phenanthrenes and Brain-Neoplasms

Etoposide (VP-16) is the topoisomerase 2 (Top2) inhibitor used for treating of glioma patients however at high dose with serious side effects. It induces DNA double-strand breaks (DSBs). These DNA lesions are repaired by non-homologous DNA end joining (NHEJ) mediated by DNA-dependent protein kinase (DNA-PK). One possible approach to decrease the toxicity of etoposide is to reduce the dose while maintaining the anticancer potential. It could be achieved through combined therapy with other anticancer drugs. We have assumed that this objective can be obtained by (1) a parallel topo2 α inhibition and (2) sensitization of cancer cells to DSBs. In this work we investigated the effect of two Top2 inhibitors NK314 and VP-16 in glioma cell lines (MO59 K and MO59 J) sensitized by DNA-PK inhibitor, NU7441. Cytotoxic effect of VP-16, NK314 alone and in combination on human glioblastoma cell lines, was assessed by a colorimetric assay. Genotoxic effect of anticancer drugs in combination with NU7441 was assessed by comet assay. Cell cycle distribution and apoptosis were analysed by flow cytometry. Compared with VP-16 or NK314 alone, the combined treatment significantly inhibited cell proliferation. Combination treatment was associated with a strong accumulation of DSBs, modulated cell cycle phases distribution and apoptotic cell death. NU7441 potentiated these effects and additionally postponed DNA repair. Our findings suggest that NK314 could overcome resistance of MO59 cells to VP-16 and NU7441 could serve as sensitizer to VP-16/NK314 combined treatment. The combined tripartite approach of chemotherapy could reduce the overall toxicity associated with each individual therapy, while concomitantly enhancing the anticancer effect to treat human glioma cells. Thus, the use of a tripartite combinatorial approach could be promising and more efficacious than mono therapy or dual therapy to treat and increase the survival of the glioblastoma patients.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Chromones; DNA-Activated Protein Kinase; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Inhibitors; Etoposide; Glioblastoma; Humans; Morpholines; Phenanthrenes

A rapid and reproducible method with high selectivity was developed for simultaneous determination of a promising anti-brain tumor agent CAT3 and its two metabolites PF403 and GLU-PF403 in mouse plasma and brain. An economic deproteinization with septuple acetonitrile (v/v) was applied to pretreat the samples in this study. All analytes were well retained and separated on a CAPCELL CORE PC (2.7 μm, 2.1 mm I.D. × 150 mm, SHISEIDO Technologies) column with an eluting solvent of acetonitrile /water containing 0.1 % formic acid (v/v) at the flow rate of 0.2 mL per minute. The detection was carried out on a Q Exactive high resolution mass spectrometer equipped with a HESI ion source in parallel reaction monitoring (PRM) mode. The corresponding transitions for quantitation were 434.23→ 70.07 for CAT3, 350.17→70.07 for PF403, 526.21→70.07 for GLU-PF403, 364.19→70.07 for IS-1 and 625.18→317.07 for IS-2, respectively. A well-linear fit curve was achieved among the range of 0.1∼50 ng/mL for CAT3, 0.2∼100 ng/mL for PF403 and 2.5∼600 ng/mL for GLU-PF403 both in mouse plasma and brain homogenate. The intra-/inter-day accuracies of three analytes were within ±14.5 % and precisions were below to 13.44 %. The mean values of recovery of three compounds in mouse plasma and brain homogenate were among 98.06 ∼ 118.63 % and 81.04∼108.69 %. The analytes in NaF-treated ice cold blood of mouse was stable within tested 30 min. Plasma and brain homogenate samples had no obvious changes during all storage, sample treatment and analytic process of mouse plasma sample. The reproducible and reliable method was well employed to the research of CAT3 pharmacokinetic characteristics in mouse plasma and brain after a single intragastric administration at dose of 10 mg/kg.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Brain; Brain Neoplasms; Chromatography, High Pressure Liquid; Drug Screening Assays, Antitumor; Humans; Indolizidines; Limit of Detection; Mice; Phenanthrenes; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Tissue Distribution

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Asarum; Brain Neoplasms; Caspases; Cell Line, Tumor; Cell Nucleus; Enzyme Activation; Humans; Molecular Structure; Neuroblastoma; Phenanthrenes; Plant Roots; X-Ray Diffraction

