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Drug Target Insights 2007: 2 197–207 197
REVIEW
Correspondence: Ningaraj, N.S., Department of Pediatric Neurooncology and Molecular Pharmacology,
Hoskins Center, Curtis and Elizabeth Anderson Cancer Institute, Memorial Health University Medical Center,
Mercer University Medical School, 4700 Waters Avenue, Savannah, GA 31404, U.S.A.Tel: +1 9123500958;
Fax: +1 9123501269; Email: NingaNa1@memorialhealth.com
Please note that this article may not be used for commercial purposes. For further information please refer to the copyright
statement at http://www.la-press.com/copyright.htm
Targeted Brain Tumor Treatment-Current Perspectives
Ningaraj N.S1, Salimath B.P2, Sankpal U.T1, Perera R1 and Vats T1
1Department of Pediatric Neurooncology and Molecular Pharmacology,
Hoskins Center, Curtis and Elizabeth Anderson Cancer Institute, Memorial Health University Medical
Center, Mercer University Medical School, 4700 Waters Avenue, Savannah, GA 31404, U.S.A.
2Department of Biotechnology, University of Mysore, Mysore 570006, Karnataka, India.
Abstract: Brain tumor is associated with poor prognosis. The treatment option is severely limited for a patient with brain
tumor, despite great advances in understanding the etiology and molecular biology of brain tumors that have lead to break-
throughs in developing pharmaceutical strategies, and ongoing NCI/Pharma-sponsored clinical trials. We reviewed the
literature on molecular targeted agents in preclinical and clinical studies in brain tumor for the past decade, and observed
that the molecular targeting in brain tumors is complex. This is because no single gene or protein can be affected by single
molecular agent, requiring the use of combination molecular therapy with cytotoxic agents. In this review, we briefl y discuss
the potential molecular targets, and the challenges of targeted brain tumor treatment. For example, glial tumors are associ-
ated with over-expression of calcium-dependent potassium (KCa) channels, and high grade glioma express specifi c KCa
channel gene (gBK) splice variants, and mutant epidermal growth factor receptors (EGFRvIII). These specifi c genes are
promising targets for molecular targeted treatment in brain tumors. In addition, drugs like Avastin and Gleevec target the
molecular targets such as vascular endothelial cell growth factor receptor, platelet-derived growth factor receptors, and
BRC-ABL/Akt. Recent discovery of non-coding RNA, specifi cally microRNAs could be used as potential targeted drugs.
Finally, we discuss the role of anti-cancer drug delivery to brain tumors by breaching the blood-brain tumor barrier. This
non-invasive strategy is particularly useful as novel molecules and humanized monoclonal antibodies that target receptor
tyrosine kinase receptors are rapidly being developed.
Abbreviations: BBB: blood-brain barrier; BTB: blood-tumor barrier; KCa: calcium-dependent potassium channels; NS-1619/
NS 004: 1,3-dihydro-1-5-(trifl uoromethyl)-2H benzimidazol-2-one; HBMVEC: human brain microvascular endothelial cells;
FACS: fl uorescence activated cell sorting; PDGFR: platelet-derived growth factor receptor; RTKIs: receptor tyrosine kinase
inhibitors; EGFR: epidermal growth factor receptor; EGFRvIII: variant III of the human EGFR; gBK channel: glioma spe-
cifi c spice variant of KCa channel gene; KATP: ATP sensitive potassium channels; Minoxidil sulfate (MS: KATP channel agonist);
Trastuzumab (Herceptin, Her-2 inhibitor, Genentech Inc.).
Keywords: Brain tumor, BBB, drug delivery, therapeutic targets in brain tumors
Introduction
Brain tumor
Nearly 20,000 new primary brain tumors and about 200,000 metastatic brain tumor cases are reported
each year in the U.S.A. (Levin, 2007). The overall survival of these patients is dismal and the majority of
survivors suffer disabling toxicities from their treatments. Standard treatment for brain tumors includes
combination of surgery, radiation therapy, and chemotherapy. Brain tumor poses unique challenges due
to its distinct biology, genetics, treatment response, and survival. Despite extensive characterization of the
brain tumor pathways, molecularly targeted approach is not available to brain tumor patients. Future
research in brain tumors needs to focus on strategies for improving drug delivery, disruption of
blood-brain-barrier (BBB), and molecular profi ling of tumors. In addition, careful studies are needed to
delineate pathways that aid and abate brain tumor progression. Identifi cation of potential markers (genes
and proteins) for targeted therapy will defi nitely help the clinicians to design the treatment accordingly.
Usually, after surgical treatment, brain tumor recurs, severely shortening life expectancy (Friedman, Kerby
and Calvert, 2000). Conventional treatments using radiation and intravenous chemotherapy are not sucessful
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because the cancer cells develop resistance to treat-
ment. Anti-cancer drugs fail to penetrate the BBB
in suffi cient quantities (Pardridge, 2001), allowing
cancer cells to develop resistance to these agents.
Therefore, understanding the biochemical regulation
of the BBB (Fig. 1) in normal and tumor-invaded
brain is of great importance to develop therapeutics
that breach or circumvent BBB and directly target
brain tumor cells (Ningaraj, 2006). The focus is now
on the targeted cancer therapies (Butowski and
Chang, 2005) that complement conventional treat-
ments and reduce the drug resistance in cancer cells
and the toxicity in normal brain (Newton, 2003).
Novel cancer therapies include anti-angiogenic
agents, immunotherapy, bacterial agents, viral
oncolysis, cyclin-dependent kinases and receptor
tyrosine kinase inhibitors (RTKIs), anti-sense
agents, gene therapy, microRNA (miRNA), and
combinations of various methods (Butowski and
Chang, 2005).
Chemotherapy
Chemotherapy is a form of targeted therapy where
cytotoxic drugs act on multiplying tumor cells. The
drugs can also be used as sensitizers to augment the
effects of radiation therapy. Chemotherapeutic
drugs can be delivered directly to brain tumors
through a polymer wafer implant such as a biode-
gradable wafer soaked with BCNU (Carmustine).
Besides BCNU, several other chemotherapy drugs
are used to treat brain tumors, which are adminis-
tered by various routes. The chemotherapeutic
drugs taken orally include Temozolomide (TMZ,
Temodar), procarbazine (Matulane), and lomustine
(CCNU). The intravenously administered drugs
include vincristine (Oncovin or Vincasar PFS),
cisplatin (Platinol), carmustine (BCNU, BiCNU),
Carboplatin (Paraplatin), while Methotrexate
(Rheumatrex or Trexall) may be taken orally, by
injection, or intrathecally. Treating brain tumors
with chemotherapy can be diffi cult because the
brain is protected by BBB, which keeps out harmful
substances such as bacteria and chemotherapeutic
drugs. Among many cytotoxic agents in the clini-
cian’s arsenal, Temozolomide (TMZ) has shown
some promise in treatment of low grade gliomas
(Friedman, Kerby and Calvert, 2000), however, the
effect on patient survival was modest (Balana et al.
