glioblastoma anti tumor action of cyclosporin a and functionally related drugs

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Chapter 25 Glioblastoma: Anti-tumor Action of Cyclosporin A and Functionally Related Drugs Bozena Kaminska, Magdalena Tyburczy, Konrad Gabrusiewicz, and Malgorzata Sielska Abstract Human malignant gliomas are highly resistant to current therapeutic approaches. Major signaling pathways that have been identified as playing important roles in glioblastomas are: the PTEN/PI3K/Akt/mTOR and the Ras/Raf/MEK/ERK signaling cascades, which support cell invasion, sur- vival and prevent apoptosis. In the face of tumor resistance to apoptosis, novel agents which can over- come resistance or/and affect cell survival by non- apoptotic mechanisms such as necrosis, senescence, autophagy and mitotic catastrophe, are highly desir- able. The present chapter focuses on anti-tumor action of cyclosporin A (CsA) and rapamycin that besides their well known immunosuppressive abilities appear to be multitarget kinase inhibitors and moderately effective anti-tumor agents in glioblastomas in vitro, in vivo and in clinical trials. A compelling evidence shows that cyclosporin A induces growth arrest and programmed cell death in cultured rat and human glioblastoma cells. The molecular mechanism involves accumulation of a cell cycle inhibitor – p21Cip1/Waf1, even in the absence of functional p53 tumor sup- pressor. In C6 glioma cells with functional TP53 and PTEN tumor suppressors CsA treatment up-regulates fasL expression, activates p53 and intrinsic mitochon- drial death pathway, while in human glioblastoma cells with defects in either TP53 or PTEN, none B. Kaminska () Laboratory of Transcription Regulation, Department of Cell Biology, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland e-mail: [email protected] of those effects were observed. Molecular analysis revealed that CsA, trough yet unknown mechanisms, down-regulates PI3K/Akt and mTOR signaling path- ways in glioblastoma cells, and interferes with pro- invasive activity of tumor-infiltrating microglia. A sys- temically applied CsA significantly reduced growth of intracranial gliomas, tumor invasion and angio- genesis. Pharmacological inhibitors of the mTOR pathway: rapamycin, temsirolimus, everolimus and AP23573 were tested as potential targeted drugs in human glioblastoma cultures and in animal mod- els. However, rapamycin and derivatives show mod- erate efficacy in patients with recurrent glioblas- toma multiforme, they deserve further clinical studies, particularly in combination with PI-3K pathway inhibitors. Defects of innate and adaptive immunity are common in glioblastoma patients contributing to a lack of effective anti-tumor responses. Thus, immunosuppressants such as CsA, rapamycin and its derivatives may be an effective novel strategy to treat drug-resistant gliomas or complement apoptosis based-therapies. Keywords Glioblastomas · Anti-tumor drugs · Immunosuppressants · Apoptosis · Rapamycin · Inhibitors Introduction Glioblastomas are the most frequent and devas- tating primary malignant brain tumor in adults. Glioblastomas are highly resistant to current therapeutic approaches, in which surgery is followed 241 M.A. Hayat (ed.), Tumors of the Central Nervous System, Volume 2, DOI 10.1007/978-94-007-0618-7_25, © Springer Science+Business Media B.V. 2011

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Page 1: Glioblastoma Anti Tumor Action of Cyclosporin a and Functionally Related Drugs

Chapter 25

Glioblastoma: Anti-tumor Action of Cyclosporin Aand Functionally Related Drugs

Bozena Kaminska, Magdalena Tyburczy, Konrad Gabrusiewicz,and Malgorzata Sielska

Abstract Human malignant gliomas are highlyresistant to current therapeutic approaches. Majorsignaling pathways that have been identified asplaying important roles in glioblastomas are: thePTEN/PI3K/Akt/mTOR and the Ras/Raf/MEK/ERKsignaling cascades, which support cell invasion, sur-vival and prevent apoptosis. In the face of tumorresistance to apoptosis, novel agents which can over-come resistance or/and affect cell survival by non-apoptotic mechanisms such as necrosis, senescence,autophagy and mitotic catastrophe, are highly desir-able. The present chapter focuses on anti-tumor actionof cyclosporin A (CsA) and rapamycin that besidestheir well known immunosuppressive abilities appearto be multitarget kinase inhibitors and moderatelyeffective anti-tumor agents in glioblastomas in vitro,in vivo and in clinical trials. A compelling evidenceshows that cyclosporin A induces growth arrest andprogrammed cell death in cultured rat and humanglioblastoma cells. The molecular mechanism involvesaccumulation of a cell cycle inhibitor – p21Cip1/Waf1,even in the absence of functional p53 tumor sup-pressor. In C6 glioma cells with functional TP53 andPTEN tumor suppressors CsA treatment up-regulatesfasL expression, activates p53 and intrinsic mitochon-drial death pathway, while in human glioblastomacells with defects in either TP53 or PTEN, none

B. Kaminska (�)Laboratory of Transcription Regulation, Department of CellBiology, Nencki Institute of Experimental Biology, 02-093Warsaw, Polande-mail: [email protected]

of those effects were observed. Molecular analysisrevealed that CsA, trough yet unknown mechanisms,down-regulates PI3K/Akt and mTOR signaling path-ways in glioblastoma cells, and interferes with pro-invasive activity of tumor-infiltrating microglia. A sys-temically applied CsA significantly reduced growthof intracranial gliomas, tumor invasion and angio-genesis. Pharmacological inhibitors of the mTORpathway: rapamycin, temsirolimus, everolimus andAP23573 were tested as potential targeted drugs inhuman glioblastoma cultures and in animal mod-els. However, rapamycin and derivatives show mod-erate efficacy in patients with recurrent glioblas-toma multiforme, they deserve further clinical studies,particularly in combination with PI-3K pathwayinhibitors. Defects of innate and adaptive immunityare common in glioblastoma patients contributingto a lack of effective anti-tumor responses. Thus,immunosuppressants such as CsA, rapamycin andits derivatives may be an effective novel strategy totreat drug-resistant gliomas or complement apoptosisbased-therapies.

