Nucleolin Expression Research Articles

Nucleolin Improves Heart Function During Recovery From Myocardial Infarction by Modulating Macrophage Polarization.

Nucleolin has multiple functions within cell survival and proliferation pathways. Our previous studies have revealed that nucleolin can significantly reduce myocardial ischemia-reperfusion injury by promoting myocardial angiogenesis and reducing myocardial apoptosis. In this study, we attempted to determine the role of nucleolin in myocardial infarction (MI) injury recovery and the underlying mechanism. Male BALB/c mice aged 6-8 weeks were used to set up MI models by ligating the left anterior descending coronary artery. Nucleolin expression in the heart was downregulated by intramyocardial injection of a lentiviral vector expressing nucleolin-specific small interfering RNA. Macrophage infiltration and polarization were measured by real-time polymerase chain reaction, flow cytometry, and immunofluorescence. Cytokines were detected by enzyme-linked immunosorbent assay. Nucleolin expression in myocardium after MI induction decreased a lot at early phase and elevated at late phase. Nucleolin knockdown impaired heart systolic and diastolic functions and decreased the survival rate after MI. Macrophage infiltration increased in the myocardium after MI. Most macrophages belonged to the M1 phenotype at early phase (2 days) and the M2 phenotype increased greatly at late phase after MI. Nucleolin knockdown in the myocardium led to a decrease in M2 macrophage polarization with no effect on macrophage infiltration after MI. Furthermore, Notch3 and STAT6, key regulators of M2 macrophage polarization, were upregulated by nucleolin in RAW 264.7 macrophages. Lack of nucleolin impaired heart function during recovery after MI by reducing M2 macrophage polarization. This finding probably points to a new therapeutic option for ischemic heart disease.

Overexpression of Nucleolin is a Potential Prognostic Marker in Endometrial Carcinoma.

PurposeNucleolin (NCL) is a multifunctional protein with oncogenic properties. NCL expression levels have been linked to the outcomes of various malignancies, but the clinical value of NCL in patients with endometrial carcinoma (EC) remains unclear. Here, the expression of NCL in EC tissues and its associations with patient outcomes were assessed.Patients and MethodsData on NCL mRNA expression in EC and adjacent nonneoplastic tissues from The Cancer Genome Atlas (TCGA) were analyzed. In addition, NCL protein expression in 82 endometroid endometrial adenocarcinoma tissues and 15 non-malignant tissues was detected by immunohistochemistry.ResultsElevated NCL expression was markedly correlated with serous endometrial carcinoma (P<0.001), advanced stage (P=0.029), and grade 3 (P<0.001). High NCL levels were associated with poorer overall survival (OS) and disease-free survival (DFS) compared with intermediate or low NCL levels (OS: P=0.001, DFS: P=0.006). The multivariate Cox proportional hazards model showed that NCL expression was an independent poor prognostic factor for DFS (HR=1.282, CI=1.027–1.601, P=0.028). A similar correlation between high expression levels of NCL and unfavorable DFS was found in endometrioid endometrial adenocarcinoma (HR=1.411, CI=1.083–1.840, P=0.011). Positive extra-nuclear NCL expression (HR=3.377, 95% CI=1.029–11.186, P=0.046) and low nuclear NCL expression (HR=0.233, 95% CI=0.068–0.796, P=0.020) were independent prognostic factors for DFS in endometrioid endometrial adenocarcinoma.ConclusionHeterotopic NCL is a potential prognostic biomarker for EC. Inhibiting the distribution of NCL from the nucleus to the cytoplasm and membrane may be a promising therapeutic strategy to improve outcomes in patients with EC with high NCL expression.

Targeting nucleolin by RNA G-quadruplex-forming motif

A high level of nucleolin (NCL) expression is often associated with a poor prognosis of patients with lung cancer (LC), suggesting that NCL can be used as a possible biomarker. NCL has been shown to display a marked preference for the binding to G-quadruplexes (G4).Here, we investigate the formation of an RNA quadruplex structure in a sequence found in the human precursor pre-MIR150 with the potential to recognize NCL. Circular dichroism (CD) spectra of pre-MIR150 G4-forming sequence (designated by rG4) indicate the formation of a parallel quadruplex structure in KCl or when complexed with the well-known G4 ligand PhenDC3. The thermal stability of rG4 is very high, and further increases in the presence of PhenDC3. The binding affinities of rG4 to PhenDC3 and NCL RBD1,2 are similar with KD values in the nanomolar range. PAGE results suggest the formation of a ternary quadruplex-ligand-protein complex (rG4-PhenDC3-NCL RBD1,2), indicative that PhenDC3 does not prevent the binding of rG4 to NCL RBD1,2. Finally, rG4 can recognize NCL-positive cells and, when fluorescently labeled, can be used as a probe for this protein. ELISA experiments indicate altered NCL expression patterns in liquid biopsies of LC patients in a non-invasive manner, potentially helping the diagnosis, prognosis, and patient response to treatment. Hence, labeled rG4 could be used as a detection probe of LC in liquid biopsies.