2004). The problem is that glioblastoma multiforme
(GBM) exhibits varying responses to TMZ (Hirose,
Berger and Pieper, 2001), and in some cases
gliomas have increased O6-methyl guanine methyl
transferase (MGMT) activity, which results in
complete resistance to TMZ (Bocangel et al. 2002).
The clinical utility of TMZ against all types of brain
tumors remains limited due to its BTB penetration
(some authors claim TMZ metabolite (MTIC)
concentration in CSF to be as high as 30%), which
demand repeated high doses to achieve in vivo
therapeutically effective concentrations in brain
tumors (Yung et al. 1999), and different phenotypes
and genotypes that render some form of resistance
against TMZ (Kanzawa et al. 2003). Most
importantly, an extensive literature search and
preliminary work on BBB/BTB penetration of TMZ
did not convince us that suffi cient amount of drug
penetrates the BBB or BTB to elicit anti-tumor
effect. To circumvent the penetration problem,
chemotherapy drugs can be delivered by
Figure 1. A schematic representation of normal and abnormal blood-brain barriers. We reason that genes in brain cancers/vasculature are
distinct from normal brain/vasculature. They are attractive targets for the design of therapies that can penetrate the BTB and selectively kill brain
cancer cells. We are studying the genes that direct the formation of the normal and abnormal (cancer) human brain vasculature, and with this
knowledge develop new treatment strategies for brain tumor patients. EC: endothelial cells; TJ: tight junction proteins; N: nucleus.
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Drug Target Insights 2007: 2
intratumoral route or by drug impregnated wafers
to attain higher concentration of drugs in the tumor
cells, but the procedures are highly invasive.
Targeted brain tumor treatment
The human genome project has raised the expecta-
tion of the development of novel therapies for brain
tumor because the conventional treatment strate-
gies have not yielded any significant clinical
outcome. Brain tumor treatment differs according
to the grade and location of the tumor. Hence,
combination of surgery, chemotherapy, and radio-
therapy can be used in treating brain tumor patients
(Stupp et al. 2005). Most promising anti-cancer
drugs for pediatric and adult patients that are effec-
tive against cancers outside the brain have failed
against brain tumors in clinical trails, in part, due
to poor penetration across the BBB. For instance,
aberrant expression of src family kinase (LCK)
(Fabian et al. 2005) or mutation of c-KIT are
involved in the pathogenesis of many cancers.
Studies using imatinib mesylate (STI 571, Gleevec,
Novartis, U.S.A.), an inhibitor of the tyrosine
kinases BRC-ABL, c-KIT, and PDGFR, have
shown signifi cant response in patients with chronic
myelogenous leukemia (CML) and gastrointestinal
stromal tumor (GIST). Clinical trials were recently
conducted to test the effi cacy of Gleevec in brain
tumors (Reardon et al. 2005; Wen et al. 2006;
Pollack et al. 2007). Gleevec is an effective agent
that targets specifi c gene/protein in cancer cells
without harming normal cells and tissues. Drugs
like Gleevec and Temozolomide attack abnormal
chemical signals or molecules inside the cells or
on the surface of the cells that have enabled brain
tumor cells to escape the normal growth controls.
Therefore, combating many forms of cancer will
probably require a variety of targeted drugs used
in combination, as cancer involves different types
of dysfunctional genes and no single or two drugs
will be sufficient. Some cancers, particularly
primary and metastatic brain tumors of the breast
and lung are diffi cult to treat because they are
caused by multiple signaling pathways that are
running amok, rather than just one, as observed in
CML and GIST (Butowski and Chang, 2005).
Gleevec may potentially target the above mentioned
oncogenes in brain tumor (Holdhoff et al. 2005)
provided it penetrates the BBB (Leis et al. 2004).
Careful molecular studies would identify the stem
cell factor/c-kit pathways in pediatric brain tumors,
which might be the target of Gleevec. Characterizing
the genetic and proteomic events that play a role
in the biology of these tumors may allow molecular
sub-typing which could lead to the development
of novel therapeutic strategies, including treatment
with Gleevec or with potassium channel modula-
tors targeting tumor and tumor blood vessel endo-
thelial cells (Ningaraj, 2006).
Targeting brain tumors
Targeting tumor and tumor blood vessel-specific
marker(s) is a good strategy to control tumor
growth (Robinson et al. 2003). It is, however,
critical to study whether tumor-specific drug
delivery has the potential to minimize toxicity
to normal tissues, and to improve the bioavail-
ability of cytotoxic agents to neoplasms.
Existing site-specific drug delivery systems
include delivery to endothelial receptor αvβ3,
and tumor specific antigens. Antibody conjuga-
tion to cytotoxic agents has shown promise in
achieving the goal of tumor-targeted cytotox-
icity. This approach may be limited by the small
subsets of tumors that can be targeted by these
antibodies and by poor biodistribution of these
a n t i b o d i e s i n t o s o l i d t u m o r s . A l t e r n a t i v e
approaches to target all neoplasms exploit
differences in human tumor blood vessel char-
acteristics when compared to normal brain blood
vessels (Black and Ningaraj, 2004; Ningaraj
et al. 2002; Ningaraj, Rao and Black, 2003a).
Epidermal growth factor receptor (EGFR) is
often amplified and mutated in human gliomas,
but the expression is low or undetectable in
normal brain. Recently, EGFR’s mutant isoform,
variant III of the human EGFR (EGFRvIII), is
under intensive investigation as potential
molecular target for the specific delivery of the
diagnostic and the therapeutic agents to brain
tumors (Yang et al. 2005). The therapeutic
monoclonal antibodies (MAb) targeting growth
factor pathways are being developed. The
purpose of antibody treatment of cancer is to
induce the direct or indirect destruction of
cancer cells, either by specifically targeting the
tumor or the tumor vasculature (Butowski and
Chang, 2005). Examples of therapeutic anti-
bodies which are effective in treating cancer
includes the humanized IgG antibody Herceptin
for the treatment of breast cancer, Cetuximab,
ABX-EGF, EMD 720000 and h-R3 directed at
extracellular receptor domain that inhibits the
ligand-receptor interactions. Other antibodies
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Drug Target Insights 2007: 2
like Y10 and MAb806, which are directed
towards the extracellular portion of EGFRvIII
in gliomas have also shown some activity in
clinical trials (Rich and Bigner, 2004). Suramin
(polysulfonated napthylurea), which acts by
interfering with the binding of several growth
factors-including EGF, platelet derived growth
factor (PDGF), and insulin growth factor (IGF1)
with their putative receptors, is being tested in
clinical trails. These MAbs, however, have poor
penetration into brain tumors, which results in
recurrence in brain tumor patients.
