102 Current Molecular Pharmacology, 2012, 5, 102-114

Temozolomide: Mechanisms of Action, Repair and Resistance

Jihong Zhang1, Malcolm F.G. Stevens2 and Tracey D. Bradshaw*,2

1
Faculty of Life Science, Kunming University of Science and Technology, Kunming, Yunnan, 650093, China
2
Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, NG7 2RD, UK
Abstract: Glioblastoma multiforme is the most common aggressive adult primary tumour of the central nervous system.
Treatment includes surgery, radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. TMZ is an alkylating agent
prodrug, delivering a methyl group to purine bases of DNA (O6-guanine; N7-guanine and N3-adenine). The primary
cytotoxic lesion, O6-methylguanine (O6-MeG) can be removed by methylguanine methyltransferase (MGMT; direct
repair) in tumours expressing this protein, or tolerated in mismatch repair-deficient (MMR-) tumours. Thus MGMT or
MMR deficiency confers resistance to TMZ. Inherent- and acquired resistance to TMZ present major obstacles to
successful treatment.
Strategies devised to thwart resistance and enhance response to TMZ, including inhibition of DNA repair mechanisms
which contribute to TMZ resistance, are under clinical evaluation. Depletion of MGMT prior to alkylating agent
chemotherapy prevents O6-MeG repair; thus, MGMT pseudosubstrates O6-benzylguanine and lomeguatrib are able to
sensitise tumours to TMZ. Disruption of base excision repair (BER) results in persistence of potentially lethal N7- and N3-
purine lesions contributing significantly to TMZ cytoxicity particularly when O6-MeG adducts are repaired or tolerated.
Several small molecule inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1), a critical BER protein are yielding
promising results clinically, both in combination with TMZ and as single agent chemotherapy in patients whose tumours
possess homologous recombination DNA repair defects. Another validated, but as yet preclinical protein target,
mandatory to BER is abasic (AP) endonuclease-1 (APE-1); in preclinical tests, APE-1 inhibition potentiates TMZ activity.
An alternative strategy is synthesis of a molecule, evoking an irrepairable cytotoxic O6-G lesion. Preliminary efforts to
achieve this goal are described.
Keywords: Base excision repair, glioblastoma multiforme, methyltransferase, mismatch repair, O6-methylguanine
methylguanine, temozolomide.

INTRODUCTION administration [6], but labile above pH 7, with a plasma half-

Malignant glioma is the most common adult primary
tumour of the central nervous system (CNS). Median
survival from time of diagnosis is approximately 12 – 15
months [1-2]. Glioblastoma multiforme (GBM; grade IV
astrocytoma) is the most prevalent and aggressive adult
primary brain tumour whose hallmark features include
uncontrolled cellular proliferation, diffuse infiltration,
resistance to apoptosis, robust angiogenesis and rampant
genomic instability [3]. The current standard of care for
newly diagnosed GBM patients includes surgery,
radiotherapy and adjuvant temozolomide (TMZ) treatment,
conferring a median survival time of 14.6 months compared
with 12.2 months for patients receiving radiotherapy alone.
Although TMZ (Temodar; Schering-Plough Corporation)
offers some hope to GBM patients, a best 5-year survival
rate of only 9.8% is achieved [4-5].
TEMOZOLOMIDE PRODRUG ACTIVATION
TMZ, a small (194 Da) lipophilic molecule (Fig. 1), is an
orally available monofunctional DNA alkylating agent of the
imidazotetrazine class. TMZ acts as a prodrug, stable at
acidic pH values, a property which permits oral
*Address correspondence to this author at the Centre for Biomolecular
Sciences, School of Pharmacy, University of Nottingham, NG7 2RD, UK;
Tel: +44(0)115 9513419; Fax: +44(0)115 9515102;
E-mail: tracey.bradshaw@nottingham.ac.uk
life of 1.8 hours at pH 7.4 [7]. Thus, TMZ is rapidly
absorbed intact, but then undergoes spontaneous breakdown
to form monomethyl triazene 5-(3-methyltriazen-1-yl)-
imidazole-4-carboxamide (MTIC). MTIC further reacts with
water to liberate 5-aminoimidazole-4-carboxamide (AIC)
and the highly reactive methyldiazonium cation (Fig. 1). The
active species methyldiazonium cation preferentially
methylates DNA at N7 positions of guanine in guanine rich
regions (N7-MeG; 70%), but also methylates N3 adenine
(N3-MeA; 9%) and O6 guanine residues (O6-MeG; 6%) [8-
9].
There is a narrow pH window close to physiological pH
at which the whole process of TMZ prodrug activation to
methyl group transfer can occur. Brain tumours possess a
more alkaline pH compared with surrounding healthy tissue,
a situation which favours prodrug activiation preferentially
within tumour tissue [10].
Thus TMZ is used to treat (but not exclusively) brain
tumours, imparting significant therapeutic benefit to GBM
patients [4].
TEMOZOLOMIDE CYTOTOXICITY
Temozolomide cytotoxicity is primarily mediated
through O6-MeG, a carcinogenic, mutagenic and toxic lesion
[11-14]. Direct repair of O6-MeG by the suicide enzyme
methylguanine-DNA methyltransferase (MGMT) removes
the methyl adduct, restoring guanine (Fig. 2). Unrepaired

1874-4672/12 $58.00+.00 © 2012 Bentham Science Publishers

Temozolomide: Mechanisms of Action, Repair and Resistance Current Molecular Pharmacology, 2012, Vol. 5, No. 1 103

H2NOC
H2NOC H N
N N CH3
N H2O N
N
N N - CO2 N H+
CH3 NH

H2NOC
NH2
NuH
N + CH3N2 NuCH3 + N2 + H+
NH
(Nu = nucleophilic centre on DNA)

Fig. (1). Structure and activation route of prodrug temozolomide.

