Isatin inhibits LSD1 regulated autophagy through SESN2-mTOR signaling pathway in neuroblastoma

: Neuroblastoma (NB) is common in the pediatric tumors with low cure rate and high mortality, drug resistance makes chemotherapy more difficult, it threaten to children's health seriously. 1H-indole-2, 3-dione (isatin) is a second-hand of derivative of anticarcinogen indirubin which had been shown to be a monoamine oxidase inhibitor and have antitumor activity, in this study, we explained that SH-SY5Y cells was add to isatin at a dose-dependent level ranging from 50 to 200 µmol/L were capable of inhibiting tumor cell growth, included triggering apoptosis and inhibiting proliferation, invasion and metastasis, and assessed the potential anticancer capability of isatin on the induction of autophagy. The experimental result showed that cell SH-SY5Y disposed with isatin could induce autophagy effectively, at the same time, it also help suppress the growth of tumor cells. Furthermore, the article indicated that the induction of autophagy of isatin-mediated occurred in inhibition-lysine-specific demethylase 1(LSD1) and sestrin2 (SESN2) dependent dependent manner. Furthermore, we identified that SH-SY5Y cells after treating with isatin induced SESN2 expression via a mTOR-dependent signal pathway, which mechanism is that isatin inhibit LSD1-enzyme activation and combine the promoter regions of SESN2, so that given rise to a significant transcriptional induction of SESN2. In addition, western blot analysis indicated that SH-SY5Y cells with isatin-exposed can regulated activity of LC-3, Beclin1 and p62 which are correlated with autophagy. Collectively, all the results from this study illistrated that adding to isatin could occur to induce autophagy and restrain NB cell SH-SY5Y growth through mTOR-dependent transcriptional induction of SESN2, this supplied a new mechanism for understanding the anti-tumor ability of isatin on NB. Taken together, this paper demonstrate that isatin is a promising candidate for treating NB. treatment with 0 µmol/L, 50 µmol/L, 100 µmol/L, 200 µmol/L isatin, the invasion of cells were obviously inhibited. b SH-SY5Y cells were treatment with 0 µmol/L, 50 µmol/L, 100 µmol/L, 200 µmol/L isatin, the migration of cells was obviously inhibited.

deterioate and metastasize, and it appear to resistance to antitumor drug therapy [3]. At present, there have recurrence and poor prognosis after therapy in many high-risk NB [4]. Hence it is importance to investigate a new drug to therapy NB. With the application of natural antineoplastic drugs in clinical, the antitumor function of the indole compounds increasingly aroused people's concern. Indoline-2,3-dione or indole-1H-2,3-dione(isatin), is a wellknown natural product which found in plants of genus isatis and in couropita guianancis aubl [5]. The first extraction of isatin was as an oxidation product of indigo, and Kekule proposed its its structure, and it was isolated as a metabolic derivative of adrenaline in humans too [5,6]. In recent years, isatin has received more and more attention from researchers. From previous research reports, isatin has the advantages of small molecular weight and low toxicity, as a derivative of the anticarcinogen indirubin, isatin has many beneficial biological activities, including antifungal, antibacterial anticancer capabilities [7,8]. From what has been discussed above, isatin had its characteristics and advantage which can be potential to exploit as a natural small molecule compounds anti-tumor drug.
Previous studies have shown that isatin is an endogenous indole which can suppress monoamine oxidase B (MAOB) and can cause the death of SH-SY5Y cell in a method of dose-dependent and time-dependent [9,10].
However, the mechanism involved anti-tumor activity of isatin is incompletely understood.
The occurrence and development of NB is related with the expression of protein and regulation of signaling pathways. The report suggested that the monoamine oxidase (MAO) expression was likely to relate to the neoplasia, after depriving growth factor, in various neuronal cells there occur to apoptotic cell death atfter the activity of MAO [11,12]. Up to date, some reports proved that the alterations of epigenetic was able to regulate autophagy, such as histone methylation and acetylation [13][14][15]. However, it had not explained the mechanisms that the cancer associated alterations of epigenetic could regulate autophagy. The lysine-specific demethylase1(LSD1), is an epigenetic enzyme, also as an amine oxidase which can promote lysine demethylation in a flavin adenine dinucleotide dependent oxidative reaction and removes mono-and dimethyl groups from lysine K4, and in specific circumstances, K9 on histone H3 [16][17][18]. Lately, there has been reported that the neuron specific isoform LSD1 has a new substrate specificity, targeting histone H4 Lys 20 [19]. Moreover, there are some non-histone proteins are targeted by LSD1, like E2F1, DNMT and p53 [20][21][22]. LSD1 has been proved to have important roles in many aspects of cell biology, like cell differentiation, cell proliferation and mobility [23][24][25].In addition, LSD1 gene is highly expressed in many types tumors, the overexpression of LSD1 is often associated with malignancy and poor prognosis. According to report, LSD1 has some effect on keeping the malignant phenotype and undifferentiated of NB cells [26]. It has reportly involved in the progression of malignant in various tumors, including ER-negative breast cancer and primary NB [26,27], and poorly differentiated NB [28]. LSD1 as a kind of MAO, it was be bound up with apoptosis, metastasis and invasion of neoplasm. As a kind of epigenetic marker, the overexpression of LSD1 is one of the marker of tumor progression [29,30]. A lot of evidence indicated that in the incidence and development of tumors LSD1 had an critical role and can be a new target gene for tumor treatment. According to the above, as a member of MAO inhibitor, isatin might had an effect on LSD1 directly, therefore, LSD1 is likely to be a potential target of isatin for tumor inhibition.
Sestrin2 (SESN2), a protein which has characteristic with highly evolutionarily conserved, participated in many physiological functions of cells，including regulating autophagy with downregulating the protein kinase in signaling pathway of mammalian target of rapamycin (mTOR) [31]. According to literature reports , LSD1 can combine Sesn2 promoter regions so as to regulate SESN2 expression, thereby inhibiting the activation of mTOR, finally, by regulating the activity of autophagy -related proteins to play a role in cancer inhibition [32]. In summary, SESN2 is also an novel target of curing the cancer by regulating the autophagy mTOR signaling pathway. Previous reports have suggested that from the microarray data, SH-SY5Y cells were treatment with isatin and untreatment controls were closely related to the mTOR signaling pathway, and connected with the genes of mTOR downstream which taken part in autophagy [33]. Form the results of this article, it offered an new mechanism to understand the inhibitory effect of isatin on NB.
All these evidences support the potential antitumor function of the new drug named isatin on NB cells, and the mechanism was likely to be inhibited LSD1 regulated autophagy through SESN2-mTOR signaling pathway. Taken together, isatin has the potential to be developed as an anticancer drug to inhibit neuroblastoma.

