Evidence for new targets and synergistic effect of metronomic celecoxib/fluvastatin combination in pilocytic astrocytoma
© Mercurio et al.; licensee BioMed Central Ltd. 2013
Received: 03 May 2013
Accepted: 04 May 2013
Published: 20 May 2013
Pilocytic astrocytomas occur predominantly in childhood. In contrast to the posterior fossa location, hypothalamo-chiasmatic pilocytic astrocytomas display a worse prognosis often leading to multiple surgical procedures and/or several lines of chemotherapy and radiotherapy to achieve long-term control. Hypothalamo-chiasmatic pilocytic astrocytomas and cerebellar pilocytic astrocytomas have a distinctive gene signature and several differential expressed genes (ICAM1, CRK, CD36, and IQGAP1) are targets for available drugs: fluvastatin and/or celecoxib.
Quantification by RT-Q-PCR of the expression of these genes was performed in a series of 51 pilocytic astrocytomas and 10 glioblastomas: they were all significantly overexpressed in hypothalamo-chiasmatic pilocytic astrocytomas relative to cerebellar pilocytic astrocytomas, and CRK and ICAM1 were significantly overexpressed in pilocytic astrocytomas versus glioblastomas.
We used two commercially available glioblastoma cell lines and three pilocytic astrocytoma explant cultures to investigate the effect of celecoxib/fluvastatin alone or in combination. Glioblastoma cell lines were sensitive to both drugs and a combination of 100 μM celecoxib and 240 μM fluvastatin was the most synergistic. This synergistic combination was used on the explant cultures and led to massive cell death of pilocytic astrocytoma cells.
As a proof of concept, a patient with a refractory multifocal pilocytic astrocytoma was successfully treated with the fluvastatin/celecoxib combination used for 18 months. It was well tolerated and led to a partial tumor response.
This study reports evidence for new targets and synergistic effect of celecoxib/fluvastatin combination in pilocytic astrocytoma. Because it is non-toxic, this new strategy offers hope for the treatment of patients with refractory pilocytic astrocytoma.
KeywordsHypothalamo-chiasmatic pilocytic astrocytoma Celecoxib Fluvastatin Target gene Synergy Drug repositioning Metronomic chemotherapy
Pilocytic astrocytomas (PA) are the most frequent gliomas in childhood. According to the World Health Organization, most of them are grade I and are characterized by an excellent prognosis. PA arise preferentially in the cerebellum, and the optic pathway. Other locations such as brainstem, medulla or brain hemispheres are also observed. Several clinico-pathological factors have been associated with a negative impact on outcome. They include incomplete surgery, the pilomyxoid variant astrocytomas (PMA grade II), young age and a hypothalamo-chiasmatic (H/C) location [1–3]. H/C PA usually carry a dismal prognosis with a high frequency of relapse leading to iterative surgery, often associated with further postoperative treatment that remains poorly successful. The strong negative impact of the H/C location on outcome is influenced by several factors including inability to perform complete resection, the high frequency of the PMA variant in this location and the young age of the patients. Recent studies have shown a wide range of mechanisms for deregulating the ERK/MAPK pathway in PA, including NF1 deletion and mutation, KIAA1549/BRAF fusion, SRGAP3/RAF1 fusion and BRAF V600E activating mutation [4–6]. These findings suggest that PA exhibiting BRAF alterations might benefit from BRAF signalling pathway inhibitors. However, not all PA demonstrate BRAF alterations and could thus benefit from this kind of treatment. This is particularly true for those arising in a H/C location, as they show a lower frequency of BRAF alteration [5, 7]. Given the chronic nature of PA in the H/C location, there is a need for long term treatments that display low toxicity and do not impair the patients’ quality of life by further damaging cognitive function (especially in young children) . Therefore the treatment of H/C PA still remains a major therapeutic challenge. Strategies relying on metronomic chemotherapy  or drug repositioning  alone or in combination  seem to be well suited for low grade glioma. Moreover, one important factor hampering the development of new targeted therapies for these tumors is the relative lack of cell lines derived from PA. Therefore, one aim of the present study was to establish cell cultures of excised tumor tissue from PA–bearing patients in order to have suitable models to test their sensitivity against various drugs.
We have previously reported that H/C PA have a genetic signature distinct from that of their cerebellar counterparts with a high expression of genes involved in invasion and cell cycle . Interestingly, among the genes overexpressed in H/C PA, we found some genes that are the targets of already available non-toxic drugs: statins and celecoxib. These include CRK (v-crk avian sarcoma virus CT10 oncogene homologue), CD36, IQGAP1, and ICAM1. Celecoxib is a non-steroidal anti-inflammatory drug which is an inhibitor of cyclooxygenase 2 (COX-2). It has potent antitumor activity through the induction of apoptosis  but can also act through COX-2-independent mechanisms. It interferes with cellular adhesion machinery by dose-dependently decreasing ICAM-1 and VCAM-1 expression in human colon adenocarcinoma HT29 cells . It also promotes anoikis (cell death secondary to the deregulation of focal adhesion complexes and loss of cell attachment to the extracellular matrix) by deregulating the focal adhesion assembly protein CRK-associated substrate P130CAS . P130CAS is a tyrosine-phosphorylated protein that interacts with the SH2 domain of v-Crk .
The statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) are a class of drugs that inhibit the rate-limiting step in the cholesterol biosynthetic pathway and are commonly used for the treatment of hypercholesterolemia. However, increasing clinical evidence suggests that statins can also be used in cancer prevention and treatment [17, 18]. The antitumor effect of statins is not fully elucidated but involves major biological mechanisms such as inhibition of cell proliferation, promotion of apoptosis and inhibition of angiogenesis . Interestingly, one of the statin targets is CD36, a scavenger receptor that is expressed by numerous cells including platelets, mononuclear phagocytes and endothelial cells and that we have found highly expressed in H/C PA. On microvascular endothelial cells, CD36 is a receptor of thrombospondin-1 and functions as a negative regulator of angiogenesis. On monocyte/macrophages it is a receptor for long-chain fatty acids and facilitates their transport into the cells . It has been shown that pivastatin inhibits CD36 expression on murine macrophages . IQGAP1, one of the IQGAP family members, binds to numerous proteins involved in tumorigenesis including the RhoGTPases Cdc42 and Rac1 that are also statin targets [21–23]. Lastly, statins can induce apoptosis via inhibition of p-ERK1/2 pathway, which is activated in PA with KIAA1549-BRAF fusion gene .
In the present study, we have confirmed the over-expression of ICAM1, CRK, CD36, and IQGAP1 transcripts in H/C PA versus cerebellar PA in a larger series of tumors, and we also showed the expression of these targets in GBM cell lines. Because of the lack of cell lines derived from patients with PA, we have used two GBM cell lines (U87-MG and U118) and a PA explant model that we have previously described  to assess the cytotoxic effect of fluvastatin and celecoxib and to determine their synergistic effect. Lastly, we report the anti-tumoral effect of celecoxib-fluvastatin combination in a refractory multifocal PA in a child.
Expression of target genes of celecoxib and fluvastatin in human tumors
ICAM1, CRK, CD36, IQGAP1 and COX2 genes have been described in the literature as target genes for celecoxib and fluvastatin drugs. Here we analyzed their expression by RT-Q-PCR in a series of PA and GBM, in U87-MG and U118 GBM cells lines, in the initial excised tumor of three patients with PA from which we derived in vitro explants cultures, and in a surgical specimen from the case report (arising from the second surgical resection because mRNA obtained from the initial specimen was of poor quality and not suitable for RT-Q-PCR).
Expression in PA versus GBM: to go further, quantification of ICAM1, CRK, CD36, IQGAP1 and COX-2 transcripts was also performed in GBM. Results are reported in Figure 1b and revealed higher ICAM1 and CRK mRNA levels in PA compared to GBM samples (p = 0.002 and p < 0.0001 respectively) (see also Additional file 1b). The remaining transcripts were not differentially expressed.
Expression in U87-MG and U118 cell lines, PA-NAV, PA-GAS and PA-PET initial tumor specimen and in the excised tumor of the case report: we quantified the ICAM1, CRK, CD36, and IQGAP1 mRNA expression in our in vitro models and in the surgical specimen of the case report. All transcripts were readily detected (see Additional file 1c for detailed relative expression ratio values).
Overall, these results showed that the target genes ICAM1, CRK, CD36, and IQGAP1 were expressed as transcripts in H/C PA but also, at lower levels, in cerebellar PA and in GBM.
In vitro efficacy of the fluvastatin-celecoxib combination in GBM human cell lines
Because of the lack of commercially available cell lines derived from patients with PA, we used two GBM cell lines (U87-MG and U118) expressing the target genes of interest to assess the cytotoxic effect of fluvastatin and celecoxib.
Synergistic effects of fluvastatin and celecoxib combination in U87-MG and U118 cell lines
Thus, the in vitro synergistic effects of celecoxib-fluvastatin combination in human GBM cells rely on induction of both apoptosis and cell proliferation decrease.
In vitro effects of fluvastatin and celecoxib on PA explant cells
In this study, because PA cell lines were unavailable for analysis, we made use of a PA explant culture model. Explant culture allows the maintenance of cells in their microenvironment and, as we previously described , it is highly accurate for the study of human brain gliomas because it recapitulates in vivo findings regarding cell migration and cell proliferation.
In untreated controls, explants and cell growth around the explants were “unaffected” (Figure 4b, A). When used alone, 240 μM fluvastatin treatment had little effect on explants but most cells around them mainly became round and lost their adhesion. This state represented the state “affected +” (Figure 4b, D). Celecoxib treatment (100 μM) mainly induced the “affected ++” state: damage to both explants and cell growth (Figure 3b, E). Explants treated with the combination (100 μM of celecoxib and 240 μM of fluvastatin) were totally disrupted and scattered, and represented the “detached” state (Figure 3b, F).
These observations, validated in 3 different PA explant cultures, confirmed that fluvastatin potentiated celecoxib action leading to a massive induction of apoptosis in PA cells.
Case report: treatment with the metronomic celecoxib/fluvastatin combination
A 4 year old girl was referred to our department as she developed a cachexia syndrome over several months. A brain computed tomography scan demonstrated three brain lesions: one in the suprasellar area, one in the third ventricle area and one in an infratentorial area, together with major hydrocephaly. Pathological examination of surgical biopsies from the posterior fossa and 3rd ventricle demonstrated a PA, with KIAA1549-BRAF fusion but no BRAFV600E mutation.