Aptamer-based approaches to detect nucleolin in prostate cancer

We have investigated the expression of nucleolin (NCL) in liquid biopsies of prostate cancer (PCa) patients and healthy controls to determine its correlation with tumor prognosis. To detect NCL we used a modified AS1411 aptamer designated by AS1411-N5. In presence of NCL, AS1411-N5 increases the fluorescence by assuming a G-quadruplex (G4) structure, while in the absence of NCL the fluorescence signal remains quenched. The structural characterization of AS1411-N5 was performed by biophysical studies, which demonstrated the formation of G4 parallel conformation in the presence of 100 mM K+ and the ability to recognize NCL with high affinity (KD = 138.1 ± 5.5 nM). Furthermore, the clinical relevance of NCL in PCa liquid biopsies was assessed by using an NCL-based ELISA assay. The protein was measured in the peripheral blood mononuclear cells (PBMCs) cell lysate of 158 individuals, including PCa patients and healthy individuals. The results depicted a remarkable increase of NCL levels in the PBMC's lysate of PCa patients (mean of 626.1 pg/mL whole blood) when compared to healthy individuals (mean of 198.5 pg/mL whole blood). The ELISA results also provided evidence for the usefulness of determining NCL levels in advanced PCa stages. Furthermore, a microfluidic assay showed the ability of AS1411-N5 in recognizing NCL in spiked human plasma samples.

Nucleolin regulates the proliferation of vascular smooth muscle cells in atherosclerotic via Aurora B.

Vascular smooth muscle cells (VSMCs) play a significant role in atherosclerosis. As a multifunctional protein, nucleolin (NCL) is involved in many important physiological and pathological processes. In this study, we aimed to investigate the role of nucleolin in VSMCs proliferation and cell cycle. The expression of nucleolin increased in VSMCs of mice with aortas advanced plaques. With the left common carotid‐artery ligation‐injury model, immunofluorescence staining revealed that nucleolin and Ki67 expression increased in VSMCs in mice left carotid artery compared with right carotid artery after surgery. POVPC or ox‐LDL up‐regulated nucleolin mRNA and protein expression in a dose‐ and time‐dependent manner in HAVSMCs. POVPC (5μg/ml) or ox‐LDL (50μg/ml) promoted the proliferation of HAVSMCs. Nucleolin ablation relieved the pro‐proliferation role of VSMCs. The cell cycle assay and cell ability results showing that POVPC or ox‐LDL increased the proliferation, but nucleolin ablation inhibited the proliferation of HAVSMCs. And nucleolin ablation can prevent DNA replication at S phase and induce cell cycle arrest in S phase. The bioinformatics database predicts protein‐protein interactions with nucleolin and aurora B. Nucleolin overexpression and ablation affected the expression of aurora B. These findings indicate for the first time that nucleolin actively involved the proliferation of VSMCs via aurora B.

Nucleolin as a potential biomarker for canine malignant neoplasia

Human nucleolin (NCL) is a multifunctional protein that is involved in diverse pathological processes. Recent evidences have shown that NCL is markedly overexpressed on the surface of most human cancer cells when compared to normal cells, being overexpressed in several malignant cells. Based on the exposed, the purpose of this pilot study is to investigate the expression pattern of NCL in canine malignant neoplasia and control groups. NCL expression at both messenger RNA and protein levels in the subcellular fractions were respectively detected by RT-PCR and western blotting, allowing to infer the NCL positivity rate in canine neoplasia. The identity of NCL amplicons obtained by RT-PCR was confirmed by Sanger sequencing and found to correspond to Canis lupus familiaris. Using flow cytometry, the blood cells expressing NCL from canine neoplasms were also identified using several cell surface markers and their levels quantified. These results showed that NCL expressed in lymphocytes, monocytes and neutrophils in dogs with malignant neoplasia is higher (> 50%) when compared with the control group. We found an increased expression of surface and cytoplasmic NCL in canine malignant neoplasia group, while nuclear NCL is predominantly found in the control group. Overall, this study discloses and identifies for the first time the presence of NCL in canine blood.

