A Novel Role of the TRPM4 Ion Channel in Exocytosis.

Under physiological conditions, the widely expressed calcium-activated TRPM4 channel conducts sodium into cells. This sodium influx depolarizes the plasma membrane and reduces the driving force for calcium entry. The aberrant expression or function of TRPM4 has been reported in various diseases, including different types of cancer. TRPM4 is mainly localized in the plasma membrane, but it is also found in intracellular vesicles, which can undergo exocytosis. In this study, we show that calcium-induced exocytosis in the colorectal cancer cell line HCT116 is dependent on TRPM4. In addition, the findings from some studies of prostate cancer cell lines suggest a more general role of TRPM4 in calcium-induced exocytosis in cancer cells. Furthermore, calcium-induced exocytosis depends on TRPM4 ion conductivity. Additionally, an increase in intracellular calcium results in the delivery of TRPM4 to the plasma membrane. This process also depends on TRPM4 ion conductivity. TRPM4-dependent exocytosis and the delivery of TRPM4 to the plasma membrane are mediated by SNARE proteins. Finally, we provide evidence that calcium-induced exocytosis depends on TRPM4 ion conductivity, not within the plasma membrane, but rather in TRPM4-containing vesicles

Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells, with Limited Impact on Cancer Hallmark Functions.

Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca2+ activated monovalent cation channel that contributes to the pathophysiology of several diseases. For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells. Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays. To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na+. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype. In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells

ADAM17-silencing influence on MC38 cells metastasis in in vitro model

ADAM17 jest transmembranową metaloproteinazą dysintegrynową. Funkcją tego enzymu jest ścinanie licznych cytokin, czynników wzrostu, receptorów i cząsteczek adhezyjnych z powierzchni błony komórkowej. ADAM17 bierze udział w kontrolowaniu odpowiedzi immunologicznej organizmu, wzrostu i różnicowania komórek, ale jego podwyższona ekspresja może być związana ze stanami patologicznymi takimi jak proces nowotworzenia. Rola jaką ADAM17 odgrywa w promocji nowotworów to między innymi stymulacja proliferacji i migracji komórek. Celem niniejszej pracy było zbadanie wpływu wyciszenia ADAM17 na potencjał przerzutowania komórek mysiego raka okrężnicy MC38 w modelu in vitro. Pierwszym etapem pracy było określenie wpływu ADAM17 na aktywność migracyjną komórek MC38. Badania potwierdziły, że wyciszenie ADAM17 w komórkach MC38 obniża aktywność migracyjną. Kolejnym zadaniem było określenie wpływu ADAM17 na produkcję aktywnych metaloproteinaz macierzy zewnątrzkomórkowej, które mogą brać udział w procesie ruchu komórek. Jak się okazało wyciszenie ADAM17 istotnie obniża produkcję MMP-2 i MMP-9. Ponadto sprawdzono czy ADAM17 ma wpływ na wydzielanie tlenku azotu przez komórki MC38 po stymulacji cytokinami. Wyniki wskazują, że komórki z wyciszonym ADAM17 produkują mniejsze ilości tlenku azotu w porównaniu do komórek kontrolnych, co jest skorelowane z ekspresją indukowalnej syntazy tlenku azotu. Podjęto również próbę określenia czy komórki MC38 są zdolne do produkcji IL-6, jednak wyniki tego testu były negatywne. Ostatecznie sprawdzono czy cytokiny INF-γ i TNF-α mają wpływ na żywotność komórek MC38 i czy można zaobserwować różnice pomiędzy komórkami z wyciszonym ADAM17 i kontrolnymi. Uzyskane wyniki wskazują, na wrażliwość komórek na użyte cytokiny, jednak nie jest to skorelowane z ekspresją ADAM17.ADAM17 is a transmembrane disintegrin mettaloprotease. This enzyme function is shedding of many cytokines, growth factors, receptors and adhesion molecules from the cell surface. ADAM17 is involved in control of immune response, cells growth and differentiation, however its high expression is bound up with the process of tumor development. The role of ADAM17 in tumor progression among others, is stimulation of cells proliferation and migration. The aim of this study was to determine the influence of silencing of ADAM17 on the metastasis potential of mouse colon cancer cells MC38 in in vitro model. The first part of this work was to examine ADAM17 influence on cells migration activity. The results confirm that silencing of ADAM17 decreases motility of MC38 cells. The next aim was to determine the effect of ADAM17 silencing on the production of active matrix metalloproteainases, which can take part in the process of cell migration. The outcome of this experiment shows that silencing of ADAM17 lowers the production of MMP-2 and MMP-9.Furthermore it was examined if ADAM17 has any influence of nitric oxide production by MC38 cells under cytokine stimulation. The results demonstrate that silencing of ADAM17 decreases the level of nitric oxide produced by MC38 cells, which is correlated with the expression of inducible nitric oxide synthase. It was also checked if MC38 cells are able to produce interleukin-6, with the negative results.Finally, it was determined if cytokines, INF-γ and TNF-α, have influence on MC38 cells viability and if there are differences between cells with silenced ADAM17 and control. It was shown that this two cytokines significantly decrease MC38 viability, although it is not dependent on ADAM17 expression

The battle with uneven opponent – Sudden Infant Death Syndrome

SIDS is one of the biggest problems of modern medicine. In the diagnosis of SIDS, we take into account all possible diseases that may be the cause of death, as well as factors indicating an accident or murder. The etiology of SIDS is not yet known. There are several pathogenetic concepts, most of which refer to pathophysiological changes associated with nervous system hypoplasia. The most important risk factors include the effects of tobacco smoke, obstetric history, and incorrect sleep position. The role of risk factors in the pathogenesis of SIDS and their interdependence is still the subject of many studies. There are many theories developed on this subject, but none have been supported by scientific research and which is extremely difficult to carry out in this group of newborns. In most cases, medical help finds a newborn already dead, so it is difficult to say what is the main cause or marker of cot death. A considerable success in preventing the onset of sudden infant death syndrome turned out to be educational campaigns for parents - in order to follow up, an information leaflet was prepared with the basic recommendations in the prevention of SIDS. Among the parents of newborn children there are still many controversial opinions about risk factors in the onset of sudden infant death syndrome, the article contains and explains the meaning of individual activities that are considered to predispose to SIDS

Ranges of the cytokine concentration between experiments.

