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/lp/springer-journals/cudraflavone-b-induces-human-glioblastoma-cells-apoptosis-via-er-VVmnGu4Nz5
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- Springer Journals
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- Copyright © The Author(s) 2023
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- 1471-2202
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- 10.1186/s12868-023-00778-4
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Abstract
Background Glioblastoma (GBM) is the most common malignant intracranial tumor with a low survival rate. How- ever, only few drugs responsible for GBM therpies, hence new drug development for it is highly required. The natural product Cudraflavone B (CUB) has been reported to potentially kill a variety of tumor cells. Currently, its anit-cancer effect on GBM still remains unknown. Herein, we investigated whether CUB could affect the proliferation and apopto - sis of GBM cells to show anti-GBM potential. Results CUB selectively inhibited cell viability and induced cell apoptosis by activating the endoplasmic reticulum stress (ER stress) related pathway, as well as harnessing the autophagy-related PI3K/mTOR/LC3B signaling pathway. Typical morphological changes of autophagy were also observed in CUB treated cells by microscope and scanning electron microscope (SEM) examination. 4-Phenylbutyric acid (4-PBA), an ER stress inhibitor, restored the CUB-caused alteration in signaling pathway and morphological change. Conclusions Our finding suggests that CUB impaired cell growth and induced cell apoptosis of glioblastoma through ER stress and autophagy-related signaling pathways, and it might be an attractive drug for treatment of GBM. Keywords GBM, Endoplasmic reticulum stress, Unfolded protein response, Autophagy Background GBM is the most common adult brain tumor (55%) with a high malignancy degree [1]. The common therapeutic strategies of GBM include surgery, radiation therapy, and chemotherapy [2]. Even though tremendous trials have *Correspondence: been made to treat GBM [3, 4], the prognosis of GMB Yuanshuai Zhou patients is still poor. The median survival time of newly zhouys@sibet.ac.cn Yanxiang Liu diagnosed patients is less than 15 months. liu-yanxiang@foxmail.com Recently, several natural product-derived compounds School of Biomedical Engineering (Suzhou), Division of Life Sciences including flavonoids have drawn more attention as and Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China potential agents for adjuvant GBM targeted therapies Jiangsu Key Lab of Medical Optics, Suzhou Institute of Biomedical [5, 6]. The preliminary experiments suggest that natural Engineering and Technology, Chinese Academy of Sciences, Keling Road products could harness the GBM cells via inducing apop- No.88, Suzhou 215163, China Institute of Clinical Medicine Research, Suzhou Science & Technology tosis, increasing reactive oxygen species (ROS), inhibit- Town Hospital, Suzhou 215153, China ing metastasis and regulating unfolded protein response Institute of Biomedical and Genetic Engineering, Islamabad, Pakistan 5 (UPR)[7–9]. Moreover, the natural products, Apigenin- Department of Pathology, Suzhou Science & Technology Town Hospital, No.1, Li Jiang Road, High-Tech District, Suzhou 215153, China 7-O-β-d-(-6′′-p-coumaroyl)-glucopyranoside (APG), © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Pan et al. BMC Neuroscience (2023) 24:10 Page 2 of 10 blockage of cell cycle sub-G1 and exhibited an inhibition caused severe and persistent ER stress in tumor cells effect on Monoamine oxidase (MAO) activity in mice leading to cell death [10, 11]. [16]. All these findings indicated that CUB may have the Cudraflavone B (CUB) is a flavonoid compound found