wogonin and Glioma

Malignant gliomas are the most common and most aggressive primary brain tumor, and for which differentiation therapy has emerged as a promising candidate strategy. In this study, we used in vitro and in vivo assays to examine the differentiation effects of wogonin, a major active constituent of Scutellaria baicalensis, on glioma C6 and U251 cells. We found that wogonin can suppress cell proliferation and induce G0/G1 arrest under a concentration-dependent manner. Wogonin also triggered significant reduction in the G1 cell-cycle regulatory proteins cyclin D1, cyclin-dependent kinase 2 and 4 along with overexpression of cell-cycle inhibitory proteins p27. Immunofluorescence and western blot analysis indicated that wogonin increased the expression of lineage-specific differentiation marker glial fibrillary acidic protein (GFAP). In mechanisms, we verified that wogonin significantly diminished the phosphorylated level of protein kinase B (AKT), and maintenance of low β-catenin expression level was dependent on glycogen synthase kinase 3β (GSK3β) activation at Ser9. Blocking GSK3β/β-catenin pathway was required for wogonin-induced proliferation inhibition and terminal differentiation by using canonical activator lithium chloride (LiCl) and inhibitor dickkopf-1 (Dkk1). Moreover, intravenous administration of wogonin delayed the growth of C6 glioma in the intracranial tumor model. These findings provide the evidence and mechanistic support for wogonin-based differentiation therapies for malignant glioblastoma. Furthermore, inhibition of GSK3β/β-catenin pathway may be a key and requisite factor in glioma differentiation.

Topics: Animals; Antineoplastic Agents, Phytogenic; beta Catenin; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Drugs, Chinese Herbal; Flavanones; Glial Fibrillary Acidic Protein; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Neoplasm Transplantation; Nerve Tissue Proteins; Neurons; Random Allocation; Rats; Rats, Sprague-Dawley; Resting Phase, Cell Cycle; Signal Transduction

A number of studies have implicated tumor-induced T(reg) cell activity in the sub-optimal response to therapeutic vaccines. Development of neo-adjuvant strategies targeting T(reg) cells is therefore imperative. Scutellaria extracts or constituent flavonoids have shown encouraging efficacy against various tumors, including gliomas, in both pre-clinical and clinical studies. We report here, for the first time, that Scutellaria ocmulgee leaf extract (SocL) and flavonoid wogonin could inhibit TGF-β1-induced T(reg) activity in malignant gliomas. F344 rats, subcutaneously transplanted with F98 gliomas, were treated with SocL. There was a significant inhibition of intra-tumoral TGF-β1 and T(reg) cell frequency as well as peripheral blood TGF-β1 levels in SocL-treated animals compared to the controls. SocL extract and wogonin also inhibited glioma-induced, TGF-β1-mediated T(reg) activity in vitro. SocL extract and wogonin also inhibited the secretion of IL-10 in T(reg) culture; whereas the level of IL-2 was either unchanged or marginally enhanced. We also observed an inhibition of Smad-3, GSK-3β and ERK1/2 signaling by SocL and wogonin in T(reg) cells, while phosphorylation of P38 MAPK was considerably enhanced, indicating that SocL or wogonin could inhibit the T cells' response to TGF-β1 via modulation of both Smad and non-Smad signaling pathways. Overall, this study suggests that Scutellaria can potentially reverse tumor-mediated immune suppression via inhibition of TGF-β1 secretion as well as via inhibition of T cells' response to TGF-β1. This may provide an opportunity for developing a novel adjuvant therapeutic strategy for malignant gliomas, combining Scutellaria with immunotherapy and chemo/radio-therapeutic regimen, which could potentially improve the disease outcome.

Topics: Animals; Cell Line, Tumor; Flavanones; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Interleukin-10; Interleukin-2; MAP Kinase Signaling System; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Plant Extracts; Plant Leaves; Rats; Scutellaria; Signal Transduction; Smad3 Protein; T-Lymphocytes, Regulatory; Transforming Growth Factor beta1

Glioma is the most common primary adult brain tumor with poor prognosis because of the ease of spreading tumor cells to other regions of the brain. Cell apoptosis is frequently targeted for developing anti-cancer drugs. In the present study, we have assessed wogonin, a flavonoid compound isolated from Scutellaria baicalensis Georgi, induced ROS generation, endoplasmic reticulum (ER) stress and cell apoptosis. Wogonin induced cell death in two different human glioma cells, such as U251 and U87 cells but not in human primary astrocytes (IC 50 > 100 μM). Wogonin-induced apoptotic cell death in glioma cells was measured by propidine iodine (PI) analysis, Tunnel assay and Annexin V staining methods. Furthermore, wogonin also induced caspase-9 and caspase-3 activation as well as up-regulation of cleaved PARP expression. Moreover, treatment of wogonin also increased a number of signature ER stress markers glucose-regulated protein (GRP)-78, GRP-94, Calpain I, and phosphorylation of eukaryotic initiation factor-2α (eIF2α). Treatment of human glioma cells with wogonin was found to induce reactive oxygen species (ROS) generation. Wogonin induced ER stress-related protein expression and cell apoptosis was reduced by the ROS inhibitors apocynin and NAC (N-acetylcysteine). The present study provides evidence to support the fact that wogonin induces human glioma cell apoptosis mediated ROS generation, ER stress activation and cell apoptosis.