Abstract 3146: Changes in expression of nuclear protein nucleolin detects malignant changes in the fimbria and offers a potential marker for early detection of ovarian cancer

Abstract Introduction: Ovarian cancer (OVCA) remains a fatal malignancy of women as in most cases it is detected at late stages. Fimbria of the fallopian tube has been suggested to be the origin of high grade serous carcinoma (HGSC) and lack of information on the malignant transformation of fimbrial surface epithelial (FSE) cells is a significant barrier to the development of an early detection test for OVCA. The nucleus of a cell has long been used in the pathology for the detection of malignancy. Tumor-associated changes in the nucleus are accompanied by a rearrangement in the nuclear matrix proteins and shedding of these proteins into the circulation. These shed proteins or autoantibodies against them represent markers of early changes for early detection of OVCA. Goals: The goal of this study was to examine the changes in the serum prevalence of nucleolin, a nuclear protein, in association with malignant changes in the FSE cells and ovarian malignant development. Due to the difficulty in accessing the fimbria from OVCA patients, we used laying hen models of spontaneous OVCA. Methods: This was an exploratory pilot study with subjects/patients and a laying hen model of spontaneous OVCA. In the first study, expression of nucleolin was examined in the fimbrial tissues from subjects (BRCA1 mutated) with risk of OVCA development who underwent prophylactic surgery (n=5, 40-50 years old) and ovarian tumors from OVCA patients at early stage (n=5, 60-70 years old). In the second study, serum, fimbrial and ovarian tissues from healthy laying hens (n=10, 4-year old), hens with fimbrial tumors (n=5) and hens with OVCA at late stage (n=10) were also used. Expression of nucleolin was examined by immunohistochemistry, Western blotting, proteomic (MS-MS) and gene-expression studies. Significant differences (P&amp;lt;0.05) in the intensity of nucleolin expression among normal as well as fimbrial and ovarian tumor groups were determined. Results: Intense expression for nucleolin was observed in the fimbrial surface epithelial (FSE) cells from subjects with BRCA1 mutation and in malignant cells in ovarian tumors from OVCA patients. Similar patterns of nucleolin expression were also detected in fimbrial tumors and ovarian tumors at late stage in hens. Two-dimensional Western blotting detected nucleolin protein of approximately 76kDa in fimbrial tumors in hens. MS-MS study showed spectra for nucleolin indicative of its serum prevalence in hens with fimbrial tumors and ovarian tumors. Semi-quantitative and quantitative PCR showed strong amplification for nucleolin gene expression in fimbrial and ovarian tumors in hens as compared with normal fimbria. Conclusion: Expression of nucleolin increases in association with malignant transformation in the fimbria and ovarian tumor development. Serum prevalence of nucleolin suggests that it may be used as a marker of early changes associated with OVCA development and may constitute a member of a panel of markers for early detection of OVCA. Support: NIH/NCI: CA210370 Citation Format: Elizabeth A. Paris, Itzel Lazcano, Pincas Bitterman, Janice Bahr, Sanjib Basu, Animesh Barua. Changes in expression of nuclear protein nucleolin detects malignant changes in the fimbria and offers a potential marker for early detection of ovarian cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3146.