Kinase inhibitors
Kinase inhibitors show great promise as a new
class of therapeutics to control gliomas. The
specifi city of RTKIs, including those that are in
clinical use or in development widely varies, and
is not strongly correlated with chemical structure
of the identity of the intended target. Many novel
interactions were recently identifi ed (Fabian et al.
2005). EGFR and PDGFR are abnormal genes
identifi ed in gliomas (Rich and Bigner, 2004),
whose expression is linked to an increased rate of
tumor cell proliferation, resistance to chemo-
therapy, invasion, and apoptosis, and hence
decreased survival in patients with malignant
gliomas. PDGF ligands bind to PDGFRs to induce
phosphorylation and activation of downstream
signaling pathways such as RAS, MAPK, and Akt.
Therefore, therapies using Gleevec, Suramin, and
MAbs are directed at PDGFR to control glioma
growth. PDGFR inhibitors may also provide addi-
tional benefit by blocking pericytes-assisted
angiogenesis (Bergers et al. 2003). Clinical trials
with EGFR and PDGFR inhibitors have shown
promise for glioma therapy, although their ability
to penetrate BBB in suffi cient amounts is largely
unknown. We transiently opened the BTB with
KCa and ATP-sensitive potassium (KATP) channel
agonists (Black and Ningaraj, 2004; Ningaraj et al.
2002; Ningaraj, Rao and Black, 2003a,b; Rao and
Ningaraj, 2001) to increase the delivery of Gleevec
and Herceptin to human glioma xenografts grown
in murine brains.
KCa channels in gliomas
Membrane ion channels are essential for cell
proliferation and appears to play a role in the
development of cancer (Ningaraj, 2006). The
KCa channels are highly expressed in gliomas
(Weaver, Liu and Sontheimer, 2004) supporting
the hypothesis that these channels play an
important role in brain tumor growth and
possibly the progression of low grade anaplastic
astrocytomas (grade II) to a deadly high grade
GBM (WHO grade IV). In addition, studies have
shown that modulation of the biological function
of KCa channels with specific inhibitors atten-
uate glioma growth (Rao and Ningaraj, 2001).
Another study showed that the activation of
intermediate KC a channels with its opener
caused down-regulation of these channels and
attenuated the non-excitable cell growth and its
proliferation (Kraft et al. 2003). We showed that
chronic activation of KCa channels with its
specific openers NS-1619 and NS-004 elicited
apoptosis in vitro and in vivo (Rao and Ningaraj,
2001). However, the role of KCa channels in
progression from a treatable low grade to an
untreatable high grade glioma in pediatric as
well as in adult patients is not fully understood.
Recently, glioma KCa/BK channels (gBK) splice
variant of the KCNMA1 gene was characterized
by enhanced sensitivity to intracellular calcium
levels (Weaver, Bomben and Sontheimer, 2006).
The study also showed that the expression of
functional gBK channels appears to be regulated
in a growth-factor-dependent manner. It is well
established that EGF activates EGFR. Several
molecular agents targeting EGFR are under-
going clinical trails as potential therapies in
neurooncology (Rich and Bigner, 2004). For
example, ZD1839 (Iressa) an orally active,
selective EGFR-tyrosine kinase inhibitor has
anti-tumor activity against malignant human
cancer cell lines (31). Glioma cells also show
up-regulation and constitutive activation of Her-
2 neu, and its expression which correlates posi-
tively with aggressive malignancy (Mellinghoff
et al. 2005). A correlation has been demonstrated
for the expression of gBK/KCa channels and
Her-2 neu, which implies gBK/KCa channels as
a downstream target for Her-2 neu signaling
(Olsen et al. 2004). How KCa channel modulates
EGFR tyrosine kinase or vice versa is poorly
understood. It appears to occur via changes in
intracellular calcium levels without change in
channel expression or phosphorylation (Weaver,
Bomben and Sontheimer, 2006). In a transgenic
glioma mouse model, a loss of EGFR overex-
pression was observed by EGFRvIII introduction
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Drug Target Insights 2007: 2
(Gullick, 2001). This model of high grade
glioma is useful in evaluating targeted molecular
therapies in brain tumor.
Targeting angiogenesis in brain
tumors
Angiogenesis plays a crucial role in malignant
primary brain tumor growth. Several preclinical
and clinical studies have confirmed that the
vascular endothelial cell growth factor (VEGF)
and the bFGF bind to their receptors to promote
glioma growth. VEGFR is expressed in human
high grade glioma but not found in normal brain.
Increased concentration of angiogenic factors and
their receptors is correlated with tumor vascula-
ture and malignant human gliomas. Furthermore,
it is shown that the endogenous inhibitor of angio-
genesis, thrombospondin-1 (TSP-1) is produced
by normal brain and low grade gliomas, but is
completely absent in high grade gliomas. The
GBM is among the most “endothelial rich” brain
tumors studied. In children with brain tumors,
microvascular density correlates with tumor
recurrence, and patient mortality. As tumor vascu-
larity is highly correlated with disease outcome
in neuroblastoma, novel therapeutic that targets
the vascular endothelium is a suitable clinical trial
target candidate. The molecules like VEGF, bFGF,
PDGF as well as endothelial integrins are linked
to advanced malignancy, which provided the
rationale for developing anti-angiogenic therapies
in brain tumors. The potential of anti-angiogenic
therapy in human brain tumors is demonstrated
in experimental brain tumor models. A wide range
of anti-angiogenic agents such as endogenous
angiogenesis inhibitors, synthetic angiogenic
inhibitors, antibodies, and anti-angiogenic gene
therapy are investigated with radiation therapy.
Anti-angiogenic drugs have low potential for
toxicity and resistance because they specifi cally
target endothelial cells. The potential of anti-
angiogenic agents to augment the anti-tumor
activity of standard cytotoxic chemotherapeutic
agents is being investigated (Bernsen and van der
Kogel, 1999; Reijneveld, Voest and Taphoorn,
2000; Takano et al. 2004).
The evidence for glioma anti-angiogenesis
therapy, with or without chemotherapy has been
described in several preclinical animal models.
