stilbenes and Glioblastoma

Autophagy, cellular senescence, programmed cell death and necrosis are key responses of a cell facing a stress. These effects are partly interconnected, but regulation of their mutual interactions is not completely clear. That regulation seems to be especially important in cancer cells, which have their own program of development and demand more nutrition and energy than normal cells. Glioblastoma multiforme (GBM) belongs to the most aggressive and most difficult to cure cancers, so studies on its pathogenesis and new therapeutic strategies are justified. Using an animal model, it was shown that autophagy is required for GBM development. Temozolomide (TMZ) is the key drug in GBM chemotherapy and it was reported to induce senescence, autophagy and apoptosis in GBM. In some GBM cells, TMZ induces small toxicity despite its significant concentration and GBM cells can be intrinsically resistant to apoptosis. Resveratrol, a natural compound, was shown to potentiate anticancer effect of TMZ in GBM cells through the abrogation G2-arrest and mitotic catastrophe resulting in senescence of GBM cells. Autophagy is the key player in TMZ resistance in GBM. TMZ can induce apoptosis due to selective inhibition of autophagy, in which autophagic vehicles accumulate as their fusion with lysosomes is blocked. Modulation of autophagic action of TMZ with autophagy inhibitors can result in opposite outcomes, depending on the step targeted in autophagic flux. Studies on relationships between senescence, autophagy and apoptosis can open new therapeutic perspectives in GBM.

Topics: Animals; Antineoplastic Agents, Alkylating; Apoptosis; Autophagy; Brain Neoplasms; Cellular Senescence; Dacarbazine; Disease Models, Animal; DNA Damage; Glioblastoma; Humans; Mice; Resveratrol; Stilbenes; Temozolomide

Glioblastoma is the most common and the deadliest brain cancer. The aim of this study was to analyze the impact of resveratrol and its five analogs: 3,4,4'-trimethoxy, 3,4,2'-trimethoxy, 3,4,2',4'-tetramethoxy, 3,4,2',6'-tetramethoxy and 3,4,2',4',6'-pentamethoxy-trans-stilbenes (MS) on human glioblastoma T98G cells.. The Parallel Artificial Membrane Permeation Assay (PAMPA) was used for the prediction of blood-brain barrier penetration ability of the tested stilbenes (PAMPA-BBB). MTT test was applied to analyze the cytotoxicity of the compounds, whereas their ability to inhibit Wnt/β-catenin target genes expression was verified using qPCR. The potential DNA demethylating properties of the analyzed compounds were tested by Methylation-Sensitive High Resolution Melting (MS-HRM). Cell cycle distribution was tested using Fluorescence-Activated Cell Sorting (FACS), whereas apoptosis was analyzed using FITC Annexin V/propidium iodide double staining assay and Western blot.. High blood-brain barrier permeability coefficient was obtained for both resveratrol as well as methoxy-stilbenes. Their ability to downregulate the expression of Wnt/β-catenin pathway-related genes was confirmed. The 4'-methoxy substituted derivatives showed higher activity, whereas 3,4,4'-tri-MS was the most potent Wnt/β-catenin pathway inhibitor. None of the compounds affected DNA methylation level of MGMT, SFRP1, or RUNX3, despite inducing moderate changes in the level of expression of epigenetic modifiers DNMT3B and TET1-3. Importantly, treatment with 3,4,4'-tri-MS and 3,4,2',4'-tetra-MS led to cycle arrest in the S phase and induced apoptosis.. Both, resveratrol, as well as its synthetic methoxy-derivatives, should be further studied as promising adjuvants in glioblastoma treatment.

Topics: Antioxidants; Apoptosis; beta Catenin; Cell Cycle Checkpoints; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Resveratrol; Stilbenes; Tumor Cells, Cultured; Wnt1 Protein

Glioblastoma multiforme (GBM) is characterized by aggressive infiltration and terrible lethality. The overwhelming majority of chemotherapeutic drugs fail to exhibit the desired treatment effects. Polydatin (PD), which was initially extracted from Polygonum cuspidatum, is distinguished for its outstanding cardioprotective, hepatoprotective, and renal protective effects, as well as significant anticancer activities. However, the anti-GBM effect of PD is unclear.. Cell proliferation and apoptosis after PD intervention were estimated using MTT, colony formation and flow cytometry assays in vitro, while wound-healing and Transwell assays were applied to assess cell migration and invasion. In addition, the anti-GBM effects of PD in vivo were detected in the subcutaneous tumor model of nude mice. Moreover, Western blot, immunofluorescence and immunohistochemical staining assays were employed to elaborate the relevant molecular mechanisms.. The present study demonstrated that PD repressed cell proliferation, migration, invasion and stemness and promoted apoptosis in GBM cells. Moreover, by correlating the molecular characteristics of cancer cells with different sensitivities to PD and employing diverse analytical methods, we ultimately verified that the cytotoxicity of PD was related to EGFR-AKT/ERK1/2/STAT3-SOX2/Snail signaling pathway inhibition, in which multiple components were vital therapeutic targets of GBM.. This work demonstrated that PD could inhibit proliferation, migration, invasion and stemness and induce apoptosis by restraining multiple components of the EGFR-AKT/ERK1/2/STAT3-SOX2/Snail signaling pathway in GBM cells.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; ErbB Receptors; Glioblastoma; Glucosides; Humans; MAP Kinase Signaling System; Mice, Inbred BALB C; Mice, Nude; Proto-Oncogene Proteins c-akt; Signal Transduction; Snail Family Transcription Factors; SOXB1 Transcription Factors; STAT3 Transcription Factor; Stilbenes

Glioblastoma (GBM) is a deadly brain tumor with a current mean survival of 12-15 months. Despite being a potent anti-cancer agent, the turmeric ingredient curcumin (C) has limited anti-tumor efficacy in vivo due to its low bioavailability. We have reported earlier a strategy involving the use two other polyphenols, epicatechin gallate (E) from green tea and resveratrol (R) from red grapes at a unique, synergistic molar ratio with C (C:E:R: 4:1:12.5, termed TriCurin) to achieve superior potency against HPV+ tumors than C alone at C:E:R (μM): 32:8:100 (termed 32 μM+ TriCurin). We have now prepared liposomal TriCurin (TrLp) and demonstrated that TrLp boosts activated p53 in cultured GL261 mouse GBM cells to trigger apoptosis of GBM and GBM stem cells in vitro. TrLp administration into mice yielded a stable plasma concentration of 210 nM C for 60 min, which, though sub-lethal for cultured GL261 cells, was able to cause repolarization of M2-like tumor (GBM)-associated microglia/macrophages to the tumoricidal M1-like phenotype and intra-GBM recruitment of activated natural killer cells. The intratumor presence of such tumoricidal immune cells was associated with concomitant suppression of tumor-load, and apoptosis of GBM and GBM stem cells. Thus, TrLp is a potential onco-immunotherapeutic agent against GBM tumors.