Abstract 5152: SJP1604, a nucleolin-targeted therapeutic agent for relapsed/refractory acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are cancers characterized by the rapid growth of abnormal white blood cells. Although the standard first-line therapy leads to high rates of remission, approximately 30% of the treated patients are refractory and more than 50% of them face a relapse because of the occurrence of drug resistance and high toxicity. Therefore, there is a strong unmet medical need for the next-generation targeted therapy to overcome drug resistance and reduce toxicity in AML/MDS. Here, we designed an aptamer-drug conjugate (SJP1604) specific for nucleolin, which is highly expressed only on the cell membrane of cancer cells including AML cells. We identified that SJP1604 (APTA-16) inhibited the growth of AML cells and drug-resistant AML cells in vitro. SJP1604 presented outstanding anti-leukemic efficacy in vivo via syngeneic mouse model and xenograft mouse model. In addition, the synergistic effects were observed upon combination treatmeant with venetoclax in vitro. We also figured out that nucleolin as a clinical biomarker was significantly highly expressed on relapsed/refractory (R/R) AML patient-derived bone marrow cells ex vivo. SJP1604 selectively targeted cancer cells by nucleolin-binding on cell membrane with its high targeting ability and plasma stability owing to its unique conformational property, therefore, it demonstrated less cytotoxicity in vitro/in vivo and a superior therapeutic index based on AUC comparison in pharmacokinetic studies. The mechanism of the anti-leukemic efficacy of SJP1604 was also studied. First of all, SJP1604 decreased the expression of nucleolin and inactivated NFκB signaling pathway via dephosphorylation of NFκB, which resulted in diminished expression of DNMT1 followed by restored p15 expression. In addition, SJP1604 increased p53 expression and decreased bcl-2 expression in the protein and mRNA level. These could be one of potential mechanism of anti-leukemogenesis of SJP1604. In conclusion, these findings suggest that SJP1604 targeting nucleolin could be developed as a first-in-class drug for relapsed/refractory AML and MDS. GLP safety/toxicology studies of SJP1604 have been completed and clinical trials will be initiated in 2020. Citation Format: Jihyun Um, Dohyeong Lee, Yongbin Park, Sung Hwan Moon, Soo Jin Lee, Min-Hyo Ki. SJP1604, a nucleolin-targeted therapeutic agent for relapsed/refractory acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5152.

Nucleolin Mediates LPS-induced Expression of Inflammatory Mediators and Activation of Signaling Pathways

SummaryIn this study, we investigated the effects of nucleolin on lipopolysaccharide (LPS)-induced activation of MAPK and NF-KappaB (NF-κB) signaling pathways and secretion of TNF-α, IL-1β and HMGB1 in THP-1 monocytes. Immunofluorescence assay and Western blotting were used to identify the nucleolin expression in cell membrane, cytoplasm and nucleus of THP-1 monocytes. Inactivation of nucleolin was induced by neutralizing antibody against nucleolin. THP-1 monocytes were pretreated with anti-nucleolin antibody for 1 h prior to LPS challenge. The irrelevant IgG group was used as control. Secretion of inflammatory mediators (TNF-α, IL-1β and HMGB1) and activation of MAPK and NF-κB/I-κB signaling pathways were examined to assess the effects of nucleolin on LPS-mediated inflammatory response. Nucleolin existed in cell membrane, cytoplasm and nucleus of THP-1 monocytes. Pretreatment of anti-nucleolin antibody significantly inhibited the LPS-induced secretion of TNF-α, IL-1β and HMGB1. P38, JNK, ERK and NF-κB subunit p65 inhibitors could significantly inhibit the secretion of IL-1β, TNF-α and HMGB1 induced by LPS. Moreover, the phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65) was significantly increased after LPS challenge. In contrast, pretreatment of anti-nucleolin antibody could significantly inhibit the LPS-induced phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65). However, the irrelevant IgG, as a negative control, had no effect on LPS-induced secretion of TNF-α and IL-1β and phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65). We demonstrated that nucleolin mediated the LPS-induced activation of MAPK and NF-κB signaling pathways, and regulated the secretion of inflammatory mediators (TNF-α, IL-1β and HMGB1).

Nucleolin and nucleophosmin expression patterns in pulmonary adenocarcinoma invading the pleura and in pleural malignant mesothelioma.

BackgroundVisceral pleural invasion (VPI) in adenocarcinoma of the lung is considered a poor prognostic factor. The purpose of this study was to analyze nucleolin and nucleophosmin expression in pulmonary adenocarcinoma (PA) with VPI and in pleural malignant mesothelioma.MethodsThe study was conducted on the basis of 19 pathologically‐confirmed cases of adenocarcinoma of the lung and 29 cases of epithelioid malignant mesothelioma. The nucleolin and nucleophosmin expression was assessed immunohistochemically and analyzed with image analysis software.ResultsNucleolin expression was lower while nucleophosmin was higher in pleural invasion of pulmonary adenocarcinoma than in the central part of the tumor. Differences in subpopulations of cells with different expression of proteins studied were also found. Malignant mesothelioma showed lower nucleolin expression than adenocarcinoma of the lung but no differences in nucleophosmin expression were found.ConclusionsThe results of our study suggested that lower nucleolin and higher nucleophosmin expression may be related to higher invasiveness of adenocarcinoma of the lung. Differences in nucleolin expression between pulmonary adenocarcinoma and malignant mesothelioma indicate another aspect of biology of these pleura‐invading cancers that requires further study.Key pointsSignificant findings of the studyDifferences in nucleolin and nucleophosmin expression in pleura invading pulmonary adenocarcinoma indicate the involvement of these proteins in its locoregional spread while differences in nucleolin expression between pulmonary adenocarcinoma and malignant mesothelioma suggest another aspect of biology of these cancers.What this study addsThis is the first study on nucleolin and nucleophosmin expression in pleural malignant mesothelioma and pleura‐invading pulmonary adenocarcinoma. Our findings may assist in understanding the mechanisms of locoregional spread of adenocarcinoma and differences between these two pleura‐invading cancers.

