Strongylocentrotus nudos Egg Polysaccharide induces autophagy and apoptosis in leukaemia cells by regulating mitochondrial function

In this study, we investigated the ability of the Polysaccharide from the Eggs of Strongylocentrotus nudus (SEP) to regulate cellular autophagy and apoptosis in leukaemia cells. Human acute myeloid leukaemia (AML) cells (HL60) and murine AML cells (L1210) treated with SEP were used to assess viability using Cell Counting Kit‐8, cytotoxicity by measuring lactate dehydrogenase release, the generation of reactive oxygen species (ROS) by DCFH‐DA staining. In addition, we utilized a mouse model of leukaemia in which L1210 cells were injected into DBA/2 mice by sub‐axillary injection. Treatment with SEP decreased cell viability, increased in cytotoxicity and increased the release of ROS in a dose‐dependent manner. SEP treatment was also associated with the activation of pro‐apoptotic proteins cleaved caspase‐3, cleaved caspase‐9 and cleaved poly (ADP‐ribose) polymerase (PARP). Activation of the apoptotic pathway led to the release of cytochrome C (CytoC) into the cytosol of the cell resulting in decreased membrane potential. The effect of SEP treatment was depended on the activation of the nuclear factor kappa‐B (NF‐κB) signalling pathway as SEP treatment led to an increase in NF‐κB phosphorylation, and inhibition of NF‐κB signalling using PDTC blocked SEP‐mediated activation of apoptosis. Treatment with SEP also prolonged survival time in our leukaemia mouse model and was associated with diminished tumour volume, increased leucocyte and lymphocyte proliferation, promoted pro‐inflammatory factor release in serum and enhanced immune function. Taken together, these data suggest that SEP inhibits the progression of leukaemia by initiating mitochondrial dysfunction, autophagy, and apoptosis via the NF‐κB signalling pathway.


| INTRODUC TI ON
Acute myeloid leukaemia (AML) originates in hematopoietic stem cells, and is characterized by leukaemia cell proliferation and infiltration as well as fever, infection and bleeding. 1 Normal hematopoiesis is hindered due to the increased proliferation of primitive myeloid cells in the bone marrow while differentiation and apoptosis are suppressed. 2 Moreover, AML has a high mortality and recurrence rate, which makes AML the most common haematological malignancy. 3 Recent progress in identifying anti-apoptotic pathways in AML primary cells has led to the development of several new drugs currently moving through the drug development pipeline, either as individual therapies or in combination with conventional chemotherapy regimens. 4 Nevertheless, there is still significant need for the identification of additional effective treatments for AML.
Cells initiate the process of autophagy in response to stressful situations, such as nutrient deprivation, as a means of self-preservation. During autophagy, the cell begins to breakdown non-essential molecules to prolong cell survival. When cells have exhausted superfluous proteins, the cell may initiate apoptosis or cell death. 5 Autophagy plays an important role in regulating the growth, differentiation and death of cells. 6 AML cells display limited myeloid differentiation, inhibition of apoptosis in progenitor cells and enhanced self-renewal capacity. 7 As autophagy promotes AML cell survival, targeting autophagy in leukaemia cells may provide new methods for the treatment of AML.
Several polysaccharides exhibit anti-tumour effects by changing the biochemical properties of cell membranes, inducing tumour cell differentiation and apoptosis, or affecting signal transmission between tumour cells. 8 Several studies have documented the ability of exogenous polysaccharides to alter the biology of normal organisms as well as exert beneficial therapeutic effects in diseased organisms. 9 At present, hundreds of polysaccharides are known to have definitive drug effects. 10 Strongylocentrotus nudus egg polysaccharide (SEP) is a water-soluble polysaccharide isolated from the eggs of Strongylocentrotus nudus. 11 Preliminary in vitro experiments show that SEP can significantly promote the proliferation of splenic lymphocytes. 12 In addition, SEP inhibits tumour growth by affecting telomerase activity and angiogenesis. 12,13 To date, few studies have investigated a relation between SEP and AML. We used SEP to treat human and mouse AML cell lines and study its effect on proliferation, apoptosis, creation of reactive oxygen species, markers of autophagy, and in vivo tumour growth. Because the nuclear factor kappa-B (NF-κB) pathway plays an important role in the occurrence of AML, we studied the impact of SEP treatment on the regulation of immune and inflammatory responses. 14 While the role of NF-κB in the progression of AML is not fully understood, NF-κB is constitutively expressed in AML cells and leukaemia stem cells in many patients. 15 Our study identifies a mechanism by which SEP regulates AML autophagy-related phenomena and may represent a potential therapeutic in the treatment of AML.
Leukaemia cells were seeded into 96-well plates with a final volume of 100 μL culture medium/well. After 12-hour culture, fresh medium containing 50, 100 or 200 μg/mL of SEP was added and the same volume of medium without SEP was used as the background control.
After SEP was added, 10 μL/well CCK-8 solution was also added to plates and the plates were allowed to incubate for 72 hours. After additional 4 hours incubation at 37°C, each well was examined for absorbance at 450 nm using a 96-well plate reader (ThermoFisher Scientific, Waltham, MA, USA). Eight repeated experiments were used for each group.

