Nucleolin Expression Research Articles

Abstract 344: SJP1604, a novel targeted therapeutic agent for AML (acute myeloid leukemia) including standard therapy-resistant AML

Abstract Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are cancers characterized by the rapid growth of abnormal white blood cells. Generally, cytarabine (Ara-C), azacitidine, and decitabine are treated as the standard first-line chemotherapy for AML/MDS. Although the 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 need for the next-generation targeted therapy to overcome drug resistance and reduce toxicity in AML/MDS. Here, we designed an aptamer-nucleoside drug conjugate (SJP1604) via utilizing easy and continuous synthesis specific for nucleolin, which is highly expressed only on the cell membrane of cancer cells. In particular, AML, CLL (chronic lymphoblastic leukemia) and ALL (acute lymphoblastic leukemia) cells are known to express the highest mRNA level of nucleolin among various cancer cells, relatively. SJP1604 can be delivered into cancer cells with its high targeting ability and plasma stability via its unique conformational property. In this study, we verified that SJP1604 selectively targeted cancer cells by nucleolin-binding on the cancer cell membrane in the drug uptake assay. Owing to the selectivity, SJP1604 demonstrated less cytotoxicity on murine bone marrow cells in vitro and mouse white blood cells in vivo. SJP1604 also exhibited a long-lasting plasma stability in 50% human plasma up to 48 hours in contrast to unstability of general aptamers. Furthermore, SJP1604 inhibited the growth of various human AML/MDS cancer cells. Interestingly, SJP1604 showed the significantly reduced IC50 values with the decreased expression level of nucleolin in cytarabine-, azacitidine- and decitabine-resistant MOLM-13 while cytarabine caused no effect on the cell growth and the expression of nucleolin of the same resistant cell lines. Injection of SJP1604 led to tumor regression and improved survival rate in MOLM-13 xenograft mouse model and C1498 syngeneic mouse model. In ex vivo study, we figured out that the expression level of nucleolin is highly up-regulated in AML patient-derived bone marrow cells and the level is much higher in relapsed/refractory (R/R) AML patient-derived bone marrow cells. Moreover, SJP1604 also significantly reduced the colony forming unit (CFU) of R/R AML patient-derived bone marrow cells compared to that of AS1411 treatment. In conclusion, these findings suggest that SJP1604 could be developed as a first-in-class drug for novel targeted therapy of AML/MDS with less drug toxicity as well as as a remarkable orphan drug for overcoming drug resistance of AML/MDS in which distinctive solutions do not exist up until recently. Citation Format: Jihyun Um, Dohyeong Lee, Jisun Oh, Yongbin Park, Sung Hwan Moon, Su Jin Lee, Min-Hyo Ki, Eui Hwan Cho. SJP1604, a novel targeted therapeutic agent for AML (acute myeloid leukemia) including standard therapy-resistant AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 344.

PB2104 INHIBITION OF NUCLEOLIN REVERSED BORTEZOMIB RESISTANCE VIA THE REGULATION OF NF‐κB PATHWAY IN MULTIPLE MYELOMA CELLS

