SKNMC Cells Research Articles

Experimental verification about treatment of Bu-Shen-Yi-Jing-Fang in Alzheimer’s disease by the analysis of the feasible signaling pathway of network pharmacology

ContextBu-shen-yi-jing-fang (BSYJF) has been reported to reduce amyloid-β (Aβ)1–42 deposition in the brain of APP/PS1 mice and ameliorate cognitive function. However, its neuroprotective mechanism remains unclear.ObjectiveThis study aims to investigate whether BSYJF exerts a protective effect on Aβ1–42-induced oxidative stress injury and explore its possible mechanism.Materials and methodsThe platform databases TCMSP, Swiss, TTD, DrugBank, and GeneCards were used to mine the targets of Alzheimer’s disease (AD) and BSYJF. The platform databases STRING and Metascape were used to build the interaction network of the target protein, and Cytoscape software was used to analyze this network and screen out the key pathways. Aβ1–42-treated SKNMC cells were established to verify the mechanism of BSYJF and the key proteins. The downstream proteins and antioxidants as well as apoptosis and ferroptosis of the PI3K/AKT/Nrf2 signaling pathway were validated using an in vitro SKNMC cell model experiment. The expression levels of related proteins were detected using Western blotting. Flow cytometry and immunofluorescence staining were used to analyze apoptosis and ferroptosis.ResultsKyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis considered the key signal pathways, mainly involving the PI3K/AKT signaling pathway. Experimental validation demonstrated that BSYJF treatment markedly increased the activity of the PI3K/AKT pathway, which could exert anti-AD effects.ConclusionsOur data provided compelling evidence that the protective effects of BSYJF might be associated with their regulation of the PI3K/AKT/Nrf2 signaling pathway. These studies offered a potential therapy for natural herbal medicine treatment of AD.

Catalpol alleviates amyloid- generation and neuronal oxidative stress injury via activating the Keap1-Nrf2/ARE signaling pathway in the immortalized lymphocytes from patients with late-onset Alzheimer's disease and SKNMC cells co-culture model.

To assess the effect of catalpol, the major bioactive constituents of Rehmannia glutinosa, on our Alzheimer's disease (AD) in vitro model. We employed the immortalized lymphocytes (lymphoblastoid cell line, LCL) from late-onset AD patients and co-cultured "them" to mimic the pathological process of late-onset AD and investigated the effect of catalpol on our AD in vitro model. In the co-culture model, AD-derived LCL triggered excessive Aβ1-42 in SKNMC cells due to its high levels of oxidative stress and resulted in neuronal oxidative stress injury through inhibiting Keap1-Nrf2/ARE signaling pathway. Treatment with catalpol and N-acetylcysteine (NAC), an antioxidant, prevented the AD LCL-induced Aβ1-42 overproduction and reduced the level of β-site amyloid precursor protein cleaving enzyme-1 (BACE1) and amyloid precursor protein (APP)-C99. Catalpol and NAC also enhanced the antioxidant capacity and reduced apoptosis in SKNMC cells co-cultured with AD LCL. The anti-oxidative effect of catalpol was antagonized by ML385, the Nrf2 inhibitor. Therefore, we speculate that the antioxidant and anti-apoptotic effects of catalpol are mediated by activating the Keap1-Nrf2/ARE signaling pathway. Catalpol affects the anti-Aβ generation and the antioxidative and antiapoptotic properties in the AD co-cultured model. So, it might be a novel natural drug and offer a potential therapeutic approach for AD.

Network pharmacology analysis and experimental validation of Anemarrhenae Rhizoma in treating Alzheimer's disease.

To explore the mechanism of Anemarrhenae Rhizoma in treatment of Alzheimer's Disease (AD). The active ingredients and targets of Anemarrhenae Rhizoma for treatment of AD were screened with network pharmacology methods, the protein-protein interaction (PPI) network was constructed and the core targets were analyzed. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriching analysis was performed. The peripheral blood lymphocytes were extracted and lymphoblastoid cell lines (LCL) were constructed and an in vitro cell model of LCL-SKNMC was established. MTT and CCK-8 methods were used to quantify SKNMC/LCL cells, 2 ´, 7 ´-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect reactive oxygen species (ROS), and immunofluorescence staining was used to detect the generation of Aβ1-42 in a co-cultured model. Western blotting was used to detect protein expression in the co-culture model. The lifespan of N2 nematodes was observed under oxidative stress, normal state, and heat stress; ROS generated by N2 nematodes was detected by DCFH-DA probes. The paralysis time of CL4176 N2 nematodes was evaluated by paralysis assay, and Aβ deposition in the pharynx was detected by Thioflavin S staining. Through network pharmacology, 15 potential active ingredients and 103 drug-disease targets were identified. PPI analysis showed that the Anemarrhenae Rhizoma might play anti-AD roles through albumin, Akt1, tumor necrosis factor, epidermal growth factor receptor (EGFR), vascular endothelial growth factor A (VEGFA), mammalian target of rapamycin (mTOR), amyloid precursor protein (APP) and other related targets. KEGG analysis showed that the pharmacological effects of Anemarrhenae Rhizoma might involve the biological processes of Alzheimer's disease, endocrine resistance, insulin resistance; and neuroactive ligand-receptor interaction, phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway, calcium signaling pathway, AGE-RAGE signaling pathway in diabetes complications, neurotrophic factor signaling pathway and others. The in vitro cell experiments showed that Anemarrhenae Rhizoma was able to reduce the production of ROS and Aβ1-42 (both P<0.01), inhibit the expression of β-secretase 1 (BACE1), APP and Aβ1-42 proteins (all P<0.05), up-regulate the expression of p-PI3K/PI3K, p-AKT/AKT, p-GSK3β/GSK3β in SKNMC cells (all P<0.05). The in vivo studies further confirmed that Anemarrhenae Rhizoma prolonged the lifespan of C. elegans under stress and normal conditions, reduced the accumulation of ROS and the toxicity of Aβ deposition. Anemarrhenae Rhizoma may reduce the production of Aβ in AD and inhibit its induced oxidative stress, which may be achieved by regulating the PI3K/Akt/GSK-3β pathway.

