The production of nitric oxide is responsible for melatonin- and LPS-induced cell death.

<p>(A) Quantification of NO production in cerebellar granule cells incubated with LPS (100 ng/mL, 24 h), melatonin (100 nM, 24 h) or melatonin + LPS. (B,C) Effect of the specific iNOS inhibitor (1400W, 1 µM) on the survival of granule cells cultured incubated with LPS (100 ng/mL, 24 h) or melatonin (100 nM, 24 h). * p<0.05%, ** p<0.01 and *** p<0.001 compared to control and # p<0.05 compared to LPS group (n = 3 per point).</p

Activation of nuclear factor-kappa B (NF-κB) in cultured cerebellar cells.

<p>(A) Super-shift assay performed with nuclear extracts from naïve cultured cerebellar cells incubated with antibodies against p50 and RelA subunits. The super-shift indicates the presence of the dimers p50/p50 and p50/RelA. (B) Time course for NF-κB activation by melatonin (100 nM) showing a transient reduction in the nuclear content of p50/p50 and p50/RelA followed by a significant increase in the nuclear content of p50/RelA (C) Concentration-response curve for melatonin (10 nM–1 µM, 15 min). (D) LPS (100 ng/mL, 15) induced NF-κB nuclear translocation in the presence or absence of melatonin (10 nM–1 µM, 15 min). For quantification the whole experiment present in graphs B, C and D was done in one gel, repeated 5 to 8 times. The values are expressed as percentage of the image detected in control conditions. * p<0.05% compared to control and # p<0.05 compared to LPS group (n = 5–8 per point). Symbols – squares: p50/RelA; triangles: p50/p50.</p

Quantification of the expression of iNOS.

<p>(A) LPS (100 ng/mL, 60 min) induced the expression of iNOS in the presence or absence of melatonin (30–300 nM, 60 min) (B) Time course of cells cultured with melatonin (100 nM), LPS (100 ng/mL) and melatonin + LPS. * p<0.05 and ** p<0.01 compared to control and # p<0.05 compared to LPS group (n = 4–6 per point). The number of cases refers to independent samples obtained in different days and from different cultures.</p

Dual effect of melatonin on the viability of cultured cerebellar cells.

<p>(A) Viability concentration-response curve for cells treated with LPS (30–300 ng/mL) incubated for 24 h; (B) Viability concentration-response curve for cells treated with melatonin (0.1 nM–1 µM) in the presence or absence of LPS (100 ng/mL), incubated for 24 hours. Each well contained 10⁵ cells at the beginning of the experiment. * p<0.05; **p<0.01 compared to control (n = 8–10 per point).</p

Plasmatic membrane toll-like receptor expressions in human astrocytomas

<div><p>Toll-like receptors (TLRs) are the first to identify disturbances in the immune system, recognizing pathogens such as bacteria, fungi, and viruses. Since the inflammation process plays an important role in several diseases, TLRs have been considered potential therapeutic targets, including treatment for cancer. However, TLRs’ role in cancer remains ambiguous. This study aims to analyze the expression levels of plasmatic cell membrane TLRs (TLR1, TLR2, TLR4, TLR5, and TLR6) in human astrocytomas the most prevalent tumors of CNS different grades (II-IV). We demonstrated that TLR expressions were higher in astrocytoma samples compared to non-neoplastic brain tissue. The gene and protein expressions were observed in GBM cell lines U87MG and A172, proving their presence in the tumor cells. Associated expressions between the known heterodimers TLR1-TLR2 were found in all astrocytoma grades. In GBMs, the mesenchymal subtype showed higher levels of TLR expressions in relation to classical and proneural subtypes. A strong association of TLRs with the activation of cell cycle process and signaling through canonical, inflammasome and ripoptosome pathways was observed by <i>in silico</i> analysis, further highlighting TLRs as interesting targets for cancer treatment.</p></div

Immunofluorescence of TLR1, TLR2, TLR4, TLR5, and TLR6 in GBM cell lines.

<p>A172 (A) and U87MG (B). TLR1, TLR4, and TLR6 are stained in red, TLR2 and TLR5 in green, and nuclei in blue by DAPI. The presence of all five TLRs was detected in both cell lines. Expression of TLR5 was more intense in A172 compared to U87MG. TLR4 and TLR5 positivity were detected in both tumor lineage cells nuclei. Magnification of 400x.</p

<i>TLR1</i>, <i>TLR2</i>, <i>TLR4</i>, <i>TLR</i>5, and <i>TLR</i>6 expression levels in astrocytomas of different malignant grades.

<p>(A) The analyzed samples consisted of 22 non-neoplastic (NN) cases, 26 astrocytoma grade II (AGII) cases, 18 astrocytoma grade III (AGIII) cases, and 96 glioblastoma (GBM) cases. Data are represented by box and whisker plots, with the median represented by the line in the middle of the boxes, and top and bottom boxes represent the first and third quartiles. qRT-PCR values are normalized by three housekeeping genes (<i>HPRT</i>, <i>GUSB</i>, <i>TBP</i>). For statistical analysis, Kruskal-Wallis and Dunn’s tests were applied, wherein (*) <i>p</i> < 0.05 when compared to NN cases and (†) <i>p</i> < 0.05 when compared to AGII (Dunn test), all the genes present <i>p</i><0.01 (Kruskal-Wallis). (B) Correlation between <i>TLR2</i>-<i>TLR1</i>, <i>TLR2</i>-<i>TLR6</i>, <i>TLR2</i>-<i>TLR4</i>, <i>TLR2</i>-<i>TLR5</i>, <i>TLR4</i>-<i>TLR1</i>, <i>TLR4</i>-<i>TLR5</i>, and <i>TLR4</i>-<i>TLR6</i> are demonstrated in GBM cases. Statistical analysis was made by the Spearman-rho correlation, and <i>p</i><0.05 were considered significative.</p

Heatmap with major genes of the TLR signaling pathways from the TCGA dataset.

<p>RPKM gene expression levels are normalized by z-scores, and comparatively up-regulated RNA expression values are presented in red and down-regulated values in blue. Mean values are in white. TLRs downstream signaling pathways: canonical, ripoptosome, and inflammasome pathways are activated in mesenchymal GBM subtype. Genes of unrelated pathways were added to show their randomic expression levels, including a microglia marker.</p

Imunohistochemistry for TLR1, TLR2, TLR4, TLR5, and TLR6 in GBM cases.

<p>Positive immunolabelling of GBM tumor cells for TLRs are demonstrated at 600x magnification of tumor tissues, and at 800x magnification in a multinucleated GBM tumor cell. The distribution of the Immunolabeling score (ILS) for these TLRs in five GBM cases was presented as a dispersion graph, where the black dots represent the mean ILS obtained by the two independent investigators for each individual tumor case, and the horizontal bar represent the mean ILS for each receptor. TLR4 and TLR5 positive staining were detected in tumor nuclei.</p

Used primer sequences.

<p>Used primer sequences.</p