scholarly journals Quantitative phosphoproteomic analysis reveals chemoresistance-related proteins and signaling pathways induced by rhIL-6 in human osteosarcoma cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Rui Zhang ◽  
Huan Wang ◽  
Erliang Li ◽  
Yonghong Wu ◽  
Yanhua Wen ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathways ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Immunohistochemical Staining ◽  
Pivotal Role ◽  
Osteosarcoma Cells ◽  
Clinical Specimens ◽  
Human Osteosarcoma ◽  
Human Osteosarcoma Cells

Abstract Background IL-6 plays a pivotal role in resistance to chemotherapeutics, including lobaplatin. However, the underlying mechanisms are still unclear. This study was to investigate the changes in phosphoproteins and their related signaling pathways in the process of IL-6-induced chemoresistance to lobaplain in osteosarcoma cells. Methods We performed a quantitative phosphoproteomic analysis of the response of SaOS-2 osteosarcoma cells to recombinant human IL-6 (rhIL-6) intervention prior to lobaplatin treatment. The cells were divided into the control group (Con), the lobaplatin group (Lob), and the rhIL-6-and-lobaplatin group (IL-6). Three biological replicates of each group were included. The differentially expressed phosphoproteins were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Netphos 3.1 was used for the prediction of kinases, and STRING was used for the visualization of protein–protein interactions. The conserved motifs surrounding the phosphorylated residues were analyzed using the motif-x algorithm. Western blot analysis was performed to verify the differential expression of p-FLNC, its predicted kinase and the related signaling pathway. The results of the bioinformatic analysis were validated by immunohistochemical staining of clinical specimens. Results In total, 3373 proteins and 12,183 peptides, including 3232 phosphorylated proteins and 11,358 phosphorylated peptides, were identified and quantified. Twenty-three significantly differentially expressed phosphoproteins were identified in the comparison between the IL-6 and Lob groups, and p-FLNC ranked second among these phosphoproteins. GO and KEGG analyses revealed the pivotal role of mitogen-activated protein kinase signaling in drug resistance induced by rhIL-6. Four motifs, namely, -SPxxK-, -RxxSP-, -SP-, and -SPK-, demonstrated higher expression in the IL-6 group than in the Lob group. The western blot analysis results verified the higher expression of p-FLNC, AKT1, and p-ERK and the lower expression of p-JNK in the IL-6 group than in the Con and Lob groups. The immunohistochemical staining results showed that p-FLNC, AKT1 and p-ERK1/2 were highly expressed in platinum-resistant clinical specimens but weakly expressed in platinum-sensitive specimens, and platinum-resistant osteosarcoma specimens demonstrated weak expression of p-JNK. Conclusions This phosphoproteomic study is the first to reveal the signature associated with rhIL-6 intervention before lobaplatin treatment in human osteosarcoma cells. p-FLNC, AKT1, and MAPK signaling contributes to resistance to lobaplatin in osteosarcoma SaOS-2 cells and may represent molecular targets to overcome osteosarcoma chemoresistance.

scholarly journals Kaempferol suppresses cell metastasis via inhibition of the ERK-p38-JNK and AP-1 signaling pathways in U-2 OS human osteosarcoma cells

2013 ◽  
Vol 30 (2) ◽  
pp. 925-932 ◽  
Author(s):  
HUI-JYE CHEN ◽  
CHUNG-MING LIN ◽  
CHAO-YING LEE ◽  
NAI-CHEN SHIH ◽  
SHU-FEN PENG ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Osteosarcoma Cells ◽  
Human Osteosarcoma ◽  
Cell Metastasis ◽  
Human Osteosarcoma Cells

scholarly journals Fucoxanthin Inhibits Myofibroblast Differentiation and Extracellular Matrix Production in Nasal Polyp-Derived Fibroblasts via Modulation of Smad-Dependent and Smad-Independent Signaling Pathways

Marine Drugs ◽  
2018 ◽  
Vol 16 (9) ◽  
pp. 323 ◽  
Author(s):  
Hyun Jung ◽  
Dae-Sung Lee ◽  
Seong Park ◽  
Jung Choi ◽  
Won-Kyo Jung ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathways ◽  
Nasal Polyp ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Type I ◽  
Molecular Signaling ◽  
Tgf Β1

