Correlation of <i>LOX</i>, <i>HIF1A</i> and <i>BMP1</i> expression levels in astrocytomas of different malignant grades.

<p>Correlation was assessed in pilocytic astrocytomas (AGI: A, B and C), low-grade astrocytomas (AGII: D, E and F), anaplastic astrocytomas (AGIII: G, H and I) and glioblastomas (GBM: J, K and L). Statistically significant values were obtained in AGI cases for <i>BMP1</i> and <i>HIF1A</i> correlation (C) and in GBM cases for <i>LOX</i> and <i>BMP1</i> correlation (J), <i>LOX</i> and <i>HIF1A</i> correlation (K) and <i>HIF1A</i> and <i>BMP1</i> expression levels (L). In AGIII samples, <i>HIF1A</i> and <i>LOX</i> expression levels were negatively correlated (H). The statistically significant correlations are shown in red. Correlations between gene expression values were assessed using the non-parametric Spearman-rho correlation test.</p

LOX expression and localization in astrocytomas and non-neoplastic brain tissues.

<p>Immunohistochemistry was performed in 6 non-neoplastic brain tissues (NN), 6 pilocytic astrocytoma (AGI), 6 low-grade astrocytoma (AGII), 6 anaplastic astrocytoma (AGIII), and 6 glioblastoma (GBM) cases. The images show representative cases of each type of sample (400x magnification for all cases). Arrows indicate the endothelial cells staining.</p

LOX immunostaining analysis in astrocytomas and non-neoplastic brain tissues.

<p>The graphs illustrate semi-quantitative immunohistochemistry labeling scores (ILS) that refer to the product of staining intensity and the percentage of positive cells stained with anti-LOX in the cytoplasm (A), nucleus (B) and endothelial cells (C). The horizontal bars show the median ILS of each group. The difference in protein expression among the groups was statistically significant for nuclear staining (<i>p</i> = 0.01) and endothelial cell staining (<i>p</i> = 0.0008) as determined by a Kruskal-Wallis test. A post-hoc Dunn’s test was used to calculate the differences in expression among the groups (*<i>p</i><0.05; **<i>p</i><0.001; ***<i>p</i><0.0001).</p

Effect of <i>LOX</i> knockdown on the invasive phenotype and anchorage-independent growth behavior of GBM cell lines.

<p>Evaluation of the invasion (A and B) and anchorage-independent growth (C and D) of U87MG and A172 cell lines after <i>LOX</i> silencing with siRNA were compared to the non-targeted control (NTC). A total of 2.5 x 10⁴ cells were seeded in the upper chamber of Transwell inserts. Cells in the lower chamber were fixed and stained with crystal violet after 18 hours of incubation. Graphs represent the number of migrated cells per field in each condition (means ± standard errors of the means) in the two independent experiments conducted in duplicate. The images show representative fields of U87MG (A) and A172 (B) cells that invaded and crossed the insert (40x magnification). Soft agar colony formation assays were performed for both U87MG (C) and A172 (D) cell lines with 1 x 10³ cells. Graphs represent the mean number of colonies ± SD of two independent experiments conducted in duplicate (Mann-Whitney test, **<i>p</i><0.001; ***<i>p</i><0.0001).</p

Gene expression levels in astrocytomas of all malignant grades relative to non-neoplastic samples.

<p>Transcript levels of <i>LOX</i> (A), <i>BMP1</i> (B), and <i>HIF1A</i> (C) were determined in 19 non-neoplastic brain tissue samples (NN), 23 pilocytic astrocytomas (AGI), 26 low-grade astrocytomas (AGII), 18 anaplastic astrocytomas (AGIII) and 84 glioblastomas (GBM). Total RNA was reverse-transcribed into cDNA and analyzed by quantitative real-time PCR (RT-qPCR) using the SYBR Green method. The differences in expression levels among the groups were statistically significant for <i>LOX</i>, <i>BMP1</i> and <i>HIF1A</i> (Kruskal-Wallis test, <i>p</i><0.0001). A post-hoc Dunn’s test was used to calculate the differences in expression between NN and each astrocytoma group (*<i>p</i><0.05; **<i>p</i><0.001; ***<i>p</i><0.0001). <i>IDH1</i> mutational status was determined for diffusely infiltrating cases and the distribution of <i>LOX</i> (D), <i>BMP1</i> (E) and <i>HIF1A</i> (F) expression levels in <i>IDH1</i> wild-type and mutated background. Horizontal bars show the median of each group. AGII and GBM cases with <i>IDH1</i>-mutated presented lower <i>LOX</i> expression levels when compared to <i>IDH1</i> wild-type cases. AGIII cases with wild-type <i>IDH1</i> presented lower <i>HIF1A</i> expression levels when compared to <i>IDH1</i>-mutated cases (Mann-Whitney test, * <i>p</i> <0.05 and t test **, <i>p</i> <0.005).</p

LOX Expression and Functional Analysis in Astrocytomas and Impact of <i>IDH1</i> Mutation

<div><p>Lysyl oxidase (LOX) is involved in vital biological processes such as cell motility, cell signaling and gene regulation. Deregulation of this protein can contribute to tumor formation and progression. Although it is known that LOX is involved in invasion, proliferation and tumor migration in other types of tumors, studies of LOX in astrocytomas of different grades are scarce. The purpose of our study was to characterize <i>LOX</i>, <i>BMP1</i> and <i>HIF1A</i> expression by real-time PCR in astrocytomas with WHO grades I to IV compared to non-neoplastic brain tissue. <i>IDH1</i> mutational status was determined by PCR and sequencing. LOX protein expression was also analyzed by immunohistochemistry. LOX functional analyses were performed using siRNA knockdown and the specific inhibitor BAPN in two glioblastoma cell lines. The expression levels of <i>LOX</i>, <i>BMP1</i> and <i>HIF1A</i> were correlated and analyzed according to <i>IDH1</i> mutation status and to the clinical end-point of overall survival of glioblastoma patients. The results demonstrate that increased expression and activity of <i>LOX</i>, <i>BMP1</i> and <i>HIF1A</i> were positively correlated with the malignant grade of astrocytomas. LOX protein expression also increased according to the degree of malignancy, with localization in the cytoplasm and nucleus and staining observed in endothelial cells. Glioblastoma with a mutation in <i>IDH1</i> expressed lower levels of LOX in the nucleus, and <i>IDH1</i>-mutated cases showed lower <i>LOX</i> expression levels when compared to wild-type <i>IDH1</i> cases. <i>LOX</i> knockdown and inhibition by BAPN in U87MG and A172 cell lines affected migration, invasion and soft agar colony formation. Taken together, these results corroborate the role of LOX in the migration, invasion and angiogenesis of astrocytomas. Furthermore, <i>LOX</i> expression is influenced by <i>IDH1</i> mutational status. This work provides new insights for researchers aiming to design targeted therapies to control astrocytomas.</p></div