potential to treat GBM. However, the experimental evi- in a variety of plants (Fig. 1a), first extracted and puri - dence is still lacking, and the underlying mechanisms fied from Cephalotaxus fortune [12]. It has been applied remain elusive. to anti-aging in clinical due to its distinct antioxidant Based on these conditions, we engaged in the research activity [13]. Furthermore, studies have shown that CUB for CUB in GBM. Here, we investigated whether CUB induced apoptosis in human oral cancer cells and mela- can affect the growth of GBM and explore the molecular noma cells in vivo [14, 15]. In addition, CUB lead to a Fig. 1 CUB increases apoptosis of GBM cells. a The structure of CUB. b The effects of CUB on cell viability of U87, U251 GBM cell lines and normal human astrocytes cells. c, d Clone formation assay of U87 c and U251 d cells treated with CUB. e Apoptosis measurements of GBM cells with CUB (10, 20 μM) incubation for 12(Additional file: Fig. S1a), 24 h. Data represents the mean ± SEM (n = 3). Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, and ***P < 0.001 P an et al. BMC Neuroscience (2023) 24:10 Page 3 of 10 mechanisms underlying its anti-GBM activity. We first San Jose, CA, USA) and analyzed using the Flowjo soft- tested the effect of CUB on the viability and prolifera - ware (Tree Star; Ashland, OR, USA). tion in U87, U251 and normal human astrocyte cell lines. Next, transcriptome sequencing was used to identify the gene expression changes in CUB-treated cells. Finally, Western blot analysis functional and mechanism experiments were conducted Cells were lysed in Radio Immuno Precipitation Assay to further verify the molecular mechanisms underlying (RIPA) buffer and the cell extraction was collected the anticancer effects of CUB in GBM. after centrifugation. Cell extraction was mixed with 5 × sodium dodecyl sulfate polyacrylamide gel electro- Materials and methods pheresis (SDS-PAGE) loading buffer (Beyotime, Shang - Cell lines and cell culture hai, China), then boiled at 98 ℃ for 10 min. Proteins Human GBM cell lines and normal human were separated by SDS–polyacrylamide gel electropho- astrocytes(NHA), U87 and U251, were obtained from resis then transferred to 0.22 μm Polyvinylidene Fluo- the Chinese Academy of Sciences Cell Bank (Shanghai, ride (PVDF) membranes (Millipore, Boston, MA, USA). China). Cells were cultured in high glucose Dulbecco’s Membranes were blocked for 2 h in Tris-buffered saline modified Eagle’s medium (DMEM) (Hyclone, USA) sup - containing 5% non-fat dry milk (Biosharp, China), then plied with 10% fetal bovine serum (FBS) (Gibco, USA) cut it into several blots to hybridized with the indicated and 100 μM streptomycin, 100 U/mL penicillin. All cells primary antibodies (1:1000 dilution) at 4 ℃ overnight. were cultured at 37 ℃ with 5% CO and 95% humid- Horseradish peroxidase secondary antibody (1:2000) was ity in a constant temperature incubator. CUB (CUB, incubated for approximately 2 h at room temperature C25H24O6, Relative molecular mass: 420.5, purity > 94%, then detected by the immobile western chemilumines- yellow powder) was purchased from WuXi PharmaTech cent horseradish peroxidase (HRP) substrate (Super ECL (WuXi, China), which was dissolved in dimethyl sulphox- Detection, Yeasen, China) (ABclonal, China). ide (DMSO) at 30 mM as stored concentration and was diluted in DMEM for usage. Real‑time PCR RNA was extracted after 24 h post-drug treatment. The Cell viability total RNA of samples was extracted using EZ-press RNA Cell viability was detected using a cell-titer blue kit (Pro- Purification Kit (EZBioscience, USA). A 4 × Reverse mega, USA). U87 and U251 cells (1 × 10 cells/well) were Transcription Master Mix (EZBioscience) was used for seeded in 96-well plates and cultured for 24 h, then cells reverse