Keywords Glioblastomas · Anti-tumor drugs ·Immunosuppressants · Apoptosis · Rapamycin ·Inhibitors

Introduction

Glioblastomas are the most frequent and devas-tating primary malignant brain tumor in adults.Glioblastomas are highly resistant to currenttherapeutic approaches, in which surgery is followed

241M.A. Hayat (ed.), Tumors of the Central Nervous System, Volume 2,DOI 10.1007/978-94-007-0618-7_25, © Springer Science+Business Media B.V. 2011

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by radiotherapy with concomitant and adjuvantchemotherapy. The prognosis remains poor witha median survival in the range of 12–15 months(Clarke et al., 2010). Common genetic abnormalitiesin glioblastoma are associated with multiple molecularmechanisms involved in drug resistance, includingdrug detoxification, aberrant activation or suppressionof cellular signal transduction pathways, deficienciesin tumor suppressors, apoptosis mediators and deathligand/receptor signaling. Significantly high frequencyof alterations in cell cycle regulators such as the TP53tumor suppressor and the p16INK4A cyclin dependentkinase inhibitor results in reduced sensitivity to amajority of anti-cancer drugs (Ohgaki and Kleihues,2009).

Major signaling pathways that have been identifiedas playing important roles in glioblastomas are: thePTEN/PI3K/Akt/mTOR and the Ras/Raf/MEK/ERKsignaling cascades, which support cell proliferation,survival, invasion, and prevent apoptosis (McCubreyet al., 2006). Components of these pathways arefrequently mutated, aberrantly expressed or consti-tutively activated in glioblastomas. Monoclonal anti-bodies or small molecular-weight kinase inhibitorstargeting specific pathways are the most commonclasses of agents in cancer treatment. However,highly selective or specific blocking of only oneof the kinases has been associated with limited orsporadic responses. Therefore, multitargeted kinaseinhibitors and combinations of single-target kinaseinhibitors should be more effective to overcometherapeutic resistance. Some agents could be usedtogether with radiation, chemotherapy, or immunother-apy to enhance treatment efficacy. Furthermore, inthe face of tumor resistance to apoptosis, novelagents which can overcome resistance or/and inducecell death by non-apoptotic mechanisms such asnecrosis, senescence, autophagy (type II programmedcell death) and mitotic catastrophe, are highlydesirable.

The present chapter focuses on anti-tumor actionof drugs such as cyclosporin A and rapamycinthat besides their well known immunosuppressiveabilities appear to be kinase inhibitors and poten-tial anti-tumor agents in glioblastomas. The presentchapter summarizes a rationale and results of pre-clinical/clinical studies of these agents in therapy ofglioblastomas.

Mechanisms of Cyclosporin A InducedCell Death in Rat C6 Glioma Cells

Mode of Immunosuppressant Action

Cyclosporin A (CsA), FK506 (tacrolimus, Prograf)and rapamycin (sirolimus) are short polypeptideswhich have revolutionized transplantology due to abil-ity to block the activation of lymphocytes and otherimmune system cells. CsA, FK506 and rapamycin bindto specific intracellular proteins called immunophilins:CsA binds to cyclophilin, FK506 and rapamycinbind to FKBP (FK506-binding protein). Drug-immunophilin complexes bind to a regulatory subunitof calcineurin and inhibit its activity. Calcineurin isa calcium- and calmodulin-dependent threonine/serinephosphatase. CsA and FK506 exert immunosuppres-sive effects by inhibiting of calcineurin-mediateddephosphorylation of NFAT (nuclear factor of acti-vated T cells), thus preventing transcriptional induc-tion of several cytokines and their receptors genes(Fig. 25.1). NFAT family proteins are transcriptionfactors that regulate the expression of a variety oftarget genes and are implicated in many functions,including cell growth, survival, invasion and angiogen-esis (Mancini and Toker, 2009). Several members ofNFAT family were detected in C6 glioma cells point-ing to a new mechanism of transcription regulation inglioma cells. A transient receptor potential 6 (TRPC6)which is required for the development of the aggres-sive glioblastoma phenotype and causes a sustainedelevation of intracellular calcium, is coupled to activa-tion of the calcineurin-NFAT pathway. Pharmacologicinhibition of this pathway reduced the develop-ment of the aggressive glioblastoma phenotype underhypoxia.

Molecular Mechanisms of Pro-apoptoticAction of Cyclosporin A in C6 Glioma Cells

It was reported by Mosieniak et al. (1997) thatcyclosporin A at concentrations at the range of 30–60 μM inhibits proliferation of rat C6 glioma cellsand induces cell death. CsA-induced cell death was an