The anti-angiogenic function of TSP-1 is known
for a long time. The TSP-1 transfected glioma
cells lacked VEGF expression ability, which
supports the rationale for using VEGF and bFGF
antibodies in clinical trails. Anti-angiogenic
d r u g , T h a l i d o m i d e e x h i b i t s s y n e r g i s t i c
anti-glioma activity when combined with DNA
alkylating agent Temozolomide, and increased
median survival from 63 weeks to 103 weeks
compared to Thalidomide only group (Baumann
et al. 2004). While evaluating anti-angiogenic
drugs for clinical development, it is important
to analyze if such drugs penetrate the BBB, and
survive p-glycoprotein-mediated drug efflux
system. At present, there is a great deal of interest
in combination therapy using conventional cyto-
toxic therapy with chemotherapeutics and radio-
therapy. Anti-angiogenic agents like TNP-470,
angiostatin, DC 101, SU5416, anti-VEGF and
VEGF-R antibodies and VEGF monoclonal
antibody A4.6.1, tyrosine kinase inhibitors,
COX-2 inhibitors, and anti-EGFR inhibitors are
used in combination with radiation. The synthetic
fumagillin analogue, TNP-470 was shown to
interfere with angiogenesis through inhibition of
endothelial cell proliferation and migration in
murine and human neuroblastoma xenograft
model. Now it is being evaluated in phase
I/II clinical trials. In brain tumor models, TNP-
470 and Minocycline together increased 9L
glioma sensitivity to BCNU and andriamycin
(Shusterman et al. 2001), while Lund, Bastholm
and Kristjansen, (2000) found that TNP-470
increased radiation sensitivity of human U87
glioblastoma xenografts. A phase II study with
anti-angiogenic monoclonal antibody bevaci-
zumab (Avastin) and anti-cytokine Irinotecan in
brain tumor patients is also being conducted
(NCT00381797). Endogenous inhibitors of
angiogenesis such as angiostatin, endostatin,
PEX, pigment epithelial-derived factor, and
thrombospondin (TSP-1&2) are shown to be
effi cacious. They exert their effects through
multiple mechanisms, including induction of
apoptosis of micro vascular endothelial cells,
inhibition of proliferation of endothelial cells,
inhibition of function, and regulation of proan-
giogenic molecules. These endogenous inhibi-
tors offer a novel treatment option because they
are unlikely to trigger a host immune response.
Angiostatin, a proteolytic fragment of plasmin-
ogen inhibits angiogenesis and attenuate the
growth of primary and metastatic tumors. Angi-
ostatin was effectively used in combination with
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Drug Target Insights 2007: 2
fractional radiation therapy in human glioma
models (Mauceri et al. 1998; Rege, Fears and
Gladson, 2005). Recently, gene therapy has hit
a snag, but offers a promising alternate treatment
strategy.
Brain tumors are attractive for gene therapy
because the brain is an immunologically privileged
organ, and the BBB provides a natural immuno-
logical barrier. Mice when treated with a retrovirus
encoding a dominant negative mutant of the VEGF
receptor Flk-1 resulted in reduced tumor growth
and decreased blood vessel density. Recombinant
adeno-associated virus (AAV) vector with the
angiostatin gene was used to reduce tumor growth
and angiogenesis in a C6 glioma model. Anti-
angiogenic therapy using Semliki Forest Virus
(SFV) carrying endostatin gene significantly
reduced the tumor growth in animals. Therefore,
gene therapy with endostatin delivered via SFV
may be a viable treatment strategy for brain tumors
(Ma et al. 2002; Yamanaka et al. 2003). Although,
the gene therapy in general is in its infancy, it
provides an alternate strategy to treat hard-to treat
brain tumors.
Epigenetic genes as brain tumor
targets
Epigenetic events are genetic modifi cations (DNA
methylation and covalent histone modifi cations) that
are heritable through cell division, which affect gene
expression without causing changes to the DNA
coding sequence. Cancer cells exhibit global
hypomethylation of the genome accompanied by
region-specifi c hypermethylation events. The hypo-
methylation mainly occurs in the repetitive sequences
leading to genomic instability and tumor formation.
Aberrant hypermethylation occurs at CpG islands
found in the promoter region of genes, which is
usually associated with the transcriptional silencing
of that gene (Baylin et al. 2001). DNA methylation
changes (Palanichamy, Erkkinen and Chakravarti,
2006), particularly CpG island hypermethylation is
frequent, early, and common event (as common as
mutations) in many types of cancers leading to the
inactivation of tumor suppressor genes.
Several genetic changes have been identifi ed in
AAs and GBMs involving heterozygous deletion
of 19q13, inactivation/deletion of tumor suppressor
genes namely p16INK4A (Hegi et al. 1997),
p14ARF (Ichimura et al. 2000), RB1 (Ichimura
et al. 1996), PTEN and p53 gene (Mashiyama et al.
1991) and amplifi cation of EGFR gene (Libermann
et al. 1985). Epigenetic research in glioma patho-
genesis revealed several epigenetic genes silenced
by promoter CpG island hypermethylation,
such as, cell cycle regulatory proteins RB1
(Nakamura et al. 1996), p16INK4A (Costello et al.
1996; Fueyo et al. 1996), myelin related gene
EMP3 (Alaminos et al. 2005), and matrix metal-
loproteinases inhibitor TIMP3 (Bachman et al.
1999). Comprehensive whole-genome microarray
studies using inhibitors of epigenetic modifi cation
have identified several genes including CST6
(putative metastatic suppressor), BIK (apoptosis
inducer), TSPYL5 (unknown function), BEX1, and
BEX2 (uncharacterized function) as putative tumor
suppressors that are frequently methylated in
primary gliomas (Kim et al. 2006; Foltz et al.
2006). Another genome-wide study using restric-
tion landmark genomic scanning has identifi ed as
many as 1500 CpG islands to be aberrantly meth-
ylated in low grade gliomas (Costello et al. 2000).
These studies have highlighted a role for DNA
methylation in gliomagenesis. To date very few
genetic assays are available to accurately provide
information regarding patient prognosis or
response to therapy. It has been hypothesized that
aberrant DNA methylation plays a key role in
tumor initiation. Therefore identifying such
modifi cations helps in early detection of cancer,
and might also provide information regarding the
mechanisms that control glioma progression
(Costello, 2003). In addition to being a diagnostic
marker, DNA methylation can also serve as an
useful prognostic marker as shown by the
methylation of the DNA repair gene, MGMT, in
gliomas. Epigenetic silencing of the gene (involved
in the repair of DNA damaged by alkylating agents)
is associated with the increased survival in patients
treated with alkylating drug Temozolomide
(Esteller et al. 2000; Komine et al. 2003). Current
laboratory studies are aimed at discovering novel
methylation markers in tumor tissue as well as in
the patient’s body fl uids (Belinsky et al. 2006;
Cairns et al. 2001). Since the primary DNA
sequence of epigenetically modifi ed genes remains
intact, it is possible to reactivate genes using
inhibitors of DNA methylation or histone modifi -
cations (Daskalakis et al. 2002; Plumb et al. 2000).
