GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy

Recent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.     

Targeting HMGB1-mediated autophagy as a novel therapeutic strategy for osteosarcoma

Autophagy is a catabolic process critical to maintaining cellular homeostasis and responding to cytotoxic insult. Autophagy is recognized as "programmed cell survival" in contrast to apoptosis or programmed cell death. Upregulation of autophagy has been observed in many types of cancers and has been demonstrated to both promote and inhibit antitumor drug resistance depending to a large extent on the nature and duration of the treatment-induced metabolic stress as well as the tumor type. Cisplatin, doxorubicin and methotrexate are commonly used anticancer drugs in osteosarcoma, the most common form of childhood and adolescent cancer. Our recent study demonstrated that high mobility group box 1 protein (HMGB1)-mediated autophagy is a significant contributor to drug resistance in osteosarcoma cells. Inhibition of both HMGB1 and autophagy increase the drug sensitivity of osteosarcoma cells in vivo and in vitro. Furthermore, we demonstrated that the ULK1-FIP200 complex is required for the interaction between HMGB1 and BECN1, which then promotes BECN1-PtdIns3KC3 complex formation during autophagy. Thus, these findings provide a novel mechanism of osteosarcoma resistance to therapy facilitated by HMGB1-mediated autophagy and provide a new target for the control of drug-resistant osteosarcoma patients.     

Targeting HMGB1-mediated autophagy as a novel therapeutic strategy for osteosarcoma

Autophagy is a catabolic process critical to maintaining cellular homeostasis and responding to cytotoxic insult. Autophagy is recognized as “programmed cell survival” in contrast to apoptosis or programmed cell death. Upregulation of autophagy has been observed in many types of cancers and has been demonstrated to both promote and inhibit antitumor drug resistance depending to a large extent on the nature and duration of the treatment-induced metabolic stress as well as the tumor type. Cisplatin, doxorubicin and methotrexate are commonly used anticancer drugs in osteosarcoma, the most common form of childhood and adolescent cancer. Our recent study demonstrated that high mobility group box 1 protein (HMGB1)-mediated autophagy is a significant contributor to drug resistance in osteosarcoma cells. Inhibition of both HMGB1 and autophagy increase the drug sensitivity of osteosarcoma cells in vivo and in vitro. Furthermore, we demonstrated that the ULK1-FIP200 complex is required for the interaction between HMGB1 and BECN1, which then promotes BECN1-PtdIns3KC3 complex formation during autophagy. Thus, these findings provide a novel mechanism of osteosarcoma resistance to therapy facilitated by HMGB1-mediated autophagy and provide a new target for the control of drug-resistant osteosarcoma patients.     

Manganese activates autophagy to alleviate endoplasmic reticulum stress–induced apoptosis via PERK pathway

Overexposure to manganese (Mn) is neurotoxic. Our previous research has demonstrated that the interaction of endoplasmic reticulum (ER) stress and autophagy participates in the early stage of Mn‐mediated neurotoxicity in mouse. However, the mechanisms of ER stress signalling pathways in the initiation of autophagy remain confused. In the current study, we first validated that ER stress–mediated cell apoptosis is accompanied by autophagy in SH‐SY5Y cells. Then, we found that inhibiting ER stress with 4‐phenylbutyrate (4‐PBA) decreased ER stress–related protein expression and reduced cell apoptosis, whereas blocking autophagy with 3‐methyladenine (3‐MA) increased cell apoptosis. These data indicate that protective autophagy was activated to alleviate ER stress–mediated apoptosis. Knockdown of the protein kinase RNA‐like ER kinase (PERK) gene inhibited Mn‐induced autophagy and weakened the interaction between ATF4 and the LC3 promoter. Our results reveal a novel molecular mechanism in which ER stress may regulate autophagy via the PERK/eIF2α/ATF4 signalling pathway. Additionally, Mn may activate protective autophagy to alleviate ER stress–mediated apoptosis via the PERK/eIF2α/ATF4 signalling pathway in SH‐SY5Y cells.     

