butylidenephthalide and Glioblastoma

Intramedullary spinal glioblastoma multiforme (GBM) tends to recur within 11 months of surgical resection, even after adjuvant chemoradiation therapy. Treatment options for recurrent spinal GBM are often limited. (Z)-n-butylidenephthalide [(Z)-BP] is a natural compound that induces apoptosis, antiproliferation, anti-invasion and antistemness effects in GBM cells. The Cerebraca wafer consists of (Z)-BP within a biodegradable wafer that can be implanted in the parenchyma of the central nervous system to treat high-grade glioma. We present a 44-year-old woman with a recurrent spinal GBM who underwent microscopic surgical tumor excision under fluorescein sodium guidance and intraoperative neurophysiologic monitoring. Four Cerebraca wafers were implanted into the cord and intradural space during the operation. MRI revealed that both tumor volume and spinal cord edema had decreased 4 days after surgery; both had substantially decreased 16 months after surgery. Neurologic functions and quality of life were improved after salvage therapy. No adverse events were reported. Cerebraca wafer implantation during surgical re-excision of spinal GBM may be a novel therapeutic approach for reduction of the tumor size and subsequent spinal cord edema with no toxicity to the spinal cord.

Topics: Adult; Brain Neoplasms; Cervical Vertebrae; Delayed-Action Preparations; Female; Glioblastoma; Humans; Phthalic Anhydrides; Polymers; Quality of Life; Spinal Cord Neoplasms

Although butylidenephthalide (BP) is an efficient anticancer drug, its poor bioavailability renders it ineffective for treating drug-resistant brain tumors. However, this problem is overcome through the use of noninvasive delivery systems, including intranasal administration. Herein, the bioavailability, drug stability, and encapsulation efficiency (EE, up to 95%) of BP were improved by using cyclodextrin-encapsulated BP in liposomal formulations (CDD1). The physical properties and EE of the CDD1 system were investigated via dynamic light scattering, transmission electron microscopy, UV-Vis spectroscopy, and nuclear magnetic resonance spectroscopy. The cytotoxicity was examined via MTT assay, and the cellular uptake was observed using fluorescence microscopy. The CDD1 system persisted for over 8 h in tumor cells, which was a considerable improvement in the retention of the BP-containing cyclodextrin or the BP-containing liposomes, thereby indicating a higher BP content in CDD1. Nanoscale CDD1 formulations were administered intranasally to nude mice that had been intracranially implanted with temozolomide-resistant glioblastoma multiforme cells, resulting in increased median survival time. Liquid chromatography-mass spectrometry revealed that drug biodistribution via intranasal delivery increased the accumulation of BP 10-fold compared to oral delivery methods. Therefore, BP/cyclodextrin/liposomal formulations have potential clinical applications for treating drug-resistant brain tumors.

Topics: Animals; Antineoplastic Agents; Biological Availability; Brain; Brain Neoplasms; Cyclodextrins; Drug Delivery Systems; Glioblastoma; Liposomes; Male; Mice, Inbred BALB C; Mice, Nude; Phthalic Anhydrides; Tissue Distribution

Glioblastoma (GBM) is one of the most common and aggressive types of brain tumor. Due to its highly recurrent rate and poor prognosis, the overall survival time with this type of tumor is only 20-21 months. Recent knowledge suggests that its recurrence is in part due to the presence of cancer stem cells (CSCs), which display radioresistant, chemoresistant, self-renewal and tumorigenic potential. Enhancers of Zeste 2 (EZH2) and AXL receptor tyrosine kinase (AXL) are both highly expressed in GBM. Additionally, they are an essential regulator involved in CSCs maintenance, migration, invasion, epithelial-to-mesenchymal transition (EMT), stemness, metastasis and patient survival. In this study, we used a small molecule, n-butylidenephthalide (BP), to assess the anti-GBM stem-like cells potential, and then tried to find out the associated genes involved with regulation in migration and invasion. We demonstrated that BP reduced the expression of AXL and stemness related genes in a dose-dependent manner. The migratory and invasive capabilities of GBM stem-like cells could be reduced by AXL/EZH2. Finally, in the overexpression of AXL, EZH2 and Sox2 by transfection in GBM stem-like cells, we found that AXL/EZH2/TGF-ꞵ1, but not Sox2, might be a key regulator in tumor invasion, migration and EMT. These results might help in the development of a new anticancer compound and can be a target for treating GBM.

