Assessment of citalopram and escitalopram on neuroblastoma cell lines. Cell toxicity and gene modulation

International audienceSelective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of-107 (p<2.26 10-7),-24.1 (p<5.6 10-9) and-17.7 (p<1.2 10-7). CCNE1, AURKA, IGF2, MYCN and ERBB2 were more moderately down-regulated by both molecules. Glioma markers E2F1, DAPK1 and CCND1 were down-regulated. Citalopram displayed more powerful action with broader and distinct spectrum of action than escitalopram

SCO-Spondin Derived Peptide NX210 Induces Neuroprotection In Vitro and Promotes Fiber Regrowth and Functional Recovery after Spinal Cord Injury

International audienceIn mammals, the limited regenerating potential of the central nervous system (CNS) in adults contrasts with the plasticity of the embryonic and perinatal periods. SCO (subcommissural organ)-spondin is a protein secreted early by the developing central nervous system, potentially involved in the development of commissural fibers. SCO-spondin stimulates neuronal differentiation and neurite growth in vitro. NX210 oligopeptide was designed from SCO-spondin's specific thrombospondin type 1 repeat (TSR) sequences that support the main neurogenic properties of the molecule. The objective of this work was to assess the neuroprotective and neuroregenerative properties of NX210 in vitro and in vivo for the treatment of spinal cord injury (SCI). In vitro studies were carried out on the B104 neuroblastoma cell line demonstrating neuroprotection by the resistance to oxidative damage using hydrogen peroxide and the measure of cell viability by metabolic activity. In vivo studies were performed in two rat models of SCI: (1) a model of aspiration of dorsal funiculi followed by the insertion of a collagen tube in situ to limit collateral sprouting; white matter regeneration was assessed using neurofilament immunostaining; (2) a rat spinal cord contusion model to assess functional recovery using BBB scale and reflex testing. We demonstrate for the first time that NX210 (a) provides neuroprotection to oxidative stress in the B104 neuroblastoma cells, (b) stimulates axonal regrowth in longitudinally oriented neofibers in the aspiration model of SCI and (c) significantly improves functional recovery in the contusive model of SCI

<i>In vivo</i> effects of NX210 on neuritic regrowth in a rat model of SCI.

<p>The injury was performed by aspirating the dorsal funiculi and the dorsal horns of the spinal gray matter. A collagen tube filled with the vehicle (A) or NX210 (B and C) was placed into the site of injury. Neuritic regrowth was detected using neurofilament immunostaining. (A) In the vehicle group (n = 5), the collagen tube remains empty without neuritic regrowth (*). (B and C): in the NX210 group (n = 5), neuritic regrowth (arrow) is clearly observed inside the collagen tube, mostly from the caudal end of the lesion. Neofibers display a longitudinal arrangement. (D): At the surface of the growing processes, NF was co-localized with laminin, a marker of the basal lamina. r: rostral; c: caudal; Scale bar: 250 μm.</p

Effects of NX210 on functional recovery after SCI: open arena test.

<p>(A) Percentage of time spent in central cells. (B) Path length. NX210-treated rats (n = 8, red) showed significant improvement in the percentage of time spent in central cells and path length at post-injury time points compared to vehicle-treated rats (n = 8, blue) and the difference between treated and vehicle groups increased throughout the study for the percentage of time spent in central cells. Data are presented as group mean ± SEM, * p<0.05.</p

Effects of NX210 on functional recovery after SCI: body weight.

<p>A contusion was performed with the NYU/MASCIC impactor. The body weight was measured once a week over the month of the study. NX210-treated group (n = 8) in solid red line, vehicle group (n = 8) in blue dashed line. NX210 treatment induces a significant increase in body weight from D1, reaches the pre-injury value at D7 and keeps increasing until the end of the study. In sharp contrast, the body weight of the control group decreases in the first week, starts to increase at D7, reaches the pre-injury value at D14 but remains inferior to the body weight of the treated group throughout the study. Data are presented as mean ± SEM. Significantly different from baseline value, ** p<0.05 and *** p<0.001.</p

Effects of NX210 on functional recovery after SCI: BBB score.

<p>Functional recovery was assessed once a week over the month of the study using the open-field locomotion test and the BBB score. NX210-treated group (n = 8) in solid red line, vehicle group (n = 8) in blue dashed line. (A) The NX210-treated group BBB score was significantly higher in the last two weeks of observation compared to the vehicle group. BBB score continues to improve at the end of the study whereas in the control group, the BBB does not significantly improve from D14 to the end of the study. (B) The percentage of animals reaching a score superior to 14, corresponds to a complete forelimb – hind limb coordination. In the NX210-treated group, the animals reached a score >14 as early as the second week post-injury. The number of animals reaching a BBB score of 14 continuously increases until the end of the study reaching 62% of the NX210-treated group. In the vehicle-treated group, a score>14 is reached by only one animal at the fourth week post-injury. Data are presented as mean ± SEM, p<0.05.</p

Effects of NX210 on functional recovery after SCI: reflex testing.

<p>Data are expressed as the percentage of animals providing a normal response to the tests (A) Toe spread. (B) Paw placement. Compared to pre-injury values, NX210-treated rats (n = 8) and vehicle-treated rats (n = 8) showed a significant impairment in both tests the day following the injury. However, at D21 and D28 post-injury, the NX210-treated group showed a significant improvement. Considering toe spread reflex, animals of the vehicle group reach a plateau at D21 whereas in the NX210 treated group, all the animals recover at the end of the study. Data are presented as group mean ± SEM. * significantly different from vehicle group, p<0.05.</p

Neuroprotective effect of NX210 against H₂O₂ using a B104 cell line.

<p>Neuroprotective effect of NX210 at 100 and 250 μg/ml was assessed by the resistance of a B104 neuroblastoma cell line after 18 hours of exposure to H₂O₂. (A) Morphological observation using phase-contrast microscopy shows normal features of B104 cells in the control group. Exposure to H₂O₂ alone decreases B104 cell viability, normal B104 cells are partly replaced by numerous cellular debris. Treatment with NX210 250 μg/ml significantly prevents H₂O₂-induced cell death. (B) Viability of B104 cells treated with NX210 was assessed using the WST-1 assay. H₂O₂-induced cell death was significantly prevented in NX210-treated cultures at the concentration of 250 μg/ml. Each experiment was performed in triplicate and repeated 4 times. Data represent the percentage of live cells relative to controls (no treatment) and are presented as mean values +/- SEM. * significantly different from H₂O₂-treated cells, p<0.001. Scale bar  =  100 μm.</p