transcription reaction at 42 ℃ for 15 min, 95 ℃ were treated with different concentrations of CUB for for 30 S. The 2 × SYBR Green qPCR Master Mix (EZBio- 48 h. To detect cell viability, 20 μl cell-titer blue reagents science) was used to perform qPCR following this pro- were diluted in 100 μl DMEM medium. In reverse experi- tocol: denaturation (5 min, 95 ℃), and 40 amplification ments, cells were pre-treated with 4-PBA, 3-MA or CQ cycles (10 s at 95 ℃, and 30 s at 60 ℃) by using the Strata for 12 h before CUB treatment. Cell viability was quanti- Gene Mx3000p (Agilent Technologies, Inc., Santa Clara, fied using Odyssey CLx Infrared Imaging System. CA). Each gene of interest was normalized to glycer- aldehyde phosphate dehydrogenase (GAPDH) and the Cloning formation assay fold change was compared relative to the control sample. Cells were seeded in 6 well plates (1 × 10 cells/well) and Each assay was performed in triplicate and experiments exposed to 5 μM CUB for 14 days. To stain the colony, were repeated in at least three pooled cell samples. cells were washed three times by PBS, then fixed with 4% polyformaldehyde (PFA) for 30 min and subsequently stained with 1% crystal violet for 5 min. Colonies were SEM examination quantified using Odyssey CLx Infrared Imaging System. Cells were fixed by 4% glutaraldehyde and postfixed in 1% OsO in 0.1 M cacodylate buffer for 2 h. After being Apoptosis analysis 4 stained with 1% Millipore-filtered uranyl acetate, the U87 and U251 cells were seeded in 6 well plates (4 × 10 samples were then dehydrated in increased concentra- cells per well) and cultured for 24 h to achieve 90–100% tions of ethanol, then infiltrated and embedded in epoxy confluence. After reagents treatment, annexin V-pro - resin (ZXBR, Spon 812). Electron photomicrographs of pidium iodide (AV-PI) staining was conducted to assess GBM cell ultra-structures were taken with a scanning apoptosis rate following the manufacturer’s instructions electron microscope (JEM-1200EX II, JEOL; Tokyo, (Beyotime, Shanghai, China). Cell apoptosis was detected Japan). by flow cytometry (FC) (BD Biosciences, AccuriTM C6; Pan et al. BMC Neuroscience (2023) 24:10 Page 4 of 10 Transferase‑mediated deoxyuridine triphosphate‑biotin Aldrich, USA), when red fluorescence was emited in nick end labeling (TUNEL) analysis the acidic secondary lysosomes and diffuse green fluo - TUNEL staining was performed using a TUNEL apop- rescence in the cytoplasm [21]. Cells were incubated tosis detection kit (Servicebio, Wuhan). After CUB 5 μg/ml AO for 15 min, and observed under a Nikon treatment, cells were immersed in 4% PFA solution microscope (Tokyo, Japan). for 15 min then washed with PBS. Next, proteinase K solution (20 μg/ml) was added for 30 min they washed Statistical analysis with PBS. A 50-μl Equilibration Buffer was added to The data were analyzed using GraphPad Prism 7.0. All each sample for 10 min. Then added 56 μl TdT-labeled data presented as means ± SEM. Statistical analysis was reaction mixture (the mixture was compounded: conducted by one-way analysis of variance (ANOVA). Recombinant TdT enzyme:CF640-dUTP Labeling The statistical significances labeled as *P < 0.05, **P < 0.01, Mix: Equilibration Buffer = 1 µl:5 µl:50 µl (1:5:50). The ***P < 0.001. cells were placed in a wet box for 60 min at 37 °C then washed with Phosphate Buffered Saline (PBS). Images were taken under fluorescence microscopy (Leica, Results Japan) and five different fields were randomly selected. CUB inhibited cell growth and induced apoptosis of GBM Three independent assays were conducted. The cell cells. numbers were calculated using ImageJ-5.0 software To determine the effect of CUB (Fig. 1a) on GBM cells, (Windows, 64-bit Java 1.8.0_112). The TUNEL-posi - two GBM cell lines U87 and U251 were treated with dif- tive rate equals the number of TUNEL-positive cells ferent concentrations of CUB (5, 10, 20, 40, 80 μM) for divided by the total cell number × 100%. 