Complete surgical resection was not indicated and she underwent chemotherapy according to the BB-SFOP protocol  from June 2002 to October 2003. After initial stabilization, in 2004, a control MRI (magnetic resonance imaging) demonstrated tumor progression in the 3rd ventricle and the appearance of medullar metastasis. In January 2008, 4 years after completion of treatment, she demonstrated local tumor progression of 3 lesions together with a major infiltration of the brainstem. The three tumors then grew further and turned into a real H/C tumor. She then received 7 cycles of oral temozolomide  that failed to control the disease. In July 2008, she underwent a partial surgical resection. In December 2008, she developed a new local and spinal progression and was treated according to standard chemotherapy published by Packer and colleagues  but developed a severe carboplatin allergic reaction after the first carboplatin infusion leading to cessation of the treatment. Because the parents refused standard alternative treatment requiring intraveinous drugs and because there was no short term functional risk, we proposed to initiate in 2009 a new strategy relying on the combination of fluvastatin and celecoxib based on preclinical and previous clinical reports. Celexoxib was administered per os at the dose of 200 mg twice daily as published in several metronomic paediatric protocols [29, 30] and fluvastatin per os once daily for 2 weeks every 4 weeks with increasing dosage starting at 2 mg/kg/day to 8 mg/kg/day .
However, eight months after stopping treatment, a degradation of her neurological status was observed. The cerebral and spinal MRI did not show any progression of the disease. Surgery was not feasible and the parents ruled out radiotherapy. Thus in September 2011, because her neurological status worsened, it was decided to initiate a new relevant treatment, at parents’ request, with the combination of irinotecan-bevacizumab, to avoid radiotherapy and aim at a rapid response as recently described . Clinical improvement was noted after a month and she is now able to walk again with a decrease in size of the tumor.
On the basis of gene expression data, in vitro and preliminary clinical data, we report here the potential use of fluvastatin, a cholesterol lowering agent, and celecoxib, an anti-inflammatory agent, in the clinical management of PA refractory to conventional treatments.
The treatment of some PA, especially H/C PA, usually requires multiple surgery and/or several lines of chemotherapy and/or radiotherapy to achieve long term control . The intrinsic toxicity of chemotherapy contributes to the burden of treatment and more specifically to the neurocognitive alteration of these patients. As proposed recently, new modalities of treatment relying on metronomic scheduling  and drug repositioning can lead to long term treatment that could turn malignant disease in chronic disease while displaying only limited toxicity [33, 34].
We have previously reported that H/C PA have a distinct genetic signature, as compared to their cerebellar counterparts, with a high expression of genes involved in invasion and cell cycle . Among the over-expressed genes in H/C PA, we found that CRK, CD36, IQGAP1 and ICAM1, could be targeted by already available non-toxic drugs such as statins and celecoxib. These compounds were not initially used as anticancer agents, but drug repositioning studies, that aim at unveiling new therapeutic properties for “old” agents, revealed their anticancer effects [18, 35].
Celecoxib is a non-steroidal anti-inflammatory drug that is an inhibitor of cyclooxygenase 2 (COX-2) and has many anticancer properties. Interestingly, celecoxib has already been used in several clinical studies including paediatric metronomic protocols [29, 30]. Our in vitro data confirms these findings as celecoxib demonstrates anti-tumor activity in 2 GBM cell lines and 3 PA explants cultures.
Fluvastatin was also identified as a drug that could target genes of interest and therefore we hypothesized that it could be another potential agent for the treatment of H/C PA. Our in vitro data confirmed our hypothesis showing activity with IC50 in the range of 500 μM to 900 μM for GBM. This result is in accordance with previous studies reporting the effect of celecoxib in other GBM cell lines [36–38]. Most interestingly, a previous paediatric phase I study determined the maximum tolerated dose of fluvastatin given for 14 days every 4 weeks and reported disease stabilization for over 20 months in 2 of the 5 patients with anaplasic astrocytoma . Ferris and colleagues also conducted a case–control study to investigate statin and/or non-steroidal anti-inflammatory drug (NSAID) therapy and risk of glioma [23, 39]. They reported that the use of statin and NSAID was also significantly inversely related to glioma risk, confirming the role of Ras/Rho GTPases or inflammatory cytokines in gliomagenesis.
The combination of celecoxib and fluvastatin revealed strong synergy when evaluating their role in vitro, since, using the Chou and Talalay method, the obtained CI was <1. Indeed, combining the IC50 celecoxib concentration with a concentration of fluvastatin below the single drug IC50 triggered massive cell death (approximately 99%), therefore strengthening the potential interest of this combination.
Steady state plasma levels of celecoxib following twice daily 250 mg/m2 celecoxib intake in children led to peak concentrations of 1400 μg/L +/− 700 and 2800 μg/L +/− 1500 respectively if celecoxib was taken without or with food . Siekmeier and colleagues  reported that fluvastatin levels following standard (1 to 2 mg/kg/day) doses could reach 100 μg/L. Since increasing doses lead to increased peak and area under curve (AUC), the fluvastatin doses (8 mg/kg/day) recommended by the phase I trial indicate that IC50 concentrations of fluvastatin are clinically achievable. In addition, Sierra and colleagues  and Dembo and colleagues  have respectively shown that statins (including fluvastatin) and COX-2 inhibitors (including celecoxib), could penetrate blood–brain-barrier and reach the central nervous system.
