naphthoquinones and Glioma

Ferroptosis has attracted increasing interest in cancer therapy. Emerging evidences suggest that naturally occurring naphthoquinones exhibit potent anti-glioma effects via various mechanisms.. The anti-glioma effects of plumbagin were evaluated by in vitro and in vivo experiments. Anti-glioma mechanism of plumbagin was studied by proteomics, flow cytometry, MDA assay, western blot, and RT-PCR. Gene knockdown/overexpression, molecular docking, PharmMappper database, and coimmunoprecipitation were used to study the targets of plumbagin.. Plumbagin showed higher blood-brain barrier penetration ability than that of lapachol and shikonin and elicited significant growth inhibitory effects in vitro and in vivo. Ferroptosis was the main mechanism of plumbagin-induced cell death. Mechanistically, plumbagin significantly downregulated the protein and mRNA levels of xCT and decreased GPX4 protein levels. NAD(P)H quinone dehydrogenase 1 (NQO1) was revealed as a plumbagin predictive target using PharmMappper database and molecular docking. Plumbagin enhanced NQO1 activity and decreased xCT expression, resulting in NQO1-dependent cell death. It also induced GPX4 degradation via the lysosome pathway and caused GPX4-dependent cell death.. Plumbagin inhibited in vitro and in vivo glioma growth via targeting NQO1/GPX4-mediated ferroptosis, which might be developed as a novel ferroptosis inducer or anti-glioma candidate.

Topics: Cell Line, Tumor; Ferroptosis; Glioma; Humans; Molecular Docking Simulation; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Phospholipid Hydroperoxide Glutathione Peroxidase

Shikonin induces glioma cell death via necroptosis, a caspase-independent programmed cell death pathway that is chiefly regulated by receptor-interacting serine/threonine protein kinase1 (RIP1) and 3 (RIP3). Chromatinolysis is considered as one of the key events leading to cell death during necroptosis. It is usually accompanied with nuclear translocation of AIF and formation of γ-H2AX. Cyclophilin A (CypA) is reported to participate in the nuclear translocation of AIF during apoptosis. However, it remains unclear whether CypA contributes to necroptosis and regulation of chromatinolysis. In this study, our results revealed for the first time that shikonin promoted time-dependent CypA activation, which contributed to nuclear translocation of AIF and γ-H2AX formation. In vitro studies showed that knockdown of CypA by siRNA or inhibition of CypA by its specific inhibitor, cyclosporine A (CsA), not only significantly mitigated shikonin-induced glioma cell death, but also prevented chromatinolysis. Mechanistically, activated CypA targeted mitochondria and triggered mitochondrial superoxide overproduction, which then promoted AIF translocation from mitochondria into the nucleus by depolarizing the mitochondria and intensified the formation of γ-H2AX by promoting intracellular accumulation of ROS. Additionally, the CypA in the nucleus can form DNA degradation complexes with AIF and γ-H2AX, which also promote the execution of chromatinolysis. Thus, we demonstrate that CypA contributes to shikonin-induced glioma cell necroptosis and promotion of chromatinolysis.

Topics: Apoptosis; Apoptosis Inducing Factor; Cyclophilin A; Glioma; Humans; Naphthoquinones; Necroptosis; Receptor-Interacting Protein Serine-Threonine Kinases

Shikonin, one of the main active ingredients of Chinese herbal medicine Lithospermum erythrorhizon, has been widely used to treat various disease including virus infection and inflammation in clinical. Its anti-tumor activity has been recorded in "Chinese herbal medicine". Recently, some studies about its anti-glioma effects have been reported. However, little is known about the molecular pharmacological activity of Shikonin in glioma.. This study aimed to systematically uncover and validate the pharmacological mechanism of Shikonin against glioma.. Network pharmacology approach, survival analysis, and Pearson co-expression analysis were performed to uncover and test the pharmacological mechanisms of Shikonin in glioma. Apoptosis assay, Caspase-3 activity assay and immunoblot analysis were practiced to validate the mechanisms.. Network pharmacology results suggested, anti-glioma effect of Shikonin by interfering endoplasmic reticulum (ER) stress-mediated tumor apoptosis targeting Caspase-3, and Bax/Bak-induced mitochondrial outer membrane permeabilization (MOMP) triggering cancer cell apoptosis. Survival analysis suggested the association of CASP3 with glioma (P < 0.05). Pearson correlation analysis indicated possible interaction of CASP3 with PERK through positive feedback regulation. Shikonin or in combination with 14G2a induced cell apoptosis in oligodendroglioma Hs683 cells in a dose-dependent manner with at a maximum apoptosis rate of 33%-37.5%, and 73%-77% respectively. Immunoblot analysis showed that Shikonin increased Caspase-3 activity to about 4.29 times, and increased 9 times when it combined with 14G2a. Shikonin increased also the expression levels of the proteins PERK and CHOP by about 4.4 and 5.6 folds, respectively, when it combined with 14G2a.. This study highlights the pharmacological mechanisms of Shikonin in the induction of tumor apoptosis in glioma cells.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Membrane Permeability; Endoplasmic Reticulum Stress; Gene Regulatory Networks; Glioma; Humans; Mitochondrial Membranes; Naphthoquinones