Topics: Animals; Biomarkers, Tumor; Catechin; Cell Line, Tumor; Curcumin; Disease Models, Animal; Drug Combinations; Drug Synergism; Glioblastoma; Humans; Immunophenotyping; Killer Cells, Natural; Liposomes; Lymphocytes, Tumor-Infiltrating; Macrophages; Mice; Microglia; Neoplastic Stem Cells; Resveratrol; Stilbenes; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Although our previous study revealed lumbar punctured resveratrol could remarkably prolong the survival of rats bearing orthotopic glioblastomas, it also suggested the administration did not completely suppress rapid tumour growth. These evidences led us to consider that the prognosis of tumour-bearing rats may be further improved if this treatment is used in combination with neurosurgery. Therefore, we investigated the effectiveness of the combined treatment on rat orthotopic glioblastomas.. Rat RG2 glioblastoma cells were inoculated into the brains of 36 rats. The rats were subjected to partial tumour removal after they showed symptoms of intracranial hypertension. There were 28 rats that survived the surgery, and these animals were randomly and equally divided into the control group without postoperative treatment and the LP group treated with 100 μl of 300 μM resveratrol via the LP route. Resveratrol was administered 24 h after tumour resection in 3-day intervals, and the animals received 7 treatments. The intracranial tumour sizes, average life span, cell apoptosis and STAT3 signalling were evaluated by multiple experimental approaches in the tumour tissues harvested from both groups.. The results showed that 5 of the 14 (35.7%) rats in the LP group remained alive over 60 days without any sign of recurrence. The remaining nine animals had a longer mean postoperative survival time (11.0 ± 2.9 days) than that of the (7.3 + 1.3 days; p < 0.05) control group. The resveratrol-treated tumour tissues showed less Ki67 labelling, widely distributed apoptotic regions, upregulated PIAS3 expression and reduced p-STAT3 nuclear translocation.. This study demonstrates that postoperative resveratrol administration efficiently improves the prognosis of rat advanced orthotopic glioblastoma via inhibition of growth, induction of apoptosis and inactivation of STAT3 signalling. Therefore, this therapeutic approach could be of potential practical value in the management of glioblastomas.

Topics: Animals; Apoptosis; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Intracranial Hypertension; Neoplasm Recurrence, Local; Prognosis; Rats; Resveratrol; Signal Transduction; STAT3 Transcription Factor; Stilbenes

Adenocarcinoma and glioblastoma cell lines express α7- and α9α10-containing nicotinic acetylcholine receptors (nAChRs), whose activation promotes tumor cell growth. On these cells, the triethylammoniumethyl ether of 4-stilbenol MG624, a known selective antagonist of α7 and α9α10 nAChRs, has antiproliferative activity. The structural analogy of MG624 with the mitocan RDM-4'BTPI, triphenylphosphoniumbutyl ether of pterostilbene, suggested us that molecular hybridization among their three substructures (stilbenoxy residue, alkylene linker, and terminal onium) and elongation of the alkylene linker might result in novel antitumor agents with higher potency and selectivity. We found that lengthening the ethylene bridge in the triethylammonium derivatives results in more potent and selective toxicity toward adenocarcinoma and glioblastoma cells, which was paralleled by increased α7 and α9α10 nAChR antagonism and improved ability of reducing mitochondrial ATP production. Elongation of the alkylene linker was advantageous also for the triphenylphosphonium derivatives resulting in a generalized enhancement of antitumor activity, associated with increased mitotoxicity.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Glioblastoma; Humans; Reactive Oxygen Species; Receptors, Nicotinic; Stilbenes

Arginine auxotrophy constitutes the Achilles' heel for several tumors, among them glioblastoma multiforme (GBM). Hence, arginine-depleting enzymes such as arginine deiminase (ADI) from Streptococcus pyogenes are promising for treatment of primary and maybe even refractory GBM. Based on our previous study in which ADI-susceptibility was shown on a panel of patient-derived GBM cell lines, we here aimed at deciphering underlying molecular mechanisms of ADI-mediated growth inhibition. We found that ADI (35 mU/mL) initially induces a cellular stress-response that is characterized by upregulation of genes primarily belonging to the heat-shock protein family. In addition to autophagocytosis, we show for the first time that senescence constitutes another cellular response mechanism upon ADI-treatment and that this bacterial enzyme is able to act as radiosensitizer (¼ cases). Long-term treatment schedules revealed no resistance development, with treated cells showing morphological signs of cell stress. Next, several combination strategies were employed to optimize ADI-based treatment. Simultaneous and sequential S. pyogenes ADI-based combinations included substances acting at different molecular pathways (curcumin, resveratrol, quinacrine, and sorafenib, 2 × 72 h treatment). Adding drugs to GBM cell lines (n = 4, including a matched pair of primary and recurrent GBM in one case) accelerated and potentiated ADI-mediated cytotoxicity. Autophagy was identified as the main cause of tumor growth inhibition. Of note, residual cells again showed classical signs of senescence in most combinations. Our results suggest an alternative treatment regimen for this fatal cancer type which circumvents many of the traditional barriers. Using the metabolic defect in GBM thus warrants further (pre-) clinical evaluation.

Topics: Autophagy; Bacterial Proteins; Cell Line, Tumor; Cell Survival; Cellular Senescence; Curcumin; Gamma Rays; Glioblastoma; Heat-Shock Proteins; Humans; Hydrolases; Quinacrine; Recombinant Proteins; Resveratrol; Stilbenes; Streptococcus pyogenes; Superoxide Dismutase; Up-Regulation

Glioblastoma-initiating cells play crucial roles in the origin, growth, and recurrence of glioblastoma multiforme. The elimination of glioblastoma-initiating cells is believed to be a key strategy for achieving long-term survival of glioblastoma patients due to the highly resistant property of glioblastoma-initiating cells to temozolomide. Resveratrol, a naturally occurring polyphenol, has been widely studied as a promising candidate for cancer prevention and treatment. Whether resveratrol could enhance the sensitivity of glioblastoma-initiating cells to temozolomide therapy has not yet been reported. Here, using patient-derived glioblastoma-initiating cell lines, we found that resveratrol sensitized glioblastoma-initiating cells to temozolomide both in vitro and in vivo. Furthermore, we showed that resveratrol enhanced glioblastoma-initiating cells to temozolomide-induced apoptosis through DNA double-stranded breaks/pATM/pATR/p53 pathway activation, and promoted glioblastoma-initiating cell differentiation involving p-STAT3 inactivation. Our results propose that temozolomide and resveratrol combination strategy may be effective in the management of glioblastoma patients, particularly for those patients who have been present with a high abundance of glioblastoma-initiating cells in their tumors and show slight responsiveness to temozolomide.

Topics: Apoptosis; Ataxia Telangiectasia Mutated Proteins; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Dacarbazine; Drug Synergism; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Neoplastic Stem Cells; Phosphorylation; Resveratrol; Signal Transduction; Stilbenes; Temozolomide; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Glioblastoma (GBM) is the most common malignant brain tumor, and glioma stem cells (GSCs) are considered a major source of treatment resistance for glioblastoma. Identifying new compounds that inhibit the growth of GSCs and understanding their underlying molecular mechanisms are therefore important for developing novel therapy for GBM.. We investigated the potential inhibitory effect of isorhapontigenin (ISO), an anticancer compound identified in our recent investigations, on anchorage-independent growth of patient-derived glioblastoma spheres (PDGS) and its mechanism of action.. ISO treatment resulted in significant anchorage-independent growth inhibition, accompanied with cell cycle G0-G1 arrest and cyclin D1 protein downregulation in PDGS. Further studies established that cyclin D1 was downregulated by ISO at transcription levels in a SOX2-dependent manner. In addition, ISO attenuated SOX2 expression by specific induction of miR-145, which in turn suppressed 3'UTR activity of SOX2 mRNA without affecting its mRNA stability. Moreover, ectopic expression of exogenous SOX2 rendered D456 cells resistant to induction of cell cycle G0-G1 arrest and anchorage-independent growth inhibition upon ISO treatment, whereas inhibition of miR-145 resulted in D456 cells resistant to ISO inhibition of SOX2 and cyclin D1 expression. In addition, overexpression of miR-145 mimicked ISO treatment in D456 cells.. ISO induces miR-145 expression, which binds to the SOX2 mRNA 3'UTR region and inhibits SOX2 protein translation. Inhibition of SOX2 leads to cyclin D1 downregulation and PDGS anchorage-independent growth inhibition. The elucidation of the miR-145/SOX2/cyclin D1 axis in PDGS provides a significant insight into understanding the anti-GBM effect of ISO compound.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; MicroRNAs; SOXB1 Transcription Factors; Stilbenes