Oncogenic gene TRIM10 confers resistance to cisplatin in osteosarcoma cells and activates the NF-κB signaling pathway

Deregulation of tripartite motif (TRIM) family proteins contribute to multiple biological processes such as neurodegeneration, development, inflammation, cell survival, apoptosis, and carcinogenesis. However, the biological function and molecular mechanism of TRIM family proteins in osteosarcoma chemoresistance remain unclear. In the current study, we found the protein expression of TRIM10 was markedly overexpressed in cisplatin resistance's osteosarcoma tissues and TRIM10 overexpression was inversely correlated with osteosarcoma patient survival. Furthermore, overexpression of TRIM10 confers cisplatin resistance on osteosarcoma cells; however, repressing TRIM10 sensitized osteosarcoma cell lines to cisplatin cytotoxicity in vitro. Mechanically, TRIM10 upregulated the nuclear levels of p65, thereby activating canonical NF-κB signaling. Taken together, our results suggest that TRIM10 contributed to cisplatin resistance in osteosarcoma cells, and targeting the TRIM10/p65 axis may represent a promising strategy to enhance cisplatin response in osteosarcoma patients with chemoresistance.     

Inactivated Sendai virus strain Tianjin induces apoptosis and autophagy through reactive oxygen species production in osteosarcoma MG-63 cells

Sendai virus strain Tianjin, a novel genotype of Sendai virus, has been proven to possess potent antitumor effect on certain cancer cell types although inactivated by ultraviolet (UV). This study was carried out to investigate the in vitro anticancer properties of UV-inactivated Sendai virus strain Tianjin (UV-Tianjin) on human osteosarcoma cells and the underlying molecular mechanism. Our studies demonstrated UV-Tianjin significantly inhibited the viability of human osteosarcoma cell lines and triggered apoptosis through activation of both extrinsic and intrinsic pathways in MG-63 cells. Meanwhile, autophagy occurred in UV-Tianjin-treated cells. Blockade of autophagy with 3-methyladenine remarkably attenuated the inhibition of cell proliferation by UV-Tianjin, suggesting that UV-Tianjin-induced autophagy may be contributing to cell death. Furthermore, UV-Tianjin induced reactive oxygen species (ROS) production, which was involved in the execution of MG-63 cell apoptosis and autophagy, as evidenced by the result that treatment of N-acetyl-L-cysteine, a ROS scavenger, attenuated both apoptosis and autophagy. In addition, inhibition of apoptosis promoted autophagy, whereas suppression of autophagy attenuated apoptosis. Our results suggest that UV-Tianjin triggers apoptosis and autophagic cell death via generation of the ROS in MG-63 cells, which might provide important insights into the effectiveness of novel strategies for osteosarcoma therapy.     

TYRO3 promotes chemoresistance via increased LC3 expression in pancreatic cancer

Pancreatic cancer (PC) is an aggressive malignancy with few treatment options, and improved treatment strategies are urgently required. TYRO3, a member of the TAM receptor tyrosine kinase family, is a known oncogene; however, the relationship between TYRO3 expression and PC chemoresistance remains to be elucidated. We performed gain- and loss-of-function experiments on TYRO3 to examine whether it is involved in chemoresistance in PC cells. TYRO3 knockdown decreased cell viability and enhanced apoptosis following treatment of PC cells with gemcitabine and 5-fluorouracil (5-FU). In contrast, no such effects were observed in TYRO3-overexpressing PC cells. It is known that autophagy is associated with cancer chemoresistance. We then examined effects of TYRO3 on autophagy in PC cells. TYRO3 overexpression increased LC3 mRNA levels and induced LC3 puncta in PC cells. Inhibition of autophagy by chloroquine mitigated cell resistance to gemcitabine and 5-FU. In a xenograft mouse model, TYRO3 silencing significantly increased sensitivity of the cells to gemcitabine and 5-FU. To further investigate the involvement of autophagy in patients with PC, we immunohistochemically analyzed LC3 expression in the tissues of patients who underwent pancreatectomy and compared it with disease prognosis and TYRO3 expression. LC3 expression was negatively and positively correlated with prognosis and TYRO3 expression, respectively. Furthermore, LC3- and TYRO3-positive patients had a significantly worse prognosis among patients with PC who received chemotherapy after recurrence. These results indicated that the TYRO3-autophagy signaling pathway confers PC resistance to gemcitabine and 5-FU, and could be a novel therapeutic target to resolve PC chemoresistance.     

