Autophagy In Neuroblastoma Cells Research Articles

Piceatannol induces caspase-dependent apoptosis by modulating intracellular reactive oxygen species/mitochondrial membrane potential and enhances autophagy in neuroblastoma cells.

The aim of this study was to evaluate the anticancer effects of piceatannol, a natural stilbenoid, on human neuroblastoma cells. In order to accomplish this goal, we performed various cellular assays, including the XTT cell proliferation assay for cell viability, colony formation assay for colony formation capacity, FITC Annexin V and cell death detection kit for apoptosis, matrigel invasion assay for invasion capacity, intracellular reactive oxygen species (ROS) red dye for intracellular ROS levels, TMRM staining method for mitochondrial membrane potential (MMP), and the CYTO-ID autophagy detection kit for autophagy. Furthermore, we analyzed the expression levels of genes associated with apoptosis and autophagy using RT-qPCR. Based on our findings, piceatannol exhibited cytotoxic effects on neuroblastoma cells. Besides, treatment with piceatannol at both 50 and 100 μM concentrations for 72 h decreased colony formation, induced apoptosis and autophagy, inhibited cell invasion, decreased MMP, and increased ROS levels in SH-SY5Y cells. In addition, we observed significant upregulation in the expression levels of CASP8, BECLIN, ATG5, ATG7, and MAPILC3A genes between the two doses. These results suggest that piceatannol enhances autophagic activity and induces caspase-dependent apoptosis, indicating its potential as a therapeutic agent against neuroblastoma cells.

Suppression of endoplasmic reticulum stress-dependent autophagy enhances cynaropicrin-induced apoptosis via attenuation of the P62/Keap1/Nrf2 pathways in neuroblastoma.

Autophagy has dual roles in cancer, resulting in cellular adaptation to promote either cell survival or cell death. Modulating autophagy can enhance the cytotoxicity of many chemotherapeutic and targeted drugs and is increasingly considered to be a promising cancer treatment approach. Cynaropicrin (CYN) is a natural compound that was isolated from an edible plant (artichoke). Previous studies have shown that CYN exhibits antitumor effects in several cancer cell lines. However, it anticancer effects against neuroblastoma (NB) and the underlying mechanisms have not yet been investigated. More specifically, the regulation of autophagy in NB cells by CYN has never been reported before. In this study, we demonstrated that CYN induced apoptosis and protective autophagy. Further mechanistic studies suggested that ER stress-induced autophagy inhibited apoptosis by activating the p62/Keap1/Nrf2 pathways. Finally, in vivo data showed that CYN inhibited tumour growth in xenografted nude mice. Overall, our findings suggested that CYN may be a promising candidate for the treatment of NB, and the combination of pharmacological inhibitors of autophagy may hold novel therapeutic potential for the treatment of NB. Our paper will contribute to the rational utility and pharmacological studies of CYN in future anticancer research.

Matrine induces autophagy in human neuroblastoma cells via blocking the AKT-mTOR pathway

Neuroblastoma (NB) is one of the most common malignant solid tumors in children. Despite significant advances in the treatment strategy, the long-term survival rate of NB patients is only 50%. Developing new agents for NB patients deserves attention. Recent research indicates that matrine, a natural quinolizidine alkaloid component extracted from the traditional Chinese medicine Sophora root, is widely used for various diseases, including antitumor effects against a variety of cancers. However, the effect of matrine on NB is unknown. Herein, we found that matrine exerted antiproliferative activity in human NB cells in dose- and time-dependent manner. Matrine triggered autophagy in NB cells by blocking the AKT-mTOR signaling pathway and suppressing the phosphorylation of AKT and mTOR. 3-Methyladenine (3-MA), a PI3K inhibitor, protected against matrine-induced inhibition of cell proliferation, further supporting that the antitumor activity of matrine was at least partly autophagy-dependent. In vivo, matrine reduced tumor growth of SK-N-DZ cells in a dose-dependent manner. Matrine treatment significantly declined the phosphorylation of AKT and mTOR and enhanced the LC3 II/GAPDH ratio in NB xenografts. Altogether, our work uncovered the molecular mechanism underlying matrine-induced autophagy in NB and provided implications for matrine as a potential therapeutic agent against NB.