Clinical trials using DNA methylation and histone
deacetylase inhibitors, which reactivate silenced
genes in cancers, are in various development
stages. The DNA methyltransferase inhibitors,
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Drug Target Insights 2007: 2
5-azacytidine (Vidaza) and 5-aza-2′-deoxycytidine
(Decitabine), are used with reasonable success in
the treatment of hematologic malignancies
(Lubbert, 2000), but have limited success in solid
tumors. Combination of HDAC inhibitors with
DNA methyltransferase inhibitors appear to syner-
gistically induce the expression of silenced genes
(Cameron et al. 1999). However, these drugs have
drawbacks such as extreme instability, serious side
effects, and sometimes these drugs at high doses
may promote malignant transformation. Alterna-
tive approaches include the use of siRNA targeted
against the DNA methyltransferase enzyme (Goffi n
and Eisenhauer, 2002) and developing stable small
molecule inhibitors that can overcome the BBB.
Small interfering RNA (siRNA)
to target brain tumor gene(s)
The siRNA directs the targeted destruction of
mRNA encoding a specific protein, in a process
known as RNA interference (RNAi). This
process stops translation of the targeted mRNA
into protein, effectively silencing the gene.
RNAi is a recent discovery, identified in
mammalian cells in 2001, but it has rapidly
advanced into practical technique, and is being
used increasingly to investigate mammalian
gene function. Tools are available to induce
RNAi in cell lines, intact tissue preparations,
and even in in vivo. Depending on the method
used, loss of gene expression may be transient
or sustained, enabling a wide range of functions
to be investigated. The RNAi is a powerful
technique that can be used to produce targeted
knockout of genes in mammalian cells (Gurney
and Hunter, 2005). Its applications potentially
include identification of protein function in
health and disease, identification of novel genes,
and drug target validation. Effective RNAi
requires an appropriate siRNA sequence to be
designed and an efficient method for delivering
the siRNA to the cells of interest. Since not all
potential siRNA sequences are effective, it is
important to verify the loss of gene expression
by measuring the level of protein remaining.
Limitations for delivering siRNA are one of the
main obstacles to produce efficient RNAi, espe-
cially in intact tissue preparations. A successful
in vitro method for targeted RNAi against the
TASK-1 potassium channel gene (Gurney and
H u n t e r, 2 0 0 5 ) w a s d e s c r i b e d . I n c r e a s i n g
evidence show that microRNA (miRNA) repre-
sent a new class of genes involved in oncogen-
esis (Ciafre et al. 2005).
miRNAs as druggable targets
The miRNAs are non-coding, double stranded
RNA molecules with an average size of 22 bp, and
serve as posttranscriptional regulators of gene
expression in higher eukaryotes. The miRNAs play
an important role in development and other cellular
processes by hybridizing to complementary target
mRNA transcripts and destabilizing the latter by
preventing their translation (Ambros, 2003; Bartel
and Bartel, 2003; Bartel, 2004). Although a few
hundred miRNAs have been discovered in a variety
of organisms, little is known about their cellular
functions. They have been implicated, among
others, in regulation of developmental timing and
pattern formation, restriction of differentiation
potential, regulation of insulin secretion, resistance
to viral infection, and in genomic rearrangements
associated with carcinogenesis or other genetic
disorders, such as the fragile X syndrome. Recent
evidence suggests that the number of unique
miRNA genes in human ranges from 1000 to
20,000. It is estimated that 20%–30% of all human
mRNA genes are miRNA targets, and hence special
attention has been given to miRNAs as candidate
drug targets in brain tumor.
Several recent reviews and research articles
have illustrated the involvement of miRNAs in
cancer (Calin and Croce, 2006a, 2006b; Jannot
and Simard, 2006; Kent and Mendell, 2006;
Jovanovic and Hengartner, 2006; Dalmay and
Edwards, 2006; Hutvagner, 2006; Osada and
Takahashi, 2006; Zhang and Coukos, 2006).
Therefore, we will restrict this section to the
general concepts. In a recent study the miRNA
expression levels in GBM was investigated
(Ciafre et al. 2005; Chan, Krichevsky and Kosik,
2005). The analysis of both glioblastoma tissues
and glioblastoma cell lines showed a signifi cantly
altered miRNA expression. The most interesting
miRNA is miR-21, which is signifi cantly up-
regulated in glioblastoma. In another study,
knockdown of miRNA-21 in cultured glioblas-
toma cells triggers activation of caspases that
leads to increased apoptotic cell death (Chan,
Krichevsky and Kosik, 2005). These data suggest
that aberrantly expressed miR-21 may contribute
to the malignant phenotype by blocking expression
204
Ningaraj et al
Drug Target Insights 2007: 2
of critical apoptosis-related genes. A set of
brain-enriched miRNAs, miR-128, miR-181a,
miR-181b, and miR-181c, are down-regulated in
glioblastoma is also discovered (Ciafre et al. 2005;
O’Driscoll, 2006). One of the early works that
demonstrates miRNAs as potential candidate drug
target was performed by obstructing the adipocyte
differentiation process in human primary adipocytes
(Esau et al. 2004). Major hurdles are expected before
a miRNA-based drug is successfully developed
against cancer. In fairness this is only the beginning
of the impact of the discovery of miRNA on under-
standing the brain tumor etiology, and developing
cancer treatment strategies.
Anti-cancer drug delivery to brain
tumor
Drug delivery in the treatment of brain tumors is
a crucial consideration in the development of anti-
cancer agent because the delivery of all substances
into the brain is tightly regulated by BBB. Brain
tumor cells diffuse into the normal brain and are
protected by intact BBB (Rich and Bigner, 2004),
where anti-cancer drug delivery is very critical.