Given that both celecoxib and fluvastatin had already been used in children with cancer, that their combination might be synergistic  and had already been tested in vitro and in vivo in other tumor models [45, 46], we decided to use this combination for a teenage girl with a refractory relapsing multifocal PA. She had previously refused standard cytotoxic chemotherapy following several lines of treatment with limited success and severe carboplatin allergy. While the celecoxib/fluvastatin combination was effective on the H/C lesion after several months of treatment with a progressive decrease in contrast enhancement that was evidenced on MRI, no similar effect was obtained on the spinal metastasis. These differences in anti-tumoral effect might be explained by tumor heterogeneity between the primary tumor and spinal metastasis. Alternatively, both agents can display anti-angiogenic properties and the reduction in contrast enhancement in the primary lesion suggests that the celecoxib/fluvastatin combination may at least in part work through angiogenesis inhibition. Therefore, different tumoral angiogenic patterns may be associated with different localizations of the disease. Lastly, if the tumoral microenvironment can change upon localization in the tumor, the inflammatory infiltrate in the primary tumor may be more senstitive to the anti-inflammatory effect of the metronomic treatment. Although a spontaneous decrease in size of the low grade glioma could not be ruled out, epidemiological, genetic and functional data indicate a potential role for combined therapy of fluvastatin and celecoxib in the treatment of refractory relapsing multifocal PA.
In conclusion, on the basis of genetic data, we identified genes that are differentially expressed in H/C PA versus cerebellar PA, but also in PA versus GBM. We then tested in vitro the single drug and combination effects of fluvastatin and celecoxib on both GBM cell lines and PA explant cultures. This strategy led to the identification of potentially new, non-toxic, long-term treatments for patients with refractory PA, whatever their location. More experiments are mandatory to explore the underlying mechanism of action of this combination. A phase I trial establishing the maximum tolerated dose of this combination in children with H/C PA is planned.
Fifty-one pilocytic astrocytomas (PA) and 10 glioblastomas (GBM) were included in this study. Among the 51 PA, 27 were located in the cerebellum, 17 in the H/C location (optic pathway), 2 in the cerebral hemisphere, 3 in the medulla and 2 in the brainstem. BRAF status (BRAFV600E mutation and KIAA1549-BRAF fusion) was known for 38/51 : 2/38 displayed BRAFV600E mutation and 29/38 PA displayed KIAA1549-BRAF fusion. Only one patient was diagnosed with neurofibromatosis type 1 (NF1). Seven pilomyxoid astrocytomas were included in this study: 2/7 from the cerebellum and 5/7 were from the H/C region.
Forty-four PA and 10 GBM were collected at our hospital (Assistance Publique-Hôpitaux de Marseille, Marseille, France) and 7 PA samples were obtained from the Department of Pathology, University of Cambridge. Mean age at diagnosis was 7 years for PA (range: 1 year to 19 years, and a median age of 6 years) and mean age at diagnosis was 60 years for GBM (range: 44 years to 73 years, and a median age of 59 years).
In addition, three PA specimens from posterior fossa location, obtained from 3 additional young patients (6, 9 and 10 years old), were also used for explant culture. BRAF status was also known for these tumor samples: none of them displayed BRAFV600E mutation but they all had KIAA1549-BRAF fusion gene.
Tumor specimens were obtained after written consent and according to a protocol approved by the local institutional review board and ethics committee and conducted according to national regulations. All frozen samples were stored in the Assistance Publique-Hôpitaux de Marseille tumor bank (authorization number 2008–70). Histological review of the frozen samples (DFB) confirmed the neoplastic nature of the tissue and demonstrated lack of normal residual tissue in samples used for RT-Q-PCR techniques.
Total RNA was extracted using TRI Reagent (Sigma-Aldrich, Paris, France), an improved version of the single-step total RNA isolation reagent developed by Chomczynski and Sacchi , according to the manufacturer’s instructions. RNA was analyzed on the spectrophotometer Nanodrop and Agilent 2100 bioanalyzer (Agilent Technologies, Massy, France). Only samples with no evidence of ribosomal peak degradation and RIN values ranging between 8.0 and 10.0 were considered as high quality intact RNA. Before use, RNA samples were treated with 1U ribonuclease-free deoxyribonuclease (Roche Applied Science, Meylan, France) at 37°C for 20 min.
Total RNA (1 μg) DNA-free was reverse-transcribed into cDNA using 1 μg of random hexamers and Superscript II reverse transcriptase as recommended by the manufacturer (Invitrogen Life Technologies, Cergy Pontoise, France).
Real-time quantitative PCR (RT-Q-PCR)
Sequence of primers used in RT-Q-PCR
F : 5’-CTACCACATCCAAGGAAGGCA-3’
R : 5’-TTTTTCGTCACTACCTCCCCG-3’
F : 5’-CAAATTCCATGGCACCGTC-3’
R : 5’-CCCACTTGATTTTGGAGGGA-3’
F : 5’-CCACACTGTGCCCATCTACG-3’
R : 5’-AGGATCTTCATGAGGTAGTCAGTCAG-3’
F : 5'-TGCAAGTCCTGATGTTTCAGA-3'
R : 5'-TGGCTTGACCAATAGGTTGAC-3'
F : 5'-AGAACAGACCAGATACAAGGCGA--3'
R : 5'-CTTAGGCAATCCAATCTCATCCA-3'
F : 5'-GGAGTGATTCTCAGGCAGGA-3'
R : 5'-TCCCGGATTCTCAAGATGTC-3'
F : 5'-AGCTTCTCCTGCTCTGCAAC-3'
R : 5'-CATTGGAGTCTGCTGGGAAT-3'
QUANTITECT (REF :QT00040586)
GBM cell lines
The human U87-MG and U118 GBM cell lines (American Type Culture Collection, Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal calf serum (FCS), 50 U/ml penicillin, 50 μg/ml streptomycin and 5 mM sodium pyruvate (all purchased at Invitrogen Life Technologies) and they were maintained at 37°C in a 5% CO2 and 95% air atmosphere.
