High frequency of BRCA1, but not CHEK2 or NBS1 (NBN), founder mutations in Russian ovarian cancer patients

<p>Abstract</p> <p>Background</p> <p>A significant portion of ovarian cancer (OC) cases is caused by germ-line mutations in BRCA1 or BRCA2 genes. BRCA testing is cheap in populations with founder effect and therefore recommended for all patients with OC diagnosis. Recurrent mutations constitute the vast majority of BRCA defects in Russia, however their impact in OC morbidity has not been yet systematically studied. Furthermore, Russian population is characterized by a relatively high frequency of CHEK2 and NBS1 (NBN) heterozygotes, but it remains unclear whether these two genes contribute to the OC risk.</p> <p>Methods</p> <p>The study included 354 OC patients from 2 distinct, geographically remote regions (290 from North-Western Russia (St.-Petersburg) and 64 from the south of the country (Krasnodar)). DNA samples were tested by allele-specific PCR for the presence of 8 founder mutations (BRCA1 5382insC, BRCA1 4153delA, BRCA1 185delAG, BRCA1 300T>G, BRCA2 6174delT, CHEK2 1100delC, CHEK2 IVS2+1G>A, NBS1 657del5). In addition, literature data on the occurrence of BRCA1, BRCA2, CHEK2 and NBS1 mutations in non-selected ovarian cancer patients were reviewed.</p> <p>Results</p> <p>BRCA1 5382insC allele was detected in 28/290 (9.7%) OC cases from the North-West and 11/64 (17.2%) OC patients from the South of Russia. In addition, 4 BRCA1 185delAG, 2 BRCA1 4153delA, 1 BRCA2 6174delT, 2 CHEK2 1100delC and 1 NBS1 657del5 mutation were detected. 1 patient from Krasnodar was heterozygous for both BRCA1 5382insC and NBS1 657del5 variants.</p> <p>Conclusion</p> <p>Founder BRCA1 mutations, especially BRCA1 5382insC variant, are responsible for substantial share of OC morbidity in Russia, therefore DNA testing has to be considered for every OC patient of Russian origin. Taken together with literature data, this study does not support the contribution of CHEK2 in OC risk, while the role of NBS1 heterozygosity may require further clarification.</p

Lactaptin induces p53-independent cell death associated with features of apoptosis and autophagy and delays growth of breast cancer cells in mouse xenografts.

Lactaptin, the proteolytic fragment of human milk kappa-casein, induces the death of various cultured cancer cells. The mechanisms leading to cell death after lactaptin treatment have not been well characterized. In this study the in vivo and in vitro effects of a recombinant analogue of lactaptin (RL2) were examined. Following treatment with the recombinant analogue of lactaptin strong caspase -3, -7 activation was detected. As a consequence of caspase activation we observed the appearance of a sub-G1 population of cells with subdiploid DNA content. Dynamic changes in the mRNA and protein levels of apoptosis-related genes were estimated. No statistically reliable differences in p53 mRNA level or protein level were found between control and RL2-treated cells. We observed that RL2 constitutively suppressed bcl-2 mRNA expression and down regulated Bcl-2 protein expression in MDA-MB-231 cells. We demonstrated that RL2 penetrates cancer and non-transformed cells. Identification of the cellular targets of the lactaptin analogue revealed that α/β-tubulin and α-actinin-1 were RL2-bound proteins. As the alteration in cellular viability in response to protein stimulus can be realized not only by way of apoptosis but also by autophagy, we examined the implications of autophagy in RL2-dependent cell death. We also found that RL2 treatment induces LC3-processing, which is a hallmark of autophagy. The autophagy inhibitor chloroquine enhanced RL2 cytotoxicity to MDA-MB-231 cells, indicating the pro-survival effect of RL2-dependent autophagy. The antitumour potential of RL2 was investigated in vivo in mouse xenografts bearing MDA-MB-231 cells. We demonstrated that the recombinant analogue of lactaptin significantly suppressed the growth of solid tumours. Our results indicate that lactaptin could be a new molecule for the development of anticancer drugs

