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Isatin Inhibits the Invasion of Human Neuroblastoma SH‑SY5Y Cells, Based on Microarray Analysis

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Isatin Inhibits the Invasion of Human Neuroblastoma SH‑SY5Y Cells, Based on Microarray Analysis 1700 MOLECULAR MEDICINE REPORTS 20: 1700-1706, 2019 Isatin inhibits the invasion of human neuroblastoma SH‑SY5Y cells, based on microarray analysis LI ZHANG1,2*, WENYAN SUN1,3*, YI CAO1, LIN HOU1, CHUANXIA JU2 and XUEFENG WANG3 1Department of Biochemistry, Medical College; 2Experimental Center for Undergraduates of Pharmacy, School of Pharmacy; 3Institute of Metabolic Diseases, Qingdao University, Qingdao, Shandong 266021, P.R. China Received March 22, 2018; Accepted April 24, 2019 DOI: 10.3892/mmr.2019.10378 Abstract. Neuroblastoma is the fourth most common type of treatment for high-risk NB. New therapeutic approaches need extracranial malignant solid tumor in children. Isatin had been to be developed (2). demonstrated to have inhibitory effects on neuroblastoma Clinical trial results have demonstrated that multi-target tumors in vivo and in vitro. The aim of the present study was to inhibitor drugs are more effective compared with single-target investigate the molecular mechanism related to the anti-inva- drugs during cancer treatment (5). 1H-indole-2, 3-dione sion effect of isatin on SH-SY5Y cells using microarray (isatin) is a derivative of the anti-cancer drug indirubin, which analysis. The microarray data identified a number of genes exhibits beneficial biological activities, including antibacterial, to be differentially upregulated or downregulated between antifungal and antitumor properties (6-8). Isatin was initially isatin-treated cells and untreated controls. A large number of considered to be an inhibitor of monoamine oxidase-b and has these genes were associated with the mTOR signaling pathway. been used to treat Parkinson's disease (9,10). Recent studies The differentially expressed genes involved in the mTOR have reported that isatin inhibits cell proliferation and invasion signaling pathway were verified further, as well as their down- in human neuroblastoma SH-SY5Y cells (11,12). stream genes associated with autophagy. The results of the mTOR has been identified as a key molecule in tumori- present study provided an insight into the potential inhibitory genesis and cancer progression (13). Increasing evidence mechanism of isatin on neuroblastoma metastasis. has identified the mTOR pathway as a relevant target for the suppression of tumorigenesis; thus, inhibition of mTOR Introduction may be a promising method for targeting human malignan- cies (14). The aim of the present study was to investigate the Neuroblastoma (NB) is the most common extracranial solid underlying molecular mechanisms of the inhibitory effect of cancer in childhood and infancy, with a mortality rate of 15%, isatin on migration and invasion in SH-SY5Y cells, which are and an incidence between 5.9 and 10.5 per million children associated with the mTOR pathway. under 15 years of age (1). The majority of NB cases are meta- static, and thus are associated with a poor prognosis and a Materials and methods high mortality rate (2). A total of 20-50% of high-risk cases do not respond adequately to high-dose chemotherapy and Cell culture. Human neuroblastoma SH-SY5Y cells (human are progressive or refractory (3). Novel treatments using new origin) were purchased from Peking Union Medical agents and combinations against NB are available in phase I College. The cell line had been STR authenticated (ATCC or II clinical trials, but the outcomes remain poor (4). Single no. CRL‑2266). Cells were cultured in DMEM (HyClone; GE targeted agents are unlikely to be sufficient for long-term Healthcare Life Sciences) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C in a humidified atmosphere with 5% CO2. Isatin was dissolved in 0.1% dimethyl sulfoxide and added to 6-well plates at concen- trations of 50, 100, 200 and 300 µM, which were determined based on our previous study (11), when the cells had reached Correspondence to: Professor Lin Hou, Department of ~70% confluence. The cells (~2x106/well) were harvested Biochemistry, Medical College, Qingdao University, 38 Dengzhou following isatin treatment at 37˚C for 48 h for further analysis. Road, Qingdao, Shandong 266021, P.R. China E‑mail: [email protected] Cell migration and invasion assay. Cells (1x106) were seeded *Contributed equally in 6-well culture plates and exposed to isatin (200 µM) with or without rapamycin (10 µM) at 37˚C for 48 h. The concentration Key words: isatin, neuroblastoma, invasion, mTOR signaling of isatin was determined according to previous results, which pathway demonstrated that 200 µM isatin did not induce necrosis of SH-SY5Y cells (11). A wound healing assay was performed to monitor cell invasion, as previously described (11). For the ZHANG et al: IsATIN INHIBIts THE INVASION OF HUMAN NEUROBLAstOMA SH-SY5Y CELLS 1701 Transwell invasion assay, isatin-treated cells were seeded in expressed as fold change compared with control. The 2-ΔΔCq Boyden chambers (EMD Millipore) with and without Matrigel, method was employed