In this study, we developed an AS1411 aptamer/hyaluronic acid-bifunctionalized microemulsion co-loading shikonin and docetaxel (AS1411/SKN&DTX-M). Such microemulsion was capable of penetrating the blood-brain barrier (BBB), targeting CD44/nucleolin-overexpressed glioma, and inhibiting the orthotopic glioma growth. AS1411/SKN&DTX-M showed a spherical morphology with a diameter around 30 nm and rapidly released drugs in the presence of hyaluronidase and mild acid. In the U87 cellular studies, AS1411/SKN&DTX-M elevated the cytotoxicity, enhanced the cellular uptake, and induced the cell apoptosis. In the artificial blood-brain barrier model, the transepithelial electrical resistance was decreased after the treatment with AS1411/SKN&DTX-M and thereby of increasing the apparent permeability coefficient. Furthermore, AS1411/SKN&DTX-M showed a strong inhibition against the formation of cancer stem cell-enriched U87 cell spheroids, in which the expression of CD133 was downregulated significantly. In the biodistribution studies, AS1411/SKN&DTX-M could selectively accumulate in the brains of orthotopic luciferase-transfected U87 glioma tumor-bearing nude mice. Importantly, AS1411/SKN&DTX-M exhibited the overwhelming inhibition of glioma growth of orthotopic luciferase-transfected U87 glioma models and reached the longest survival period among all the treatments. In summary, the codelivery of shikonin and docetaxel using bifunctionalization with hyaluronic acid and AS1411 aptamer offers a promising strategy for dual drug-based combinational antiglioma treatment.

Topics: Animals; Antineoplastic Agents; Apoptosis; Aptamers, Nucleotide; Cell Line; Cell Line, Tumor; Docetaxel; Drug Delivery Systems; Emulsions; Glioma; Humans; Hyaluronic Acid; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Naphthoquinones; Nucleolin; Oligodeoxyribonucleotides; Phosphoproteins; RNA-Binding Proteins; Tissue Distribution

Chromatinolysis refers to enzymatic degradation of nuclear DNA and is regarded as one of the crucial events leading to cell death. Mixed-lineage kinase domain-like protein (MLKL) has been identified as a key executor of necroptosis, but it remains unclear whether MLKL contributes to necroptosis via regulation of chromatinolysis. In this study, we find that shikonin induces MLKL activation and chromatinolysis in glioma cells in vitro and in vivo, which are accompanied with nuclear translocation of AIF and γ-H2AX formation. In vitro studies reveal that inhibition of MLKL with its specific inhibitor NSA or knockdown of MLKL with siRNA abrogates shikonin-induced glioma cell necroptosis, as well as chromatinolysis. Mechanistically, activated MLKL targets mitochondria and triggers excessive generation of mitochondrial superoxide, which promotes AIF translocation into nucleus via causing mitochondrial depolarization and aggravates γ-H2AX formation via improving intracellular accumulation of ROS. Inhibition of nuclear level of AIF by knockdown of AIF with siRNA or mitigation of γ-H2AX formation by suppressing ROS with antioxidant NAC effectively prevents shikonin-induced chromatinolysis. Then, we found that RIP3 accounts for shikonin-induced activation of MLKL, and activated MLKL reversely up-regulates the protein level of CYLD and promotes the activation of RIP1 and RIP3. Taken together, our data suggest that MLKL contributes to shikonin-induced glioma cell necroptosis via promotion of chromatinolysis, and shikonin induces a positive feedback between MLKL and its upstream signals RIP1 and RIP3.

Topics: Animals; Apoptosis Inducing Factor; Cell Line, Tumor; DNA Fragmentation; DNA, Neoplasm; Gene Expression Regulation, Neoplastic; Glioma; Humans; Mice; Mitochondria; Naphthoquinones; Necroptosis; Protein Kinases; Rats; Reactive Oxygen Species; Xenograft Model Antitumor Assays

Shikonin, a traditional Chinese medicine, has been identified as being capable of inducing apoptosis in various tumors, including glioma, and is thus considered to be a promising therapeutic agent for tumor therapy. However, little is known about the molecular mechanism of shikonin in glioma. The present study investigated the influence of shikonin on the proliferation and apoptosis of glioma cells U251 and U87MG and explored the potential molecular mechanisms. It was identified that shikonin was able to induce apoptosis in human glioma cells in a time‑ and dose‑dependent manner, and a decreased expression level of cluster of differentiation (CD)147 was observed in shikonin‑treated U251 and U87MG cells. Knockdown of CD147 inhibited U251 and U87MG cell growth, whereas CD147 overexpression enhanced cell growth and decreased shikonin‑induced apoptosis. Additionally, an increased expression level of CD147 suppressed the elevated production of reactive oxygen species and mitochondrial membrane potential levels induced by shikonin. The data indicated that shikonin‑induced apoptosis in glioma cells was associated with the downregulation of CD147 and the upregulation of oxidative stress. CD147 may be an optional target of shikonin‑induced cell apoptosis in glioma cells.