Abstract 2569: Development of potent dual PDK1/AurA kinase inhibitors for Ewing sarcoma therapy

Abstract Background. It has been estimated that more than 50% of cancers are related to the dysregulation of the PI3K-PDK1-Akt signaling axis. Within this pathway, phosphoinositide-dependent kinase-1 (PDK1) and Aurora kinase A (AurA) have key roles. PDK1 is a phosphorylation-mediated activator of Akt, the main apoptotic regulator, thereby acting at the crossroads of several oncogenic pathways. Overexpression and/or aberrant activation of AurA have been functionally linked to neoplastic transformation principally through the development of centrosome amplification and chromosomal instability. In this study, the molecular mechanisms involved in the dual inhibitory capacity of PDK1- and AurA-targeting OXID-pyridonyl compounds were explored in a panel of tumor cell lines carrying PI3KCA, K-RAS, PTEN, RB-1, EGFR and/or TP53 mutations Materials and Methods. To evaluate the enzymatic inhibitory activity, the oxindole-based derivatives were subjected to FRET-based Z′-Lyte assay (Invitrogen™) against PDK1 and AurA. All in vitro assays for ADME-Tox profiling were performed as published previously[1]. Anti-proliferative effects of OXID-pyridonyl compounds (SA16, IB35, VI8, VI18) were assessed by soft agar clonogenic formation and MTT assays after 72-h exposures with increasing concentrations (from 0.014 to 30 µM) in a panel of 15 cell lines. Protein levels were analyzed by Western Blot using commercial antibodies. OSU-03012 (PDK-1 inhibitor) and MK-5108 (AurA inhibitor) were used as reference compounds. Results. Among the four evaluated compounds, SA16 displayed nanomolar inhibitory potency against both PDK1 and AurA. In vitro, drugs SA16 and VI8 showed to exert anti-tumor activity in the 6647, TC71, SKNMC Ewing sarcoma (ES) cells lines and in the HCT116 colon carcinoma-derived cells (IC50s values between 2.6 and 25 µM after 72-h exposures), when tested in a panel of 15 cancer cell lines. These results were confirmed by the evaluation of 3D-clonogenic capacity in soft agar. The effect of 1 µM SA16 at the protein level was evaluated after 5 to 120 min exposure in the ES cell line SKNMC. No modulation of Aur-A protein or PDK-1 phosphorylation (Ser241) were observed in SKNMC cells, whereas a decrease of downstream PDK1 targets such as p-AKT, p-RSK2, and p-p44/42 MAPK was observed after 30 min of exposure. In addition, SA16 displayed acceptable in vitro ADME-Tox properties. Our results on structural insights into the mode of binding[2], in vitro drug-intake kinetics and in vivo PK appear to indicate that SA16 presents adequate features as a scaffold for the design of PDK1/AurA dual-target molecules. Conclusion. We report the synthesis of novel first-in-class 2-oxindole-based derivatives as dual PDK1-AurA kinase inhibitors as a novel scaffold for the design and synthesis of a new multitarget strategy for Ewing sarcoma.[1] Runfola M. et al. Data Brief 29 (2020) 105206.[2] Sistito S. et al. Eu J Med Chem 226 (2021) 11389. Citation Format: Maria Eugenia Riveiro, Samuel Huguet, Ramiro Vazquez, Roberta Frapolli, Guido Puricelli, Simona Sestito, Olivier Madar, Keyvan Rezai, Gianpiero Garau, Simona Rapposelli. Development of potent dual PDK1/AurA kinase inhibitors for Ewing sarcoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2569.