to analyze the relative expression of and the assay was performed as previously described (11). genes (22). Cells were counted in at least five fields under an inverted light microscope (magnification, x10/x20). All experiments were Protein extraction and western blot analysis. Total protein performed in triplicate. was extracted from SH-SY5Y cells using RIPA lysis buffer (Beyotime Institute of Biotechnology) following isatin treat- Microarray analysis. Microarray expression analysis was ment for 48 h , a n d t h e p r o t e i n c o n c e n t r a t i o n w a s q u a n t i fi e d u s i ng performed using total RNA from SH-SY5Y cells that were a bicinchoninic acid protein assay. Proteins (20-40 µg/lane) incubated at 37˚C with or without 200 µM isatin for 48 h. were separated on SDS-polyacrylamide gels (8-12%) and Total RNA was extracted using TRIzol® (Beyotime Institute transferred onto PVDF membranes. Membranes were blocked of Biotechnology). An Affymetrix Microarray kit (Thermo with 5% bovine serum albumin for 2 h at room temperature, Fisher Scientific, Inc.) was used for the gene expression and then incubated with the following primary antibodies analysis. Raw data intensities were quantile-normalized, and diluted in 5% BSA: β-actin (1:3,000; cat. no. ab8227; Abcam), genes exhibiting significantly higher intensities compared AMPK (1:500; cat. no. ab131512; Abcam), phosphorylated with the background and a fold-change (FC)>1.5 following (p)‑AMPK (1:500; cat. no. ab131357; Abcam), mTOR (1:300; isatin treatment, compared with the control, were selected. cat. no. ab32028; Abcam), p-mTOR (1:5,000; cat. no. ab109268; This selection uncovered a total of 429 genes. Expression Abcam), microtubule associated protein 1 light chain 3α values of the genes were rescaled to mean 0 and SD 1, and (LC3; 1:1,000; cat. no. 3868; Cell Signaling Technology), hierarchical clustering was performed using Ward's algo- Beclin-1 (1:1,000; cat. no. 3738; Cell Signaling Technology) rithm (15). The number of clusters was determined using the and p62 (1:1,000; cat. no. 5114; Cell Signaling Technology) Akaike Information Criterion (16). A Mann-Whitney test was at 4˚C overnight, washed with TBS with Tween‑20, and subse- performed to discriminate clusters of genes with differential quently incubated with horseradish peroxidase-conjugated expression levels between the two groups. Differentially goat anti-rabbit immunoglobulin G secondary antibodies expressed genes were selected based on FC in average gene (1:1,000; cat. no. HS101-01; Beijing Transgen Biotech Co., expression with P<0.05, as determined by Student's t-test. Ltd.) for 1 h at room temperature. Protein expression levels Gene Ontology (GO) enrichment (‘biological process’, were detected using the Enhanced Chemiluminescence Plus ‘cellular component’ and ‘molecular function’) (17,18) and kit (Wuhan Boster Biological Technology, Ltd.) and densito- Kyoto Encyclopedia of Genes and Genomes (KEGG) path- metric analysis was performed using Quantity One analysis ways (19-21) of differentially expressed genes were analyzed software (version 4.52; Bio-Rad Laboratories, Inc.). using the Database for Annotation, Visualization and Integrated Discovery Bioinformatics Resources, version 6.7 Statistical analysis. Each experiment was performed at least (http://david.abcc.ncifcrf.gov). Subsequent selection of three times. Data are expressed as the mean ± SD. Multiple enriched GO terms was performed based on the calculated comparisons were performed by one-way ANOVA followed q-values using the threshold q<0.05. by a least significant difference or Tamhane's T2 post hoc test in SPSS 22.0 (IBM Corp.). P<0.05 was considered to indicate Reverse transcription‑qPCR (RT‑qPCR). Total RNA a statistically significant difference. of SH-SY5Y cells was extracted using TRIzol and was reverse transcribed to cDNA using TransScript™ Reverse Results Transcriptase (Beijing Transgen Biotech Co., Ltd.) at 42˚C for 15 min and 85˚C for 5 sec. Then, qPCR was performed Isatin treatment affects gene expression in SH‑SY5Y cells. To independently three times using SYBR Green mix (Takara investigate the underlying molecular events of the anti-inva- Bio, Inc.). Gene expression was analyzed using the following sive activity of isatin in SH-SY5Y cells, RNA was extracted primers: β-actin forward, 5'-GCG TGA​CAT TAA​GGA​ from SH-SY5Y cells (untreated or treated with 200 µM isatin CAA​GC‑3' and reverse, 5'‑CCA​CGT​CAC​ACT TCA​TGA​ for 48 h) for Affymetrix cDNA microarray analysis. The TGG-3'; mTOR forward, 5'‑CTG GGA​CTC​AAA​TGT GTG GeneChip results revealed 284 differentially upregulated and CAG TTC‑3' and reverse, 5'-GAA​CAA​TAG GGT GAA​TGA​ 145 downregulated genes between cells treated with isatin and TCC​GGG-3'; ribosomal protein S6 kinase B1 (RPS6KB1) controls (FC>1.5; Fig. 1A). GO term analysis demonstrated forward, 5'-GGT GGA​GTT TGG GAG​CAT TA‑3' and reverse, that the differentially expressed genes were involved in redox 5'-TGT GAG GTA​GGG AGG​CAA​AT-3'; eukaryotic transla- activity, binding and transcription function, cell metabolism tion initiation factor 4E‑binding protein 1 (EIF4EBP1) and transport (data not shown).
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