Nucleolin and Nucleophosmin Expression in Gleason 3 and Gleason 4 Prostate Cancer With Seminal Vesicles Invasion (pT3b).

The aim of this study was to analyze the expression of nucleolin (NCL) and nucleophosmin (NPM) in prostate adenocarcinoma and in its loco-regional spread in the form of seminal vesicle invasion (SVI). The study was performed on tissue microarrays of 40 cases of Gleason 3+4 pT3b prostate cancers including tissue samples from SVI. The expression of NCL and NPM was detected immunohistochemically and analyzed with image analysis software. The expression of NCL and NPM were higher in cancer cells within a prostate gland than in SVI. Gleason 4 pattern showed higher expression of NPM than Gleason 3 pattern cells. Differences in nuclear NCL and NPM expression in cancer cells between the prostate gland and SVI may indicate involvement of these proteins in loco-regional spread of adenocarcinoma of the prostate. Differences in NPM expression in Gleason 3 and Gleason 4 pattern suggest involvement of this protein in the differentiation of prostate cancer.

LncRNA H19 involved in myocardial ischaemic preconditioning via post-translational control of nucleolin expression

LncRNA H19 involved in myocardial ischaemic preconditioning via post-translational control of nucleolin expression

PolyG mitigates silica-induced pulmonary fibrosis by inhibiting nucleolin and regulating DNA damage repair pathway

Polyguanylic acid potassium salt (PolyG) has an anti-fibrotic G-quadruplex (G4) structure. It could inhibit the expression of nucleolin, a protein involved in cell proliferation and apoptosis. However, its role in regulating nucleolin in silicosis is still unknown. After instillation of 50 μl of crystalline silica suspension (50 mg/ml) into the trachea of C57BL/6 mice, we show that nucleolin expression is upregulated in mouse pulmonary tissue following the treatment with silica and that PolyG, which were injected 2.5 mg/kg body weight into mice by abdomen, could alleviate pulmonary fibrosis through inhibiting the expression of nucleolin. Further, we demonstrated that the expression of the DNA double-strand break (DSB) marker, γ-H2AX, increased in response to silica treatment. PolyG could efficiently reduce the protein expression of γ-H2AX and decreased the level of fibrosis-related genes, such as Col1a1 and Col3a1, as well as the levels of fibrosis-associated proteins α-SMA and vimentin in the lungs of silica-treated mice. These findings show that PolyG could regulate nucleolin and DNA damage repair to control fibrotic response in experimental silicosis and provide a new target for preventive intervention.

LncRNA H19 is involved in myocardial ischemic preconditioning via increasing the stability of nucleolin protein.

Myocardial ischemic preconditioning (IP) is defined as a brief period of myocardial ischemia/reperfusion (I/R) that significantly reduces injury during the subsequent exposure to long-term I/R. However, the underlying mechanisms of myocardial IP are yet to be elucidated. This study investigated the expression and roles of long noncoding RNA (lncRNA) H19 in myocardial IP in vitro and in vivo. LncRNA H19 expression levels were analyzed by quantitative reverse-transcription polymerase chain reaction, cell viability was determined by the Cell Counting Kit-8 assay, apoptosis was evaluated based on the caspase 3 activity, and RNA immunoprecipitation was performed to examine the interaction between lncRNA H19 and nucleolin. The results of this study showed that lncRNA H19 expression was significantly upregulated in mouse hearts subjected to myocardial IP, in rat H9C2 cells exposed to H2 O2 preconditioning (H2 O2 -PC), and in neonatal rat cardiomyocytes subjected to hypoxia preconditioning. H19 knockdown abrogated the H2 O2 -PC-mediated protection in cardiomyocytes evidenced by the decreased cell viability and increased caspase-3 activity. Conversely, H19 overexpression enhanced the protective role of H2 O2 -PC in cardiomyocytes. In addition, H19 overexpression increased the expression of nucleolin, whereas H19 ablation abrogated H2 O2 -PC-induced upregulation of nucleolin in cardiomyocytes. Furthermore, H19 overexpression increased the stabilization of nucleolin; an interaction between H19 and nucleolin was identified using the RNA-protein interaction studies. Furthermore, nucleolin small interfering RNA relieved the protective role of lncRNA H19. These findings demonstrated that the lncRNA H19 is involved in myocardial IP via increasing the stability of nucleolin protein and lncRNA H19 may represent a potential therapeutic target for the treatment of the myocardial injury.