Cell culture and treatments
Human neuroblastoma (SH-SY5Y )cells which were buy from Peking Union Medical College. DMEM (HyClone; GE Healthcare Life Sciences) were used to culture cells with 10% fetal bovine serum (BI; California, USA) supplemented at 37˚C in a humidified atmosphere with 5% CO2. The drug (Isatin) was dissolved in 0.1% dimethyl sulfoxide, than put it to culture dish at concentrations of 0, 50, 100, 200μmol/L, after the cells had grow to about~70% proportion. The cells were harvested after treatment with isatin at 37˚C for 48h for further application and analysis.

CCK-8 assay
It used CCK-8 assay to detect the inhibition rate and toxicity of isatin on SH-SY5Y cells. A 96-well plate was used to culture the SH-SY5Y cells steady with a final volume of 100µl of complete culture medium, the different concentrations (0, 25, 50, 100, 200, 400, 800µmol/L) of isatin(five wells for each concentration) were treated with SH-SY5Ycells when the it grow to about 60% and incubated for 24h/48h /72h at 37˚C in a humidified atmosphere with 5%CO2. Aftertreatment, 10µl CCK-8 solution was added to each well and incubated at 37°C for 2h. Then the absorbance of the samples was measured at 450nm with a Microplate Reader (SynergyH1; Bio Tek, Vermont, USA). Each independent experiment was run for three times.
According to the formula to calculate isatin inhibition rate of SH-SY5Y cells and IC50(used SPSS Statistics).

Cell morphological changes of the experiment
The 6-well plate were used to culture SH-SY5Y cells, when it spread to 60% in the total volume of 2 ml complete medium, exposed with isatin in different concentrations(0, 50, 100, 200 µmol/L) for 48 h, the cells morphology changes was observed by electron microscope, the pictures were taken by microscope.

Flow cytometry to detect cell apoptosis
Flow cytometry (Annexin V APC Apoptosis Detection kit) was used to determine cells apoptosis. 100 μl 1× Binding Buffer used to suspend with approximately 2 ×10 6 cells, and disposed of 5μl Annexin V APC. Then, added 300μl 1× Binding Buffer to centrifuge for 5 min at 1000rpm and threw away the supernatant, the addition of 10 mL PI dye were put to inincubate for another 5 min. The BD Accuri C6 Software was used to analysis the results.

Flow cytometry to detect cell cycle
Flow cytometry was used to determine cell cycle. SH-SY5Y cells (3×10 6 /mL) were starved for 48 h treatment with isatin, then fixed with 70% ethanol overnight. Next, mixed 200ul 1 × PBS with 2μl RNAase (0.25mg/ml), then hatched at 37℃ and stained cells with 500 mL propidium iodide (PI; Roche, Switzerland) in a dark environment. Detected on machine (BD Accuri C6 Software) and analyzed the results with Flowjo software.