Glioblastoma multiforme (GBM, astrocytoma grade IV) is the most common malignant primary brain tumor in adults. Addressing the shortage of effective treatment options for this cancer, we explored repurposing of existing drugs into combinations with potent activity against GBM cells. We report that the phytoalexin pterostilbene is a potentiator of two drugs with previously reported anti-GBM activity, the EGFR inhibitor gefitinib and the antidepressant sertraline. Combinations of either of these two compounds with pterostilbene suppress cell growth, viability, sphere formation and inhibit migration in tumor GBM cell (GC) cultures. The potentiating effect of pterostilbene was observed to a varying degree across a panel of 41 patient-derived GCs, and correlated in a case specific manner with the presence of missense mutation of EGFR and PIK3CA and a focal deletion of the chromosomal region 1p32. We identify pterostilbene-induced cell cycle arrest, synergistic inhibition of MAPK activity and induction of Thioredoxin interacting protein (TXNIP) as possible mechanisms behind pterostilbene's effect. Our results highlight a nontoxic stilbenoid compound as a modulator of anticancer drug response, and indicate that pterostilbene might be used to modulate two anticancer compounds in well-defined sets of GBM patients.

Topics: Aged; Aged, 80 and over; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; DNA Copy Number Variations; Drug Synergism; Female; Gefitinib; Gene Expression Profiling; Gene Knockdown Techniques; Glioblastoma; Humans; Male; MAP Kinase Signaling System; Middle Aged; Mutation; Phenotype; Protein Kinase Inhibitors; Quinazolines; Stilbenes; Transcriptome

Trans-resveratrol suppresses glioblastoma growth in vitro, but its effects on intracranial glioblastomas remain untested. Resveratrol crosses the blood-brain barrier, and lumbar puncture (LP) greatly increases its bioavailability in rat brains; therefore, we investigated the effectiveness of LP-administered resveratrol on orthotopic rat glioblastomas. Twenty-four tumor-bearing rats were separated into two groups: Group 1 receiving 100 μl saline containing 0.3% DMSO and Group 2 receiving 100 μl resveratrol (300 μM). Treatments started 3 days after transplantation in 2-day intervals until death. Intracranial drug availabilities, tumor sizes, average life spans and the impacts on STAT3 signaling, apoptosis and autophagy rates were evaluated. MRI imaging revealed that average tumor size in the LP group (495.8 ± 22.3 mm2) was smaller than the control groups (810.3 ± 56.4 mm2; P<0.05). The mean survival time in the LP group (22.2 ± 2.1 d) was longer than control animals (16.0 ± 1.8 d; P<0.05). LP resveratrol-treated glioblastomas showed less Cyclin D1 staining, enhanced autophagy with up-regulated LC3 and Beclin1 expression, and widely distributed apoptotic foci around tumor capillaries with suppressed STAT3 expression and nuclear translocation. In conclusion, LP-delivered resveratrol efficiently inhibited orthotopic rat glioblastoma growth by inactivating STAT3 signaling and enhancing autophagy and apoptosis.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Cell Line, Tumor; Cell Proliferation; Cell Survival; Glioblastoma; Magnetic Resonance Imaging; Rats; Resveratrol; Signal Transduction; Spinal Puncture; STAT3 Transcription Factor; Stilbenes

The purpose of this study was to evaluate the effect of resveratrol on cytogenetic damages of iododeoxyuridine (IUdR) and x-ray megavoltage radiation (6 MV) in spheroid model of U87MG glioblastoma cancer cell line using clonogenic and alkaline comet assay. Cells were cultured as spheroids (350 µm) that were treated with 20 μM resveratrol, 1 μM IUdR and 2 Gy of 6 MV x-ray. After treatment, viability of the cells, colony forming ability and the induced DNA damages were examined using trypan blue dye exclusion, colonogenic and alkaline comet assay, respectively. Our results showed that resveratrol could significantly reduce the colony number and induce the DNA damages of the cells treated with IUdR in combination with 6 MV x-ray radiation. That results indicated that resveratrol as an inhibitor of hypoxia inducible factor 1 alpha (HIF-1α) protein in combination with IUdR as a radiosensitizer enhanced the radiosensitization of glioblastoma spheroid cells.

Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Survival; Comet Assay; DNA Damage; Dose-Response Relationship, Drug; Glioblastoma; Humans; Idoxuridine; Resveratrol; Stilbenes; Tetrazolium Salts; Thiazoles; Trypan Blue; X-Rays

Resveratrol possesses anti-tumor activities against central nervous system (CNS) tumors in vitro but has not yet been used clinically due to its low bioavailability, particularly in the CNS. This study thus aimed to elucidate brain bioavailability of trans-resveratrol by monitoring brain concentrations and dwell times following administration of resveratrol through intragastric, intraperitoneal, external carotid artery/ECA and intrathecal routes. In parallel, we evaluated the biological responses of rat RG2 glioblastoma cells as well as RG2-formed rat intracranial glioblastomas treated with resveratrol via intrathecal administration. The results revealed that resveratrol was detected in rat brains except when administered systemically. Intrathecal administration of reseveratrol led to abundant apoptotic foci and increased staining of the autophagy proteins, LC-3 and Beclin-1 and shrinkage of the intracranial tumors. In conclusion, the BBB penetrability of resveratrol is remarkably increased by intracthecal administration. Regular short-term resveratrol treatments suppress growth and enhance autophagic and apoptotic activities of rat RG2 glioblastoma cells in vitro and in vivo. Therefore, intrathecal administration of resveratrol could be an optimal intervention approach in the adjuvant management of brain malignancies.

Topics: Analysis of Variance; Animals; Anticarcinogenic Agents; Brain; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Routes; Female; Gene Expression Regulation, Neoplastic; Glioblastoma; Glucuronosyltransferase; Male; Rats; Rats, Sprague-Dawley; Resveratrol; Stilbenes

Invasion and metastasis of glioblastoma-initiating cells (GICs) are thought to be responsible for the progression and recurrence of glioblastoma multiforme (GBM). A safe drug that can be applied during the rest period of temozolomide (TMZ) maintenance cycles would greatly improve the prognosis of GBM patients by inhibiting GIC invasion. Resveratrol (RES) is a natural compound that exhibits anti-invasion properties in multiple tumor cell lines. The current study aimed to evaluate whether RES can inhibit GIC invasion in vitro and in vivo. GICs were identified using CD133 and Nestin immunofluorescence staining and tumorigenesis in non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. Invasive behaviors, including the adhesion, invasion and migration of GICs, were determined by tumor invasive assays in vitro and in vivo. The activity of matrix metalloproteinases (MMPs) was measured by the gelatin zymography assay. Western blotting analysis and immunofluorescence staining were used to determine the expression of signaling effectors in GICs. We demonstrated that RES suppressed the adhesion, invasion and migration of GICs in vitro and in vivo. Moreover, we proved that RES inhibited the invasion of GICs via the inhibition of PI3K/Akt/NF-κB signal transduction and the subsequent suppression of MMP-2 expression.