Topics: Apoptosis; Basigin; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Down-Regulation; Glioma; Humans; Medicine, Chinese Traditional; Membrane Potential, Mitochondrial; Naphthoquinones; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering

Shikonin (SHK) is a highly liposoluble naphthoquinone pigment has recently been investigated as a potential antiglioma agent. However, shikonin shows several limitations like poor aqueous solubility, short half-life and non-selective biodistribution. Herein, we have developed a nanoparticles (NPs) prepared from PEG-PLGA using an emulsion solvent evaporation method. Nanoparticle surfaces were modified by coating with lactoferrin (Lf) to improve the crossing of the blood brain barrier and targeting of glioma cells via receptor-mediated path-ways. X-ray powder diffraction and differential scanning calorimetry analysis revealed the amorphous nature of SHK encapsulated within the NPs. Moreover, the drug-loaded NPs exhibit narrow size distribution and high encapsulation efficiency. The in vitro release experiments of the NPs exhibited sustained release for more than 72h. When compared to free SHK and SHK/NPs, in vivo study demonstrated higher brain concentration of SHK, indicating a significant effect of Lf coated NPs on brain targeting. Accordingly, these findings provide evidence for the potential of Lf-modified NPs as a targeted delivery system for brain glioblastomas treatment.

Topics: Animals; Brain; Cell Line, Tumor; Drug Carriers; Drug Delivery Systems; Glioma; Humans; Lactoferrin; Nanoparticles; Naphthoquinones; Polyesters; Polyethylene Glycols; Rats; Tissue Distribution

RIP1 and RIP3 are necroptosis initiators, but their roles in regulation of glycolysis remain elusive. In this study, we found shikonin activated RIP1 and RIP3 in glioma cells in vitro and in vivo, which was accompanied with glycolysis suppression. Further investigation revealed that shikonin-induced decreases of glucose-6-phosphate and pyruvate and downregulation of HK II and PKM2 were significantly prevented when RIP1 or RIP3 was pharmacologically inhibited or genetically knocked down with SiRNA. Moreover, shikonin also triggered accumulation of intracellular H

Topics: Animals; Cell Line, Tumor; Cysteine; Gene Expression Regulation, Neoplastic; Glioma; Glutathione; Glycolysis; Humans; Hydrogen Peroxide; Mice; Naphthoquinones; Nuclear Pore Complex Proteins; Rats; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Xenograft Model Antitumor Assays

Plumbagin is a natural naphthoquinone constituent isolated from the roots of medicinal plant Plumbago zeylanica L., and has demonstrated anti-proliferative and anti-invasion activities in various cancer cells. However, its effect on the migration and invasion of glioma cells has not been elucidated. Therefore, human glioma U87 and U251 cells were treated with plumbagin at 1.0 and 2.0 μM for 24 h, and cell migration and invasion were assessed with scratch wound healing and invasion assays. The results showed that plumbagin significantly inhibited the migration and invasion of U87 and U251 cells, suppressed the activity and expression of MMP-2/-9, and inhibited the nuclear translocation of transcription factors Sp1 in the U87 and U251 cells. Moreover, plumbagin reduced the level of p-PI3K and p-Akt in these cells. The combined treatment with plumbagin and PI3K/Akt agonist insulin-like growth factor-1 (IGF-1) reversed plumbagin-mediated inhibitory effects on MMP-2/-9 expression, cell migration and invasion. These findings suggest that the plumbagin-induced inhibition of glioma cell migration and invasion is closely associated with the downregulation of MMP-2/-9 expression and activity, and suppression of PI3K/Akt signaling pathway activation. Thus, plumbagin might be a potential anti-invasive agent in the treatment of glioma.

Topics: Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Down-Regulation; Glioma; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Naphthoquinones; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

Necroptosis is a type of programmed necrosis regulated by receptor interacting protein kinase 1 (RIP1) and RIP3. Necroptosis is found to be accompanied by an overproduction of reactive oxygen species (ROS), but the role of ROS in regulation of necroptosis remains elusive. In this study, we investigated how shikonin, a necroptosis inducer for cancer cells, regulated the signaling leading to necroptosis in glinoma cells in vitro. Treatment with shikonin (2-10 μmol/L) dose-dependently triggered necrosis and induced overproduction of intracellular ROS in rat C6 and human SHG-44, U87 and U251 glioma cell lines. Moreover, shikonin treatment dose-dependently upregulated the levels of RIP1 and RIP3 and reinforced their interaction in the glioma cells. Pretreatment with the specific RIP1 inhibitor Nec-1 (100 μmol/L) or the specific RIP3 inhibitor GSK-872 (5 μmol/L) not only prevented shikonin-induced glioma cell necrosis but also significantly mitigated the levels of intracellular ROS and mitochondrial superoxide. Mitigation of ROS with MnTBAP (40 μmol/L), which was a cleaner of mitochondrial superoxide, attenuated shikonin-induced glioma cell necrosis, whereas increasing ROS levels with rotenone, which improved the mitochondrial generation of superoxide, significantly augmented shikonin-caused glioma cell necrosis. Furthermore, pretreatment with MnTBAP prevented the shikonin-induced upregulation of RIP1 and RIP3 expression and their interaction while pretreatment with rotenone reinforced these effects. These findings suggest that ROS is not only an executioner of shikonin-induced glioma cell necrosis but also a regulator of RIP1 and RIP3 expression and necrosome assembly.