We showed that improved drug delivery in human
glioma xenograft models (Ningaraj, 2006) has the
potential to be extrapolated to patients with brain
tumors for better control of the disease. In this
direction, our laboratory is developing methods
for high-throughput screening of RTKIs for selec-
tive delivery to brain tumors, simultaneously
monitor dosing, delivery, and pharmacological
effi cacy of RTK inhibitors in animal brain tumor
models. The challenges and opportunities of the
biochemical modulation of BBB for selective drug
delivery to brain tumor was reviewed recently
(Ningaraj, 2006). We showed that intravenously
administered, potassium channel agonists increase
TMZ (Fig. 2) and Her-2 MAb (Herceptin) (Ning-
araj, Rao and Black, 2003b) delivery across the
BTB to elicit anti-tumor activity and increase
survival in nude mice with intracranially implanted
human glial tumor. Our study suggested that the
BTB allows a small amount of TMZ into brain
tumors. Potassium channel agonist-mediated
biochemical modulation signifi cantly increased
BTB permeability allowing greater amounts of
Figure 2. Quantitative increases in BTB permeability. A signifi cant increase in the mean Ki for [
14C]-Temozolomide (TMZ) after i.v. infusion
of 100 µg/kg/min for 15 min of NS-1619 and MS compared to a vehicle-treated group was observed. The increase in [14C]-TMZ uptake in
tumor center was signifi cant although a slight increase in uptake of the radiotracer was observed in the brain tissue-surrounding tumor. No
[14C]-TMZ uptake in contralateral normal brain, which served as internal control, was observed in all the groups. Data are presented as mean
± S.D (N = 6), ***P < 0.001 versus vehicle-treated group. Precaution was taken to avoid necrotic area during the Ki measurement by com-
paring the QAR brain section with a corresponding H&E stained serial brain tumor section.
205
Targeted Brain Tumor Treatment
Drug Target Insights 2007: 2
TMZ, selectively to reach brain tumor and brain
tissue surrounding tumor, which represents prolif-
erating edges of tumor where the BBB may be
intact (Pardridge et al. 1992). Furthermore, we
showed that Trastuzumab combined with TMZ
co-administered with potassium channel agonists
signifi cantly increased survival rates in mice with
intracranial GBM xenograft (unpublished data).
These results are consistent with our earlier study,
where we showed that potassium channel activator
(Minoxidil sulfate: MS) infusion selectively
enhanced carboplatin delivery to tumor tissue
without increasing delivery to normal brain
(Ningaraj, Rao and Black, 2003b). MS co-infusion
with carboplatin in rats resulted in tumor regression,
significantly increasing survival (Black and
Ningaraj, 2004). The ability to deliver Her-2 neu
targeting drug Herceptin (Trastuzumab) by potas-
sium channel-mediated BTB modulation in human
xenografts may be clinically useful because GBMs
frequently have altered receptor tyrosine kinase
genes (Fuller and Bigner, 1992), including Her-2
neu that is over expressed in about 17%–20% of
GBM patients (Forseen et al. 2002) resulting in
poor prognosis and patient survival. A molecular
target-based therapy using Trastuzumab and
Pertuzumab (Omnitarg, 2C4) is developed by
Genentech Inc., for brain tumor, but their delivery
across the BTB remains a major concern.
Molecular medicine
To conclude, the future of molecular targeted
therapy is to achieve customized treatment strategy
for brain tumor patients, where individual patient
treatment will be based on the molecular profi le of
the disease. The information based on the changing
levels of active genes/proteins inside tumor cells in
response to an anti-cancer drug, could help physi-
cians to determine early in the treatment whether a
drug works effectively or not. Researchers have
identifi ed gene/protein markers that are useful in
individualizing treatment in prostate, breast, and
ovarian cancer patients. Although, brain tumor tissue
is heterogeneous, the genetic profi ling of tumor
tissue gives valuable molecular information. As a
case in point, high-throughput gene profi ling of
brain tumor biopsy samples by gene array technique
can be compared with genomic data generated using
tumor samples to predict whether patients would
benefit from anti-cancer drug (like Gleevec)
treatment or by potassium channel modulation in
tumor and tumor vascular endothelial cells.
Acknowledgments
We thank Robert Bishop, Ph.D., Schering-Plough
Research Institute, Kenilworth, New Jersey for
kindly providing radiolabeled and non-radiolabeled
Temozolomide. We also acknowledge American
Cancer Society award to NSN.
References
Ambros, V. 2003. MicroRNA pathways in fl ies and worms: growth, death,
fat, stress, and timing. Cell, 113:673–6.
Alaminos, M. et al. 2005. EMP3, a myelin-related gene located in the
critical 19q13.3 region, is epigenetically silenced and exhibits features
of a candidate tumor suppressor in glioma and neuroblastoma.
Cancer Res., 65:2565–71.
Bachman, K.E. et al. 1999. Methylation-associated silencing of the tissue
inhibitor of metalloproteinase-3 gene suggest a suppressor role in
kidney, brain, and other human cancers. Cancer Res., 59:798–802.
Balana, C.et al. 2004. Phase II study of temozolomide and cisplatin as primary
treatment prior to radiotherapy in newly diagnosed glioblastoma multi-
forme patients with measurable disease. A study of the Spanish Medical
Neuro-Oncology Group (GENOM). J. Neurooncol., 70:359–69.
Bartel, B. and Bartel, D.P. 2003. MicroRNAs: at the root of plant develop-
ment? Plant Physiol., 132:709–17.
Bartel, D.P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and
function. Cell, 116:281–97.
Baumann, F. et al. 2004. Combined thalidomide and temozolomide treat-
ment in patients with glioblastoma multiforme. J. Neurooncol.,
67:191–200.
Baylin, S.B. et al. 2001. Aberrant patterns of DNA methylation, chromatin forma-
tion and gene expression in cancer. Hum. Mol. Genet., 10:687–92.
Bernsen, H.J. and van der Kogel, A.J. 1999. Antiangiogenic therapy in brain
tumor models. J. Neurooncol., 45:247–55.
Bergers, G. et al. 2003. Benefi ts of targeting both pericytes and endothelial
cells in the tumor vasculature with kinase inhibitors. J. Clin. Invest.,
111:1287–95.
Belinsky, S.A. et al. 2006. Promoter hypermethylation of multiple genes in
sputum precedes lung cancer incidence in a high-risk cohort. Cancer
Res., 66:3338–44.
Black, K.L. and Ningaraj, N.S. 2004. Modulation of brain tumor capillaries
for enhanced drug delivery selectively to brain tumor. Cancer Control,
11:165–73.
Bocangel, D.B. et al. 2002. Multifaceted resistance of gliomas to temozolo-
mide. Clin. Cancer Res., 8:2725–34.
Butowski, N. and Chang, S.M. 2005. Small molecule and monoclonal an-
tibody therapies in neurooncology. Cancer Control, 12:116–24.
Cameron, E.E. et al. 1999. Synergy of demethylation and histone deacety-
lase inhibition in the re-expression of genes silenced in cancer. Nat.
Genet., 21:103–7.
Cairns, P. et al. 2001. Molecular detection of prostate cancer in urine by
GSTP1 hypermethylation. Clin. Cancer Res., 7:2727–30.