Topics: Animals; Cell Adhesion; Cell Line, Tumor; Cell Survival; Dacarbazine; Down-Regulation; Glioblastoma; Humans; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Neoplasm Transplantation; NF-kappa B; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Resveratrol; Signal Transduction; Stilbenes; Temozolomide

Tumor growth is not solely a consequence of autonomous tumor cell properties. Rather, tumor cells act upon and are acted upon by their microenvironment. It is tumor tissue biology that ultimately determines tumor growth. Thus, we developed a compound library screen for agents that could block essential tumor-promoting effects of the glioblastoma (GBM) perivascular stem cell niche (PVN). We modeled the PVN with three-dimensional primary cultures of human brain microvascular endothelial cells in Matrigel. We previously demonstrated stimulated growth of GBM cells in this PVN model and used this to assay PVN function. We screened the Microsource Spectrum Collection library for drugs that specifically blocked PVN function, without any direct effect on GBM cells themselves. Three candidate PVN-disrupting agents, Iridin, Tigogenin and Triacetylresveratrol (TAR), were identified and evaluated in secondary in vitro screens against a panel of primary GBM isolates as well as in two different in vivo intracranial models. Iridin and TAR significantly inhibited intracranial tumor growth and prolonged survival in these mouse models. Together these data identify Iridin and TAR as drugs with novel GBM tissue disrupting effects and validate the importance of preclinical screens designed to address tumor tissue function rather than the mechanisms of autonomous tumor cell growth.

Topics: Animals; Brain Neoplasms; Cell Communication; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Endothelial Cells; Female; Glioblastoma; Humans; Mice, Nude; Phytotherapy; Plant Extracts; Protamines; Resveratrol; Small Molecule Libraries; Spirostans; Stilbenes; Survival Analysis; Tumor Burden; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The schweinfurthins have potent antiproliferative activity in multiple glioblastoma multiforme (GBM) cell lines; however, the mechanism by which growth is impeded is not fully understood. Previously, we demonstrated that the schweinfurthins reduce the level of key isoprenoid intermediates in the cholesterol biosynthetic pathway. Herein, we describe the effects of the schweinfurthins on cholesterol homeostasis. Intracellular cholesterol levels are greatly reduced in cells incubated with 3-deoxyschweinfurthin B (3dSB), an analog of the natural product schweinfurthin B. Decreased cholesterol levels are due to decreased cholesterol synthesis and increased cholesterol efflux; both of these cellular actions can be influenced by liver X-receptor (LXR) activation. The effects of 3dSB on ATP-binding cassette transporter 1 levels and other LXR targets are similar to that of 25-hydroxycholesterol, an LXR agonist. Unlike 25-hydroxycholesterol, 3dSB does not act as a direct agonist for LXR α or β. These data suggest that cholesterol homeostasis plays a significant role in the growth inhibitory activity of the schweinfurthins and may elucidate a mechanism that can be targeted in human cancers such as GBM.

Topics: Anticholesteremic Agents; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter 1; Biological Transport; Cell Line, Tumor; Cell Survival; Cholesterol; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Glioblastoma; Glutathione Transferase; Humans; Hydroxycholesterols; Hydroxylation; Kinetics; Liver X Receptors; Neoplasm Proteins; Orphan Nuclear Receptors; Protein Interaction Domains and Motifs; Recombinant Fusion Proteins; Stilbenes

Glioblastoma is a malignant human cancer that confers a dismal prognosis. Ionizing radiation (IR) is applied as the standard treatment for malignant gliomas. However, radiotherapy remains merely palliative because of the existence of glioma stem cells (GSCs), which are regarded as highly radioresistant "seed" cells. In this study, the effect and possible mechanisms of radiotherapy in combination with resveratrol (Res) were investigated in a radioresistant GSC line, SU-2. Our results showed that Res inhibited SU-2 proliferation and enhanced radiosensitivity as indicated by clonogenic survival assay. We also observed a decrease in the expression of neural stem cell marker CD133, which indicated that treatment with Res and IR induced SU-2 cell differentiation. In addition, the combination of Res with IR significantly increased autophagy and apoptosis levels in both in vitro cells and nude mouse model. Finally, Res significantly attenuated the repair of radiation-induced DNA damage. Taken together, the present study demonstrated that the significant radiosensitization ability of Res both in vitro and in vivo was attributed to its synergistic antitumor effects, including inhibition of self-renewal and stemness, induction of autophagy, promotion of apoptosis, and prevention of DNA repair. Therefore, Res may function as a radiation sensitizer for malignant glioma treatment.

Topics: AC133 Antigen; Animals; Antigens, CD; Apoptosis; Autophagy; Biomarkers, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Disease Models, Animal; DNA Damage; Glioblastoma; Glycoproteins; Humans; Male; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Peptides; Radiation Tolerance; Radiation-Sensitizing Agents; Resveratrol; Stilbenes; Tumor Cells, Cultured

Glioblastomas, the most common form of primary brain tumors, are the fourth cause of death by cancer in adults. Increasing evidences suggest that glioblastoma resistance to existing radio- and chemotherapies rely on glioblastoma stem cells (GSCs). GSCs are endowed with a unique combination of stem-like properties alike to normal neural stem cells (NSCs), and of tumor initiating properties. The natural polyphenol resveratrol is known to exert opposite actions on neural cells according to their normal or cancerous status. Here, we used resveratrol to explore the molecular mechanisms differing between GSCs and NSCs. We observed a dual action of resveratrol on GSCs: resveratrol blocked GSC proliferation up to 150 μM and induced their necrosis at higher doses. On the opposite, resveratrol had no effect on NSC behavior. To determine the mechanisms underlying resveratrol effects, we focused our attention on the family of NAD-dependent deacetylases sirtuins (SIRT). A member of this family, SIRT1, has been repetitively shown to constitute a preferential resveratrol target, at least in normal cells. Western blot analysis showed that SIRT1 and SIRT3 were expressed by both GSCs and NSCs whereas SIRT2 expression was restricted to GSCs. Pharmacological blockade of SIRT2 activity or down-regulation of SIRT2 expression with siRNAs counteracted the inhibitory effect of resveratrol on cell proliferation. On the contrary, inhibition of SIRT2 activity or expression did not counteract GSC necrosis observed in presence of high doses of resveratrol. Our results highlight SIRT2 as a novel target for altering GSC properties.

Topics: Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Glioblastoma; Humans; Necrosis; Neoplastic Stem Cells; Resveratrol; RNA, Small Interfering; Sirtuin 2; Stilbenes; Structure-Activity Relationship; Tumor Cells, Cultured

Vineatrol(®) 30 is a grapevine-shoot extract, which contains resveratrol as well as considerable amounts of so-called resveratrol oligomers such as hopeaphenol and r2-viniferin. In this study, we analysed whether the two above-mentioned resveratrol oligomers were able to inhibit the growth of the canine glioblastoma cell line D-GBM and the canine histiocytic sarcoma cell line DH82, compared their potency to inhibit tumour cell growth with that of resveratrol and determined whether the induction of apoptosis via caspase 9 and 3/7 activation underlies the tumour cell growth-inhibiting effect of hopeaphenol and r2-viniferin. Vineatrol(®) 30, resveratrol, hopeaphenol and r2-viniferin inhibited the growth of D-GBM and DH82 cells in a concentration-dependent manner, whereby hopeaphenol and r2-viniferin were more potent than resveratrol itself in inhibiting the growth of the canine tumour cell lines. Moreover, the anti-proliferative effect of both resveratrol oligomers in D-GBM cells is based on their capacity to induce caspase 9 and 3/7 activation.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Dogs; Glioblastoma; Histiocytic Sarcoma; Molecular Structure; Phenols; Polyphenols; Resveratrol; Stilbenes