| Detection of intracellular reactive oxygen species (ROS) using fluorescence spectroscopy
The intracellular production of ROS was measured utilizing the dichloro-dihydro-fluorescein diacetate (DCFH-DA) dye assay. With oxidant, DCFH was transformed into fluorescent 2,7-dichlorofluorescein (DCF). Cells were incubated with 50, 100 or 200 μg/mL of SEP for 24h, followed by 10 μmol/L DCFH-DA treatment for 30 minutes. Fluorescence intensity was evaluated using a fluorescence microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. 16

| Cytotoxicity as measured by Lactate dehydrogenase (LDH) release
The amount of LDH released by cells was detected using the Cytotoxicity Detection Kit (C0016, Beyotime). Cells were treated with 50, 100 or 200 μg/mL of SEP for 24 hours, centrifuged at 400 g for 5 minutes and the supernatant was removed. The LDH release reagent was diluted 1:10 in phosphate buffer saline (PBS) and then added to the pellet. One hour later, samples were centrifuged and 120 μL supernatant per well was transferred to a new plate and the plate was read on a plate reader (Thermo Fisher Scientific, Waltham, MA, USA) at an absorbance of 490 nm.

| Flow cytometry
Initiation of apoptosis in leukaemia cells was tested using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis kit (C1062L, Beyotime) and measuring staining using flow cytometry. Briefly, cells were detached with trypsin and then centrifuged at 100 g, for 5 minutes at room temperature and the residual medium and trypsin was removed. Afterwards, 50, 100 or 200 μg/ mL of SEP were added to cells and they were allowed to incubate for 24 hours. Cells were harvested the following day and the cell pellet was washed twice with PBS and resuspended in 500 μL loading buffer. FITC-Annexin V and PI were then added to the cells (5 μL and 10 μL, respectively) in dark at room temperature for 5 minutes, ings for bedding at 25 ± 2°C with a 12-hour light/dark cycle, receiving standard laboratory feed, ad libitum. We injected 1 × 10 6 L1210 cells subcutaneously into the sub-axillary region of each mouse. 19 The day after injection of AML cells, the mice were injected with either saline (negative control group), 20 mg/kg cyclophosphamide (CTX) (CTX-treated group) or SEP at a 10 or 40 mg/kg. Ten days after inoculation, all mice were anaesthetized with 3% pentobarbital sodium and weighed. Blood samples were harvested via cardiac puncture before mice were killed. The animals were observed for mortality throughout the treatment period and mean survival time (days) were calculated using the following formula: [(TC)/C] × 100%, where T was the number of survival days of the treated groups and C was the survival days of the control group.

| Leucocyte and lymphocyte proliferation assay
Murine blood samples were collected and held on ice until the leucocyte and lymphocyte cell counts could be analysed using an auto blood analyzer (CA620-VET, Sweden). Briefly, double distilled water was used as the blank control and a standard reference material as a quality control. The blood samples were shaken gently and placed under needle to measure leucocyte and lymphocyte cell counts.

| Microarray analysis
L1210 cells were seeded in 100 mm cell culture dishes (1 × 10 7 /dish) and cultured overnight. Cells were removed from the plates and centrifuged at 250 g for 5 minutes. After removing the supernatant, the cells were treated with 100 μg/mL SEP treatment for 24 hours. The NC samples were treated with a volume of medium equal to that used for the SEP-treated samples. Total RNA was extracted from 5 × 10 6 cells and purified by phenol-chloroform-isoamyl alcohol extraction (25:24:1). Spectrophotometry and capillary electrophoresis (Agilent 2100 Bioanalyzer, USA) were utilized to confirm RNA purity using the A260/A280 ratio. RNA was processed according to the Affymetrix's instructions and hybridized to Affymetrix HG-U133A oligonucleotide microarrays. Raw data were processed by Microarray Suite 5.0. All probe scales were set to a constant value of 500 for each microarray. Principal component analysis (PCA) was adopted to analyse the gene profiles of the control and SEP-treated group using Cluster 3.0. We selected genes with a fold change of at least 2 between the two groups for further analysis and these genes were subjected to Gene Ontology (GO) analysis and KEGG functional annotation using DAVIE database (https://david.ncifc rf.gov/ home.jsp). Visualization was performed by using R and Cytoscape. 21
Lysates were centrifuged at 7000 g for 10 minutes at 4°C. We then
Mitochondrial membrane potential changes were calculated by the red to green fluorescence ratio. 16

| Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted using TRIzol (Invitrogen Life Technologies, Carlsbad, CA, USA), and the quality was tested using agarose gel elec- For all experiments, P < .05 was considered statistically significant.

