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. 2018 May 20;16(5):172.
doi: 10.3390/md16050172.

Anti-Tumorigenic and Anti-Metastatic Activity of the Sponge-Derived Marine Drugs Aeroplysinin-1 and Isofistularin-3 against Pheochromocytoma In Vitro

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Anti-Tumorigenic and Anti-Metastatic Activity of the Sponge-Derived Marine Drugs Aeroplysinin-1 and Isofistularin-3 against Pheochromocytoma In Vitro

Nicole Bechmann et al. Mar Drugs. .

Abstract

Over 10% of pheochromocytoma and paraganglioma (PPGL) patients have malignant disease at their first presentation in the clinic. Development of malignancy and the underlying molecular pathways in PPGLs are poorly understood and efficient treatment strategies are missing. Marine sponges provide a natural source of promising anti-tumorigenic and anti-metastatic agents. We evaluate the anti-tumorigenic and anti-metastatic potential of Aeroplysinin-1 and Isofistularin-3, two secondary metabolites isolated from the marine sponge Aplysina aerophoba, on pheochromocytoma cells. Aeroplysinin-1 diminished the number of proliferating cells and reduced spheroid growth significantly. Beside these anti-tumorigenic activity, Aeroplysinin-1 decreased the migration ability of the cells significantly (p = 0.01), whereas, the invasion capacity was not affected. Aeroplysinin-1 diminished the high adhesion capacity of the MTT cells to collagen (p < 0.001) and, furthermore, reduced the ability to form spheroids significantly. Western Blot and qRT-PCR analysis showed a downregulation of integrin β1 that might explain the lower adhesion and migration capacity after Aeroplysinin-1 treatment. Isofistularin-3 showed only a negligible influence on proliferative and pro-metastatic cell properties. These in vitro investigations show promise for the application of the sponge-derived marine drug, Aeroplysinin-1 as anti-tumorigenic and anti-metastatic agent against PPGLs for the first time.

Keywords: Aeroplysinin; Isofistularin; cancer progression; cell adhesion molecules; hypoxia; integrin β1; marine sponges; metastasis; pheochromocytoma and paraganglioma.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic view: fresh collected 15 cm large A. aerophoba demosponge that grow under marine ranching conditions and the chemical structure of its bioactive secondary metabolites Aeropysinin-1 and Isofistularin-3.
Figure 2
Figure 2
Anti-proliferative activity of Aeroplysinin-1 and Isofistularin-3 on pheochromocytoma cells in monolayer culture. (A) Aeroplysinin-1 decreased the viability of all three pheochromocytoma cell lines significantly after 24 h treatment. Isofistularin-3 only affected the viability of the mouse pheochromocytoma cells, whereas, the PC12 rat pheochromocytoma cells was not affected up to a concentration of 100 µM under normoxic and hypoxic conditions. Cultivation under hypoxia increased the necessary effective concentration to reduce the viability to 50% (EC50). (B) Furthermore, the effect on the number of proliferating cells was analyzed under normoxic and hypoxic conditions. Treatment with 1 µM Aeroplysinin-1 reduced the number of proliferating cells in all three cell lines in trend. Under hypoxic conditions (1% oxygen), pheochromocytoma cells stopped cell division. Four to five independent experiments were performed (n = 4–14). Average ± standard error of the mean (SEM); Analysis of variance (ANOVA) and Bonferroni post hoc test comparison vs. control * p < 0.05, ** p < 0.01.
Figure 3
Figure 3
Influence of Aeroplysinin-1 and Isofistularin-3 on cell death related pathways. Induction of apoptosis by the treatment with Aeroplysinin-1 and Isofistularin-3 was analyzed by the measurement of the relative caspase-3 and caspase-7 activity in (A) MPC, (B) MTT, and (C) PC12 cells. Moreover, the impact on the (DG) gene expression levels of the MTT cells was determined by qRT-PCR. (E) Beside apoptosis, necrosis and autophagy are also forms of cell death regulated by several genes. (F) The analyzed markers for necrosis Ppia (cyclophilin A), Ppid (cyclophilin D), Ripk and Bnip3 were not affected by the treatment with Aeroplysinin-1. (G) Furthermore, no difference of the Becn1 expression level was detectable after Aeroplysinin-1 treatment. Three to four independent experiments (n = 3–4). Average ± SEM; ANOVA and Bonferroni post hoc test comparison vs. control * p < 0.05 or ** p < 0.01.
Figure 4
Figure 4
Spheroid growth inhibition by a single or fractionated treatment with Aeroplysinin-1. (A) Spheroids were treated with different concentrations Aeroplysinin-1 after completed spheroid formation (day 4) and growth was monitored over a time period of 14 days. Furthermore, (B) a fractionated treatment on day four, 8, 11, and 15 took place. Three to four independent experiments (n = 15–48). Average ± SEM; ANOVA and Bonferroni post hoc test comparison vs. control * p < 0.05, ** p < 0.01; or vs. 5 µM Aeropylysinin-1 # p < 0.05. Arrows mark the different treatment time points. Scale bar: 200 µm.
Figure 5
Figure 5
Aeroplysinin-1 influences the pro-metastatic behavior of MTT cells. Impact of different concentrations of Aeroplysinin-1 on (A) MTT cell migration and (B) invasion were analyzed in Boyden-Chamber assays (with (B) or without (A) Matrigel coating) after 24 h. Furthermore, the adhesion capacity of MTT cells to (C) collagen and (D) fibronectin after 24 h treatment with Aeroplysinin-1 was determined. (E) Spheroid formation was tracked over four days and the spheroid diameter was analyzed four days after seeding. Three independent experiments (n = 12–18). Average ± SEM; ANOVA and Bonferroni post hoc test comparison vs. control * p < 0.05 or ** p < 0.01. Scale bar: 200 µm.
Figure 6
Figure 6
Regulation of cell adhesion molecules in mouse pheochromocytoma cells (MTT) by Aeroplysinin-1. The impact of 24 h treatment with Aeroplysinin-1 on the gene expression was confirmed by qRT-PCR and the protein expression was analyzed by western blot analysis. Three to four independent experiments were performed (n = 3–4) and a representative section of the immunochemical detection is shown. Treatment with 10 µM Aeroplysinin-1 diminished the expression of integrin β1 (A) significantly, whereas, the gene expression of Itga1 (B) and Itga3 (C) was not affected. Furthermore, Aeroplysinin-1 had no impact on the protein expression of the calcium-dependent cadherins. Average ± SEM; ANOVA and Bonferroni post hoc test comparison vs. control ** p < 0.01.

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