Figure 2 - uploaded by Aurélie Henry
Content may be subject to copyright.
OPN inhibition affects the survival of GBM cells and delays DNA double-strand break repair. A. Representative picture of U251-MG cells transiently transfected with siRNAs directed against OPN (siRNA OPN#1 and #3) and exposed to a single dose of 2 Gy. Cells were then submitted to clonogenic assay. Note the difference in term of colonies number when cells are depleted in OPN. B. Surviving fraction of U87-MG, U87-MG vIII and U251-MG was measured in OPN depleted cells compared to control after an exposition to a growing dose of irradiation ranging from 0 to 6 Gy. OPN depletion was validated by western blot. Data are presented as mean ± SEM of one representative experiment, n=3 representing 6 wells for each condition. C. U87-MG cells were transiently transfected with siRNAs directed against OPN (siRNA OPN#1 and #3). After 48 hours, monolayers of transfected cells were exposed to a single dose of irradiation with 2 Gy and harvested 3 hours post irradiation for comet assay. Representative cells and comet cells were imaged using epifluorescence microscopy (left panel). White arrows point to cells with comet's tail. The number of cells with comet's tail was counted for each condition (right panel). Scale bar = 50 μM. D. The tail DNA content and the olive tail moment were calculated, as described under Material and Methods section, in order to evaluate the amount of DNA fragments under each condition. E. U87-MG cells were transiently transfected with siRNAs directed against OPN (siRNA OPN#1 and #3). The number of cells with comet's tail was counted at 1h, 3h and 12h postirradiation (2Gy). Data are presented as mean ± SEM of one representative experiment, n=3 representing 6 wells for each condition. ***, P<0.001. **, P<0.01. *, P<0.05. Immunoblot data were normalized for β-actin.
Source publication
Glioblastoma (GBM) represents the most aggressive and common solid human brain tumor. We have recently demonstrated the importance of osteopontin (OPN) in the acquisition/maintenance of stemness characters and tumorigenicity of glioma initiating cells. Consultation of publicly available TCGA database indicated that high OPN expression correlated wi...
Contexts in source publication
Context 1
... performed clonogenic assays on transiently OPN-depleted cells after exposure to growing doses of irradiation (0 to 6 Gy). We observed that OPN- depleted GBM cells formed fewer colonies compared to control as shown for U251-MG cells transfected with two siRNAs specifically directed against OPN (siRNA OPN#1 and #3) and exposed to 2 Gy ( Figure 2A). Non-irradiated control cells showed a decreased number of colonies for U87-MG and U87-MG vIII upon OPN depletion while U251-MG cells did not (Supplementary Figure S1C). ...
Context 2
... control cells showed a decreased number of colonies for U87-MG and U87-MG vIII upon OPN depletion while U251-MG cells did not (Supplementary Figure S1C). The calculation of cell survival fractions further demonstrated that OPN-depleted GBM cells were significantly sensitized to irradiation for all GBM cell lines analyzed when compared to control cells ( Figure 2B). Consistent with their known exacerbated radioresistance [21], control and OPN-depleted U87-MG vIII showed a higher viability than parental U87-MG cells. ...
Context 3
... with their known exacerbated radioresistance [21], control and OPN-depleted U87-MG vIII showed a higher viability than parental U87-MG cells. OPN efficient depletion is shown in Figure 2B. These results indicate for the first time that OPN inhibition consistently induced GBM cells radiosensitivity. ...
Context 4
... assess DNA damage response in OPN depleted GBM cells, we performed single-cell gel electrophoresis comet assay. Three hours after a single exposure to 2 Gy-irradiation, OPN-depleted cells (siRNA OPN#1 and #3) showed a higher proportion of cells with comet's tail than control cells as shown and quantified in Figure 2C. Measurement of tail DNA content and the olive tail moment confirmed that OPN-deficient cells had significantly more DNA fragments compared to control cells (P<0.0001) ( Figure 2D). ...
Context 5
... hours after a single exposure to 2 Gy-irradiation, OPN-depleted cells (siRNA OPN#1 and #3) showed a higher proportion of cells with comet's tail than control cells as shown and quantified in Figure 2C. Measurement of tail DNA content and the olive tail moment confirmed that OPN-deficient cells had significantly more DNA fragments compared to control cells (P<0.0001) ( Figure 2D). Next, we addressed the question whether DNA damage was persistent with time in OPN-depleted cells when compared to control cells. ...
Context 6
... this purpose, we counted the number of cells with comet's tail in non-irradiated cells and after 1h, 3h and 12h post 2 Gy-irradiation. We observed that OPN-deficient U87-MG cells presented a significantly higher number of cells with comet's tail than control cells up to 12h post irradiation ( Figure 2E). The number of cells with comet's tail in non-irradiated cells did not show any significant difference between OPN-depleted and control cells. ...
