Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression

<p>Abstract</p> <p>Background</p> <p>Neuroblastoma is a severe pediatric tumor, histologically characterised by a variety of cellular phenotypes. One of the pharmacological approaches to neuroblastoma is the treatment with retinoic acid. The mechanism of action of retinoic acid is still unclear, and the development of resistance to this differentiating agent is a great therapy problem.</p> <p>Doublecortin, a microtubule-associated protein involved in neuronal migration, has recently been proposed as a molecular marker for the detection of minimal residual disease in human neuroblastoma. Nevertheless, no information is available on the expression of doublecortin in the different cell-types composing human neuroblastoma, its correlation with neuroblastoma cell motility and invasiveness, and the possible modulations exerted by retinoic acid treatment.</p> <p>Methods</p> <p>We analysed by immunofluorescence and by Western blot analysis the presence of doublecortin, lissencephaly-1 (another protein involved in neuronal migration) and of two intermediate filaments proteins, vimentin and neurofilament-68, in SK-N-SH human neuroblastoma cell line both in control conditions and under retinoic acid treatment. Migration and cell invasiveness studies were performed by wound scratch test and a modified microchemotaxis assay, respectively.</p> <p>Results</p> <p>Doublecortin is expressed in two cell subtypes considered to be the more aggressive and that show high migration capability and invasiveness.</p> <p>Vimentin expression is excluded by these cells, while lissencephaly-1 and neurofilaments-68 are immunodetected in all the cell subtypes of the SK-N-SH cell line. Treatment with retinoic acid reduces cell migration and invasiveness, down regulates doublecortin and lissencephaly-1 expression and up regulates neurofilament-68 expression. However, some cells that escape from retinoic acid action maintain migration capability and invasiveness and express doublecortin.</p> <p>Conclusion</p> <p>a) Doublecortin is expressed in human neuroblastoma cells that show high motility and invasiveness;</p> <p>b) Retinoic acid treatment reduces migration and invasiveness of the more aggressive cell components of SK-N-SH cells;</p> <p>c) The cells that after retinoic acid exposure show migration and invasive capability may be identified on the basis of doublecortin expression.</p

International descriptive and interventional survey for oxycholesterol determination by gas- and liquid-chromatographic methods

Increasing numbers of laboratories develop new methods based on gas-liquid and high-performance liquid chromatography to determine serum concentrations of oxygenated cholesterol metabolites such as 7α-, 24(S)-, and 27-hydroxycholesterol. We initiated a first international descriptive oxycholesterol (OCS) survey in 2013 and a second interventional survey 2014 in order to compare levels of OCS reported by different laboratories and to define possible sources of analytical errors. In 2013 a set of two lyophilized serum pools (A and B) was sent to nine laboratories in different countries for OCS measurement utilizing their own standard stock solutions. In 2014 eleven laboratories were requested to determine OCS concentrations in lyophilized pooled sera (C and D) utilizing the same provided standard stock solutions of OCS. The participating laboratories submitted results obtained after capillary gas-liquid chromatography-mass selective detection with either epicoprostanol or deuterium labelled sterols as internal standards and high-performance liquid chromatography with mass selective detection and deuterated OCS as internal standard. Each participant received a clear overview of the results in form of Youden-Plots and basic statistical evaluation in its used unit. The coefficients of variation of the concentrations obtained by all laboratories using their individual methods were 58.5–73.3% (survey 1), 56.8–60.3% (survey 2); 36.2–35.8% (survey 1), 56.6–59.8, (survey 2); 61.1–197.7% (survey 1), 47.2–74.2% (survey 2) for 24(S)-, 27-, and 7α-hydroxycholesterol, respectively. We are surprised by the very great differences between the laboratories, even under conditions when the same standards were used. The values of OCS's must be evaluated in relation to the analytical technique used, the efficiency of the ample separation and the nature of the internal standard used. Quantification of the calibration solution and inappropriate internal standards could be identified as major causes for the high variance in the reported results from the different laboratories. A harmonisation of analytical standard methods is highly needed

First international descriptive and interventional survey for cholesterol and non-cholesterol sterol determination by gas- and liquid- chromatography–Urgent need for harmonisation of analytical methods

Serum concentrations of lathosterol, the plant sterols campesterol and sitosterol and the cholesterol metabolite 5α-cholestanol are widely used as surrogate markers of cholesterol synthesis and absorption, respectively. Increasing numbers of laboratories utilize a broad spectrum of well-established and recently developed methods for the determination of cholesterol and non-cholesterol sterols (NCS). In order to evaluate the quality of these measurements and to identify possible sources of analytical errors our group initiated the first international survey for cholesterol and NCS. The cholesterol and NCS survey was structured as a two-part survey which took place in the years 2013 and 2014. The first survey part was designed as descriptive, providing information about the variation of reported results from different laboratories. A set of two lyophilized pooled sera (A and B) was sent to twenty laboratories specialized in chromatographic lipid analysis. The different sterols were quantified either by gas chromatography-flame ionization detection, gas chromatography- or liquid chromatography-mass selective detection. The participants were requested to determine cholesterol and NCS concentrations in the provided samples as part of their normal laboratory routine. The second part was designed as interventional survey. Twenty-two laboratories agreed to participate and received again two different lyophilized pooled sera (C and D). In contrast to the first international survey, each participant received standard stock solutions with defined concentrations of cholesterol and NCS. The participants were requested to use diluted calibration solutions from the provided standard stock solutions for quantification of cholesterol and NCS. In both surveys, each laboratory used its own internal standard (5α-cholestane, epicoprostanol or deuterium labelled sterols). Main outcome of the survey was, that unacceptably high interlaboratory variations for cholesterol and NCS concentrations are reported, even when the individual laboratories used the same calibration material. We discuss different sources of errors and recommend all laboratories analysing cholesterol and NCS to participate in regular quality control programs

Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression-5

<p><b>Copyright information:</b></p><p>Taken from "Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression"</p><p>http://www.biomedcentral.com/1471-2407/8/30</p><p>BMC Cancer 2008;8():30-30.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254429.</p><p></p> the starting (0 hr after scratch) and the end (24 hr after scratch) point of the analysis. In control the distance between the borderlines becomes significantly shorter 24 hr after wounding, while it is still elevated in RA treated samples. IF DCX analysis performed both in control and in RA treated cells 24 hr after wounding. In control samples the majority of invading cells are DCX(red). Even if the migration rate of the treated cells is reduced, few of them move in and invade the scratched area; all the cells still migrating after 6 days RA treatment are DCX(inset and of panel are magnified in panel ). Nuclei are counterstained with DAPI (blue)

Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression-8

<p><b>Copyright information:</b></p><p>Taken from "Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression"</p><p>http://www.biomedcentral.com/1471-2407/8/30</p><p>BMC Cancer 2008;8():30-30.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254429.</p><p></p>nels and ) clearly shows the heterogeneity of the cell population. The triple IF for DCX (), LIS1 () and TUB () shows an immunoreactive signal for DCX only in the N-type and in some I-type cells. The anti-LIS1 antibody stains all the cell types. When co-expressed by the same cell, TUB and DCX colocalize both in the cell body and along the neuritic processes (see inset 1 of panel ). The triple IF for DCX (), NF-68 () and VIM () shows that none of the DCXcells is immunoreactive for VIM. Vimentin is present merely in the S-type and in some I-type DCXcells. Some other I-type cells do express neither DCX nor VIM (arrow in the inset 2 of panel ). The NF-68 protein is expressed by all the cell subtypes

Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression-1

<p><b>Copyright information:</b></p><p>Taken from "Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression"</p><p>http://www.biomedcentral.com/1471-2407/8/30</p><p>BMC Cancer 2008;8():30-30.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254429.</p><p></p>s RA treatment (-). PC analysis (panels and ) shows morphological changes induced by RA. After 6 days RA treatment the DCXcells are few () in comparison to the control (), and they have the morphology of undifferentiated I-type cells. On the contrary is increased the number of VIMcells and the differentiated N-type VIMcells do not show immunoreactivity for DCX (). Bar graphs show the decrease in the number of DCXcells and the increase in the number of VIMcells after RA treatment. Bars represent mean ± standard error, N = 8, double star = p < 0.01. Neither in control nor in 6 days RA treated samples there are DCX/VIMcells immunopositive for both DCX and VIM, but as previously observed in control conditions (see fig.1), some DCX/VIMcells are present after RA treatment (see the cell marked with *, panel ). As in the controls (), NF-68 localizes in all RA treated SK-N-SH cells (). It is important to note that after the first 2 days of treatment it is possible to observe N-type cells (white arrows) that start to express VIM while DCX is disappearing

Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression-0

<p><b>Copyright information:</b></p><p>Taken from "Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression"</p><p>http://www.biomedcentral.com/1471-2407/8/30</p><p>BMC Cancer 2008;8():30-30.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254429.</p><p></p>nels and ) clearly shows the heterogeneity of the cell population. The triple IF for DCX (), LIS1 () and TUB () shows an immunoreactive signal for DCX only in the N-type and in some I-type cells. The anti-LIS1 antibody stains all the cell types. When co-expressed by the same cell, TUB and DCX colocalize both in the cell body and along the neuritic processes (see inset 1 of panel ). The triple IF for DCX (), NF-68 () and VIM () shows that none of the DCXcells is immunoreactive for VIM. Vimentin is present merely in the S-type and in some I-type DCXcells. Some other I-type cells do express neither DCX nor VIM (arrow in the inset 2 of panel ). The NF-68 protein is expressed by all the cell subtypes

Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression-2

<p><b>Copyright information:</b></p><p>Taken from "Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression"</p><p>http://www.biomedcentral.com/1471-2407/8/30</p><p>BMC Cancer 2008;8():30-30.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254429.</p><p></p>P) and analyzed by WB. DCX is a phosphoprotein and appears as a doublet that resolves to a single band after CIP treatment (+CIP) in both C and RA samples, indicating that the slower-migrating species is phosphorylated DCX (P-DCX)

Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression-4

<p><b>Copyright information:</b></p><p>Taken from "Retinoic acid reduces human neuroblastoma cell migration and invasiveness: effects on DCX, LIS1, neurofilaments-68 and vimentin expression"</p><p>http://www.biomedcentral.com/1471-2407/8/30</p><p>BMC Cancer 2008;8():30-30.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254429.</p><p></p>ithdrawal and 12 days) were analyzed by immunoblotting using an anti-DCX specific antibody; membranes were reprobed with anti-tubulin as a loading control. Quantification of DCX protein amount by scanning densitometry : data are means ± standard error, N = 8, and are expressed as a percentage of the control (100%). After 2 or 4 days of RA treatment DCX levels are significantly decreased, but to a lesser extent than after a 6 days exposure to RA. Lengthening SK-N-SH exposure to RA up to 12 days does not modify the magnitude of the DCX reduction observed after 6 days of treatment. Moreover, in cells treated with RA for 6 days and then grown for 6 additional days in medium without RA (6 days+6), DCX protein amount remains at low levels; single star = p < 0.05; double star = p < 0.01; C, control, RA, samples treated with RA 10 μM