O6-MeG mispairs with thymine (not cytosine) during DNA
replication, alerting DNA mismatch repair (MMR) [15-16].
MMR exclusively recognises the mispaired thymine on the
daughter strand and excises it, yet O6-MeG persists in the
template strand. Therefore, futile cycles of thymine re-
insertion and excision result in persistent DNA strand
breaks, causing replication fork collapse [17]. G2/M cell
cycle arrest is triggered, occurring in the second cell cycle
following treatment [18-20] via ATR/CHK1-dependent
signalling [21]; ultimately, apoptosis ensues [22] (Figs. 3 and
4). A good response to TMZ therefore requires functional
MMR and low levels of MGMT.
The quantitatively more abundant N7-MeG and N3-MeA
lesions are rapidly repaired by DNA base excision repair
(BER; Figs. 4 and 5). N7-MeG appears not to be markedly
cytotoxic: in contrast, N3-MeA lesions are lethal if not
intercepted [23].
Therefore, the most important DNA repair systems
impacting the mechanism of action and cytotoxicty of TMZ
are MGMT (direct repair), MMR and BER (Figs. 4 and 5).
DNA REPAIR MECHANISMS CONTRIBUTING TO
TEMOZOLOMIDE RESISTANCE
Direct Repair
MGMT (O6-Alkylguanine-DNA alkyltransferase; AGT)
repairs O6-alkylguanine adducts in a single step,
independently of any other protein or cofactors (Fig. 2). It is
a small protein (22 kDa) present in both the cytoplasm and
nucleus. Upon DNA alkylation, a shift towards more nuclear
localisation may facilitate the repair process [24]. MGMT is
able to repair not only O6-MeG, but also guanine residues
with longer O6-alkyl adducts such as ethyl, chloroethyl,
hydroxyethyl, n-propyl, n-butyl, and more bulky cyclic
lesions conferred by benzyl or pyridyloxobutyl groups, but
with diminishing efficiency as adduct size increases [25-27].
The O6-alkyl group is transferred from guanine to the active
site cysteine residue (Cys 145) of MGMT in a
stoichiometric, auto-inactivating reaction, thereby repairing
DNA and inactivating MGMT [28]. MGMT binds damaged
substrate DNA in the minor groove, the target base is then

MGMT MGMT

Cysteine 145 Cysteine 145

CH3
H2NOC
N
N CH3
N O
N N O
CH3 6 6
N N
N HN
O
H H
TMZ N
H2N N H2N N N

d Ribose d Ribose

O6-methylguanine guanine
Fig. (2). Repair of O6-methylguanine adducts by O6-methylguanine-DNA methyltransferase.
104 Current Molecular Pharmacology, 2012, Vol. 5, No. 1 Zhang et al.

A B
600

500 MGMT

SNB19M

U373V

U373M
SNB19V
400
GI50 (mm)

300

200

100

0
S N B 19V S N B 19M U 373V U 373M

Cell Line

C Control 24 h 48 h 72 h 120 h

Fig. (3). A) Effect of temozolomide on glioblastoma multiforme cell growth. Cells were seeded at a density of 650 per well. After 24 h,
temozolomide was introduced. At the time of drug addition and following 7 days incubation, MTT assays were performed to determine cell
growth.
B) Expression of methylguaninemethyltransferase protein in SNB19M and U373M cells. Western blot assays were performed following
separation of protein from whole cell lysates.
C) Effect of temozolomide on SNB19V cell cycle.

MGMT+ G survival

O6-Me-G
MMR+ cytotoxicity
MGMT-
TMZ MMR-

N7-,N3-Me-Purine mutations tolerated

BER survival

Fig. (4). Key DNA repair mechanisms influencing cellular response to temozolomide.