Topics: Animals; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Dose-Response Relationship, Drug; Glioma; Humans; Mitochondria; Naphthoquinones; Necrosis; Nuclear Pore Complex Proteins; Oxidative Stress; Protein Serine-Threonine Kinases; Rats; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Signal Transduction; Time Factors

Malignant gliomas are aggressive and life-threatening tumours that still show a poor prognosis: the current therapeutic approach based on surgical resection and chemotherapy combined with radiotherapy does not provide a satisfactory chance of long-term survival to patients. Natural bioactive compounds represent a precious source of molecules with antiproliferative activity, potentially effective also against glioma cells. Among these, Juglone is a known allelopathic compound extracted from the eastern black walnut (Juglans nigra) whose antimitotic effect has been extensively described in mammalian cells. We investigated the antiproliferative effect of a synthetic derivative of this natural compound, 2-(2,4-dihydroxyphenyl)-8-hydroxy-1,4-naphthoquinone (DiNAF), in rat glioma cells. We compared this molecule and its effect with the natural reference compound and with newly synthesised derivatives to build a preliminar structure-activity relationship. Biological assays and NMR-based redox experiments confirmed that DiNAF is a promising lead and supported the hypothesis of a redox mechanism underlying its cytotoxic activity.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Glioma; Juglans; Magnetic Resonance Spectroscopy; Mitosis; Models, Molecular; Naphthoquinones; Rats; Rats, Wistar; Structure-Activity Relationship

Because the anti-apoptotic protein Bcl-xL is overexpressed in glioma, one might expect that inhibiting or silencing this gene would promote tumor cell killing. However, our studies have shown that this approach has limited independent activity, but may tip the balance in favor of apoptosis induction in response to other therapeutic interventions. To address this issue, we performed a pharmacological screen using a panel of signaling inhibitors and chemotherapeutic agents in Bcl-xL silenced cells. Although limited apoptosis induction was observed with a series of inhibitors for receptor tyrosine kinases, PKC inhibitors, Src family members, JAK/STAT, histone deacetylase, the PI3K/Akt/mTOR pathway, MAP kinase, CDK, heat shock proteins, proteasomal processing, and various conventional chemotherapeutic agents, we observed a dramatic potentiation of apoptosis in Bcl-xL silenced cells with the survivin inhibitor, YM155. Treatment with YM155 increased the release of cytochrome c, smac/DIABLO and apoptosis inducing-factor, and promoted loss of mitochondrial membrane potential, activation of Bax, recruitment of LC3-II to the autophagosomes and apoptosis in Bcl-xL silenced cells. We also found an additional mechanism for the augmentation of apoptosis due to abrogation of DNA double-strand break repair mediated by Rad51 repression and enhanced accumulation of γH2AX. In summary, our observations may provide a new insight into the link between Bcl-xL and survivin inhibition for the development of novel therapies for glioma. © 2016 Wiley Periodicals, Inc.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; bcl-X Protein; Cell Line, Tumor; Cytochromes c; DNA Damage; Gene Silencing; Glioma; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Membrane Potential, Mitochondrial; Naphthoquinones; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Survivin; TOR Serine-Threonine Kinases

Survival of patients with glioblastoma remains within the range of several months despite advances in therapeutic options. We have already shown that 5-hydroxy-1,4-naphthalenedione (juglone) exerts antiproliferative, anti-invasive, and cytotoxic effects on glioma C6 cells. Here, we further revealed that juglone is relatively selective to glioma cells as compared to the normal glial culture, and investigated its mechanisms of action. The incubation of glioma C6 cells with juglone generated high levels of reactive oxygen species (ROS). The produced ROS accounted for the anticancer effect of juglone as antioxidant N-acetylcysteine reduced both cytotoxic and antiproliferative activities of juglone. Furthermore, high resolution respirometry revealed that juglone decreased oxygen consumption mainly by affecting pyruvate/malate- and glutamate/malate-induced mitochondrial respiration. The inhibition of respiratory complex I by amytal decreased juglone-triggered generation of ROS and diminished its anticancer activity. Thus, our results indicate that juglone generates ROS through interaction with respiratory complex I in glioma C6 cells, and, in turn, induces the anticancer effects.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Electron Transport; Glioma; Humans; Mitochondria; Mitochondrial Membranes; Naphthoquinones; Rats, Wistar; Reactive Oxygen Species

Shikonin has been reported to induce glioma cell death via necroptosis, a type of programmed necrosis primarily mediated by RIP1 and RIP3. Although RIP1 and RIP3 are found to regulate some features of necrosis such as energy depletion and cellular membrane disruption, it remains unclear whether RIP1 and RIP3 could modulate DNA double strand breaks (DSBs), which is a crucial event leading to chromatinolysis. In this study, we used glioma cell lines and mice model of xenograft glioma to investigate the roles of RIP1 and RIP3 in shikonin-induced DNA DSBs. We found that shikonin induced upregulation of RIP1 and RIP3, necrosome formation and DNA DSBs in vitro and in vivo. In vitro investigation showed that the DNA DSBs and the reduction of cellular viabilities induced by shikonin were both prevented when RIP1 or RIP3 was pharmacologically inhibited by specific inhibitor or genetically knocked down with siRNA. Then, we proved that suppression of intracellular ROS with antioxidant NAC inhibited DNA DSBs caused by either hydrogen peroxide or shikonin, suggesting that ROS played a crucial role in regulation of DNA DSBs of glioma cells induced by shikonin. Further, we found that RIP1 and RIP3 regulated shikonin-induced overproduction of ROS via causing excessive generation of mitochondrial superoxide and depletion of GSH, indicating that ROS was the downstream signal of RIP1 and RIP3. Taken together, we demonstrated that RIP1 and RIP3 contributed to shikonin-induced DNA DSBs in glioma cells via increase of intracellular ROS levels.