Hsa_circ_0013401 Accelerates the Growth and Metastasis and Prevents Apoptosis and Autophagy of Neuroblastoma Cells by Sponging miR-195 to Release PAK2.

Background Increased levels of circRNAs have been identified in a variety of cancers. However, the specific functions and mechanisms of circRNAs in neuroblastoma (NB) have not been fully explored. Methods The levels of hsa_circ_0045997, hsa_circ_0080307, hsa_circ_0013401, hsa_circ_0077578, and microRNA-195 were confirmed by RT-qPCR in NB. Gain- and loss-of-function assays and rescue experiments were conducted to determine the influence of hsa_circ_0013401, miR-195, and P21-activated kinase 2 (PAK2) on the proliferation, apoptosis, autophagy, migration, and invasion of NB cells. Regulatory gene targets were validated by the luciferase assay. A xenograft mouse model was used to determine the in vivo effects of hsa_circ_0013401. Results hsa_circ_0013401 was highly expressed, miR-195 was lowly expressed, and there was a negative correlation between hsa_circ_0013401 and miR-195 in NB. The inhibitory effects of hsa_circ_0013401 knockdown suppressed the proliferation, migration, and invasion and induced the apoptosis and autophagy of NB cells by targeting miR-195 to downregulate PAK2 expression. Luciferase reporter assays showed that miR-195 was a direct target of hsa_circ_0013401, and PAK2 was the downstream target gene of miR-195. In vivo studies showed that hsa_circ_0013401 promotes tumor formation. Conclusions hsa_circ_0013401 induced NB progression through miR-195 to enhance PAK2. Therefore, we might highlight a novel regulatory axis (hsa_circ_0013401/miR-195/PAK2) in NB.

LncRNA NORAD accelerates the progression and doxorubicin resistance of neuroblastoma through up-regulating HDAC8 via sponging miR-144-3p

The dysregulation of non-coding RNAs (ncRNAs) often caused aberrant cell behaviors. In the present study, we focused on the role of long noncoding RNA (lncRNA) non-coding RNA activated by DNA damage (NORAD) in the development of neuroblastoma (NB). The enrichment of NORAD, miRNA-144-3p (miR-144-3p) and histone deacetylase 8 (HDAC8) was measured by quantitative real time polymerase chain reaction (qRT-PCR). The proliferation, chemoresistance, apoptosis, metastasis and autophagy of NB cells were determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, transwell migration and invasion assays and Western blot assay, respectively. The target relationship between miR-144-3p and NORAD or HDAC8 was predicted by Starbase software and validated through dual-luciferase reporter assay, RIP and RNA-pull down assays. The protein expression of HDAC8 was measured by Western blot assay. Murine xenograft model was used to verify the function of NORAD in vivo. We found that the level of NORAD was up-regulated in NB tissues and cells, and the level of NORAD was negatively correlated with the prognosis of NB patients. NORAD promoted the proliferation, metastasis and doxorubicin (DOX) resistance while inhibited the apoptosis and autophagy of NB cells. MiR-144-3p was a target of NORAD in NB cells, and NORAD accelerated the progression and DOX resistance of NB through sponging miR-144-3p. HDAC8 was a direct target of miR-144-3p in NB cells, and miR-144-3p suppressed the progression of NB through down-regulating HDAC8. NORAD up-regulated the expression of HDAC8 through sponging miR-144-3p in NB cells. NORAD accelerated the growth of NB tumors at least partly through miR-144-3p/HDAC8 signaling in vivo. In conclusion,NORAD promoted the progression and DOX resistance of NB through miR-144-3p/HDAC8 axis in vivo and in vitro.