Abstract 1294: Dual targeting of PDK1 and Aurora A using first-in class OXID-pyridonyl compounds in preclinical models of Ewing sarcoma

Abstract Background. PDK1, is a master activator of multiple prosurvival and oncogenic protein kinases such as AKT, PKC, RSK and SGK families in oncology[1]. On the other hand, overexpression and aberrant activation of Aurora-A kinase (AurA) have been functionally linked to oncogenic transformation mainly through development of centrosome amplification and chromosomal instability[2]. In this study, the molecular mechanisms at the simultaneous PDK1/AurA inhibition using OXID-pyridonyl[3] compounds were explored in panel of tumor cell lines carrying different oncogenic mutations (PI3KCA, K-RAS, PTEN, RB-1, EGFR and TP53). Materials and Methods. Established human cancer cell lines from breast, glioblastoma, renal prostate, colon, lung and Ewing sarcoma were studied. Anti-proliferative effects of OXID-pyridonyl compounds (SA16, IB35, VI8, VI18) were assessed by soft agar clonogenic formation and MTT assays after 72h-exposure with increasing concentrations (from 0.014 to 30 µM). Protein levels were analyzed by Western Blot using commercial antibodies. OSU-03012 (PDK1 inhibitor) and MK-5108 (AurA inhibitor) were used as reference compounds to investigate the functional crosstalk between the two pathways in sensitive cancer cell lines. Results. We assessed anti-proliferative activity of single-agents SA16, IB35, VI8 and VI18 in a panel of 15 cancer cell lines and observed that three Ewing sarcoma and one colon carcinoma cell lines were sensitive (6647,TC71, SKNMC and HCT116), with IC50s values between 2.6 and 25 µM after 72h-exposure, whereas all the studied cell lines were resistant to IB35 and VI18. These results were confirmed by soft agar clonogenic formation studies. AurA, PDK-1, p-PDK1 ser241, AKT, p-AKT ser473 protein levels were characterized but no differences in their basal levels were observed between OXID-pyridonyl compounds -sensitive or -resistant cell lines. SA16 effect at the protein level was evaluated after 5 to 120 min in sensitive cell lines. In SKNMC, TC71 and HCT116 cells, we didn't observe a modulation of AurA or p-PDK1 ser241 while a decrease of p-AKT was observed in SKNMC and HCT116 cells. In addition, a decrease of the p-p44/42 MAPK was observed after 30 min-exposure in SKNMC cells. On the other hand, p-RB1, p-Cdc2, P21 and C-MYC were not regulated at the protein level with SA16 treatment up to 120 min-exposure. In resistant cell lines, U87MG and PC3, both p-PDK1 ser241 and AurA levels were not modulated up to 2h of treatment with SA16. Conclusion. Here we report the characterization of two potent first-in-class dual PDK1-AurA inhibitors, SA16 and VI8. Our findings suggest that innovative PDK1/AurA dual-target molecules which could represent an attractive lead scaffold for the design and synthesis of a new multitarget strategy for Ewing sarcoma. [1] Nagashima K et al. J Biol Chem. 2011. [2] Yan M et al. Med Res Rev. 2016. [3] Sestito, S et al. Proceedings 2019. Citation Format: Maria Eugenia Riveiro, Simona Rapposelli, Samuel Huguet, Ramiro Vazquez, Mohamed Bekradda, Guido Puricelli, Simona Sestito, Olivier Madar, Keyvan Rezai. Dual targeting of PDK1 and Aurora A using first-in class OXID-pyridonyl compounds in preclinical models of Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1294.

Abstract 6158: The relevance of endoglin and MMP14 to the metastatic potential of Ewing sarcoma cells

Abstract Background: Ewing sarcoma (ES) is a developmental bone/soft tissue neoplasia, for which mesenchymal stem cell (MSC) is the putative cell of origin. Although the outcome of ES patients bearing primary tumors has improved significantly, patients with metastatic ES still have a poor prognosis. The MSC marker endoglin/CD105 (ENG) and the ENG-shedding protease MMP14 have been, respectively, described as cell migration and invasion regulators. In our attempt to better understand the mechanism of ES progression, we are focusing on the role of ENG and MMP14 in ES cells. Methods: In vitro ENG-knockdown (ENG-KD; RM82 and SKNMC cell lines) and ENG-overexpressing (pENG; TC71 cell lines) models were generated by shRNA technology and pDisplay vectors, respectively, and confirmed by qRT-PCR, western blotting and flow cytometry. In vitro MMP14-knockout (MMP14-KO; RM82 and SKNMC) models were generated by CRISPR-Cas9 and confirmed by western blotting and Sanger sequencing. Transwell migration/invasion, adhesion, clonogenicity and proliferation assays were performed in vitro. Actin filament organization was evaluated by immunofluorescence. Extracellular vesicles (EVs) were isolated by ultracentrifugation and density gradient separation. Transcriptomic profiling was performed by Clarion S Human arrays followed by Gene Set Enrichment Analysis. HiPathia tool was used to recode the transcriptomic data into measurements of cell activity by applying mechanistic models. Reverse Phase Protein Array (RPPA) analysis was evaluated on the ENG-KD RM82 model. ENG-targeting immunoprecipitations (IP) from wild type RM82 protein extracts were analyzed by mass spectrometry (MS). Results: The impaired clonogenic potential of ENG-KD RM82 and SKNMC models suggests a role of ENG on the stemness capacity of ES, without affecting the proliferation rate. The reduced migratory and adhesive phenotype of the ENG-KD RM82 model and the transcriptomic profiling of both ENG-KD RM82 and SKNMC cells, suggest a role of ENG in ES cell migration and adhesion. The altered organization of actin filaments in ENG-KD and pENG models could support a role of ENG in actin filament remodeling. The transcriptomic profiling of ENG-KD models and the RPPA results underscore the regulation of FAK signaling by ENG. The IP-MS analysis of ENG partners identifies Integrin-Linked Kinase-Associated serine/threonine Phosphatase (ILKAP) as one of the main interactors of ENG in RM82 cells, suggesting its possible association with the focal adhesion member ILK. ENG is enriched in EVs from different ES cell lines. The transcriptomic profiling of MMP14-KO SKNMC cells highlights the involvement of MMP14 activity in the extracellular matrix organization of ES cells. Conclusion: ENG favors a pro-migratory phenotype in ES cells. These findings help to better understand the molecular landscape of tumoral cells from ES patients, and might have potential clinical implications. Citation Format: Pilar Puerto-Camacho, Ana Teresa Amaral, Juan Diaz-Martin, Alfonso Bolado-Carrancio, Carmen Jordan-Perez, Carmen Salguero-Aranda, Marina Esteban-Medina, Inmaculada Alamo-Alvarez, Joaquin Dopazo, Carmelo Bernabeu, Adam Byron, Valerie G. Brunton, Enrique de Álava. The relevance of endoglin and MMP14 to the metastatic potential of Ewing sarcoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6158.