24 h. The Cell Titer-Blue results showed that GBM cells viability decreased significantly after CUB treatment, compared with normal human astrocytes (Fig. 1b). It Immunofluorescence (IF) staining also suggests that CUB suppressed cell survival in a dose- Immunofluorescence staining was performed in GBM dependent manner, while the major differences between cell lines growing in dishes of NEST cell culture (China). tumor cells and normal cells were observed at 10 and According to a standard protocol, ice-cold 4% PFA served 20 μM in 24 h. In addition, cloning formation assays con- as fixative. Primary antibodies were rabbit polyclonal Abs firmed that CUB inhibited GBM cell survival ((Fig. 1c, d). against human activating transcription factor 4 (ATF4) Furthermore, we validated the CUB’s effect on apoptosis and C/EBP-homologous protein (CHOP) (Abclonal). of the GBM cells. GBM cells were treated with CUB as Nuclei were stained with DAPI (4,6-diamidino-2′- indicated, then stained with Annexin V and PI for further phenylindol, 5 µg/ml). Secondary Abs were goat anti-rab- flow cytometry analysis. CUB-induced cell apoptosis was bit Alexa Fluor 488 IgG and goat anti-mouse Alexa Fluor found in a dose-dependent and time-dependent manner 647 (Invitrogen, Camarillo, CA). in both U87 and U251 cells (Fig. 1e, Additional file 1: Fig. S1a). Transcriptome sequencing and expression analysis U87 cells were treated with CUB then the total RNA was extracted as whole transcriptome libraries, and CUB activated ER stress pathways in human GBM cells deep sequenced by Novogene Bioinformatics Technol- Knowing that CUB could use to harness GBM cells, ogy Cooperation (Beijing, China). The protocol of dif - the underlying mechanism is still unknown. To address ferential expression analyses of genomics and Feature that, an RNA-seq analysis was employed. U87 cells were Counts of genomic features were described accordingly treated with 10 μM CUB for 24 h. Then the total RNA in previous study [17–20]. of treated and untreated cells was extracted for the tran- scriptome test. RNA-seq analysis revealed that the gene mRFP‑GFP‑LC3 transfection expression levels of ER stress and UPR pathways were The adenovirus probe mRFP-GFP-LC3 was used to significantly higher after CUB treatment (Fig. 2a, b). We examine autophagic flux. The transfected cells were next checked a group of ER stress-related genes. The incubated with 4% paraformaldehyde. Autophagic flux expression level of phosphorylation of eukaryotic initia- was observed under Nikon microscope (Tokyo, Japan). tion factor-2α (eIF2α), CHOP and ER-stress sensor inosi- tol-requiring enzyme 1(IRE1) were increased upon CUB treatment in a dose-dependent manner (Fig. 2c). To con- Acridine orange (AO) staining firm this, we detected the ER stress-related protein level, Cell apoptosis and lysosomal membrane permeabil- CHOP and p-eIF2α. Indeed, it is very consistent with the ity were evaluated by the AO staining assay (Sigma result of real-time PCR (Fig. 2d). P an et al. BMC Neuroscience (2023) 24:10 Page 5 of 10 In addition, immune fluorescence assays demonstrated cytotoxic effects of CUB (Additional file 1: Fig. S4b). that CUB cloud active ATF4 expression in U87 cells Taken together, our results indicate that CUB promotes (Fig. 2e). In conclusion, our findings indicated that CUB autophagy to induce cell death through inhibition of triggers severe ER stress via PERK-eIF2α-ATF4 pathways. PI3K/AKT/mTOR signaling. CUB induced autophagy and inhibited PI3K/mTOR/LC3B 4‑PBA could rescue the CUB triggered ER stress pathway and autophagy‑related phenotype ER stress often lead to a dramatic