shikonin and Glioma

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

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

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

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

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

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