Elimination of MYCN-Amplified Neuroblastoma Cells by Telomerase-Targeted Oncolytic Virus via MYCN Suppression

Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system. High-risk NB is characterized by MYCN amplification and human telomerase reverse transcriptase (hTERT) rearrangement, contributing to hTERT activation and a poor outcome. For targeting hTERT-activated tumors, we developed two oncolytic adenoviruses, OBP-301 and tumor suppressor p53-armed OBP-702, in which the hTERT promoter drives expression of the viral E1 gene for tumor-specific virus replication. In this study, we demonstrate the therapeutic potential of the hTERT-driven oncolytic adenoviruses OBP-301 and OBP-702 using four human MYCN-amplified NB cell lines (IMR-32, CHP-134, NB-1, LA-N-5) exhibiting high hTERT expression. OBP-301 and OBP-702 exhibited a strong antitumor effect in association with autophagy in NB cells. Virus-mediated activation of E2F1 protein suppressed MYCN expression. OBP-301 and OBP-702 significantly suppressed the growth of subcutaneous CHP-134 tumors. Thus, these hTERT-driven oncolytic adenoviruses are promising antitumor agents for eliminating MYCN-amplified NB cells via E2F1-mediated suppression of MYCN protein.

LncRNA SNHG7 enhances chemoresistance in neuroblastoma through cisplatin-induced autophagy by regulating miR-329-3p/MYO10 axis.

Neuroblastoma (NB) is a type of extracranial solid tumor that usually occurs in children. Drug resistance has become a major obstacle in NB chemotherapy. Long non-coding RNA small nucleolar RNA host gene 7 (SNHG7) is an oncogene in many cancers, including NB. This study aimed to investigate the role of SNHG7 in cisplatin sensitivity of NB and the underlying mechanism. Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability, and the IC50 of cisplatin was calculated. The protein levels of autophagy markers were measured by Western blot assay. The levels of SNHG7, miR-329-3p and myosin X (MYO10) were examined by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot assay. The interaction among SNHG7, miR-329-3p and MYO10 was confirmed by Dual-Luciferase reporter assay. Cisplatin curbed the viability of NB cells in a dose-dependent manner and facilitated autophagy in NB cells. Silencing of SNHG7 reduced cisplatin resistance and suppressed cisplatin-induced autophagy. SNHG7 was a sponge of miR-329-3p and modulated chemosensitivity and autophagy by regulating miR-329-3p. In addition, SNHG7 upregulated MYO10 by sponging miR-329-3p. MYO10 restored the effect of miR-329-3p on cisplatin sensitivity and autophagy. Moreover, suppression of autophagy blocked SNHG7-induced cisplatin resistance. Depletion of SNHG7 potentiated cisplatin sensitivity through inhibition of autophagy by modulating miR-329-3p/MYO10 axis, providing a new therapeutic approach to overcome cisplatin resistance in NB chemotherapy.

Inhibition of JNJ-26481585-mediated autophagy induces apoptosis via ROS activation and mitochondrial membrane potential disruption in neuroblastoma cells.

Neuroblastoma (NB) is the common pediatric tumor of the sympathetic nervous system characterized by poor prognosis. Owing to the challenges such as high tumor heterogeneity, multidrug resistance, minimal residual disease, etc., there is an immediate need for exploring new therapeutic strategies and effective treatments for NB. Herein, in the current study, we explored the unexplored response of NB cells to the second-generation histone deacetylase inhibitor (HDACi) JNJ-26481585(JNJ) and the lysosomotropic agent, Chloroquine (CQ) alone and upon JNJ/CQ treatment as a plausible therapeutic. We identify that while JNJ alone induced autophagy in NB cells, JNJ/CQ treatment decreased the viability and proliferation of NB cells in vitro by switching from autophagy to apoptosis. Further we found that autophagy inhibition by CQ pre-treatment led to the generation of ROS and a decrease in the mitochondrial membrane potential (MMP) that subsequently caused caspase-3-mediated apoptotic cell death in NB cells. Corroborating the above observations, we found that the ROS scavenger N-acetylcysteine (NAC) countered caspase-3 activity and the cells were rescued from apoptosis. Finally, these observations establish that JNJ/CQ treatment resulted in cell death in NB cells by triggering the formation of ROS and disruption of MMP, suggesting that modulation of JNJ-induced autophagy by CQ represents a promising new therapeutic approach in NB.

