Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Abstract
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.
The Youth Scholars Foundation for the Basic Research and Cultivation of Zhengzhou University, the Youth Innovation Fund of the First Affiliated Hospital of Zhengzhou University
FIGURE 1. SEP inhibited cell growth and induced apoptosis in leukaemia cell lines. A, CCK‐8 measurement of L1210 and HL‐60 cell viability. B, Detection of ROS in L1210 and HL‐60 cells using DCFH‐DA fluorescent probe labelling. C, Measurement of LDH activity as an indicator of cytotoxicity in L1210 and HL‐60 cells. D, Apoptosis rates of L1210 and HL‐60 cells using flow cytometry. Experiments represent three or more replicates. Data are shown as mean ± standard deviation. Comparisons between multiple groups were conducted using one‐way ANOVA followed by Tukey's post hoc analysis. * indicates P < .05 compared with the control group
FIGURE 2. SEP extended survival in a mouse model of leukaemia by enhancing immune function. A, Survival time of the mice that received control, CTX or SEP treatment followed by inoculation with L1210 cells (n = 15). B, The ratio of tumour weight to mouse weight for each treatment group (n = 10). C, Quantitation of leucocytes and lymphocytes (n = 10). D, ELISA analysis of TNF‐α, IL‐6 and IFN‐γ for each treatment group. Experiments represent three or more replicates. Data are shown as mean ± standard deviation. Comparisons between multiple groups were conducted using one‐way ANOVA followed by Tukey's post hoc analysis. * indicates P < .05 compared with the control group
FIGURE 3. Microarray analysis of differentially expressed genes. A, PCA analysis scatter plot depicting the degree of dispersion between genes from control samples and SEP‐treated samples. B, Comparison of differential gene expression between control and SEP‐treated samples. C, GO analysis of differentially expressed genes. D, Predicted network of interacting genes based on GO and KEGG analysis. E, Flowchart of microarray and informatics analysis
FIGURE 4. SEP induces apoptosis in L1210 cells via interfering mitochondrial function. A, Western blot analysis of cleaved caspase‐3, cleaved caspase‐9 and cleaved PARP after 24 h treatment with SEP compared to untreated control. B, Western blot analysis of p‐NF‐κB and NF‐κB in L1210 cells after treatment with SEP at various concentrations. C, ELISA analysis of TNF‐α, IL‐6 and IFN‐γ expression in L1210 cells after treatment with SEP at various concentrations for 24 h. D, Western blot analysis of cleaved caspase‐3, cleaved caspase‐9 and cleaved PARP after treatment with SEP (200 µg/mL) and with or without the addition of NF‐κB inhibitor PDTC for 24 h. E, L1210 cells were treated with SEP at various concentrations for 24 h, stained with JC‐1 dye and observed with an epifluorescent microscope. Scale bar = 25 μm. F, L1210 cells were treated with SEP at various concentrations for 24 h, subjected to mitochondrial and cytoplasmic separation, and the CytoC content in the mitochondria and cytosol was measured by Western blot. Experiments represent three or more replicates. Data are shown as mean ± standard deviation. Comparisons between multiple groups were conducted using one‐way ANOVA followed by Tukey's post hoc analysis. * indicates P < .05 compared with the control group
FIGURE 5. SEP‐induced L1210 cell apoptosis through NF‐κB‐dependent autophagy. A, L1210 cells were treated with various concentrations of SEP for 24 h, or SEP (200 μg/mL) with/without NF‐κB inhibitor PDTC followed by analysis of LC3‐II and BECN1 expression using Western blot analysis. B, L1210 cells were treated with different concentrations of SEP (50, 100 and 200 μg/mL) followed by extraction of total RNA. The mRNA levels of ATG3, ATG5, ATG7, ATG12 and BECN1 were measured by RT‐qPCR. C, L1210 cells were incubated with SEP (200 μg/mL) with/without autophagy inhibitor 3‐MA (2 mmol/L), followed by flow cytometry with annexin V and PI staining. Experiments represent three or more replicates. Data are shown as mean ± standard deviation. Comparisons between multiple groups were conducted using one‐way ANOVA followed by Tukey's post hoc analysis. * indicates P < .05 compared with the control group
FIGURE 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
Agarwal,
Differentiation of leukemic blasts is not completely blocked in acute myeloid leukemia.
2019, Pubmed
Agarwal,
Differentiation of leukemic blasts is not completely blocked in acute myeloid leukemia.
2019,
Pubmed
B'chir,
The eIF2α/ATF4 pathway is essential for stress-induced autophagy gene expression.
2013,
Pubmed
Bai,
Overcoming resistance to mitochondrial apoptosis by BZML-induced mitotic catastrophe is enhanced by inhibition of autophagy in A549/Taxol cells.
2018,
Pubmed
Baldwin,
Regulation of cell death and autophagy by IKK and NF-κB: critical mechanisms in immune function and cancer.
2012,
Pubmed
Bosman,
Constitutive NF-κB activation in AML: Causes and treatment strategies.
2016,
Pubmed
Bowlin,
Participation of T-lymphocytes in the curative effect of a novel synthetic polyamine analogue, N,N'-bis[3-(ethylamino)propyl]-1,7-heptanediamine, against L1210 leukemia in vivo.
1991,
Pubmed
Cao,
MLKL mediates apoptosis via a mutual regulation with PERK/eIF2α pathway in response to reactive oxygen species generation.