*<p>The values represent the lowest average and the highest average cytokine concentrations estimated in ADAM17-silenced tumor lysates and in sera from mice bearing ADAM17-silenced tumor taken from three independent experiment (at least 5 mice per group). Concentrations of cytokines in the sera of control healthy mice (pg/ml): TNF – 5.0±1.4; IFNγ – 1.5±0.5; MCP-1 – 20.6±4.6.</p

Expression of growth factors and growth factor receptors by MC38CEA colon carcinoma.

<p>(<b>A</b>) RT-PCR analysis of the expression of TGFα, EGFR and ErbB4 in MC38CEA cells in culture, in MC38CEA tumors and in positive controls: murine brain endothelial cells (MBE) and murine breast cancer cell line (4T1) and murine brain. cDNA from: M – mock-transfected cells, TM – mock-transfected tumors, S – ADAM17-silenced cells, TS – ADAM17-silenced tumors. Amplification of cDNA coding for elongation factor 2 (EF2) was performed as a control of samples' quality. (<b>B</b>) Analysis of the effect of exogenous EGF on ³H-thymidine incorporation into MC38CEA or 4T1 cells (positive control). * – <i>P</i><0.01 <i>vs.</i> control. (<b>C</b>) Quantitative RT-PCR analysis of ErbB2 and ErbB3 expression in ADAM17-silenced (S2, S3) and mock-transfected (M1, M2, M3) cell lines and in control cell lines, MBE and 4T1 as well as in murine tissues. (<b>D</b>) The levels of NRG-1 released to the medium from ADAM17-silenced (S2, S3) and mock-transfected (M1, M3) cells measured by ELISA. * – <i>P</i><0.01 <i>vs.</i> M1 and M3. (<b>E</b>) Western blotting analysis of ErbB2 phosphorylation in ADAM17-silenced (S2, S3), mock-transfected (M1, M2, M3) and wild-type (wt) MC38CEA cells incubated in the serum-free medium alone or in the medium enriched in exogenous rmNRG-1β. GAPDH was used as a loading control. Quantification of Western blot signals is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050791#s2" target="_blank">Materials and Methods</a> and is based on at least 3 independent experiments for a given cell line. * – <i>P</i><0.01 <i>vs.</i> the same cell line incubated w/o rmNRG-1β; & – <i>P</i><0.001 <i>vs.</i> M1. All data in panels A, B, C and D come from at least 3 independent experiments performed in duplicates.</p

Effect of ADAM17 silencing on proliferation and viability of MC38CEA cells.

<p>The cells were grown in serum-free or serum-low DMEM and their growth and viability were monitored daily using the MTT assay. Bars represent average values ± SD from 3 independent experiments performed in sextuplicates.</p

Analysis of ADAM17-silencing in MC38CEA cells.

<p>(<b>A</b>) Quantitative RT-PCR analysis of ADAM17 mRNA levels in the wild-type MC38CEA (WT) and in the MC38CEA cell lines stably transfected with control shRNA (M1, M2, M3), or with ADAM17 shRNAs (S2, S3, S35). * – <i>P</i><0.01 <i>vs.</i> WT and each M cell line. (<b>B</b>) The levels of TNF released to the medium from mock-transfected or ADAM17-silenced MC38CEA cell lines incubated in the absence or presence of PMA, measured by ELISA. Bars represent average values ± SD from 3 independent experiments performed in duplicates. * – <i>P</i><0.01 <i>vs.</i> unstimulated cells.</p

Effect of ADAM17 silencing on cell motility.

<p>(<b>A</b>) Exemplary trajectories of migrating wild type (WT), mock-transfected (M3) and ADAM17-silenced (S35) MC38CEA cells displayed in circular diagrams drawn with the initial point of each trajectory placed at the origin of the plot. Each panel shows the trajectories of 50 cells. The variables of cell movement are presented below diagrams. CME, coefficient of movement efficiency; *<i>P</i><0.05 (<i>vs.</i> wild type and mock-transfected cells). Data are from one representative experiment. Comparable results were obtained in the second experiment with M3 and S35 as well as in two experiments, in which the movements of WT, M2, and S3 were analyzed. (<b>B, C</b>) Wound healing assay (<b>B</b>) Representative wound images. Micrographs taken at 0 and 24 h after scratch wounding of cell monolayers. (<b>C</b>) Quantification of the results of two independent experiments performed in duplicates for each cell line. The lengths of cell displacement are expressed as a difference between average pre- and post-healing wound. * – <i>P</i><0.01 <i>vs.</i> M3. (<b>D</b>) Influence of rmNRG-1β on mock-transfected- and ADAM17-silenced MC38CEA cells – representative wound images. Wounded monolayers were incubated for 22 h in serum-free medium in the absence (control) or in the presence of rmNRG-1β. (<b>E</b>) Quantification of the results from a single experiment representative of three performed. * – <i>P</i><0.01 <i>vs.</i> the same cell line incubated w/o rmNRG-1β.</p