Flexible Strain-Sensitive Silicone-CNT Sensor for Human Motion Detection

This article describes the manufacturing technology of biocompatible flexible strain-sensitive sensor based on Ecoflex silicone and multi-walled carbon nanotubes (MWCNT). The sensor demonstrates resistive behavior. Structural, electrical, and mechanical characteristics are compared. It is shown that laser radiation significantly reduces the resistance of the material. Through laser radiation, electrically conductive networks of MWCNT are formed in a silicone matrix. The developed sensor demonstrates highly sensitive characteristics: gauge factor at 100% elongation −4.9, gauge factor at 90° bending −0.9%/deg, stretchability up to 725%, tensile strength 0.7 MPa, modulus of elasticity at 100% 46 kPa, and the temperature coefficient of resistance in the range of 30–40 °C is −2 × 10−3. There is a linear sensor response (with 1 ms response time) with a low hysteresis of ≤3%. An electronic unit for reading and processing sensor signals based on the ATXMEGA8E5-AU microcontroller has been developed. The unit was set to operate the sensor in the range of electrical resistance 5–150 kOhm. The Bluetooth module made it possible to transfer the received data to a personal computer. Currently, in the field of wearable technologies and health monitoring, a vital need is the development of flexible sensors attached to the human body to track various indicators. By integrating the sensor with the joints of the human hand, effective movement sensing has been demonstrated

Identification of RL2-interacting proteins A and B.

<p>Protein identification was based on the SwissProt database using MALDI spectra of protein typsin lysates.</p

RL2 regulates activity of apoptosis-related genes.

<p><b>A.</b> MDA-MB-231 cells were treated with RL2 (0.2 mg/mL) for the indicated time followed by total RNA isolation. Levels of transcripts were analysed by real time RT PCR with specific primers and were presented as relative values normalized to the level of GAPDH mRNA. n = 3; the error bars represent standard deviations. The asterisks indicate significant difference from control (0 h), *p<0.05, or between indicated groups, **p<0.05, ns - not significant difference between groups (p>0.05). <b>B</b>. MDA-MB-231 cells were treated with RL2 (0.2 mg/mL) followed by preparation of whole-cell lysates, which were then subjected to Western blot with the indicated antibodies. Tubulin was used as the internal control. Results shown are representative of five independent experiments.</p

Enhanced cytotoxic outcome in combination of RL2 with CQ.

<p>MDA-MB-231 cells were used as a model. <b>A</b>. Dose-dependence of CQ cytotoxicity was calculated using MTT data of three independent experiments. Shown are mean values ± SD. <b>B</b>. Cells were treated with different concentrations of RL2 in the presence or absence of CQ, and after 48 h cell viability was measured by MTT assay. <b>C</b>. Cells were incubated with RL2 (0.2 mg/mL), CQ (5 μM) or a combination for 24 h and the percentage of cells with active caspase -3,-7 was analysed using flow cytometry. Shown are mean values ± SD of three independent experiments. The asterisks indicate significant difference from control (*, p<0.05). <b>D</b>. Cells were incubated with RL2 (0.2 mg/mL), CQ (5 μM) or a combination for 24–48 h. Cathepsin D activity was analysed using a fluorescence substrate (labeled with methyl cumaryl amide) by fluorimetry as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093921#s2" target="_blank">Materials and methods</a>’ section. RFU – relative fluorescence units per microgram protein of sample. Shown are mean values ± SD of three independent experiments. The asterisks indicate significant difference between indicated groups (**, p<0.05), ns - not significant difference between groups (p>0.05).</p

Separation of proteins of RL2-bound fraction.

<p>Coomassie stained gel. RL2-bound fractions of MCF-7 lysates were prepared as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093921#s2" target="_blank">Materials and methods</a>’ section and then were separated by 15% SDS-PAGE gel. Bands indicated (*) were excised followed by in-gel trypsin digestion for mass spectrum analysis. Other bands were not investigated. Shown is representative gel of two independent experiments. Lane 1 – fraction eluted from non-modified Sepharose (control); Lane 2 – fraction eluted from RL2-modified Sepharose with 300 mM NaCl, flow rate was 1 mL/min and detection wavelength was 280 nm. M – molecular weights marker (14.4–116.0 kDa).</p

RL2 treatment induces autophagic changes in MDA-MB-231 cells.

<p><b>A.</b> Cells were treated with RL2 (0.2 mg/mL) or non-treated (control) for 8 h and fixed with 4% paraformaldehyde followed by immunostaining with anti-LC3A/B primary and FITC-conjugated secondary antibody. Images are representative of two independent experiments. <b>B</b>. MDA-MB-231 cells were treated with RL2 (0.2 mg/mL) for various time points (0, 2, 6 and 24 h). Total lysates were prepared and subjected to Western blot analysis. Shown are representative blots of five independent experiments.</p