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.

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 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.

scholarly journals Qualitative and Quantitative Analysis of IDH1 Mutation in Progressive Gliomas by Allele-Specific qPCR and Western Blot Analysis

2019 ◽  
Vol 18 ◽  
pp. 153303381982839 ◽  
Author(s):  
Moritz Perrech ◽  
Lena Dreher ◽  
Gabriele Röhn ◽  
Pantelis Stavrinou ◽  
Boris Krischek ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Western Blot ◽  
Real Time ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Idh1 Mutation ◽  
World Health ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Health Organization

To date, diagnosis of IDH1 mutation is based on DNA sequencing and immunohistochemistry, methods limited in terms of sensitivity and ease of use. Recently, the diagnosis of IDH1 mutation by real-time polymerase chain reaction was introduced as an alternative method. In this study, real-time polymerase chain reaction was validated as a tool for detection of IDH1 mutation, and expression levels were analyzed for correlation with course of the disease. A total of 113 tumor samples were obtained intraoperatively from 84 patients with glioma having a diagnosis of diffuse glioma (World Health Organization II), anaplastic glioma (World Health Organization III), secondary glioblastoma ± chemotherapy, primary glioblastoma ± chemotherapy (World Health Organization IV). Tumor samples were snap frozen and processed for sectioning and RNA and protein isolation. Presence of IDH1 mutation was determined by DNA sequencing. Hereafter, quantitative expression of IDH1 messenger RNA was assessed using real-time polymerase chain reaction with specific primers for IDH1 mutation and –wt; protein expression was verified by Western Blot analysis and immunohistochemistry. Additionally, 19 samples of low-grade glioma and their consecutive high-grade glioma were analyzed at different time points of the disease. IDH1 mutation was identified in 63% of samples by DNA sequencing. In correlation with the real-time polymerase chain reaction results, a cutoff value was determined. Above this threshold, sensitivity and specificity of real-time polymerase chain reaction in detecting IDH1 mutation were 98% and 94%, respectively. Quantitative analysis revealed that IDH1 mutation expression is upregulated in secondary glioblastoma (mean ± standard error of mean: 3.52 ± 0.55) compared to lower grade glioma (II = 1.54 ± 0.22; III = 1.67 ± 0.23). In contrast, IDH1 wt expression is upregulated in all glioma grades (concentration >0.1) compared to control brain tissue (0.007 ± 0.0016). Western Blot analysis showed a high concordance to both sequencing and real-time polymerase chain reaction results in qualitative analysis of IDH1 mutation status (specificity 100% and sensitivity 100%). Moreover, semiquantitative protein expression analysis also showed higher expression levels of mutated IDH1 in secondary glioblastoma. In our study, real-time polymerase chain reaction and Western Blot analysis were found to be highly efficient methods in detecting IDH1 mutation in glioma samples. As cost-effective and time-saving methods, real-time polymerase chain reaction and Western Blot analysis may therefore play an important role in IDH1 mutation analysis in the future. IDH1 mutation expression level was found to correlate with the course of disease to a certain extent. Yet, clinical factors as recurrent disease or prior radiochemotherapy did not alter IDH1 mutation expression level.

Relationship of pro-angiogenic factor Cyr61to colorectal cancer development and prognosis.

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 446-446 ◽  
Author(s):  
M. Baek ◽  
S. Bae ◽  
D. Jeong
Keyword(s):  
Colorectal Cancer ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Clinicopathological Parameters ◽  
Colorectal Cancers ◽  
Colorectal Carcinomas ◽  
Over Expression ◽  
Normal Tissues ◽  
Cancer Tissues