Cell viability assay on GBM cell lines
Celecoxib (Sigma-Aldrich) was reconstituted in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and fluvastatin (Sigma-Aldrich) in sterile water then diluted in culture media before use.
Cytotoxic effect of celecoxib and/or fluvastatin on U87-MG and U118 cell lines was evaluated by assessing cell metabolic capacity, which reflects viability, using the MTT kit (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich). The assays were conducted in quadruplicate with blank controls containing culture media only.
U87-MG and U118 cell lines (3.103 cells/well) were seeded in 96-well plates. After 48 hr (subconfluency), cells were treated with serial concentrations of fluvastatin (30; 60; 120; 240; 365; 490; 610; 730; 850; 975 μM), celecoxib (0; 26; 52; 65; 78; 91; 104; 117; 131; 183; 210; 236; 260; 288; 314 μM) and their combinations in 100 μl of culture media. In both cell lines, the concentrations of fluvastatin in combined treatments tested were 240 and 480 μM and the concentrations of celecoxib were 50, 80, 100, 130 and 260 μM.
At 48 h after treatment, 10 μl of MTT reagent (1/10) was added to each well and incubated for 4 h at 37°C. Then, the reduced formazan crystals were dissolved in iso-propanol and absorbance was measured at 562 nm on a microtiter ELISA plate reader. The cell growth inhibitory activity was obtained by subtracting the absorbance of the blank controls and expressed as percentage of cell growth inhibition as compared to untreated controls (medium and drug diluents).
The IC50 values of both drugs for the 2 GBM cell lines were determined. Then, synergistic interaction between fluvastatin and celecoxib was analysed using the combination index (CI) values that were calculated with the Calcusyn software based on the Chou and Talalay method . The CI theorem provides quantitative definition for additive effects (CI=1), synergisms (CI<1) and antagonisms (CI>1) in drug combinations.
Cell cycle analysis
Cell cycle analysis was performed by PI staining of permeabilized cells and flow cytometry (FACS Calibur; BD Biosciences). A total of 10 000 events were counted for each sample. Data were analyzed with FlowJo software (Celeza GmbH, Olten, Switzerland) choosing the Dean-Jet-Fox model analysis.
Quantification of cell proliferation was performed by KI67 staining. After permeabilization, cells were incubated with KI67 antibody (1/25) (Dako, Glostrup) for 30 min at 4°C. Then, cells were incubated with the secondary antibody (anti-mouse IgG FITC, 1/100) (Jackson immunoresearch, West Grove, USA). Cells were analysed by flow cytometry (FACS Calibur; BD Biosciences) and data were analyzed using CellQuest Pro analysis software.
Annexin V/PI double staining
Apoptotic cells were quantified by Annexin V/PI double staining assay using the FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Le Pont de Claix, France) as recommended by the manufacturer. The cells were analysed by flow cytometry using a FACS Calibur flow cytometer (BD Biosciences) within 1 hour and data were analyzed by CellQuest Pro analysis software.
Establishment of PA explant cultures
Three PA samples named PA-NAV, PA-GAS and PA-PET were collected after surgery in DMEM supplemented with 10% FCS, 50 U/ml penicillin, 50 μg/ml streptomycin and 5 mM sodium pyruvate (all purchased at Invitrogen Life Technologies). Tumors were processed as previously described . Briefly, tissues were washed, dissected, automatically sectioned using a McIlwain tissue chopper (Campden Instruments, Loughborough, England) and cut into 500-μm3 pieces in DMEM 10% FCS, and plated on glass coverslips (12-mm diameter) precoated with poly-(L)-lysine (10 μg/ml, Sigma-Aldrich). The explant pieces were maintained in DMEM 10% FCS. Medium was supplemented with 0.4% methylcellulose (Sigma-Aldrich). Explant cultures were incubated at 37°C in a 5% CO2 and 95% air atmosphere during maximum 40 days and were fed every 3 days. Expression of GFAP and A2B5 were systematically analyzed on culture explants by immunofluorescent staining, as previously described  in order to confirm the glial nature of cultured cells.
Cell viability assay on PA explant cells
Cytotoxic effect of celecoxib and fluvastatin and synergistic interaction between both drugs were tested on PA explants from PA-NAV, PA-GAS and PA-PET. Briefly, after 10 days of culture, explants were treated with fluvastatin (240 μM), celecoxib (100 μM) and their combination (celecoxib 100 μM + fluvastatin 240 μM). At 48 h after treatment, explant cultures behavior was analyzed by phase contrast microscopy (Leica).
The association of the results of RT-Q-PCR with diagnosis (H/C PA versus cerebellar PA and PA versus GBM) and KI67 quantification was assessed by the non parametric Mann–Whitney test using IBM SPSS PASW statistics 17.0. A p value <0.05 was considered significant.
- H/C PA:
Hypothalamo-Chiasmatic Pilocytic Astrocytomas
Real-Time Quantitative Polymerase Chain Reaction
(3-(4,5-dimethylthiazol-2yl)-diphenyl tetrazolium bromide)
Dulbecco's Modified Eagle Medium
Fetal Calf Serum
Magnetic Resonance Imaging
Non-Steroidal Anti-Inflammatory Drug
This work was supported by Institut National Contre le Cancer (grant INCa-DGOS-Inserm 6038), PACA Canceropole, Institutional grants (INSERM, Aix-Marseille University), Amélie la Vie, Ln13 la Vie and The Brain Tumour Charity (S. Lambert’s funding, UK). We thank the Societé Française de Lutte contre les Cancers et les Leucémies de l’Enfant et de l’Adolescent (SFCE) and the Association pour la Recherche sur les Tumeurs Cérébrales (ARTC-Sud) for their financial support. Frozen samples were provided by the AP-HM Tumor Bank (Authorization Number 2008–70) and we are grateful to Peter Collins (Department of Pathology, University of Cambridge, Cambridge, England) for providing 7 tumor samples. We are grateful to Anderson Loundou (Epidemiology Unit, DRRC, Marseille) for his help in statistical analyses and to Charles Prévot (INSERM, U911, Marseille) for his help in flow cytometry experiments.
