Angiotensin II Induces Differentiation of Human Neuroblastoma Cells by Increasing MAP2 and ROS Levels

Figure 1. Morphological changes in neuroblastoma cells after Ang II treatment. Cells were incubated with Ang II (600 nM) for 24 h, and cell size changes were visualized by bright-field microscopy. Changes in cell size were also analyzed by flow cytometry. (a) Untreated neuroblastoma cells visualized in bright-field. (b) Ang II-treated neuroblastoma cells visualized in bright field. Changes in cell size and shape (b) were observed in cells exposed to Ang II when compared to untreated (a). (c) Representative flow cytometric histogram of untreated (dark peak) and Ang II-treated (gray peak) neuroblastoma cells. (d) Cell size significantly decreased after incubation with Ang II (dark bar) when compared to untreated (open bar). Each bar represents the average of five experiments. Vertical lines at each bar represent SEM (∗∗∗p < 0.001). FSC: forward scatter.

Figure 2. Effect of Ang II-treated neuroblastoma cells on MAP2 and NF-H expression levels. Cells were incubated with Ang II (600 nM) for 24 h, and the levels of MAP2 and NF-H proteins were measured by flow cytometry using specific monoclonal antibodies. (a) MAP2 levels increased significantly when the cells were incubated with Ang II compared to untreated. (b) No significant differences were observed in the levels of NF-H when the cells were incubated with Ang II compared to untreated. Each bar represents the average of four experiments. Vertical lines at each bar represent SEM (∗p < 0.05). MFI: mean fluorescence intensity.

Figure 3. Effect of Ang II in the generation of intracellular ROS and cell viability. Human neuroblastoma cells were cultured in the presence of Ang II (600 nM) for 24 h, and intracellular ROS levels and the percentage of healthy, apoptotic, or necrotic cells were analyzed by flow cytometry. (a) Ang II increased significantly the intracellular ROS levels in neuroblastoma cells when compared to untreated. (b) No significant differences were observed in the percentage of healthy, apoptotic, or necrotic cells in Ang II-treated neuroblastoma cells when compared to untreated. Each bar represents the average of three experiments. Vertical lines at each bar represent SEM (∗p < 0.05). MFI: mean fluorescence intensity.

Figure 4. Effect of apocynin on neuroblastoma MAP2 expression levels and ROS production induced by Ang II. Cells were incubated with Ang II (600 nM) alone or Ang II plus apocynin (500 μM) for 24 h, and the levels of MAP2 and intracellular ROS were quantified by flow cytometry. A significant increase was observed on MAP2 (a) and intracellular ROS levels (b) when the cells were incubated with Ang II alone (dark gray bar) compared to untreated (open bar). Both MAP2 and intracellular ROS levels decreased significantly when the cells were exposed to Ang II plus apocynin (dark bar) compared to those exposed to Ang II alone (dark gray bar). No significant differences were observed in the levels of MAP2 (a) or intracellular ROS (b) between cells that were exposed to Ang II plus apocynin (dark bar) and untreated (open bar) or apocynin alone (light gray bar). Each bar represents the average of four experiments. Vertical lines at each bar represent SEM (∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). MFI: mean fluorescence intensity.

Figure 5. Effect of selective AT1 or AT2 receptor blockers on neuroblastoma MAP2 expression levels induced by Ang II. Cells were incubated with Ang II (600 nM) alone, Ang II plus Losartan (10−7 M), or Ang II plus PD123.319 (10−7 M) for 24 h, and the levels of MAP2 were quantified by flow cytometry. A significant increase was observed in MAP2 levels when the cells were incubated with Ang II alone compared to untreated. The levels of MAP2 decreased significantly when the cells were exposed to Ang II plus PD123.319 compared to those exposed to Ang II alone. However, no significant differences were observed when the cells were incubated with Ang II plus Losartan compared to those exposed to Ang II alone. Each bar represents the average of four experiments. Vertical lines at each bar represent SEM (∗∗∗∗p < 0.0001). MFI: mean fluorescence intensity.

Figure 6. Effect of Ang II metabolism inhibitors on neuroblastoma MAP2 expression levels. Cells were incubated with Ang II (600 nM) alone, Ang II plus AP-N inhibitor (10−7 M), or Ang II plus MLN-4760 (10−7 M) for 24 h, and the levels of MAP2 were quantified by flow cytometry. A significant increase was observed on MAP2 levels when the cells were incubated with Ang II alone compared to untreated. However, no significant differences were observed in the levels of MAP2 when the cells were exposed to Ang II alone or Ang II plus AP-N inhibitor or MLN-4760. Each bar represents the average of four experiments. Vertical lines at each bar represent SEM (∗∗∗∗p < 0.0001). MFI: mean fluorescence intensity.