Tablet cloning
SH-SY5Y cells (≈1×10 6 ) were used of trypsin digestion and inoculated in 6well plates with proper density (500 cells per / hole), cultured at 37˚C and added to isatin with different concentration(0, 50, 100, 200μmol/L) when the cells appear to stick wall, keep on culturing cells when cloning appear visible to the naked eye; methanol fix for 10min; Crystal violet staining for 10 min.

DAPI staining to detect apoptosis
SY5Y cells were cultured in the 24-well plate treatment like previous for 48h.
Remove the medium washed with 1 × PBS;add 100μl methyl alcohol to fixate cells for 5min; depleting methyl alcohol and washed with 1 × PBS, add in 500 μl DAPI staining fluid for 5min at room temperature; Discarding the dyeing liquid and washed with 1 × PBS for 3min for 3 times; Each group were taken using fluorescence microscope at × 200 for three times.

Quantitative RT-PCR assay.
The extraction of total RNA of SH-SY5Y cells were used with trizol reagent (Solarbio, Beijing, China). The template cDNA was used a reverse transcription kit (Transgene, Beijing, China) to construct, and qPCR with Trans Start Probe RT-PCR Super Mix (Transgene, Beijing, China). The data were obtained on Bio-Rad (California, USA) One-Step Plus system. The primer sequences used are shown in Table 1.

Gene
Forward Reverse

Statistical analysis
All experimental data were performed three times and used the mean ± standard deviation (SD) to calculate. The data were statistically analyzed by one-way variance (ANOVA) and pairwise comparison by using statistical software (GraphPad Prism 6), P < 0.05 was considered statistically significant.

Isatin inhibited the proliferation of SH-SY5Y cells
The results of CCK-8 assay showed that isatin could suppress the proliferation of SH-SY5Y cells, and the inhibition rate is increasingly dosedependent of isatin. After the test we found that the most obvious inhibition effect of SH-SY5Y was treatment with isatin for 48h (Fig.1a). The IC50 of isatin is 271.31μmol/L calculated by software (SPSS Statistics 7). According to the results we selected 0, 50, 100 and 200μmol/L concentrations for experimental study. Tablet cloning found that after adding to isatin the formation of the colony number of SH-SY5Y cells on 6-well plate less and less compared with the control group, it suggested that isatin could control the proliferation of SH-SY5Y cells (Fig.1b).

Cell morphological changed of the experiment
After treating with isatin for 48 h, the SH-SY5Y cells morphology changed that the nucleus appeared pyknosis and cells fragment became increased, the number of living cells decreased significantly with more doses of isatin (Fig.2).
Therefore we think that isatin can obviously inhibit growth of SH-SY5Y cells.

Flow cytometry to detect cell cycle and apoptosis
After treating with isatin for 48 h. It's found that cell cycle of SH-SY5Y cells were changed by using flow cytometry to detect. The proportion of S phase decreased significantly, cells obviously were block in phase G1 (Fig.3a c).
Flow cytometry to detect cell cycle apoptosis, the number of living cells decreased obviously with more doses of isatin (Fig. 3b d). The proportion of the early apoptotic cells and the late apoptosis cells were more and more with isatin increased. From the results, we speculate that isatin can obviously increase the SH-SY5Y cell apoptosis rate.

DAPI staining to detect apoptosis
After SH-SY5Y cells were treatment with isatin for 48h, DAPI staining to detect cell apoptosis. Compared with the blank control group, cell morphology changed after join isatin (Fig. 4), the morphology changed more obvious with the increase of concentration, we can see cytoplasmic concentrated from the picture, and the nucleus had become more shrunk, shiny and turn round, these suggested that the apoptosis of SH-SY5Y cells were increased, The results showed that isatin can promote apoptosis, which is more obvious with the increase of drug concentration.

Isatin suppressed invasion and migration of SH-SY5Y cells
After cells were treatment with isatin for 48 h, the scratch test of SH-SY5Y cell was taken to detect migration ability. The results showed that the scratches healed more slowly in the treatment group than in the control group (Fig. 5b).
Using transwell detected the ability of cell invasion. The results showed that with the concentration of the drug was higher, the ability of cells through the small room is more and more low compared with controls. All the above experimental results showed that isatin had a significant inhibitory effect on the invasion and migration of neuroblastoma cells (Fig. 5a).

Quantitative RT-PCR assay.
After SH-SY5Y cells treated as before, we detected the mRNA relative expression of LSD1, SESN2, p53, Beclin1, P62 and LC3, from the results

Isatin can inhibit the enzyme activity of LSD1
It is reported that isatin can restrain activity of MAO. Because of LSD1 is one of MAO so we guess if isatin as one of MAO inhibitor can work on LSD1 directly. The results from qPCR and the western blot were found that the mRNA relative expression level of LSD1 occurred to decline after treatment with isatin, similarly, the relative expression level of the protein decreased (Fig. 7a). These results showed that isatin as MAOI can directly inhibit the expression of LSD1 enzyme acted like as an inhibitor.

