Ivermectin induces autophagy-mediated cell death through the AKT/mTOR signaling pathway in glioma cells

Abstract Glioma is one of the most common types of primary brain tumors. Ivermectin (IVM), a broad-spectrum antiparasitic drug, has been identified as a novel anticancer agent due to its inhibitory effects on the proliferation of glioma cells in vitro and in vivo. However, the ability of IVM to induce autophagy and its role in glioma cell death remains unclear. The main objective of the present study was to explore autophagy induced by IVM in glioma U251 and C6 cells, and the deep underlying molecular mechanisms. In addition, we examined the effects of autophagy on apoptosis in glioma cells. In the present study, transmission electron microscopy (TEM), immunofluorescence, Western blot and immunohistochemistry were used to evaluate autophagy activated by IVM. Cell viability was measured by 3-(4,5-dimethylthiazol2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and colony formation assay. The apoptosis rate was detected by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Meanwhile, autophagy inhibition was achieved by using chloroquine (CQ). U251-derived xenografts were established for examination of IVM-induced autophagy on glioma in vivo. Taken together, the results of the present study showed that autophagy induced by IVM has a protective effect on cell apoptosis in vitro and in vivo. Mechanistically, IVM induced autophagy through AKT/mTOR signaling and induced energy impairment. Our findings show that IVM is a promising anticancer agent and may be a potential effective treatment for glioma cancers.

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miR-182-5p and miR-378a-3p regulate ferroptosis in I/R-induced renal injury

Cell Death and Disease ◽

10.1038/s41419-020-03135-z ◽

2020 ◽

Vol 11 (10) ◽

Author(s):

Chenguang Ding ◽

Xiaoming Ding ◽

Jin Zheng ◽

Bo Wang ◽

Yang Li ◽

...

Keyword(s):

Cell Death ◽

Renal Injury ◽

Molecular Mechanisms ◽

Ischemia Reperfusion ◽

Kidney Injury ◽

Luciferase Reporter ◽

Renal Tubular Cell ◽

In Cells

Abstract Renal tubular cell death is the key factor of the pathogenesis of ischemia/reperfusion (I/R) kidney injury. Ferroptosis is a type of regulated cell death (RCD) found in various diseases. However, the underlying molecular mechanisms related to ferroptosis in renal I/R injury remain unclear. In the present study, we investigated the regulatory role of microRNAs on ferroptosis in I/R-induced renal injury. We established the I/R-induced renal injury model in rats, and H/R induced HK-2 cells injury in vitro. CCK-8 was used to measure cell viability. Fe2+ and ROS levels were assayed to evaluate the activation of ferroptosis. We performed RNA sequencing to profile the miRNAs expression in H/R-induced injury and ferroptosis. Western blot analysis was used to detect the protein expression. qRT-PCR was used to detect the mRNA and miRNA levels in cells and tissues. We further used luciferase reporter assay to verify the direct targeting effect of miRNA. We found that ischemia/reperfusion-induced ferroptosis in rat’s kidney. We identified that miR-182-5p and miR-378a-3p were upregulated in the ferroptosis and H/R-induced injury, and correlates reversely with glutathione peroxidases 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) expression in renal I/R injury tissues, respectively. In vitro studies showed that miR-182-5p and miR-378a-3p induced ferroptosis in cells. We further found that miR-182-5p and miR-378a-3p regulated the expression of GPX4 and SLC7A11 negatively by directly binding to the 3′UTR of GPX4 and SLC7A11 mRNA. In vivo study showed that silencing miR-182-5p and miR-378a-3p alleviated the I/R-induced renal injury in rats. In conclusion, we demonstrated that I/R induced upregulation of miR-182-5p and miR-378a-3p, leading to activation of ferroptosis in renal injury through downregulation of GPX4 and SLC7A11.