- Fernandez C, Figarella-Branger D, Girard N, Bouvier-Labit C, Gouvernet J: Paz Paredes A, Lena G: Pilocytic astrocytomas in children: prognostic factors–a retrospective study of 80 cases. Neurosurgery 2003, 53: 544–553. discussion 554–545 10.1227/01.NEU.0000079330.01541.6EView ArticlePubMedGoogle Scholar
- Qaddoumi I, Sultan I, Gajjar A: Outcome and prognostic features in pediatric gliomas: a review of 6212 cases from the Surveillance, Epidemiology, and End Results database. Cancer 2009, 115: 5761–5770. 10.1002/cncr.24663PubMed CentralView ArticlePubMedGoogle Scholar
- Tihan T, Ersen A, Qaddoumi I, Sughayer MA, Tolunay S, Al-Hussaini M, Phillips J, Gupta N, Goldhoff P, Baneerjee A: Pathologic characteristics of pediatric intracranial pilocytic astrocytomas and their impact on outcome in 3 countries: a multi-institutional study. Am J Surg Pathol 2011, 36: 43–55.View ArticleGoogle Scholar
- Cin H, Meyer C, Herr R, Janzarik WG, Lambert S, Jones DT, Jacob K, Benner A, Witt H, Remke M: Oncogenic FAM131B-BRAF fusion resulting from 7q34 deletion comprises an alternative mechanism of MAPK pathway activation in pilocytic astrocytoma. Acta Neuropathol 2011, 121: 763–774. 10.1007/s00401-011-0817-zView ArticlePubMedGoogle Scholar
- Hawkins C, Walker E, Mohamed N, Zhang C, Jacob K, Shirinian M, Alon N, Kahn D, Fried I, Scheinemann K: BRAF-KIAA1549 fusion predicts better clinical outcome in pediatric low-grade astrocytoma. Clin Cancer Res 2011, 17: 4790–4798. 10.1158/1078-0432.CCR-11-0034View ArticlePubMedGoogle Scholar
- Jones DT, Kocialkowski S, Liu L, Pearson DM, Backlund LM, Ichimura K, Collins VP: Tandem duplication producing a novel oncogenic BRAF fusion gene defines the majority of pilocytic astrocytomas. Cancer Res 2008, 68: 8673–8677. 10.1158/0008-5472.CAN-08-2097PubMed CentralView ArticlePubMedGoogle Scholar
- Horbinski C, Hamilton RL, Nikiforov Y, Pollack IF: Association of molecular alterations, including BRAF, with biology and outcome in pilocytic astrocytomas. Acta Neuropathol 2010, 119: 641–649. 10.1007/s00401-009-0634-9View ArticlePubMedGoogle Scholar
- Padovani L, Andre N, Constine LS, Muracciole X: Neurocognitive function after radiotherapy for paediatric brain tumours. Nat Rev Neurol 2012, 8: 578–588. 10.1038/nrneurol.2012.182View ArticlePubMedGoogle Scholar
- Pasquier E, Kavallaris M, Andre N: Metronomic chemotherapy: new rationale for new directions. Nat Rev Clin Oncol 2010, 7: 455–465. 10.1038/nrclinonc.2010.82View ArticlePubMedGoogle Scholar
- Andre N, Padovani L, Pasquier E: Metronomic scheduling of anticancer treatment: the next generation of multitarget therapy? Future Oncol 2011, 7: 385–394. 10.2217/fon.11.11View ArticlePubMedGoogle Scholar
- André N, Banavali S, Snihur Y, Pasquier E: Time for metronomics in low and middle income countries? Lancet Oncol In Press
- Tchoghandjian A, Fernandez C, Colin C, El Ayachi I, Voutsinos-Porche B, Fina F, Scavarda D, Piercecchi-Marti MD, Intagliata D, Ouafik L: Pilocytic astrocytoma of the optic pathway: a tumour deriving from radial glia cells with a specific gene signature. Brain 2009, 132: 1523–1535. 10.1093/brain/awp048View ArticlePubMedGoogle Scholar
- Jendrossek V: Targeting apoptosis pathways by Celecoxib in cancer. Cancer Lett
- Gallicchio M, Rosa AC, Dianzani C, Brucato L, Benetti E, Collino M, Fantozzi R: Celecoxib decreases expression of the adhesion molecules ICAM-1 and VCAM-1 in a colon cancer cell line (HT29). Br J Pharmacol 2008, 153: 870–878.PubMed CentralView ArticlePubMedGoogle Scholar
- Casanova I, Parreno M, Farre L, Guerrero S, Cespedes MV, Pavon MA, Sancho FJ, Marcuello E, Trias M, Mangues R: Celecoxib induces anoikis in human colon carcinoma cells associated with the deregulation of focal adhesions and nuclear translocation of p130Cas. Int J Cancer 2006, 118: 2381–2389. 10.1002/ijc.21662View ArticlePubMedGoogle Scholar
- Sakai R, Iwamatsu A, Hirano N, Ogawa S, Tanaka T, Mano H, Yazaki Y, Hirai H: A novel signaling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner. EMBO J 1994, 13: 3748–3756.PubMed CentralPubMedGoogle Scholar
- Gauthaman K, Fong CY, Bongso A: Statins, stem cells, and cancer. J Cell Biochem 2009, 106: 975–983. 10.1002/jcb.22092View ArticlePubMedGoogle Scholar