Topics: Animals; Cell Line, Tumor; Disease Models, Animal; DNA Breaks, Double-Stranded; Glioma; Heterografts; Mice; Naphthoquinones; Nuclear Pore Complex Proteins; Reactive Oxygen Species; Receptor-Interacting Protein Serine-Threonine Kinases; RNA-Binding Proteins; Up-Regulation

Lapachol is a natural naphthoquinone compound that possesses extensive biological activities. The aim of this study is to investigate the inhibitory effects of lapachol on rat C6 glioma both in vitro and in vivo, as well as the potential mechanisms.. The antitumor effect of lapachol was firstly evaluated in the C6 glioma model in Wistar rats. The effects of lapachol on C6 cell proliferation, apoptosis and DNA damage were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)/ phenazinemethosulfate (PMS) assay, hoechst 33358 staining, annexin V-FITC/PI staining, and comet assay. Effects of lapachol on topoisomerase I (TOP I) and topoisomerase II (TOP II) activities were detected by TOP I and TOP II mediated supercoiled pBR322 DNA relaxation assays and molecular docking. TOP I and TOP II expression levels in C6 cells were also determined.. High dose lapachol showed significant inhibitory effect on the C6 glioma in Wistar rats (P < 0.05). It was showed that lapachol could inhibit proliferation, induce apoptosis and DNA damage of C6 cells in dose dependent manners. Lapachol could inhibit the activities of both TOP I and II. Lapachol-TOP I showed relatively stronger interaction than that of lapachol-TOP II in molecular docking study. Also, lapachol could inhibit TOP II expression levels, but not TOP I expression levels.. These results showed that lapachol could significantly inhibit C6 glioma both in vivo and in vitro, which might be related with inhibiting TOP I and TOP II activities, as well as TOP II expression.

Topics: Animals; Antineoplastic Agents, Phytogenic; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; DNA Damage; DNA Topoisomerases, Type I; DNA Topoisomerases, Type II; Dose-Response Relationship, Drug; Glioma; Molecular Docking Simulation; Naphthoquinones; Rats; Rats, Wistar; Topoisomerase Inhibitors; Xenograft Model Antitumor Assays

Survivin has multiple functions in the progression of cancer. The aim of the present study was to investigate the influence of YM155, a specific survivin inhibitor, on the biological behavior of U87 glioblastoma cells. The proliferative activity and growth rate of U87 cells were determined by colony formation assay and mononuclear cell direct cytotoxicity assay (MTT assay). The reconstituted basement membrane penetrating capacity was determined by cell invasion assay. The cell movement and migratory capacity were detected by wound-healing repair assay. We found that inhibition of survivin by YM155 dramatically decreased the invasive and metastatic capacities of the cells, suppressed the proliferation, decelerated the rate of growth, and reduced the number of clones on soft agar. In conclusion, YM155 has the potential to be used in the clinical treatment of GBM.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Glioma; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Naphthoquinones; Neurons; Survivin

Plumbagin, a natural quinonoid constituent isolated from the root of medicinal plant Plumbago zeylanica L, has exhibited anti-tumor and anti-proliferative activities in various tumor cell lines as well as in animal tumor models. However, its anticancer effects and the mechanisms underlying its suppression of glioma cell growth have not been elucidated. Oncogenic transcription factor Forkhead Box M1 (FOXM1) has garnered particular interest in recent years as a potential target for the prevention and/or therapeutic intervention in glioma, nevertheless, less information is currently available regarding FOXM1 inhibitor. Here, we reported that plumbagin could effectively inhibit cell proliferation, migration and invasion and induce apoptosis of glioma cells. Cell cycle assay showed that plumbagin induced G2/M arrest. Interestingly, we found that plumbagin decreased the expression of FOXM1 both at mRNA level and protein level. Plumbagin also inhibited the transactivation ability of FOXM1, resulting in down-regulating the expression of FOXM1 downstream target genes, such as cyclin D1, Cdc25B, survivin, and increasing the expression of p21(CIP1) and p27(KIP1). Most importantly, down-regulation of FOXM1 by siFOXM1 transfection enhanced plumbagin-induced change in viability. On the contrary, over-expression of FOXM1 by cDNA transfection reduced plumbagin-induced glioma cell growth inhibition. These results suggest that plumbagin exhibits its anticancer activity partially by inactivation of FOXM1 signaling pathway in glioma cells. Our findings indicate that plumbagin may be considered as a potential natural FOXM1 inhibitor, which could contribute to the development of new anticancer agent for therapy of gliomas.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Naphthoquinones; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Transfection

This work was aimed to the development of a set of new naphtoquinone derivatives that can act against glioma. The compounds were tested in order to find out their ability to inhibit the growth of glioma cells, and the results of these assays were correlated with electrochemical analysis and NMR-based reoxidation kinetic studies, suggesting that a redox mechanism underlies and may explain the observed biological behavior. In addition to a full description of the synthetic pathways, electrochemistry, NMR and single crystal X-ray diffraction data are provided.