Topoisomerase II (Topo2) inhibitors in combination with cisplatin represent a common treatment modality used for glioma patients. The main mechanism of their action involves induction of DNA double-strand breaks (DSBs). DSBs are repaired via the homology-dependent DNA repair (HRR) and non-homologous end-joining (NHEJ). Inhibition of the NHEJ or HRR pathway sensitizes cancer cells to the treatment. In this work, we investigated the effect of three Topo2 inhibitors-etoposide, NK314, or HU-331 in combination with cisplatin in the U-87 human glioblastoma cell line. Etoposide as well as NK314 inhibited Topo2 activity by stabilizing Topo2-DNA cleavable complexes whereas HU-331 inhibited the ATPase activity of Topo2 using a noncompetitive mechanism. To increase the effectiveness of the treatment, we combined cisplatin and Topo2 inhibitor treatment with DSB repair inhibitors (DRIs). The cells were sensitized with NHEJ inhibitor, NU7441, or the novel HRR inhibitor, YU238259, prior to drug treatment. All of the investigated Topo2 inhibitors in combination with cisplatin efficiently killed the U-87 cells. The most cytotoxic effect was observed for the cisplatin + HU331 treatment scheme and this effect was significantly increased when a DRI pretreatment was used; however, we did not observed DSBs. Therefore, the molecular mechanism of cytotoxicity caused by the cisplatin + HU331 treatment scheme is yet to be evaluated. We observed a concentration-dependent change in DSB levels and accumulation at the G2/M checkpoint and S-phase in glioma cells incubated with NK314/cisplatin and etoposide/cisplatin. In conclusion, in combination with cisplatin, HU331 is the most potent Topo2 inhibitor of human glioblastoma cells.

Topics: Apoptosis; Benzamides; Brain Neoplasms; Cannabidiol; Cell Cycle; Cell Line, Tumor; Chromones; Cisplatin; DNA Breaks, Double-Stranded; DNA Repair; Etoposide; Glioblastoma; Humans; Morpholines; Phenanthrenes; Sulfonamides; Topoisomerase II Inhibitors

Among primary brain cancers, gliomas are the most deadly and most refractory to current treatment modalities. Previous reports overwhelmingly support the role of the RNA-binding protein Hu antigen R (HuR) as a positive regulator of glioma disease progression. HuR expression is consistently elevated in tumor tissues, and a cytoplasmic localization appears essential for HuR-dependent oncogenic transformation. Here, we report HuR aggregation (multimerization) in glioma and the analysis of this tumor-specific HuR protein multimerization in clinical brain tumor samples. Using a split luciferase assay, a bioluminescence resonance energy transfer technique, and site-directed mutagenesis, we examined the domains involved in HuR multimerization. Results obtained with the combination of the split HuR luciferase assay with the bioluminescence resonance energy transfer technique suggested that multiple (at least three) HuR molecules come together during HuR multimerization in glioma cells. Using these data, we developed a model of HuR multimerization in glioma cells. We also demonstrate that exposing glioma cells to the HuR inhibitor tanshinone group compound 15,16-dihydrotanshinone-I or to the newly identified compound 5 disrupts HuR multimerization modules and reduces tumor cell survival and proliferation. In summary, our findings provide new insights into HuR multimerization in glioma and highlight possible pharmacological approaches for targeting HuR domains involved in cancer cell-specific multimerization.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cell Transformation, Neoplastic; ELAV-Like Protein 1; Furans; Glioma; Humans; Neoplasm Proteins; Phenanthrenes; Protein Aggregation, Pathological; Protein Domains; Quinones

