Effect Of Nucleolin Research Articles

Effect of Nucleolin on Lymphoma Proliferation by Regulating Thymidine Kinase 1

To investigate the mechanism of nucleolin (NCL) involved in lymphoma proliferation by regulating thymidine kinase 1 (TK1). Twenty-three patients with diffuse large B-cell lymphoma (DLBCL) were selected and divided into initial treatment group (14 cases) and relapsed/refractory group (9 cases). Serum TK1 and C23 protein in peripheral blood mononuclear cells were detected. Cell models of CA46-NCL-KD (CA46-NCL-knockdown) and CA46-NCL-KNC (CA46-NCL-knockdown negative control) were established by lentivirus vector mediated transfection in Burkitt lymphoma cell line CA46. The half maximal inhibitory concentration (IC50) of CA46-NCL-KD, CA46-NCL-KNC, and CA46 to adriamycin were detected by cell proliferation assay (MTS). The expression of NCL mRNA and protein in CA46-NCL-KD and CA46-NCL-KNC cells were dectected by Q-PCR and Western blot, respectively. The cell cycle of CA46-NCL-KD, CA46-NCL-KNC, and CA46 cells were detected by flow cytometry. The expression of TK1 protein in CA46-NCL-KD and CA46-NCL-KNC cells was detected by an enhanced chemiluminescence (ECL) dot blot assay. The level of serum TK1 in the initial treatment group was 0.43(0-30-1.01) pmol/L, which was lower than 10.56(2.19-14.99) pmol/L in the relapsed/refractory group (P<0-01), and the relative expression level of NCL protein in peripheral blood was also significantly lower. The IC50 of CA46-C23-KD cells to adriamycin was (0.147±0.02) μg/ml, which was significantly lower than (0.301±0.04) μg/ml of CA46-C23-KNC cells and (0.338±0.05) μg/ml of CA46 cells (P<0.05). Compared with CA46-NCL-KNC cells, the expression of NCL mRNA and protein, TK1 protein decreased in CA46-NCL-KD cells, and the proportion of S phase and G2/M phase also decreased, while G0/G1 phase increased in cell cycle. The increased expression of NCL in DLBCL and CA46 cells indicates low sensitivity to drug. NCL may participate in regulation of lymphoma proliferation by affecting TK1 expression, thereby affecting the drug sensitivity.

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).

Effect of nucleolin on adriamycin resistance via the regulation of B-cell lymphoma 2 expression in Burkitt's lymphoma cells.

Nucleolin (NCL, C23) is an important nucleocytoplasmic multifunctional protein. Due to its multifaceted profile and high expression in cancer, NCL is considered to be a marker of drug resistance associated with chemotherapy. However, the biochemical mechanisms in which NCL suppresses drug sensitivity in several cancers have yet to be fully elucidated. This study aims to explore the effect of NCL on drug sensitivity and its potential mechanism in CA46 Burkitt's lymphoma (BL) cells. CA46 BL cells were transfected with lentiviruses carrying the NCL gene (CA46‐NCL‐overexpression, CA46‐NCL‐OE), or shRNA sequences that target the endogenous NCL gene (CA46‐NCL‐knockdown, CA46‐NCL‐KD). Adriamycin (ADM) IC50 levels for CA46‐NCL‐overexpressed (OE), CA46‐NCL‐OE control (OEC), CA46‐NCL‐knockdown (KD), and CA46‐NCL‐KD control (KDC) cells were 0.68 ± 0.06 μg/ml, 0.68 ± 0.06 μg/ml, 0.68 ± 0.06 μg/ml, and 0.30 ± 0.04 μg/ml, respectively. Apoptosis rates were significantly increased following NCL KD, whereas the opposite effect was noted in OE cells. A significant reduction of B‐cell lymphoma 2 (Bcl‐2) mRNA and protein levels in KD cells was observed, while OE cells displayed the opposite effect. The stability of Bcl‐2 mRNA was influenced by NCL levels, the half‐life of which was extended after NCL‐OE, whereas it was reduced in KD cells. Finally, results of RNA‐immunoprecipitation assays indicated that NCL could bind to Bcl‐2 mRNA in CA46 cells. Taken together, these results suggested that NCL could mediate Bcl‐2 expression and stability, and thus enhance ADM resistance in CA46 BL cells.

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.

Abstract B17: Targeting the oncogenic TBX3 and its co-factors in anti-cancer drug development