The dual role of BI 2536, a small-molecule inhibitor that targets PLK1, in induction of apoptosis and attenuation of autophagy in neuroblastoma cells.

Neuroblastoma (NB) is the most common extra-cranial solid tumor in childhood with the overall 5 years' survival less than 40%. Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase expressed during mitosis and over expressed in multiple cancers, including neuroblastoma. We found that higher PLK1 expression related to poor outcome of NB patients. BI2536, a small molecule inhibitor against PLK1, significantly reduced cell viability in a panel of NB cell lines, with IC50 less than 100 nM. PLK1 inhibition by BI 2536 treatment induced cell cycle arrest at G2/M phase and cell apoptosis in NB cells. Realtime PCR array revealed the PLK1 inhibition related genes, such as BIRC7, TNFSF10, LGALS1 and DAD1 et al. Moreover, autophagy activity was investigated in the NB cells treated with BI 2536. BI 2536 treatment in NB cells increased LC3-II puncta formation and LC3-II expression. Formation of autophagosome induced by BI 2536 was observed by transmission electron microscopy. However, BI2536 abrogated the autophagic flux in NB cells by reducing SQSTM1/p62 expression and AMPKαT172 phosphorylation. These results provide new clues for the molecular mechanism of cell death induced by BI 2536 and suggest that BI 2536 may act as new candidate drug for neuroblastoma.

Long Non Coding RNA SNHG16 Facilitates Proliferation, Migration, Invasion and Autophagy of Neuroblastoma Cells via Sponging miR-542-3p and Upregulating ATG5 Expression.

BackgroundNeuroblastoma (NB) is a heterogeneous pediatric malignant tumor with many biological and clinical characteristics. Long non-coding RNA small nucleolar RNA host gene 16 (SNHG16) plays vital role in the development of NB. However, the potential mechanism of SNHG16 in the progression of NB is rarely reported.MethodsThe expression levels of SNHG16, miR-542-3p and autophagy-related gene 5 (ATG5) were measured with quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation, migration and invasion of NB cells were determined using 3-(4, 5-dimethylthiazol-2-YI)-2, 5-diphenyltetrazolium bromide (MTT) or transwell assay. Protein levels of ATG5, microtubule-associated protein A1/1B-light chain3 (LC3-I/II) and p62 were detected by Western blot analysis. The interaction between miR-542-3p and SNHG16 or ATG5 was predicted by starBase and confirmed by dual luciferase reporter assay. Xenograft mice models were constructed to confirm the role of SNHG16 in vivo.ResultsSNHG16 was upregulated in NB tissues and cells and associated with clinical stage and poor prognosis of NB. Knockdown of SNHG16 impeded proliferation, migration, invasion and autophagy of NB cells in vitro, and suppressed tumor growth in vivo. Interestingly, SNHG16 mediated ATG5 expression through sponging miR-542-3p in NB cells. Moreover, miR-542-3p downregulation reversed the inhibitory effects of SNHG16 silencing on proliferation, migration, invasion and autophagy of NB cells. Besides, ATG5 overturned the regulatory effects on proliferation, migration, invasion and autophagy of NB cells induced by SNHG16 or miR-542-3p knockdown.ConclusionSNHG16 facilitated proliferation, migration, invasion and autophagy of NB cells via sponging miR-542-3p and upregulating ATG5 expression in NB.

