Download Inhibitors of topoisomerases as anticancer drugs: Problems and

Indian Journal of Experimental Biology
Vol. 42, July 2004, pp. 649-659
Review Article
Inhibitors of topoisomerases as anticancer drugs: Problems and prospects
B S Dwarakanath l *, Divya Khaitan l & Rohit Mathur l • 2
'Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Mazumdar Marg, Delhi 110054, India
2Dr B R Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India
DNA topoisomerases, which solve topological problems associated with various DNA transactions, are the targets of
many therapeutic agents. Various topoisomerase inhibitors especially, topo-poisons, camptothecin (topo-I) and etoposide
(topo-ll) are some of the drugs that are used in the current treatment protocols, particularly for the treatment of leukemia
(AML, ALL etc). However, tumor resistance, normal and non-specific tissue cytotoxicity are the limitations for successful
development of these drugs as one of the primary therapeutic agents for the treatment of tumors in vitro. This brief review
presents the current understanding about cytotoxicity development and outlines various approaches to overcome the limitations for enhancing the efficacy of topo-poison based anticancer drugs.
Keywords: Camptothecin, Cleavable complex, 2-Deoxy-D-glucose, Etoposide, Topoisomerases
IPC Code: Int Cl 7 A61 K
Topoisomerases are ubiquitous and evolutionarily
conserved nuclear enzymes, which modulate the
topological state of DNA by passing an intact DNA
helix through a transient single (topo-I) or double
(topo-II) strand break, facilitating vital nuclear transactions such as transcription, replication, chromosome
segregation, mitotic division etc l . Since the level of
topoisomerases is generally elevated in malignant
(cancer) and actively growing cells, these enzymes
have been the specific targets of several anti-tumor
drugs2 • Currently used topo~somerase inhibitors exert
their cytotoxic effects either by stabilizing the covalent complexes between the enzyme and DNA
(cleavable complex) or by inhibiting the catalytic activitl A (Fig. I). Despite the development of specific
and more effective inhibitors, with enhanced clinical
activities, drug resistance by tumors as well as lack of
specificity resulting in normal tissue toxicity has continued to limit the success of cancer therapy using
topoisomerase poisons. Therefore, there is a great
need to develop newer approaches, which selectively
enhance the drug-induced toxicity in cancer cells.
One of the approaches to achieve this objective
would be to synthesize novel, sequence specific DNA
ligands, with associated topoisomerase inhibitory aCtivity, that exhibit differential effects between tumor
*Correspondent author
Fax (+91) 11-2391-9509, [email protected]
and no~al cells5•6. On the other hand, approaches
that selectively modify the drug accumulation and
consequences of drug-induced primary lesions in tumor cells, while sparing cells from the normal tissue,
could be equally effective. Pre-target events including
intracellular drug accumulation and distribution, drugtarget interactions influenced by enzyme levels, sequence specificity and activity, status of nuclear organization etc., and post-target events like macromolecular synthesis, DNA repair, cell cycle perturbations
and apoptosis, collectively contribute to the druginduced cytotoxicity 7.8. Therefore, an understanding
of the molecular mechanisms involved in the repair/reversal of cleavable complexes as well as the
interactions of topoisomerases with DNA repair machinery, besides the induction of cell cycle perturbations and apoptosis will greatly facilitate the optimal
use of topoisomerases as anticancer drugs.
Topoisomerases as molecular targets for anticancer therapy-The long stretch of DNA in eukaryotic
cells is packed very efficiently to fit into nucleus as a ·
result; the chromosomal DNA is twisted extensively.
Therefore selected regions of DNA need to become
sufficiently untangled and relaxed to allow vital nuclear transactions viz., transcription, replication,
chromosome strand separation, mitotic division, recombination ' and chromatin remodeling 9 • Topoi-
INDIAN J EXP BIOL, JULY 2004
650
somerases maintain the topology and regulate the dynamics of DNA structure by not only relaxing, unknotting, interlinking of strands, decatenation of
gapped or nicked DNA circles but also by performing
catenation and knotting to facilitate the condensation
and packing of DNA into higher order structure 10. In
addition, these enzymes fine-tune the steady-state
level of DNA supercoiling both to facilitate protein ·
interactions with DNA and to prevent excessive supercoiling that is deleterious I. Topoisomerases are
known that specifically relax only negative supercoils,
that relax supercoils of both signs or that introduce
either negative (bacterial DNA gyrase) or positive
supercoils into DNA (reverse gyrase) 1I. These enzymes accomplish this feat by either passing one
strand (type-I subfamily) or by passing a region of
duplex from the same or a different molecule through
a double strand gap generated in DNA (type-II subfamily) followed by religation of the strand break.