flipped out of the helix and bound to MGMT, altering the
conformation of the DNA binding domain allowing
alkylated MGMT to be detached from DNA and degraded
through the ubiquitin/proteasomal system [15, 29].
MGMT protects cells from carcinogens; however, it is
also able to protect cancer cells from chemotherapeutic
alkylating agents such as TMZ. Tissue expression is
variable, with high protein expression in liver and lower
expression in haematopoietic tissues and brain [30-31].
Tumour MGMT expression is immensely variable, highest
levels are found in breast, ovarian and lung tumours,
whereas lowest activity is observed in gliomas, pancreatic
Temozolomide: Mechanisms of Action, Repair and Resistance
TMZ
O6-MeG
DR MMR
MGMT MSH2-MSH6
MSH2-MSH3
(Mutsα and β)
MLH1-PMS2
MLH1-PMS1
(MutlLα and β)
Nucleases (EXO1)
RFC, PCNA,
RPA, DNA Pol
Ligase
Fig. (5). Summary of proteins involved in DNA repair pathways activated by temozolomide-induced DNA lesions.
carcinomas and malignant melanomas [32]. Hence, TMZ
treatment of metastatic malignant melanoma has been under
clinical evaluation [33] and is now an approved indication in
certain territories. In gliomas, however, MGMT activity
varying 300-fold has been reported [34], and a strong
positive correlation exists between MGMT activity and
alkylating agent resistance [35-37] in vivo and in vitro, as
exemplified in Fig. (3). MGMT transfected SNB19 and
U373 GBM cells are > 13- and > 5-fold more resistant to
TMZ challenge than their isogenic, vector control transfected
partner cell lines, possessing negligible and low MGMT
activity respectively. The MGMT gene is located on
chromosome 10q26 and frequent loss of chromosome 10
observed in 60% – 85% glioma cases is thought to be related
to low protein expression [38]. Mutations of MGMT and
protein phosphorylation, responsible for MGMT inactivation
have also been detected in human tumours [39]. However,
loss of MGMT activity is most frequently a consequence of
MGMT promoter methylation [40-42]. Gene inactivation by
promoter methylation is a common epigenetic phenomenon
in tumourigenesis [43]. Methylation, mediated by 5`-
methylcytosine methyltransferase, takes place on the
cytosine of CpG islands. Hypermethylation of CpG islands
in the MGMT promoter region prevents transcription factor
binding, silencing the gene [41, 44]. MGMT methylation has
been detected in 45% - 70% high grade gliomas [45-46].
Clinical evidence has revealed that patients with MGMT
promoter methylation respond better than those without
promoter methylation to radiotherapy treatment with either
BCNU or TMZ [47-49]. The correlation between MGMT
promoter methylation extent and clinical response to
alkylating agents means that MGMT promoter methylation is
a good predictive marker of response to alkylating agent
chemotherapy.
DNA Mismatch Repair
Mismatch repair (MMR; Fig. 5) is the recognition and
correction of mispaired bases and insertion/deletion loops
Current Molecular Pharmacology, 2012, Vol. 5, No. 1 105
N7-MeG N3-MeA
BER
Glycosylase
APE-1
PARP
short patch long patch
Polβ Pol
XRCC1 PCNA
Ligase III FEN1
Ligase 1
(resulting from gains or losses of short repeat units within
microsatellite sequences) generated during DNA synthesis.
MutS
(comprising MSH2 and MSH6) or MutS
(comprising MSH2 and MSH3) complexes recognise and
bind to mismatch lesions. MutS
binds base to base
mismatches and insertion/deletion mismatch loops of one or
two nucleotides. Muts has little affinity for base to base
mismatches [50-51], but is involved in repair of loops of up
to 16 nucleotides. In man, Muts
is predominantly
implicated in DNA damage signalling. The MSH2/MSH6
heterodimer undergoes an ATP-dependent conformational
change and recruits the MLH1/PMS2 heterodimer, which
coordinates the interplay between the mismatch recognition
complex and additional proteins including exonuclease 1,
helicases, proliferating cell nuclear antigen, single strand
DNA binding protein, DNA polymerase
and , necessary
for removal and replacement of the mismatched DNA base.
MMR plays a critical role in correction of replicative
mismatches that have escaped polymerase proofreading, and