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Current translational potential and underlying molecular mechanisms of necroptosis

Cell Death and Disease ◽

10.1038/s41419-019-2094-z ◽

2019 ◽

Vol 10 (11) ◽

Cited By ~ 14

Author(s):

Tamás Molnár ◽

Anett Mázló ◽

Vera Tslaf ◽

Attila Gábor Szöllősi ◽

Gabriella Emri ◽

...

Keyword(s):

Cell Death ◽

Protein Kinase ◽

Posttranslational Modifications ◽

Molecular Mechanisms ◽

Inflammatory Diseases ◽

Immune Surveillance ◽

Kinase Domain ◽

Tumor Formation

Abstract Cell death has a fundamental impact on the evolution of degenerative disorders, autoimmune processes, inflammatory diseases, tumor formation and immune surveillance. Over the past couple of decades extensive studies have uncovered novel cell death pathways, which are independent of apoptosis. Among these is necroptosis, a tightly regulated, inflammatory form of cell death. Necroptosis contribute to the pathogenesis of many diseases and in this review, we will focus exclusively on necroptosis in humans. Necroptosis is considered a backup mechanism of apoptosis, but the in vivo appearance of necroptosis indicates that both caspase-mediated and caspase-independent mechanisms control necroptosis. Necroptosis is regulated on multiple levels, from the transcription, to the stability and posttranslational modifications of the necrosome components, to the availability of molecular interaction partners and the localization of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). Accordingly, we classified the role of more than seventy molecules in necroptotic signaling based on consistent in vitro or in vivo evidence to understand the molecular background of necroptosis and to find opportunities where regulating the intensity and the modality of cell death could be exploited in clinical interventions. Necroptosis specific inhibitors are under development, but >20 drugs, already used in the treatment of various diseases, have the potential to regulate necroptosis. By listing necroptosis-modulated human diseases and cataloging the currently available drug-repertoire to modify necroptosis intensity, we hope to kick-start approaches with immediate translational potential. We also indicate where necroptosis regulating capacity should be considered in the current applications of these drugs.

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Activation of autophagy during farnesyl pyrophosphate synthase inhibition is mediated through PI3K/AKT/mTOR signaling

Journal of International Medical Research ◽

10.1177/0300060519875371 ◽

2019 ◽

Vol 48 (4) ◽

pp. 030006051987537

Author(s):

Jie Han ◽

Chaoyang Huang ◽

Jiukun Jiang ◽

Dongmei Jiang

Keyword(s):

Molecular Mechanisms ◽

Cellular Proliferation ◽

Umbilical Vein ◽

Mtor Signaling ◽

Farnesyl Pyrophosphate ◽

Farnesyl Pyrophosphate Synthase ◽

Acridine Orange Staining ◽

Autophagy Induction

Objectives Autophagy is divided into three phases: autophagosome engulfment of intracellular organelles and proteins, autophagosome fusion with lysosomes, and autolysosome degradation. The farnesyl pyrophosphate synthase inhibitor ibandronate (IBAN) has in vivo cardioprotective properties, potentially via anti-oxidant effects. Whether autophagy is involved in the cardioprotective effect of IBAN remains unexplored. Methods Human umbilical vein endothelial cells (HUVECs) were treated in vitro with IBAN to assess autophagy induction. Lysosomal activation and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling were assessed using a LysoTracker assay, acridine orange staining and western blotting. An MTS assay was used to assess cellular proliferation. Autophagy was inhibited using chloroquine or RNA silencing of autophagy-related 7 (Atg7) expression. Results IBAN induced autophagy in HUVECs. Moreover, IBAN activated lysosomal function, which is pivotal to autophagy induction. PI3K/AKT/mTOR activity was inhibited in IBAN-treated HUVECs, indicating the involvement of this pathway in IBAN-induced autophagy. Inhibition of autophagy using either chloroquine or Atg7 siRNA potentiated inhibition of HUVEC growth by IBAN, suggesting the involvement of non-autophagy pathways in the antiproliferative effects of IBAN. Conclusions These findings provide insights into the role of autophagy in the cardioprotective effects of IBAN and the molecular mechanisms underlying autophagy induction by IBAN.