GX15\u2013070 (Obatoclax), a Bcl-2 family proteins inhibitor engenders apoptosis and pro-survival autophagy and increases Chemosensitivity in neuroblastoma

BackgroundNeuroblastoma (NB) is a frequent pediatric tumor associated with poor prognosis. The disregulation of Bcl-2, an anti-apoptotic protein, is crucial for the tumoral development and chemoresistance. Autophagy is also implicated in tumor cell survival and chemoresistance. The aim of our study was to demonstrate therapeutic efficiency of GX 15–070, a pan-Bcl-2 family inhibitor, used alone and in combination with conventional drugs or with hydroxychloroquine (HCQ), an autophagy inhibitor.MethodsFive neuroblastoma cell lines were tested for the cytotoxic activity of GX 15–070 alone or in combination with cisplatin, doxorubicin, HCQ or Z-VAD-FMK a broad-spectrum caspase inhibitor. Apoptosis and autophagy levels were studied by western-blot and FACS. Orthotopic injections were performed on NOD/LtSz-scid/IL-2Rgamma null mice that were treated with either GX 15–070 alone or in combination with HCQ.ResultsSynergistic cytotoxicity was observed for the drug combination in all of the 5 neuroblastoma cell lines tested, including MYCN amplified lines and in cancer stem cells. GX 15–070 significantly increased apoptosis and autophagy in neuroblastoma cells as evidenced by increased levels of the autophagy marker, LC3-II. Inhibition of autophagy by HCQ, further increased the cytotoxicity of this combinatorial treatment, suggesting that autophagy induced by these agent plays a cytoprotective role. In vivo, GX 15–070 combined with HCQ significantly decreased the growth of the tumor and the number of distant metastases.ConclusionsBased on the synergistic effect of HCQ and GX 15–070 observed in this study, the combination of these two drugs may be utilized as a new therapeutic approach for neuroblastoma.

Abstract 2207: The dual role of BI 2536, a small-molecule inhibitor that targets PLK1, in induction of apoptosis and attenuation of autophagy in neuroblastoma cells

Abstract Neuroblastoma(NB) is the most common extra-cranial solid tumor in childhood with the overall survival less than 40%. Polo-like kinase 1 (PLK1) is a serine/threonine-protein kinase expressed during mitosis and overexpressed in multiple cancers, including neuroblastoma. We found the PLK1 expression related to the outcome of NB patients. BI2536, a small molecule inhibitor against PLK1, significantly reduced cell viability in a panal of NB cell lines, with IC50 less than 100 nM. PLK1 inhibition by BI 2536 treatment induced cell cycle arrest at G2/M phase and cell apoptosis in NB cells. Realtime PCR array revealed the PLK1-regulated genes, such as BIRC7, TNFSF10, LGALS1 and DAD1 et al. Moreover, autophagy activity was investigated in the NB cells treated with BI 2536. BI 2536 treatment in NB cells increased LC3-II puncta formation and LC3-II expression. Formation of autophagosome induced by BI 2536 was observed by TEM. However, BI2536 abrogated the autophagic flux in NB cells by reducing SQSTM1/p62 expression and AMPKαT172 phosphorylation. These results provide new clues for the molecular mechanism of cell death induced by BI 2536 and suggest that BI 2536 may act as new candidate drug for neuroblastoma. Citation Format: Zhiheng Li. The dual role of BI 2536, a small-molecule inhibitor that targets PLK1, in induction of apoptosis and attenuation of autophagy in neuroblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2207.

MiR-34a inhibits progression of neuroblastoma by targeting autophagy-related gene 5

