cell migration and invasion was assessed using wound healing and Transwell assays. The rate of wound healing was significantly slower in the SH-SY5Y cells treated with isatin compared with the untreated cells. At 48 h, wound coverage was almost complete in the control group but a noticeable gap remained in the all isatin-treated groups (P<0.01; Fig. 3A and B). Furthermore, isatin also significantly decreased the invasion capacity of the SH-SY5Y cells through the Matrigel-coated Transwell insert (Fig. 3C and D) by 35.22±4.21, 42.14±1.58 and 83.42±3.67% at 200 µM (P<0.01), the same level of significance was also observed at 50 and 100 µM (P<0.01).

LSD1 is an oncogene that inhibits the tumor suppressor p53 by demethylating the lysine 370 residue. Consistent with this, LSD1 is overexpressed in neuroblastoma tissues and cell lines, and is associated with the grade of tumor malignancy (17). Isatin significantly decreased the expression of LSD1 mRNA (P<0.01, 100 and 200 µmol/l vs. control) and protein (P<0.05, 50 µmol/l vs. control; P<0.01, 100 and 200 µmol/l vs. control), and increased that of p53 mRNA (P<0.05, 50 µmol/l vs. control; P<0.01, 100 and 200 µmol/l vs. control) and protein (P<0.01 vs. control) (Fig. 4A-E). In addition, isatin also upregulated the levels of p53Me2 (P<0.05, 50 µmol/l vs. control; P<0.01, 100 and 200 µmol/l vs. control) (Fig. 4F and G). Given that LSD1 specifically demethylates histone H3K4 and transcriptionally inhibits the target genes (23), the levels of H3K4Me1 and H3K4Me2 were also analyzed, demonstrating that isatin treatment significantly upregulated H3k4Me1 (P<0.01, 100 and 200 µmol/l vs. control) and H3K4Me2 (P<0.01, 100 and 200 µmol/l vs. control) (Fig. 4H-J). Furthermore, the down-stream pro-apoptotic protein p21 was significantly increased by isatin (P<0.01 vs. control) (Fig. 4H and K) while the p53 destabilizing MDM2 (P<0.05, 50 µmol/l vs. control; P<0.01, 100 and 200 µmol/l vs. control) and antiapoptotic Bcl-2 were downregulated (P<0.05 vs control) (Fig. 4L, M, O, P and R). TGFβ1 may activate LSD1 via the ERK/NF-κB pathway (23). Consistent with the aforementioned results, isatin not only decreased the levels of TGFβ1 (P<0.05, 50 µmol/l vs. control; P<0.01, 100 and 200 µmol/l vs. control) protein in neuroblastoma cells (Fig. 4Q and S) but also inhibited the co-precipitation of LSD1 and phosphorylated (p-)NF-κB and decreased the expression level of p-NF-κB (Fig. 4T). Taken together, these data demonstrated that isatin inhibits the expression of TGFβ1/NF-κB/LSD1.

The Luc-SH-SY5Y cells were injected in nude mice to establish an in vivo neuroblastoma model, and metastasis was tracked using real-time fluorescence imaging. Luc labeling has no direct effect on cell proliferation (Fig. S1). As shown in Fig. 5, the untreated tumor-bearing mice showed strong fluorescence signals in the cervical vertebrae, spine, scapula, pelvic bone and extremities of long bones, indicating that distant metastasis of the tumor cells had occurred. The fluorescence intensities decreased significantly in the ISA and CTX-treated groups, with a more substantial inhibition in the latter (P<0.01), and were weakest in the CTX+ISA group and limited the brain, spine and pelvis. The CTX dose in the combination regimen was half of that in the monotherapy group, which indicated a synergistic effect of combining CTX and ISA. The nude mice were weighed once every 2 days. No significant changes were found in the body weight of the nude mice in each group (Fig. S2).

A statistical analysis on the fluorescence signal intensity of bone metastasis in each group was made, as shown in Fig. 6A. The results showed that compared with the model group, the fluorescence intensity of bone metastasis in the other three groups was significantly decreased (P<0.01 vs. control; Fig. 6B). Compared with the ISA group, the fluorescence intensity of bone metastases in the CTX group and the combination group was reduced, but there was no statistical difference. There was no significant difference between CTX group and combination group.