| SEP promotes apoptosis and prevents leukaemia cell proliferation
SEP is thought to suppress tumorigenesis by enhancing immune function, but the underlying mechanism is unclear. [11][12][13]26,27 Previous studies indicated that SEP may stimulate T lymphocyte 11 and macrophage function, 27 as well as up-regulate IL-2, TNF-α and IFN-γ expression. 13,26 The CCK-8 assay was utilized to measure the effects of SEP on the cell growth and viability of HL60 and L1210 cells.
Increasing concentrations of SEP progressively reduced cell viability of L1210 cells ( Figure 1A). Furthermore, treatment with SEP also led to dose-dependent increases in intracellular ROS levels compared with untreated cells ( Figure 1B). Measurement of lactate dehydrogenase (LDH) as a marker of cytotoxicity indicated that there was more cell death in SEP-treated AML cells than in untreated cells ( Figure 1C). Annexin V and Propidium iodide staining followed by flow cytometry also suggested that SEP promoted apoptosis in SEPtreated AML cell lines ( Figure 1D). Taken together, these results indicate that SEP inhibited leukaemia cell growth and induced apoptosis in leukaemia cells.

| SEP modulated immune responses to prolong the survival of leukaemic mice
To demonstrate the anti-leukaemic effects of SEP in vivo, we inoculated DBA/2 mice with L1210 cells by sub-axillary injection. 28 As CTX is a chemotherapeutic agent widely used in the clinical treatment of leukaemia, it was chosen to serve as a positive control in our mouse model. 29 When the survival curves associated with each treatment group were compared, treatment with SEP extended the survival time of leukaemic mice in a dose-dependent fashion ( Figure 2A). Notably, compared with control group, both CTX and SEP significantly reduced tumour size in vivo ( Figure 2B).

Microarray analysis
We compared the transcriptomes of leukaemia cells that were or were not treated with SEP using the GeneChip Microarray assay.
PCA analysis showed significant segregation of SEP-treated and non-treated samples ( Figure 3A), and a Venn diagram was created to illustrate any overlap of differentially regulated genes ( Figure 3B). GO and KEGG pathway analysis using the DAVID database were carried out to identify the potential biological functions of the differentially expressed genes ( Figure 3C). The results suggest that SEP treatment primarily affects cell cycle, DNA replication and cell apoptosis-related biological processes.
In addition, KEGG pathway analysis also identified adhesion and mitotic nuclear division as being regulated by SEP. In L1210 cells, SEP activated apoptosis and adhesion and regulated cell survival, immunity and metabolism. Analysis of overlapping pathways by GO and KEGG analysis identified pro-apoptotic and anti-tumour functions of SEP. Our results showed a dose-dependent increase in cleaved caspase-3, caspase-9, and PARP after SEP treatment ( Figure 4A). SEP also increased the phosphorylation of NF-κB p65 ( Figure 4B) and the expression of inflammatory cytokines TNF-α, IL-6 and IFN-γ ( Figure 4C). In addition, the effect of SEP on cleaved caspase-3, caspase-9 and PARP expression was eliminated by addition of PDTC, a specific inhibitor of the NF-κB pathway ( Figure 4D). SEP treatment led to a decrease in mitochondrial membrane potential ( Figure 4E), an increase in CytoC released into the cytosol and a concomitant reduction in CytoC in mitochondria ( Figure 4F). Similar results were observed in the human AML cell line, HL-60 ( Figure S2). SEP treatment stimulated the release of TNF-α, IL-6 and IFN-γ and increased mitochondrial membrane potential, which were suppressed by the addition of NF-κB inhibitor ( Figure S3). In summary, SEP treatment lowered mitochondrial membrane potential, triggered the release of mitochondrial CytoC, and activated caspase-3 and caspase-9

| SEP induces ROS-mediated mitochondrial apoptosis by activating the NF-κB signalling pathway
promoting apoptosis and that SEP function was dependent on the NF-κB signal pathway.

| SEP regulates autophagy to promote leukaemia cell survival
The connection between autophagy and apoptosis is still not well understood. Autophagy is a well-studied process, which regulates cytoplasm and organelle turnover. Cells use autophagy as a survival mechanism during extreme conditions such as nutrient deprivation, but it may also be involved in non-apoptotic cellular death pathways. 33 Previous reports suggest that dexamethasone treatment induces apoptosis in leukaemia cells by activating acute promyelocytic leukaemia protein and Akt-dependent autophagy leading to mitochondrial dysfunction. 34 As SEP-induced mitochondrial dysfunction was the key cause of leukaemia cell apoptosis, we decided to investigate whether SEP promoted leukaemia cell apoptosis by activating autophagy. Treatment of murine AML L1210 cells with SEP led to concentration-dependent increases in LC3-II and BECN1 expression, demonstrating that SEP could activate the autophagy pathway ( Figure 5A, upper). Activation of autophagy was blocked by treatment with the NF-κB inhibitor PDTC ( Figure 5A Figure S4). Cumulatively, our data suggest that the SEP induction of apoptosis is mediated by NF-κB activation of autophagy.