Topics: Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Crystallography, X-Ray; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Glioma; Humans; Models, Molecular; Molecular Structure; Naphthoquinones; Structure-Activity Relationship

Plumbagin is a natural compound that is isolated from the root of the medicinal plant Plumbago zeylanica L. Based on a previous in vitro study performed by our group, which demonstrated the effectiveness of plumbagin against glioma cells, we further ascertained whether plumbagin exhibits the same effectiveness against glioma cell xenografts in nude mice. Our results revealed that tumor volume was reduced by 54.48% in the plumbagin-treated group compared with the controls. Furthermore, there were no obvious signs of toxicity as assessed by the organ sizes and cell morphologies of the mice that were treated with plumbagin. Immunofluorescence assays further revealed that plumbagin significantly inhibited glioma cell proliferation and induced cell apoptosis. Importantly, we also determined that the expressions of FOXM1 and its downstream target effectors, including cyclin D1 and Cdc25B, were down-regulated in the treated group, while the expressions of p21 and p27 were increased; the latter findings corroborate the results of our previous in vitro study. Taken together, these findings indicate that plumbagin may be a natural downregulator of FOXM1 with potential therapeutic effectiveness for the treatment of gliomas.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Female; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression; Glioma; Humans; Mice, Nude; Molecular Targeted Therapy; Naphthoquinones; Neoplasm Transplantation; Phytotherapy; Plumbaginaceae

Shikonin is a quinone-containing natural product that induces the apoptotic death of some cancer cell lines in culture through increasing intracellular reactive oxygen species (ROS). Quinone-based drugs have shown potential in the clinic, making shikonin an interesting compound to study. Our previous study found that shikonin induces apoptosis in neuroblastoma by induction of ROS, but its mechanism of action and scope of activity are unknown. In this study, we investigated the mode of oxidative stress of shikonin in human glioma cells. ROS induction by shikonin was of mitochondrial origin, as demonstrated by detection of superoxide with MitoSOX Red. Pre-incubation of shikonin with inhibitors of different complexes of the respiratory chain suggested that shikonin-induced ROS production occurred via complex II. In addition, NADPH oxidase and lipooxygenase are two other main ROS-generated sites in shikonin treatment. ROS production by shikonin resulted in the inhibition of nuclear translocation of Nrf2. Stable overexpression of Nrf2 in glioma cells inhibited ROS generation by shikonin. ROS generation from mitochondrial complex II, NADPH oxidase and lipooxygenase is likely the primary mechanism by which shikonin induces apoptosis in glioma cells. These findings also have relevance to the development of certain ROS producers as anti-cancer agents. These, along with shikonin have potential as novel chemotherapeutic agents on human glioma.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Cytochromes c; Cytosol; Glioma; Humans; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species; Superoxides

Induction of apoptosis by the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising antitumor therapy. However, not all tumor cells are sensitive to TRAIL, highlighting the need for strategies to overcome TRAIL resistance. Inhibitor of apoptosis family member survivin is constitutively activated in various cancers and blocks apoptotic signaling. Recently, we demonstrated that YM-155 [3-(2-methoxyethyl)-2-methyl-4,9-dioxo-1-(pyrazin-2-ylmethyl)-4,9-dihydro-3H-naphtho[2,3-d]imidazol-1-ium bromide], a small molecule inhibitor, downregulates not only survivin in gliomas but also myeloid cell leukemia sequence 1 (Mcl-1), and it upregulates proapoptotic Noxa levels. Because Mcl-1 and survivin are critical mediators of resistance to various anticancer therapies, we questioned whether YM-155 could sensitize resistant glioma cells to TRAIL. To address this hypothesis, we combined YM-155 with TRAIL and examined the effects on cell survival and apoptotic signaling. TRAIL or YM-155 individually induced minimal killing in highly resistant U373 and LNZ308 cell lines, but combining TRAIL with YM-155 triggered a synergistic proapoptotic response, mediated through mitochondrial dysfunction via activation of caspases-8, -9, -7, -3, poly-ADP-ribose polymerase, and Bid. Apoptosis induced by combination treatments was blocked by caspase-8 and pan-caspase inhibitors. In addition, knockdown of Mcl-1 by RNA interference overcame apoptotic resistance to TRAIL. Conversely, silencing Noxa by RNA interference reduced the combined effects of YM-155 and TRAIL on apoptosis. Mechanistically, these findings indicate that YM-155 plays a role in counteracting glioma cell resistance to TRAIL-induced apoptosis by downregulating Mcl-1 and survivin and amplifying mitochondrial signaling through intrinsic and extrinsic apoptotic pathways. The significantly enhanced antitumor activity of the combination of YM-155 and TRAIL may have applications for therapy of malignant glioma.

Topics: Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Cell Line, Tumor; Cell Survival; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Gene Knockdown Techniques; Glioma; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Naphthoquinones; Protein Conformation; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Survivin; TNF-Related Apoptosis-Inducing Ligand