Background:The proteasome inhibitor bortezomib has remarkable efficacy against multiple myeloma (MM). However, intrinsic and acquired resistance to bortezomib could limit its efficacy. The potential resistance mechanisms have yet to be clarified. The nucleo‐cytoplasmic protein nucleolin (NCL) is high expression in several tumors, especially in resistance cells, that may act in the drug resistance. Previously we first observed the higher expression of NCL in KM3 bortezomib resistant cells (KBTZ) than its parental KM3 MM cells, indicating the role of NCL in bortezomib resistance and needing further research to elucidate.Aims:To investigate the potential mechanism(s) by which NCL regulates bortezomib resistance in MM cell line.MethodsKM3 MM cells and its bortezomib resistant cells KBTZ were transfected with lentiviruses carrying the shRNA sequences that target the endogenous NCL gene and control scrambled gene respectively. Post‐transfected cells were named as followed: KM3‐NCL‐Knockdown(KM3‐NCL‐KD), KM3‐NCL‐Scramble(KM3‐NCL‐Scr), KBTZ‐NCL‐Knockdown(KBTZ‐NCL‐KD), KBTZ‐NCL‐Scramble(KBTZ‐NCL‐Scr). Effect of NCL on cell viability and cells drug sensitivity were measured. Cell cycle related proteins and drug resistance related molecules expression level were evaluated. Related signaling pathways were also examined by western blot.Results:NCL silencing significantly inhibited cell proliferation and colony formation ability in KM3 and KBTZ cells. Colony sizes and numbers were both decreased in NCL silenced cells. Colony formation rates in KM3‐NCL‐KD, KM3‐NCL‐Scr, KBTZ‐NCL‐KD, KBTZ‐NCL‐Scr were 9.33% ± 0.47%, 27.22% ± 1.85%, 7.16% ± 0.24% and 28.22% ± 3.57%, respectively (P < 0.05). Moreover, NCL silencing arrested cell cycle transition in G0/G1, resulted in an increasing trend in cell apoptosis. CyclinD, cyclinE, CDK2 proteins were downregulated and P21 protein was upregulated after knockdown of NCL. Notably, NCL silencing increased the sensitivity to bortezomib in KM3 cells and prominently reversed the resistance to bortezomib in KBTZ cells. IC50of bortezomib were 17.29 ± 3.35 ng/mlin KM3‐NCL‐Scr cells and 10.13 ± 1.5 ng/mlin KM3‐NCL‐KD cells (P < 0.05), indicating a 1.7‐fold increase of bortezomib sensitivity after NCL knockdown. IC50of bortezomib were 590.78 ± 147.92 ng/mlin KBTZ‐NCL Scr cells and 63.48 ± 13.16 ng/mlin KBTZ‐NCL KD cells (P < 0.05), meaning a 10.8‐fold reversal of bortezomib resistance after NCL knockdown (Figure 1). Besides, compared with Scr cells, both KM3‐ and KBTZ‐NCL‐KD cells displayed lessrhodamine‐123 effluxion, suggesting that NCL silencing suppressed drug efflux activity. Knockdown of NCL declined ATP‐binding cassette transporter proteins MRP1, LRP, BCRP at mRNA levels and protein levels. Defined mRNA expression level of KM3‐NCL‐Scr and KBTZ‐NCL‐Scr cells equal to 1, relative mRNA expression of MRP1, LRP, BCRPwere 0.56 ± 0.26, 0.13 ± 0.03, 0.13 ± 0.09 in KM3‐NCL‐KD cells and 0.69 ± 0.11, 0.35 ± 0.07, 0.53 ± 0.20 in KBTZ‐NCL‐KD cells, respectively. Furthermore,downregulated expression of P‐NF‐κB, P‐IκBα, IKKα and P‐IKKα protein, decreased intranuclear NF‐κB level and upregulated expression of IκBα protein were observed in both KM3‐ and KBTZ–NCL‐KD cells, indicating that NCL may act by regulating activity of NF‐κB signaling pathway.Summary/Conclusion:In conclusion, our resultssuggested that inhibition of NCL reversed bortezomib resistance via the regulation of NF‐κB pathway in multiple myeloma cells, indicatingthe novel molecular understanding of bortezomib resistance and the role of NCL in MM resistance, and the emerging marker of NCL as a potential target.image

Nucleolin promotes AngII‑induced phenotypic transformation of vascular smooth muscle cells via interaction with tropoelastin mRNA.

The current study aimed to clarify the role of nucleolin in the phenotypic transformation of vascular smooth muscle cells (VSMCs) and to preliminarily explore its underlying mechanism. The spatial and temporal expression patterns of nucleolin, and the effects of angiotensin II (Ang II) on the expression of VSMC phenotypic transformation markers, α-smooth muscle-actin, calponin, smooth muscle protein 22α and osteopontin were investigated. The effects of nucleolin on VSMC phenotypic transformation and the expression of phenotypic transformation-associated genes, tropoelastin, epiregulin and fibroblast growth factor 2 (b-FGF), were determined. Protein-RNA co-immunoprecipitation was used to investigate the potential target genes regulated by the nucleolin in phenotypic transformation of VSMCs. Finally, the stability of tropoelastin mRNA and the effects of nucleolin on the expression of tropoelastin were assayed. The results revealed that Ang II significantly promoted the phenotypic transformation of VSMCs. The expression of nucleolin was gradually upregulated in VSMCs treated with Ang II at different concentrations for various durations. Ang II induced nucleolin translocation from the nucleus to cytoplasm. Additionally, Ang II significantly promoted the phenotypic transformation of VSMCs. Overexpression and silencing of nucleolin regulated the expressions of tropoelastin, epiregulin and b-FGF. There was an interaction between tropoelastin mRNA and nucleolin protein, promoting the stability of tropoelastin mRNA and enhancing the expression of tropoelastin at the protein level. Upregulation of nucleolin had an important role in Ang II-induced VSMC phenotypic transformation, and its underlying mechanism may be through interacting with tropoelastin mRNA, leading to its increased stability and protein expression. The findings provide a new perspective into the regulatory mechanism of VSMC phenotypic transformation.