Nasal polyps (NPs) are a multifactorial disorder associated with a chronic inflammatory state of the nasal mucosa. Fucoxanthin (Fx) is a characteristic orange carotenoid obtained from brown algae and has diverse immunological properties. The present study investigated whether Fx inhibits fibrosis-related effects in nasal polyp-derived fibroblasts (NPDFs) and elucidated the molecular signaling pathways involved. The production of collagen type I (Col-1) was investigated in NP tissue via immunohistochemistry and western blot analysis. NPDFs were treated with transforming growth factor (TGF)-β1 (1 ng/mL) in the presence or absence of Fx (5–30 µM). The levels of α-smooth muscle actin (α-SMA), Col-1, and phosphorylated (p)-Smad 2/3, signal protein-1 (SP-1), MAPKs (mitogen-activated protein kinases), and Akt were measured by western blot analysis. The expression of Col-1 was detected in NP tissues. TGF-β1 stimulated the production of α-SMA and Col-1, and stimulated the contraction of collagen gel. However, pretreatment with Fx attenuated these effects. Furthermore, these inhibitory effects were mediated through modulation of both Smad 2/3 and Akt/SP-1 signaling pathways in TGF-β1-induced NPDFs. The results from the present study suggest that Fx may be a novel anti-fibrotic agent for the treatment of NP formation.

scholarly journals Investigation into imbalance of Th1/Th2 cells in cirrhotic, hypersplenic rats

2019 ◽  
Vol 48 (3) ◽  
pp. 030006051988944 ◽  
Author(s):  
Yunfu Lv ◽  
Yejuan Li ◽  
Ning Liu ◽  
Yonghong Dong ◽  
Jie Deng
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chemokine Receptors ◽  
Immunohistochemical Staining ◽  
Th2 Cells ◽  
Intragastric Administration ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Chemokine Receptor Ccr3

Objectives To evaluate the Th1/Th2 cell profile in spleens of cirrhotic and hypersplenic rats by investigating the expression of Th1-associated chemokine receptors CXCR3, CCR5 and Th2-associated chemokine receptor CCR3. Methods Experimental liver cirrhosis and hypersplenism were induced in rats by the intragastric administration of carbon tetrachloride (CCl4; 40% solution [0.3 ml/100g, twice/week for 8 weeks]) and confirmed by pathology and hemogram. Presence of the three chemokine receptors was investigated by real-time polymerase chain reaction (RT-PCR), immunohistochemical staining, and western blot analysis. Results By comparison with control animals (n=10), RT-PCR demonstrated that CXCR3 and CCR5-mRNA levels were significantly elevated in the hypersplenic rats (n=26) and CCR3-mRNA levels were lower. Immunohistochemical staining showed that by comparison with controls, the mean density of the Th1-associated CXCR3 and CCR5 receptors was significantly increased but there was no difference between groups in Th2-associated CCR3 receptors. Western blot analysis showed that by comparison with controls, hypersplenic rats had higher levels of CXCR3 and CCR5 protein but lower levels of CCR3 protein. Conclusions The abnormal expression of Th1-associated chemokine receptors in spleens of rats with cirrhosis and hypersplenism induced by CCL4 suggests that a functional imbalance between Th1/Th2 cells may play a role in the pathogenesis of hypersplenism.

Monoclonal Antibody Against ROR1 in Chronic Lymphocytic Leukemia Cells Induced Apoptosis Via PI3-Kinase/AKT/CREB Pathway

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1769-1769
Author(s):  
Amir Hossein Daneshmanesh ◽  
Mohammad Hojjat-Farsangi ◽  
Asa Sandin ◽  
Abdul Salam Khan ◽  
Ali Moshfegh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Signaling Pathways ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Pi3 Kinase ◽  
Signaling Proteins ◽  
Downstream Signaling ◽  
Induced Apoptosis ◽  
Creb Pathway

