scholarly journals Ribonucleic acid-binding protein CPSF6 promotes glycolysis and suppresses apoptosis in hepatocellular carcinoma cells by inhibiting the BTG2 expression

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Yang Liu ◽  
Hongbo Zou ◽  
Qichao Xie ◽  
Lan Zou ◽  
Rui Kong ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Rna Binding ◽  
Binding Protein ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Glucose Assay ◽  
Normal Hepatocyte ◽  
Hcc Cells

AbstractHepatocellular carcinoma (HCC) is currently the sixth most common malignancy and the second major cause of tumor-related deaths in the world. This study aimed to investigate the role of cleavage and polyadenylation factor-6 (CPSF6) and B-cell translocation gene 2 (BTG2) in regulating the glycolysis and apoptosis in HCC cells. The RNA and protein expression of CPSF6 and BTG2 in normal hepatocyte and HCC were, respectively, detected by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis and Western blot analysis. The viability and apoptosis of transfected Huh-7 cells were, respectively, analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. The expression of apoptosis-related proteins and HK-2 in transfected Huh-7 cells was also detected by Western blot analysis. The levels of glucose and lactate in the culture supernatant of transfected Huh-7 cells were, respectively, detected with the glucose assay kit and lactate assay kit. The interaction of CPSF6 and BTG2 was confirmed by RNA binding protein immunoprecipitation (RIP) assay. As a result, CPSF6 expression was increased while BTG2 expression was decreased in Huh-7 cells. Interference with CPSF6 suppressed the viability and glycolysis, and promoted the apoptosis of Huh-7 cells. Furthermore, CPSF6 interacted with BTG2 and interference with CPSF6 upregulated the BTG2 expression and inhibited the protein kinase B (AKT)/extracellular signal-regulated kinase (ERK)/nuclear factor (NF)-κB pathway. Interference with BTG2 could partially reverse the above cell changes caused by interference with CPSF6. In conclusion, CPSF6 inhibited the BTG2 expression to promote glycolysis and suppress apoptosis in HCC cells by activating AKT/ERK/NF-κB pathway.

scholarly journals Silencing the Expression of Cyclin G1 Enhances the Radiosensitivity of Hepatocellular Carcinoma in vitro and in vivo by Inducing Apoptosis

2020 ◽  
Author(s):  
Gang Xu ◽  
Shanshan Bu ◽  
Xiushen Wang ◽  
Hong Ge
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Cyclin G1 ◽  
Related Proteins ◽  
Hcc Cells

Abstract Background: Radiotherapy plays an important role in the treatment of hepatocellular carcinoma (HCC). Cyclin G1 was a novel member of the cyclin family, and it is abnormally expressed in HCC. The aim of this study was to investigate the role of cyclin G1 in the radiotherapy of HCC cells. Methods: The expression of cyclin G1 was silenced by transfection of cyclin G1-siRNA into HepG2 cells, and the expression of cyclin G1 mRNA and protein was measured by qRT-PCR and western blot analysis. The proliferation was analysed by MTT assay, and the radiosensitivity of HCC cells was detected by using a colony formation assay and a xenograft tumour model. The expression of apoptosis-related proteins (Bcl-2 and Bax) was detected by western blot analysis. Results: The expression of cyclin G1 mRNA and protein in HepG2-cyclin G1-siRNA cells is significantly decreased compared with that in HepG2 cells. Silencing the expression of cyclin G1 inhibits the proliferation of HCC cells and enhances the radiosensitivity of HepG2 cells in vitro and in vivo. HepG2-cyclin G1-siRNA significantly decreases Bcl-2 expression and increases Bax expression in cells.Conclusion: Silencing the expression of cyclin G1 enhances the radiosensitivity of HCC cells in vitro and in vivo, and the mechanism is probably related to the regulation of apoptosis-related proteins.