Icariside II enhances cisplatin-induced apoptosis by promoting endoplasmic reticulum stress signalling in non-small cell lung cancer cells

Although cisplatin is the most effective first-line drug in the management of advanced non-small cell lung cancer (NSCLC), drug resistance remains a major clinical challenge. There is increasing evidence that icariside II (IS) exhibits antitumour activity in a variety of cancers. In the current study, we investigated the anticancer effects of icariside II combined with cisplatin and elucidated the underlying mechanism in NSCLC. Here, we showed that cotreatment with IS and cisplatin inhibited cell proliferation and induced cellular apoptosis. Using mRNA sequencing (mRNA-seq), we identified differentially expressed genes (DEGs) in which there was an enrichment in PERK-mediated unfolded protein response (UPR) signalling. The western blot results revealed that IS activated endoplasmic reticulum (ER) stress, including three branches of UPR signalling, PERK, IRE1 and ATF6, and the downstream PERK-eIF2α-ATF4-CHOP pathway, thus potentiating the apoptosis induced by cisplatin. In addition, the combination of IS with cisplatin significantly reduced xenograft tumour growth in C57BL/6 and BALB/c nude mice in vivo. Notably, the combination therapy displayed no evident toxicity. Taken together, IS enhances cisplatin-induced apoptosis partially by promoting ER stress signalling in NSCLC, suggesting that combination treatment with IS and cisplatin is a novel potential therapeutic strategy for NSCLC.     

Mutation of kri1l causes definitive hematopoiesis failure via PERK-dependent excessive autophagy induction

Dysregulation of ribosome biogenesis causes human diseases, such as Diamond-Blackfan anemia, del (5q-) syndrome and bone marrow failure. However, the mechanisms of blood disorders in these diseases remain elusive. Through genetic mapping, molecular cloning and mechanism characterization of the zebrafish mutant cas002, we reveal a novel connection between ribosomal dysfunction and excessive autophagy in the regulation of hematopoietic stem/progenitor cells (HSPCs). cas002 carries a recessive lethal mutation in kri1l gene that encodes an essential component of rRNA small subunit processome. We show that Kri1l is required for normal ribosome biogenesis, expansion of definitive HSPCs and subsequent lineage differentiation. Through live imaging and biochemical studies, we find that loss of Kri1l causes the accumulation of misfolded proteins and excessive PERK activation-dependent autophagy in HSPCs. Blocking autophagy but not inhibiting apoptosis by Bcl2 overexpression can fully rescue hematopoietic defects, but not the lethality of kri1l^cas002 embryos. Treatment with autophagy inhibitors (3-MA and Baf A1) or PERK inhibitor (GSK2656157), or knockdown of beclin1 or perk can markedly restore HSPC proliferation and definitive hematopoietic cell differentiation. These results may provide leads for effective therapeutics that benefit patients with anemia or bone marrow failure caused by ribosome disorders.     

Protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α pathway attenuates tumor necrosis factor alpha-induced apoptosis in nucleus pulposus cells by activating autophagy

Cellular loss induced by tumor necrosis factor alpha (TNF-α) contributes to the pathogenesis of intervertebral disc (IVD) degeneration. Cellular stress induced by TNF-α activates several processes to restore cell homeostasis. These processes include autophagy, endoplasmic reticulum stress, and related unfolded protein response (UPR). However, the effect and mechanism of UPR and autophagy regulated by TNF-α in IVD degeneration (IDD) remain unclear. The effect of autophagy on biological changes in nucleus pulposus cells (NPCs) also remains elusive. In this study, rat NPCs were cultured with TNF-α in the presence or absence of the UPR or autophagy pathway small-interfering RNAs. The associated genes and proteins were evaluated through immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses to monitor UPR and autophagy signaling and identify the regulatory mechanism of autophagy by the UPR pathway. Trypan blue exclusion assay, cell flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, qRT-PCR, and western blot analyses were performed to examine the apoptosis of NPCs. The results showed that the acute exposure of TNF-α induced the apoptosis of rat NPCs and activated the protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α (PERK/eIF2α) pathway of UPR and initiated autophagy. Silencing the PERK/eIF2α pathway or inhibiting autophagy enhanced the apoptosis of NPCs. Interference of the PERK/eIF2α pathway suppressed the autophagy of rat NPCs under TNF-α stimulation. Taken together, the PERK/eIF2α pathway reinforces the survival of NPCs under TNF-α stimulation by activating autophagy. Therefore, PERK/eIF2α-dependent autophagy could be a novel biological therapeutic target for IDD.     

Uncoupling Proteostasis and Development in Vitro with a Small Molecule Inhibitor of the Pancreatic Endoplasmic Reticulum Kinase,PERK

Loss-of-function mutations in EIF2AK3, encoding the pancreatic endoplasmic reticulum (ER) kinase, PERK, are associated with dysfunction of the endocrine pancreas and diabetes. However, to date it has not been possible to uncouple the long term developmental effects of PERK deficiency from sensitization to physiological levels of ER unfolded protein stress upon interruption of PERK modulation of protein synthesis rates. Here, we report that a selective PERK inhibitor acutely deregulates protein synthesis in freshly isolated islets of Langerhans, across a range of glucose concentrations. Acute loss of the PERK-mediated strand of the unfolded protein response leads to rapid accumulation of misfolded pro-insulin in cultured beta cells and is associated with a kinetic defect in pro-insulin processing. These in vitro observations uncouple the latent role of PERK in beta cell development from the regulation of unfolded protein flux through the ER and attest to the importance of the latter in beta cell proteostasis.     