Abstract Abstract 1769 Background: Phosphoinositide 3-kinase (PI3K)/AKT cascade regulates cell survival, proliferation and differentiation in a variety of cells. In CLL cells PI3K pathway is constitutively activated leading to AKT activation and phosphorylation of cAMP response element-binding protein (CREB). CREB is a transcription factor overexpressed and constitutively phosphorylated in a variety of cancers and seems to have a role in tumor pathobiology. There is a great need to develop novel strategies for targeted therapy in CLL. Monoclonal antibodies (mAbs) specifically targeting leukemic cells might be a rewarding approach. ROR1 is a type I transmembrane receptor tyrosine kinase belonging to one of the twenty families of receptor tyrosine kinases (RTKs). ROR1 is overexpressed on CLL cells but not in white blood cells of healthy donors. ROR1 is constitutively phosphorylated in CLL and siRNA transfection induced apoptosis. We have developed a unique anti-ROR1 mAb directed against CRD (cysteine-rich domain) of the extracellular region of ROR1 capable of inducing direct apoptosis of primary CLL cells. Our anti-CRD mAb induced dephosphorylation of the ROR1 molecule. Aims: To study the apoptotic effect of an anti-ROR1 CRD mAb and effects on downstream signaling pathways involved in CLL, specially the PI3-kinase/AKT/CREB pathway using primary CLL cells. Methods: Using a peptide-based mouse mAb generation method we produced several mAbs against the three extracellular domains of ROR1. In the current study we used one of the best anti-ROR1 antibodies, an anti-CRD mAb raised against the CRD region of ROR1 (Daneshmanesh et al., Leukemia. 2012 Jun;26(6):1348-55). Flow cytometry was used for surface staining of ROR1. Primary CLL cells were incubated with the anti-ROR1 CRD mAb and apoptosis was detected by the MTT assay and Annexin V/propidium iodide (flow cytometry) methods in a 24 h assay. Antibody untreated and treated cell lysates were prepared and subjected to Western blot analysis for identification of signaling molecules involved in apoptosis induced by the anti-ROR1 CRD mAb. We analysed total and phosphorylated levels of the following signaling proteins: AKT, p-AKT, PI3K, p-PI3K, CREB, p-CREB, ERK, p-ERK, PKC and p-PKC. Phosphoproteins were measured before incubation with the mAb and after 20 min-2 h. Results: ROR1 surface expression was detected on 80–85% of the CLL cells. The frequency of apoptotic cells induced by the anti-CRD mAb was in the range of 45–50% which is in accordance with our previous reports (see above). Time kinetics experiments using anti-ROR1 CRD mAb incubated with primary CLL cells revealed dephosphorylation of ROR1 downstream signaling molecules. We analysed the following molecules known to be involved in CLL: PKC, PI3-kinase and ERK1/2. After co-culturing CLL cells with the anti-ROR1 CRD mAb, Western blot analysis showed decreased level of phosphorylated AKT in treated compared to untreated samples. No changes in the phosphorylation levels of ERK1/2 and PKC proteins were seen. Furthermore, we analysed the PI3-kinase protein which is upstream of AKT, and noticed that in CLL cells treated with the anti-ROR1 CRD mAb, the phosphorylation intensity of PI3-kinase p85 isoform has decreased but not p55 isoforrn. Moreover, we also studied the CREB phosphorylation in treated and untreated CLL samples and detected dephosphorylation of CREB in treated as compared to untreated samples. Conclusion: Incubation of CLL cells with an anti-ROR1 CRD mAb induced apoptosis of primary CLL cells. Apoptosis was preceded by dephosphorylation within 2 h of PI3-kinase, AKT and CREB proteins indicating deactivation of these signaling proteins by the anti-ROR1 mab. In untreated CLL cells no effect on phosphorylation of these proteins was noted. Furthermore our ROR1 mAb did not dephosphorylate PKC or ERK. Our data may suggest that activation of CREB molecule might occur via the PI3K/AKT pathway and may be a survival signal in CLL cells associated with the aberrant expression of ROR1. The constitutive phosphorylation of PKC and ERK1/2 seen in CLL might not be related to the overexpression of ROR1. Further studies are warranted for a better understanding of signaling pathways associated with ROR1 and the downstream signaling effects of ROR1 targeting drugs. Disclosures: No relevant conflicts of interest to declare.

Effect of nicotine on chemoresistant phenotype as mediated through Src-dependent Id1 expression in pancreatic adenocarcinoma cells.