Abstract B19: Identification of physiologically relevant EWS-FLI1 target genes in Ewing’s sarcoma via CRISPRa screening

Abstract Ewing’s sarcoma is an aggressive pediatric bone and soft-tissue cancer with a pathognomonic chromosomal translocation t(11;22) resulting in expression of EWS-FLI1, an “undruggable” fusion protein acting as a transcriptional modulator. Identification and ranking of repressed EWS-FLI1 target genes essential for cancer cell survival will potentially provide much-needed insights to develop novel therapeutic strategies. We performed a CRISPR activation (CRISPRa) dropout screen in Ewing cells. We generated a clonal SKNMC cell line homogenously expressing the synergistic activation mediator (SAM) CRISPRa system to functionally interrogate repressed EWS-FLI1 target genes. The systems functionality was tested using CD44, a surface marker absent on the surface of Ewing cells. We found robust and stable expression of CD44 after introduction of promoter-targeting gRNAs. The library of repressed EWS-FLI1 target genes, named LIBerty, was constructed to target 872 genes bioinformatically selected from publicly available silenced EWS-FLI1 RNA-Seq datasets as well as genes identified as repressed signature genes in Ewing’s sarcoma via meta-analysis with 3,777 unique guideRNAs. LIBerty was lentivirally delivered into SAM SKNMC cells and data from four biologic replicates were gathered at three time points: three, ten, and 21 days after infection. Cells were harvested and samples were prepared for next-generation sequencing (NGS) via PCR. The NGS data were evaluated with the analysis tool PinAPL-Py. Preliminary analysis of the screen revealed efficient selection of the positive control genes BAD and BBC3. In addition, high-ranking hits included CDKN1A and CDKN1C, both cell cycle regulators and known tumor-suppressor genes. Furthermore, we identified PCDH7 and TGFBR2, two genes already known from the literature to play a role in Ewing’s sarcoma. The presence of validated genes amid highest-ranking candidates confirms robustness of the conducted CRISPRa screen. Further analysis and validation of previously unexplored targets and pathways is currently ongoing. Our CRISPRa screen revealed both known as well as previously unknown EWS-FLI1 repressed genes whose absence of function is essential for tumor cell survival. Citation Format: Vadim Saratov, Qui A. Ngo, Gloria Pedot, Felix K. Niggli, Beat W. Schaefer. Identification of physiologically relevant EWS-FLI1 target genes in Ewing’s sarcoma via CRISPRa screening [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B19.

N-(5-(Trifluoromethyl)-1,3,4-Thiadiazol-2-Yl)Benzamide and Benzothioamide Derivatives Induce Apoptosis Via Caspase-Dependent Pathway

New 1,3,4-thiadiazole-based compounds were designed, synthesized, and their anticancer effects were assessed by MTT assay against PC3 (prostate cancer), HT-29 (colon cancer), and SKNMC (neuroblastoma) cell lines. The results were compared to that of doxorubicin. According to MTT assay, some of the synthesized compounds exhibit higher cytotoxic activity (IC 50 , μM range) than doxorubicin against PC3 and SKNMC cells but not HT29 cells. According to the analysis of structure – activity relationship, compounds with methoxy group as an electron donating moiety rendered higher activity than nitro group as an electron withdrawing group. Compound 4d with ortho position of methoxy moiety activated caspases 3 and 9 in both PC3 and HT-29 cell lines.