The Antitumor Effects of Britanin on Hepatocellular Carcinoma Cells and its Real-Time Evaluation by In Vivo Bioluminescence Imaging

2020 ◽  
Vol 20 (9) ◽  
pp. 1147-1156
Author(s):  
Hanrui Li ◽  
GeTao Du ◽  
Lu Yang ◽  
Liaojun Pang ◽  
Yonghua Zhan
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Tumor Size ◽  
Hepg2 Cells ◽  
Western Blot Analysis ◽  
Bioluminescence Imaging ◽  
Antitumor Effects ◽  
In Vivo Bioluminescence Imaging

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

scholarly journals The ErbB2/Neu/HER2 receptor is a new calmodulin-binding protein

2004 ◽  
Vol 381 (1) ◽  
pp. 257-266 ◽  
Author(s):  
Hongbing LI ◽  
Juan SÁNCHEZ-TORRES ◽  
Alan del CARPIO ◽  
Valentina SALAS ◽  
Antonio VILLALOBO
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Tyrosine Kinases ◽  
Western Blot Analysis ◽  
Binding Protein ◽  
Signalling Pathways ◽  
Breast Adenocarcinoma ◽  
Dependent Manner ◽  
Her2 Receptor

We have demonstrated previously that the EGFR (epidermal growth factor receptor) is a calmodulin (CaM)-binding protein. To establish whether or not the related receptor ErbB2/Neu/HER2 also binds CaM, we used human breast adenocarcinoma SK-BR-3 cells, because these cells overexpress this receptor thus facilitating the detection of this interaction. In the present paper, we show that ErbB2 could be pulled-down using CaM–agarose beads in a Ca2+-dependent manner, as detected by Western blot analysis using an anti-ErbB2 antibody. ErbB2 was also isolated by Ca2+-dependent CaM-affinity chromatography. We also demonstrate using an overlay technique with biotinylated CaM that CaM binds directly to the immunoprecipitated ErbB2. The binding of biotinylated CaM to ErbB2 depends strictly on the presence of Ca2+, since it was prevented by the presence of EGTA. Moreover, the addition of an excess of free CaM prevents the binding of its biotinylated form, demonstrating that this was a specific process. We excluded any interference with the EGFR, as SK-BR-3 cells express considerably lower levels of this receptor, and no detectable EGFR signal was observed by Western blot analysis in the immunoprecipitated ErbB2 preparations used to perform the overlay assays with biotinylated CaM. We also demonstrate that treating living cells with W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide], a cell-permeant CaM antagonist, down-regulates ErbB2 phosphorylation, and show that W7 does not interfere non-specifically with the activity of ErbB tyrosine kinases. We also show that W7 inhibits the phosphorylation (activation) of both ERK1/2 (extracellular-signal-regulated kinases 1 and 2) and Akt/PKB (protein kinase B), in accordance with the inhibition observed in ErbB2 phosphorylation. In contrast, W7 treatment increased the phosphorylation (activation) of CREB (cAMP-response-element-binding protein) and ATF1 (activating transcription factor-1), two Ca2+-sensitive transcription factors that operate downstream of these ErbB2 signalling pathways, most likely because of the absence of calcineurin activity. We conclude that ErbB2 is a new CaM-binding protein, and that CaM plays a role in the regulation of this receptor and its downstream signalling pathways in vivo.

scholarly journals RNA Binding Protein HuR Promotes Autophagosome Formation by Regulating Expression of Autophagy-Related Proteins 5, 12, and 16 in Human Hepatocellular Carcinoma Cells

2019 ◽  
Vol 39 (6) ◽  
Author(s):  
Eunbyul Ji ◽  
Chongtae Kim ◽  
Hoin Kang ◽  
Sojin Ahn ◽  
Myeongwoo Jung ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Autophagic Flux ◽  
Autophagosome Formation ◽  
Cellular Autophagy ◽  
Related Proteins ◽  
Cellular Components ◽  
Hcc Cells