Topics: Axl Receptor Tyrosine Kinase; Biomarkers; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Self Renewal; Enhancer of Zeste Homolog 2 Protein; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Models, Biological; Neoplastic Stem Cells; Phenotype; Phthalic Anhydrides; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumour. The neoplasms are difficult to resect entirely because of their highly infiltration property and leading to the tumour edge is unclear. Gliadel wafer has been used as an intracerebral drug delivery system to eliminate the residual tumour. However, because of its local low concentration and short diffusion distance, patient survival improves non-significantly. Axl is an essential regulator in cancer metastasis and patient survival. In this study, we developed a controlled-release polyanhydride polymer loading a novel small molecule, n-butylidenephthalide (BP), which is not only increasing local drug concentration and extending its diffusion distance but also reducing tumour invasion, mediated by reducing Axl expression. First, we determined that BP inhibited the expression of Axl in a dose- and time-dependent manner and reduced the migratory and invasive capabilities of GBM cells. In addition, BP downregulated matrix metalloproteinase activity, which is involved in cancer cell invasion. Furthermore, we demonstrated that BP regulated Axl via the extracellular signal-regulated kinases pathway. Epithelial-to-mesenchymal transition (EMT) is related to epithelial cells in the invasive migratory mesenchymal cells that underlie cancer progression; we demonstrated that BP reduced the expression of EMT-related genes. Furthermore, we used the overexpression of Axl in GBM cells to prove that Axl is a crucial target in the inhibition of GBM EMT, migration and invasion. In an in vivo study, we demonstrated that BP inhibited tumour growth and suppressed Axl expression in a dose-dependent manner according to a subcutaneous tumour model. Most importantly, in an intracranial tumour model with BP wafer in situ treatment, we demonstrated that the BP wafer not only significantly increased the survival rate but also decreased Axl expression, and inhibited tumour invasion. These results contribute to the development of a BP wafer for a novel therapeutic strategy for treating GBM invasion and increasing survival in clinical subjects.

Topics: Animals; Axl Receptor Tyrosine Kinase; Cell Line, Tumor; Cell Movement; Drug Delivery Systems; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Mice; Neoplasm Invasiveness; Neoplasm Metastasis; Phthalic Anhydrides; Polymers; Proto-Oncogene Proteins; Receptor Protein-Tyrosine Kinases; Xenograft Model Antitumor Assays

The natural compound n-butylidenephthalide (BP) can pass through the blood-brain barrier to inhibit the growth of glioblastoma multiforme tumors. However, BP has an unstable structure that reduces its antitumor activity and half-life in vivo.. The aim of this study is to design a drug delivery system to encapsulate BP to enhance its efficacy by improving its protection and delivery.. To protect its structural stability against protein-rich and peroxide solutions, BP was encapsulated into a lipo-PEG-PEI complex (LPPC). Then, the cytotoxicity of BP/LPPC following preincubation in protein-rich, acid/alkaline, and peroxide solutions was analyzed by MTT. Cell uptake of BP/LPPC was also measured by confocal microscopy. The therapeutic effects of BP/LPPC were analyzed in xenograft mice following intratumoral and intravenous injections.. When BP was encapsulated in LPPC, its cytotoxicity was maintained following preincubation in protein-rich, acid/alkaline, and peroxide solutions. The cytotoxic activity of encapsulated BP was higher than that of free BP (~4.5- to 8.5-fold). This increased cytotoxic activity of BP/LPPC is attributable to its rapid transport across the cell membrane. In an animal study, a subcutaneously xenografted glioblastoma multiforme mouse that was treated with BP by intratumoral and intravenous administration showed inhibited tumor growth. The same dose of BP/LPPC was significantly more effective in terms of tumor inhibition.. LPPC encapsulation technology is able to protect BP's structural stability and enhance its antitumor effects, thus providing a better tool for use in cancer therapy.