Evaluation of the Cytotoxic and Apoptogenic Effects of Glabridin and Its Effect on Cytotoxicity and Apoptosis Induced by Doxorubicin Toward Cancerous Cells

Purposes: In the present study, we tried for the first time to examine the anti-proliferative and anti-apoptogenic effect of Glabridin (Glab) toward three groups of cancer cells (SKNMC, H1299, and A2780). Furthermore, the possibility of co-administration of Glab with doxorubicin (DOX) to these cells was also examined to find out whether Glab can potentiate the cytotoxic effect of this chemotherapy agent. Methods: Different cellular assays (MTT, caspase-3 activity, MMP, RT-PCR analysis) were carried out on the cancer cells treated with Glab. Results: Cellular toxicity assay revealed that Glab can potentially reduce the viability of these cells with IC50 concentrations up to 10, 12, and 38 μM toward A2780, SKNMC, and H1299 cell lines, respectively. The results of MMP and caspase-3 activity assays, in association with the results corresponding to the BAX and Bcl-2 gene expressions, altogether revealed that Glab can exert apoptogenic effect on these cells. The intrinsic mitochondrial pathway was found to be the main mechanism, in which Glab induced apoptosis toward H1299 cells and SKNMC cells, while the apoptosis mechanism for A2780 cells could be probably through extrinsic pathway. Glab also potentiated the cytotoxic effect of DOX and its accumulation in H1299 cell line. Conclusion: The results of this study revealed the promising cytotoxic role of Glab on different carcinoma cells. These data also suggested that co-chemotherapy method using Glab could be effective for treatment of cancer, but further in-vivo and clinical studies are still needed to assure these results.

Polypyrrole nanotube modified by gold nanoparticles for improving the neural microelectrodes

High selectivity and low impedance are preferred properties for neural microelectrodes. The localized and controlled release of drugs from the nanostructured coatings may reduce brain tissue responses for chronic recordings. Here, polypyrrole (PPy) nanotube was chemically synthesized inside an alumina template. The formation of nanotube was confirmed by scanning electron microscopy (SEM) of the cross-section of templates. Then, PPy nanotube was loaded with dexamethasone (Dex) as a dopant during the polymerization process up to 93%. Successful loading of Dex molecules into the PPy nanotube was verified by the Fourier-transform infrared spectroscopy (FTIR). Gold nanoparticles were synthesized via the reduction of Au3+ ions on the surface of PPy nanotubes and were confirmed by transmission electron microscopy (TEM). The anodic electrophoretic deposition (EPD) method was successfully applied for deposition of 0.1 mg of the synthesized nanostructures on the surface of neural microelectrode in 20 min. Electrochemical impedance spectroscopy (EIS) showed a tenfold decrease in the impedance of the coated microelectrode as compared with the bare one. In vitro studies on U87MG cells demonstrated that the release of Dex from the coating reduced effectively the number of reactive astrocytes without any toxic side effects on SK-NMC and PC12 neuronal cells. Results also indicated that the release of Dex from the carrier can only occur by the electrical stimulation.

A new Alzheimer's disease cell model using B cells to induce beta amyloid plaque formation and increase TNF alpha expression

Different cell models have been developed for the study of Alzheimer's disease (AD) pathways. The neuronal dysfunction and cell death mechanisms that are commonly found in this disease are due to the production of high levels of cytokines and the formation of amyloid plaques. In the cell model introduced in the present study, the production of these two important factors is induced by using B cells from an AD patient.The B cells of an Alzheimer's patient and a normal control were immortalized by using EBV (Epstein-Barr virus) to produce a lymphoblastoid cell line (LCL). The amount of TNF alpha cytokine was evaluated at the RNA and protein levels by RT-PCR and ELISA, respectively. The AD LCL was cultured with SKNMC cells with and without treatment of TNF alpha siRNA. Amyloid plaque formation was monitored by Congo-red staining and microscopy.The amount of TNF alpha cytokine was significantly increased in the AD LCL compared to the normal LCL. The AD LCL induced the formation of amyloid plaques in SKNMC cells. The AD LCL treated with TNF alpha siRNA and co-cultured with SKNMC cells decreased the size and number of amyloid plaques in SKNMC cells.This cellular model is an appropriate model for studying Alzheimer's disease and the mechanisms related to it, as well as for research on cytokine inhibitors, especially TNF alpha inhibitors.

The Shc protein RAI promotes an adaptive cell survival program in hypoxic neuroblastoma cells.

Neuroblastoma (NB) is a highly malignant pediatric solid tumor where a hypoxic signature correlates with unfavorable patient outcome. The hypoxia-inducible factor (HIF)-1α plays an important role in NB progression, contributing to cell proliferation and invasiveness. RAI belongs to the Shc family proteins, it is mainly neuron specific and protects against cerebral ischemia. RAI is also expressed in several NB cell lines, where it promotes cell survival. In this work, hypoxia differently affected cell survival and pro-apoptotic program in two NB cell lines, either expressing RAI (SKNBE) or not (SKNMC). RAI expression appeared to promote NB cell survival and to reduce some pro-apoptotic markers under hypoxia. Accordingly, the RAI silencing in SKNBE cells resulted in a reduction of cell survival and HIF-1α expression. Furthermore, using SKNMC cells stably expressing RAI, we defined a role of RAI in NB cell responses to hypoxia. Of interest, in hypoxic SKNMC cells expressing RAI HIF-1α protein levels were higher than in control cells. This was associated with a) an increased cell survival; b) an increased expression of anti-apoptotic markers; c) a pro-autophagic and not pro-apoptotic phenotype; and d) an increased metabolic activity. We may conclude that RAI plays an important role in hypoxic signaling in NB cells and the interplay between RAI and HIF-1α may be relevant in the protection of NB cells against hypoxia. Our results may contribute to a further understanding the physiology of NB cells and the molecular mechanisms involved in their survival, with important implications in NB progression.