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Fig. 25.1 Mechanism of immunosuppressant action (a) Immu-nosuppressants bind to immunophilines: CsA to cyclophilin(cyc) and FK506 to FKBP (FK506-binding protein); subse-quently complexes bind to the calcineurin and inhibit its activity.Calcineurin is a calcium- and calmodulin-dependent phos-phatase which dephosphorylates NFAT transcription factorsallowing them to translocate to the nucleus, where NFAT pro-teins in cooperation with other transcription factors (f.e. AP-1)regulate the expression of genes coding for chemokines,cytokines and their receptors. The immunosuppressant-immunophilin complex may interfere with MAPK signaling

pathways. (b) Summary of pro-apoptotic mechanisms inducedby CsA in C6 glioma cells. CsA induces activation of JNK(c-Jun amino-terminal kinase) and MKK3 (MAP kinase-activated protein kinase)-p38 MAPK signaling pathways. Itleads to activation of AP-1 and transcriptional up-regulation ofFasL (ligand Fas) which binds receptor Fas expressed on gliomacells and induces cell death. Activation of p38 MAPK signalingleads to accumulation of p53 and induction of p53-dependentexpression of pro-apoptotic genes involved in mitochondrialdeath pathway

active process requiring expression of new genes andproteins, with typical features of apoptosis: oligonu-cleosomal DNA fragmentation and caspase 3 activa-tion (Mosieniak et al., 1997; Pyrzynska et al., 2000).Two major mechanisms responsible for induction of

apoptotic death by CsA were identified. Apoptotic celldeath induced by CsA was associated with a persis-tent activation of mitogen activated protein kinases(MAPK), in particular c–Jun N-terminal kinase (JNK)and p38 MAPK. Prolonged activation of JNK led

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to accumulation of phosphorylated c-Jun and ATF-2 (main substrates of JNK) and formation of theAP-1 transcription factor followed by transcriptionalactivation of the Fas Ligand expression (Pyrzynskaet al., 2000). Further studies with promoter constructsdepleted of DNA binding sites for particular tran-scription factors revealed that activation of the FasLgene promoter was only partially AP-1-dependentand collaborative action of other transcription factorswas required for promoter activation. It was demon-strated that CsA down-regulates Akt signaling to facil-itate activation of Forkhead family members resultingin transcriptional activation of the FasL expression(Ciechomska et al., 2003). Down-regulation of Akt sig-naling was necessary to permit Forkhead transcriptionfactor translocation to the nucleus and pre-requisiteto transcriptional activation of the FasL expression.Treatment of glioma cells with lower doses of CsA(1–10 μM) was sufficient to reduce Akt phosphory-lation and sensitized cells to doxorubicin, and UVCtreatments (unpublished).

Another event resulting from prolonged activa-tion of MAP kinases, in particular p38 MAPK, wasaccumulation of the tumor suppressor p53 in gliomacells. The p53-family of transcription factors consistsof three genes – p53, p63, and p73 sharing signif-icant structural and functional similarities. p53 is apotent inducer of apoptosis and tumor suppression.Many anti-cancer agents, from traditional chemo- andradiation therapies to more recently developed smallmolecules, exert their effects by enhancing the anti-proliferative effects of p53 and transactivating p63/p73proteins. In normal cells the p53 is expressed atlow, constitutive level and localized predominantly incytoplasm. The latent form of p53 is stabilized andactivated by posttranslational modifications. The acti-vation of p53 occurs in response to DNA damage orstress such as hypoxia, nutrients or nucleotide depriva-tion. p53-mediated cell cycle arrest is largely broughtabout by induction of p21Waf1, an inhibitor of cyclin-dependent kinases. Activation of p53 may also resultin apoptosis via transcriptional activation of a num-ber of pro-apoptotic proteins including Bax, Fas, p85,IGF-BP3, and PIG3 and apoptotic protease activatingfactor-1 (apaf-1).

It was reported by Pyrzynska et al. (2002) that CsAtreatment results in up-regulation of p53 protein leveland its accumulation in cell nuclei. Concomitantly,the levels of p21Waf1 and Bax proteins increased,

and Bcl-xL decreased in CsA-treated glioma cells.Bax protein translocated to mitochondria, as revealedby immunofluorescence and double staining with amitochondrial marker, and likely induced mitochon-drial apoptotic pathway. Contribution of p53 to CsA-induced cell death was further confirmed in experi-ments, in which glioma cells stably transfected with amutant p53 (p53Val135) failed to increase p21 and Baxprotein levels and were less sensitive to CsA-inducedapoptosis. Also primary fibroblasts from p53-/-knockout mice were significantly more resistant toCsA-induced apoptosis compared to their correspond-ing counterparts containing functional p53 (Pyrzynskaet al., 2002).

Accumulation and activity of p53 can be regu-lated by phosphorylation that occurs at several Ser andThr residues, and a number of cellular kinases havebeen proposed to directly phosphorylate p53, includingcasein kinase I, casein kinase II, double-stranded-RNA-dependent protein kinase, ATM, CDK7, DNA-activated protein kinase, Jun-NH2 kinase and p38MAP kinase. The induction of cell death by CsA wasassociated with a persistent activation of MKK3-p38MAP kinase signaling pathway. Overexpression of adominant negative form of MKK3, an upstream acti-vator of p38, abrogated phosphorylation of p38 MAPKand p53 accumulation (Pyrzynska et al., unpublished).Together, the compelling evidence demonstrate that theapoptotic program activated by CsA can be mediatedby multiple pathways: via activation of p53 transcrip-tion factor and p53-mediated apoptosis, and throughup-regulation of FasL expression by JNK-AP-1 andForkhead (Fig. 25.1).

Cyclosporin A Induces Cell Deathor Growth Arrest and Senescenceof Human Glioblastoma Cells

In rat C6 glioma cells apoptotic cell death induced byCsA was associated with induction of intrinsic deathpathway: p53- and mitochondria-dependent, as well asextrinsic, death ligand-dependent pathway. However,the experiments performed on glioma cells lacking afunctional p53, namely the cell line stably expressingthe temperature-sensitive p53 mutant, indicated that upto 20% of cells died even in the absence of functional

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p53, suggesting also a p53-independent mode of CsAaction.