A smart ATP-responsive chemotherapy drug-free delivery system using a DNA nanostructure for synergistic treatment of breast cancer in vitro and in vivo

This study demonstrated a chemotherapy drug-free delivery system for breast cancer treatment based on a simple DNA nanostructure composed of sequence 1 containing ATP and AS1411 aptamers and sequence 2 containing antimiR-21. The DNA nanostructure was used for co-delivery of KLA peptide and antimiR-21 as antiapoptotic agents. These therapeutic agents could not be internalised into eukaryotic cells freely which is one of the great features of this targeting platform. The presented delivery system was ATP-responsive, leading to disassembly of the DNA nanostructure in high ATP concentration of cancer cells and restoration of the function of antimiR-21 in these cells. The DNA nanostructure was associated with high cellular uptake by MCF-7 and 4T1 cells due to expression of nucleolin as target of AS1411 on their plasma membranes, while the developed targeting platform could not be internalised into CHO cells because of lack of the active targeting moiety on their surfaces. Furthermore, the results showed that co-delivery of antimiR-21 and KLA peptide using the DNA nanostructure could efficiently prohibit tumour growth in vitro and in vivo and induce a synergistic anticancer activity. Thus, this work provides a new ATP-responsive nanotargeting delivery system and synergistic chemotherapy drug-free regimen for cancer treatment.

Abstract A101: Nucleolin: A novel cell surface protein for neuroblastoma targeted therapy

Abstract Background: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Since the use of targeted therapies is severely limited by the lack of specific receptors, chemotherapy and radiotherapy remain the main treatment choices, which, nevertheless expose high-risk NB patients to unacceptable toxicity and to the development of drug resistance. Nucleolin (NCL), a multifunctional protein involved in both physiological and pathological conditions, is overexpressed and partially localized on the cell surface of tumor cells of adult cancers. Little is reported about NCL in pediatric tumors, including NB. Methods: To test the expression of NCL as a potential and targetable cell surface marker in NB, human NB cell lines were evaluated in vitro and after injection in mice, by Imaging Flow Cytometry (IFC) and immunohistochemistry (IHC) analyses, respectively. Cell binding and internalization of rhodamine-labeled nanocarriers, decorated with the NCL-recognizing F3 peptide (Rhod-F3-SL) were evaluated in vitro by FACS and confocal microscopy. The cytotoxic potential of a doxorubicin (DXR)-loaded, pH-sensitive nanocarrier and decorated with the F3 peptide was evaluated in vitro, in terms of inhibition of NB cells proliferation and induction of cell death by CSFE and MTS assays, respectively. Orthotopic and pseudometastatic animal models of human NB, mimicking the growth and spread of NB in patients, were finally enrolled to examine in vivo the anti-tumor activity of DXR-loaded, pH-sensitive nanocarriers decorated with F3 peptide. Results: IFC analyses demonstrated that NCL is expressed at different extent on the surface of all NB cells analyzed. Notably, IHC evaluations displayed NCL staining on both tumor and endothelial tumor cells in well-established NB xenografts. Moreover, in vitro binding studies showed that Rhod-F3-SL strongly associated to NB cells, exhibiting a specific co-localization with cell surface NCL. The cellular distribution of Rhod-F3-SL also confirms the binding specificity, showing an increased selective internalization of NCL-targeted nanoparticles, compared to that obtained with untargeted formulations. Finally, F3-targeted, DXR-loaded, pH-sensitive nanocarriers resulted significantly more effective, in terms of inhibition of cell proliferation and reduction of cell viability in vitro, and in terms of delay of tumor growth in each NB animal model tested, when compared to DXR-loaded, pH-sensitive untargeted liposomes. Conclusion: Our findings demonstrate that NCL represents a novel cell surface protein to be targeted by “drug”-loaded nanoparticles as an innovative therapeutic strategy for high-risk NB. Citation Format: Brignole Chiara, Veronica Bensa, Genny Del Zotto, Silvia Bruno, Nuno A Fonseca, Ana F Cruz, Daniela Di Paolo, Patrizia Perri, Vera Moura, Laura Emionite, Michele Cilli, Mirco Ponzoni, Joao N Moreira, Fabio Pastorino. Nucleolin: A novel cell surface protein for neuroblastoma targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A101. doi:10.1158/1535-7163.TARG-19-A101