Context 7
... next validated our observations in IPTG- inducible shOPN U87-MG clones before their subsequent use in in vivo experiments. IPTG-inducible shOPN U87-MG cells were maintained in a medium containing IPTG during 2, 5 and 10 days and the efficiency of OPN silencing was assessed at the mRNA level (Supplementary Figure S2). Accordingly, IPTG addition during 4 days induced a significant decrease of OPN level as assessed by western blot ( Figure 4B). ...
Similar publications
Glioblastoma multiforme (GBM) is a brain tumor with high mortality and recurrence rate. Radiotherapy and chemotherapy after surgery are the main treatment options available for GBM. However, patients with glioblastoma have a grave prognosis. The major reason is that most GBM patients are resistant to radiotherapy. UBA1 is considered an attractive p...
Citations
... Blocking the OPN gene in combination with irradiation led to the decreased viability of breast cancer cells and induction of apoptosis, which highlights the role of OPN in the response to ionizing radiation [15]. In glioblastoma, OPN inhibition resulted in increased radiosensitivity and tumor size reduction in vivo [16]. It was found that autophagy-induced OPN suppression abrogated the radioresistance of NSCLC cells [17]. ...
Osteopontin (OPN)-CD44 signaling plays an important role in promoting tumor progression and metastasis. In cancer, OPN and CD44 overexpression is a marker of aggressive disease and poor prognosis, and correlates with therapy resistance. In this study, we aimed to evaluate the association of single nucleotide polymorphisms (SNPs) in the OPN and CD44 genes with clinical outcomes in 307 non-small cell lung cancer (NSCLC) patients treated with radiotherapy or chemoradiotherapy. The potential impact of the variants on plasma OPN levels was also investigated. Multivariate analysis showed that OPN rs11730582 CC carriers had a significantly increased risk of death (p = 0.029), while the CD44 rs187116 A allele correlated with a reduced risk of locoregional recurrence (p = 0.016) in the curative treatment subset. The rs11730582/rs187116 combination was associated with an elevated risk of metastasis in these patients (p = 0.016). Furthermore, the OPN rs1126772 G variant alone (p = 0.018) and in combination with rs11730582 CC (p = 7 × 10−5) was associated with poor overall survival (OS) in the squamous cell carcinoma subgroup. The rs11730582 CC, rs187116 GG, and rs1126772 G, as well as their respective combinations, were independent risk factors for unfavorable treatment outcomes. The impact of rs11730582-rs1126772 haplotypes on OS was also observed. These data suggest that OPN and CD44 germline variants may predict treatment effects in NSCLC.
... Blocking the OPN gene in combination with irradiation led to decreased viability of breast cancer cells and induction of apoptosis, which highlights the role of OPN in the response to ionizing radiation [15]. In glioblastoma, OPN inhibition resulted in increased radiosensitivity and tumor size reduction in vivo [16]. It was found that autophagy-induced OPN suppression abrogated radioresistance of NSCLC cells [17]. ...
Osteopontin (OPN)-CD44 signaling plays an important role in promoting tumor progression and metastasis. In cancer, OPN and CD44 overexpression is a marker of aggressive disease and poor prognosis, and correlates with therapy resistance. In this study, we aimed to evaluate the association of single nucleotide polymorphisms (SNPs) in the OPN and CD44 genes with clinical outcomes in 307 non-small cell lung cancer (NSCLC) patients treated with radiotherapy or chemoradiotherapy. The potential impact of the variants on plasma OPN levels was also inves-tigated. Multivariate analysis showed that OPN rs11730582 CC carriers had a significantly in-creased risk of death (p = 0.029), while the CD44 rs187116 A allele correlated with reduced risk of locoregional recurrence (p = 0.016) in the curative treatment subset. The rs11730582/rs187116 combination was associated with an elevated risk of metastasis in these patients (p = 0.016). Fur-thermore, the OPN rs1126772 G variant alone (p = 0.018) and in combination with rs11730582 CC (p = 7x10-5) was associated with poor OS in the squamous cell carcinoma subgroup. The rs11730582 CC, rs187116 GG and rs1126772 G, as well as their respective combinations, were in-dependent risk factors for unfavorable treatment outcomes. The impact of rs11730582-rs1126772 haplotypes on OS was also observed. These data suggest that OPN and CD44 germline variants may predict treatment effects in NSCLC.