Neuroblastoma (NB) is a common pediatric malignancy with high mortality in childhood. Although many attentions have been gained, novel biomarkers for NB diagnosis and prognosis are still needed. microRNAs (miRNAs) played important roles in NB progression and miR-34a is a tumor suppressor in NB. However, the mechanism that underlies miR-34a regulating proliferation, migration, invasion and autophagy in NB remains poorly understood. In this study, cell proliferation was investigated by MTT and colony assay. Cell apoptosis was measured by caspase 3 activity assay. Cell migration and invasion were detected by trans-well analysis. Autophagy was measured via GFP-LC3 puncta fluorescence assay and western blots (WB). The expression of miR-34a was examined by quantitative real-time PCR (qRT-PCR). The regulatory effect of miR-34a on autophagy-related gene 5 (ATG5) was detected by qRT-PCR and WB. The interaction between miR-34a and ATG5 was probed by luciferase activity and RNA immunoprecipitation (RIP) assay. Results showed that miR-34a expression was inhibited in NB tissues and cells with low survival rate. Addition of miR-34a suppressed cell proliferation, migration, invasion and autophagy but promoted apoptosis in NB cells, whereas miR-34a deficiency played opposite roles in NB progression. Intriguingly, ATG5 was directly targeted by miR-34a. Moreover, ATG5 restoration attenuated miR-34a-mediated inhibitory effect on proliferation, apoptosis, migration, invasion and autophagy. These results indicated miR-34a suppressed proliferation, apoptosis, migration, invasion and autophagy in NB cells by targeting ATG5, providing a novel therapeutic avenue for NB treatment.

Knockdown of galectin-1 facilitated cisplatin sensitivity by inhibiting autophagy in neuroblastoma cells

Neuroblastoma (NB) is a type of solid extracranial tumor that usually occurs in babies and children. Chemotherapy is a common method for NB treatment, however, the drug resistance exerts during the chemotherapy of NB. Galectin-1 is a member of galectin family and plays a potent role in the development of chemotherapy and radiotherapy resistance. However, the effect of galectin-1 on cisplatin resistance in NB remains unknown. The present study aimed to investigate the role of galectin-1 in cisplatin resisitance and the potential mechanism. Human neuroblastoma SH-SY5Y and SK-N-SH cells were treated with cisplatin and/or galectin-1/siRNA targeting galectin-1 (si-Gal-1). The cell viability was measured by MTT assay. The IC50 values for cisplatin of neuroblastoma cells were calculated. The expression levels of autophagy markers including microtubule-associated protein light chain 3 (LC3B), Beclin-1, and p62 were detected by western blot. We found that cisplatin inhibited cell viability of SH-SY5Y and SK-N-SH in a dose-dependent manner. Cisplatin induced the ratio of LC3B-II/LC3B-I and Beclin-1 expression, and inhibited the p62 expression. Knockdown of galectin-1 decreased the IC50 for cisplatin of SH-SY5Y and SK-N-SH cells and inhibited cisplatin-induced autophagy. Moreover, inhibition of autophagy suppressed galectin-1-induced increase in IC50 for cisplatin. In conclusion, galectin-1 knockdown enhanced cisplatin sensitivity of neuroblastoma cells by inhibiting autophagy. The findings might provid a novel therapeutic target to overcome cisplatin resistance in chemotherapy of NB.

Sphingadienes show therapeutic efficacy in neuroblastoma in vitro and in vivo by targeting the AKT signaling pathway.

Neuroblastoma is a childhood malignancy that accounts for approximately 15% of childhood cancer deaths. Only 20-35% of children with metastatic neuroblastoma survive with standard therapy. Identification of more effective therapies is essential to improving the outcome of children with high-stage disease. Sphingadienes (SD) are growth-inhibitory sphingolipids found in natural sources including soy. They exhibit chemopreventive activity in mouse models of colon cancer, where they mediate cytotoxicity by inhibiting key pro-carcinogenic signaling pathways. In this study, the effect of SD on neuroblastoma was analyzed. Low micromolar concentrations of SD were cytotoxic to transformed and primary neuroblastoma cells independently of N-Myc amplification status. SD induced both caspase-dependent apoptosis and autophagy in neuroblastoma cells. However, only inhibition of caspase-dependent apoptosis protected neuroblastoma cells from SD-mediated cytotoxicity. SD also inhibited AKT activation in neuroblastoma cells as shown by reduced phosphorylated AKT levels. Pre-treatment with insulin attenuated SD-mediated cytotoxicity in vitro. SD-loaded nanoparticles (NP) administered parenterally to immunodeficient mice carrying neuroblastoma xenografts resulted in cytotoxic levels of SD in the circulation and significantly reduced tumor growth compared to vehicle-treated controls. Analysis of tumor extracts demonstrated reduced AKT activation in tumors of mice treated with SD-NP compared to controls treated with empty NP. Our findings indicate SD are novel potential chemotherapeutic agents that promote neuroblastoma cell death and reduce tumorigenicity in vivo.