loss of MMR results in a dramatic increase in insertion/
deletion mutations, particularly in repetitive sequence
microsatellite DNA. Indeed, microsatellite instability (MSI)
is a recognised surrogate biomarker for the loss of MMR
function [52].
MMR is of clinical significance in several cancers
including endometrial, ovarian, gastric and particularly
colorectal cancers. Indeed, MMR deficiency has been
observed in 15% - 20% of sporadic colorectal tumours [53-
54] as well as some breast, prostate, bladder, head and neck
cancers [55]. Colorectal cancer studies have demonstrated
hereditary and sporadic MMR gene mutations responsible
for MSI. In hereditary nonpolyposis colorectal cancer
(HNPCC), germ line mutations in MLH1 or MSH2 cause
microsatellite repeat replication errors to persist [56].
Somatic MMR gene mutations may be the result of
epigenetic gene silencing via methylation of the MLH1
promoter [57-58]. Aberrantly methylated hMLH1 promoter
DNA has been reported in the sera of 9/19 patients with
106 Current Molecular Pharmacology, 2012, Vol. 5, No. 1
microsatellite unstable colon carcinoma [59]. MMR
mutations allow microsatellite insertions/deletions which
cause inactivating frameshift mutations within tumour
suppressor coding regions, critical genes in cell cycle
regulation and cancer prevention.
MMR status influences the response of cells to genotoxic
agents, indeed, methylating agent cytotoxicity induced by
TMZ requires functional MMR. HCT 116 (MLH1 mutant)
and DLD1 (MSH6 mutant) MMR deficient colon carcinoma
cells are resistant to TMZ treatment (GI50 > 500 μM) [60].
MMR-deficient cells are reported to be up to 100-fold less
sensitive to methylating agents compared with their MMR
proficient counterparts [21, 61]. In such cells, O6-MeG-
thymine mispairs are not recognised, O6-MeG lesions are
tolerated, cells continue cycling, surviving at the expense of
extensive mutagenesis [61].
Base Excision Repair
Base excision repair (BER) is the major pathway
involved in removal and repair of non-bulky damaged
nucleotides, abasic sites and DNA single strand breaks
generated by reactive oxygen species, ionising radiation and
alkylating agents [62]. N7- and N3- purines, methylated by
TMZ are repaired by BER (Figs. 4 and 5). Damaged bases
are recognised by lesion-specific glycosylases that hydro-
lytically cleave the N-glycosidic bond, generating an abasic
(apurinic/apyrimidinic; AP) site. AP endonuclease (APE-1)
then cleaves the phosphodiester backbone on the 5`side of
the AP site, leaving 3`-OH and 5`-deoxyribose phosphate
(dRp) termini at the DNA strand break. The terminal 5`dRp
residue is removed by exonuclease or DNA-deoxyribopho-
sphodiesterase, leaving a nucleotide gap. Further repair can
proceed via two pathways: short patch involving
replacement of one nucleotide; and long patch involving gap
filling of 2 – 10 nucleotides. In short patch BER, the single
nucleotide gap is filled by DNA polymerase and the nick
sealed by the DNA ligase III/X-ray repair cross-
complementing 1 (XRCC1) heterodimer [63]. In long patch
BER, the DNA strand may become displaced, creating a
flap. Flap endonuclease-1 (FEN-1) cleaves the flap and DNA
ends are sealed by DNA ligase I [64].
A protein key to successful DNA damage signaling and
BER is poly(ADP-ribose) polymerase-1 (PARP-1).
Constitutively expressed, but activated in response to DNA,
damage, PARP-1 modifies nuclear proteins by poly(ADP-
ribosyl)ation. PARP-1 enzyme (113 kDa), encoded by the
ADP-ribosyl transferase (ADPRT) gene, possesses 3
domains. The DNA binding domain, possessing 2 zinc finger
structures and a nuclear localisation sequence at the N-
terminus, recognises DNA single and double strand nicks
(SSBs and DSBs). The automodification domain mediates
protein/protein interactions, and at the C-terminus is the
active site catalytic domain. In response to DNA damage,
PARP-1 binds to DNA SSB (or DSB) and cleaves
–
nicotnamide adenine dinucleotide (NAD+) releasing
nicotinamide and ADP-ribose. PARP uses NAD+ to catalyse
auto-(and other protein) poly(ADP-ribosyl)ation. Long,
branched ADP-ribose polymers attract recruitment of the
BER protein complex comprising XRCC1, DNA polymerase