The intensity of the transfer fluorescence signal in the main organs of each group of nude mice was analyzed, as shown in Fig. 6C. The results showed that compared with the model group, the fluorescence intensity of the main organs (heart, liver, lung, kidney and spleen) in the other three groups were significantly decreased (P<0.01; Fig. 6D). Compared with the ISA group, the fluorescence intensity of the main organs in the CTX group and the combination group were significantly decreased (P<0.01; Fig. 6D). There was no significant difference between CTX group and combination group. However, no visible solid tumor tissues were found in any of the mice.

The levels of the angiogenic VEGF, and pro-metastatic MMP2 and MMP9 in the sera of the differentially-treated tumor-bearing mice were measured. The expression of VEGF is positively correlated with tumor microvessel density. It can accelerate tumor invasion and early metastasis by promoting tumor angiogenesis to meet the nutrient and oxygen supply needed by rapid growth tumor (24). VEGF levels were significantly decreased in the ISA and ISA+CTX groups compared with the model (P<0.01) and CTX groups (P<0.05) (Fig. 7A). MMPs aid tumor cell invasion and metastasis by degrading the extracellular matrix (25), MMP2 decreased significantly in ISA and CTX+ISA group (P<0.01, vs. control or CTX), simultaneously, MMP9 has obviously decreased in ISA and CTX+ISA group (P<0.01, vs. control, P<0.05, vs. CTX). Taken together, these data suggested that isatin might inhibit tumor invasion, metastasis and angiogenesis by targeting the MMPs and VEGF.

The potential adverse effects of ISA were assessed in terms of oxidative stress, renal function and liver function. The results of SOD activity showed that the CTX group was significantly lower compared with the other three groups (P<0.05 CTX+ISA vs. CTX, P<0.01 ISA vs. CTX). There was no significant difference in SOD activity between the model, CTX and CTX+ISA groups (Fig. 8A). The activity of GSH-PX enzyme in CTX+ISA group was significantly higher compared with control (P<0.01), compared with CTX group, GSH-PX activity was higher in ISA and CTX+ISA group (P<0.01) (Fig. 8B). The serum MDA content of the nude mice in CTX group was significantly higher compared with other groups (P<0.01; Fig. 8C).

The BUN content of the CTX group was significantly higher compared with the other three groups (P<0.01 vs. control and CTX+ISA groups, P<0.05 vs. ISA group; Fig. 8D). There was no significant difference in BUN content between model and CTX+ISA group (Fig. 8D). The results showed that the concentration of creatinine (CREA) in the CTX group was dignificantly higher compared with other group (P<0.05, vs. control or ISA, P<0.01 vs. CTX+ISA; Fig. 8E). In addition, the concentration of serum bilirubin in the CTX group was significantly higher compared with that in the model group (P<0.05), and total bilirubin in the ISA and CTX+ISA groups was significantly lower compared with that in the CTX group (P<0.05 and P<0.01, respectively; Fig. 8F). Serum AKP results showed that the AKP activity in the control, ISA and CT+ISA groups was significantly lower compared with that in the CTX group (P<0.05 vs. control or ISA, P<0.01, vs.CTX+ISA; Fig. 8G). In addition, the γ-GT activity of the CTX group was also significantly higher compared with other groups (P<0.05 vs. ISA, P<0.01 vs. control or CTX+ISA; Fig. 8H).

Since isatin is a monoamine oxidase inhibitor and LSD1 is a monoamine oxidase [Triazole-dithiocarbamate based selective lysine specific demethylase 1 (LSD1) inactivators inhibit gastric cancer cell growth, invasion, and migration, 10.1021/jm401002r], the binding ability was predicted using molecular docking of isatin to the LSD1 crystal structure. As shown in Fig. 9, isatin bound to the surrounding amino acids (colored green) in LSD1 protein via hydrophobic interactions. In addition, electrostatic interactions were also seen at the ligand-receptor binding site (purple amino acids of LSD1). Finally, hydrogen bonds were formed between two carbonyl groups of isatin and the NH of Glu801 and OH of Ser289. It was hypothesized that isatin can bind to LSD1 with high affinity and inhibit its function.