Inhibitors of PARP-1(Poly(ADP-ribose) polymerase-1) act by competing with NAD(+), the enzyme physiological substrate, which play a protective role in many pathological conditions characterized by PARP-1 overactivation. It has been shown that PARP-1 also promotes tumor growth and progression through its DNA repair activity. Since angiogenesis is an essential requirement for these activities, we sought to determine whether PARP inhibition might affect rat brain microvascular endothelial cells (GP8.3) migration, stimulated by C6-glioma conditioned medium (CM). Through wound-healing experiments and MTT analysis, we demonstrated that PARP-1 inhibitor PJ-34 [N-(6-Oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide] abolishes the migratory response of GP8.3 cells and reduces their viability. PARP-1 also acts in a DNA independent way within the Extracellular-Regulated-Kinase (ERK) signaling cascade, which regulates cell proliferation and differentiation. By western analysis and confocal laser scanning microscopy (LSM), we analyzed the effects of PJ-34 on PARP-1 expression, phospho-ERK and phospho-Elk-1 activation. The effect of MEK (mitogen-activated-protein-kinase-kinase) inhibitor PD98059 (2-(2-Amino-3-methoxyphenyl)-4 H-1-benzopyran-4-one) on PARP-1 expression in unstimulated and in CM-stimulated GP8.3 cells was analyzed by RT-PCR. PARP-1 expression and phospho-ERK activation were significantly reduced by treatment of GP8.3 cells with PJ-34 or PD98059. By LSM, we further demonstrated that PARP-1 and phospho-ERK are coexpressed and share the same subcellular localization in GP8.3 cells, in the cytoplasm as well as in nucleoplasm. Based on these data, we propose that PARP-1 and phospho-ERK interact in the cytosol and then translocate to the nucleus, where they trigger a proliferative response. We also propose that PARP-1 inhibition blocks CM-induced endothelial migration by interfering with ERK signal-transduction pathway.

Topics: Active Transport, Cell Nucleus; Animals; Brain; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Culture Media, Conditioned; Endothelial Cells; ets-Domain Protein Elk-1; Extracellular Signal-Regulated MAP Kinases; Glioma; Microvessels; Paracrine Communication; Phenanthrenes; Phosphorylation; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Binding; Protein Kinase Inhibitors; Rats; Signal Transduction

Neuroblastoma is the most common cancer in infants and the fourth most common cancer in children. Our previous study showed that PF403 had a potent antitumor ability. In the present study, we evaluated the anti-neuroblastoma property of PF403 and investigated the underlying mechanisms. MTT assay, colony formation assay and flow cytometry assay were used to assess cytotoxicity of PF403 on SH-SY5Y cells. Transwell assay was chosen to estimate the anti-invasion ability of PF403 on neuroblastoma cells. The protein expression was detected by western blot analysis. The SH-SY5Y brain xenograft model was used to assess in vivo antitumor activity of PF403. PF403-mediated SH-SY5Y cell death was found to be dose- and time-dependent, and PF403 was able to limit invasion and metastasis of neuroblastoma cells. MRI and pathology analysis proved that the pro-drug of PF403, CAT3, inhibited SH-SY5Y cells in vivo. PF403 decreased expression of phosphorylated FAK, MMP-2 and MMP-9 proteins, and downregulated the activity of PI3K/AKT and Raf/ERK pathways, followed by regulation of the proteins expression of Bcl-2 family, activated caspase-3, -9 and PARP and initiation of apoptosis of neuroblastoma cells. PF403 exerted cytotoxicity against SH-SY5Y neuroblastoma cell both in vitro and in vivo, and inhibited its invasion ability, suggesting PF403 has potential as a new anticancer drug for the treatment of neuroblastoma.

Topics: Animals; Antineoplastic Agents; Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Indolizidines; Indolizines; Mice; Neoplasm Invasiveness; Neuroblastoma; Phenanthrenes; Prodrugs; Xenograft Model Antitumor Assays

Malignant gliomas (MGs) are among the most aggressive types of cancers in the human brain. Frequent tumor recurrence caused by a lack of effective therapeutic approaches results in a poor prognosis. Signal transducer and activator of transcription 3 (STAT3), an oncogenic protein, is constitutively activated in MGs and predicts a poor clinical outcome. STAT3 therefore is considered to be a promising target for the treatment of MGs. Cryptotanshinone (CTS), the main bioactive compound from the root of Salvia miltiorrhiza Bunge, has been reported to have various pharmacological effects. However, little is known about its function in MG cells. In this study, we evaluated the effect of CTS on the proliferation of human glioma cell lines (T98G and U87). Our results revealed that CTS significantly suppresses glioma cell proliferation. The phosphorylation of STAT3 Tyr705, but not Ser727, was inhibited by CTS, and STAT3 nuclear translocation was attenuated. Overexpression of constitutively active mutant STAT3C reversed the inhibitory effect of CTS, while knockdown STAT3 showed a similar inhibitory effect as CTS treatment. Following the downregulation of STAT3-regulated proteins cyclinD1 and survivin, cell cycle progression significantly arrested in G1/G0 phase. These results indicate that CTS may be a potential antiproliferation agent for the treatment of MGs and that its mechanism may be related to the inhibition of STAT3 signaling.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; G1 Phase; Glioma; Humans; Mutant Proteins; Phenanthrenes; Phosphorylation; Phosphotyrosine; Rats; RNA, Small Interfering; S Phase; Signal Transduction; STAT3 Transcription Factor