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 216-216
Author(s):  
J. G. Trevino ◽  
S. R. Pillai ◽  
S. P. Chellappan
Keyword(s):  
Pancreatic Cancer ◽  
Western Blot ◽  
Cell Viability ◽  
Cancer Cells ◽  
Signaling Pathways ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Pancreatic Tumor ◽  
Pancreatic Cancer Cells ◽  
Dependent Pathway

216 Background: The signaling pathways contributing to DNA-binding protein inhibitor Id1 expression and chemoresistance in pancreatic cancer remain unknown. Id1 plays a role in pancreatic tumor progression with tumor-promoting effects of nicotine regulating protein tyrosine kinase Src activation and Id1 expression, both associated with chemoresistance in other systems. We hypothesize Id1 expression regulates chemoresistance in pancreatic cancer through a nicotine-promoting Src-dependent pathway. Methods: We probed pancreatic cancer cell lines (L3.6pl, PANC-1, Mia-PaCa-2) for innate gemcitabine chemoresistance with cell viability MTT assay and western blot analysis of PARP cleavage programmed cell death. Gemcitabine-sensitive cells were exposed to rising gemcitabine concentrations to establish a resistant subtype, L3.6plGemRes. Protein analysis and mRNA expression were determined by western blot analysis and RT-PCR respectively. Induction of Src phosphorylation or Id1 expression was performed with nicotine (1 μM). Results: Inhibition of c-Src expression was performed with short-interfering RNA (siRNA). Nicotine-induced Src phosphorylation and Id1 expression. Inhibition of Src by siRNA resulted in decreased nicotine-induced Id1 expression. Inhibition of Src and Id1 expression by siRNA in innate or established gemcitabine resistant pancreatic cancer cells resulted in gemcitabine sensitization. To determine if nicotine contributes to gemcitabine chemoresistance, we exposed gemcitabine-sensitive cells to nicotine with subsequent exposure to gemcitabine IC50, 250 ng/ml, and cell viability assays confirmed a 2-fold increase in cell prolilferation and a 4.5-fold reduction in apoptosis. Further, nicotine induced phosphorylation of key signaling enzymes involved in proliferation and apoptosis, Erk1/2 and Akt respectively. Conclusions: In summary, we demonstrate that Id1, through a nicotine-promoting Src-dependent pathway, is necessary for establishment of a chemoresistant phenotype in pancreatic cancer cells. Understanding the signaling pathways involved in pancreatic tumor chemoresistance will lead to therapies resulting in improved tumor responses. No significant financial relationships to disclose.

C/EBPβ Expression in ALK+ Anaplastic Large Cell Lymphomas (ALCL) Is Regulated by Stat3 Signaling Pathway.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3570-3570
Author(s):  
Natasa Anastasov ◽  
Martina Rudelius ◽  
Margit Klier ◽  
Therese Dau ◽  
Daniela Angermeier ◽  
...  
Keyword(s):  
Western Blot ◽  
Signaling Pathways ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Chromosomal Translocation ◽  
Large Cell ◽  
Facs Analysis ◽  
Akt Inhibitor ◽  
Factor C

Abstract Background: ALK+ ALCL is characterized by the t(2;5) chromosomal translocation, resulting in the expression of a fusion protein called NPM-ALK. We recently reported the abnormal expression of the transcription factor C/EBPβ in ALCL, and demonstrated that C/EBPβ expression is dependent on NPM-ALK kinase activity. However, it is unclear how this signal is transduced. The aim of this study is to investigate the different signaling pathways that have been implicated in NPM/ALK signaling to elucidate their role in the expression of C/EBPβ. Materials and methods: To analyze the different signaling pathways induced by NPM-ALK, Ba/F3 cells were transfected with an NPM-ALK kinase-inhibitable construct (NPM-ALK-ATP-Abl). Imatinib was used to block NPM-ALK activity. Highly effective shRNA sequences (>85% knockdown) were identified for AKT, mTOR, and Stat3 proteins using a specific lacZ reporter fusion assay in HEK-293T cells, and corroborated by Western blot analysis. Each of these shRNAs were cloned into a lentiviral transfer vector carrying GFP as a reporter gene, which enables the detection of infected cells by FACS analysis. Three ALK+ ALCL cell lines were analyzed (SUDHL-1, Karpas 299 and Ki-JK), using appropriate controls. Western Blot analysis and qRT-PCR were performed to quantitate the knockdown effect. These studies were supplemented with pharmacological inhibitors: rapamycin, MAPK inhibitors (U0126 and PD98059) and AKT inhibitor (Calbiochem). The effect of Stat3, AKT, mTOR and MAPK knockdown on proliferation and cell viability was analyzed by MTT assay and FACS analysis. Results: Ba/F3 cells transfected with NPM-ALK-ATP-Abl construct resulted in induction of C/EBPβ expression and phosphorylation of Stat3, AKT and MAPK with no changes observed in mTOR phosphorylation. The opposite effect was observed when the NPM-ALK-ATP-Abl activity was inactivated with Imatinib. The infection rates of the specific shRNA constructs in the three ALK+ALCL cell lines were almost 100%. Downregulation of Stat3 in ALK+ALCL cells inhibited C/EBPβ at mRNA and protein level with impairment in cell proliferation and viability. In contrast, downregulation of AKT and mTOR showed no changes in C/EBPβ expression, whereas their downstream targets (rpS6 and 4E-BP1) phosphorylations were inactivated. These results were corroborated with rapamycin and AKT pharmacological inhibitory studies. MEK inhibitors (U0126 and PD98059) blocked the ERK1/2 phosphorylation reflected in growth retardation and its downstream target TSC2 phosphorylation without changing the expression of C/EBPβ. However, the phosphoThr-235 C/EBPβ was deactivated, confirming the importance of ERK1/2 in the phosphorylation and activation of C/EBPβ. Conclusions: In this study, we demonstrated that the induction of C/EBPβ expression by NPM-ALK correlates with the phosphorylation of AKT, MAPK and Stat3. However, only the downregulation of Stat3 has influence on C/EBPβ mRNA and protein expression, whereas MAPK is important for the phosphorylation and modulation of CEBPβ function. The downregulation of C/EBPβ, as a consequence of Stat3 inhibition has an important effect on cell growth and survival.