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Iron toxicity, lipid peroxidation and ferroptosis after intracerebral haemorrhage

Stroke and Vascular Neurology ◽

10.1136/svn-2018-000205 ◽

2019 ◽

Vol 4 (2) ◽

pp. 93-95 ◽

Cited By ~ 17

Author(s):

Jieru Wan ◽

Honglei Ren ◽

Jian Wang

Keyword(s):

Lipid Peroxidation ◽

Cell Death ◽

Intracerebral Haemorrhage ◽

Neuronal Death ◽

Molecular Mechanisms ◽

Apoptotic Cell ◽

Iron Toxicity ◽

Secondary Brain Injury

Intracerebral haemorrhage (ICH) is a devastating type of stroke with high mortality and morbidity. However, we have few options for ICH therapy and limited knowledge about post-ICH neuronal death and related mechanisms. In the aftermath of ICH, iron overload within the perihaematomal region can induce lethal reactive oxygen species (ROS) production and lipid peroxidation, which contribute to secondary brain injury. Indeed, iron chelation therapy has shown efficacy in preclinical ICH studies. Recently, an iron-dependent form of non-apoptotic cell death known as ferroptosis was identified. It is characterised by an accumulation of iron-induced lipid ROS, which leads to intracellular oxidative stress. The ROS cause damage to nucleic acids, proteins and lipid membranes, and eventually cell death. Recently, we and others discovered that ferroptosis does occur after haemorrhagic stroke in vitro and in vivo and contributes to neuronal death. Inhibition of ferroptosis is beneficial in several in vivo and in vitro ICH conditions. This minireview summarises current research on iron toxicity, lipid peroxidation and ferroptosis in the pathomechanisms of ICH, the underlying molecular mechanisms of ferroptosis and the potential for combined therapeutic strategies. Understanding the role of ferroptosis after ICH will provide a vital foundation for cell death-based ICH treatment and prevention.

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A NUP98-HOXD13 Leukemic Fusion Gene Leads To Aberrant Actin Localization In Dysplastic Megakaryocytes

Blood ◽

10.1182/blood.v122.21.2486.2486 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2486-2486

Author(s):

David L. Caudell ◽

Benjamin Okyere ◽

Jacob Cawley ◽

Abdul Gafoor A. Puthiyaveetil ◽

Bettina Heid

Keyword(s):

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Hematopoietic Malignancy ◽

Glass Slides ◽

Transmission Electron ◽

Platelet Release ◽

Actin Localization

Abstract Myelodysplastic syndrome (MDS) is a hematopoietic malignancy characterized by peripheral cytopenias due to bone marrow (BM) failure. Megakarypoiesis, megakaryocyte (MK) production, and platelet release are impaired in in some cases of MDS. Patients often have fewer, but larger circulating platelets, which have abnormal demarcation membrane systems (DMS); the DMS, which determines the number and size of platelets released, is dependent on actin formation. However, the precise role of actin during megakaryopoiesis is poorly understood. Transgenic mice that express the fusion gene NUP98-HOXD13 (NHD13) is a model for MDS and have dysplastic MKs in BM, and macro platelets in circulation. We hypothesized that expression of NHD13 disrupts actin localization during megakaryopoiesis resulting in reduced platelet release and macro platelet formation. To test the hypothesis, BM from wild type (WT) and NHD13 mice were flushed and cultured in media supplemented with Thrombopoietin for 5 days. Following in vitro propagation, MKs were harvested over a discontinuous gradient for downstream experiments. Sternums were also fixed in paraformaldehyde, stained with hematoxylin and eosin, and evaluated by light microscopy to analyze MK morphology in vivo. NHD13 BM contained many dysplastic MKs. Harvested MKs and BM cores from one femur were processed and analyzed by transmission electron microscopy (TEM) and the ultrastructural properties of the DMS detailed. TEM of MKs showed NHD13 leads to formation of an irregular DMS along with abnormal distribution of unusually large granules in MK cytoplasm. Cultured MKs were also cytospun onto glass slides, labeled with fluorescent-tagged F-actin and Myosin IIa and the cytoskeleton visualized by confocal microscopy. WT MKs in vitro had two phenotypes: (1) MKs with myosin and actin evenly dispersed in the cytoplasm and (2) MK with actin predominantly in the periphery of the cytoplasm. In contrast, transgenic MKs displayed only the former phenotype suggesting that actin localization is impaired in NHD13 MKs. Finally, MKs were stimulated with estrogen and adhered to fibrinogen matrices to determine their proplatelet formation functionality. Our results showed impaired proplatelets formation in NHD13 MKs. These data suggest that expression of NHD13 leads to aberrant actin localization leading to dysplastic MK differentiation and macro platelet release. Understanding molecular mechanisms of abnormal megakaryopoiesis in MDS is important as many MDS patients die of hemorrhagic complications. Further studies using this model system will provide a platform for translational research and should reveal potential therapeutic targets in MDS, leading to improved patient care/survival. Disclosures: No relevant conflicts of interest to declare.