Dendropanoxide Induces Autophagy through ERK1/2 Activation in MG-63 Human Osteosarcoma Cells and Autophagy Inhibition Enhances Dendropanoxide-Induced Apoptosis

Anticancer effects of dendropanoxide (DP) newly isolated from leaves and stem of Dendropanax morbifera Leveille were firstly investigated in this study. DP inhibited cell proliferation and induced apoptosis in dose- and time-dependent manner in MG-63 human osteosarcoma cells, which was dependent on the release of cytochrome c to the cytosol and the activation of caspases. Moreover, the DP-treated cells exhibited autophagy, as characterized by the punctuate patterns of microtubule-associated protein 1 light chain 3 (LC3) by confocal microscopy and the appearance of autophagic vacuoles by MDC staining. The expression levels of ATG7, Beclin-1 and LC3-II were also increased by DP treatment. Inhibition of autophagy by 3-methyladenine (3-MA) and wortmannin (Wort) significantly enhanced DP-induced apoptosis. DP treatment also caused a time-dependent increase in protein levels of extracellular signal-regulated kinase 1 and 2 (ERK1/2), and inhibition of ERK1/2 phosphorylation with U0126 resulted in a decreased DP-induced autophagy that was accompanied by an increased apoptosis and a decreased cell viability. These results indicate a cytoprotective function of autophagy against DP-induced apoptosis and suggest that the combination of DP treatment with autophagy inhibition may be a promising strategy for human osteosarcoma control. Taken together, this study demonstrated for the first time that DP could induce autophagy through ERK1/2 activation in human osteosarcoma cells and autophagy inhibition enhanced DP-induced apoptosis.     

Testis developmental related gene 1 regulates the chemosensitivity of seminoma TCam‐2 cells to cisplatin via autophagy

We previously identified testis developmental related gene 1 (TDRG1), a gene implicated in proliferation of TCam‐2 seminoma cells. Recent evidence has revealed that autophagy influences the chemosensitivity of cancer cells to chemotherapy. However, whether TDRG1 protein regulates autophagy in seminoma cells and influences their sensitivity to cis‐dichlorodiammine platinum (CDDP) remains unknown. In this study, we used TCam‐2 cells and male athymic BALB/c nude mice with xenografts of TCam‐2 cells to investigate autophagy, cell viability, apoptosis and the p110β/Rab5/Vps34 (PI3‐kinase Class III) pathway under the conditions of TDRG1 overexpression or knockdown and with or without CDDP treatment. We found that TDRG1 upregulation promoted autophagy in both TCam‐2 cells and seminoma xenografts via p110β/Rab5/Vps34 activation. Inhibition of autophagy reduced cell viability and promoted apoptosis during CDDP treatment of TCam‐2 cells. Similarly, TDRG1 knockdown inhibited autophagy, reduced cell viability and promoted apoptosis during CDDP treatment of TCam‐2 cells. TDRG1 knockdown inhibited tumour growth and promoted apoptosis in TCam‐2 cell xenografts, whereas TDRG1 overexpression had the opposite effect. According to these results, we propose that high expression of TDRG1 promotes autophagy through the p110β/Rab5/Vps34 pathway in TCam‐2 cells. TDRG1 overexpression promotes autophagy and leads to CDDP resistance, whereas TDRG1 knockdown inhibits autophagy and promotes chemosensitivity to CDDP both in vivo and in vitro. This study has uncovered a novel role of TDRG1 in reducing chemoresistance during CDDP treatment and provides potential therapeutic strategies for the treatment of human seminoma.     