Temozolomide (TMZ) is the most widely used drug to treat glioblastoma (GBM), which is the most common and aggressive primary tumor of the Central Nervous System and one of the hardest challenges in oncotherapy. TMZ is an alkylating agent that induces autophagy, apoptosis and senescence in GBM cells. However, therapy with TMZ increases survival after diagnosis only from 12 to 14.4 months, making the development of combined therapies to treat GBM fundamental. One candidate for GBM therapy is Resveratrol (Rsv), which has additive toxicity with TMZ in several glioma cells in vitro and in vivo. However, the mechanism of Rsv and TMZ additive toxicity, which is the aim of the present work, is not clear, especially concerning cell cycle dynamics and long term effects.. Glioma cell lines were treated with Rsv and TMZ, alone or in combinations, and the induction and the role of autophagy, apoptosis, cell cycle dynamics, protein expression and phosphorylation status were measured. We further evaluated the long term senescence induction and clonogenic capacity.. As expected, temozolomide caused a G2 cell cycle arrest and extensive DNA damage response. Rsv did not reduced this response, even increasing pATM, pChk2 and gammaH2Ax levels, but abrogated the temozolomide-induced G2 arrest, increasing levels of cyclin B and pRb(S807/811) and reducing levels of pWee1(S642) and pCdk1(Y15). This suggests a cellular state of forced passage through G2 checkpoint despite large DNA damage, a scenario that may produce mitotic catastrophe. Indeed, the proportion of cells with high nuclear irregularity increased from 6 to 26% in 48 h after cotreatment. At a long term, a reduction in clonogenic capacity was observed, accompanied by a large induction of senescence.. The presence of Rsv forces cells treated with TMZ through mitosis leading to mitotic catastrophe and senescence, reducing the clonogenic capacity of glioma cells and increasing the chronic effects of temozolomide.

Topics: Antineoplastic Agents, Alkylating; Antineoplastic Agents, Phytogenic; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Autophagy; CDC2 Protein Kinase; Cell Cycle Proteins; Cell Line, Tumor; Cellular Senescence; Checkpoint Kinase 2; Cyclin B; Dacarbazine; DNA Damage; Drug Synergism; G2 Phase Cell Cycle Checkpoints; Glioblastoma; Histones; Humans; Mitosis; Nuclear Proteins; Phosphorylation; Protein-Tyrosine Kinases; Resveratrol; Stilbenes; Temozolomide; Time Factors

To further elucidate the correlation of resveratrol sensitivities with biotransformation activities of human and rat glioblastoma cells for personalized anti-glioblastoma therapy.. Resveratrol sensitivity of human U251 and rat RG2 and C6 glioblastoma cells was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide/MTT, flow cytometry, and TUNEL assays. The metabolic patterns of those cell lines were analyzed by high-performance liquid chromatography/HPLC coupled with tandem mass spectrum/MS/MS, and high-resolution mass spectrometry/HRMS. Immunocytochemical staining and Western blotting were employed to check resveratrol metabolic enzyme expression.. Both rat RG2 and C6 and human U251 glioblastoma cells are sensitive to 100 μM resveratrol in terms of growth arrest and increased apoptotic fraction. The main resveratrol metabolite in U251 cells is monosulfate biotransformed by sulfotransferases/SULTs and in RG2 and C6 cells is monoglucuronide generated by UDP-glucuronosyltransferase/UGT. Both metabolites show lesser therapeutic efficacy. Although brain-associated UGTs (UGT1A6, 2B7, and 8) and SULTs (SULT1A1, 1C2, and 4A1) are expressed in rat and human glioma cells, the overall level of UGTs is predominant in the rat and SULTs in human glioblastoma cells. In similar to SULT expression pattern, UGT1A6, 2B7, and 8 are frequently downregulated (84.6 %, 82/97; 90.7 %, 88/97; 80.4 %, 78/97) in human glioblastoma tissues.. Our results suggest (1) the decreased resveratrol biotransforming activity in rat and human resveratrol-sensitive glioblastoma cells; (2) the discrepant resveratrol metabolic patterns between human and rat glioblastoma cells; (3) the more powerful anti-glioblastoma efficacy of trans-resveratrol rather than resveratrol monoglucuronide or monosulfate; and (4) the value of RG2 and C6 cells in establishing resveratrol-based rat in vivo therapeutic model.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Brain; Cell Line, Tumor; Cell Proliferation; Enzyme Induction; Glioblastoma; Glucuronides; Glucuronosyltransferase; Humans; Male; Metabolic Detoxication, Phase II; Neoplasm Proteins; Nerve Tissue Proteins; Neurons; Rats; Rats, Wistar; Resveratrol; Species Specificity; Stilbenes; Sulfotransferases; Sulfuric Acid Esters

Glioblastoma multiforme (GBM) is the most aggressive brain tumor in adults, and despite state-of-the-art treatment, survival remains poor and novel therapeutics are sorely needed. The aim of the present study was to identify new synergistic drug pairs for GBM. In addition, we aimed to explore differences in drug-drug interactions across multiple GBM-derived cell cultures and predict such differences by use of transcriptional biomarkers.. We performed a screen in which we quantified drug-drug interactions for 465 drug pairs in each of the 5 GBM cell lines U87MG, U343MG, U373MG, A172, and T98G. Selected interactions were further tested using isobole-based analysis and validated in 5 glioma-initiating cell cultures. Furthermore, drug interactions were predicted using microarray-based transcriptional profiling in combination with statistical modeling.. Of the 5 × 465 drug pairs, we could define a subset of drug pairs with strong interaction in both standard cell lines and glioma-initiating cell cultures. In particular, a subset of pairs involving the pharmaceutical compounds rimcazole, sertraline, pterostilbene, and gefitinib showed a strong interaction in a majority of the cell cultures tested. Statistical modeling of microarray and interaction data using sparse canonical correlation analysis revealed several predictive biomarkers, which we propose could be of importance in regulating drug pair responses.. We identify novel candidate drug pairs for GBM and suggest possibilities to prospectively use transcriptional biomarkers to predict drug interactions in individual cases.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carbazoles; Cell Line, Tumor; Drug Interactions; Gefitinib; Glioblastoma; Humans; Quinazolines; Sertraline; Stilbenes

Arginine deprivation, either by nutritional starvation or exposure to ADI-PEG20, induces adaptive transcriptional upregulation of ASS1 and ASL in glioblastoma multiforme ex vivo cultures and cell lines. This adaptive transcriptional upregulation is blocked by neoplasia-specific CpG island methylation in either gene, causing arginine auxotrophy and cell death. In cells with methylated ASS1 or ASL CpG islands, ADI-PEG20 initially induces a protective autophagic response, but abrogation of this by chloroquine accelerates and potentiates cytotoxicity. Concomitant methylation in the CpG islands of both ASS1 and ASL, observed in a subset of cases, confers hypersensitivity to ADI-PEG20. Cancer stem cells positive for CD133 and methylation in the ASL CpG island retain sensitivity to ADI-PEG20. Our results show for the first time that epigenetic changes occur in both of the two key genes of arginine biosynthesis in human cancer and confer sensitivity to therapeutic arginine deprivation. We demonstrate that methylation status of the CpG islands, rather than expression levels per se of the genes, predicts sensitivity to arginine deprivation. Our results suggest a novel therapeutic strategy for this invariably fatal central nervous system neoplasm for which we have identified robust biomarkers and which overcomes the limitations to conventional chemotherapy imposed by the blood/brain barrier.