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Chloroquine enhances temozolomide cytotoxicity in malignant gliomas by blocking autophagy

Neurosurgical FOCUS ◽

10.3171/2014.9.focus14504 ◽

2014 ◽

Vol 37 (6) ◽

pp. E12 ◽

Cited By ~ 63

Author(s):

Encouse B. Golden ◽

Hee-Yeon Cho ◽

Ardeshir Jahanian ◽

Florence M. Hofman ◽

Stan G. Louie ◽

...

Keyword(s):

Molecular Mechanisms ◽

Malignant Gliomas ◽

Glioma Cells ◽

In Vivo Model ◽

Autophagosome Formation ◽

Individual Drug ◽

In Vivo Experiments ◽

Associated Proteins

Object In a recent clinical trial, patients with newly diagnosed glioblastoma multiforme benefited from chloroquine (CQ) in combination with conventional therapy (resection, temozolomide [TMZ], and radiation therapy). In the present study, the authors report the mechanism by which CQ enhances the therapeutic efficacy of TMZ to aid future studies aimed at improving this therapeutic regimen. Methods Using in vitro and in vivo experiments, the authors determined the mechanism by which CQ enhances TMZ cytotoxicity. They focused on the inhibition-of-autophagy mechanism of CQ by knockdown of the autophagy-associated proteins or treatment with autophagy inhibitors. This mechanism was tested using an in vivo model with subcutaneously implanted U87MG tumors from mice treated with CQ in combination with TMZ. Results Knockdown of the autophagy-associated proteins (GRP78 and Beclin) or treatment with the autophagy inhibitor, 3-methyl adenine (3-MA), blocked autophagosome formation and reduced CQ cytotoxicity, suggesting that autophagosome accumulation precedes CQ-induced cell death. In contrast, blocking autophagosome formation with knockdown of GRP78 or treatment with 3-MA enhanced TMZ cytotoxicity, suggesting that the autophagy pathway protects from TMZ-induced cytotoxicity. CQ in combination with TMZ significantly increased the amounts of LC3B-II (a marker for autophagosome levels), CHOP/GADD-153, and cleaved PARP (a marker for apoptosis) over those with untreated or individual drug-treated glioma cells. These molecular mechanisms seemed to take place in vivo as well. Subcutaneously implanted U87MG tumors from mice treated with CQ in combination with TMZ displayed higher levels of CHOP/GADD-153 than did untreated or individual drug-treated tumors. Conclusions Taken together, these results demonstrate that CQ blocks autophagy and triggers endoplasmic reticulum stress, thereby increasing the chemosensitivity of glioma cells to TMZ.