and DNA ligase III, FEN-1 to progress repair (and serve as
an energy source for ligation) [65-66]. Release of
Zhang et al.
polyribosylated PARP from the DNA lesion allows access of
essential BER proteins. Thus, PARP facilitates efficient
DNA repair and survival of cells subjected to mild genotoxic
stress [67]. Inhibition of PARP increases the frequency of
DNA strand breaks, accordingly PARP deficient cells are
hypersensitive to carcinogenic agents [68].
As noted, the majority of DNA lesions generated by
TMZ are N7-MeG and N3-MeA, comprising 80-85% and 8-
18% of total alkyl adducts respectively. These lesions,
rapidly and efficiently repaired by BER, become highly
cytotoxic when BER is disrupted [69]. PARP inhibition
enhances the cytotoxicty of base lesions normally repaired
by BER, and indeed, enhances TMZ cytotoxicity in vitro and
in vivo [70-72]. TMZ cytotoxicity is primarily manifest via
O6-MeG adducts. However, when O6-MeG lesions are
either repaired by MGMT or tolerated following MMR
disruption, N-Me purine adducts become significant, and
then inhibition of BER enhances TMZ therapeutic efficacy
[70]. Therefore, disruption of BER by PARP inhibition
provides a means to overcome resistance that frequently
develops as a consequence of selection of MMR deficient
cells during therapy [73].
Acquired Drug Resistance
Tumours initially sensitive to chemotherapy often
develop resistance – acquired resistance. Acquired drug
resistance emerges, by Darwinian evolution, as a
consequence of selective pressure in the presence of
chemotherapeutic agent. Acquired chemo-resistance may be
a consequence of drug-induced genetic and epigenetic
changes in neoplastic cells, inducing and selecting genes
which confer a survival advantage, or result from selection
of pre-existing resistant cell clones [74]. In initially
heterogenous tumours, chemotherapy eliminates drug-
sensitive malignant cells allowing survival of drug-resistant
cells and chemo-resistant cancer stem cells which may
advance to seed more (acquired) resistant tumours. Indeed,
within glioblastomas and medullbalstomas, populations of
cells with stem cell-like properties, associated with tumour-
initiating capacity and resistance to therapy have been
identified [2, 75-76]. Thus tumours demonstrating resistance
to multiple chemotherapeutic agents whose mechanisms of
action are distinct may emerge [77]. Such multidrug
resistance is a major factor contributing to treatment failure.
Mechanisms conferring resistance to chemotherapeutic
agents include decreased cellular drug uptake, increased drug
efflux by membrane pumps actively expelling chemotherapy
agents, intracellular drug inactivation, alteration of drug
target by mutation, inactivation or over-expression, enhanced
repair of drug-induced DNA damage or suppression of repair
resulting in tolerance to DNA lesions and alteration of
apoptosis-related genes [78-80]. The consequence of such
measures is survival of malignant cells resistant to drug-
induced apoptosis
MECHANISMS OF GLIOMA CHEMORESISTANCE
AND ACQUIRED DRUG RESISTANCE TO TEMO-
ZOLOMIDE
Although TMZ is first line chemotherapy for glioma
patients, inherent and acquired resistance, conferred by
Temozolomide: Mechanisms of Action, Repair and Resistance
multiple mechanisms, results in treatment failure. Intrinsic
glioma resistance may be a consequence of the presence
within tumours of multi-drug resistance proteins (MRP)1,
MRP3, MRP5, and glutathione-S-transferase (GST)
[81].
It has recently confirmed that expression of MDR1/ABCB1
encoding multi-drug resistant p-glycoprotein negatively
impacts sensitivity to TMZ [82]. TMZ, a small lipophilic
molecule is able to cross the blood brain barrier (BBB), an
obstacle to delivery of most agents to brain tumours.
Furthermore, gliomas are naturally resistance to apoptosis as
a consequence of phosphate and tensin homologue on
chromosome 10 (PTEN) tumour suppressor mutation and
constitutively active Akt/PI3K/mTOR/NF-kB signaling [83].
Inherent resistance or tolerance to TMZ treatment may be
conferred, as discussed, by MGMT activity, or MMR
deficiency respectively. Thus adaptive alterations in DNA
repair pathways play critical roles in development of
resistance to TMZ. Many studies have now demonstrated a
robust inverse relationship between MGMT expression and
inherent sensitivity to TMZ in vivo and in vitro [48, 49, 84,
85] (Fig. 2). It has also been reported that acquired TMZ
resistance attributed to enhanced MGMT activity arises
frequently in glioma cell lines and xenografts [86]. Zhang
and coworkers [87] report that up-regulation of MGMT
expression in U373 GBM cells exposed to incremental
concentrations of TMZ confers > 4-fold resistance to TMZ
in this TMZ-acquired resistant cell line. MGMT is an
inducible DNA repair gene which can be up-regulated by not
only alkylating agents but also ionising radiation and
glucocorticoids [88]. GBM MGMT activity has been
compared in newly diagnosed patients, and recurrent GBM
patients receiving combined radio- and alkylating agent-
(TMZ, chloroethylnitrosourea) therapy. MGMT activity in
untreated tumour samples was 37 fmol/mg protein, but in
recurrent GBM MGMT activity of 182 fmol/mg protein was
determined [85], a consequence either of selection of
MGMT-expressing cells, or induction of MGMT by
alkylating agents.
The relatively low incidence of MSI in high grade
gliomas would suggest that MMR deficiency is rarely
involved in intrinsic resistance to alkylating agents in this
disease [89]. However, a large body of data implicates MMR
deficiency in development of tolerance to O6-MeG lesions,
and therefore resistance to TMZ [90, 91]. Somatic mutations
harboured within MMR proteins MSH2, MLH1 and MSH6
in glioma cell lines, GBM xenografts and patient tumour
samples after treatment with TMZ is associated with TMZ
tolerance regardless of MGMT expression [87, 92, 93].
Analysis of a large number of clinical samples revealed loss
of MSH6 protein in a subset of recurrent GBM patients