446 Background: Angiogenic factorCysteine-rich 61 (Cyr61) is a member of the CCN protein family that has been implicated in diverse biological processes such as cell adhesion, proliferation, angiogenesis, and tumorigenesis. An altered expression of Cyr61 is found to be associated with several human cancers. However, the correlation of expression of Cyr61 protein and clinical features of colorectal cancer remains unknown. Methods: Cyr61 expression in colorectal cancer and normal tissues was evaluated by Western blot analysis. Immunohistochemical staining was carried out using tissue microassay (TMA) to examine the expression status of Cyr61. Correlations of Cyr61 over-expression with various clinicopathologic factors were also determined. Statistical analysis was performed to explore the links between expression of the Cyr61 and clinicopathological parameters. Results: On Western blot analysis Cyr61 up-regulation was observed in colorectal cancer tissues (17/21, 80.9%). In 234 colorectal cancers, tumor tissue microarray revealed significantly up-regulated Cyr61 protein expression in colorectal cancer tissues versus normal tissues adjacent to tumor. Cyr61 expression was high in 136 of 234 cases of colorectal carcinomas (58.1%). Cyr61 over-expression was significantly associated with TNM stage (p=0.012) and regional lymph node involvement (p=0.018). Kaplan-Meier survival analysis showed that over-expression of Cyr61 was related to poor survival of colorectal cancer patients (p=0.031). But significant associations were not found between CYR61 expression versus tumor grade, age and gender. Conclusions: Our results suggest that Cyr61 is highly expressed in colorectal carcinomas and Cyr61 may play a role in the progression of colorectal cancers. Also, Cyr61 might be a new molecular marker to predict the prognosis and serve as valuable targets for therapeutic intervention of patients with colorectal carcinoma. No significant financial relationships to disclose.

Proteasome Inhibitor MG132 Enhances Sensitivity to Cisplatin on Ovarian Carcinoma Cells In Vitro and In Vivo

2016 ◽  
Vol 26 (5) ◽  
pp. 839-844 ◽  
Author(s):  
Na Guo ◽  
Zhilan Peng ◽  
Jiawen Zhang
Keyword(s):  
Polymerase Chain Reaction ◽  
Western Blot ◽  
Reverse Transcription ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Methyl Thiazolyl Tetrazolium ◽  
Combination Group ◽  
Chain Reaction ◽  
Methyl Thiazolyl Tetrazolium Assay ◽  
Polymerase Chain

BackgroundPlatinum-based combination chemotherapy after surgery is considered a standard treatment; therefore, any recent drug development should be new, effective, and low toxic, and should have a synergistic effect with platinum. This study aimed to observe the growth of SKOV3 cells after treatment with cisplatin by combining with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) and to investigate the effect of the relationship between MG132 and cisplatin combination.Materials and MethodsCell growth was detected by methyl thiazolyl tetrazolium assay after treatment with MG132 at 0.5, 1.5, 2.5, 3.5, and 5.0 μg/mL concentrations for 24, 48, and 72 hours; with cisplatin at 1.0, 2.0, 3.0, 4.0, and 5.0 μg/mL concentrations; and with combination with MG132 at 1.5 μg/mL for 24 hours. The apoptotic rates of cells were detected by a flow cytometer after cisplatin treatment at 1.0, 2.0, 3.0, and 4.0 μg/mL concentrations and that combined with MG132 at 1.5 μg/mL concentration for 12, 24, and 36 hours. A total of 20 BALB/c (nu/nu) female nude mice (age, 4–6 weeks; body weight, 17–19 g) were divided into 4 groups: control, MG132, cisplatin, and combination groups. The expression of Caspase3 and Beclin1 was detected by Western blot analysis and reverse transcription-polymerase chain reaction after treatment with 3.0 μg/mL of the cisplatin group and combined treatment with 1.5 μg/mL of MG132 group for 24 hours, respectively.ResultsMethyl thiazolyl tetrazolium assay demonstrated the inhibitory rates, and the flow cytometery showed that the apoptotic rates in the combination group were higher than those in the cisplatin group (P < 0.01). Western blot analysis and reverse transcription-polymerase chain reaction detected that Caspase3 and Beclin1 at a relative quantity in the combination group were higher than those in the cisplatin group (P < 0.05).ConclusionsMG132 has a synergistic antitumor effect by combining with cisplatin, and it is expected to be an effective antitumor drug for platinum-resistant refractory ovarian cancer.

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 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 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.

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