- Hindler K, Cleeland CS, Rivera E, Collard CD: The role of statins in cancer therapy. Oncologist 2006, 11: 306–315. 10.1634/theoncologist.11-3-306View ArticlePubMedGoogle Scholar
- Silverstein RL, Febbraio M: CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior. Sci Signal 2009, 2: re3. 10.1126/scisignal.272re3PubMed CentralView ArticlePubMedGoogle Scholar
- Han J, Zhou X, Yokoyama T, Hajjar DP, Gotto AM Jr, Nicholson AC: Pitavastatin downregulates expression of the macrophage type B scavenger receptor, CD36. Circulation 2004, 109: 790–796. 10.1161/01.CIR.0000112576.40815.13View ArticlePubMedGoogle Scholar
- Lin SJ, Chen YH, Lin FY, Hsieh LY, Wang SH, Lin CY, Wang YC, Ku HH, Chen JW, Chen YL: Pravastatin induces thrombomodulin expression in TNFalpha-treated human aortic endothelial cells by inhibiting Rac1 and Cdc42 translocation and activity. J Cell Biochem 2007, 101: 642–653. 10.1002/jcb.21206View ArticlePubMedGoogle Scholar
- Roy M, Kung HJ, Ghosh PM: Statins and prostate cancer: role of cholesterol inhibition vs. prevention of small GTP-binding proteins. Am J Cancer Res 2011, 1: 542–561.PubMed CentralPubMedGoogle Scholar
- Gaist D, Andersen L, Hallas J, Toft Sorensen H, Schroder HD, Friis S: Use of statins and risk of glioma: a nationwide case–control study in Denmark. Br J Cancer
- Yanae M, Tsubaki M, Satou T, Itoh T, Imano M, Yamazoe Y, Nishida S: Statin-induced apoptosis via the suppression of ERK1/2 and Akt activation by inhibition of the geranylgeranyl-pyrophosphate biosynthesis in glioblastoma. J Exp Clin Cancer Res 2011, 30: 74. 10.1186/1756-9966-30-74PubMed CentralView ArticlePubMedGoogle Scholar
- Colin C, Baeza N, Bartoli C, Fina F, Eudes N, Nanni I, Martin PM, Ouafik L, Figarella-Branger D: Identification of genes differentially expressed in glioblastoma versus pilocytic astrocytoma using Suppression Subtractive Hybridization. Oncogene 2006, 25: 2818–2826. 10.1038/sj.onc.1209305View ArticlePubMedGoogle Scholar
- Grill J, Sainte-Rose C, Jouvet A, Gentet JC, Lejars O, Frappaz D, Doz F, Rialland X, Pichon F, Bertozzi AI: Treatment of medulloblastoma with postoperative chemotherapy alone: an SFOP prospective trial in young children. Lancet Oncol 2005, 6: 573–580. 10.1016/S1470-2045(05)70252-7View ArticlePubMedGoogle Scholar
- Khaw SL, Coleman LT, Downie PA, Heath JA, Ashley DM: Temozolomide in pediatric low-grade glioma. Pediatr Blood Cancer 2007, 49: 808–811. 10.1002/pbc.21270View ArticlePubMedGoogle Scholar
- Packer RJ, Ater J, Allen J, Phillips P, Geyer R, Nicholson HS, Jakacki R, Kurczynski E, Needle M, Finlay J: Carboplatin and vincristine chemotherapy for children with newly diagnosed progressive low-grade gliomas. J Neurosurg 1997, 86: 747–754. 10.3171/jns.1997.86.5.0747View ArticlePubMedGoogle Scholar
- Andre N, Abed S, Orbach D, Alla CA, Padovani L, Pasquier E, Gentet JC, Verschuur A: Pilot study of a pediatric metronomic 4-drug regimen. Oncotarget 2011, 2: 960–965.PubMed CentralView ArticlePubMedGoogle Scholar
- Andre N, Rome A, Coze C, Padovani L, Pasquier E, Camoin L, Gentet JC: Metronomic etoposide/cyclophosphamide/celecoxib regimen given to children and adolescents with refractory cancer: a preliminary monocentric study. Clin Ther 2008, 30: 1336–1340. 10.1016/S0149-2918(08)80059-8View ArticlePubMedGoogle Scholar
- Lopez-Aguilar E, Sepulveda-Vildosola AC, Rivera-Marquez H, Cerecedo-Diaz F, Valdez-Sanchez M, Villasis-Keever MA: Security and maximal tolerated doses of fluvastatin in pediatric cancer patients. Arch Med Res 1999, 30: 128–131. 10.1016/S0188-0128(98)00018-9View ArticlePubMedGoogle Scholar
- Couec ML, Andre N, Thebaud E, Minckes O, Rialland X, Corradini N, Aerts I: Marec Berard P, Bourdeaut F, Leblond P: Bevacizumab and irinotecan in children with recurrent or refractory brain tumors: toxicity and efficacy trends. Pediatr Blood Cancer 2012, 59: 34–38. 10.1002/pbc.24066View ArticlePubMedGoogle Scholar
- Andre N, Pasquier E: For cancer, seek and destroy or live and let live? Nature 2009, 460: 324.View ArticlePubMedGoogle Scholar
