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

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.

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

Technology in Cancer Research & Treatment ◽

10.1177/1533033819828396 ◽

2019 ◽

Vol 18 ◽

pp. 153303381982839 ◽

Cited By ~ 1

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.

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Discrepancy Between Polymerase Chain Reaction Assay and Western Blot Analysis in the Assessment of CagA Status in Dyspeptic Patients

Helicobacter ◽

10.1046/j.1523-5378.2001.00019.x ◽

2001 ◽

Vol 6 (2) ◽

pp. 130-135 ◽

Cited By ~ 4

Author(s):

Omero Alessandro Paoluzi ◽

Pina Rossi ◽

Carla Montesano ◽

Sabrina Bernardi ◽

Emanuela Carnieri ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Western Blot ◽

Blot Analysis ◽

Polymerase Chain Reaction Assay ◽

Western Blot Analysis ◽

Chain Reaction ◽

Polymerase Chain ◽

Caga Status

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Short-term application of dexamethasone on stem cells derived from human gingiva reduces the expression of RUNX2 and β-catenin

Journal of International Medical Research ◽

10.1177/0300060517701035 ◽

2017 ◽

Vol 45 (3) ◽

pp. 993-1006 ◽

Cited By ~ 3

Author(s):

Bo-Bae Kim ◽

Minji Kim ◽

Yun-Hee Park ◽

Youngkyung Ko ◽

Jun-Beom Park

Keyword(s):

Stem Cells ◽

Polymerase Chain Reaction ◽

Mesenchymal Stem Cells ◽

Western Blot ◽

Blot Analysis ◽

Chain Reaction ◽

Short Term ◽

Mrna Sequencing ◽

Human Gingiva ◽

Polymerase Chain

Objective Next-generation sequencing was performed to evaluate the effects of short-term application of dexamethasone on human gingiva-derived mesenchymal stem cells. Methods Human gingiva-derived stem cells were treated with a final concentration of 10−7 M dexamethasone and the same concentration of vehicle control. This was followed by mRNA sequencing and data analysis, gene ontology and pathway analysis, quantitative real-time polymerase chain reaction of mRNA, and western blot analysis of RUNX2 and β-catenin. Results In total, 26,364 mRNAs were differentially expressed. Comparison of the results of dexamethasone versus control at 2 hours revealed that 7 mRNAs were upregulated and 25 mRNAs were downregulated. The application of dexamethasone reduced the expression of RUNX2 and β-catenin in human gingiva-derived mesenchymal stem cells. Conclusion The effects of dexamethasone on stem cells were evaluated with mRNA sequencing, and validation of the expression was performed with qualitative real-time polymerase chain reaction and western blot analysis. The results of this study can provide new insights into the role of mRNA sequencing in maxillofacial areas.

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