treated with TMZ, and that this loss was associated with
progressive disease during TMZ therapy [90]. MSH6 protein
down-regulation was observed in SNB19 GBM cells
previously exposed to increasing concentrations of TMZ,
proliferating in medium spiked with 100 μM TMZ. These
cells demonstrated 7.8-fold acquired resistance to TMZ
compared with their TMZ-naive parent cell line [87]. Studies
undertaken by Yip et al. [93] confirmed that MSH6
inactivation in vitro results in increased resistance to TMZ;
reconstitution of MSH6 expression restored TMZ sensitivity.
Current Molecular Pharmacology, 2012, Vol. 5, No. 1 107
It has been corroborated in vivo that GBM MSH mutations,
selected during TMZ treatment correlate with TMZ
resistance. MSH6 mutations, and/or absent protein
expression were neither found in pre-treatment GBM nor
radiotherapy post-treatment GBM, but were detected in
approximately half of recurrent GBM patients treated with
TMZ and radiotherapy, strongly indicating that MSH6
alterations in the tumour cell genome are associated with
alkylating agent therapy and resistance. Indeed, MSH6
mutations have been found in 26% recurrent GBM cases
following alkylating agent chemotherapy [93].
GBM patients possessing tumour MGMT promoter
methylation respond well to alkylating agent therapy initially
[49], however, alkylating agent treatment of such MGMT-
deficient GBM confers a strong selective pressure to lose
MMR function, resulting in O6-MeG tolerance and
resistance to cell death [94]. Recent comprehensive genomic
characterisation of human GBM genes revealed that initial
methylation of the MGMT promoter in conjunction with
alkylating agent treatment leads to a shift in the mutation
spectrum affecting mutations at MMR gene loci. Thus,
patients who initially respond to therapy may evolve not
only treatment resistance but also an MMR-defective
hypermutator phenotype.
N-Me purine lesions are repaired by BER, a major
contributor to cellular resistance to TMZ [95]. The
importance of BER was confirmed in SNB19 human
glioblastoma cells generated to possess acquired resistance
to TMZ. In these cells, O6-MeG lesions were tolerated
following loss of MSH6 MMR protein. The PARP inhibitor
NU1025 partially restored sensitivity to TMZ (3.5x) in these
cells. Moreover, transcription of NTHL1 gene, encoding a
key BER protein, possessing both abasic endonuclease and
N-glycosylase activity, was elevated > 5-fold [87]. Up-
regulation of APE-1 (Ref-1) is characteristic of human
gliomas, contributing to TMZ resistance [96]. APE-1 activity
is also able to promote resistance to radiotherapy plus TMZ
or BCNU in medulloblastoma and primitive neuroectoderm
tumours [97]. Reduction in APE-1 protein and its
endonuclease activity using APE-1-directed antisense
oligonucleotides in SNB19 GBM cells has been shown to
decrease resistance to TMZ and BCNU. Hence, APE-1 may
present both a predictive and prognostic marker for
alkylating agent treatment [98], and a potential therapeutic
target [99].
Elucidation of mechanisms of tumour resistance to TMZ
has helped steer therapeutic strategies attempting to
overcome resistance. In vitro tissue culture studies are
integral to understanding molecular mechanisms responsible
for resistance. Exposure of tumour cells to TMZ selects cell
populations with acquired resistance or tolerance to this
agent, disrupting or usurping the same molecular
mechanisms which underlie acquired resistance to TMZ
emerging clinically [85, 87, 90]. Comprehensive
understanding of such molecular mechanisms may reveal
novel targets for therapy, guide rational chemotherapy
combination regimens to modulate resistance, or lead to
development of novel TMZ analogues to surmount
resistance mechanisms.
108 Current Molecular Pharmacology, 2012, Vol. 5, No. 1
OVERCOMING RESISTANCE TO TMZ
Inhibition of MGMT
As direct repair of O6-MeG adducts by MGMT protein
has a major impact on alkylating agent resistance clinically,
a number of therapeutic approaches have been explored to
modulate MGMT activity and enhance drug response [100].
The potent non-toxic inhibitors of MGMT O6-benzyl
guanine (O6-BG) and O6-(4-bromothenyl)guanine
(lomeguatrib; PaTrin-2; Fig. 6), have been used in clinical
trials to deplete MGMT before administration of alkylating
agent therapy [44, 101, 102]. O6-BG acts as a
pseudosubstrate and binds to MGMT, covalently transferring
the benzyl moiety to the active site cysteine residue 145,
causing its irreversible inactivation. O6-BG is not
incorporated into the DNA of living cells, reacting directly
with both cytoplasmic and nuclear MGMT [102]. Pre-
treatment of tumour cells containing high MGMT levels with
O6-BG enhances TMZ activity in vitro and in vivo, but has
little effect on tumour cells possessing low or undetectable
MGMT levels [14].
Lomeguatrib is an orally bioavailable potent
pseudosubstrate for MGMT. Covalent transfer of the
bromothenyl group to the active site cysteine inactivates
MGMT. Lomeguatrib has shown promising activity in
sensitising a variety of human tumour xenografts to the
growth-inhibitory effects of O6-alkylating agents, including
TMZ and 1,3-bis-(2-chloroethyl)-1-nitrosurea, at the expense
of only limited additional toxicity [103, 104]. A Phase I
clinical trial combining lomeguatrib and TMZ [105] led to a
randomised Phase II study in 100 patients with metastatic
melanoma [106]. In this study, the efficacy of combination
treatment was similar to that of TMZ treatment alone in
terms of response rates and median time to disease
progression. However, the lomeguatrib schedule adopted
permitted rapid recovery of tumour MGMT within 24 h.
Subsequent clinical trials established a lethal strategy to target cancers with specific DNA repair
pharmacodynamically effective schedule and doses of
lomeguatrib which effects complete and consistent MGMT
depletion in melanoma, CNS, prostate and colorectal
tumours [107, 108]. Moreover, significantly higher levels of
O6-MeG adducts were present in PBMC DNA following
A B
O
N
N