change of cell morphol- To better understand the roles of ER stress signaling in ogies [22]. Indeed, after treatment with CUB for 24 h, CUB-mediated ER stress and autophagy, we investi- GBM cells exhibited swelling, cytoplasmic vacuolization gated whether abolish the ER stress-related pathways and cellular tentacles retraction (Fig. 3a). Among these, could reverse the CUB-mediated phenotype. The GBM the cytoplasmic vacuoles accumulation is one of the typi- cells were pre-treated with 4-PBA (an ER stress inhibi- cal characteristics of cell autophagy [23, 24]. To verify tor) for 12 h before CUB treatment. Then, we checked this, we used scanning SEM to characterize vacuole for- the ER stress- and autophagy-related protein levels. As mation in U87 and U251 cells treated with 20 μM CUB expected, the ER stress inhibitor 4-PBA counteracted the for 12, 24 h. We observed the typical autophagy phe- expression change of p-eIF2α, CHOP and Akt/mTOR/ notype, the double-membrane structure of autophagic LC3B caused by CUB (Fig. 4a, b). Correspondingly, ER vacuoles and large cytoplasmic vacuoles on electron stress vacuoles reduced distinctly in 4-PBA pretreated micrographs (Fig. 3c, Additional file 1: Fig. S1b). To fur- cells (Fig. 4c). In addition, 4-PBA increased the viabil- ther confirm whether CUB could cause autophagy in ity of GBM cells, which was decreased by CUB treat- GBM cells, we performed mRFP-GFP-LC3 transfection ment (Fig. 4d). To further verify the effect of 4-PBA on assay and AO staining. According to the confocal fluo - cell apoptosis, we conducted TUNEL assay. In line with rescence imaging, CUB treatment increased the LC3 II cell viability results, 4-PBA counteracted CUB in apop- level in U87 and U251 cells, as indicated by increased tosis (Additional file 1: Fig. S7a). These observations con - GFP + mRFP + /GFP + mRFP- ratio and raised AO fluo - firmed that CUB-induced apoptosis via activating the ER rescence intensity (Additional file 1: Fig. S3a, b), as well stress related pathways. as increased ratio of LC3-II/ LC3I (Fig. 3b). Additionally, p-mTOR, p-p70 and p-Akt were down-regulated while Discussion mTOR, p70 and Akt were unchanged (Fig. 3b). Taken Natural products produced from the plants are impor- together, these data suggested that CUB activated the tant sources of screening leading compounds in cancer autophagy in GBM cells with classical morphological treatment. To date, more than half of the FDA-approved changes and harnessed PI3K/Akt/mTOR/LC3B signaling anti-tumor drugs come from natural products and their pathway. derivatives [25]. In this study, we revealed that natural 3-Methyladenine(3-MA), PI3K inhibitor, was selected flavonoid CUB suppressed cell growth and promoted cell to inhibit the autophagy. To verify if ER stress-induced apoptosis in GBM. Moreover, we investigated the under- autophagy cause cell death, we performed the pre- lying molecular mechanisms, and found that CUB stim- treatment of 3-MA for 4 h to detect the autophagic flux ulates ER stress-dependent autophagy in human GBM and AO staining. We observed that 3-MA suppressed cells via the PERK/eIF2 pathway. Inhibition of CUB- autophagic flux and prevented CUB-induced cell apop - induced ER stress can prevent autophagy and restored tosis (Additional file 1: Fig. S3a, b). To further verify cell vitality. Together, these data suggested that CUB is a CUB could induce autophagy-dependent cell death, we potential drug candidate for GBM therapy. added another autophagy inhibitor chloroquine (CQ). Temozolomide (TMZ) was used to treat GBM for over CQ pretreatment reversed the inhibition of PI3K/Akt/ a decade, but its treatment benefits are limited due to mTOR pathway by CUB with increased phosphoryla- resistance [26]. CUB has been reported to show anti- tion levels of mTOR