... High serum OPN level is found as a poor prognostic indicator in GBMs (Sreekanthreddy et al. 2010). Henry et al. revealed that OPN plays a role in the initiation of DNA repair in response to irradiation in GBM cells and hence induce GBM radioresistance (Henry et al. 2016). Le et al. (2003) identified osteopontin as a potential hypoxia marker. ...
Hypoxia is a prevalent hallmark of many malignant neoplasms. The aim was to assess the serum hypoxia biomarkers HIF-1α, VEGF, osteopontin, erythropoietin, caveolin-1, GLUT-1, and LDH pre- and post-radiotherapy in patients with brain tumors. The study was conducted on 120 subjects were divided into two groups: group I: 40 healthy volunteers as control group. Group II: 80 brain tumor patients were subdivided into glioblastoma subgroup: 40 glioblastoma patients, meningioma subgroup: 40 malignant meningioma patients. Two venous blood samples were collected from every patient prior to and following RT and one sample from controls. Biomarkers were assayed by ELISA. In glioblastoma subgroup, HIF-1α, VEGF, and LDH were significantly increased after RT. On the contrary, these biomarkers were significantly decreased after RT in malignant meningioma subgroup. Osteopontin was significantly increased after RT in both subgroups. Regarding erythropoietin, it was significantly decreased in both subgroups when compared to before RT. Caveolin-1 showed a significant increase in glioblastoma subgroup after RT comparing to before RT. GLUT-1 was significantly increased after RT in both subgroups comparing to before RT. Association of significant elevation of hypoxia biomarkers either pre- or post-RT with aggressive tumor such as glioblastoma indicates that, they are markers of malignancy and may have a role in tumor development and progression.
... CD44 and osteopontin (CD44 ligand secreted from myeloid-and tumor cells) was proposed as a promising immune checkpoint in human colon carcinoma [22] and moreover implicated in the pathogenesis of several cancer types including GBMs [23]. GBM cellderived osteopontin increased tumor invasion and radiation resistance in vitro and in vivo [24,25]. In microglia and macrophages, osteopontin enhanced activation and induced a pro-tumorigenic phenotype [26]. ...
Glioblastoma is the most common primary malignant brain tumor in adults with an overall survival of only 14.6 months. Hypoxia is known to play a role in tumor aggressiveness but the influence of hypoxia on the immune microenvironment is not fully understood. The aim of this study was to investigate the expression of immune‐related proteins in normoxic and hypoxic tumor areas by digital spatial profiling. Tissue samples from 10 glioblastomas were stained with a panel of 40 antibodies conjugated to photo‐cleavable oligonucleotides. The free oligo‐tags from normoxic and hypoxic areas were hybridized to barcodes for digital counting. Differential expression patterns were validated by Ivy Glioblastoma Atlas Project (GAP) data and an independent patient cohort. We found that CD44, Beta‐catenin and B7‐H3 were upregulated in hypoxia, whereas VISTA, CD56, KI‐67, CD68 and CD11c were downregulated. PD‐L1 and PD‐1 were not affected by hypoxia. Focusing on the checkpoint‐related markers CD44, B7‐H3 and VISTA, our findings for CD44 and VISTA could be confirmed with Ivy GAP RNA sequencing data. Immunohistochemical staining and digital quantification of CD44, B7‐H3 and VISTA in an independent cohort confirmed our findings for all three markers. Additional stainings revealed fewer T cells and high but equal amounts of tumor‐associated microglia and macrophages in both hypoxic and normoxic regions. In conclusion, we found that CD44 and B7‐H3 were upregulated in areas with hypoxia whereas VISTA was downregulated together with the presence of fewer T cells. This heterogeneous expression should be taken into consideration when developing novel therapeutic strategies.
... This protein is a pro-inflammatory and largely associated with cancer pathophysiology, cell adhesion, migration, tumour progression, metastasis development and resistance to treatment [18]. GBM patients have positive association between OPN expression and malignancy grade besides OPN serum level was a poor prognostic marker for GBM patients [19]. ...
... In another research stated that high OPN expression was associated with poor survival in GBM patients treated with radiotherapy. Also same researchers indicated that OPN depletion makes GBMCs more susceptible to radiation and DNA damage accumulation after irradiation is higher in these cells than in control cells [19]. In our experiments, we observed OPN expression in GBMCs both in day 1 and day 12. ...