H2N N N
H
Fig. (6). Structures of A) O6-benzylguanine and B) lomeguatrib.
Zhang et al.
lomeguatrib / TMZ combination therapy compared with
TMZ treatment alone. However, myelosuppression remains a
prohibitive limiting side effect to the use of MGMT
inhibitors and alkylating agent combination cancer
chemotherapy. This is a consequence of low MGMT
expression within bone marrow which renders this tissue
susceptible to cytotoxicity [109]. To protect haematopoeitic
cells during chemotherapy, the strategy of gene transfer of
mutant MGMT cDNA, encoding protein resistant to
inactivation has been developed [36, 110, 111]. A phase I
clinical study of such myelosuppressive gene therapy is
underway in the U.S.
INHIBITION OF BER
Poly(ADP-ribose)polymerase Inhibition
N-Me purines, rapidly repaired by BER, contribute little
to TMZ-induced toxicity in the absence of MGMT and
presence of proficient MMR. However, when O6-MeG is
repaired (MGMT) or tolerated (MMR-), and BER is
disrupted by PARP inhibition, N7-MeG and N3-MeA
contribute significantly to TMZ cytotoxicity [72, 112]. It was
to test the hypothesis that PARP inhibition could potentiate
TMZ activity that PARP inhibitor AG 014699 first entered
clinical trials in cancer patients [113-116]. The combination
revealed increased response rates and median time to
progression compared with TMZ treatment alone. ABT 888
demonstrated broad in vivo activity in combination with
TMZ in diverse tumours [117]. At least 8 PARP inhibitors
are currently undergoing Phase I, II, or III clinical
evaluation, either in combination with chemotherapeutic
agents or alone, for treatment of malignant solid tumours
[112, 113, 117]. The chemical structures of three such small
molecule inhibitors of PARP-1: olaparib, ABT 888 and BSI-
201 are shown in Fig. (7). It was hypothesised that single
agent PARP inhibitor therapy would provide a synthetic
defects. Indeed, PARP inhibition demonstrates substantial
single agent antitumour activity, possessing a wide
therapeutic index in homologous DNA repair-defective
tumours such as those arising in BRCA1 and BRCA2
mutation carriers; responses to olaparib (KU-0059436;
AZD2281) have been observed in Phase I and II studies
Temozolomide: Mechanisms of Action, Repair and Resistance
A F
O
N B
NH
O
H2N
C
Fig. (7). Structures of PARP inhibitors A) olaparib, B) ABT 888 and C) BSI-201 under clinical evaluation.
[117-119]. PARP inhibitor efficacy is also being assessed for
treatment of triple negative metastatic breast, and PTEN
mutant tumours [120].
Inhibiton of APE-1
Pharmacological inhibition of BER using PARP
inhibitors either alone or in combination with chemotherapy
such as TMZ has shown promise in clinical trials. However,
acquired resistance to PARP inhibitors is inevitably
beginning to emerge; indeed up-regulation of homologous
recombination repair to compensate for diminished BER has
been identified [121]. Obligatory intermediates of BER are
potentially cytotoxic AP sites, processed by APE-1. Abbots
and Madhusudan [122] highlight data that validate APE-1 as
an anticancer target. Indeed, small molecule inhibitors of
APE-1 are able to potentiate alkylating agent cytotoxicty in
preclincial models. The topoisomerase II inhibitor
lucanthrone also inhibits APE-1, causing accumulation of
AP sites [123] potentiating TMZ cytotoxicty [124].
Methoxyamine (MA) binds directly to AP sites and
indirectly inhibits APE-1 by preventing AP site processing
by APE-1; thus, AP sites accumulate. MA potentiates the
cytotoxicty of a wide variety of DNA damaging agents in
vitro and in xenograft studies [124-126] in both MMR
proficient and deficient tumour cells. A phase 1 clinical
study combining MA with TMZ in advanced solid tumours
is currently underway.
APE-1 is a multifunctional enzyme possessing a DNA
repair domain and a redox domain. It is often referred to as
Ref-1 and is involved in redox-sensitive activation of
transcription factors including P53, NFkB, AP1 and HIF-1
.
Thus, synthesis of small molecules which directly target
APE-1 may hold wide therapeutic benefit in the cancer
arena.
Current Molecular Pharmacology, 2012, Vol. 5, No. 1 109
O
N
H2N O
N H3C
N HN
O H
NO2
DESIGN OF IMIDAZOTETRAZINE ANALOGUES TO
CIRCUMVENT MGMT REPAIR
The TMZ molecule acts as a methyl group delivery
vehicle imparting its toxic lode on O6-G. Primary inherent
TMZ resistance, conferred by MGMT, leads to
stoichiometric removal of O6-methyl adducts restoring
guanine. Logically, one could postulate that synthesis of an
imidazotetrazine analogue might be possible, that would
deliver an O6-G adduct not recognised by MGMT. In vitro
results of such synthetic efforts are summarised in Table 1
[60].
Imidazotetrazine (series A) TMZ and its ring-opened
triazene (series B) MTIC reveal growth inhibitory activity
(GI50 < 100 μM) against vector control GBM cell lines
SNB19V and U373V; however, activity is abrogated in their
MGMT transfected SNB19M and U373M isogenic partners.
Compounds possessing a large lipophilic R group (1) and (2)
are much less active, whereas the trimethylsilymethyl
derivative (3) shows a pattern of activity similar to that of
TMZ.
Compounds with a small more polar group, e.g.
chloromethyl (5) or MOM (6) are more potent than TMZ
against vector control cell lines and also retain their activity
against isogenic MGMT overexpressing variants. Similarly
the imidazotetrazine esters (7 and 9) demonstrate a “flat”
distribution of activity across the four GBM cell lines; ring-
opened triazene counterparts (8 and 10) are approximately
equiactive to their cyclic imidazotetrazine precursors. These
results imply that the new imidazotetrazine TMZ analogues
are ring-opened to create DNA alkylating species which
generate cytotoxic lesions irreparable by MGMT. However,
in vivo, imidazotetrazine esters would be substrates for
plasma esterases, and the corresponding imidazotetrazine
carboxylic acid 11 of methyl ester 7, for example reveals
110 Current Molecular Pharmacology, 2012, Vol. 5, No. 1 Zhang et al.