Calin, G.A. and Croce, C.M. 2006a. Genomics of chronic lymphocytic
leukemia microRNAs as new players with clinical signifi cance.
Semin. Oncol., 33:167–73.
Calin, G.A. and Croce, C.M. 2006b. MicroRNAs and chromosomal abnor-
malities in cancer cells. Oncogene, 25:6202–10.
Chan, J.A., Krichevsky, A.M. and Kosik, K.S. 2005. MicroRNA-21 is an
antiapoptotic factor in human glioblastoma cells. Cancer Res.,
65:6029–33.
Ciafre, S.A. et al. 2005. Extensive modulation of a set of microRNAs in primary
glioblastoma. Biochem. Biophys. Res. Commun., 334:1351–8.
Costello, J.F. et al. 1996. Silencing of p16/CDKN2 expression in human
gliomas by methylation and chromatin condensation. Cancer Res.,
56:2405–10.
Costello, J.F. et al. 2000. Aberrant CpG-island methylation has non-random
and tumour-type-specifi c patterns. Nat. Genet., 24:132–8.
206
Ningaraj et al
Drug Target Insights 2007: 2
Costello, J.F. 2003. DNA methylation in brain development and glioma-
genesis. Front Biosci., 8:s175–84.
Dalmay, T. and Edwards, D.R. 2006. MicroRNAs and the hallmarks of
cancer. Oncogene, 25:6170–5.
Daskalakis, M. et al. 2002. Demethylation of a hypermethylated P15/INK4B
gene in patients with myelodysplastic syndrome by 5-Aza-2′-
deoxycytidine (decitabine) treatment. Blood, 100:2957–64.
Fabian, M.A. et al. 2005. A small molecule-kinase interaction map for
clinical kinase inhibitors. Nat. Biotechnol., 23:329–36.
Forseen, S.E. et al. 2002. Identifi cation and relationship of HER-2/neu
overexpression to short-term mortality in primary malignant brain
tumors. Anticancer Res., 22:1599–602.
Foltz, G. et al. 2006. Genome-wide analysis of epigenetic silencing identi-
fi es BEX1 and BEX2 as candidate tumor suppressor genes in malig-
nant glioma. Cancer Res., 66:6665–74.
Friedman, H.S., Kerby, T. and Calvert, H. 2000. Temozolomide and treat-
ment of malignant glioma. Clin. Cancer Res., 6:2585–97.
Fuller, G.N. and Bigner, S.H. 1992. Amplifi ed cellular oncogenes in neoplasms
of the human central nervous system. Mutat. Res., 276:299–306.
Fueyo, J. et al. 1996. Hypermethylation of the CpG island of p16/CDKN2
correlates with gene inactivation in gliomas. Oncogene,
13:1615–9.
Gullick, W.J. 2001. Update on HER-2 as a target for cancer therapy: alter-
native strategies for targeting the epidermal growth factor system in
cancer. Breast Cancer Res., 3:390–4.
Goffi n, J. and Eisenhauer, E. 2002. DNA methyltransferase inhibitors-state
of the art. Ann. Oncol., 13:1699–716.
Gurney, A.M. and Hunter, E. 2005. The use of small interfering RNA to
elucidate the activity and function of ion channel genes in an intact
tissue. J. Pharmacol. Toxicol. Methods, 51:253–62.
Hegi, M.E. et al. 1997. Hemizygous or homozygous deletion of the chromo-
somal region containing the p16INK4a gene is associated with amplifi cation
of the EGF receptor gene in glioblastomas. Int. J. Cancer., 73:57–63.
Hirose, Y. Berger, M.S. and Pieper, R.O. 2001. p53 effects both the duration
of G2/M arrest and the fate of temozolomide-treated human glioblas-
toma cells. Cancer Res., 61:1957–63.
Hutvagner, G. 2006. MicroRNAs and cancer: issue summary. Oncogene,
25:6154–5.
Ichimura, K. et al. 1996. Human glioblastomas with no alterations of the
CDKN2A (p16INK4A, MTS1) and CDK4 genes have frequent
mutations of the retinoblastoma gene. Oncogene., 13:1065–72.
Ichimura, K. et al. 2000. Deregulation of the p14ARF/MDM2/p53 pathway
is a prerequisite for human astrocytic gliomas with G1-S transition
control gene abnormalities. Cancer Res., 60:417–24.
Jannot, G. and Simard, M.J. 2006. Tumour-related microRNAs functions
in Caenorhabditis elegans. Oncogene, 25:6197–201.
Jovanovic, M. and Hengartner, M.O. 2006. miRNAs and apoptosis: RNAs
to die for. Oncogene, 25:6176–87.
Kanzawa, T. et al. 2003. Inhibition of DNA repair for sensitizing resistant
glioma cells to temozolomide. J. Neurosurg., 99:1047–52.
Kent, O.A. and Mendell, J.T. 2006. A small piece in the cancer puzzle: microRNAs
as tumor suppressors and oncogenes. Oncogene, 25:6188–96.
Kim, T.Y. et al. 2006. Epigenomic profi ling reveals novel and frequent
targets of aberrant DNA methylation-mediated silencing in malignant
glioma. Cancer Res., 66:7490–501.
Komine, C. et al. 2003. Promoter hypermethylation of the DNA repair gene
O6-methylguanine-DNA methyltransferase is an independent predic-
tor of shortened progression free survival in patients with low grade
diffuse astrocytomas. Brain Pathol., 13:176–84.
Kraft, R. et al. 2003. BK channel openers inhibit migration of human glio-
ma cells. Pfl ugers Arch., 446:248–55.
Leis, J.F. et al. 2004. Central nervous system failure in patients with
chronic myelogenous leukemia lymphoid blast crisis and Philadelphia
chromosome positive acute lymphoblastic leukemia treated with
imatinib (STI-571). Leuk Lymphoma., 45:695–8.
Levin, V.A. 2007. Are gliomas preventable? Recent Results Cancer Res.,
174:205–5.
Libermann, T.A. et al. 1985. Amplifi cation, enhanced expression and pos-
sible rearrangement of EGF receptor gene in primary human brain
tumours of glial origin. Nature, 313:144–7.
Lund, E.L., Bastholm, L. and Kristjansen, P.E. 2000. Therapeutic synergy
of TNP-470 and ionizing radiation: effects on tumor growth, vessel
morphology, and angiogenesis in human glioblastoma multiforme
xenografts. Clin. Cancer Res., 6:971–8.
Lubbert, M. 2000. DNA methylation inhibitors in the treatment of leuke-
mias, myelodysplastic syndromes and hemoglobinopathies: clinical
results and possible mechanisms of action. Curr. Top Microbiol.
Immunol., 249:135–64.