ABSTRACT Autophagy is a process of lysosomal self-degradation of cellular components by forming autophagosomes. Autophagosome formation is an essential process in autophagy and is fine-tuned by various autophagy-related gene (ATG) products, including ATG5, ATG12, and ATG16. Although several reports have shown that numerous factors affect multiple levels of gene regulation to orchestrate cellular autophagy, the detailed mechanism of autophagosome formation still needs further investigation. In this study, we demonstrate that the RNA binding protein HuR (human antigen R) performs an essential function in autophagosome formation. We observe that HuR silencing leads to inhibition of autophagosome formation and autophagic flux in liver cells. Ribonucleoprotein immunoprecipitation (RIP) assay allows the identification of ATG5, ATG12, and ATG16 mRNAs as the direct targets of HuR. We further show that HuR mediates the translation of ATG5, ATG12, and ATG16 mRNAs by binding to their 3′ untranslated regions (UTRs). In addition, we show that HuR expression positively correlates with the levels of ATG5 and ATG12 in hepatocellular carcinoma (HCC) cells. Collectively, our results suggest that HuR functions as a pivotal regulator of autophagosome formation by enhancing the translation of ATG5, ATG12, and ATG16 mRNAs and that augmented expression of HuR and ATGs may participate in the malfunction of autophagy in HCC cells.

Adult and fetal human mesangial cells interact with specific laminin domains

1991 ◽  
Vol 261 (4) ◽  
pp. F688-F695
Author(s):  
B. S. Weeks ◽  
J. B. Kopp ◽  
S. Horikoshi ◽  
F. B. Cannon ◽  
M. Garrett ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Mesangial Cell ◽  
Mesangial Cells ◽  
Cell Attachment ◽  
Binding Protein ◽  
Laminin Binding Protein ◽  
Laminin Binding

Mesangial cells are centrally located pericytes in the renal glomerulus. They are surrounded by an extracellular matrix and directly contact the glomerular basement membrane in vivo. Because these interactions are critical for renal development and function, we have studied human mesangial cell interactions with laminin, a major adhesive component of basement membranes present in the extracellular matrix of the mesangium. Human fetal and adult mesangial cell attachment was stimulated by both laminin and the laminin-derived synthetic peptides YIGSR-NH2, CQAGTFALRGDNPQG-NH2, and CIKVAVS-NH2. Furthermore, mesangial cells spread on laminin as well as on both the RGD-containing and CIKVAVS peptides. When added in solution, all three peptides inhibited mesangial cell attachment to laminin, and the latter two peptides inhibited mesangial cell spreading on laminin. Laminin affinity column chromatography demonstrated several low-molecular-mass laminin-binding proteins ranging from between 35 and 42 kDa, which predominated in fetal mesangial cells, whereas a higher molecular mass laminin-binding protein of 65 kDa was predominant in adult mesangial cells. Western blot analysis with an anti-32-kDa laminin-binding protein antibody showed increased expression of both 31- and 42-kDa proteins in fetal mesangial cells when compared with the adult. The antisera to the 32-kDa laminin-binding protein also inhibited fetal mesangial spreading on the CIKVAVS peptide. Western blot analysis with an anti-67-kDa laminin-binding protein antibody revealed a 110-kDa protein in adult mesangial cells that was not present in fetal mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals A FUS-LATS1/2 Axis Inhibits Hepatocellular Carcinoma Progression via Activating Hippo Pathway

2018 ◽  
Vol 50 (2) ◽  
pp. 437-451 ◽  
Author(s):  
Le Bao ◽  
Lei Yuan ◽  
Pengfei Li ◽  
Qingyun Bu ◽  
Aijun Guo ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Cell Viability ◽  
Rna Binding ◽  
Binding Protein ◽  
Hippo Pathway ◽  
Rna Binding Protein ◽  
Luciferase Reporter ◽  
Western Blots ◽  
Qrt Pcr