The protective autophagy activated by GANT-61 in MYCN amplified neuroblastoma cells is mediated by PERK.

The proto-oncogene MYC can trigger the unfolded protein response (UPR). The double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK), one of three primary branches of the UPR, is a key regulator of autophagy, promoting tumorigenesis. Upon activation of PERK, there is an increase in phosphorylation of the eukaryotic initiation factor-2 alpha (eIF2α), which in turn, activates the transcription factor-4 (ATF4), responsible for an increased expression of LC3, a common autophagy marker. PERK is repressed upon GLI1 and GLI2 induction. GANT-61 is an inhibitor of GLI1 and GLI2, known to reduce autophagy in MYCN non-amplified, but not in MYCN amplified neuroblastoma (NB) cells. In our study, we tested the effect of the joint administration of a PERK inhibitor (GSK2606414) and the GLI inhibitor GANT-61 to MYCN amplified and MYCN non-amplified NB cells. Our results suggest that inhibition of PERK impairs GANT-61 induced autophagy in NB cells with MYCN amplification, but had no effect on the MYCN non-amplified NB cells. In summary, PERK seems to be a good therapeutic target for NB. Inhibition of PERK reduces autophagy in MYCN amplified NB cells, thus amplifying the efficacy of the GLI inhibitor GANT-61 in reducing proliferation of this type of cancer cells.

Lysine-specific demethylase LSD1 regulates autophagy in neuroblastoma through SESN2-dependent pathway

Autophagy is a physiological process, important for recycling of macromolecules and maintenance of cellular homeostasis. Defective autophagy is associated with tumorigenesis and has a causative role in chemotherapy resistance in leukemia and in solid cancers. Here, we report that autophagy is regulated by the lysine-specific demethylase LSD1/KDM1A, an epigenetic marker whose overexpression is a feature of malignant neoplasia with an instrumental role in cancer development. In the present study, we determine that two different LSD1 inhibitors (TCP and SP2509) as well as selective ablation of LSD1 expression promote autophagy in neuroblastoma cells. At a mechanistic level, we show that LSD1 binds to the promoter region of Sestrin2 (SESN2), a critical regulator of mTORC1 activity. Pharmacological inhibition of LSD1 triggers SESN2 expression that hampers mTORC1 activity, leading to enhanced autophagy. SESN2 overexpression suffices to promote autophagy in neuroblastoma cells, while loss of SESN2 expression reduces autophagy induced by LSD1 inhibition. Our findings elucidate a mechanism whereby LSD1 controls autophagy in neuroblastoma cells through SESN2 transcription regulation, and we suggest that pharmacological targeting of LSD1 may have effective therapeutic relevance in the control of autophagy in neuroblastoma.

Suberoylanilide hydroxamic acid sensitizes neuroblastoma to paclitaxel by inhibiting thioredoxin-related protein 14-mediated autophagy.

Paclitaxel is not as effective for neuroblastoma as most of the front‐line chemotherapeutics due to drug resistance. This study explored the regulatory mechanism of paclitaxel‐associated autophagy and potential solutions to paclitaxel resistance in neuroblastoma. The formation of autophagic vesicles was detected by scanning transmission electron microscopy and flow cytometry. The autophagy‐associated proteins were assessed by western blot. Autophagy was induced and the autophagy‐associated proteins LC3‐I, LC3‐II, Beclin 1, and thioredoxin‐related protein 14 (TRP14), were found to be upregulated in neuroblastoma cells that were exposed to paclitaxel. The inhibition of Beclin 1 or TRP14 by siRNA increased the sensitivity of the tumor cells to paclitaxel. In addition, Beclin 1‐mediated autophagy was regulated by TRP14. Furthermore, the TRP14 inhibitor suberoylanilide hydroxamic acid (SAHA) downregulated paclitaxel‐induced autophagy and enhanced the anticancer effects of paclitaxel in normal control cancer cells but not in cells with upregulated Beclin 1 and TRP14 expression. Our findings showed that paclitaxel‐induced autophagy in neuroblastoma cells was regulated by TRP14 and that SAHA could sensitize neuroblastoma cells to paclitaxel by specifically inhibiting TRP14.