Objectives: The aim of this study is to investigate the radiosensitivity of Glioblastoma multiforme (GBM; U87 MG) and astrocyte (SVG p12) cell lines in vitro through the signalling pathways. Methods: GBM and astrocytes were treated with 2, 4, 6, and 8 gray of ionized radiation, followed by a clonogenic assay. The effective dose of radiation was determined as 2 gray. Immunofluorescence technics selected to analyse the macrophage migration inhibiting factor (MIF), nuclear factor of activated T-cells cytoplasmic 2 (NFATc2), osteopontin (OPN), mammalian target of rapamycin (mTOR) and stage-specific embryonic antigen-1 (SSEA-1). Additionally, p53 and cell cycle assays were performed. Results: On day 1, astrocytes showed decreased expression of MIF, OPN and mTOR and increased expression of SSEA-1 in the test group after 2 gray radiation. GBM showed decreased expression of p53 and mTOR, but increased expression of NFATc2. The results of MIF expression were found higher in GBM compared to astrocytes on day 1. Interestingly, on day 12, increased expression of SSEA-1, OPN and p53 were observed in both cell lines’ test groups. Further analysis showed that all control groups of GBM and astrocytes were significantly accumulated in the S phase. After radiotherapy application, percentage of GBM in G0/G1 phases and especially in G2/M phases increased; conversely, in the S phase it decreased. Moreover, percentage of astrocytes increased in the S phase and decreased in G0/G1 phases and in G2/M phases. Conclusions: This combination of findings suggests that as a result of the radiotherapy effect, GBM started to accumulate on check points. The central question in this study focused on changes in molecular protein expression in cancer cells after radiotherapy, particularly key signalling pathways of tumorigenesis and a new possible point of view for treating such diseases.
... OPN is overexpressed in hypoxia, [29] associated with CSCs in periarteriolar niches and is suggested to promote tumor recurrence by supporting migration of the CSCs out of these niches [30]. Furthermore, OPN plays an essential role for tumorigenicity, maintenance of stemness of CSCs [31] and is involved in DNA damage repair post irradiation promoting radio-resistance of glioma cells [32]. ...
Background
Despite of a multimodal approach, recurrences can hardly be prevented in glioblastoma. This may be in part due to so called glioma stem cells. However, there is no established marker to identify these stem cells.
Methods
Paired samples from glioma patients were analyzed by immunohistochemistry for expression of the following stem cell markers: CD133, Musashi, Nanog, Nestin, octamer-binding transcription factor 4 (Oct4), and sex determining region Y-box 2 (Sox2). In addition, the expression of osteopontin (OPN) was investigated. The relative number of positively stained cells was determined. By means of Kaplan–Meier analysis, a possible association with overall survival by marker expression was investigated.
Results
Sixty tissue samples from 30 patients (17 male, 13 female) were available for analysis. For Nestin, Musashi and OPN a significant increase was seen. There was also an increase (not significant) for CD133 and Oct4. Patients with mutated Isocitrate Dehydrogenase-1/2 (IDH-1/2) status had a reduced expression for CD133 and Nestin in their recurrent tumors. Significant correlations were seen for CD133 and Nanog between OPN in the primary and recurrent tumor and between CD133 and Nestin in recurrent tumors. By confocal imaging we could demonstrate a co-expression of CD133 and Nestin within recurrent glioma cells. Patients with high CD133 expression had a worse prognosis (22.6 vs 41.1 months, p = 0.013). A similar trend was seen for elevated Nestin levels (24.9 vs 41.1 months, p = 0.08).
Conclusions
Most of the evaluated markers showed an increased expression in their recurrent tumor. CD133 and Nestin were associated with survival and are candidate markers for further clinical investigation.
... OPN is overexpressed in hypoxia, [29] associated with CSCs in periarteriolar niches and is suggested to promote tumor recurrence by supporting migration of the CSCs out of these niches [30]. Furthermore, OPN plays an essential role for tumorigenicity, maintenance of stemness of CSCs [31] and is involved in DNA damage repair post irradiation promoting radio-resistance of glioma cells [32]. ...
Background: Despite of a multimodal approach, recurrences can hardly be prevented in glioblastoma. This may be in part due to so called glioma stem cells. However, there is no established marker to identify these stem cells.
Methods: Paired samples from glioma patients were analyzed by immunohistochemistry for expression of the following stem cell markers: CD133, Musashi, Nanog, Nestin, octamer-binding transcription factor 4 (Oct4), and sex determining region Y-box 2 (Sox2). In addition, the expression of osteopontin (OPN) was investigated. The relative number of positively stained cells was determined. By means of Kaplan-Meier analysis, a possible association with overall survival by marker expression was investigated.