Poly(ADP-ribose) polymerases (PARPs) are recognized as key regulators of cell survival or death. PARP-1 is essential to the repair of DNA single-strand breaks via the base excision repair pathway. The enzyme may be overactivated in response to inflammatory cues, thus depleting cellular energy pools and eventually causing cell death. Accordingly, PARP-1 inhibitors, acting by competing with its physiological substrate NAD(+), have been proposed to play a protective role in a wide range of inflammatory and ischemia/reperfusion-associated diseases. Recently, it has also been reported that PARP-1 regulates proinflammatory mediators, including cytokines, chemokines, adhesion molecules, and enzymes (e.g., iNOS). Furthermore, PARP-1 has been shown to act as a coactivator of NF-κB- and other transcription factors implicated in stress/inflammation, as AP-1, Oct-1, SP-1, HIF, and Stat-1. To further substantiate this hypothesis, we tested the biomolecular effects of PARP-1 inhibitors DPQ and PJ-34 on human glioblastoma cells, induced to a proinflammatory state with lipopolysaccharide and Interferon-γ. PARP-1 expression was evaluated by laser scanning confocal microscopy immunofluorescence (LSM); nitrite production, LDH release and cell viability were also determined. LSM of A-172, SNB-19 and CAS-1 cells demonstrated that DPQ and PJ-34 downregulate PARP-1 expression; they also cause a decrease of LDH release and nitrite production, while increasing cell viability. Similar effects were caused in all three cell lines by N-mono-methyl-arginine, a well known iNOS inhibitor, and by L-carnosine and trehalose, two antioxidant molecules. These results demonstrate that, similar to other well characterized drugs, DPQ and PJ-34 reduce cell inflammation and damage that follow PARP-1 overexpression, while they increase cell survival: this suggests their potential exploitation in clinical Medicine.

Topics: Anti-Inflammatory Agents; Biomarkers; Brain Neoplasms; Carnosine; Cell Line, Tumor; Cell Survival; Coloring Agents; Down-Regulation; Fluorescent Antibody Technique; Glioblastoma; Humans; Isoquinolines; L-Lactate Dehydrogenase; Microscopy, Confocal; Nitrites; Phenanthrenes; Piperidines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Tetrazolium Salts; Thiazoles; Trehalose

Malignant glioma is the most devastating and aggressive tumor in brain, characterized by rapid proliferation and diffuse invasion. Chemotherapy and radiotherapy are the pivotal strategies after surgery; however, high drug resistance of malignant glioma and the blood-brain barrier usually render chemotherapy drugs ineffective. Here, we find that triptolide, a small molecule with high lipid solubility, is capable of inhibiting proliferation and invasion of malignant glioma cells effectively. In both investigated malignant glioma cell lines, triptolide repressed cell proliferation via inducing cell cycle arrest in G0/G1 phase, associated with downregulation of G0/G1 cell cycle regulators cyclin D1, CDK4, and CDK6 followed by reduced phosphorylation of retinoblastoma protein (Rb). In addition, triptolide induced morphological change of C6 cells through downregulation of protein expression of MAP-2 and inhibition of activities of GTPases Cdc42 and Rac1/2/3, thus significantly suppressing migratory and invasive capacity. Moreover, in an in vivo tumor model, triptolide delayed growth of malignant glioma xenografts. These findings suggest an important inhibitory action of triptolide on proliferation and invasion of malignant glioma, and encourage triptolide as a candidate for glioma therapy.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Blotting, Western; Brain Neoplasms; cdc42 GTP-Binding Protein; Cell Cycle; Cell Movement; Cell Proliferation; Cyclin-Dependent Kinases; Diterpenes; Epoxy Compounds; Female; Flow Cytometry; Glioma; Mice; Mice, Nude; Neoplasm Invasiveness; Phenanthrenes; rac GTP-Binding Proteins; rac1 GTP-Binding Protein; RAC2 GTP-Binding Protein; Rats; Retinoblastoma Protein; Tumor Cells, Cultured