Cordycepin induces apoptosis and autophagy in human neuroblastoma SK-N-SH and BE(2)-M17 cells

Cordycepin, also termed 3'-deoxyadenosine, is a derivative of the nucleoside adenosine that represents a potential novel class of anticancer drugs targeting the 3' untranslated region of RNAs. Cordycepin has been reported to induce apoptosis in certain cancer cell lines, but the effects of cordycepin on human neuroblastoma cells have not been studied. In the present study, an MTT assay revealed that cordycepin inhibits the viability of neuroblastoma SK-N-SH and BE(2)-M17 cells in a dose-dependent manner. In addition, cordycepin increases the early-apoptotic cell population of SK-N-SH cells, as determined by fluorescence-activated cell sorting analysis. The induction of apoptosis in neuroblastoma cells by cordycepin was further confirmed by western blotting, which revealed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase 1 in the SK-N-SH and BE(2)-M17 cells. Cordycepin also induced the formation of a punctate pattern of light-chain 3 (LC3)-associated green fluorescence in the SK-N-SH cells transfected with a pEGFP-LC3 vector. Furthermore, western blotting revealed cleavage of LC3 A/B in cordycepin-treated neuroblastoma SK-N-SH cells. Taken together, the results indicate that cordycepin significantly increases apoptosis and autophagy in neuroblastoma cells, and may therefore be a drug candidate for neuroblastoma therapy, but requires additional evaluation.

Inhibition of autophagy potentiates the efficacy of Gli inhibitor GANT-61 in MYCN-amplified neuroblastoma cells.

BackgroundAberrant Hedgehog (Hh) signaling is often associated with neuroblastoma (NB), a childhood malignancy with varying clinical outcomes due to different molecular characteristics. Inhibition of Hh signaling with small molecule inhibitors, particularly with GANT-61, significantly suppresses NB growth. However, NB with MYCN amplification is less sensitive to GANT-61 than those without MYCN amplification.MethodsAutophagic process was examined in two MYCN amplified and two MYCN non-amplified NB cells treated with GANT-61. Subsequently, chemical and genetic approaches were applied with GANT-61 together to evaluate the role of autophagy in GANT-61 induced cell death.ResultsHere we show that GANT-61 enhanced autophagy in MYCN amplified NB cells. Both an autophagic inhibitor 3-methyladenine (3-MA) and genetic disruption of ATG5 or ATG7 expression suppressed GANT-61 induced autophagy and significantly increased apoptotic cell death, whereas pre-treatment with an apoptotic inhibitor, Z-VAD-FMK, rescued GANT-61 induced cell death and had no effect on the autophagic process. In the other hand, GANT-61 barely induced autophagy in MYCN non-amplified NB cells, but overexpression of MYCN in MYCN non-amplified NB cells recapitulated GANT-61 induced autophagy seen in MYCN amplified NB cells, suggesting that the level of GANT-61 induced autophagy in NB cells is related to MYCN expression level in cells.ConclusionAberrant Hh signaling activation as an oncogenic driver in NB renders inhibition of Hh signaling an effective measure to suppress NB growth. However, our data suggest that enhanced autophagy concomitant with Hh signaling inhibition acts as a pro-survival factor to maintain cell viability, which reduces GANT-61 efficacy. Besides, MYCN amplification is likely involved in the induction of the pro-survival autophagy. Overall, simultaneous inhibition of both Hh signaling and autophagy could be a better way to treat MYCN amplified NB.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-768) contains supplementary material, which is available to authorized users.