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.
J Cell Mol Med
2021 Jan 01;251:272-283. doi: 10.1111/jcmm.15995.
Show Gene links
Show Anatomy links
Strongylocentrotus nudos Egg Polysaccharide induces autophagy and apoptosis in leukaemia cells by regulating mitochondrial function.
Wang C
,
Li M
,
Li L
,
Shen X
,
Liu Y
,
Wang S
.
???displayArticle.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.
???displayArticle.pubmedLink???
33289295
???displayArticle.pmcLink???PMC7810953 ???displayArticle.link???J Cell Mol Med ???displayArticle.grants???[+]
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