Topics: Apoptosis; Arginine; Argininosuccinate Lyase; Argininosuccinate Synthase; Autophagy; Azacitidine; Central Nervous System Neoplasms; Chloroquine; CpG Islands; Decitabine; DNA Methylation; Epigenomics; Glioblastoma; Humans; Hydrolases; Polyethylene Glycols; RNA Interference; RNA, Small Interfering; Stilbenes; Tumor Cells, Cultured; Up-Regulation

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Patients diagnosed with GBM have a poor prognosis, and it has been reported that tumor malignancy and GBM recurrence are promoted by STAT3 signaling. As resveratrol (RV), a polyphenol in grapes, is reported to be a potent and non-toxic cancer-preventive compound, the aim of this study was to investigate the therapeutic effect and molecular mechanisms of RV on GBM-derived radioresistant tumor initiating cells (TIC). Firstly, our results showed that primary GBM-CD133(+) TIC presented high tumorigenic and radiochemoresistant properties as well as increased protein levels of phosphorylated STAT3. We consistently observed that treatment with shRNA-STAT3 (sh-STAT3) or AG490, a STAT3 inhibitor, significantly inhibited the cancer stem-like cell properties and radioresistance of GBM-CD133(+) in vitro and in vivo. Furthermore, treatment of GBM-CD133(+) with 100 µM RV induced apoptosis and enhanced radiosensitivity by suppressing STAT3 signaling. Microarray results suggested that RV or AG490 inhibited the stemness gene signatures of GBM-CD133(+) and facilitated the differentiation of GBM-CD133(+) into GBM-CD133(-) or astrocytoma cells. Finally, xenotransplant experiments indicated that RV or sh-STAT3 therapy could significantly improve the survival rate and synergistically enhance the radiosensitivity of radiation-treated GBM-TIC. In summary, RV can reduce in vivo tumorigenicity and enhance the sensitivity of GBM-TIC to radiotherapies through the STAT3 pathway.

Topics: Aged; Animals; Antineoplastic Agents, Phytogenic; Astrocytoma; Brain Neoplasms; Chemoradiotherapy; Female; Glioblastoma; Humans; Male; Mice; Mice, SCID; Middle Aged; Resveratrol; Signal Transduction; STAT3 Transcription Factor; Stilbenes; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Glioblastoma multiforme (GBM) is the most common intracranial tumor, with a dismal prognosis. Although temozolomide (TMZ)-based chemotherapy following neurosurgery has been proven to be effective, not all patients benefit clinically because of TMZ resistance. Given that protein expression of O(6)-methylguanine-DNA-methyltransferase (MGMT) is the most important determinant of TMZ resistance, great efforts have been made to suppress it by regulating MGMT-related transcription factors. The study presented here demonstrates that resveratrol, a natural polyphenol, is able to reverse TMZ resistance of glioblastoma T98G cells which have relatively high MGMT activity. The data showed that combination treatment with TMZ and resveratrol resulted in an enhanced antitumor potential of TMZ, decreased the 50% inhibiting concentration (IC50) of TMZ and increased the induction of apoptosis in TMZ-resistant T98G cells. Hoechst 33258 staining revealed increased apoptotic morphology, such as chromatin aggregation and nuclear and cytoplasmic condensation, in cells receiving combination treatment. Western blot analysis manifested a significant decreased intracellular content and nuclear translocation of NF-κB and increased cleavage of caspase-3 in cells exposed to combination treatment, compared to those in cells treated with TMZ alone. In addition, recombinant expression of NF-κB subunit p65 remarkably promoted nuclear translocation of NF-κB and abolished the TMZ-resistance reversal induced by combination treatment, suggesting an underlying NF-κB-dependent mechanism. Our study improved the knowledge on the mechanism of TMZ resistance and suggested a novel strategy for TMZ-based chemotherapy in glioblastoma patients.

Topics: Antineoplastic Agents, Alkylating; Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Line, Tumor; Dacarbazine; DNA Methylation; DNA Modification Methylases; DNA Repair Enzymes; Down-Regulation; Drug Combinations; Drug Resistance, Neoplasm; Glioblastoma; Humans; NF-kappa B; Resveratrol; Stilbenes; Temozolomide; Tumor Suppressor Proteins

Schweinfurthins are potent inhibitors of cancer cell growth, especially against human central nervous system tumor lines such as SF-295 cells. However, the mechanisms through which these compounds impede cell growth are not fully understood. In an effort to understand the basis for the effects of schweinfurthins, we present a fluorescent schweinfurthin, 3-deoxyschweinfurthin B-like p-nitro-bis-stilbene (3dSB-PNBS), which displays biological activity similar to that of 3-deoxyschweinfurthin B (3dSB). These two schweinfurthins retain the unique differential activity of the natural schweinfurthins, as evidenced by the spindle-like morphological changes induced in SF-295 cells and the unaltered appearance of human lung carcinoma A549 cells. We demonstrate that incubation with 3dSB or 3dSB-PNBS results in cleavage of poly-ADP-ribose polymerase (PARP) and caspase-9, both markers of apoptosis. Coincubation of 3dSB or 3dSB-PNBS with the caspase-9 inhibitor (Z)-Leu-Glu(O-methyl)-His-Asp(O-methyl)-fluoromethylketone prevents PARP cleavage. Therapeutic agents that induce apoptosis often activate cellular stress pathways. A marker for multiple stress pathways is the phosphorylation of eukaryotic initiation factor 2α, which is phosphorylated in response to 3dSB and 3dSB-PNBS treatment. Glucose-regulated protein 78 and protein disulfide isomerase, both endoplasmic reticulum chaperones, are up-regulated with schweinfurthin exposure. Using the fluorescent properties of 3dSB-PNBS and dimethoxyphenyl-p-nitro-bis-stilbene (DMP-PNBS), a control compound, we show that the intracellular levels of 3dSB-PNBS are higher than those of Rhodamine 123 or DMP-PNBS in SF-295 and A549 cells.

Topics: Apoptosis; Caspase 9; Cell Line, Tumor; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Eukaryotic Initiation Factor-2; Fluorescent Dyes; Glioblastoma; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Membrane Proteins; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Disulfide-Isomerases; Stilbenes; Up-Regulation

Resveratrol has been regarded as a promising candidate for cancer prevention and treatment. The present study was to investigate the impact of resveratrol on the antitumor effects of temozolomide (TMZ), a standard treatment regiment of glioblastoma (GBM), in vitro and in vivo.. We found that the combination of resveratrol and TMZ significantly resulted in G(2)/M cell cycle arrest by flow cytometry, triggered a robust increase in expression of astrocyte differentiation marker glial fibrillary acid protein (GFAP), downregulated the expression of matrix metalloproteinase-9 (MMP-9) by immunohistochemistry and western blot analysis as well as inhibited cell migration by scratch wound assay. Further study revealed that TMZ in combination with resveratrol remarkably increased reactive oxygen species (ROS) production, which serves as an upstream signal for AMP-activated protein kinase (AMPK) activation. Subsequently, activated AMPK inhibited mTOR signaling and downregulated antiapoptosis protein Bcl-2, which was contributed to the additive antiproliferation effects of combination treatment. In an orthotopic xenograft model of GBM, TMZ plus resveratrol treatment significantly reduced the volume of tumor, which was confirmed by decreased expression of Ki-67, a marker of proliferation index.. Our findings demonstrate for the first time that resveratrol can enhance TMZ-mediated antitumor effects in GBM in vitro and in vivo, via ROS-dependent AMPK-TSC-mTOR signaling pathway.

Topics: AMP-Activated Protein Kinases; Animals; Antineoplastic Agents, Alkylating; Calcium-Binding Proteins; Cell Line, Tumor; Dacarbazine; Drug Synergism; Drug Therapy, Combination; Female; Glioblastoma; Humans; Mice; Mice, Inbred BALB C; Mice, Knockout; Mice, Nude; Reactive Oxygen Species; Resveratrol; Signal Transduction; Stilbenes; Temozolomide; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Glioblastoma multiforme (GBM) cells show different responses to resveratrol, for unknown reasons. Our data from human medulloblastoma cells and primary cultures of rat brain cells revealed an inverse correlation of sulfonation activity with resveratrol sensitivities, providing a clue to the underlying mechanisms of the variable sensitivities of GBM cells to resveratrol. In this study, we found that U251 cells were sensitive and LN229 cells were insensitive to resveratrol. Thus, these two cell lines were taken as comparable models for elucidating the influence of sulfonation activities on resveratrol sensitivity. HPLC showed identical resveratrol metabolic patterns in both cell lines. LC/MS and high-resolution mass MS analyses further demonstrated that resveratrol monosulfate generated by sulfotransferases (SULTs) was the major metabolite of human GBM cells. The levels of brain-associated SULT (SULT1A1, SULT1C2, and SULT4A1) expression in U251 cells were lower than those in LN229 cells, suggesting the inverse relationship of SULT-mediated sulfonation activity with high intracellular resveratrol bioavailability and resveratrol sensitivity of human GBM cells. Furthermore, immunohistochemical staining revealed reductions in expression of the three brain-associated SULTs in 72.8%, 47.5% and 66.3% of astrocytomas, respectively. Therefore, the levels of brain-associated SULTs and sulfonation activity mediated by them could be important parameters for evaluating the potential response of human GBM cells to resveratrol, and may have value in the personalized treatment of GBMs with resveratrol.

Topics: Antineoplastic Agents, Phytogenic; Arylsulfotransferase; Blotting, Western; Brain Neoplasms; Chromatography, High Pressure Liquid; Drug Resistance, Neoplasm; Flow Cytometry; Glioblastoma; Humans; Immunoenzyme Techniques; Mass Spectrometry; Neoplasm Grading; Real-Time Polymerase Chain Reaction; Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stilbenes; Sulfonic Acids; Sulfotransferases; Tissue Array Analysis; Tumor Cells, Cultured

Malignant glioblastoma represents a challenge in the chemotherapy of brain tumors, because of its aggressive behavior characterized by chemoresistance, infiltrative diffusion, and high rate of recurrence and death. In this study, we used cultured human U87MG cells and primary human glioblastoma cultures to test the anticancer properties of resveratrol (RV), a phytoalexin abundantly present in a variety of dietary products. In U87MG cells, 100 μM RV elicited cell growth arrest by 48 h and bax-mediated cell toxicity by 96 h and greatly limited cell migration and invasion through matrigel. Both in U87MG cells and in primary glioblastoma cultures, the chronic administration of RV (100 μM for up to 96 h) decreased the expression of nestin (a brain (cancer) stem cells marker) but increased that of glial acidic fibrillary protein (a mature glial cell marker) and of βIII-tubulin (a neuronal differentiation marker). Chronic treatment with RV increased the proportion of cells positive for senescence-associated β-galactosidase activity. This is the first report showing the ability of RV to induce glial-like and neuronal-like differentiation in glioblastoma cells. The beneficial effects of chronic RV supplementation lasted up to 96 h after its withdrawal from the culture medium. The present findings support the introduction of pulsed administration of this food-derived molecule in the chemotherapy regimen of astrocytomas.

Topics: Antineoplastic Agents, Phytogenic; Brain Neoplasms; Cell Differentiation; Cell Division; Cell Line, Tumor; Glioblastoma; Humans; Microscopy, Fluorescence; Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; Stilbenes; Wound Healing

Mathematical modeling techniques have been widely employed to understand how cancer grows, and, more recently, such approaches have been used to understand how cancer can be controlled. In this manuscript, a previously validated hybrid cellular automaton model of tumor growth in a vascularized environment is used to study the antitumor activity of several vascular-targeting compounds of known efficacy. In particular, this model is used to test the antitumor activity of a clinically used angiogenesis inhibitor (both in isolation, and with a cytotoxic chemotherapeutic) and a vascular disrupting agent currently undergoing clinical trial testing. I demonstrate that the mathematical model can make predictions in agreement with preclinical/clinical data and can also be used to gain more insight into these treatment protocols. The results presented herein suggest that vascular-targeting agents, as currently administered, cannot lead to cancer eradication, although a highly efficacious agent may lead to long-term cancer control.

Topics: Algorithms; Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Antineoplastic Agents, Alkylating; Antineoplastic Agents, Phytogenic; Antineoplastic Combined Chemotherapy Protocols; Bevacizumab; Blood Vessels; Computer Simulation; Cytotoxins; Dacarbazine; Glioblastoma; Humans; Models, Biological; Neoplasms; Stilbenes; Temozolomide; Treatment Outcome

Prognosis of patients with glioblastoma (GBM) remains very poor, thus making the development of new drugs urgent. Resveratrol (Rsv) is a natural compound that has several beneficial effects such as neuroprotection and cytotoxicity for several GBM cell lines. Here we evaluated the mechanism of action of Rsv on human GBM cell lines, focusing on the role of autophagy and its crosstalk with apoptosis and cell cycle control. We further evaluated the role of autophagy and the effect of Rsv on GBM Cancer Stem Cells (gCSCs), involved in GBM resistance and recurrence. Glioma cells treated with Rsv was tested for autophagy, apoptosis, necrosis, cell cycle and phosphorylation or expression levels of key players of these processes. Rsv induced the formation of autophagosomes in three human GBM cell lines, accompanied by an upregulation of autophagy proteins Atg5, beclin-1 and LC3-II. Inhibition of Rsv-induced autophagy triggered apoptosis, with an increase in Bax and cleavage of caspase-3. While inhibition of apoptosis or autophagy alone did not revert Rsv-induced toxicity, inhibition of both processes blocked this toxicity. Rsv also induced a S-G2/M phase arrest, accompanied by an increase on levels of pCdc2(Y15), cyclin A, E and B, and pRb (S807/811) and a decrease of cyclin D1. Interestingly, this arrest was dependent on the induction of autophagy, since inhibition of Rsv-induced autophagy abolishes cell cycle arrest and returns the phosphorylation of Cdc2(Y15) and Rb(S807/811), and levels of cyclin A, and B to control levels. Finally, inhibition of autophagy or treatment with Rsv decreased the sphere formation and the percentage of CD133 and OCT4-positive cells, markers of gCSCs. In conclusion, the crosstalk among autophagy, cell cycle and apoptosis, together with the biology of gCSCs, has to be considered in tailoring pharmacological interventions aimed to reduce glioma growth using compounds with multiple targets such as Rsv.