Table 1. Effect of Imidazotetrazine Anlogues (Series A and B) on GBM Cell Growth

H2NOC
N
N H2NOC
N H
N N N N
R N R
N
O NH
A B

Cell Lines GI50 (μM)

Compound Series R R
SNB19V SNB19M U373V U373M

TMZ A Me Me 35.7 ± 12.0 469.9 ± 88.7 68.0 ± 32.0 368.7 ± 86.1

MTIC B Me Me 50.9 ± 17.9 472.1 ± 88.7 93.7 ± 39.7 397.9 ± 33.7
1 A CH2Ph CH2Ph 140.5 ± 79.8 180.0 ± 86.3 165.0 ± 107.8 177.7 ± 133.7
2 A CH2CH2 Ph CH2CH2 Ph 98.6 ± 26.2 85.9 ± 13.7 114.7 ± 91.0 91.1 ± 38.3
3 A CH2Si(Me) 3 CH2Si(Me) 3 28.4 ± 5.9 302.4 ± 69.3 45.6 ± 14.5 249.5 ± 72.1
4 A CH2CF3 CH2CF3 39.9 ± 5.9 52.4 ± 11.5 102.5 ± 32.9 94.5 ± 24.0
5 A CH2Cl CH2Cl 34.6 ± 14.1 31.0± 7.9 29.3 ± 7.8 24.4 ± 1.0
6 A CH2OMe CH2OMe 30.8 ± 9.0 32.9 ± 9.2 26.0 ± 5.0 33.1 ± 9.3
7 A CH2CO2 Me CH2CO2 Me 47.5 ± 17.5 49.0 ± 14.7 32.1 ± 21.9 46.4 ± 16.7
8 B CH2CO2 Me CH2CO2 Me 47.7 ± 6.1 49.7 ± 7.3 32.6 ± 20.5 46.4 ± 9.8
9 A CH2CO2 Et CH2CO2 Et 52.4 ± 4.5 61.1 ± 5.2 63.2 ± 10.7 55.6 ± 6.5
10 B CH2CO2 Et CH2CO2 Et 33.6 ± 2.6 37.4 ± 11.9 30.3 ± 20.4 23.4 ± 12.2
11 A CH2CO2H CH2CO2H 339.7 ± 130.5 297.7 ± 68.4 269.0 ± 205.0 198.0± 91.1

poor activity against vector control and MGMT transfected ABBREVIATIONS

SNB19 and U373 GBM cell lines (GI50 > 195 μM).
ABCB1 = ATP-binding cassette protein B1
CONCLUDING REMARKS ADPRT = ADP-ribosyltransferase
AGT = alkylguanine DNA alkyltransferase
TMZ offers hope to newly diagnosed GBM patients.
However, in tumours expressing MGMT, the primary AIC = 5-aminoimidazole-4-carboxamide
cytotoxic lesion is stoichiometrically removed. These
AP = abasic
tumours are inherently resistant to TMZ.
APE-1 = AP endonuclease-1
While potentiation of TMZ activity can be conferred by
agents that deplete MGMT, adverse, therapy-limiting BBB = blood brain barrier
complications emerge. The majority of lesions induced by BCNU = 1,3-bis-(2-chloroethyl)-1-nitrosourea
TMZ are subject to rapid BER; interruption of BER by (Carmustine)
PARP inhibition is also able to potentiate TMZ response.
However, in tumours initially responsive to TMZ therapy, BER = base excision repair
acquired resistance or tolerance to treatment evolves, tumour CNS = central nervous system
cells cunningly adapt and modify DNA repair machinery,
MGMT and BER proteins are up-regulated, MMR is DSB = DNA double strand break
disabled, exposing DNA, oblivious to TMZ-induced lesions, dRp = 5`-deoxyribose phosphate
vulnerable to further mutagenic events promoting a more
resistant and aggressive GBM cancer cell. It is thus evident GBM = glioblastoma multiforme
that formidable therapeutic challenges persist, and GST = glutathione-S-transferase
development of novel agents is vital to augment current
HNPCC = hereditary nonpolyposis colorectal cancer
treatment of this intractable disease.
MDR = multidrug resistance
ACKNOWLEDGEMENTS MGMT = methylguanine DNA methyltransferase
We thank Marc Hummersone and the chemists of MMR = DNA mismatch repair
Pharminox Ltd synthesis of novel imidazotetrazine and
MRP = multidrug resistance protein
imidazotriazene molecules.
Temozolomide: Mechanisms of Action, Repair and Resistance
MSI = microsatellite instability
MTIC = 5-(3-methyltriazen-1-yl)-imidazole-4-
carboxamide
NAD+ =
-nicotinamide adenine dinucleotide
N3-MeA = N3-methyladenine
N7-MeG = N7-methylguanine
O6-MeG = O6-methylguanine
PARP-1 = poly(ADP-ribose)polymerase-1
PTEN = phosphatise and tensin homologue
SSB = DNA single strand break
TMZ = temozolomide
XRCC1 = X-ray repair cross-complementing 1
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PMID: 22122467
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