LSD1-depentent epigenetic induced SESN2 expression represses the mTOR pathway
The results of Western Blot showed that SH-SY5Y cells after adding to isatin 48 h, SESN2 relative protein expression was increased (Fig. 7a), and mTOR relative protein expression reduced (Fig. 7b), the result explained that the mechanism of isatin inhibiting SH-SY5Y cells must be relevant to SESN2 and mTOR. Here, it identified that as an LSD1-repressed gene SESN2 participated in mTORC1 signal routing from KEGG. From the report SESN2 promoter was directly bound and repressed by LSD1. Pharmacological inhibition of LSD1 triggers a structural modeling in the chromatin surrounding the TSS of the SESN2 gene, leading to transcriptional activation of SESN2 expression. Previous studies (the microarray data) shown that isatin-treated cells and untreated controls were associated with the mTOR signaling pathway [34]. These results suggested that isatin had a significant inhibitory effect on NB cells, and the mechanism was likely to involve SESN2.

Autophagy related protein expression changed after treating with isatin
The results of Western Blot showed that SH-SY5Y cells after adding to isatin for 48 h, the protein levels of LC3, Beclin1and P62 had changed (Fig. 7b).
The mTOR pathway play an important role in the occurrence of autophagy.
Including LC3 is also a marker associated with autophagy. It is reported that LC3-II gene expression in the membrane of autophagosomes reflected the level of autophagy [35,36]. The experimental results showed that LC3 protein expression level increased, which mean the level of autophagy increased.
P62 expression is also an important marker to reflect autophagy. P62 was related to autophagy level negatively [37]. The results of Western Blot showed that after adding to isatin like before, the protein relative expression of P62 of SH-SY5Y cell was decreased. The antitumor effect of autophagy on tumor was induced by certain ATG protein by report, such as Beclin1 [38]. The results of Western Blot showed that SH-SY5Y cells treatment with isatin like above, the protein relative expression of P62 was increased. The results of this study revealed that autophagy was related with isatin, which provided a novel mechanism for isatin to act on NB. .

4.Discussion
Isatin is a derivative of anticarcinogen indirubin, it exhibited many beneficial biological activities, including antifungal, antibacterial anticancer capabilities.
The CCK8 results had revealed that the SH-SY5Y cells were deal with isatin at doses ranging from 50 to 200 µmol/L could be able to inhibit tumor cell Autophagy is an important process that senses the changes of intracellular environment, and it make cell contribution to acclimatize oneself to adverse environments, such as energy insufficiency, nutrient depletion, and other cellular stress environments [39]. Autophagy play dual roles in tumor and defective autophagy is associated with cancer [40,41]. Autophagy can be used as a neoplam inhibitor by preventing the accumulation of damaged proteins and organelles, or by inhibiting apoptosis and accelerating the survival pathway of cancer growth [35,42,43]. The results from qPCR and Western Blot from our present study showed that treatment with relatively doses of isatin could regulate the expression of autophagy-related proteins, this suggested that isatin was likely to activate autophagy in NB cells SH-SY5Y, other experiments still proved that the apoptosis of NB cells SH-SY5Y increased after dealing with isatin, thus, the SH-SY5Y cells underwent apoptosis and autophagy after the addition of isatin, and apoptosis and autophagy promoted each other and jointly restrained the growth of NB cells.
However, how isatin actived autophagy in NB SH-SY5Y cells and the mechanisms involved are unclear, so the molecular mechanism underlying relationship between autophagy and tumor still need further investigated.
Lately, some reports display that epigenetic alterations were related to autophagy, such as the methylation and acetylation of histone [14,38]. There has been no assessment for the mechanism that regulation of autophagy by there have also occur in apoptosis, so as to under the action of autophagy and apoptosis, inhibiting tumor cell growth. But the mechanism of isatin inducing autophagy and apoptosis remains to be further investigated, and further animal and clinical trials are needed to develop isatin into a clinical cancer drug, So there's a lot of research and work that needs to be done.
Nevertheless, this paper provides a new approach to the mechanism of isatin inhibits neuroblastoma, and a theoretical basis for the anti-tumor effect of isatin on NB, finally demonstrating the potential of isatin to develop anticancer drugs for neuroblastoma and other tumors.

Conclusions
Isatin inhibits the growth of neuroblastoma, possibly by activating autophagy and by acting in conjunction with apoptosis, demonstrating the potential of isatin to be a chemotherapy agent for neuroblastoma.

Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.