Mashiyama, S. et al. 1991. Detection of p53 gene mutations in human brain
tumors by single-strand conformation polymorphism analysis of
polymerase chain reaction products. Oncogene., 6:1313–8.
Mauceri, H.J. et al. 1998. Combined effects of angiostatin and ionizing
radiation in antitumour therapy. Nature, 394:287–91.
Ma, H.I. et al. 2002. Intratumoral gene therapy of malignant brain tumor in
a rat model with angiostatin delivered by adeno-associated viral
(AAV) vector. Gene Ther., 9:2–11.
Mellinghoff, I.K. et al. 2005. Molecular determinants of the response of
glioblastomas to EGFR kinase inhibitors. N. Engl. J. Med.,
353:2012–24.
Nakamura, M. et al. 1996. Promoter hypermethylation of the RB1 gene in
glioblastomas. Lab Invest., 81:77–82.
Newton, H.B. 2003. Molecular neuro-oncology and development of
targeted therapeutic strategies for brain tumors. Part 1: Growth
factor and Ras signaling pathways. Expert Rev. Anticancer Ther.,
3:595–614.
Ningaraj, N.S. et al. 2002. Regulation of blood-brain tumor barrier
permeability by calcium-activated potassium channels. J. Pharmacol.
Exp. Ther., 301:838–51.
Ningaraj, N.S., Rao, M.K. and Black, K.L. 2003a. Adenosine 5′-triphos-
phate-sensitive potassium channel-mediated blood-brain tumor
barrier permeability increase in a rat brain tumor model. Cancer
Res., 63:8899–911.
Ningaraj, N.S. 2006. Drug delivery to brain tumours: challenges and prog-
ress. Expert Opin. Drug Deliv., 3:499–509.
O’Driscoll L. 2006. The emerging world of microRNAs. Anticancer Res.,
26:4271–8.
Olsen, M.L., Weaver, A.K., Ritch, P.S., Sontheimer, H. 2004. Modulation
of glioma BK channels via erbB2, 81:2179–89.
Osada, H. and Takahashi, T. 2006. MicroRNAs in biological processes and
carcinogenesis. Carcinogenesis, 28:2–12.
Pardridge, W.M. et al. 1992. Blood-brain barrier and new approaches to
brain drug delivery. West J. Med., 156:281–6.
Pardridge, W.M. 2001. Drug targeting, drug discovery, and brain drug
development. In: Brain drug targeting: The future of brain drug devel-
opment. Cambridge: Cambridge University Press, pp. 1–13.
Plumb, J.A. et al. 2000. Reversal of drug resistance in human tumor xeno-
grafts by 2′-deoxy-5-azacytidine-induced demethylation of the
hMLH1 gene promoter. Cancer Res., 60:6039–44.
Pollack, I.F. et al. 2007. Phase I trial of imatinib in children with newly
diagnosed brainstem and recurrent malignant gliomas: a Pediatric
Brain Tumor Consortium report. Neuro-oncol., 9:145–60.
Rao, M. and Ningaraj, N. 2001. Activation of Calcium-dependent potas-
sium channels elicits selective glioma cell death. In: AACR-NCI-
EORTC.
Rege, T.A., Fears, C.Y. and Gladson, C.L. 2005. Endogenous inhibitors of
angiogenesis in malignant gliomas: nature’s antiangiogenic therapy.
Neuro-oncol., 7:106–21.
Reijneveld, J.C., Voest, E.E. and Taphoorn, M.J. 2000. Angiogenesis in malig-
nant primary and metastatic brain tumors. J. Neurol., 247:597–608.
Reardon, D.A. et al. 2005. Phase II study of imatinib mesylate plus
hydroxyurea in adults with recurrent glioblastoma multiforme.
J. Clin. Oncol., 23:9359–68.
Rich, J.N. and Bigner, D.D. 2004. Development of novel targeted therapies in
the treatment of malignant glioma. Nat. Rev. Drug Discov., 3:430–46.
207
Targeted Brain Tumor Treatment
Drug Target Insights 2007: 2
Robinson, S.P. et al. 2003. Tumour dose response to the antivascular agent
ZD6126 assessed by magnetic resonance imaging. Br. J. Cancer.,
88:1592–7.
Saito, Y. and Jones, P.A. 2006. Epigenetic Activation of Tumor Suppressor
MicroRNAs in Human Cancer Cells. Cell Cycle, 5:2220–22.
Shusterman, S. et al. 2001. The angiogenesis inhibitor tnp-470 effectively
inhibits human neuroblastoma xenograft growth, especially in the
setting of subclinical disease. Clin. Cancer Res., 7:977–84.
Stupp, R. et al. 2005. Radiotherapy plus concomitant and adjuvant temo-
zolomide for glioblastoma. N. Engl. J. Med., 352:987–96.
Takano, S. et al. 2004. Angiogenesis and antiangiogenic therapy for malig-
nant gliomas. Brain Tumor Pathol., 21:69–73.
Thomas, M. 2003. Epidermal growth factor receptor tyrosine kinase inhibitors:
application in non-small cell lung cancer. Cancer Nurs., 26: 21S–25S.
Weaver, A.K., Bomben, V.C. and Sontheimer, H. 2006. Expression and
function of calcium-activated potassium channels in human glioma
cells. Glia., 54:223–33.
Weaver, A.K., Liu, X. and Sontheimer, H. 2004. Role for calcium-activated
potassium channels (BK) in growth control of human malignant
glioma cells. J. Neurosci. Res., 78:224–34.
Wen, P.Y. et al. 2006. Phase I/II study of imatinib mesylate for recurrent
malignant gliomas: North American Brain Tumor Consortium Study
99–08. Clin. Cancer Res., 12:4899–907.
Yamanaka, R. et al. 2003. Induction of an antitumor immunological response
by an intratumoral injection of dendritic cells pulsed with geneti-
cally engineered Semliki Forest virus to produce interleukin-18
combined with the systemic administration of interleukin-12.
J. Neurosurg., 99:746–53.
Yang, W. et al. 2005. Development of a syngeneic rat brain tumor model
expressing EGFRvIII and its use for molecular targeting studies with
monoclonal antibody L8A4. Clin. Cancer Res., 11:341–50.
Yung, W.K. et al. 1999. Multicenter phase II trial of temozolomide in
patients with anaplastic astrocytoma or anaplastic oligoastrocy-
toma at first relapse. Temodal Brain Tumor Group. J. Clin. Oncol.,
17:2762–71.
Zhang, L. and Coukos, G. 2006. MicroRNAs: A New Insight into Cancer
Genome. Cell Cycle, 5:2216–19.
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>>
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice