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Neuroblastoma is the most common extracranial nervous system tumor in children. It presents with a spectrum of clinical prognostic measures ranging from benign growths that regress spontaneously to highly malignant, treatment evasive tumors affiliated with increased mortality rates. MYCN amplification is commonly seen in high-risk neuroblastoma, rendering it highly malignant and recurrence prone. In our current study, we investigated the therapeutic potential of small molecule inducers of TRAIL, ONC201, and ONC206 in MYCN-amplified IMR-32 and non-MYCN-amplified SK-N-SH human neuroblastoma cell lines. Our results exhibit potent antitumor activity of ONC201 and ONC206 via a novel inhibition of EGF-induced L1CAM and PDGFRβ phosphorylation in both cell lines. Drug treatment significantly reduced cellular proliferation, viability, migration, invasion, tumorsphere formation potential, and increased apoptosis in both cell lines. The protein expression of tumorigenic NMYC, Sox-2, Oct-4, FABP5, and HMGA1 significantly decreased 48 h post-drug treatment, whereas cleaved PARP1/caspase-3 and γH2AX increased 72 h post-drug treatment, compared with vehicle-treated cells in the MYCN-amplified IMR-32 cell line. We are the first to report this novel differential protein expression after ONC201 or ONC206 treatment in human neuroblastoma cells, demonstrating an important multitarget effect which may yield added therapeutic benefits in treating this devastating childhood cancer.

Inhibiting pro-inflammatory cytokine activity can reverse inflammation mediated dysfunction of islet grafts. Human amniotic epithelial cells (hAECs) possess regenerative, immunomodulatory and anti-inflammatory properties. We hypothesized that hAECs could protect islets from cellular damage induced by pro-inflammatory cytokines. To verify our hypothesis, hAEC monocultures, rat islets (RI), or RI-hAEC co-cultures where exposed to a pro-inflammatory cytokine cocktail (Interferon γ: IFN-γ, Tumor necrosis factor α: TNF-α and Interleukin-1β: IL-1β). The secretion of anti-inflammatory cytokines and gene expression changes in hAECs and viability and function of RI were evaluated. The expression of non-classical Major Histocompatibility Complex (MHC) class I molecules by hAECs cultured with various IFN-γ concentrations were assessed. Exposure to the pro-inflammatory cocktail significantly increased the secretion of the anti-inflammatory cytokines IL6, IL10 and G-CSF by hAECs, which was confirmed by upregulation of IL6, and IL10 gene expression. HLA-G, HLA-E and PDL-1 gene expression was also increased. This correlated with an upregulation of STAT1, STAT3 and NF-κB1gene expression levels. RI co-cultured with hAECs maintained normal function after cytokine exposure compared to RI cultured alone, and showed significantly lower apoptosis rate. Our results show that exposure to pro-inflammatory cytokines stimulates secretion of anti-inflammatory and immunomodulatory factors by hAECs through the JAK1/2 – STAT1/3 and the NF-κB1 pathways, which in turn protects islets against inflammation-induced damages. Integrating hAECs in islet transplants appears as a valuable strategy to achieve to inhibit inflammation mediated islet damage, prolong islet survival, improve their engraftment and achieve local immune protection allowing reducing systemic immunosuppressive regimens. Graphical Abstract This study focuses on the cytoprotective effect of isolated hAECs on islets exposed to pro-inflammatory cytokines in vitro. Exposure to pro-inflammatory cytokines stimulated secretion of anti-inflammatory and immunomodulatory factors by hAECs putatively through the JAK1/2 – STAT1/3 and the NF-κB1 pathways. This had protective effect on islets against inflammation-induced damages. Taken together our results indicate that incorporating hAECs in islet transplants could be a valuable strategy to inhibit inflammation mediated islet damage, prolong islet survival, improve their engraftment and achieve local immune protection allowing to reduce systemic immunosuppressive regimens.

BackgroundNeuroblastoma is the most common pediatric extra-cranial nervous system tumor, originating from neural crest elements and giving rise to tumors in the adrenal medulla and sympathetic chain ganglia. Amplification of MYCN confers increased malignancy and poorer prognosis in high-risk neuroblastoma. Our SILAC proteomics analysis revealed over-expression of HSP90 in MYCN-amplified IMR-32 compared to the non-MYCN amplified SK-N-SH human neuroblastoma cells, rendering them highly resistant to therapeutic intervention.MethodsWe used cellular bio-functional (proliferation, migration/invasion, apoptosis, viability and stem-cell self-renewal) assays and Western blot analysis to elucidate the therapeutic efficacy of HSP90 inhibition with 17-AAG.Results17-AAG treatment significantly inhibited cellular proliferation, viability and migration/invasion and increased apoptosis in both cell lines. Moreover, drug treatment significantly abrogated stem-cell self-renewal potential in the MYCN-amplified IMR-32 cells. Differential tumorigenic protein expression revealed a novel mechanism of therapeutic efficacy after 17-AAG treatment with a significant downregulation of HMGA1, FABP5, Oct4, MYCN, prohibitin and p-L1CAM in SK-N-SH cells. However, we observed a significant up-regulation of p-L1CAM, MYCN and prohibitin, and significant down-regulation of Oct4, FABP5, HMGA1, p-ERK, cleaved/total caspase-3 and PARP1 in IMR-32 cells.ConclusionsHSP90 inhibition revealed a novel therapeutic mechanism of antitumor activity in MYCN-amplified neuroblastoma cells that may enhance therapeutic sensitivity.