Polyphenolic Extract of Euphorbia supina Attenuates Manganese-Induced Neurotoxicity by Enhancing Antioxidant Activity through Regulation of ER Stress and ER Stress-Mediated Apoptosis.

Manganese (Mn) is an important trace element present in human body, which acts as an enzyme co-factor or activator in various metabolic reactions. While essential in trace amounts, excess levels of Mn in human brain can produce neurotoxicity, including idiopathic Parkinson’s disease (PD)-like extrapyramidal manganism symptoms. This study aimed to investigate the protective role of polyphenolic extract of Euphorbia supina (PPEES) on Mn-induced neurotoxicity and the underlying mechanism in human neuroblastoma SKNMC cells and Sprague-Dawley (SD) male rat brain. PPEES possessed significant amount of total phenolic and flavonoid contents. PPEES also showed significant antioxidant activity in 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and reducing power capacity (RPC) assays. Our results showed that Mn treatment significantly reduced cell viability and increased lactate dehydrogenase (LDH) level, which was attenuated by PPEES pretreatment at 100 and 200 µg/mL. Additionally, PPEES pretreatment markedly attenuated Mn-induced antioxidant status alteration by resolving the ROS, MDA and GSH levels and SOD and CAT activities. PPEES pretreatment also significantly attenuated Mn-induced mitochondrial membrane potential (ΔΨm) and apoptosis. Meanwhile, PPEES pretreatment significantly reversed the Mn-induced alteration in the GRP78, GADD34, XBP-1, CHOP, Bcl-2, Bax and caspase-3 activities. Furthermore, administration of PPEES (100 and 200 mg/kg) to Mn exposed rats showed improvement of histopathological alteration in comparison to Mn-treated rats. Moreover, administration of PPEES to Mn exposed rats showed significant reduction of 8-OHdG and Bax immunoreactivity. The results suggest that PPEES treatment reduces Mn-induced oxidative stress and neuronal cell loss in SKNMC cells and in the rat brain. Therefore, PPEES may be considered as potential treat-ment in Mn-intoxicated patients.

Abstract 2008: Ewing sarcoma cells harboring a translocation that retains EWSR1 exon 8 require HNRNPH1 to express the in-frame oncogenic fusion transcript EWS-FLI1

Abstract The primary oncogenic event in ∼85% of Ewing sarcomas is a t(11:22)(q24:q12) translocation. This translocation generates a fusion gene containing the 5’ end of the EWSR1 gene and the 3’ end of the FLI1 gene referred to as EWS-FLI1. The exact genomic breakpoints within the EWSR1 and FLI1 genes vary, but typically occur within introns and require the splicing machinery to generate an in-frame EWS-FLI1 transcript. In an estimated 40% of EWS-FLI1 driven tumors, the generation of an in-frame EWS-FLI1 fusion transcript requires alternative splicing. In particular, translocations that retain exon 8 of EWSR1 generate an out-of-frame transcript unless this exon is removed. In this study, we demonstrate that Ewing sarcoma cells harboring a genomic breakpoint that retains exon 8 of EWSR1 require HNRNPH1 to express an in-frame EWS-FLI1 mature mRNA. Using a genome-wide RNAi screen, we identified several proteins involved in RNA processing as required for the activity of EWS-FLI1, including the heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1). The role of HNRNPH1 in alternative splicing led us to hypothesize that ES cells are dependent on HNRNPH1 for the expression of the EWS-FLI1 transcript. Analysis of the expression of EWS-FLI1 following HNRNPH1 silencing in Ewing sarcoma cell lines representing different translocation breakpoints and transcript isoforms showed that only Ewing sarcoma cell lines retaining EWSR1 exon 8 at a genomic level (TC32 and SKNMC cells) are dependent on HNRNPH1 expression. Silencing of HNRNPH1 in TC32 or SKNMC cells, results in the expression of an out-of-frame EWS-FLI1 transcript that cannot express the EWS-FLI1 oncogenic transcription factor. This leads to the reversal of expression of EWS-FLI1 gene targets and cell death. Ewing sarcoma cell lines that harbor a translocation upstream of EWSR1 exon 8 (TC71 and RD-ES) exhibit none of these molecular or phenotypic changes upon HNRNPH1 silencing. We next employed an RNA pull-down and PCR strategy to identify putative binding sites for HNRNPH1 on the EWS-FLI1 pre-mRNA. This analysis showed enrichment for the binding of EWSR1 exon 8 by HNRNPH1. Towards the 3’ end of EWSR1 exon 8 we identified two G-rich sequences, a motif typically bound by HNRNPH1. To determine if HNRNPH1 binds one or both of these sites, we developed an in vitro protein-RNA-oligomer binding assay. This assay confirmed the binding of HNRNPH1 to both G-rich sites in EWSR1 exon 8. Current studies are focused on using the protein-RNA-oligomer binding assay to fully map the interaction of HNRNPH1 with sequences within EWSR1 exon 8 and understand the molecular mechanism to target it. These results demonstrate a sequence-specific, breakpoint-dependent vulnerability in Ewing sarcoma that has the potential to be exploited as a therapeutic target and suggests a novel strategy to target fusion oncogenes. Citation Format: Suntae Kim, Christiane Olivero, Guillermo O. Rangel Rivera, Nirmalya Sen, Sara Haddock, Konrad Huppi, Lee J. Helman, Patrick J. Grohar, Natasha J. Caplen. Ewing sarcoma cells harboring a translocation that retains EWSR1 exon 8 require HNRNPH1 to express the in-frame oncogenic fusion transcript EWS-FLI1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2008.