and P70 (Additional file 1: Fig. S4a) proliferative activity in B16 melanoma cells, human gas- in U251 and U87 cells, and protected U87 cells from the tric carcinoma cells and human oral cancer cells with (See figure on next page.) Fig. 2 CUB activates ER stress. a, b Gene Ontology (GO) pathways a and Kyoto Encyclopedia of Genes and Genomes (KEGG) b analyzed the transcriptome sequencing of U87 GBM cells after CUB treatment. U87 cells were treated with 10 μM of CUB. c Quantitative real-time PCR (qPCR) analysis of GBM cells treated with of CUB (5,10, 20 µM) for 24 h. d Immunoblotting of the ER stress markers, p-eIF2Α and CHOP, in GBM cells (Notion: here are cropped blots, original cropped images and replicates are presented in Additional file 1: Fig. S2). e Fluorescence images of ATF4 in GBM cells exposed to CUB (10 μM) or control for 24 h. Data were mean ± SEM from three independent experiments. The statistical analysis was performed using one-way ANOVA, and the significance labeled as * P < 0.05, ** P < 0.01 and *** P < 0.001 Pan et al. BMC Neuroscience (2023) 24:10 Page 6 of 10 Fig. 2 (See legend on previous page.) P an et al. BMC Neuroscience (2023) 24:10 Page 7 of 10 Fig. 3 CUB induces GBM cell autophagy. a Morphological images of GBM cells. b Western blot analysis detected markers of the Akt/mTOR/pS70k pathway and LC3B-I, LC3-II in U251 cells treated with CUB at indicated concentration for 24 h (10 µg of the cell lysates was loaded)(Cropped blots, original cropped images and replicates are presented in Additional file 1: Fig. S5 and S6). c SEM images of U251, U87 cells treated with CUB (10, 20 µM) or DMSO for 24 h. Boxes highlight autophagic vacuoles and ER stress bubbles. Scale bars were marked in the images effective lowest concentration at the 12uM in previous cells showed high ratios of PI3K-AKT signaling path- experiments. Our studies showed that CUB induced cell way activation, U251 cells are more resistant to hypoxia apoptosis in both U87 and U251 GBM cells by ER stress- and low glucose condition [27]. The glycolysis rate of induced autophagy, here we showed that its IC50 was cells plays a vital role in mitochondrial respiration func- 10uM, which indicated it is promising for in vivo experi- tion to determine the relative contribution of autophagy, ments and clinical studies. which is consistent with the lower autophagy level that In our study, CUB induced ~ 56% cell death at the dose was observed for U251 cells compared with U87 cells of 20uM after 24 h in U87 cells, while ~ 40% cell death in in another study (Additional file 1: Fig. S1c). This may the case of U251(Fig. 1a). We found U87 displayed ear- reflect that U251 cells has a slowly autophagy response to lier response to CUB- induced ER stress as evidenced ER stress induced by CUB. by the earlier appearance of intracellular vacuoles after Although CUB shows promising anticancer-activity 12 h treatment of CUB (Additional file 1: Fig. S1c). Pre- in multiple cancers types via inducing apoptosis, our vious research reported that although U87 and U251 knowledge of its underlying molecular mechanisms is Pan et al. BMC Neuroscience (2023) 24:10 Page 8 of 10 Fig. 4 The ER stress inhibitor 4-PBA counteracts CUB. a, b Western blot detected the protein levels of p-eIF2Α, CHOP Akt/mTOR/pS70k pathway, LC3B, and β-actin in GBM cells pretreated with 4-PBA (2 mM) for 12 h before exposure to CUB (20 µM)(Cropped blots, original cropped images and replicates are presented in Additional file 1: Fig. S8 and. S9). c Morphological images of U87 cells treated with CUB (10 µM), 4-PBA (2 mM), and CUB (10 µM) with 4-PBA (2 mM) together, respectively (Cropped blots). d Cell viability of GBM cells CUB (10 µM), 4-PBA (2 mM) and CUB (10 µM) with 4-PBA (2 mM) together. The scale bars were marked in the image. All