Shikonin was reported to induce necroptosis in leukemia cells, but apoptosis in glioma cell lines. Thus, it is needed to clarify whether shikonin could cause necroptosis in glioma cells and investigate its underlying mechanisms.. Shikonin and rat C6 glioma cell line and Human U87 glioma cell line were used in this study. The cellular viability was assayed by MTT. Flow cytometry with annexin V-FITC and PI double staining was used to analyze cellular death modes. Morphological alterations in C6 glioma cells treated with shikoinin were evaluated by electronic transmission microscopy and fluorescence microscopy with Hoechst 33342 and PI double staining. The level of reactive oxygen species was assessed by using redox-sensitive dye DCFH-DA. The expressional level of necroptosis associated protein RIP-1 was analyzed by western blotting.. Shikonin induced cell death in C6 and U87 glioma cells in a dose and time dependent manner. The cell death in C6 and U87 glioma cells could be inhibited by necroptosis inhibitor necrotatin-1, not by pan-caspase inhibitor z-VAD-fmk. Shikonin treated C6 glioma cells presented electron-lucent cytoplasm, loss of plasma membrane integrity and intact nuclear membrane in morphology. The increased ROS level caused by shikonin was attenuated by necrostatin-1 and blocking ROS by anti-oxidant NAC rescued shikonin-induced cell death in both C6 and U87 glioma cells. Moreover, the expressional level of RIP-1 was up-regulated by shikonin in a dose and time dependent manner as well, but NAC suppressed RIP-1 expression.. We demonstrated that the cell death caused by shikonin in C6 and U87 glioma cells was mainly via necroptosis. Moreover, not only RIP-1 pathway, but also oxidative stress participated in the activation of shikonin induced necroptosis.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Shape; Cell Survival; Glioma; Humans; Medicine, Chinese Traditional; Naphthoquinones; Necrosis; Nuclear Pore Complex Proteins; Rats; Reactive Oxygen Species; RNA-Binding Proteins

Gliomas, the most malignant form of brain tumors, contain a small subpopulation of glioma stem cells (GSCs) that are implicated in therapeutic resistance and tumor recurrence. Topoisomerase I inhibitors, shikonin and topotecan, play a crucial role in anti-cancer therapies. After isolated and identified the GSCs from glioma cells successfully, U251, U87, GSCs-U251 and GSCs-U87 cells were administrated with various concentrations of shikonin or topotecan at different time points to seek for the optimal administration concentration and time point. The cell viability, cell cycle and apoptosis were detected using cell counting kit-8 and flow cytometer to observe the inhibitory effects on glioma cells and GSCs. We demonstrated that shikonin and topotecan obviously inhibited proliferation of not only human glioma cells but also GSCs in a dose- and time-dependent manner. According to the IC50 values at 24 h, 2 μmol/L of shikonin and 3 μmol/L of topotecan were selected as the optimal administration concentration. In addition, shikonin and topotecan induced cell cycle arrest in G0/G1 and S phases and promoted apoptosis. The down-regulation of Bcl-2 expression with the activation of caspase 9/3-dependent pathway was involved in the apoptosis process. Therefore, the above results showed that topoisomerase I inhibitors, shikonin and topotecan, inhibited growth and induced apoptosis of GSCs as well as glioma cells, which suggested that they might be the potential anticancer agents targeting gliomas to provide a novel therapeutic strategy.

Topics: Apoptosis; Brain Neoplasms; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Glioma; Humans; Naphthoquinones; Neoplastic Stem Cells; Proto-Oncogene Proteins c-bcl-2; Topoisomerase I Inhibitors; Topotecan

Antiapoptotic proteins are commonly overexpressed in gliomas, contributing to therapeutic resistance. We recently reported that clinically achievable concentrations of the Bcl-2/Bcl-xL inhibitor ABT-737 failed to induce apoptosis in glioma cells, with persistent expression of survivin and Mcl-1. To address the role of these mediators in glioma apoptosis resistance, we analyzed the effects of YM-155, a survivin suppressant, on survival on a panel of glioma cell lines. YM-155 inhibited cell growth and downregulated survivin and Mcl-1 in a dose- and cell line-dependent manner. While U373, LN18, LNZ428, T98G, LN229, and LNZ308 cells exhibited an IC(50) of 10 to 75 nmol/L, A172 cells were resistant (IC(50) ∼ 250 nmol/L). No correlation was found between sensitivity to YM-155 and baseline expression of survivin or cIAP-1/cIAP-2/XIAP. However, strong correlation was observed between EGF receptor (EGFR) activation levels and YM-155 response, which was confirmed using EGFR-transduced versus wild-type cells. Because we postulated that decreasing Mcl-1 expression may enhance glioma sensitivity to ABT-737, we examined whether cotreatment with YM-155 promoted ABT-737 efficacy. YM-155 synergistically enhanced ABT-737-induced cytotoxicity and caspase-dependent apoptosis. Downregulation of Mcl-1 using short hairpin RNA also enhanced ABT-737-inducing killing, confirming an important role for Mcl-1 in mediating synergism between ABT-737 and YM-155. As with YM-155 alone, sensitivity to YM-155 and ABT-737 inversely correlated with EGFR activation status. However, sensitivity could be restored in highly resistant U87-EGFRvIII cells by inhibition of EGFR or its downstream pathways, highlighting the impact of EGFR signaling on Mcl-1 expression and the relevance of combined targeted therapies to overcome the multiple resistance mechanisms of these aggressive tumors.

Topics: Apoptosis; bcl-X Protein; Biphenyl Compounds; Brain Neoplasms; Cell Line, Tumor; Down-Regulation; Drug Resistance, Neoplasm; ErbB Receptors; Gene Expression Regulation, Neoplastic; Glioma; Humans; Imidazoles; Inhibitor of Apoptosis Proteins; Microtubule-Associated Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Naphthoquinones; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sulfonamides; Survivin