Background/Aims: The roles and related mechanisms of RNA binding protein FUS (fused in sarcoma/translocated in liposarcoma) are unclear in numerous cancers, including hepatocellular carcinoma (HCC). Methods: Quantitative reverse transcription PCR (qRT-PCR), western blot, cell viability, transwell migration and invasion, tumor spheres formation and in vivo tumor formation assays were used to examine the effects of FUS on HCC progression in HuH7 and MHCC97 cells. Additionally, transcriptome analysis based on RNA-sequencing data, qRT-PCR, western blots, luciferase reporter and RNA binding protein immunoprecipitation (RIP) assays were used to explore the LATS1/2 (large tumor suppressor kinases 1/2)-related mechanisms contributing to FUS functions. Finally, qRT-PCR and western blot analysis were used to detect the levels of FUS and LATS1/2 in HCC and adjacent normal tissues, and the correlation between them in HCC tissues. Results: Overexpression of FUS decreased cell viability, migration, invasion and stemness. Moreover, FUS interacted and stabilized LATS1/2 stability, and thus promoted LATS1/2 expression and activated Hippo pathway. Finally, FUS and LAST1/2 levels were positively correlated and significantly down-regulated in HCC tissues. Conclusion: We demonstrate that FUS/LATS1/2 axis inhibits HCC progression via activating Hippo pathway.

scholarly journals Paeonol Induces Protective Autophagy in Retinal Photoreceptor Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Daowei Zhang ◽  
Jiawen Wu ◽  
Jihong Wu ◽  
Shenghai Zhang
Keyword(s):  
Oxidative Stress ◽  
Western Blot ◽  
Cell Viability ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Critical Role ◽  
Cell Counting ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Retinal Photoreceptor

Background: Retinal photoreceptor (RP) cells are widely involved in retina-related diseases, and oxidative stress plays a critical role in retinal secondary damage. Herein, we investigated the effectiveness and potential mechanisms of autophagy of paeonol (Pae) in terms of oxidation resistance.Methods: The animal model was induced by light damage (LD) in vivo, whereas the in vitro model was established by H2O2 stimulation. The effectiveness of Pae was evaluated by hematoxylin and eosin, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, immunofluorescence, transmission electron microscopy, electroretinogram, and Western blot analysis in vivo, and the underlying mechanisms of Pae were assessed by Cell Counting Kit-8 assay, reactive oxygen species (ROS) assay, and Western blot analysis in 661W cells. We mainly evaluated the effects of Pae on apoptosis and autophagy.Results: Increased apoptosis of the LD-induced and decreased autophagy of RPs were mitigated by Pae treatment. Pea, which increased the expression of mitochondrial functional protein cytochrome c, reversed the decreased cell viability and autophagy induced by oxidative stress in 661W cells. Experiments showed that autophagy was downregulated in PINK1/Parkin dependent and the BNIP3L/Nix dependent pathways under H2O2 stimulation and was upregulated by Pae treatment. Pae increased the cell viability and reduced ROS levels through autophagy.Conclusion: Pretreatment with Pae preserved RP cells by enhancing autophagy, which protected retinal function.

scholarly journals Decreased expression of the human carbonyl reductase 2 gene HCR2 in hepatocellular carcinoma

2006 ◽  
Vol 11 (2) ◽  
Author(s):  
Shan Liu ◽  
Lijie Ma ◽  
Weixue Huang ◽  
Yin Shai ◽  
Xiaona Ji ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Immunohistochemical Staining ◽  
Carcinoma Cells ◽  
Expression Ratio ◽  
Normal Tissues ◽  
Polymerase Chain ◽  
Altered Gene

AbstractAltered gene expression was associated with the induction and maintenance of hepatocellular carcinoma (HCC). To determine the significance of HCR2 in HCC, here we compare the expression levels of HCR2 in carcinoma and in paired non-carcinoma tissues using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemical staining. The expression ratio (ER) of HCR2 between the tumor and paired tumor-free tissues was calculated for each case and the data was clinicopathologically analyzed. The expression of HCR2 mRNA was found to be significantly decreased in HCC tissues compared with paired normal tissues (P < 0.001). HCR2 was downregulated in 58% (n = 22) of 38 HCC patients. The ER of HCR2 was higher in Edmondson’s grade I/II carcinomas than that in Edmondson’s grade III/IV carcinomas (P < 0.05). Western blot analysis showed HCR2 to be notably depressed in carcinoma tissues in 3 out of 4 HCC patients. Immunohistochemical staining indicated most HCR2 protein accumulated in non-carcinoma cells. These results suggested that altered HCR2 expression might play roles in the carcinogenesis and progression of HCC, and it could be a clinical marker for prognosis, and a molecular target for screening potential anti-HCC drugs.

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