Topics: Apoptosis; Blotting, Western; Caspases; Cell Proliferation; Endoplasmic Reticulum Stress; Flavanones; Flow Cytometry; Glioma; Humans; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured

In an earlier study, we reported that nitric oxide is involved in lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate-induced malignant transformation via increases in metalloproteinase 9 enzyme activity and inducible nitric oxide synthase gene expression in rat glioma C6 cells, however the mechanism has remained undefined. Lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate, but not lipopolysaccharide or 12-o-tetradecanoylphorbol 13-acetate alone, induced transformation in glioma C6 cells (but not in human glioblastoma cells GBM-8401 cells) without affecting their viability. An increase in inducible nitric oxide synthase protein expression, nitric oxide production, and metalloproteinase 9 enzyme activity is identified lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-treated C6 cells, however lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate and 12-o-tetradecanoylphorbol 13-acetate (but not lipopolysaccharide) addition shows the similar inductive pattern on metalloproteinase 9 enzyme activity without affecting inducible nitric oxide synthase protein expression and nitric oxide production in GBM-8401 cells. Treatment of C6 cells with lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate increases the expression of phosphorylated extracellular regulated protein kinases and Jun N-terminal kinases, but not p38, proteins, and an addition of the extracellular regulated protein kinases inhibitor PD98059 or Jun N-terminal kinases inhibitors SP600125, but not the p38 inhibitor SB203580, significantly blocked lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced inducible nitric oxide synthase protein expression and metalloproteinase 9 enzyme activity accompanied by blocking morphological transformation in C6 cells. Among 19 structurally related flavonoids, kaempferol and wogonin exhibit significant inhibitory effects on lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced morphological transformation and colony formation, and attenuation of inducible nitric oxide synthase, phosphorylated extracellular regulated protein kinases protein expression, and metalloproteinase 9 enzyme activity was observed. 2'-OH flavone at a dose of 100 microM inhibition of lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced events via apoptosis induction is identified. Furthermore, lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate, but not lipopolysaccharide or 12-o-tetradecanoylphorbol 13-acetate, induces tumoral i

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Carcinogens; Cell Movement; Cell Transformation, Neoplastic; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Flavanones; Flavonoids; Glioma; Humans; Kaempferols; Lipopolysaccharides; MAP Kinase Signaling System; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Nitric Oxide; Nitric Oxide Synthase; Rats; Tetradecanoylphorbol Acetate

Lipopolysaccharide (LPS) from Gram-negative bacterial has been identified as an important molecule involved in the inflammatory process through inducing nitric oxide (NO) production. However, the effect of LPS in carcinogenesis is still undefined. In the present study, the biological effect of LPS was examined in 12-o-tetradecanoylphorbol 13-acetate (TPA)-treated rat glioma cells C6. Results of MTT assay showed that LPS and TPA exhibited no significant cytotoxicity in glioma C6 cells. Interestingly, transformation foci were found in LPS/TPA-treated glioma C6 cells, but not in LPS- or TPA-treated cells. The transformation foci induced by LPS/TPA were also observed in the absence of serum. It indicates that induction of transformation foci formation by LPS and TPA is independent on the serum in glioma C6 cells. Induction of iNOS gene expression and NO production was examined in LPS/TPA-treated cells, but not obvious in LPS- or TPA-treated cells. NO donor sodium nitroprusside (SNP) induces transformation in glioma C6 cells in according with elevating NO production. In addition, LPS/TPA induces metalloproteinase 9 (MMP9) activity by gelatin activity assay in gel. Wogonin and quercetin but not rutin, inhibitors of iNOS gene expression and NO production induced by LPS, showed the significant inhibition on LPS/TPA-induced transformation foci formation, accompanied by inhibiting iNOS gene expression, NO production and MMP9 activity. Results of the present study provide scientific evidences to link the inflammatory responses and carcinogenesis, and suggest that NO derived from inflammation may contribute to the progression of carcinogenesis; natural products with anti-inflammatory effects such as wogonin and quercetin possess the ability to block transformation induced by LPS/TPA.

Topics: Animals; Carcinogens; Cell Line, Tumor; Cell Survival; Cell Transformation, Neoplastic; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Induction; Enzyme Inhibitors; Flavanones; Gene Expression; Glioma; Lipopolysaccharides; Matrix Metalloproteinase 9; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Quercetin; Rats; Tetradecanoylphorbol Acetate

Inhibitory mechanism of the water extract of Scutellariae Radix on prostaglandin E2 (PGE2) release was examined in C6 rat glioma cells. Scutellariae Radix reduced a Ca2+ ionophore A23187-induced PGE2 release by inhibition of arachidonic acid (AA) liberation. Sho-saiko-to and San'o-shashin-to, which contain Scutellariae Radix, also inhibited PGE2 release. A23187 caused phosphorylation of mitrogen-activated protein kinase (MAPK), resulting in activation of cytosolic phospholipase A2 (cPLA2). Scutellariae Radix and baicalein inhibited the phosphorylation of MAPK. Baicalein, but not baicalin, inhibited A23187-induced PGE2 release. These results suggest that baicalein in Scutellariae Radix reduces AA liberation through the inhibition of the MAPK-cPLA2 pathway.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Arachidonic Acid; Calcimycin; Calcium-Calmodulin-Dependent Protein Kinases; Dinoprostone; Drugs, Chinese Herbal; Enzyme Inhibitors; Flavanones; Flavonoids; Glioma; Ionophores; Molecular Sequence Data; Phosphorylation; Prostaglandin Antagonists; Rats; Tumor Cells, Cultured