Comparative Evaluation of Cytotoxic and Apoptogenic Effects of Several Coumarins on Human Cancer Cell Lines: Osthole Induces Apoptosis in p53-Deficient H1299 Cells.

Natural products are excellent resources for finding lead structures for the development of chemotherapeutic agents. Coumarins are a class of natural compounds found in a variety of plants. In this study, we evaluated the cytotoxic potential of coumarins isolated from Prangos ferulacea (L.) Lindl. in PC3, SKNMC, and H1299 (p53 null) human carcinoma cell lines. Osthole proved to be an outstanding potent cytotoxic agent especially against PC3 cells. Isoimperatorin exhibited moderate inhibitory effect against SKNMC and PC3 cell lines. Oxypeucedanin and braylin did not display any cytotoxic activity. In the next set of experiments, the apoptotic potentials of osthole and isoimperatorin were investigated. Induction of apoptosis by isoimperatorin was accompanied by an increase in activation of caspase-3, -8, and -9 in SKNMC cells and caspase-3 and -9 in PC3 cells. Moreover, isoimperatorin induced apoptosis by upregulating Bax and Smac/DIABLO genes in PC3 and SKNMC cells. Osthole induced apoptosis by downregulating antiapoptotic Bcl-2 in only PC3 cells and upregulating the proapoptotic genes Bax and Smac/DIABLO in PC3, SKNMC, and H1299 cells. The effects of osthole on H1299 cells are important because the loss of p53 has been associated with poor clinical prognosis in cancer treatment.

Abstract 4338: The effect of opposing roles of NF-kB on TRAIL-R2 expression in TRAIL cytotoxicity in neuroblastoma cells

Abstract TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in most, but not all, cancer cells. The molecular factors regulating the sensitivity to TRAIL are still incompletely understood. The transcription factor nuclear factor-kappa B (NF-kB) has been implicated, but its exact role is controversial. In this study, we have investigated the role of NF-kB on enhancement of TRAIL cytotoxicity in neuroblastoma cells. IkB kinase (IKK) inhibitor decreased basal NF-kB activity and marginally induced cell death in neuroblastoma cell line SK-NMC. Pretreatment with IKK inhibitor synergistically increased TRAIL-mediated apoptosis via up-regulation of TRAIL- receptor2 (TRAIL-R2) expression in response to the decrease in basal NF-kB activity, indicating that basal NF-kB activity blocks TRAIL-R2 expression in SK-NMC cells. In addition, pretreatment with Cisplatin, a platinum analog and an alkylating agent widely used in neuroblstomas, showed increase in NF-kB activity and induction of apoptotic cell death in SK-NMC cells. Pretreatment with Cisplatin significantly increased TRAIL-mediated apoptosis via up-regulation of TRAIL-R2 expression through induction of NF-kB, suggesting that induced NF-kB is involved in TRAIL-R2 expression up-regulated by Cisplatin. IKK inhibitor or Cisplatin followed by TRAIL treatment increased caspase-8, -9, and -3 activation. Mcl-1 cleavage and Bid truncation. Our results implicate that basal NF-kB activity is involved in TRAIL resistance but induced NF-kB plays a role in TRAIL sensitivity, and suggest that the enhanced sensitivity of SK-NMC cells to TRAIL is associated with an increase in TRAIL-R2 expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4338. doi:10.1158/1538-7445.AM2011-4338