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Molecular mechanisms of niclosamide antitumor activity

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20156106680 ◽

2015 ◽

Vol 61 (6) ◽

pp. 680-693 ◽

Cited By ~ 10

Author(s):

E.Yu. Moskaleva ◽

V.G. Perevozchikova ◽

A.S. Zhirnik ◽

S.E. Severin

Keyword(s):

Antitumor Activity ◽

Molecular Mechanisms ◽

Anticancer Agent ◽

Intestinal Parasite ◽

Chemical Compounds ◽

Immunodeficient Mice ◽

Parasite Infections ◽

Induce Growth Inhibition

In this review the recent data regarding the antitumor activity of niclosamide and the molecular mechanisms of its antitumor activity are presented. Niclosamide has been used in the clinic for the treatment of intestinal parasite infections. In recent years in several screening investigations of various drugs and chemical compounds niclosamide was identified as a potential anticancer agent. Niclosamide not only inhibits the Wnt/b-catenin, mTORC1, STAT3, NF-kB and Notch signaling pathways, but also targets mitochondria in cancer cells to induce growth inhibition and apoptosis. A number of studies have established the anticancer activity of niclosamide in both in vitro and in vivo in xenotransplantation models using human tumors and immunodeficient mice. It is important that niclosamide is active not only against tumor cells but also cancer stem cells. Normal cells are resistant to niclosamide. The accumulated experimental data suggest niclosamide is a promising drug for the treatment of various types of cancer.

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Targeting Positive Cofactor 4 induces autophagic cell death in MYC-expressing diffuse large B cell lymphoma

10.21203/rs.3.rs-421070/v1 ◽

2021 ◽

Author(s):

Le Ma ◽

Qiang Gong ◽

Zelin Chen ◽

Yu Wang ◽

Xu Tan ◽

...

Keyword(s):

Cell Death ◽

B Cell ◽

Molecular Mechanisms ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Autophagic Cell Death ◽

Large B Cell Lymphoma ◽

Large B Cell

Abstract Background: The MYC-expressing diffuse large B-cell lymphoma (DLBCL) is one of the refractory lymphomas. The pathogenesis of MYC-expressing DLBCL is still unclear, and there is a lack of effective therapy. In this study, we have explored the clinical significance and the molecular mechanisms of transcription co-activator 4 (PC4) in MYC-expressing DLBCL.Methods: We investigated PC4 expression in 54 cases of DLBCL patients’ tissues and matched normal specimens, and studied the molecular mechanisms of PC4 in MYC-expressing DLBCL both in vitro and in vivo.Results: We reported for the first time that targeting c-Myc could induce autophagic cell death in MYC-expressing DLBCL cell lines. We next characterized that PC4 was an upstream regulator of c-Myc, and PC4 was overexpressed in DLBCL and was closely related to clinical staging, prognosis and c-Myc expression. Further, our in vivo and in vitro studies revealed that PC4 knockdown could induce autophagic cell death of MYC-expressing DLBCL. And inhibition of c-Myc mediated aerobic glycolysis and activation of AMPK / mTOR signaling pathway were responsible for the autophagic cell death induced by PC4 knockdown in MYC-expressing DLBCL. Through the CHIP, DLRTM and EMSA assay, we also found that PC4 exerted its oncogenic functions by directly binding to c-Myc promoters.Conclusions: PC4 exerts its oncogenic functions by directly binding to c-Myc promoters. Inhibition of PC4 can induce autophagic cell death of MYC-expressing DLBCL. Our study provides novel insights into the functions and mechanisms of PC4 in MYC-expressing DLBCL, and suggests that PC4 might be a promising therapeutic target for MYC-expressing DLBCL.