Nucleolin Is a Functional Binding Protein for Salinomycin in Neuroblastoma Stem Cells.

The aim of this study is to illuminate a novel therapeutic approach by identifying a functional binding target of salinomycin, an emerging anticancer stem cell (CSC) agent, and to help dissect the underlying action mechanisms. By utilizing integrated strategies, we identify that nucleolin (NCL) is likely a salinomycin-binding target and a critical regulator involved in human neuroblastoma (NB) CSC activity. Salinomycin markedly suppresses NB CD34 expression and reduces CD34+ cell population in an NCL-dependent manner via disruption of the interaction of NCL with CD34 promoter. The elevated levels of NCL expression in NB tumors are associated with poor patient survival. Altogether, these results indicate that NCL is likely a novel functional salinomycin-binding target that exhibits the potential to be a prognostic marker for NB therapy.

Study on the expression and roles of nucleolin in cardiac injury in septic mice

Objective: To explore the expression and roles of nucleolin in cardiac injury in septic mice. Methods: C57BL/6 mice (WT mice) and myocardium-specific expression of nucleolin transgenic mice (TG mice) were randomly divided into sham group (n=10, sham-operated) and sepsis group (n=15, animal model). Cecal ligation and puncture (CLP) was adopted to produce animal models of sepsis. The expression of nucleolin was detected by Western blotting analysis at 0, 12, 24, 48 hours after the operation. The 7-day survival rate, haemodynamic measurement, levels of isoenzyme of creatinekinase-MB (CK-MB) and cardiac troponin I (cTnI) in serum and levels of reactive oxygen species (ROS) and malondlaldehyde (MDA) in myocardium were evaluated 24 hours after the operation. The data were compared between groups with t test. Results: The expression of nucleolin in myocardium up-regulated significantly in WT+CLP group when compared with that in the WT+Sham group(2.57±0.34 vs 1.00±0.15, t=7.468, P<0.01). Compared with those in the WT+Sham group, the survival rate decreased (33.3% vs 100%, χ(2)=13.375, P<0.01), maximal rate of pressure development (+dp/dtmax) declined (t=4.993, P<0.01), but the serum levels of CK-MB and cTnI and the levels of ROS and MDA in myocardium increased in the WT+CLP group(t=5.031, 4.335, 3.365, 2.375, all P<0.05). Compared with that in WT+CLP group, the 7-day survival rate of mice increased in TG+CLP group (60.0% vs 33.3%, χ(2)=8.227, P=0.004), and the cardiac function improved (t=2.337, P=0.019), but the serum levels of CK-MB and cTnI and the levels of ROS and MDA in myocardium in TG+CLP group reduced significantly (t=2.127, 3.347, 2.115, 2.224,P<0.05). Conclusion: The expression of nucleolin is up-regulated in the myocardium of septic mice, and the overexpression of nucleolin can inhibit oxidative stress injury, attenuate the cardiac injury and dysfunction, and reduce mortality in septic mice.

Cytoplasmic Nucleolin Expression in Chronic Lymphocytic Leukemia

Cytoplasmic Nucleolin Expression in Chronic Lymphocytic Leukemia

Nucleolin Overexpression Confers Increased Sensitivity to the Anti-Nucleolin Aptamer, AS1411

AS1411 is an antiproliferative DNA aptamer, which binds the ubiquitous protein, nucleolin. In this study, we show that constitutive overexpression of nucleolin confers increased sensitivity to the growth inhibitory effects of AS1411. HeLa cells overexpressing nucleolin have an increased growth rate and invasiveness relative to control cells. Nucleolin overexpressing cells demonstrate increased growth inhibition in response to the AS1411 treatment, which correlates with increased apoptosis and cell cycle arrest, when compared to non-transfected cells. AS1411 induces nucleolin expression at the RNA and protein level in HeLa cells, suggesting a feedback loop with important implications for the clinical use of AS1411.

Druggable Nucleolin Identifies Breast Tumours Associated with Poor Prognosis That Exhibit Different Biological Processes.

Background: Nucleolin (NCL) is a multifunctional protein with oncogenic properties. Anti-NCL drugs show strong cytotoxic effects, including in triple-negative breast cancer (TNBC) models, and are currently being evaluated in phase II clinical trials. However, few studies have investigated the clinical value of NCL and whether NCL stratified cancer patients. Here, we have investigated for the first time the association of NCL with clinical characteristics in breast cancers independently of the different subtypes. Methods: Using two independent series (n = 216; n = 661), we evaluated the prognostic value of NCL in non-metastatic breast cancers using univariate and/or multivariate Cox-regression analyses. Results: We reported that NCL mRNA expression levels are markers of poor survivals independently of tumour size and lymph node invasion status (n = 216). In addition, an association of NCL expression levels with poor survival was observed in TNBC (n = 40, overall survival (OS) p = 0.0287, disease-free survival (DFS) p = 0.0194). Transcriptomic analyses issued from The Cancer Genome Atlas (TCGA) database (n = 661) revealed that breast tumours expressing either low or high NCL mRNA expression levels exhibit different gene expression profiles. These data suggest that tumours expressing high NCL mRNA levels are different from those expressing low NCL mRNA levels. Conclusions: NCL is an independent marker of prognosis in breast cancers. We anticipated that anti-NCL is a promising therapeutic strategy that could rapidly be evaluated in high NCL-expressing tumours to improve breast cancer management.

Phosphorylation of nucleolin is indispensable to its involvement in the proliferation and migration of non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) is one of the mostly deadly malignancies in the world. Nucleolin is a multifunctional protein that mainly regulates ribosome biogenesis but also has other functions including modulating the transcription of mRNAs and repressing RNA polymeraseII. Nucleolin is overexpressed in various cancer cells, including NSCLC cells. It can confer resistance to apoptosis and promote cell migration and blood vessel formation by directly taking part in various tumor signal transduction pathways. The activities of nucleolin are regulated mainly by intracellular localization and post-translational modifications, including phosphorylation, glycosylation, methylation, and ADP-ribosylation. Phosphorylation of nucleolin (P-nucleolin) in NSCLC cells is still not well characterized. In the present study, the levels of nucleolin and P-nucleolin were examined in lung tissue and cells and it was demonstrated that levels of the two forms of nucleolin were significantly increased in NSCLC compared with non-cancerous tissues and cells. In addition, it was demonstrated that high expression levels of nucleolin and P-nucleolin were significantly associated with poor overall survival of NSCLC patients. Doxorubicin (DOX) is a type of anthracycline that has been used in the treatment of various types of cancer, including NSCLC. Upregulation of nucleolin through exogenous expression of nucleolin promoted A549 cell proliferation and migration, while downregulation of nucleolin through expression of small interfering RNA-nucleolin attenuated A549 cell proliferation and migration. Following stimulation with DOX, A549 cell proliferation and migration decreased and the expression of P-nucleolin also decreased. In order to investigate whether P-nucleolin is indispensable to the proliferation and migration of NSCLC cells, a plasmid encoding mutant nucleolin, in which the phosphorylation site at threonine-76 was mutated to alanine, was constructed. Compared with the A549 cells transfected with wild-type nucleolin, P-nucleolin expression and cell proliferation and migration were significantly decreased in A549 cells transfected with mutant nucleolin. These results indicate that targeting P-nucleolin may be a promising strategy for treating NSCLC patients.

Increase in anti-apoptotic molecules, nucleolin, and heat shock protein 70, against upregulated LRRK2 kinase activity

ABSTRACTLeucine-rich repeat kinase 2 (LRRK2) is involved in Parkinson’s disease (PD) pathology. A previous study showed that rotenone treatment induced apoptosis, mitochondrial damage, and nucleolar disruption via up-regulated LRRK2 kinase activity, and these effects were rescued by an LRRK2 kinase inhibitor. Heat-shock protein 70 (Hsp70) is an anti-oxidative stress chaperone, and overexpression of Hsp70 enhanced tolerance to rotenone. Nucleolin (NCL) is a component of the nucleolus; overexpression of NCL reduced cellular vulnerability to rotenone. Thus, we hypothesized that rotenone-induced LRRK2 activity would promote changes in neuronal Hsp70 and NCL expressions. Moreover, LRRK2 G2019S, the most prevalent LRRK2 pathogenic mutant with increased kinase activity, could induce changes in Hsp70 and NCL expression. Rotenone treatment of differentiated SH-SY5Y (dSY5Y) cells increased LRKK2 levels and kinase activity, including phospho-S935-LRRK2, phospho-S1292-LRRK2, and the phospho-moesin/moesin ratio, in a dose-dependent manner. Neuronal toxicity and the elevation of cleaved poly (ADP-ribose) polymerase, NCL, and Hsp70 were increased by rotenone. To validate the induction of NCL and Hsp70 expression in response to rotenone, cycloheximide (CHX), a protein synthesis blocker, was administered with rotenone. Post-rotenone increased NCL and Hsp70 expression was repressed by CHX; whereas, rotenone-induced kinase activity and apoptotic toxicity remained unchanged. Transient expression of G2019S in dSY5Y increased the NCL and Hsp70 levels, while administration of a kinase inhibitor diminished these changes. Similar results were observed in rat primary neurons after rotenone treatment or G2019S transfection. Brains from G2019S-transgenic mice also showed increased NCL and Hsp70 levels. Accordingly, LRRK2 kinase inhibition might prevent oxidative stress-mediated PD progression.Abbreviations: 6-OHDA: 6-hydroxydopamine; CHX: cycloheximide; dSY5Y: differentiated SH-SY5Y; g2019S tg: g2019S transgenic mouse; GSK/A-KI: GSK2578215A kinase inhibitor; HSP70: heat shock protein 70; LDH: lactose dehydrogenase; LRRK2: leucine rich-repeat kinase 2; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; myc-GS LRRK2: myc-tagged g2019S LRRK2; NCL: nucleolin; PARP: poly(ADP-ribose) polymerase; PD: Parkinson’s disease; PINK1: PTEN-induced putative kinase 1; pmoesin: phosphorylated moesin at t558; ROS: reactive oxygen species

Nucleolin protects against doxorubicin-induced cardiotoxicity via upregulating microRNA-21.

Nucleolin is a multifunctional protein and participates in many important biological processes. Our previous study found that nucleolin protects the heart against myocardial ischemia-reperfusion injury. In this study, we aimed to investigate the role of nucleolin in doxorubicin (DOX)-induced cardiotoxicity. The expression pattern of nucleolin in hearts subjected toDOX injury was investigated, and we found that administration of DOX induced nucleolin expression significantly in vivo and in vitro. Gene transfection and RNA interference approaches were used in cardiomyocytes to investigate the function of nucleolin. Nucleolin overexpression protects cardiomyocytes against DOX-induced injury. Nucleolin-ablated cardiomyocytes become susceptible to the injury induced by DOX. The hearts of cardiac-myocyte-specific nucleolin transgenic mice are more resistant to DOX injury. Furthermore, nucleolin upregulates microRNA(miRNA)-21 expression in vivo and in vitro, and the miRNA-21 inhibitor negates the protective effect of nucleolin against injury induced by DOX. These results have demonstrated that nucleolin is involved in the regulation of DOX-induced cardiac injury and dysfunction via the regulation of miRNA-21 expression, and may be a novel therapeutic target for DOX-induced cardiotoxicity.

Abstract 1946: SJP1604, a novel nucleolin-targeted anti-cancer drug for acute myeloid leukemia using aptamer-drug conjugation technology

Abstract Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are cancers characterized by the rapid growth of abnormal white blood cells. Generally, cytarabine (Ara-C), azacitidine, and decitabine are treated as the standard first-line chemotherapy for AML/MDS. Although the 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 need for the next-generation targeted therapy to overcome drug resistance and reduce toxicity in AML/MDS. Aptamers mostly consist of oligonucleotides, which can specifically bind to their target molecules with high affinity. Here, we designed a synthetic DNA aptamer-nucleoside drug conjugate (SJP1604) specific for nucleolin, which is highly expressed only on the cell membrane of cancer cells. In particular, AML and ALL (acute lymphoblastic leukemia) cells are known to express the highest mRNA level of nucleolin among various cancer cells, relatively. Owing to the unique conformational property of SJP1604, it can be delivered into cancer cells with its high targeting ability and plasma stability. In this study, we verified that SJP1604 selectively targeted cancer cells by nucleolin-binding on the cancer cell membrane in the drug uptake assay. SJP1604 also exhibited a long-lasting plasma stability in 50% human plasma up to 48 hours in sharp contrast to unstability of general aptamers. Furthermore, SJP1604 inhibited not only the growth of human AML/MDS cell lines (MV-4-11, HL-60, MOLM-13, THP-1 and KG-1) but also drug-resistant cell lines (cytarabine-, azacitidine- and decitabine-resistant MOLM-13, cytarabine-resistant HL60 and cytarabine-resistant MV-4-11). Interestingly, SJP1604 showed the significantly reduced IC50 values with the decreased expression level of nucleolin in cytarabine-, azacitidine- and decitabine-resistant MOLM-13 while cytarabine caused no effect on the cell growth and the expression of nucleolin of the same resistant cell lines. Intravenous administration of SJP1604 (150 mg/kg) led to tumor regression and improved survival rate in MOLM-13 xenograft mouse model. In conclusion, these findings suggest that SJP1604 could be developed as a first-in-class drug for novel targeted therapy of AML/MDS with less drug toxicity as well as an orphan drug for overcoming drug resistance of AML/MDS, using aptamer-drug conjugation technology. Citation Format: Jihyun Um, Dohyeong Lee, Yongbin Park, Sung Hwan Moon, Su Jin Lee, Min-Hyo Ki, Hee Jong Shin, Eui Hwan Jo. SJP1604, a novel nucleolin-targeted anti-cancer drug for acute myeloid leukemia using aptamer-drug conjugation technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1946.