Therefore, we tested efficacy of CsA towardsthree human glioblastoma cell lines that are radio-and chemotherapy resistant, in part due to mutationsin the tumor suppressor PTEN or/and TP53 genes:T98G (mutated PTEN and TP53), U373-MG (mutatedTP53 and PTEN) and U87-MG cells (wild typeTP53/mutated PTEN). Cultured cells were exposedto increasing concentrations of CsA (Fig. 25.2) and30 μg/ml CsA affected growth of all studied gliomacells, produced dramatic changes in cell number andmorphology in 24 h. The inhibitory effect of CsA oncell growth and survival was dose-dependent, and pro-gressed with the time of exposure. CsA at the concen-tration range of 5–20 μg/ml had no visible cytotoxiceffects (Zupanska et al., 2005). Morphological alter-ations induced by 30 μg/ml CsA were characterizedby shrinkage of the cells, rounding up of the cell bodyand detachment from the bottom of the plate at 24–48 h after the treatment (Fig. 25.2). Cell death inducedby CsA was blocked by cycloheximide (a protein syn-thesis inhibitor) and showed no signs of necrosis, suchas swelling or disruption of cells. The appearanceof numerous large vacuoles was observed in CsA-treated T98G cells and to less extent in U373-MG cells(Zupanska et al., 2005).

Features of CsA-triggered cell death in humanglioblastoma cells do not fulfill all criteria of apoptosis.For example, biochemical hallmarks of apoptosis suchas phosphatidylserine exposure in the external leafletof the plasma membrane bilayer, the “ladder-like”oligonucleosomal DNA fragmentation or appearanceof the subG1 population were not detected, althoughcondensation of chromatin, deformation of nucleiand sparse TUNEL labeling were observed. Cells inCsA-treated cultures showed “bean” shaped nucleiwith condensed chromatin or nuclei with irregularclumps of dense chromatin. Such nuclear alterationsas “bean” shaped nuclei with condensed chromatinusually preceded oligonucleosomal DNA fragmenta-tion in CsA-treated rat glioma cells (Mosieniak et al.,1997). In T98G glioblastoma cells nuclear alterationswere completely abolished by cycloheximide (CHX)treatment, indicating dependency of cell death on denovo protein synthesis. Measurement of changes in themitochondrial membrane potential in response to CsAtreatment with the fluorescent probe JC-1 did not showalterations in mitochondrial potential, which excludes

a possibility of initiation of mitochondrial pathwayalong with apoptosome formation (Zupanska et al.,2005).

Western blot analysis using specific antibodies rec-ognizing intact and cleaved caspases 7, 3 and PARP(poly (ADP-ribose) polymerase-1) was employed tofurther characterize cell death induced by CsA. Thetreatment of human glioblastoma cells with 30 μMCsA resulted in activation of the caspase cascade (asevidenced by the appearance of cleaved caspase 7, 3and cleaved PARP) in T98G cells, and to lesser extentin U373-MG cells. No caspase activation was detectedin U87 glioma cells (Fig. 25.2).

Growth arrest may lead to replicative senescenceor differentiation. Senescent cells in contrast to pre-senescent, proliferating or quiescent cells expressa beta-galactosidase activity detectable at pH 6.0.Since flatten morphology of CsA-treated U87 cellsand reduction of the number of proliferating cellssuggested the induction of differentiation process, asenescence-associated β-galactosidase (SA-beta-Gal)staining was performed (Fig. 25.2). Untreated U87-MG and T98G cells demonstrated very rare cellspositive for the SA-beta-Gal staining. After 24 h ofCsA treatment the percentage of cells positive forSA-beta-Gal was doubled in U87-MG cells comparedto control, while only few T98G cells were posi-tively stained. SA-beta-Gal positive U87-MG cellsincreased in size and flattened out, thereby attainingmorphology of senescent-like cells (Zupanska et al.,2005).

Cell proliferation is controlled by cell cycleregulatory factors which include cyclins and cyclin-dependent kinases. p21WAF1/Cip1 protein is anuniversal inhibitor of cyclin kinases and plays animportant role in inhibiting cell proliferation. Thelevels of p21WAF1/Cip1 protein were increased afterexposure to 30 μM CsA in all examined glioblastomacell lines. The highest up-regulation of p21WAF1/Cip1protein was observed in T98G cells and the moderateaccumulation was detected in U373-MG and U87-MG cells. Further studies revealed transcriptionalactivation of p21WAF1/Cip1 expression preceded intime by a long-term activation of ERK1/2 signaling,subsequent c-Jun phosphorylation and accumula-tion of AP-1 complex in CsA-treated glioblastomacells. Pre-treatment with ERK pathway inhibitors oroverexpression of dominant negative mutants MKK1,ERK2 and c-Jun reduced the p21WAF1/Cip1

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Fig. 25.2 CsA induces programmed cell death or growtharrest/senescence of malignant glioblastoma cells. (a)Morphological alterations induced by CsA treatment in humanglioma cells. T98G cells cultured in DMEM with 10% fetalbovine serum, were exposed to 30 μM CsA alone or with1 μg/ml cycloheximide (CHX). Upper panel shows that a major-ity of cells lost processes and became round in CsA-treatedcultures. Lower panel shows Hoechst 33258 staining reveal-ing deformations of cell nuclei and condensation of chromatin.CHX inhibits morphological alterations in CsA-treated T98Gcells. (b) Caspase cascade activation in CsA-triggered cell death.

A representative immunoblot shows caspase activation in totalprotein extracts of human glioma cells treated with 30 μM CsA.Specific antibodies recognizing intact and cleaved caspases 7,3 and cleaved PARP (Cell Signaling, USA) were employed.(c) Detection of the Senescence-Associated beta-galactosidase(SA-beta-Gal) staining in CsA-treated U87-MG glioma cells.The substantial increase in cellular volume and the blue stainingreflecting SA-beta-Gal activity was observed in U87-MG cellstreated with CsA. Original magnification for larger photos is×10; for insets ×20

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expression, confirming involvement of this pathway.Transcriptional activation of p21WAF1/Cip1 expres-sion by CsA was independent of p53 and precededCsA-induced growth arrest in glioblastoma cells(Zupanska et al., 2007).

In conclusion, the findings above presented demon-strate an ability of CsA to induce growth arrest orprogrammed, but non-apoptotic cell death in humanglioblastoma cells at the concentration of 30 μM orhigher. Many forms of programmed cell death differentfrom “classical” apoptosis have been described by thecriteria of morphology, biochemistry, and response toapoptosis inhibitors, particularly in transformed cellswhich contain endogenous inhibitors preventing a par-ticular pathway.

Anti-tumor Effects of CsA in GliomaModels

Good cytostatic and cytotoxic efficacy of CsA in ratand human glioblastoma cultures encouraged studiesin more complex models: in organotypic brain slicecultures and in murine glioma model. Organotypicbrain slice cultures injected with glioblastoma cellsrecapitulate many features of glioblastoma and arevery useful for investigating the cellular and molecu-lar mechanisms of glioma invasion under conditionsmost analogous to those of normal brains in vivo. Itwas reported by Markovic et al. (2005) that microglialcells contribute significantly to invasion of glioma cellsin cultured brain slices. Microglia are the intrinsicimmune cells of the brain, serving principally to con-trol the innate and the adaptive immune responses inthe central nervous system, to initiate host-defenseand tissue repair mechanisms. Microglial cells areattracted towards glioma (glioma tissue consists of upto 30% of microglial cells) and microglia density ingliomas positively correlates with malignancy, inva-siveness and grading of the tumors (Watters et al.,2005). When glioma cells were injected into brainslices depleted of endogenous microglia (by incuba-tion with clodronate-filled liposomes for 96 h), theinvasiveness of the tumors was significantly decreased.Inoculation of exogenous microglia together withglioma cells into cultured brain slices increased theinfiltrative behavior of glioma cells. Experiments with

co-culture of microglia with glioma cells revealed thatsoluble factors released from glioma cells stronglystimulate metalloprotease-2 (MMP-2) activity increas-ing breakdown of extracellular matrix and therebypromoting tumor invasiveness (Markovic et al., 2005).Further studies showed that membrane type 1 metal-loproteinase (MT1-MMP) is up-regulated in glioma-associated microglia. Microglial MT1-MMP in turnactivates glioma-derived pro-MMP-2 and promotesglioma expansion. Tumor growth and invasion in exvivo model using MT1-MMP deficient brain tissue andin microglia-depleted animals were strongly reduced(Markovic et al., 2009).

It was reported by Sliwa et al. (2007) that migra-tion/invasion of fluorescently labeled GL261 gliomacells in murine brain slices significantly decreasedupon treatment with CsA, even at low concentra-tions: 1, 10 and 30 μM. Anti-invasive effects oflower, non-cytotoxic doses of CsA suggested an exis-tence of the additional mechanism of CsA actionon tumor invasion. When glioma cells were injectedinto slices devoid of endogenous microglia, theinhibitory effect of 1 μM CsA on glioma invasive-ness mostly vanished. It indicated that CsA abolishesmicroglia-promoting effects on glioma malignancy.The inhibitory action of CsA on microglia functionhas been directly demonstrated in microglia-gliomaco-cultures. Glioma-derived factors in co-culturesor glioma conditioned medium induce morphologi-cal transformation of microglial cells into amoeboidphagocytes, activate MAPK signaling pathways andproduction of some cytokines. CsA at low doses of0.1. and 1 μM blocked transformation of microglialcells stimulated by glioma-conditioned medium andabolished pro-invasive effects of microglia (Sliwaet al., 2007). A recent study points to a crucialrole of microglia-derived transforming growth fac-tor beta 1 (TGF-β1) in regulation of glioma invasion(Wesolowska et al., 2008). Blockade of TGF-β1 sig-naling by silencing of TGF-β1 type II receptor withshRNA or neutralizing anti-TGF-β1 antibody abol-ished the promoting effect of microglia on gliomainvasion.

In vivo tumor models developed by intracranialor subcutaneous implantation of glioma cell linesin rodents are used to test novel therapies. Theadvantages of these glioma models are their highlyefficient gliomagenesis, reproducible growth rates,and knowledge of tumor location. However, these

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models have been criticized for not recapitulatingall pathological features of human glioblastoma mul-tiforme (GBM), they allow to target different fea-tures of GBM, including location in the brain, inva-sion of brain parenchyma, angiogenesis, and secre-tion of immune suppressive molecules. In studiesby Sliwa et al. (2007) fluorescently labeled GL261glioma cells were implanted into the striatum ofDBA/2 J mice and developed gliomas of consider-able size in 14 days. CsA (Sandimmun, Novartis)was administrated intraperitoneally (i.p.) every sec-ond days at doses of 2 or 10 mg/kg of body weightstarting from the second day after cell inoculation.Systemically applied 2 or 10 mg/kg CsA signifi-cantly decreased tumor volumes (by 70%) with similarefficacy.

Further studies in syngenic murine models, suchas GL261 mouse glioma cells implanted to C57BL6mice, have confirmed anti-tumor action of CsA admin-istrated intraperitoneally every second day at dosesof 2 and 10 mg/kg. Immunofluorescence studieswith Iba1 antibody demonstrated that CsA blocksaccumulation and activation of glioma-associatedmicroglia confirming the in vitro data (unpublished).It was reported that macrophage–colony stimulat-ing factor (M-CSF) and other cytokines secretedby high-grade glioma cells stimulate differentia-tion of tumor-infiltrating microglia/macrophages intocells acquiring the anti-inflammatory (M2) pheno-type. M-CSF was significantly correlated with his-tological malignancy and with the proportion ofM2 microglia/macrophages in vivo. Such M2 cellsinstead of initiating immune responses support tumorgrowth and invasion. CsA with its strong effecton pro-tumorigenic activity of microglia could beeffective drug in modulation of anti-inflammatoryM2 phenotype of microglia/macrophages in gliomatherapy.

CsA has been used to block the immune reac-tion towards human glioblastoma cells transplantedto rodent brains. It was reported by Mathiesen et al.(1989) that human glioblastoma cells implanted tocerebrum of rat hosts survive longer in immunosup-pressed animals but aggressive growth of glioblas-toma cells and tumor proliferation was not observed.Human glioblastoma cells implanted into the brainof Wistar rats immunosuppressed with CsA appliedat 12 mg/kg/daily survived under such conditions butformed dense and poorly diffused tumors (Strojnik

et al., 2006). Up to now CsA has not beentested as anti-invasive/cytotoxic agent in clinicaltrials.

Anti-tumor Action of Rapamycinand Derivatives

Mechanism of Action of Rapamycin

Rapamycin (Sirolimus) is a macrolide discovered∼1970 as a product of the bacterium Streptomyceshygroscopicus in a soil sample from the Easter Island(Rapa Nui). Rapamycin was originally developed asan antifungal agent, but turned out to be a potentimmunosuppressive drug and since 1997 has beenused to prevent host-rejection in kidney transplanta-tion. An immunosuppressive effect of rapamycin isdue to the inhibition of interleukin 2 (IL-2)-mediatedT-cell proliferation and activation. The mode of actionof rapamycin is to bind the cytosolic immunophilinFK-binding protein 12 (FKBP12). This complexinhibits the mammalian target of rapamycin (mTOR)pathway by directly binding the mTOR Complex1 (mTORC1). mTOR is a kinase playing a keyrole in the regulation of cell growth and prolifer-ation by regulating ribosomal biogenesis and pro-tein translation. Among others, mTOR can be acti-vated by growth factors and hormones, which ismediated by the induction of PI-3-kinase (PI-3K).Active PI-3K generates phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] which activates kinaseAkt phosphorylating and inactivating the tuberoussclerosis complex 2 (TSC2). TSC2 acts as a GTPase-activating protein for the small GTPase RHEB (Rashomolog enriched in brain). Inhibition of TSC2 activ-ity leads to elevated RHEB-GTP levels and activationof mTORC1 which by its downstream effectors, suchas ribosomal protein S6 kinases (S6K) and 4E-bindingprotein (4E-BP1), further up-regulates ribosome bio-genesis and protein translation (Fig. 25.3). This resultsin an increase in cell size and mass, and enhancedproliferation. On the other hand, inhibition of mTORpromotes autophagy in some cell types. Autophagyis a process of degradation of organelles and macro-molecules to supply cells with nutrients and can func-tion as a cytoprotective mechanism or alternative celldeath process.

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Fig. 25.3 Rapamycin inhibits proliferation or induces cell deathof malignant glioblastoma cells. (a) mTOR signaling network.mTORC1 stimulates cell growth upon activation by growth fac-tors or insulin (for details see the text). (b) Rapamycin inhibitsthe kinase activity of mTOR measured by reduction of S6Kphosphorylation in T98G cells. Western blot analysis of thephospho-S6K (T389) level in T98G glioma cells after treatmentwith 10 nM rapamycin for 24 h. S6K served as a protein loadingcontrol. (c) Rapamycin affects proliferation of T98G cells. Cellproliferation was determined 24 h after exposure to 1, 10, 30

or 60 μM rapamycin using BrdU test. Bars represent the ratioof proliferating cells treated with the inhibitor related to con-trol cells (means ± SEM of three independent experiments, eachin triplicate). Statistical analysis was done by one-way ANOVAfollowed by Newman-Keuls test, ∗∗∗p < 0.001. (d) Rapamycininduces morphological alterations in T98G cells. Phase-contrastimages of T98G glioma cells performed 24 h after treatment with10, 30 or 60 μM rapamycin. Cell death was observed after usingthe highest drug concentration. Original magnification is ×10

Rapamycin and Its Analogs in Pre-clinicaland Clinical Trials in Glioblastomas

Enhanced or constitutive activation of thePI3K/Akt/mTOR pathway is an important factor ingliomagenesis (Guertin and Sabatini, 2007). In humanglioblastomas Akt is activated in approximately 70%of tumors due to loss of PTEN (phosphatase andtensin homolog deleted on Chromosome 10), thetumor suppressor and negative regulator of PI-3 K/Aktsignaling, and/or up-regulation of epidermal growthfactor receptor (EGFR) and platelet-derived growthfactor receptor (PDGFR) tyrosine kinases. PTENmutations are present in 20–40% of GBM. Since

mTOR is a crucial downstream component of thePTEN/Akt signaling, pre-clinical studies with phar-macological inhibitors of the mTOR pathway wereinitiated in glioblastomas. Apart from rapamycin, itsanalogs with improved pharmacokinetic propertieswere synthesized. CCI779 (temsirolimus; Wyeth),RAD001 (everolimus; Novartis) and AP23573 (Ariad)are currently being tested as potential targeted drugsin glioblastomas. Pharmacological inactivation ofmTOR decreased tumor cells proliferation and tumorsize in PTEN-deficient mice (Rajasekhar et al., 2003).Moreover, xenografts generated from PTEN nullU87 glioma cells in mice had inhibited growth andreduced proliferation rate after rapamycin treatment.

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Conversely, rapamycin did not decrease size of PTENwild-type LN229 tumors (Wei et al., 2008). Thesefindings provided grounds to initiate clinical trials ofmTOR inhibitors in glioblastomas.

The results of six studies using sirolimus orCCI-779 (a dihydroxymethyl propionic acid ester ofsirolimus) as a single agent, or sirolimus and RAD001in combination with inhibitors of EGFR in patientswith recurrent glioblastoma multiforme (GBM) areshown in Table 25.1. Treatment with CCI-779 orsirolimus alone had only a limited activity in recurrentGBM, particularly in unselected patients. In the phaseII study by Chang et al. (2005) CCI-779 was admin-istered weekly to forty-three patients with recurrentGBM (14 patients were on enzyme-inducing anti-epileptic drug). Initially CCI-779 was administered at adose of 250 mg intravenously but the dose was reducedto 170 mg because of side effects. CCI-779 was welltolerated; however, failed to demonstrate any efficacyas a single agent in patients with recurrent GBM.Despite initial disease stabilization in approximately50% of patients, the durability of response was short.In another phase II study CCI-779 was administered ina 250-mg intravenous dose weekly to sixty-five recur-rent GBM patients with ≥1 chemotherapy regimen(Galanis et al., 2005). The study reported that 20 of 65patients with recurrent GBM (36%) had radiographicimprovement. Progression-free survival at 6 monthswas 7.8% and median overall survival was 4.4 months.Median time to progression for all patients was 2.3months and was significantly longer for responders

(5.4 months) versus non-responders (1.9 months). Theauthors assessed activation of the PI3K pathway byexamining tumor specimens for total/phosphorylatedAkt and p70s6 kinase, and found p70s6 kinase stainingindices being significantly more frequent in respondersversus non-responders (Galanis et al., 2005).

It was reported by Cloughesy et al. (2008) thatrapamycin treatment leads to substantial inhibition oftumor cell proliferation in seven of 14 patients, asdemonstrated by reduction of Ki-67 staining in a sub-set of patients with PTEN loss. Inhibition of mTORsignaling was observed in tumor tissue. However,rapamycin led to the activation of Akt in some patients,which correlated with faster tumor progression theauthors concluded that rapamycin has anti-canceractivity in PTEN-deficient glioblastoma and deservesfurther clinical study alone or in combination withPI3K pathway inhibitors.

More recently, based on evidence of the synergismbetween inhibitors of mTOR and EGFR (Rao et al.,2005), several studies investigated such a combination.In two trials a partial radiological response (≥50%decrease in the product of perpendicular diametersof contrast enhancing mass without new lesions) wasseen. Doherty et al. (2006) reported a trial on 22 GBMsand six anaplastic gliomas patients treated with eithergefitinib 500 mg or erlotinib 150 mg orally/daily incombination with sirolimus administered at a dose of6 mg orally the first day followed by 4 mg orally oncedaily thereafter. Both medications were given dailyin 28-day cycles. Out of this cohort, 19% of patients

Table 25.1 Results of targeted therapy trials of mTOR inhibitors used alone or in combination with inhibitors of EGFR in recurrentglioblastomas

Study Treatment Patients (n)Radiologicalresponse (%)

Median PFS(months)

6-monthPFS (%)

Median OS(months)

Chang et al. (2005) CCI-779, 170–250 mgweekly

43 5 2.3 3 NA

Galanis et al. (2005) CCI-779, 250 mg weekly 65 0 2.3 8 4.4Cloughesy et al.

(2008)Sirolimus, 2, 5, or 10 mg

daily15 14 NA NA NA

Doherty et al.(2006)

Sirolimus, 4 mg daily;gefitinib, 500 mg, orerlotinib, 150 mg daily

22 18 3 25 NA

Kreisl et al. (2009) RAD001, 70 mg weekly;gefitinib, 250 mg daily

22 14 2.6 5 5.8

Reardon et al.(2010)

Sirolimus, 5–10 mg daily;erlotinib, 150–450 mgdaily

32 0 1.8 3 8.5

PFS, progression-free survival; OS, overall survival; NA, not available

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25 Glioblastoma: Anti-tumor Action of Cyclosporin A and Functionally Related Drugs 251

experienced a partial response and 50% had stabledisease; 6-month progression-free survival (6 M-PFS)was 25%. In a second study on 22 patients (all hadreceived prior radiation and chemotherapy) receiv-ing gefitinib (250 mg daily) and everolimus (70 mgweekly), 14% of patients had a partial response and36% of patients stable disease; median overall sur-vival was 5.8 months (Kreisl et al., 2009). Overallresponse rate was 19% and a 6 M-PFS of 25% inGBM patients. No molecular markers of response weredescribed. A retrospective review of eight consecutivenegative phase II trials in recurrent malignant gliomasfrom the M.D. Anderson Cancer Center found a 6 M-PFS for GBM of 15%. For comparison, among GBMpatients treated with temozolomide at 200 mg/m2/dayorally for the first 5 days of a 28-day cycle, PFSat 6 months was 18%; median progression-free sur-vival and median overall survival were 2.1 and 5.4months, respectively. The 6-month survival rate was46% (Brada et al., 2001).

Although the trials demonstrate moderate clinicalbenefits from the combination of two drugs, ongo-ing phase I/II studies are investigating the efficacy ofusing mTOR inhibitors with other targeted agents andchemoradiation, namely: sirolimus plus vandetanib,CCI-779 or RAD001 plus temozolomide, CCI-779plus radiation or sorafenib or bevacizumab or perifos-ine, RAD001 plus AEE788, and RAD001 plus Gleevecand hydroxyurea (http://clinicaltrials.gov).

General Considerations

A use of CsA may raise reservations due to employ-ment of drug blocking the immune system in tumorpatients, poor drug accessibility due to blood-brainbarrier or general toxicity. However, a growingevidence shows both innate and adaptive immu-nity impaired in glioblastomas (Yang et al., 2010).Glioblastomas are poorly immunogenic and do notexpress specific tumor antigens (Watters et al., 2005).Microglia infiltrating glioblastomas are converted intotumor supportive cells and contribute to tumor growth(Markovic et al., 2005, 2009; Sliwa et al., 2007).Microglial cells operate as the first line innate andadaptive immunity of the central nervous system(CNS). In the normal CNS, microglia express lowlevels of major histocompatibility complex (MHC)

class I and class II molecules and co-stimulatorymolecules such as CD86 and CD40. Upon activa-tion, microglia convert to an active phenotype, up-regulate MHC class I and class II and co-stimulatorymolecules and take part in CD4-and CD8-specificT cell responses. Furthermore, malignant glioblas-toma cells secrete chemoattractants and growth fac-tors, including monocyte chemoattractant protein1 (MCP-1), colony stimulating factor 1 (CSF-1),granulocyte-macrophage colony stimulating factor(GM-CSF), hepatocyte growth factor (HGF) andrecruit microglia. Even though glioblastoma accumu-late many microglia, macrophages and a small pop-ulation of lymphocytes, the defense mechanisms aredown-regulated. It was reported by Hussain et al.(2006) that glioblastoma-infiltrating microglia iso-lated from human tumor tissue samples do not pro-duce inflammatory cytokines: interleukin 1β (IL-1β),interleukin 6 and tumor necrosis factor α (TNF-α),cytokines critical for developing effective innateimmune responses.

On the contrary, glioma-associated microglia/macrophages might promote tumor growth by induc-ing immunosuppression in the tumor microenviron-ment. Glioblastoma cells and accumulating microgliarelease several cytokines, such as interleukin 10 (IL-10) and TGF-β which may inhibit T-cell activationand contribute to the local glioma immunosuppres-sive milieu (Watters et al., 2005). An effective anti-tumor T-cell response is decreased in glioblastomas,because expression of MHC class II and co-stimulatoryB7 molecules is reduced and microglia appear defi-cient in proper antigen presentation. Thus, potentialimmunossuppressive effects of CsA can be neglectedbecause the immune system in glioblastoma patientsis already paralyzed and anti-tumor responses are non-functioning. Although, tumors incidence in transplantrecipients is a recognized consequence, a recent exam-ination of reported cases of tumor in the transplantpopulation of the Israel Penn International TransplantTumor Registry shows that primary brain tumors,including gliomas, do not appear to be overrepresentedin the Registry, indicating that they may not arise withincreased frequency in transplant recipients (Schiff,2004).

As it has been shown in animal glioma model bySliwa et al. (2007), CsA has a systemic effect, canaffect cell interactions in tumor microenvironment andblock pro-tumorigenic activity of tumor infiltrating

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microglia/macrophages. A recent study demonstratesthat mTOR signaling controls microglial activation inresponse to cytokines and appears to play a crucial rolein regulating of microglial viability (Dello Russo et al.,2009). Thus anti-tumor action of rapamycin in vivomay be partly due to interference with cell interactionsin tumor microenvironment.

The blood-brain barrier (BBB) possesses a signifi-cant impediment for the delivery of therapeutic drugsinto the brain. In the normal adult brain, the BBBmainly consists of vascular endothelial cells, astrocytesand pericytes. In malignant glioblastomas, the bloodvessel network is broken down. Increased permeabil-ity of tumor blood vessels is induced by angiogenicfactors released by malignant glioma cells, such asvascular endothelial growth factor (VEGF) that is asso-ciated with intravascular migration of glioma cells intoblood vessels and their transfer to distant areas in thebrain via blood flow. The mechanism of vascular per-meability is used by glioma cells to facilitate migrationand invasiveness, but can be also employed to facilitatedelivery of anti-tumor medicines into the brain (Tateand Aghi, 2009).

Previous studies demonstrated that a systemic injec-tion of 100 mg/kg/i.p. CsA results in a blood con-centration of 10 μM maintained for at least 18 h(Ciechomska et al., 2005). Thus, concentrations ofCsA (2 and 10 mg/kg) tested in animal glioma modelcorrespond to blood concentrations of 0.2 or 1 μM,respectively. A permeable blood-brain barrier in braintumors may result in similar CsA concentrations in thebrain as detected in the blood. In contrast to widelyaccepted notion that CsA does not cross or poorly crossthe blood-brain barrier, studies on immunosuppressedpatients demonstrated the presence of CsA in cere-brospinal fluids suggesting that the drug is able to crossthe blood-brain barrier. Furthermore, CsA may impairthe brain endothelial barrier function by acceleratingNO production in the brain endothelial and astroglialcells.

Notably, CsA at lower micromolar concentrationsdoes not affect survival of non-transformed cells.Neurons from mixed neuronal-glial cultures devel-oped from hippocampal dentate gyrus are affectedby CsA at the concentrations higher than 8–10 μM(Kaminska et al., 2001). Astrocytes are more resistantand CsA at concentration of 40 μM or higher affectsthe survival of astrocytes from neonatal brain cultures.Altogether, these results suggest that CsA, particularly

at lower concentrations affecting pro-invasive activ-ity of microglia, could be effective anti-tumor drugwithout inducing neurotoxicity. CsA induces cell deathvia multiple mechanisms and some of them areable to prevail alterations of growth regulatory andapoptotic pathways, caused by common mutations inhuman glioblastomas. Rapamycin and its derivatives,besides the well described inhibition of mTOR path-way in glioblastoma cells, may additionally affect pro-invasive activity of glioblastoma infiltrating microglia.The unique mechanism of action of CsA and phar-macological inhibitors of the mTOR pathway justifiesfurther research on their anti-tumoral properties eitheralone or in combined therapies.

Acknowledgements This work was supported by grantP-N/024/2006.

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