Abstract Sarcomas are heterogeneous neoplasms of mesenchymal origin whose clinical management is compromised due to inadequate diagnostic markers and limited therapeutic options. While the incidence rates for sarcomas in Africa remain uncertain, there is a widely held view that they are common in black African children and adolescents. Indeed, according to a Nigerian study, soft tissue sarcomas make up as much as 11.3% of all childhood cancers. We recently reported that the transcription factor, TBX3, is overexpressed in a diverse range of sarcoma subtypes where it plays a direct oncogenic role and it has been proposed as a novel target for their treatments. Direct targeting of transcription factors for therapies however continues to represent a serious challenge and therefore the molecular mechanisms that mediate its biological activity may be more amenable as anti-cancer drug targets. To address this, affinity purifications coupled with mass spectrometry was performed to identify TBX3 protein co-factors that regulate its oncogenic activity in sarcomas. Here we describe the validation of nucleolin as TBX3 co-factor by co-immunoprecipitation assays and confocal microscopy/co-localization analysis. The effect of nucleolin on TBX3 function was determined using RNAi rescue experiments coupled with growth curve and scratch motility assays to assess cell proliferation and migration respectively. Finally, the effect of the nucleolin targeting aptamer, AS1411, on the cell viability of a range of sarcoma subtypes was tested. Results show that the overexpression of TBX3 and nucleolin are common in a number of sarcoma subtypes. We provide compelling evidence that nucleolin interacts with and co-operates with TBX3 to promote proliferation and migration of chondrosarcoma, liposarcoma and rhabdomyosarcoma cells. Furthermore, AS1411 exhibits potent anti-cancer activity in these sarcomas whilst it has no effect on normal cells. In conclusion, we propose that TBX3 and nucleolin can be used in combination as biomarkers for the diagnosis and targeted therapy of a diverse range of sarcomas which are highly aggressive and treatment resistant cancers. Citation Format: Tarryn Willmer, Shannon Smyly, Danica Smuts, Sharon Prince. Targeting the oncogenic TBX3 and its co-factors in anti-cancer drug development [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B17.

Control of AC133/CD133 and impact on human hematopoietic progenitor cells through nucleolin.

AC133 is a prominent surface marker of CD34+ and CD34- hematopoietic stem/progenitor cell (HSPC) subsets. AC133+ HSPCs contain high progenitor cell activity and are capable of hematopoietic reconstitution. Furthermore, AC133 is used for prospective isolation of tumor-initiating cells in several hematological malignancies. Nucleolin is a multifunctional factor of growing and cancer cells, which is aberrantly active in certain hematological neoplasms, and serves as a candidate molecular target for cancer therapy. Nucleolin is involved in gene transcription and RNA metabolism and is prevalently expressed in HSPCs, as opposed to differentiated hematopoietic tissue. The present study dissects nucleolin-mediated activation of surface AC133 and its cognate gene CD133, via specific interaction of nucleolin with the tissue-dependent CD133 promoter P1, as a mechanism that crucially contributes to AC133 expression in CD34+ HSPCs. In mobilized peripheral blood (MPB)-derived HSPCs, nucleolin elevates colony-forming unit (CFU) frequencies and enriches granulocyte-macrophage CFUs. Furthermore, nucleolin amplifies long-term culture-initiating cells and also promotes long-term, cytokine-dependent maintenance of hematopoietic progenitor cells. Active β-catenin, active Akt and Bcl-2 levels in MPB-derived HSPCs are nucleolin-dependent, and effects of nucleolin on these cells partially rely on β-catenin activity. The study provides new insights into molecular network relevant to stem/progenitor cells in normal and malignant hematopoiesis.

Abstract 3187: Nucleolin-induced cap-independent translational pathway involved in Sp1 accumulation in tumorigenesis.

Abstract Sp1 is a transcriptional factor to regulate numbers genes related to cell proliferation, apoptosis and tumor progression. Previous studies indicate that Sp1 is induced significantly through modulating its transcriptional activity and protein stability. To detail study the level of Sp1 protein and mRNA in tumorigenesis found that Sp1 mRNA was just increased a little, but Sp1 protein was increased dramatically, implying that enhancement of translational activity by nucleion might be important in the Sp1 accumulation in tumor progression. Here we found a Y-shift motif within the 5’-UTR of the Sp1 mRNA, and to study the contribution in Sp1 level found that the 5’-URT could enhance the translational efficiency of Sp1. Using this Y-shift motif as a probe fished the interacted proteins. Several RNA binding proteins were identified by LC/MS/MS, and nucleolin is one of them. To further address the effect of nucleolin in Sp1 induction found that nucleolin overexpression did not alter the level of Sp1 mRNA, but increased the translational activity obviously. In addition, the signal transduction pathway was also studied, and found that EGFR and Kras pathways might be involved in the induction in the cap-independent pathway in Sp1 induction in tumorigenesis. Taken the previous studies and this study together, during tumor formation, several strategies including increasing the transcriptional activity, protein stability and translational efficiency were used to induce the Sp1 level to regulate many oncogenes to enhance the cancer progression. Keywords: Nucleolin, Sp1, Cap-independent pathway, Citation Format: Tsung-I Hsu, Jan-Jong Hung, Wen-Chang Chang. Nucleolin-induced cap-independent translational pathway involved in Sp1 accumulation in tumorigenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3187. doi:10.1158/1538-7445.AM2013-3187

Nucleolin promotes secondary structure in ribosomal RNA

The effect of nucleolin on the secondary structure of RNA was studied using circular dichroism (CD). Nucleolin caused decreases in the main positive bands and shifts to higher wavelengths in the CD spectra of synthetic polynucleotides such as poly(G) and poly(A) indicating helix destabilizing activity. In contrast, nucleolin effected increases in signal and shifts to lower wavelengths of the peaks of CD spectra of ribosomal RNA, suggesting enhancement of secondary structure. Another major nucleolar RNA binding protein, B23, had helix destabilizing activity but did not enhance RNA secondary structure. It is proposed that nucleolin promotes formation of secondary structure in preribosomal RNA during the early stages of ribosome biogenesis.