Targeting Nucleolin for Reversal of Chemotherapy Resistance in Acute Lymphoblastic Leukemia

Chemotherapy resistance is considered to be the principal cause of ineffective treatment in acute lymphoblastic leukemia (ALL). Nucleolin (NCL) is high expression andplays oncogenic roles in most cancers. However, less research on the role of NCL in hematologic malignancies was noted. Our previous studies have showed that overexpression of NCL was associated with worse prognosis in the patients with acute leukemia and NCL expressionwashigher in resistant HL-60/ADR than in sensitive HL-60 cells. The potential mechanisms of NCL in chemotherapy resistance have yet to be revealed. Here we presented that expression of NCL was associated positively with chemotherapy resistance and poor prognosis in ALL. Overexpressed NCL at both mRNA and protein level was relevant to a poorer overall survival (OS) and relapse free survival (RFS), indicating NCL as an independent prognostic marker in ALL. mRNA level of NCL in de novo ALL was quantitatively higher than in complete remission(CR) status, and refractory/relapse ALL had the highest level. Upon above clinical data, we further investigated the mechanism(s) by which NCL regulated drug resistance in ALL cells. Remarkably, NCL expression was higher in resistant ALL cells relative to sensitive parental cells. When treated with ADM, NCL level was decreased in sensitive parental cells while unchanged in resistant cells. Overexpressing NCL suppressed drug sensitivity, altered drug effluxion and decreased intracellular drug accumulation, while inhibition of NCL led to a completely reversed appearance, more intracellular Adriamycin(ADM) mean fluorescence intensity (MFI) and percentage of ADM accumulated cells population. Overexpression of NCL increased significantly the IC50 of ADM. The IC50 of ADM on Jurkat-NCL-overexpression(OE), Jurkat-NCL-knockdown(KD), Molt-4-NCL-OE, Molt-4-NCL-KD, Nalm-6-NCL-OE, Nalm-6-NCL-KD were 1.362±0.271μg/ml, 0.077±0.010μg/ml, 4.863±0.733μg/ml, 0.081±0.018μg/ml, 0.237±0.042μg/ml and 0.046±0.002μg/ml, respectively (P &lt;0.05). Involvement of ATP-binding cassette (ABC) transporters was proved in NCL mediated drug resistance. Silencing NCL resulted in a decrease of P-gp, MRP1, LRP and BCRP in ALL cells, and NCL overexpression increased the MRP1, LRP and BCRP. The Akt/mTOR and ERK signaling pathways were involved in this procedure. Notably, co-IP assays confirmed the NCL-Ras, NCL-ERK and NCL-BCRP interaction. For intervention study, aptamer AS1411, a NCL inhibitor, could reduce drug resistance in ALL cell lines and primary ALL cells.Moreover, AS1411 treatment decreased BCRP protein expression. Furthermore, the ALL leukemia models that nude mice engrafted with Nalm-6 cells and NCG mice engrafted with Luc+ Nalm-6 cells were established, then treated with ADM plus AS1411 or control CRO for comparison drug sensitivity and survival. Growth of subcutaneous xenograft tumors was inhibited in those treated with AS1411 or ADM, compared to their respective controls treated with CRO or PBS. The stronger inhibition effect was observed in those treated with AS1411 combined with ADM. For Luc+Nalm-6 derived ALL model, leukemia progression was suppressed in mice treated with AS1411 and AS1411 combined with ADM. AS1411and ADM, especially combination of AS1411 and ADM, could improve survival of the leukemic mice compared to those treated with PBS. The results showed that NCL targeted by AS1411 sensitized ADM treatment and prolonged survival in vivo. In summary, our findings revealed NCL as a survival predictor and the novel role of NCL in ALL chemo-resistance. NCL may be a potential target for improving outcome in ALL. Disclosures No relevant conflicts of interest to declare.