Results: Sixty tissue samples from 30 patients (17 male, 13 female) were available for analysis. For Nestin, Musashi and OPN a significant increase was seen. There was also an increase (not significant) for CD133 and Oct4. Patients with mutated Isocitrate Dehydrogenase-1/2 (IDH-1/2) status had a reduced expression for CD133 and Nestin in their recurrent tumors. Significant correlations were seen for CD133 and Nanog between OPN in the primary and recurrent tumor and between CD133 and Nestin in recurrent tumors. By confocal imaging we could demonstrate a co-expression of CD133 and Nestin within recurrent glioma cells. Patients with high CD133 expression had a worse prognosis (22.6 vs 41.1 months, p = 0.013). A similar trend was seen for elevated Nestin levels (24.9 vs 41.1 months, p = 0.08).
Conclusions: Most of the evaluated markers showed an increased expression in their recurrent tumor. CD133 and Nestin were associated with survival and are candidate markers for further clinical investigation.
... e search for an effective gene target to enhance the sensitivity of radiotherapy is a vital and urgent issue for the therapy of GAC. Previous studies revealed that SPP1 is involved in the radiosensitivity of a variety of cancers [16,17]. In the current study, we found that SPPI overexpression was associated with poor prognosis in GAC patients, and si-SPP1 pretreatment decreased invasion and increased irradiation-induced DNA damage, cell cycle arrest, and cell death in GAC cells; the underlying mechanism involves suppression of the Wnt/β-catenin pathway. ...
Purpose. Radiotherapy has been widely applied for the treatment of locally advanced and metastatic gastric adenocarcinoma (GAC). The aberrant expression of secreted phosphoprotein 1 (SPP1) is involved in radiosensitivity in a variety of cancers. The present study aims to characterize the clinical significance of SPP1 expression in GAC and its role and underlying mechanism of radiosensitivity. Methods. The SPP1 expression in GAC tissues and pericarcinomatous tissues was determined by QRT-PCR and immunohistochemistry, and the SPP1 expression in GAC cell lines (BGC823, AGS, and SGC7901) and normal human gastric epithelial cell line (GES-1) was determined by western blot. T-test, one-way ANOVA, Cox regression model, and Kaplan–Meier plotter were applied to further assess the association between SPP1 expression and the prognosis of the patients with GAC. After irradiation and transfection with si-SPP1 combined with or without Wnt/β-catenin pathway inhibitor (XAV939), western blot, transwell, flow cytometry, and TOP-flash reporter assay were applied to detect DNA damage, invasion, apoptosis, cell cycle, and activation of Wnt/β-catenin pathway, respectively. Results. SPP1 mRNA and protein levels in GAC tissues were both dramatically higher than those in pericarcinomatous tissues. SPP1 overexpression was positively associated with tumor size, nodal status, and histological grade of GAC patients. SPP1 overexpression, depth of invasion, and nodal status were independent prognostic factors for the patients. High SPP1 expression was negatively related to the overall survival in patients with GAC. We found that SPP1 knockdown enhanced the radiosensitivity of GAC cell lines (AGS and SGC7901). Increasing H2AX phosphorylation, apoptosis and G2/M phase arrest, and decreasing invasion were observed after the administration of si-SPP1 and irradiation. Radiosensitivity of SPP1 was mainly dependent on the Wnt/β-catenin signal pathway. XAV939 could enhance these phenomena induced by irradiation combined with SPP1 knockdown. Conclusion. This study demonstrates that SPP1 suppresses Wnt/β-catenin signaling to enhance the radiosensitivity of GAC via inhibiting invasion and accelerating DNA damage, G2/M phase arrest, and apoptosis.
1. Introduction
Gastric cancer (GC) is an aggressive malignancy with an extremely poor prognosis and a high incidence [1]. The incidence of gastric adenocarcinoma (GAC) accounts for 95 percent of gastric malignant tumors [2]. Accumulated evidence has demonstrated that radiotherapy has a role in the management of neoadjuvant, adjuvant, and palliative treatment of GC [3, 4]. However, because of the low radiosensitivity of GC, no difference in survival was observed in GAC patients receiving radiotherapy [5, 6]. Therefore, it is urgent and imperative to find an effective radiosensitizer to enhance the curative effect of radiotherapy and alleviate its toxicity to the tissues and organs around the radiation field.
Secreted phosphoprotein 1 (SPP1), which is also known as bone sialoprotein 1, osteopontin (OPN), early T-lymphocyte activation 1, and Eta-1 protein, controls the cell growth, proliferation, apoptosis, and migration [7]. High plasma concentrations of SPP1 are correlated with a poor prospect for patients with head and neck cancer after radiotherapy, which can prognosticate clinically relevant hypoxia, and might determine patients who will benefit from modifying hypoxia during radiotherapy [8]. Studies have indicated that SPP1 is elevated in many malignancies, such as ovarian cancer [9], cervical cancer [10], and breast cancer [11]. And, high SPP1 expression level at the end of radiotherapy is correlated with poor survival, such as glioma [12] and non-small-cell lung cancer [13]. However, the role of SPP1 in radiosensitivity in GAC remains unclear. Consequently, the association between SPP1 and radiosensitivity in GAC needs to be investigated further.