Gemcitabine-Incorporated G-Quadruplex Aptamer for Targeted Drug Delivery into Pancreas Cancer

Gemcitabine has been considered a first-line chemotherapy agent for the treatment of pancreatic cancer. However, the initial response rate of gemcitabine is low and chemoresistance occurs frequently. Aptamers can be effectively internalized into cancer cells via binding to target molecules with high affinity and specificity. In the current study, we constructed an aptamer-based gemcitabine delivery system, APTA-12, and assessed its therapeutic effects on pancreatic cancer cells in vitro and in vivo. APTA-12 was effective in vitro and in vivo in pancreatic cancer cells with high expression of nucleolin. The results of in vitro cytotoxicity assays indicated that APTA-12 inhibited the growth of pancreatic cancer cell lines. In vivo evaluation showed that APTA-12 effectively inhibited the growth of pancreatic cancer in Capan-1 tumor-bearing mice compared to mice that received gemcitabine alone or vehicle. These results suggest that the gemcitabine-incorporated APTA-12 aptamer may be a promising targeted therapeutic strategy for pancreatic cancer.

Nucleolin is expressed in patient-derived samples and glioblastoma cells, enabling improved intracellular drug delivery and cytotoxicity

One of the major challenges in Glioblastoma (GBM) therapy relates with the existence of glioma stem-like cells (GSCs), known to be chemo- and radio-resistant. GSCs and non-stem GBM cells have the ability to interchange, emphasizing the importance of identifying common molecular targets among those cell sub-populations. Nucleolin overexpression has been recently associated with breast cancer sub-populations with different stem-like phenotype. The goal of this work was to evaluate the potential of cell surface nucleolin as a target in GBM cells.Different levels of nucleolin expression resulted in a 3.4-fold higher association of liposomes targeting nucleolin (functionalized with the nucleolin-binding F3 peptide) in U87, relative to GBM11 glioblastoma cells. Moreover, nucleolin was suggested as a potential marker in OCT4-, NANOG-positive GSC, and in the corresponding non-stem GBM cells, as well as in SOX2-positive GSC. Doxorubicin delivered by liposomes targeting nucleolin enabled a level of cytotoxicity that was 2.5- or 4.6-fold higher compared to the non-targeted counterparts. Importantly, an overexpression of nucleolin was also observed in cells of patient-derived samples, as compared with normal brain. Overall, these results suggested nucleolin as a therapeutic target in GBM.

Nucleolin in Neurons of the Human Substantia Nigra

Using immunocytochemistry and confocal laser microscopy, the distribution of nucleolin (protein C23) in neurons of the human substantia nigra was investigated. Fragments of the human midbrain (n = 8) with the verified lack of neurodegeneration were used in the study. The material was fixed in zinc ethanol formaldehyde, a special fixative that provides high preservation of antigenic determinants. New morphological data on the distribution of nucleolin in neurons of the human substantia nigra were obtained. Nucleolin was found in the nucleolus and nucleoplasm of dopaminergic neurons but not in the cytoplasm or cell surface. In the nucleolus and nucleoplasm, protein C23 is distributed irregularly in the form of some accumulations (clumps) with fuzzy borders. Moreover, nucleolin distribution varied in structurally similar neurons containing cytoplasmic neuromelanin. The Marinesco bodies typically not exhibiting nucleolin expression were identified in the nucleus, in addition to nucleoli, by confocal laser microscopy with simultaneous use of transmitted light detector. The findings can contribute to elucidation of the role of nucleolin in the specific functions of normal dopaminergic neurons in the human substantia nigra and assessment of probable involvement of C23 in the development of nucleolar stress and neurodegeneration.