2018,
Pubmed
Chen,
Protective effect of mitochondria‑targeted peptide MTP‑131 against oxidative stress‑induced apoptosis in RGC‑5 cells.
2017,
Pubmed
Cheng,
Ganoderma lucidum polysaccharides in human monocytic leukemia cells: from gene expression to network construction.
2007,
Pubmed
Dawson,
Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia.
2011,
Pubmed
Gozuacik,
Autophagy as a cell death and tumor suppressor mechanism.
2004,
Pubmed
Herold,
Isolated trisomy 13 defines a homogeneous AML subgroup with high frequency of mutations in spliceosome genes and poor prognosis.
2014,
Pubmed
Jo,
Sulforaphane induces apoptosis in T24 human urinary bladder cancer cells through a reactive oxygen species-mediated mitochondrial pathway: the involvement of endoplasmic reticulum stress and the Nrf2 signaling pathway.
2014,
Pubmed
Knapper,
A randomized assessment of adding the kinase inhibitor lestaurtinib to first-line chemotherapy for FLT3-mutated AML.
2017,
Pubmed
Laane,
Cell death induced by dexamethasone in lymphoid leukemia is mediated through initiation of autophagy.
2009,
Pubmed
Lee,
REDD-1 aggravates endotoxin-induced inflammation via atypical NF-κB activation.
2018,
Pubmed
Li,
Maca polysaccharides: A review of compositions, isolation, therapeutics and prospects.
2018,
Pubmed
Li,
Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.
1997,
Pubmed
Liang,
Preactivation of Notch1 in remote ischemic preconditioning reduces cerebral ischemia-reperfusion injury through crosstalk with the NF-κB pathway.
2019,
Pubmed
Liu,
Rice protein prolamin promotes anti-leukemia immunity and inhibits leukemia growth in vivo.
2018,
Pubmed
Liu,
Structure characterization and anti-leukemia activity of a novel polysaccharide from Angelica sinensis (Oliv.) Diels.
2019,
Pubmed
Liu,
Immunomodulatory activity of polysaccharides isolated from Strongylocentrotus nudus eggs.
2008,
Pubmed
,
Echinobase
Livak,
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
2001,
Pubmed
Ma,
Extraction, preliminary characterization and immunostimulatory activity in vitro of a polysaccharide isolated from Strongylocentrotus nudus eggs.
2014,
Pubmed
,
Echinobase
Man,
Caspase-3 controls AML1-ETO-driven leukemogenesis via autophagy modulation in a ULK1-dependent manner.
2017,
Pubmed
Martin,
Autophagy proteins suppress protective type I interferon signalling in response to the murine gut microbiota.
2018,
Pubmed
Mueller,
Autologous Transplantation for Older Adults with AML.
2018,
Pubmed
Nepstad,
The PI3K-Akt-mTOR Signaling Pathway in Human Acute Myeloid Leukemia (AML) Cells.
2020,
Pubmed
Ramonas,
Effect of dose, schedule, and route of administration on the in vivo toxicity and antitumor activity of two activated sulfhydryl derivatives of cyclophosphamide.
1980,
Pubmed
Rashid,
Muscle LIM protein/CSRP3: a mechanosensor with a role in autophagy.
2015,
Pubmed
Samudio,
UV-inactivated HSV-1 potently activates NK cell killing of leukemic cells.
2016,
Pubmed
Sciarretta,
The Role of Autophagy in the Heart.
2018,
Pubmed
Singh,
Intratumoral immunotherapy for melanoma.
2015,
Pubmed
Soiffer,
Prevention of graft-versus-host disease by selective depletion of CD6-positive T lymphocytes from donor bone marrow.
1992,
Pubmed
Wang,
Effective inhibition of a Strongylocentrotus nudus eggs polysaccharide against hepatocellular carcinoma is mediated via immunoregulation in vivo.
2011,
Pubmed
,
Echinobase
Wei,
Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.
2001,
Pubmed
Xiao,
Macrophage autophagy regulates mitochondria-mediated apoptosis and inhibits necrotic core formation in vulnerable plaques.
2020,
Pubmed
Xie,
Polysaccharide enhanced NK cell cytotoxicity against pancreatic cancer via TLR4/MAPKs/NF-κB pathway in vitro/vivo.
2019,
Pubmed
,
Echinobase
Yamashita,
How autophagy eats large mitochondria: Autophagosome formation coupled with mitochondrial fragmentation.
2017,
Pubmed
Yang,
Induction of autophagy by spermidine is neuroprotective via inhibition of caspase 3-mediated Beclin 1 cleavage.
2017,
Pubmed
Yin,
Advances in Research on Immunoregulation of Macrophages by Plant Polysaccharides.
2019,
Pubmed
Yu,
Promising Neuroprotective Function for M2 Microglia in Kainic Acid-Induced Neurotoxicity Via the Down-Regulation of NF-κB and Caspase 3 Signaling Pathways.
2019,
Pubmed
Zhang,
Curdlan sulfate-O-linked quaternized chitosan nanoparticles: potential adjuvants to improve the immunogenicity of exogenous antigens via intranasal vaccination.
2018,
Pubmed
Zhang,
A polysaccharide component from Strongylocentrotus nudus eggs inhibited hepatocellular carcinoma in mice by activating T lymphocytes.
2017,
Pubmed
,
Echinobase