data were the mean ± SEM of values from experiments performed in triplicate. * P < 0.05, ** P < 0.01 and *** P < 0.001 compared to control very limited, e.g. regulating the tumour necrosis factor in cancer cells, it needs deep understanding for the α (TNFα) and nuclear fractor-κB (NF-κB) expression, PERK/ATF4/CHOP in crosstalk between ER stress and affecting the anti-inflammatory response [28], and acti - apoptosis. vating the MAPK signaling pathway to promote apop- However, inhibition of ER stress does not fully abro- tosis [13]. Here, we observed ER stress was induced by gate cell death. This may be because excess CUB impairs CUB treatment in GBM cells. Based on transcriptome cell viability through activation of NF-κB (Additional analysis results, we demonstrated that the CUB-induced file 1: Fig. S7b). Additionally, it has shown impres- GBM cell apoptosis is dependent upon persistent ER sive anti-inflammatory and neuro-protective effects on stress that activates the PERK/ATF4/CHOP pathway. It macrophages. In GBM, tumor-associated macrophages is very well in line with that persistent ER stress causes comprise half of tumor mass and involving in tumor tumor apoptosis, provides prospects for tumor treatment progression [33]. We believe that it will be worth further strategies [22, 29]. In addition, we also observed cell cycle exploration through its promotion effect on macrophages inhibition upon CUB. This is consistent with a previous and resistance to tumors, if it can cross the blood–brain study that in response to PERK-ATF4-CHOP UPR reac- barrier. Currently, the concentrations of CUB we used are tion, eIF2α is activated to inhibit cyclin-D1 expression, close to those used in other anti-tumor reports. However, causing cell cycle G1 blocking [30]. we did not conduct in vivo experiment to assess its per- Furthermore, our results support that drug-induced meability to the blood–brain barrier and suitable admin- persistent ER stress resulted in cell death via autophagy istration concentration. We insight it will be further [31]. CUB treated cells showed the classic morphologi- explored in the next studies. cal and molecular characteristics of autophagy, such as autolysosomes formation and the membrane-binding Abbreviations LC-II. [32]. Mechanically, the result highlighted CUB- GBM Glioblastoma induced ER stress-activated autophagy through Akt/CUB Cudraflavone B APG Apigenin-7-O-β-d-(-6″-p-coumaroyl)-glucopyranoside mTOR/RPS6KB1 pathway. In line with this, the ER stress UPR Unfolded protein response inhibitor increased the activity of Akt/mTOR/RPS6KB1 ROS Reactive oxygen species signaling, leading to less autophagy flux. These results 4-PBA 4-Phenylbutyric acid MAO Monoamine oxidase highlighted this pathway as a potential mediator of CUB- NHA Normal human astrocytes induced autophagy in GBM cells. Considering the dys- FBS Fetal bovine serum regulated and reprogrammed metabolic environment DMSO Dimethyl sulphoxide. P an et al. BMC Neuroscience (2023) 24:10 Page 9 of 10 FC Flow cytometry Funding RIPA Radio immuno precipitation assay This work was supported by the Young Scientists Fund of the National Natural SDS-PAGE S odium dodecyl sulfate polyacrylamide gel electropheresis Science Foundation of China (81903028), Innovative and Entrepreneurial Tal- PVDF Polyvinylidene fluoride ent Program of Jiangsu for Guohua Shi team, the China Postdoctoral Science HRP Horseradish peroxidase Foundation (2020M671599), Projects of International Cooperation of Jiangsu GAPDH Gly ceraldehyde phosphate dehydrogenase Province (No. BZ2020004). Suzhou municipal key clinical disciplines cultivate TUNEL Transferase-mediated deoxyuridine triphosphate-biotin nick end program (SZFCXK202142), Suzhou Medical Innovation Applied Research labeling Project (SKYD2022090). PBS Phosphate