Nucleolin mediated pro‐angiogenic role of Hydroxysafflor Yellow A in ischaemic cardiac dysfunction: Post‐transcriptional regulation of VEGF‐A and MMP‐9

Hydroxysafflor Yellow A (HSYA), a most representative ingredient of Carthamus tinctorius L., had long been used in treating ischaemic cardiovascular diseases in China and exhibited prominently anticoagulant and pro‐angiogenic activities, but the underlying mechanisms remained largely unknown. This study aimed to further elucidate the pro‐angiogenic effect and mechanism of HSYA on ischaemic cardiac dysfunction. A C57 mouse model of acute myocardial infarction (AMI) was firstly established, and 25 mg/kg HSYA was intraperitoneally injected immediately after operation and given once, respectively, each morning and evening for 2 weeks. It was found that HSYA significantly improved ischaemia‐induced cardiac haemodynamics, enhanced the survival rate, alleviated the myocardial injury and increased the expressions of CD31, vascular endothelial growth factor‐A (VEGF‐A) and nucleolin in the ischaemic myocardium. In addition, HSYA promoted the migration and tube formation of human umbilical vein endothelial cells (HUVECs), enhanced the expressions of nucleolin, VEGF‐A and matrix metalloproteinase‐9 (MMP‐9) in a dose‐ and time‐dependent manner. However, down‐regulation of nucleolin expression sharply abrogated the effect mentioned above of HSYA. Further protein‐RNA coimmunoprecipitation and immunoprecipitation‐RT‐PCR assay showed that nucleolin binded to VEGF‐A and MMP‐9 mRNA and overexpression of nucleolin up‐regulated the mRNA expressions of VEGF‐A and MMP‐9 in the HUVECs through enhancing the stability of VEGF‐A and MMP‐9 mRNA. Furthermore, HSYA increased the mRNA expressions of VEGF‐A and MMP‐9 in the extract of antinucleolin antibody‐precipitated protein from the heart of AMI mice. Our data revealed that nucleolin mediated the pro‐angiogenic effect of HSYA through post‐transcriptional regulation of VEGF‐A and MMP‐9 expression, which contributed to the protective effect of HSYA on ischaemic cardiac dysfunction.

Respiratory syncytial virus prolifically infects N2a neuronal cells, leading to TLR4 and nucleolin protein modulations and RSV F protein co-localization with TLR4 and nucleolin

BackgroundRespiratory syncytial virus (RSV) infects the central nervous system, resulting in neurological symptoms. However, the precise underlying pathogenic mechanisms have not been elucidated. In the present study, the infectivity of RSV on N2a neuronal cells and the possible roles of Toll-like receptor 4 (TLR4) and nucleolin (C23) during RSV infection were investigated.MethodsWe compared two experimental groups (infected and non-infected) and monitored the RSV viral titers in the culture supernatant by a viral plaque assay. We also inspected the morphology of the nucleus in infected N2a cells. We measured the level of RSV F protein and studied its co-localization with TLR4 and nucleolin using immunofluorescence assays and laser confocal microscopy. The potential interaction of RSV F protein with TLR4 and nucleolin was examined by coimmunoprecipitation. The expression changes of TLR4, nucleolin, TLR3 and TLR7 proteins in N2a cells and IL-6 and TNF-α in the culture supernatant were investigated by Western Blot analysis and ELISA assay. Changes in neuronal cell apoptosis status was examined by flow cytometry.ResultsThe results demonstrated prolific RSV infection of N2a cells, which triggered a decrease of NeuN protein expression, coinciding with an increase of nuclear lesions, F protein expression, RSV viral titers, and late apoptotic levels of N2a cells. RSV infection induced co-localization of RSV F protein with TLR4 and nucleolin, which could potentially lead to a direct interaction. Furthermore, it was found that TLR4 and nucleolin levels increased early after infection and decreased subsequently, whereas TLR3 and TLR7 expression increased throughout RSV infection.ConclusionThe RSV Long strain can prolifically infect N2a neuronal cells, modulating the expression of TLR4 and nucleolin, as well as TLR3, TLR7 and their downstream inflammatory factors, and inducing the co-localization of the RSV F protein with TLR4 and nucleolin.