Tanshinone IIA is a derivative of phenanthrene-quinone isolated from Danshen, a widely used Chinese herbal medicine. It has antioxidant properties, cytotoxic activities against multiple human cancer cells, inducing apoptosis and differentiation of some human cancer cells. The purpose of this study is to confirm its anticancer activity on human glioma cells, and to elucidate mechanism of its activity. Human glioma cells were tested in vitro for cytotoxicity, colony formation inhibition, BrdU incorporation after treatment with tanshinone IIA. Its effect of apoptosis induction was detected through EB/AO staining, cell cycle analysis and the expressions of ADPRTL1 and CYP1A1 genes, the differentiation induction effect was investigated through morphology, mRNA and protein expressions of GFAP and nestin genes by RT-PCR and immunocytochemistry. Tanshinone IIA demonstrated a dose- and time-dependent inhibitory effect on cell growth, IC(50) was 100 ng/ml, and it significantly inhibited colony formation and BrdU incorporation of human glioma cells. After treatment with 25-100 ng/ml of tanshinone IIA, the apoptotic cells increased significantly (P < 0.01), the cells in G(0)/G(1) phase increased (P < 0.01), and decreased in S phase, ADPRTL1 and CYP1A1 mRNA expression increased 1-2 folds. The cells treated with 100 ng/ml tanshinone IIA demonstrated astrocytes or neuron-like morphology, GFAP mRNA and protein expressions increased, nestin mRNA and protein expressions decreased significantly. The findings in this study suggested that tanshinone IIA exhibited strong effects on growth inhibition and induction of apoptosis and differentiation in human glioma cells. It might serve as a novel promising differentiation-inducing and/or therapeutic agent for human gliomas, and need to be investigated further.

Topics: Abietanes; Analysis of Variance; Antineoplastic Agents, Phytogenic; Apoptosis; Brain Neoplasms; Cell Differentiation; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Glial Fibrillary Acidic Protein; Glioma; Humans; Intermediate Filament Proteins; Nerve Tissue Proteins; Nestin; Phenanthrenes; RNA, Messenger; Statistics, Nonparametric; Tumor Cells, Cultured

(1) A new human glioblastoma multiforme (GBM) cell line, WJ1, was established from the tissue derived from a 29-year-old patient diagnosed with a grade IV GBM. (2) The WJ1 cell line has been subcultured for more than 80 passages in standard culture media without feeder layer or collagen coatings. (3) GBM cells grow in vitro with distinct morphological appearance. Ultrastructural examination revealed large irregular nuclei and pseudo-inclusion bodies in nuclei. The cytoplasm contained numerous immature organelles and a few glia filaments. Growth kinetic studies demonstrated an approximate population doubling time of 60 h and a colony forming efficiency of 4.04%. The karyotype of the cells was hyperdiploid, with a large subpopulation of polyploid cells. Drug sensitivities of DDP, VP-16, tanshinone IIA of this cell line were assayed. They showed a dose- and time-dependent growth inhibition effect on the cells. (4) Orthotopic transplantation of GBM cells into athymic nude mice induced the formation of solid tumor masses about 6 weeks. The cells obtained from mouse tumor masses when cultivated in vitro had the same morphology and ultrastructure as those of the initial cultures. (5) This cell line may provide a useful model in vitro and in vivo in the cellular and molecular studies as well as in testing novel therapies for human glioblastoma multiforme.

Topics: Abietanes; Adult; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Brain Neoplasms; Cell Culture Techniques; Cell Division; Cell Line, Tumor; Cisplatin; Etoposide; Glial Fibrillary Acidic Protein; Glioblastoma; Humans; Intermediate Filament Proteins; Karyotyping; Male; Mice; Mice, Nude; Nerve Tissue Proteins; Nestin; Phenanthrenes; Xenograft Model Antitumor Assays