| D ISCUSS I ON
This study examined the ability of SEP to promote apoptosis in leukaemia cells. We utilized a leukaemia mouse model in which DBA/2 mice received sub-axillary injection of L1210 cells to study the effect of SEP treatment on the survival of mice with AML. After treatment with SEP, the mice were evaluated for their expression of pro-inflammatory cytokines TNF-α, IL-2 and IFN-γ in the serum of mice. Our results indicated that SEP could inhibit the proliferation of leukaemia cells, which was consistent with the reported role of SEP in other tumour cells. 12 Furthermore, we showed that SEP had a concentrationdependent inhibitory effect on leukaemia progression.
Apoptosis, or programmed cell death, is the process whereby cells undergo a controlled self-destruction in response to the activation of specific endogenous or exogenous signals, and under the control of a family of related genes. 35 Caspase-3 is a key enzyme involved in the mitochondrial activation of apoptosis. [36][37][38] Our results showed that as the concentration of SEP increased caspase-3 enzyme activity, suggesting that the activation of caspase-3 played an important role in activating the apoptotic pathway in leukaemic cells after SEP treatment.
Our study further found that SEP-induced leukaemia cell apoptosis through the NF-κB signalling pathway. Specifically, SEP promoted the activation of NF-κB leading to an increase in markers associated with autophagy. NF-κB is known to be an important inhibitor of apoptosis as well, promoting self-protection and adaptation, and is itself a target in the treatment of various cancers and other proliferative diseases. 39 In addition, NF-κB can inhibit cellular autophagy by activating the mammalian target of rapamycin (mTOR) pathway. 40 NF-κB is also known for its ability to regulate the expression of a variety of cytokines, growth factors and other basic cellular messenger proteins, allowing it to regulate a variety of cell functions. 41 Inhibition of NF-κB activity can promote tumour cell apoptosis. 42 Our results suggest SEP promotes mitochondrial dysfunction and apoptosis via activation of the NF-κB pathway.
Mitochondria are one of the most sensitive organelles to various injuries. 43 When cells are stimulated, the number, size and structure of mitochondria can undergo pathological changes, affecting the survival of cells. 44 Mitochondrial-mediated regulation of autophagy has both a direct and indirect relationship with the onset of tumours and represents a key regulator of quality control in tumour cells. The outcome of activation of the autophagy pathway may differ depending on the stage of tumour development. 45 In this experiment, the relationship between NF-κB and the activation of autophagy was examined by investigating the activation of LC3-II protein. NF-κB activation led to increases in LC3 and activation of autophagy.
Moreover, this study found that SEP prolonged the lifespan of mice with leukaemia by enhancing the immune system function.
Similar to SEP, APS-1II extracted with NaOH can activate peripheral blood leucocytes and lymphocytes, stimulate the secretion of cytokines TNF-α, IFN-γ and IL-2, promote the proliferation of splenocytes, enhance the phagocytic activity of peritoneal macrophages and induce a protective immune response. Regulation of these factors significantly prolongs the lifespan of L1210 tumour-bearing mice and suggests that SEP and APS-1II can act as drug candidates for the treatment of leukaemia. 20 We have shown that SEP promotes the apoptosis of AML cells using a mechanism that requires the NF-κB signalling pathway ( Figure 6). This research has highlighted new markers and therapeutic F I G U R E 6 Schematic diagram depicting how SEP-mediated induction of autophagy leads to activation of apoptosis in AML. Treatment with SEP leads to mitochondrial dysfunction resulting in the activation of caspase-3 and caspase-9 as well as the release of CytoC, which triggers apoptosis in leukaemia cells. SEP also induces cell apoptosis by enhancing phosphorylation of NF-κB and activating NF-κB signalling pathway. SEP may elevate autophagy-related protein LC3-II, ATG7 and BECN1, stimulating autophagy in leukaemia cells and eventually promoting apoptosis targets for the treatment of AML. We cannot exclude the possibility that other pathways may directly participate in or interact with the NF-κB signalling pathway to affect the progression of AML and further study is necessary to provide further insights into these pathways.

This work was supported by grants from The Youth Scholars
Foundation for the Basic Research and Cultivation of Zhengzhou University and the Youth Innovation Fund of the First Affiliated Hospital of Zhengzhou University.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data supporting the findings of this study is available within the article.