Inhibition of TNF\u03b1-interacting protein \u03b1 (Tip\u03b1)-associated gastric carcinogenesis by BTG2/TIS21 via downregulating cytoplasmic nucleolin expression

To understand the regulation of Helicobacter pylori (H. pylori)-associated gastric carcinogenesis, we examined the effect of B-cell translocation gene 2 (BTG2) expression on the biological activity of Tipα, an oncoprotein secreted from H. pylori. BTG2, the human ortholog of mouse TIS21 (BTG2/TIS21), has been reported to be a primary response gene that is transiently expressed in response to various stimulations. Here, we report that BTG2 is constitutively expressed in the mucous epithelium and parietal cells of the gastric gland in the stomach. Expression was increased in the mucous epithelium following H. pylori infection in contrast to its loss in human gastric adenocarcinoma. Indeed, adenoviral transduction of BTG2/TIS21 significantly inhibited Tipα activity in MKN-1 and MGT-40, human and mouse gastric cancer cells, respectively, thereby downregulating tumor necrosis factor-α (TNFα) expression and Erk1/2 phosphorylation by reducing expression of nucleolin, a Tipα receptor. Chromatin immunoprecipitation proved that BTG2/TIS21 inhibited Sp1 expression and its binding to the promoter of the nucleolin gene. In addition, BTG2/TIS21 expression significantly reduced membrane-localized nucleolin expression in cancer cells, and the loss of BTG2/TIS21 expression induced cytoplasmic nucleolin availability in gastric cancer tissues, as evidenced by immunoblotting and immunohistochemistry. Higher expression of BTG2 and lower expression of nucleolin were accompanied with better overall survival of poorly differentiated gastric cancer patients. This is the first report showing that BTG2/TIS21 inhibits nucleolin expression via Sp1 binding, which might be associated with the inhibition of H. pylori-induced carcinogenesis. We suggest that BTG2/TIS21 is a potential inhibitor of nucleolin in the cytoplasm, leading to inhibition of carcinogenesis after H. pylori infection.

Cell surface expression of nucleolin mediates the antiangiogenic and antitumor activities of kallistatin.

Kallistatin is a unique serine proteinase inhibitor and heparin-binding protein. A previous study conducted by our group indicated that kallistatin has antiangiogenic and antitumoral activities. In the present study, we report that kallistatin specifically binds to membrane surface-expressed nucleolin with high affinity. Antibody-mediated neutralization or siRNA-induced nucleolin knockdown results in loss of kallistatin suppression of endothelial cell proliferation and migration in vitro and tumor angiogenesis and growth in vivo. In addition, we show that kallistatin is internalized and transported into cell nuclei of endothelial cells via nucleolin. Within the nucleus, kallistatin inhibits the phosphorylation of nucleolin, which is a critical step required for cell proliferation. Thus, we demonstrate that nucleolin is a novel functional receptor of kallistatin that mediates its antiangiogenic and antitumor activities. These findings provide mechanistic insights into the inhibitory effects of kallistatin on endothelial cell growth, tumor cell proliferation, and tumor-related angiogenesis.

Targeting nucleolin for better survival in diffuse large B-cell lymphoma.

Anthracyclines have been a cornerstone in the cure of diffuse large B-cell lymphoma (DLBCL) and other hematological cancers. The ability of anthracyclines to eliminate DLBCL depends on the presence of topoisomerase-II-alpha (TopIIA), a DNA repair enzyme complex. We identified nucleolin as a novel binding partner of TopIIA. Abrogation of nucleolin sensitized DLBCL cells to TopIIA targeting agents (doxorubicin/etoposide). Silencing nucleolin and challenging DLBCL cells with doxorubicin enhanced the phosphorylation of H2AX (γH2AX-marker of DNA damage) and allowed DNA fragmentation. Reconstitution of nucleolin expression in nucleolin-knockdown DLBCL cells prevented TopIIA targeting agent-induced apoptosis. Nucleolin binding to TopIIA was mapped to RNA-binding domain 3 of nucleolin, and this interaction was essential for blocking DNA damage and apoptosis. Nucleolin silencing decreased TopIIA decatenation activity, but enhanced formation of TopIIA-DNA cleavable complexes in the presence of etoposide. Moreover, combining nucleolin inhibitors: aptamer AS1411 or nucant N6L with doxorubicin reduced DLBCL cell survival. These findings are of clinical importance because low nucleolin levels versus high nucleolin levels in DLBCL predicted 90-month estimated survival of 70% versus 12% (P<0.0001) of patients treated with R-CHOP-based therapy.