- Gatenby RA: A change of strategy in the war on cancer. Nature 2009, 459: 508–509. 10.1038/459508aView ArticlePubMedGoogle Scholar
- Patel MI, Subbaramaiah K, Du B, Chang M, Yang P, Newman RA, Cordon-Cardo C, Thaler HT, Dannenberg AJ: Celecoxib inhibits prostate cancer growth: evidence of a cyclooxygenase-2-independent mechanism. Clin Cancer Res 2005, 11: 1999–2007. 10.1158/1078-0432.CCR-04-1877View ArticlePubMedGoogle Scholar
- Sareddy GR, Geeviman K, Ramulu C, Babu PP: The nonsteroidal anti-inflammatory drug celecoxib suppresses the growth and induces apoptosis of human glioblastoma cells via the NF-kappaB pathway. J Neurooncol 2011, 106: 99–109.View ArticlePubMedGoogle Scholar
- Kang KB, Wang TT, Woon CT, Cheah ES, Moore XL, Zhu C, Wong MC: Enhancement of glioblastoma radioresponse by a selective COX-2 inhibitor celecoxib: inhibition of tumor angiogenesis with extensive tumor necrosis. Int J Radiat Oncol Biol Phys 2007, 67: 888–896. 10.1016/j.ijrobp.2006.09.055View ArticlePubMedGoogle Scholar
- Gaiser T, Becker MR, Habel A, Reuss DE, Ehemann V, Rami A, Siegelin MD: TRAIL-mediated apoptosis in malignant glioma cells is augmented by celecoxib through proteasomal degradation of survivin. Neurosci Lett 2008, 442: 109–113. 10.1016/j.neulet.2008.07.014View ArticlePubMedGoogle Scholar
- Ferris JS, McCoy L, Neugut AI, Wrensch M, Lai R: HMG CoA reductase inhibitors, NSAIDs and risk of glioma. Int J Cancer 2012, 131: E1031–1037. 10.1002/ijc.27536PubMed CentralView ArticlePubMedGoogle Scholar
- Steinbach G, Lynch PM, Phillips RK, Wallace MH, Hawk E, Gordon GB, Wakabayashi N, Saunders B, Shen Y, Fujimura T: The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis. N Engl J Med 2000, 342: 1946–1952. 10.1056/NEJM200006293422603View ArticlePubMedGoogle Scholar
- Siekmeier R, Lattke P, Mix C, Park JW, Jaross W: Dose dependency of fluvastatin pharmacokinetics in serum determined by reversed phase HPLC. J Cardiovasc Pharmacol Ther 2001, 6: 137–145. 10.1177/107424840100600205View ArticlePubMedGoogle Scholar
- Sierra S, Ramos MC, Molina P, Esteo C, Vazquez JA, Burgos JS: Statins as neuroprotectants: a comparative in vitro study of lipophilicity, blood–brain-barrier penetration, lowering of brain cholesterol, and decrease of neuron cell death. J Alzheimers Dis 2011, 23: 307–318.PubMedGoogle Scholar
- Dembo G, Park SB, Kharasch ED: Central nervous system concentrations of cyclooxygenase-2 inhibitors in humans. Anesthesiology 2005, 102: 409–415. 10.1097/00000542-200502000-00026View ArticlePubMedGoogle Scholar
- Xia L, Zhang D, Du R, Pan Y, Zhao L, Sun S, Hong L, Liu J: Fan D: miR-15b and miR-16 modulate multidrug resistance by targeting BCL2 in human gastric cancer cells. Int J Cancer 2008, 123: 372–379. 10.1002/ijc.23501View ArticlePubMedGoogle Scholar
- Gao J, Jia WD, Li JS, Wang W, Xu GL, Ma JL, Ge YS, Yu JH, Ren WH, Liu WB, Zhang CH: Combined inhibitory effects of celecoxib and fluvastatin on the growth of human hepatocellular carcinoma xenografts in nude mice. J Int Med Res 2010, 38: 1413–1427. 10.1177/147323001003800423View ArticlePubMedGoogle Scholar
- Gao J, Li JS, Xu GL, Jia WD, Ma JL, Yu JH, Ge YS: [Effects of celecoxib combined with fluvastatin on tumor growth and cell apoptosis in a xenograft model of hepatocellular carcinoma]. Zhonghua Gan Zang Bing Za Zhi 2010, 18: 900–904.PubMedGoogle Scholar
- Chappe C, Padovani L, Scavarda D, Forest F, Nanni-Metellus I, Loundou A, Mercurio S, Fina F, Lena G, Colin C, Figarella-Branger D: Dysembryoplastic neuroepithelial tumors share with pleomorphic xanthoastrocytomas and gangliogliomas BRAF mutation and expression. Brain Pathol 2013.Google Scholar
- Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987, 162: 156–159.View ArticlePubMedGoogle Scholar
- Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001, 29: e45. 10.1093/nar/29.9.e45PubMed CentralView ArticlePubMedGoogle Scholar
- Pasquier E, Ciccolini J, Carre M, Giacometti S, Fanciullino R, Pouchy C, Montero MP, Serdjebi C, Kavallaris M, Andre N: Propranolol potentiates the anti-angiogenic effects and anti-tumor efficacy of chemotherapy agents: implication in breast cancer treatment. Oncotarget 2011, 2: 797–809.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.