604 H 2S Induces Analgesia Is Mediated In Vivo Activation of μ Opioid Receptor and PI3K/AKT Pathway Activation

Background and aim. Hydrogen sulfide (H2S) functions as a neuromodulator and reverses colorectal distension (CRD)-induced pain. Opioid system is widely involved in analgesia. Whether the opioid receptors are involved in the H2S -induced analgesia is unknown. Methods. Four, graded (0.4-1.6 ml water) CRD were produced in awake rats and colorectal pain were assessed by measuring the abdominal withdrawal response (AWR) after i.p. administration of saline or Na2S, a donor of H2S (100 mMol/kg). Spinal cFos expression was also detected. To determine whether the opioid system was involved in the H2S -induced antinociception, pre-treatment with non-selective opioid antagonist naloxone (0.5 mg/kg i.p.) or selective antagonists of μ(MOR) (CTAP, 0.09 mg/kg), k (KOR) (GNTI, 0.08 mg/kg) and δ (DOR) (NTI, 4 mg/kg) opioid receptors or antisense oligodeoxynucleotides directed against specific exons of the DOR, KOR and MOR (10 μg each probe/10 μl saline). i.c.v. administered were performed and the AWR score detected. The H2S (50 μM) In Vitro effects of H2S on MOR phosphorylation, β-arrestins recruitment and MOR internalization were determined in the neuronal cell line SKNMC and compared with those obtained with the MOR agonist DAMGO (1 μ). The effects of H2S and DAMGO on PI3K/AKT pathway was also detected. Results. H2S significantly inhibited the CRD-induced pain and spinal cFos overexpression. The H2S-induced analgesia was significantly reverted by naloxone, CTAP and antisense oligodeoxynucleotides against MOR, while both DOR and KOR inhibition appeared less effective. In the SKNMC cells, both H2S and DAMGO caused the timedependent MOR phosphorylation on serine 377 and the co-immunoprecipitation between MOR and β-arrestin, indicating that H2S induced the interaction of b-arrestins with MOR. Finally, plasma membrane fraction and immunoflurescence staining demonstrated that both DAMGO and H2S caused the translocation of MOR from the plasma membrane to the cytoplasma and that MOR internalization was inhibited by pre-treating cells with CTAP (1 μM). Both DAMGO and H2S induced AKT phosphorylation. CTAP inhibited the DAMGOinduced AKT phosphorylation but not those induced by H2S. Finally, pre-treating neuronal cells with the PI3K inhibitor LY294002 caused the blockade of H2S-induced MOR internalization. Conclusions. This study provides evidence that: 1) 2Sinhibits CRD-induced pain by interacting with peripheral and central MOR; 2) H2S causes MOR phosphorylation, the association between MOR and β-arrestins and MOR internalization; 3) H2S induces AKT phosphorylation; 4) inhibition of PI3K/AKT pathway results in the blockade of H2S-induced MOR internalization.

Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA

Humanin (HN) was originally identified as an endogenous peptide that protects neuronal cells from apoptosis by mutant Alzheimer's disease genes. This 24-residue peptide has been recently shown to suppress apoptosis by interfering with activation of Bcl-2-associated X protein (Bax) in cytosol. In the present study, we showed that HN increases ATP levels in human lymphocytes, muscular TE671 cells, and neural SKN-MC cells, and protects these cells from serum deprivation-induced apoptosis. The suppressed apoptotic death of serum-deprived cells would be explained by the anti-Bax effect of HN; however, HN also increased ATP levels of serum-supplemented cells (non-apoptotic cells), in which Bax is likely to be inactive. This result suggests the presence of a certain mechanism independent of Bax inactivation to increase ATP levels of cells under non-apoptotic condition. By treatment with HN, the ATP levels of lymphocytes from patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) associated with A3243G mutant mtDNA were increased as well, suggesting that HN is able to prevent cells in MELAS from falling into ATP deficiency. Our quantitative PCR findings indicated that the HN-induced increase in ATP may not be a consequence of mitochondrial proliferation, because HN rather suppressed mtDNA replication. This suppression may be important in the treatment of affected cells in MELAS, since the mutant mtDNAs that increase during compensatory mtDNA replication for ATP deficiency cause excessive formation of reactive oxygen species, leading to further energy crisis. We thus propose that HN, which increases cellular ATP levels without inducing mtDNA replication, may be suited for the treatment of MELAS.

Cellular characteristics of neuroblastoma cells: regulation by the ELR −-CXC chemokine CXCL10 and expression of a CXCR3-like receptor

Bone marrow stroma cells secrete the chemokine CXCL12 that may support bone marrow metastasis formation by neuroblastoma cells. The present study demonstrates that bone marrow stroma cell lines also secrete CXCL10, a chemokine that was shown in the past to have anti-malignancy functions. A receptor recognized by antibodies against CXCR3 was shown to be expressed by six neuroblastoma cell lines. Further detailed analysis was performed on the NUB6 and SK-NMC neuroblastoma cells, showing that CXCL10 induced potent Erk phosphorylation in a G αi-dependent manner. The role of a CXCR3-like receptor in Erk phosphorylation was substantiated by the ability of CXCL11, another potent CXCR3 ligand, to induce Erk phosphorylation in the NUB6 and SK-NMC cells. Further characterization of CXCL10 activities indicated that CXCL10 partly inhibited the growth of the NUB6 and SK-NMC cells. Both NUB6 and SK-NMC cells did not migrate to CXCL10, although their migratory machinery was intact, as evidenced by their migration to bone marrow constituents. Altogether, these results suggest that CXCL10 interacts with a CXCR3-like receptor in neuroblastoma cell lines, raising the possibility that following the homing of the tumor cells to the bone marrow (through a CXCL10-independent mechanism), CXCL10 may partly inhibit neuroblastoma cell growth at this site.

Photolysis of intracellular caged sphingosine-1-phosphate causes Ca 2+ mobilization independently of G-protein-coupled receptors

Sphingosine-1-phosphate (S1P), the product of sphingosine kinase, activates several widely expressed G-protein-coupled receptors (GPCR). S1P might also play a role as second messenger, but this hypothesis has been challenged by recent findings. Here we demonstrate that intracellular S1P can mobilize Ca 2+ in intact cells independently of S1P-GPCR. Within seconds, S1P generated by the photolysis of caged S1P raised the intracellular free Ca 2+ concentration in HEK-293, SKNMC and HepG2 cells, in which the response to extracellularly applied S1P was either blocked or absent. Ca 2+ transients induced by photolysis of caged S1P were caused by Ca 2+ mobilization from thapsigargin-sensitive stores. These results provide direct evidence for a true intracellular action of S1P.