Bufalin Induces the Interplay between Apoptosis and Autophagy in Glioma Cells through Endoplasmic Reticulum Stress

Malignant gliomas are common primary tumors of the central nervous system. The prognosis of patients with malignant glioma is poor in spite of current intensive therapy and thus novel therapeutic modalities are necessary. Bufalin is the major component of Chan-Su (a traditional Chinese medicine) extracts from the venom of Bufo gargarizan. In this study, we evaluated the growth inhibitory effect of bufalin on glioma cells and explored the underlying molecular mechanisms. Our results showed that bufalin inhibited the growth of glioma cells significantly. Mechanistic studies demonstrated that bufalin induced apoptosis through mitochondrial apoptotic pathway. In addition, bufalin was also found to induce ER stress-mediated apoptosis, which was supported by the up- regulation of ER stress markers, CHOP and GRP78, and augmented phosphorylation of PERK and eIF2α as well as cleavage of caspase-4. Downregulation of CHOP using siCHOP RNA attenuated bufalin-induced apoptosis, further confirming the role of ER stress response in mediating bufalin-induced apoptosis. Evidence of bufalin-induced autophagy included formation of the acidic vesicular organelles, increase of autophagolysosomes and LC3-II accumulation. Further experiments showed that the mechanism of bufalin-induced autophagy associated with ATP deleption involved an increase in the active form of AMPK, decreased phosphorylation levels of mTOR and its downstream targets 4EBP1 and p70S6K1. Furthermore, TUDC and silencing of eIF2α or CHOP partially blocked bufalin-induced accumulation of LC3-II, which indicated that ER stress preceded bufalin-induced autophagy and PERK/eIF2α/CHOP signaling pathway played a major part in the process. Blockage of autophagy increased expression of ER stress associated proteins and the ratio of apoptosis, indicating that autophagy played a cytoprotective role in bufalin induced ER stress and cell death. In conclusion, bufalin inhibits glioma cell growth and induces interplay between apoptosis and autophagy through endoplasmic reticulum stress. It will provide molecular bases for developing bufalin into a drug candidate for the treatment of maglinant glioma.     

Inhibition of autophagy and enhancement of endoplasmic reticulum stress increase sensitivity of osteosarcoma Saos‐2 cells to cannabinoid receptor agonist WIN55,212‐2

WIN55,212‐2, a cannabinoid receptor agonist, can activate cannabinoid receptors, which has proven anti‐tumour effects in several tumour types. Studies showed that WIN can inhibit tumour cell proliferation and induce apoptosis in diverse cancers. However, the role and mechanism of WIN in osteosarcoma are still unclear. In this study, we examined the effect of WIN55,212‐2 on osteosarcoma cell line Saos‐2 in terms of cell viability and apoptosis. Meanwhile, we further explored the role of endoplasmic reticulum stress and autophagy in apoptosis induced by WIN55,212‐2. Our results showed that the cell proliferation of Saos‐2 was inhibited by WIN55,212‐2 in a dose‐dependent and time‐dependent manner. WIN55,212‐2‐induced Saos‐2 apoptosis through mitochondrial apoptosis pathway. Meanwhile, WIN55,212‐2 can induce endoplasmic reticulum stress and autophagy in Saos‐2 cells. Inhibition of autophagy and enhancement of endoplasmic reticulum stress increased apoptosis induced by WIN55,212‐2 in Saos‐2 cells. These findings indicated that WIN55,212‐2 in combination with autophagic inhibitor or endoplasmic reticulum stress activator may shed new light on osteosarcoma treatment. Copyright © 2016 John Wiley & Sons, Ltd.     

Regulation of Autophagy Via PERK-eIF2α Effectively Relieve the Radiation Myelitis Induced by Iodine-125

Radiation myelitis is the most serious complication in clinical radiotherapy for spinal metastases. We previously showed that ¹²⁵I brachytherapy induced apoptosis of spinal cord neurons accompanied by autophagy. In this study, we further investigated the mechanism by which ¹²⁵I radiation triggered autophagy in neural cells. We found that autophagy induced by ¹²⁵I radiation was involved in endoplasmic reticulum (ER) stress and mainly dependent on PERK-eIF2α pathway. The expressions of LC3II, ATG12 and PI3K were significantly suppressed in PERK knockout neural cells. Meanwhile, the expressions of phosphorylated-Akt s473 and caspase3/8 all significantly increased in neural cells transfected with a PERK siRNA and which enhanced apoptosis of neurons after ¹²⁵I radiation. The results were consistent with that by MTT and Annexin-FITC/PT staining. In annimal model of banna pigs with radiation myelitis caused by ¹²⁵I brachytherapy, we have successfully decreased PERK expression by intrathecal administration of the lentivirus vector. The apoptosis rate was significantly higher than that in control group and which deteriorated radiation myelitis of banna pigs. Thus, autophagy caused by ¹²⁵I radiation was mainly as an attempt of cell survival at an early stage, but it would be a self-destructive process and promoted the process of apoptosis and necrosis radiated by ¹²⁵I for more than 72 hours. The study would be useful and helpful to maximize efficiency of radiation therapy in clinical therapy.