Topics: Apoptosis; Autophagy; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Glioblastoma; Humans; Neoplastic Stem Cells; Phagosomes; Resveratrol; Stilbenes

Glioblastoma is the most malignant form of adult brain tumor and is associated with a dismal prognosis. Emerging data suggest that Notch signaling participates principally in the formation and malignant progression of glioblastoma. Resveratrol is a terpenoid that exhibits broad pro-apoptotic activity in various types of cancers, including glioblastoma. However, the effects of resveratrol on Notch signaling in glioblastomas have not yet been fully elucidated. We demonstrated that resveratrol strongly suppressed cell growth and induced apoptosis in A172 and T98G glioblastoma cells, which have low active Notch-1 expression and a heterozygous p53 mutation. Our results suggest that resveratrol significantly activates intracellular Notch-1 and restores wild-type p53 expression in a time-dependent manner. Significant de-phosphorylation of Akt, increased Bax expression, decreased Bcl-2 expression and cleavage of caspase-3 were also observed in resveratrol-induced apoptosis in glioblastoma cells. Moreover, simultaneous treatment with resveratrol and a Notch-1 inhibitor (MRK-003) partially attenuated the apoptosis and completely blocked the activation of Notch-1 and the increase in wild-type p53. This suggests that restoration of wild-type p53 expression depends on Notch-1 activation. In addition, the de-phosphorylation of Akt, increased expression of Bax and cleavage of caspase-3 were not fully reversed by MRK-003 treatment, suggesting that p53 restoration is not the only mechanism underlying resveratrol-induced apoptosis. Taken together, we confirmed the anti-proliferative and pro-apoptotic effects of resveratrol on glioblastoma cells and revealed Notch-1 activation-dependent restoration of p53 as an important causative mechanism.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Astrocytes; bcl-2-Associated X Protein; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Genes, p53; Glioblastoma; Humans; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Receptor, Notch1; Resveratrol; Signal Transduction; Stilbenes; Tumor Suppressor Protein p53

Trans-resveratrol rather than its biotransformed monosulfate metabolite exerts anti-medulloblastoma effects by suppressing STAT3 activation. Nevertheless, its effects on human glioblastoma cells are variable due to certain unknown reason(s).. Citing resveratrol-sensitive UW228-3 medulloblastoma cell line and primarily cultured rat brain cells/PBCs as controls, the effect of resveratrol on LN-18 human glioblastoma cells and its relevance with metabolic pattern(s), brain-associated sulfotransferase/SULT expression and the statuses of STAT3 signaling and protein inhibitor of activated STAT3 (PIAS3) were elucidated by multiple experimental approaches. Meanwhile, the expression patterns of three SULTs (SULT1A1, 1C2 and 4A1) in human glioblastoma tumors were profiled immunohistochemically. The results revealed that 100 µM resveratrol-treated LN-18 generated the same metabolites as UW228-3 cells, while additional metabolite in molecular weight of 403.0992 in negative ion mode was found in PBCs. Neither growth arrest nor apoptosis was found in resveratrol-treated LN-18 and PBC cells. Upon resveratrol treatment, the levels of SULT1A1, 1C2 and 4A1 expression in LN-18 cells were more up-regulated than that expressed in UW228-3 cells and close to the levels in PBCs. Immunohistochemical staining showed that 42.0%, 27.1% and 19.6% of 149 glioblastoma cases produced similar SULT1A1, 1C2 and 4A1 levels as that of tumor-surrounding tissues. Unlike the situation in UW228-3 cells, STAT3 signaling remained activated and its protein inhibitor PIAS3 was restricted in the cytosol of resveratrol-treated LN-18 cells. No nuclear translocation of STAT3 and PIAS3 was observed in resveratrol-treated PBCs. Treatment with STAT3 chemical inhibitor, AG490, committed majority of LN-18 and UW228-3 cells but not PBCs to apoptosis within 48 hours.. LN-18 glioblastoma cells are insensitive to resveratrol due to the more inducible brain-associated SULT expression, insufficiency of resveratrol to suppress activated STAT3 signaling and the lack of PIAS3 nuclear translocation. The findings from PBCs suggest that an effective anticancer dose of resveratrol exerts little side effect on normal brain cells.

Topics: Animals; Apoptosis; Arylsulfotransferase; Cell Line, Tumor; Cells, Cultured; Glioblastoma; Humans; Immunohistochemistry; Molecular Chaperones; Protein Inhibitors of Activated STAT; Rats; Resveratrol; STAT3 Transcription Factor; Stilbenes; Sulfotransferases; Tyrphostins

Erucylphosphohomocholine (ErPC3, Erufosine) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial F(O)F(1)-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the F(O) and F(1) subunits of the mitochondrial F(O)F(1)-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Deltapsi(m)). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Deltapsi(m), normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the F(O) subunit of the mitochondrial F(O)F(1)-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Deltapsi(m) as well as ROS generation by ErPC3 and TSPO.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Butylated Hydroxyanisole; Caspases; Cell Line, Tumor; Cyclosporine; Electron Transport; Enzyme Inhibitors; Erucic Acids; Glioblastoma; Humans; Membrane Potential, Mitochondrial; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Oligomycins; Phosphorylcholine; Proton-Translocating ATPases; Reactive Oxygen Species; Receptors, GABA; Stilbenes

Resveratrol (3,4',5-trihydroxystilbene) is a polyphenol synthesized by a wide variety of plant species in response to injury, UV irradiation and fungal attack. Many studies have revealed a variety of resveratrol intracellular targets whose modulation gives rise to overlapping responses leading to growth arrest and death. Many authors have reported different human cancer cell lines, treated with resveratrol at micromolar concentrations, arrested their proliferative cycle in the G1/S boundary or in the S phase and this cell cycle arrest was followed by apoptotic death. Less is known about the ability of resveratrol to modify the effect of radiation exposure in normal and cancer cells. Considering that controlled exposure to ionizing radiation is one of the most used treatments in cancer patients and that these schedules are not always effective in medical practice, as in the case of glioma patients, the testing of combined treatment protocols (resveratrol and ionizing radiation) could be of interest, opening the door to future studies which would examine the pharmacological activity of resveratrol. In this study we have looked into whether resveratrol is able to modulate cell cycle progression in human glioblastoma cells and to regulate GJs expression in cancer cells. With this aim in mind we have performed a cytofluorimetric multiparameter assay to quantify the presence of GJs in U87 glioma cells treated with resveratrol and/or X rays. We report that resveratrol induces a delay in cell cycle progression and both alone and in combination with X rays is able to enhance gap junction Intercellular Communications.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Brain Neoplasms; Cell Communication; Cell Cycle; Cell Line, Tumor; Cell Survival; Connexin 43; Gap Junctions; Glioblastoma; Humans; Microscopy, Fluorescence; Mutation; Resveratrol; Stilbenes; X-Rays

Glioblastoma is a highly malignant brain tumor with a high-invasive phenotype, so the prognosis is unfavorable, even in response to multidisciplinary treatment strategies. Obviously, therefore, a better therapeutic strategy is needed. Resveratrol has been reported to be one of the most potent chemopreventive agents inhibiting the cellular processes associated with tumor development, including initiation, promotion, and progression.. In this study we used RT-PCR, western blot and SDS-zymography to investigate the effect of resveratrol on the expression of genes and proteins involved in the extracellular matrix remodeling associated with tumor invasion in human cultured glioblastoma cells treated for 24, 48 and 72 h. We analyzed the expression of matrix metalloproteinase-2 (MMP-2), the main mediator of glioblastoma invasiveness, and the Secreted Protein Acidic and Rich in Cysteine (SPARC), involved in the regulation of cell-matrix interactions.. Our results show a dose-related decrease of MMP-2 mRNA and protein levels 72 h after resveratrol treatment, and lower SPARC gene and protein expression 72 h after resveratrol treatment. This indicates that resveratrol may influence the two major factors in the ECM remodeling occurring with tumor invasion, suggesting it may have uses as a therapeutic agent for brain tumors.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Glioblastoma; Humans; Matrix Metalloproteinase 2; Osteonectin; Resveratrol; RNA, Messenger; Stilbenes