buffered saline IF Immunofluorescence Availability of data and materials CHOP C/EBP-homologous protein The datasets generated during and analyzed during the current study are not IRE1 ER-stress sensor inositol-requiring enzyme 1 publicly available due to we propose to continue our study of this drug in eIF2α Eukaryotic initiation factor-2α GBM therapy but are available from the corresponding author on reasonable KEGG Kyotoe ncyclopedia of genes and genomes request. GO Gene ontology qPCR Quantitative real-time PCR Declarations TMZ Temozolomide TNFα Tumour necrosis factor α Ethics approval and consent to participate CQ Chloroquine Not applicable. Supplementary Information Consent for publication Not applicable. The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s12868- 023- 00778-4. Competing interests Authors state no competing interest. Additional file 1. Fig. S1. CUB induced autophagy through ER stress- dependent pathways. (a)Flow cytometry analysis results of U87, U251 cells at 12h treatment of CUB (10μM, 20μM). (b) Representative SEM Received: 18 January 2022 Accepted: 24 January 2023 images of U87, U251 cells occurred autophagy induced by CUB (10μM, 20μM). (c)Morphological images of GBM cells at 6h of CUB treatment. n = 3 per group. Means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Fig. S2. CUB activates ER stress, blots origin images and replicates of Fig2d. Fig. S3. CUB activates autophagy flux in GBM cells. (a) Confocal images and References the ratio of GFP-LC3/mRFP-LC3 at 12h. CUB (10μM), 3-MA(10mM). (b) 1. Aldape K, Brindle KM, Chesler L, Chopra R, Gajjar A, Gilbert MR, Gottardo Confocal images of AO staining at 12h. Fig. S4. CUB induced cell death N, Gutmann DH, Hargrave D, Holland EC, et al. Challenges to curing through autophagy-dependent pathways. (a) Western blotting analysis of primary brain tumours. Nat Rev Clin Oncol. 2019;16(8):509–20. U87, U251 cells at 12h treatment of CUB (0μM, 20μM) or 3-MA(1mM) and 2. Brinkman TM, Krasin MJ, Liu W, Armstrong GT, Ojha RP, Sadighi ZS, Gupta CQ(30μM) pretreatment followed treatment of CUB(20μM). (b) Autophagy P, Kimberg C, Srivastava D, Merchant TE, et al. Long-term neurocogni- inhibitors suppressed U87 cell proliferation detected by the Cell-title tive functioning and social attainment in adult survivors of pediatric Blue assay. Cells were incubated with 3-MA(1mM) or CQ (30μM) 12 h and CNS tumors: results from the St Jude lifetime cohort study. J Clin Oncol. then cultured in CUB(20μM) medium for the indicated times. Means ± 2016;34(12):1358–67. SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Fig. S5. CUB induces GBM cell 3. Le Rhun E, Preusser M, Roth P, Reardon DA, van den Bent M, Wen P, autophagy, blots origin images and replicates of Fig. 3b (U87). Fig. S6. Reifenberger G, Weller M. Molecular targeted therapy of glioblastoma. CUB induces GBM cell autophagy, blots origin images and replicates of Cancer Treat Rev. 2019;80:101896. Fig. 3b (U251). Fig. S7. CUB promoted apoptosis and NF-κB activation in 4. Moreau A, Febvey O, Mognetti T, Frappaz D, Kryza D. Contribution of U87 cells. (a) Images of TUNEL staining Green: TUNEL-positive cells, Blue: different positron emission tomography tracers in glioma management: DAPI. (b) Western Blot analysis of NF-κB pathway. All data are expressed as focus on glioblastoma. Front Oncol. 2019;9:1134. the mean ± SEM of values from experiments performed in triplicate. * P 5. Park MN, Song HS, Kim M, Lee MJ, Cho W, Lee HJ, Hwang CH, Kim S, < 0.05, ** P < 0.01 and *** P < 0.001 compared to control (Cropped blots). Hwang Y, Kang B, et al. 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Journal
BMC Neuroscience – Springer Journals
Published: Jan 31, 2023
Keywords: GBM; Endoplasmic reticulum stress; Unfolded protein response; Autophagy