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Cannabidiol Induces Apoptosis and Perturbs Mitochondrial Function in Human and Canine Glioma Cells

Frontiers in Pharmacology ◽

10.3389/fphar.2021.725136 ◽

2021 ◽

Vol 12 ◽

Author(s):

Chase Gross ◽

Dominique A. Ramirez ◽

Stephanie McGrath ◽

Daniel L. Gustafson

Keyword(s):

Cell Death ◽

Anticancer Agent ◽

Glioma Cells ◽

Fluorescent Imaging ◽

Mitochondrial Activity ◽

Dependent Manner ◽

Trpv1 Channel ◽

Pharmacologic Inhibitors ◽

Dose Dependent

Cannabidiol (CBD), the major non-psychoactive compound found in cannabis, is frequently used both as a nutraceutical and therapeutic. Despite anecdotal evidence as an anticancer agent, little is known about the effect CBD has on cancer cells. Given the intractability and poor prognoses of brain cancers in human and veterinary medicine, we sought to characterize the in vitro cytotoxicity of CBD on human and canine gliomas. Glioma cells treated with CBD showed a range of cytotoxicity from 4.9 to 8.2 μg/ml; canine cells appeared to be more sensitive than human. Treatment with >5 μg/ml CBD invariably produced large cytosolic vesicles. The mode of cell death was then interrogated using pharmacologic inhibitors. Inhibition of apoptosis was sufficient to rescue CBD-mediated cytotoxicity. Inhibition of RIPK3, a classical necroptosis kinase, also rescued cells from death and prevented the formation of the large cytosolic vesicles. Next, cellular mitochondrial activity in the presence of CBD was assessed and within 2 hours of treatment CBD reduced oxygen consumption in a dose dependent manner with almost complete ablation of activity at 10 μg/ml CBD. Fluorescent imaging with a mitochondrial-specific dye revealed that the large cytosolic vesicles were, in fact, swollen mitochondria. Lastly, calcium channels were pharmacologically inhibited and the effect on cell death was determined. Inhibition of mitochondrial channel VDAC1, but not the TRPV1 channel, rescued cells from CBD-mediated cytotoxicity. These results demonstrate the cytotoxic nature of CBD in human and canine glioma cells and suggest a mechanism of action involving dysregulation of calcium homeostasis and mitochondrial activity.

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Elesclomol-induced increase of mitochondrial reactive oxygen species impairs glioblastoma stem-like cell survival and tumor growth

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-02031-4 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Mariachiara Buccarelli ◽

Quintino Giorgio D’Alessandris ◽

Paola Matarrese ◽

Cristiana Mollinari ◽

Michele Signore ◽

...

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Cell Death ◽

Molecular Mechanisms ◽

Reactive Oxygen ◽

Strong Increase ◽

Oxygen Species ◽

Mitochondrial Reactive Oxygen Species

Abstract Background Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults, characterized by a poor prognosis mainly due to recurrence and therapeutic resistance. It has been widely demonstrated that glioblastoma stem-like cells (GSCs), a subpopulation of tumor cells endowed with stem-like properties is responsible for tumor maintenance and progression. Moreover, it has been demonstrated that GSCs contribute to GBM-associated neovascularization processes, through different mechanisms including the transdifferentiation into GSC-derived endothelial cells (GdECs). Methods In order to identify druggable cancer-related pathways in GBM, we assessed the effect of a selection of 349 compounds on both GSCs and GdECs and we selected elesclomol (STA-4783) as the most effective agent in inducing cell death on both GSC and GdEC lines tested. Results Elesclomol has been already described to be a potent oxidative stress inducer. In depth investigation of the molecular mechanisms underlying GSC and GdEC response to elesclomol, confirmed that this compound induces a strong increase in mitochondrial reactive oxygen species (ROS) in both GSCs and GdECs ultimately leading to a non-apoptotic copper-dependent cell death. Moreover, combined in vitro treatment with elesclomol and the alkylating agent temozolomide (TMZ) enhanced the cytotoxicity compared to TMZ alone. Finally, we used our experimental model of mouse brain xenografts to test the combination of elesclomol and TMZ and confirmed their efficacy in vivo. Conclusions Our results support further evaluation of therapeutics targeting oxidative stress such as elesclomol with the aim of satisfying the high unmet medical need in the management of GBM.

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