In this study, we found that high SPP1 was associated with poor prognosis in GAC patients. We observed that SPP1 knockdown pretreatment increased H2AX phosphorylation, apoptosis and G2/M phase arrest, and decreased invasion in response to irradiation in AGS and SGC7901 cells. Additionally, we noticed that SPP1 could activate the Wnt/β-catenin signal pathway in GAC cells through TOP-flash reporter assay and western blot validation, and pretreatment with Wnt/β-catenin signal pathway inhibitor (XAV939) sensitized GAC cells to IR.
2. Methods
2.1. Patients
A total of 198 tissue specimens, including 72 cases of adjacent tissues (3-4 cm from the tumor tissue) and 126 cases of gastric adenocarcinomas, were collected from patients with GAC who underwent surgery from September 2010 to September 2015 at the Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, China. The specimens were confirmed by immunohistochemistry (IHC) and included in the clinical/prognostic analysis. Participants who met the following criteria were included: histologically confirmed diagnosis of GAC, no history of prior anticancer therapies, underwent radical surgery, and complete follow-up data available. Written informed consents were signed by all participants. Ethical approval to conduct this study was obtained from the ethics committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine.
2.2. Immunohistochemistry
All specimens were routinely fixed in 10% buffered formalin and embedded in paraffin. And, the 4-μm-thick tissue sections that were cut from wax blocks were prepared on APES-coated glass slides. Slides were deparaffinized in xylene, rehydrated in graded ethanol, and immersed in 3% hydrogen peroxidase-methanol for 15 minutes to eliminate endogenous peroxidase activity. A microwave antigen retrieval procedure was performed for 5 min in 10 mM citrate buffer (pH 6.0). The sections were incubated with a primary anti-SPP1 antibody (1 : 200, ab8448, Abcam, Shanghai, China) at 4°C for overnight. After incubation with HRP-conjugated secondary antibody (1 : 50, ab6721, Abcam) at room temperature for 30 minutes, they are visualized by using 3,3′-diaminobenzidine and then counterstained with hematoxylin for 3 min.
Two experienced pathologists blinded to the clinical data were responsible for reviewing the immunoreactivity of SPP1. The score of staining intensity as well as the proportion of immunostaining positive cells was between 0–3 and 0–4, respectively. The proportions were as follows: 0, negative; 1, ≤10% positive cells; 2, >10% but ≤50% positive cells; 3, >50% but ≤75% positive cells; and 4, >75% positive cells. The staining intensity was as follows: 0, absent; 1, weak; 2, moderate; and 3, strong. The two scores were multiplied to obtain an overall protein expression score. For statistical analysis, the final evaluation criteria of SPP1 expression were as follows: 0–4 as low expression and 5–12 as high expression.
2.3. RNA Extraction and RT-qPCR
Total RNA from GAC tissues and pericarcinomatous tissue was extracted by TRIzol solution (Invitrogen, CA, USA). To synthesized cDNA, PrimeScript™ IV 1st strand cDNA Synthesis Mix (Takara, Dalian, China) was applied. The QPCR was carried out by the SYBR Premix ExTaq TM II (Takara, Dalian, China) by ABI 7900 qRT-PCR system (Applied Biosystems, Foster City, CA, United States). The cDNA was then subjected to qRT-qPCR by SYBR Premix ExTaq TM II (Takara, Dalian, China) to evaluate the relative mRNA levels of SPP1 on an Applied Biosystems real-time PCR machine (ABI 7900HT, CA, USA), and β-actin was applied as the internal control. And, the relative mRNA level was calculated by utilizing the 2−ΔΔCt method.
Primer sequence: SPP1 F: 5′-TTTGTTGTAAAGCTGCTTTTCCTC-3′ R: 5′-GAATTGCAGTGATTTGCTTTTGC-3′ β-Actin F: 5′-CTCTCTCTACCTACATCTCTACTAAAA-3′ R: 5′-AACTCTAACTCTCTCTCTAACTACTTCTC-3′
2.4. Cell Culture
Normal human gastric epithelial cell line (GES-1) and human stomach gastric adenocarcinoma cell line (AGS, SGC7901, and BGC823) were obtained from the Center for Chinese Typical Cultures Preservation, Wuhan University. All cell lines were cultured in 1640 media supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2 incubator. In the subsequent experiments, cells were pretreated with or without a β-catenin inhibitor XAV939 (10 µM, Sigma-Aldrich, Darmstadt, Germany) for 48 h and then were exposed to 4 Gy for 24 h with X-ray irradiator RS2000 (RAD SOURCE, USA).