scholarly journals Non-Genomic Action of Androgens is Mediated by Rapid Phosphorylation and Regulation of Androgen Receptor Trafficking

2017 ◽  
Vol 43 (1) ◽  
pp. 223-236 ◽  
Author(s):  
Qiong Deng ◽  
Zeng Zhang ◽  
Yong Wu ◽  
Wang-yang Yu ◽  
Jianwen Zhang ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Western Blot ◽  
Signaling Pathways ◽  
Sertoli Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Antibody Array ◽  
Membrane Association ◽  
Phosphorylation Sites ◽  
Testosterone Treatment

Background: Testosterone is critical for maintaining spermatogenesis and male fertility. The accomplishment of these processes requires the synergistic actions of the classical and non-classical signaling pathways of androgens. Methods: A murine testicular Sertoli cell line, TM4 cell was used to examine androgen actions in Sertoli cells. Western blot analysis and immunofluorescence assay were employed to study the testosterone-induced Androgen receptor (AR) translocation. Protein phosphorylation antibody array was applied to identify the phosphorylation sites under testosterone treatment, and these findings were verified by Western blot analysis. Results: We found that a physiological dose of testosterone induced fast membrane association of AR. By using a phosphorylation antibody array, several phosphorylation sites, such as MEK1/2 (Ser217/221), Akt (Ser473), and Erk1/2 (Thr202/Tyr204) were rapidly phosphorylated within 5 min of testosterone treatment. Inhibition of the MEK and Akt signaling pathways prevented AR trafficking. Blocking of AR by flutamide eliminated the stimulation effect of testosterone on kinase phosphorylation. Testosterone induced kinase Src phosphorylation, and inhibition of Src restricted AR translocation to the membrane and the nucleus. Conclusion: Findings suggested that the membrane association of AR was mediated by the MEK and Akt phosphorylation signaling pathways, which resulted in Src activation and was initiated by testosterone binding to the membrane-localized AR. This study provides new insights into the testosterone signaling pathway in Sertoli cells, which mediate spermatogenesis. In addition, the study can be used in the diagnosis and treatment of male infertility caused by disorders in spermatogenesis.

Methyl Protodioscin Induces Apoptosis in Human Osteosarcoma Cells by Caspase-Dependent and MAPK Signaling Pathways

2017 ◽  
Vol 65 (13) ◽  
pp. 2670-2676 ◽  
Author(s):  
Shun-Cheng Tseng ◽  
Tai-Shan Shen ◽  
Chia-Chieh Wu ◽  
Ing-Lin Chang ◽  
Hsin-Yao Chen ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Mapk Signaling ◽  
Osteosarcoma Cells ◽  
Human Osteosarcoma ◽  
Human Osteosarcoma Cells ◽  
Mapk Signaling Pathways
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