Topics: Animals; Antineoplastic Agents; Blood-Brain Barrier; Brain Neoplasms; Cell Line, Tumor; Drug Delivery Systems; Female; Glioblastoma; Humans; Immunohistochemistry; Injections, Intravenous; Liposomes; Male; Mice; Mice, Nude; Phthalic Anhydrides; Polyethylene Glycols; Polyethyleneimine; Protective Agents; Spectrometry, Fluorescence; Xenograft Model Antitumor Assays

Developing an effective drug for treating human glioblastoma multiform (GBM) has been investigated persistently. A pure compound butylidenephthalide (BP), isolated from Angelica sinensis, has been shown the activities to arrest the growth and initiate apoptosis of GBM in our previous reports. In this study, we further demonstrated that BP treatment accelerates the cell senescence in a dose-dependent manner in vitro and in vivo. S-phase kinase-associated protein 2 (Skp2), a proto-oncogene, is generally upregulated in cancer. We found that it was downregulated in BP-treated GBM cells. The downregulation of Skp2 is parallel with increasing p16 and p21 expression which causes G0/G1 arrest and tumor cell senescence. We also found that restoring the Skp2 protein level by exogenous overexpression prevents the BP-induced cell senescence. Therefore, the linkage between cell senescence and Skp2 expression is strengthened. Promoter binding analysis further detailed that the BP-mediated SP1 reduction might involve in the Skp2 downregulation. In summary, these results emphasize that BP-triggered senescence in GBM cells is highly associated with its control on Skp2 regulation.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cellular Senescence; Down-Regulation; Glioblastoma; Humans; Mice; Mice, Nude; Phthalic Anhydrides; Proto-Oncogene Mas; S-Phase Kinase-Associated Proteins; Sp1 Transcription Factor

In previous study, n-butylidenephthalide (BP), a natural compound from Angelica sinensis, has anti-glioblastoma multiform (GBM) cell effects. In this study, we modified BP structure to increase anti-GBM cell effects. The anti-GBM cell effects of one derivative of BP, (Z)-N-(2-(dimethylamino)ethyl)-2-(3-((3-oxoisobenzofuran-1(3H)-ylidene)methyl)phenoxy)acetamide (PCH4) were tested in vitro and in vivo.. MTT assay and PI/Annexin V assay were performed to evaluate the anti-GBM effects of PCH4. The Nur77 expression and translocation were assayed by RT-PCR and Western blot. The Nur77 siRNA was used to downregulate the Nur77 expression. The JNK inhibitor (SP600125) was used to block the JNK pathway.. The anti-GBM effect of PCH4 is four times more than BP. The IC(50) of PCH4 on DBTRG-05MG cells was 50 µg/ml. Nur77 expression and translocation from the nucleus to the cytoplasm were important in PCH4-induced apoptosis. Furthermore, the downregulation of PCH4-induced Nur77 expression by Nur77 siRNA reduced PCH4-induced apoptosis. In addition, PCH4-induced apoptosis was associated with the JNK pathway. The JNK inhibitor, SP600125, inhibited Nur77 mRNA expression and reduced PCH4-induced apoptosis.. In conclusion, PCH4, a derivative of BP, induced Nur77-mediated apoptosis via the JNK pathway and this mechanism, which is different from that of BP, may explain the increase in the anti-tumor effects on GBM.

Topics: Angelica sinensis; Animals; Apoptosis; Benzofurans; Blotting, Western; Brain Neoplasms; Cell Proliferation; DNA-Binding Proteins; Ethylamines; Flow Cytometry; Glioblastoma; Humans; Luciferases; MAP Kinase Kinase 4; Mice; Mice, Nude; Nuclear Receptor Subfamily 4, Group A, Member 1; Nuclear Receptor Subfamily 4, Group A, Member 2; Phthalic Anhydrides; Protein Transport; Receptors, Steroid; Receptors, Thyroid Hormone; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Telomerase is widely expressed in most human cancers, but is almost undetectable in normal somatic cells and is therefore a potential drug target. Using the human telomerase promoter platform, the naturally occurring compound butylidenephthalide (BP) was selected for subsequent investigation of antitumor activity in vitro and in vivo.. We treated human glioblastoma cells with BP and found a dose-dependent decrease in human telomerase reverse transcriptase (hTERT) mRNA expression and a concomitant increase in p16 and p21 expression. Because c-Myc and Sp1 are involved in transcriptional regulation of hTERT, the effect of BP on c-Myc and Sp1 expression was examined.. Using electrophoretic mobility shift assays and western blotting, we showed that BP represses hTERT transcriptional activity via downregulation of Sp1 expression. Using the telomerase repeat amplification protocol, an association between BP concentration and suppression of telomerase activity, induction of human glioblastoma senescence, and inhibition of cellular proliferation was identified. This was supported by a mouse xenograft model, in which BP repressed telomerase and inhibited tumor proliferation, resulting in tumor senescence. Overexpression of hTERT restored telomerase activity in human glioblastoma cells and overcame replicative senescence.. These findings suggest that BP inhibits proliferation and induces senescence in human glioblastomas by downregulating hTERT expression and consequently telomerase activity. This is the first study to describe regulation of telomerase activity by BP in human glioblastomas.