2.5. Transfection
The AGS and SGC7901 cells (5 × 10⁶/ml) were cultivated at 37°C under 5% CO2 water-saturated atmosphere in 1640 medium supplemented with 10% FBS. Two si-RNAs were synthesized by GenePharma (Shanghai, China) and transfected into GAC cell lines using lipofectamine 3000 reagent (siRNA: lipofectamine = 10 nM:1 µL) (Invitrogen, Shanghai, China) for 24 h. Then, serum-free transfection solution was discarded and replaced with 1640 medium supplemented with 10% FBS for further culture. After 48 h, cellular proteins can be extracted to verify the knockdown efficiency.
The siRNA sequences were as follows: Si-SPP1-1, 5′-CGAUCGAUAGUGCCGAGAAGC-3′ Si-SPP1-2, 5′-AGCUAGUCCUAGACCCUAAGA-3′
2.6. Western Blot
Total protein was isolated by RIPA lysis buffer (Abcam, Shanghai, China), and then the protein concentration was measured using a Pierce BCA assay (Abcam, Shanghai, China). Protein (40 μg) was separated on SDS-PAGE and transferred to polyvinylidene (PVDE) membrane. After being blocked with 5% nonfat milk at room temperature for 1 h, the membranes were incubated with the primary antibodies anti-SPP1 (1 : 1000, ab8448), γ-H2AX (1 : 1000, ab81299), Bax (1 : 1000, ab32503), Bcl-2 (1 : 1000, ab32124), c-JUN (1 : 1000, ab40766), c-myc (1 : 1000, ab32072), cyclin-D1 (1 : 1000, ab16663), β-catenin (1 : 1000, ab223075), and β-actin (ab8227, 1 : 2000) overnight and then probed with HRP-conjugated anti-IgG antibody (1 : 1000, ab133470, all obtained from Abcam, Shanghai, China) for 1 h. Immunoreactive bands were visualized with an enhanced chemiluminescence system (Thermo Scientific, Shanghai, China).
2.7. Transwell Assay
Total 1 × 10⁴ cells (AGS and SGC7901) were seeded into Matrigel upper chambers (BD Bioscience, USA). The lower chamber contained a complete medium. After 24 hours of culture, the cells passing through the Matrigel membrane were fixed in methanol, stained, and counted in five fields under a microscope (Olympus, Japan).
2.8. Cell Cycle Detection
A total of 2 × 10⁵ cells/well were plated into a six-well plate. Before any treatment, the cell culture medium was changed to a serum-free medium to culture the cells for 12 h, ensuring it enters a similar phase. IR with or without siRNA transfection and XAV939 treatment was carried out as described in the previous methods. After 48 h, the cells were centrifuged, collected, then were fixed in 700 µl of 75% ethanol (−20°C precooled). Subsequently, the cells were suspended in 400 µl of propidium iodide (PI) solutions (50 µg/ml) containing 100 µl of 1 mg/ml RNAse (Solarbio, Shanghai, China) for 10 min. Cellular DNA content was analyzed using flow cytometer (Accuri C6, BD Biosciences).
2.9. Cell Apoptosis Assay
A total of 2 × 10⁵ cells/well were plated in 6-well plates and incubated for 24 h. GAC cells were transfected with siRNA combined with XAV939, followed by IR (4 Gy). Subsequently, all cells were collected and stained with 100 ul of 1× binding buffer, 5 µl of Annexin V-FITC, and 5 ul of Annexin PI in the dark for 15 min at 4°C. Apoptotic cells were analyzed using a flow cytometer (BD Biosciences).
2.10. Luciferase Reporter Assay
A total of 1 × 10⁵ cells/well were plated in a 24-well plate and incubated with RPMI-1640 medium at 37°C for 24 h. Cells were transfected with a 0.8 µg TOP-flash or FOP-flash vector and 0.02 μg Renilla luciferase vector as an internal control (EMD Millipore, Billerica, MA, USA). After transfection for 24 h, cells were harvested in passive lysis buffer (Promega), and the reporter activities were assayed by Dual-Luciferase Assay System kit (Promega Corporation). Renilla reniformis luciferase expression was used for normalization.
2.11. Statistical Analysis
The significance among two or more comparisons was analyzed by a two-tailed Student’s t-test or one-way ANOVA. All data were expressed as mean ± standard deviation (SD) and a value <0.05 was indicated statistically significant.