Inhibiting nucleolin reduces inflammation induced by mitochondrial DNA in cardiomyocytes exposed to hypoxia and reoxygenation.

Background and purposeCellular debris causes sterile inflammation after myocardial infarction. Mitochondria constitute about 30 percent of the human heart. Mitochondrial DNA (mtDNA) is a damage‐associated‐molecular‐pattern that induce injurious sterile inflammation. Little is known about mtDNA's inflammatory signalling pathways in cardiomyocytes and how mtDNA is internalized to associate with its putative receptor, toll‐like receptor 9 (TLR9).Experimental ApproachWe hypothesized that mtDNA can be internalized in cardiomyocytes and induce an inflammatory response. Adult mouse cardiomyocytes were exposed to hypoxia‐reoxygenation and extracellular DNA. Microscale thermophoresis was used to demonstrate binding between nucleolin and DNA.Key resultsExpression of the pro‐inflammatory cytokines IL‐1β and TNFα were upregulated by mtDNA, but not by nuclear DNA (nDNA), in cardiomyocytes exposed to hypoxia‐reoxygenation. Blocking the RNA/DNA binding protein nucleolin with midkine reduced expression of IL‐1β/TNFα and the nucleolin inhibitor AS1411 reduced interleukin‐6 release in adult mouse cardiomyocytes. mtDNA bound 10‐fold stronger than nDNA to nucleolin. In HEK293‐NF‐κB reporter cells, mtDNA induced NF‐κB activity in normoxia, while CpG‐DNA and hypoxia‐reoxygenation, synergistically induced TLR9‐dependent NF‐κB activity. Protein expression of nucleolin was found in the plasma membrane of cardiomyocytes and inhibition of nucleolin with midkine inhibited cellular uptake of CpG‐DNA. Inhibition of endocytosis did not reduce CpG‐DNA uptake in cardiomyocytes.Conclusion and implicationsmtDNA, but not nDNA, induce an inflammatory response in mouse cardiomyocytes during hypoxia‐reoxygenation. In cardiomyocytes, nucleolin is expressed on the membrane and blocking nucleolin reduce inflammation. Nucleolin might be a therapeutic target to prevent uptake of immunogenic DNA and reduce inflammation.Linked ArticlesThis article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc

Effect of Inhibiting NCL Expression on Drug-Resistance of Chronic Myeloid Leukemia Cell Line K562 and Its Resistant Cells

To construct a K562 and adriamycin-resistant K562 (KAR) cell line with stably down-regulation of NCL (nucleolin) expression, and to investigate the effect of NCL down-regulation on the drug resistance in K562 and KAR cells. K562 and KAR cells were infected with lentivirus, and stably transfected cell clones were obtained by puromycin screening. The cell proliferation was detected by MTS assay, the cell apoptosis was detected by flow cytometry, and the expression level of drug resistance related genes was detected by real-time PCR. The K562 and KAR cells with stable down-regulation of NCL were successfully constructed. Compared with the control group, the proliferation of K562 and KAR cells with down-regulating NCL expression decreased significantly (P ＜0.05), the apoptosis of cells increased significantly (P ＜0.05), and cell resistance to adriamycin was down-regulated. Inhibition of NCL expression may increase the sensitivity of cells to adriamycin, which may be related with the promotion of apoptosis of K562 and KAR cells.

AS1411-conjugated gold nanoparticles affect cell proliferation through a mechanism that seems independent of nucleolin

G-rich oligonucleotide, AS1411, has been shown to interact with nucleolin and to inhibit cancer cell proliferation and tumor growth. This antiproliferative action is increased when AS1411 is conjugated to different types of nanoparticles. However, the molecular mechanisms are not known. In this work, we show in several cell lines that optimized AS1411-conjugated gold nanoparticles (GNS-AS1411) inhibit nucleolin expression at the RNA and protein levels. We observed an alteration of the nucleolar structure with a decrease of ribosomal RNA accumulation comparable to what is observed upon nucleolin knock down. However, the expression of genes involved in cell cycle and the cell cycle blockage by GNS-AS1411 are not regulated in the same way as that in cells where nucleolin has been knocked down. These data suggest that the anti-proliferative activity of GNS-AS1411 is not the only consequence of nucleolin targeting and down-regulation.