Naxitamab Activity in Neuroblastoma Cells Is Enhanced by Nanofenretinide and Nanospermidine

Neuroblastoma cells highly express the disialoganglioside GD2, a tumor-associated carbohydrate antigen, which is also expressed in neurons, skin melanocytes, and peripheral nerve fibers. Immunotherapy with monoclonal anti-GD2 antibodies has a proven efficacy in clinical trials and is included in the standard treatment for children with high-risk neuroblastoma. However, the strong neuro-toxicity associated with anti-GD2 antibodies administration has hindered, until now, the possibility for dose-escalation and protracted use, thus restraining their therapeutic potential. Strategies to increase the efficacy of anti-GD2 antibodies are actively sought, with the aim to enable chronic treatments that could eradicate minimal residual disease and subsequent relapses, often occurring after treatment. Here, we report that Nanofenretinide and Nanospermidine improved the expression of GD2 in neuroblastoma cells (CHP-134) and provided different effects in combination with the anti-GD2 antibody naxitamab. In particular, Nanofenretinide significantly increased the cytotoxic effect of naxitamab while Nanospermidine inhibited cell motility at extents proportional to naxitamab concentration. In neuroblastoma cells characterized by a low and heterogeneous basal expression of GD2, such as SH-SY5Y, which may represent the cell heterogeneity in tumors after chemotherapy, both Nanofenretinide and Nanospermidine increased GD2 expression in approximately 50% of cells, thus shifting the tumor population towards improved sensitivity to anti-GD2 antibodies

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines

Background: EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. Methods: Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. Results: Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. Conclusion: This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cytomorphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death

A novel compound heterozygous genotype associated with aromatic amino acid decarboxylase deficiency: Clinical aspects and biochemical studies

Aromatic amino acid decarboxylase (AADC) deficiency is a rare autosomal neurometabolic disorder caused by a deficit of AADC, a pyridoxal 5'-phosphate (PLP)-dependent enzyme, which catalyzes the synthesis of dopamine and serotonin. While many studies have highlighted the molecular defects of the homozygous pathogenic variants, so far only a study investigated heterozygous variants at protein level. Here, we report a clinical case of one AADC deficiency compound heterozygous patient bearing the A91V mutation and the novel C410G mutation. To elucidate its enzymatic phenotype, the A91V and C410G homodimers were first expressed in Escherichia coli, purified and characterized. Although both apo variants display an unaltered overall tertiary structure, they show a ̴ 20-fold decreased PLP binding affinity. The C410G mutation only causes a ̴ 4-fold decrease of the catalytic efficiency, while the A91V mutation causes a 1300-fold decrease of the kcat/Km, and changes in the holoAADC consisting in a marked alteration of the tertiary structure and the coenzyme microenvironment. Structural analyses of these mutations are in agreement with these data. Unfortunately, the C410G/A91V heterodimer was constructed, expressed and purified in rather modest amount. Anyway, measurements of decarboxylase activity indicate that its putative kcat value is lower than that predicted by averaging the kcat values of the two parental enzymes. This indicates a negative interallelic complementation between the C410G and A91V monomers. Overall, this study allowed to relate the clinical to the enzymatic phenotype of the patient and to extend knowledge in the clinical and molecular pathogenesis of AADC deficiency

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines-9

�mol/L gefitinib plus 10 nM EGF (Gb + E). ANOVA One-way analysis of variance and Tukey's Multiple Comparison Test. Caco-2. NT vs: **E, ***Cx5, *Cx10, ***Gb, ***Cx5 + E, *Cx10 + E, **Gb + E. *p < 0.05, **p < 0.01, ***p < 0.001. Each point represents a mean of quadruplicate values for each sample ± SD.<p><b>Copyright information:</b></p><p>Taken from "Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines"</p><p>http://www.biomedcentral.com/1471-2407/8/227</p><p>BMC Cancer 2008;8():227-227.</p><p>Published online 8 Aug 2008</p><p>PMCID:PMC2528013.</p><p></p

All heat maps were obtained by using an unsupervised hierarchical clustering method with a correlation distance between all the samples and between the selected genes

Heat map of genes selected from 10 nM EGF treated samples.<p><b>Copyright information:</b></p><p>Taken from "Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines"</p><p>http://www.biomedcentral.com/1471-2407/8/227</p><p>BMC Cancer 2008;8():227-227.</p><p>Published online 8 Aug 2008</p><p>PMCID:PMC2528013.</p><p></p

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines-3

Y as untreated cells. c. 10 nmol/L cetuximab treated cells. Filopodi are evident. Insert: microvilli reduction is evident. d. 10 nmol/L cetuximab plus 10 nM EGF treated cells. Filipodi and some vesicles are evident. Insert: microvilli reduction is evident. e. 1 μmol/L gefitinib treated cells. Some vesicles are evident. Insert: microvilli reduction is evident. f. 1 μmol/L gefitinib plus 10 nM EGF treated cells. Lamellipodi, some vesicles and weak contacts with nearby cells are evident. Insert: microvilli reduction is evident.<p><b>Copyright information:</b></p><p>Taken from "Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines"</p><p>http://www.biomedcentral.com/1471-2407/8/227</p><p>BMC Cancer 2008;8():227-227.</p><p>Published online 8 Aug 2008</p><p>PMCID:PMC2528013.</p><p></p

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines-5

En samples.<p><b>Copyright information:</b></p><p>Taken from "Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines"</p><p>http://www.biomedcentral.com/1471-2407/8/227</p><p>BMC Cancer 2008;8():227-227.</p><p>Published online 8 Aug 2008</p><p>PMCID:PMC2528013.</p><p></p

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines-1

<p><b>Copyright information:</b></p><p>Taken from "Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines"</p><p>http://www.biomedcentral.com/1471-2407/8/227</p><p>BMC Cancer 2008;8():227-227.</p><p>Published online 8 Aug 2008</p><p>PMCID:PMC2528013.</p><p></p

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines-0

�mol/L gefitinib plus 10 nM EGF (Gb + E). ANOVA One-way analysis of variance and Tukey's Multiple Comparison Test. Caco-2. NT vs: **E, ***Cx5, *Cx10, ***Gb, ***Cx5 + E, *Cx10 + E, **Gb + E. *p < 0.05, **p < 0.01, ***p < 0.001. Each point represents a mean of quadruplicate values for each sample ± SD.<p><b>Copyright information:</b></p><p>Taken from "Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines"</p><p>http://www.biomedcentral.com/1471-2407/8/227</p><p>BMC Cancer 2008;8():227-227.</p><p>Published online 8 Aug 2008</p><p>PMCID:PMC2528013.</p><p></p