3. Results
3.1. SPP1 Is Upregulated in Gastric Adenocarcinoma
RT-qPCR was carried out to measure SPP1 mRNA levels in GAC tissues and adjacent normal tissues. SPP1 was overexpressed in GAC tissues compared with normal gastric tissues (, Figure 1(a)). This result was confirmed by detecting the level of SPP1 protein. As shown in Figure 1(b), negative staining could be discovered in adjacent normal tissues. It was found that SPP1 protein was mainly located at the cytoplasm of the GAC cell. 169 GAC tissues (85.4%) expressed SPP1. Among the 198 GAC tissues, 110 samples (55.6%) expressed high levels, and 88 samples (44.4%) expressed low levels of SPP1.
(a)
... Focusing on apoptosis, b8 was notably associated to protection of Oligodendrocytic Progenitor Cells from H 2 O 2 -induced cell death in response to high levels of Osteopontin (OPN), a secreted ECM phosphoprotein (40). Because OPN, a candidate ligand for avb8 (41), was described as a major actor for GIC stemness, GB cell dedifferentiation and tumorigenesis (42)(43)(44), and GB cell radioresistance (45), it would be of interest to decipher the OPN role in the b8-induced molecular mechanisms to sustain GIC survival and stemness. Another thing to consider is the role of Survivin in b8-induced GIC stemness, viability, and resistance, because we and other demonstrated that this protein, markedly overexpressed in GIC, is involved in GB cell dedifferentiation, radioresistance, and faster recurrence (18). ...
Glioblastomas (GBs) are malignant brain tumors with poor prognosis despite treatment with surgery and radio/chemotherapy. These tumors are defined by an important cellular heterogeneity and notably contain a subpopulation of Glioblastoma-initiating cells (GICs), which contribute to tumor aggressiveness, resistance and recurrence. Some integrins are specifically expressed by GICs and could be actionable targets to improve glioblastoma treatment. Here, integrin β8 (ITGB8) was identified as a potential selective target in this highly tumorigenic GIC subpopulation. Using several patient-derived primocultures it was demonstrated that integrin β8 is overexpressed in GICs compared to their differentiated progeny. Furthermore, integrin β8 is also overexpressed in glioblastoma and its overexpression is correlated with poor prognosis and with the expression of several other classic stem cell markers. Moreover, inhibiting integrin β8 diminished several main GIC characteristics and features, including self-renewal ability, stemness, migration potential and tumor formation capacity. Blockade of integrin β8 integrin significantly impaired GIC cell viability via apoptosis induction. Finally, the combination of radiotherapy and integrin β8 targeting radiosensitized GICs through post-mitotic cell death.
Implications:
This study identifies integrin β8 as a new selective marker for glioblastoma-initiating cells and as a promising therapeutic target in combination with chemo-radiotherapy for the treatment of highly aggressive brain tumors.
... OPN has been widely associated with tumour progression, development of metastasis and characteristic resistance to treatment of cancer stem cells [49]. This glycoprotein has been identified as a prognostic factor in human GBM [50]. ...
Notch-1 and osteopontin (OPN) mediate angiogenesis and glioma stem-like cell (GSLC) maintenance. However, the relationship between these molecules and GSLCs during the development of glioma is unknown. We investigate the expression of Notch-1, OPN and vascular endothelial growth factor (VEGF) associated to the stemness markers nestin and CD133 in three stages of murine gliomas induced by N-ethyl-N-nitrosourea (ENU).
Notch-1 and OPN overexpress in the intermediate stage (II), which corresponds to the “angiogenesis switch”. Nestin+ cells appear in all stages of ENU-glioma but CD133 only from stage II on. In stage III, neoplastic cells expressing nestin, CD133 and nestin/CD133 reside in spheroid-like aggregates (SAs) and in the neoangiogenic border. These aggregates show Notch-1 and VEGF+ surrounding cells and a significant size and density increase with respect to stage I (3.3 ± 1.5 to 22.4 ± 6.3 µm², n° = 0.3 ± 0.1 to 4.2 ± 0.9, from stage I to stage III, respectively).
OPN expression increases in correlation to the glioma malignancy from 4.5 ± 1.8% (I) to 12.3 ± 1.2% of OPN+ cells (III). It predominates in astrocyte-like cells of the neoangiogenic border, displaying co-location with VEGF and CD133. The OPN immunopositivity distribution correlates with the CD133 distribution.
In conclusion, OPN co-expressing with CD133 contributes to the identification of GSLCs in the neoangiogenic border, while Notch-1 is present around SAs in advanced stages. The ENU-glioma, mainly in stage II, is a useful tool for assessing new antitumour therapies against these molecules.