Shikonin is the main naphthoquinone compound of the root of Lithospermum erythrorhizon. Our previous study demonstrated that shikonin possesses anticancer activity in human hepatoma cells. However, the anticancer mechanism of shikonin in human glioma cells is unclear at present. In the present study, we demonstrated that shikonin induces apoptosis in three human glioma cell lines: U87MG, Hs683, and M059K cells.. Cell cycle, generation of reactive oxygen species (ROS), depletion of glutathione (GSH), and disruption of mitochondrial transmembrane potential in shikonin-treated cells were determined by flow cytometry. Apoptosis-related proteins, catalase, and superoxide dismutase-1 (SOD-1) were determined by Western blot testing. N-acetylcysteine (NAC), pifithrin-α (PFT-α), or cyclosporin A were applied to evaluate the molecular mechanism of shikonin in apoptosis.. Shikonin induces the generation of ROS, depletion of GSH, disruption of mitochondrial transmembrane potential, upregulation of p53, and cleavage of PARP [poly(ADP-ribose) polymerase] in U87MG glioma cells. Moreover, shikonin causes catalase downregulation and SOD-1 upregulation as well as decreased Bcl-2 and increased Bax expression. Pretreatment with NAC, PFT-α, or cyclosporin A causes the recovery of shikonin-induced apoptosis. The ROS generation and GSH depletion induced by shikonin trigger mitochondrial transmembrane potential disruption. ROS production was partially dependent on the upregulation of p53 upon shikonin treatment.. These studies are the first to show that shikonin-induced apoptosis occurs through multiple pathways in human glioma cells. We conclude that shikonin may be used as a potential chemotherapeutic agent against human glioma.

Topics: Acetylcysteine; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; bcl-2-Associated X Protein; Benzothiazoles; Catalase; Cell Line, Tumor; Cyclosporine; G1 Phase Cell Cycle Checkpoints; Glioma; Glutathione; Humans; Membrane Potential, Mitochondrial; Mitochondria; Naphthoquinones; Oxidative Stress; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Superoxide Dismutase; Superoxide Dismutase-1; Toluene; Tumor Suppressor Protein p53

Autophagy is mainly responsible for the degradation of long-lived proteins and subcellular organelles. Autophagy is responsible for the non-apoptotic cell death, and plays a crucial role in regulating cellular functions. β-Lapachone is a quinone-containing compound originally obtained from the lapacho tree in South America. Here, we show that β-lapachone induces death in U87 MG cells, which is not inhibited by blockers of pan-caspase or necrosis. β-Lapachone-induced cell death gradually increased in a time-dependent manner in U87 MG cells, which were partly prevented by pretreatment of a specific inhibitor of NQO1 (dicoumarol). These results suggested that β-lapachone-induced cell death was mediated by NQO1-independent as well as NQO1-dependent cell death pathways. During progression of β-lapachone-induced cell death, translocation and processing of LC3 as well as an increase in acidic vesicular organelles, as assessed by acridine orange staining, were observed. Furthermore, β-lapachone-induced cell death was inhibited by either a knockdown of beclin-1/Atg-6 or Atg-7 gene expression or by autophagy inhibitors (3-methyl adenine or bafilomycin A1). Reactive oxygen species (ROS) were involved in β-lapachone-induced autophagic cell death of U87 MG glioma cells, because β-lapachone induced ROS production and antioxidant N-acetylcysteine (NAC) decreased autophagic cell death. Our results collectively demonstrate that ROS mediate β-lapachone-induced autophagic cell death in U87 MG glioma cells.

Topics: Adenine; Autophagy; Blotting, Western; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Flow Cytometry; Glioma; Humans; Macrolides; Microscopy, Fluorescence; NAD(P)H Dehydrogenase (Quinone); Naphthoquinones; Reactive Oxygen Species; RNA, Small Interfering

Beta-lapachone and camptothecin are structurally unrelated agents thought to inhibit topoisomerase-I activity through distinct mechanisms. We find that beta-lapachone is much more potent than camptothecin in inducing acute cytotoxic effects on human malignant glioma cells. Acute cytotoxicity induced by both drugs is apoptotic by electron microscopy, but not blocked by inhibitors of RNA or protein synthesis and not associated with changes in the expression of bcl-2, bax, p53, p21 or GADD45 proteins. In contrast, prolonged exposure of glioma cells to both drugs for 72 hr results in growth inhibition and apoptosis, with EC50 values around 1 microM. None of 7 glioma cell lines tested were resistant to either drug. LN-229 cells which have partial p53-wild-type activity show enhanced expression of p53, p21 and bax protein, whereas bcl-2 levels decrease, after exposure to camptothecin. In contrast, beta-lapachone increases bax protein expression in the absence of p53 activation. T98G cells are mutant for p53. In these cells, p53 levels do not change and p21 is not induced. bax accumulation in T98G cells is induced by both drugs, with bcl-2 levels unaltered. Surprisingly, ectopic expression of murine bcl-2 fails to abrogate the toxicity of either drug. Camptothecin, but not beta-lapachone, sensitizes human malignant glioma cells to apoptosis induced by the cytotoxic cytokines, tumor necrosis factor-alpha and CD95 ligand. Thus, both drugs have potent anti-glioma activity that may be mediated by enhanced bax expression but is not inhibited by ectopic bcl-2 expression. Camptothecin-like agents are particularly promising for immunochemotherapy of malignant glioma using cytotoxic drugs and CD95 ligand.

Topics: Apoptosis; bcl-2-Associated X Protein; Camptothecin; Cell Cycle Proteins; Cell Division; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; fas Receptor; Glioma; Humans; Immunotherapy; Naphthoquinones; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Topoisomerase I Inhibitors; Tumor Cells, Cultured; Tumor Suppressor Protein p53