Topics: Animals; Blotting, Western; Brain Neoplasms; Cell Proliferation; Cellular Senescence; Cyclin-Dependent Kinase Inhibitor p21; Electrophoretic Mobility Shift Assay; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, p16; Glioblastoma; Humans; Immunoenzyme Techniques; Mice; Mice, Nude; Phthalic Anhydrides; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Telomerase; Transcription, Genetic; Tumor Cells, Cultured

The natural compound n-butylidenephthalide (BP), which is isolated from the chloroform extract of Angelica sinensis, has been investigated for its antitumoral effects on glioblastoma multiform (GBM) brain tumors both in vitro and in vivo. To determine the mechanism of BP-induced growth arrest and apoptosis, we examined BP-induced changes in gene expression by microarray screening using human GBM brain tumor cells. This analysis identified several BP-inducible genes, including the nuclear receptors NOR-1, Nurr1, and Nur77. Among these genes, Nur77 is particularly interesting because it plays an important role in the apoptotic processes in various tumor cell lines. BP was able to increase Nur77 mRNA and protein expression in a time-dependent manner. After BP treatment in GBM 8401 cells, Nur77 translocated from the nucleus to the cytoplasm, the cytochrome c was released from the mitochondria, and caspase 3 became activated. Furthermore, using Nur77 promoter-luciferase assay, BP increased Nur77 was AP1 related. Inhibition of BP-induced Nur77 expression by Nur77 short interfering RNA blocked BP-induced apoptosis in GBM 8401 cells, suggesting that the induction of Nur77 negatively affected GBM 8401 cell survival. In summary, our results suggest that up-regulation of Nur77 may explain the antitumoral activity of BP in brain tumor cells.

Topics: Animals; Brain Neoplasms; Cell Line, Tumor; DNA-Binding Proteins; Gene Targeting; Glioblastoma; Humans; Mice; Nuclear Receptor Subfamily 4, Group A, Member 1; Phthalic Anhydrides; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Xenograft Model Antitumor Assays

The naturally-occurring compound, n-butylidenephthalide (BP), which is isolated from the chloroform extract of Angelica sinensis (AS-C), has been investigated with respect to the treatment of angina. In this study, we have examined the anti-tumor effects of n-butylidenephthalide on glioblastoma multiforme (GBM) brain tumors both in vitro and in vivo. In vitro, GBM cells were treated with BP, and the effects of proliferation, cell cycle and apoptosis were determined. In vivo, DBTRG-05MG, the human GBM tumor, and RG2, the rat GBM tumor, were injected subcutaneously or intracerebrally with BP. The effects on tumor growth were determined by tumor volumes, magnetic resonance imaging and survival rate. Here, we report on the potency of BP in suppressing growth of malignant brain tumor cells without simultaneous fibroblast cytotocixity. BP up-regulated the expression of Cyclin Kinase Inhibitor (CKI), including p21 and p27, to decrease phosphorylation of Rb proteins, and down-regulated the cell-cycle regulators, resulting in cell arrest at the G(0)/G(1) phase for DBTRG-05MG and RG2 cells, respectively. The apoptosis-associated proteins were dramatically increased and activated by BP in DBTRG-05MG cells and RG2 cells, but RG2 cells did not express p53 protein. In vitro results showed that BP triggered both p53-dependent and independent pathways for apoptosis. In vivo, BP not only suppressed growth of subcutaneous rat and human brain tumors but also, reduced the volume of GBM tumors in situ, significantly prolonging survival rate. These in vitro and in vivo anti-cancer effects indicate that BP could serve as a new anti-brain tumor drug.

Topics: Angelica sinensis; Animals; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Brain Neoplasms; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Drugs, Chinese Herbal; Glioblastoma; Humans; Male; Mice; Mice, Transgenic; NIH 3T3 Cells; Phthalic Anhydrides; Rats; Resting Phase, Cell Cycle; Survival Rate; Treatment Outcome