Sensitive Genotyping of Somatic Mutations in the EGFR, KRAS, PIK3CA, BRAF Genes from NSCLC Patients Using Hydrogel Biochips

Emelyanova, Marina; Arkhipova, Ksenia; Мазуренко, Н. Н.; Чудинов, А. В.; Demidova, Irina; Zborovskaya, I. B.; Lyubchenko, Lyudmila; Заседателев, А. С.; Nasedkina, T. V.

doi:10.1097/pai.0000000000000084

Cited by 9 publications

(5 citation statements)

References 40 publications

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“…Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, and can be further classified as nonsquamous carcinoma (including adenocarcinoma, which accounts for 40% of NSCLCs) and squamous NSCLC, which makes up 30% of NSCLC cases [ 2 – 4 ]. Inhibitors of the epidermal growth factor receptor (EGFR) are used for the treatment of NSCLC [ 5 ]. In addition, other inhibitors such as miR-99a and heat shock protein 90 (HSP 90) have been also involved in lung tumor development.…”

Section: Introductionmentioning

confidence: 99%

Anti-cancer effect of snake venom toxin through down regulation of AP-1 mediated PRDX6 expression

et al. 2015

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Snake venom toxin (SVT) from Vipera lebetina turanica contains a mixture of different enzymes and proteins. Peroxiredoxin 6 (PRDX6) is known to be a stimulator of lung cancer cell growth. PRDX6 is a member of peroxidases, and has calcium-independent phospholipase A2 (iPLA2) activities. PRDX6 has an AP-1 binding site in its promoter region of the gene. Since AP-1 is implicated in tumor growth and PRDX6 expression, in the present study, we investigated whether SVT inhibits PRDX6, thereby preventing human lung cancer cell growth (A549 and NCI-H460) through inactivation of AP-1. A docking model study and pull down assay showed that SVT completely fits on the basic leucine zipper (bZIP) region of c-Fos of AP-1. SVT (0–10 μg/ml) inhibited lung cancer cell growth in a concentration dependent manner through induction of apoptotic cell death accompanied by induction of cleaved caspase-3, -8, -9, Bax, p21 and p53, but decreased cIAP and Bcl2 expression via inactivation of AP-1. In an xenograft in vivo model, SVT (0.5 mg/kg and 1 mg/kg) also inhibited tumor growth accompanied with the reduction of PRDX6 expression, but increased expression of proapoptotic proteins. These data indicate that SVT inhibits tumor growth via inhibition of PRDX6 activity through interaction with its transcription factor AP-1.

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Section: Introductionmentioning

confidence: 99%

Anti-cancer effect of snake venom toxin through down regulation of AP-1 mediated PRDX6 expression

et al. 2015

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“…In this proof-of-concept work, it was shown that peptide hydrogel architecture provides a rational design for responsive peptide-based hydrogels. 72,73 Some biosensors built with hydrogel systems have been studied, for example, the biochips for somatic mutation in genes detection, 74 microfluidic devices sensitive to metalloproteinases that regulates the composition of an extracellular matrix and are correlated to some vascular disease, cancer progression, and bone disorders. 41 In this sense, the opportunity of using hydrogels for antigens encapsulation can motivate the design of novel devices for disease diagnosis.…”

Section: Resultsmentioning

confidence: 99%

Nanostructured Antigen-Responsive Hydrogels Based on Peptides for Leishmaniasis Detection

Souza

Kogikoski

Silva

et al. 2016

J. Braz. Chem. Soc.

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Hydrogels based on peptide nanostructures are biological entities that can be applied in a wide range of applications, such as scaffolds for tissue engineering, drug delivery, and biosensors. The aim of this research was to study peptide hydrogels based on N-(9-fluorenylmethoxycarbonyl)-L,L-diphenylalanine (Fmoc-FF) in two different media: water and phosphate buffer. These hydrogels were used for encapsulating Leishmania infantun chagasi soluble proteins. The structure of the matrices was investigated in detail through scanning and transmission electron microscopy, and small angle X-ray scattering (SAXS). The mechanical behavior of the hydrogels were assessed through rheology assays, demonstrating both the physical and chemical stability of the hydrogel scaffolds. The immunogenicity of immobilized antigens was studied using enzyme-linked immunosorbent assay (ELISA) detection after the reaction with positive and negative dog sera for Leishmania infantum chagasi. The hydrogel was efficient to encapsulate antigens, and can promote the development of novel devices that requires the storage of biomolecules under moist environmental conditions.

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“…The hybridization and image analysis were performed as described before [ 61 ]. Briefly, hybridization was performed in 40 μl of 25% formamide (Serva, Oklahoma City, USA), 6× saline-sodium phosphate-EDTA (Promega, Fitchburg, USA) and 20 μl lebeled PCR products.…”

Section: Methodsmentioning

confidence: 99%

“…The high sensitivity has been reached by combining, in one assay, the LNA PCR clamp technique and the mutation-specific hybridization on a hydrogel biochip, which allows the identification of somatic mutations in a large excess of wild type (WT) DNA. Previously, this approach has been developed and applied for the analysis of somatic mutations in EGFR, KRAS, BRAF, PI3K genes in lung cancer cells [ 61 ].…”

Section: Introductionmentioning

confidence: 99%

Detection ofBRAF,NRAS,KIT,GNAQ,GNA11andMAP2K1/2mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis

et al. 2017

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Target inhibitors are used for melanoma treatment, and their effectiveness depends on the tumor genotype. We developed a diagnostic biochip for the detection of 39 clinically relevant somatic mutations in the BRAF, NRAS, KIT, GNAQ, GNA11, MAP2K1 and MAP2K2 genes.We used multiplex locked nucleic acid (LNA) PCR clamp for the preferable amplification of mutated over wild type DNA. The amplified fragments were labeled via the incorporation of fluorescently labeled dUTP during PCR and were hybridized with specific oligonucleotides immobilized on a biochip. This approach could detect 0.5% of mutated DNA in the sample analyzed. The method was validated on 253 clinical samples and six melanoma cell lines.Among 253 melanomas, 129 (51.0%) BRAF, 45 (17.8%) NRAS, 6 (2.4%) KIT, 4 (1.6%) GNAQ, 2 (0.8%) GNA11, 2 (0.8%) MAP2K1 and no MAP2K2 gene mutations were detected by the biochip assay. The results were compared with Sanger sequencing, next generation sequencing and ARMS/Scorpion real-time PCR. The specimens with discordant results were subjected to LNA PCR clamp followed by sequencing. The results of this analysis were predominantly identical to the results obtained by the biochip assay. Infrequently, we identified rare somatic mutations.In the present study we demonstrate that the biochip-based assay can effectively detect somatic mutations in approximately 70% of melanoma patients, who may require specific targeted therapy.

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Sensitive Genotyping of Somatic Mutations in the EGFR, KRAS, PIK3CA, BRAF Genes from NSCLC Patients Using Hydrogel Biochips

Cited by 9 publications

References 40 publications

Anti-cancer effect of snake venom toxin through down regulation of AP-1 mediated PRDX6 expression

Anti-cancer effect of snake venom toxin through down regulation of AP-1 mediated PRDX6 expression

Nanostructured Antigen-Responsive Hydrogels Based on Peptides for Leishmaniasis Detection

Detection ofBRAF,NRAS,KIT,GNAQ,GNA11andMAP2K1/2mutations in Russian melanoma patients using LNA PCR clamp and biochip analysis

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