Several studies have demonstrated that topo I activity
is sensitive to physiological, environmental and
pharmacological DNA modifications and can act as a
specific mismatch and abasic site nicking enzyme l2 .
Recently, increased in vitro cleavage by topo II has
also been reported following the introduction of abasic sites into DNA substrates l3 , analogous to PARP,
which binds to single strands breaks and initiate DNA
repair l4 .
Topo II a level changes during the cell cycle
whereas, topo II ~ and topo I levels remain essentially
I I
3
TopoD
Topo I
Topo-I
"'110,,11"'"
similar throughout the cell cycle l5 ,16 and distributed
homogenously throughout the nucleus 17. More recent
studies have mapped the enzyme linked cleavable
complexes on the DNA loops bound to the nuclear
matrix at matrix attachment regions (MARs). Topo II
~ and topo I remained completely extractable
throughout the cell cycle, while scaffold associated
topo II a does not appear to be involved in catalytic
DNA turnover, although it may playa role in replication of centrioles, where it accumulates during M
phase l8 .
Levels of topoisomerases are generally elevated in
malignantly transformed (cancer) and actively growing cells 5,6 as compared to the non-malignant tissue l9 ,
which is partly attributed to the higher rate of DNA
replication, transcription and other cellular processes
in transformed cells for a higher rate of growth and
proliferation. Increased demand for a rate of cellular
proliferation increases the topo levels manifolds (up
to 200 folds), thereby allowing a differential sensitivity of tumors to drugs targeting topoisomerases as
compared to normal cells. Higher levels of topo pool
sizes has also been attributed to the over-expression
of topoisomerase gene as a result of mutation during
the process of transformation, although in most tumor
types it appears to be as a result of higher requirement
in proliferating tumor cells. However, topo pool sizes
vary among different types of tumors, albeit to a
lesser extent and this marked heterogeneity of enzyme
levels has been correlated with drug sensitivit/o-22 .
Topo-II
,cA_I~'[".·?·.·.]
,..........................·41•
......~~.~::.:~.~..]~
~ ---------~~
Fig. I-Action of topoisomerase I and II and their inhibition
DW ARAKANATH et al.: TOPOISOMERASE POISONS AS ANTICANCER AGENTS
Higher levels of topoisomerase being a selective and
differential marker of transformed cells make these
enzymes an attractive molecular target for the development of antineoplastic chemotherapy. Currently
several anti-tumor drugs like etoposide, doxorubicin,
teniposide and camptothecin specifically target topoisomerase enzyme2.
Types of topoisomerase inhibitors and development
of cytotoxicity--Topoisomerase inhibitors include
catalytic inhibitors of the enzyme and topoisomerase
poisons. Topoisomerase poisons exert their cytotoxic
effects by stabilizing the covalent complexes between
enzyme and DNA (cleavable complex). These compounds interfere in the religation step of the enzyme
catalysis, thereby, leaving the DNA strand breaks unligated4, lO. 23. The protein-DNA strand breaks thus created are not efficiently repaired and induce apoptosis24. On the other hand, the catalytic inhibitors inhibit
the enzyme catalysis activity by not allowing the enzyme to function itself and therefore do not allow topoisomerases to create strand break, Several compounds, membrone, suramin, bisdioxopiperazines
(ICRF), are known catalytic inhibitors of topoisomerase, while epipodophyllotoxins like etoposide (topo-II
inhibitor) and camptothecin (topo-I inhibitor) are well
known topoisomerase poisons 2. However, these compounds differ in the mechanism of their action as well
as the cytotoxicity, for example, ICRF-193 does not
have a significant cytotoxicity, while topoisomerase
poisons like etoposide and camptothecin (CPT) have
considerable amount of cytotoxicity. Not only the
concentrations of DNA-protein cross links, but their
life-time and stability also contribute to the cytotoxicity, Hypersensitive response to CPT induced by
Cockayne's syndrome has been correlated with the
increased life-time of double strand breaks (DSBs)
and cleavable complexes, predominantly at the site of
replication in nascent DNA 25 , Although, cleavable
complexes are formed in all phases of cell cycle, Sphase cells are found to be more sensitive particularly
to topo poisons26 . There is enough evidence that nascent DNA binds to the nuclear matrix is an important
site of cleavable complex formation, and these complexes inhibit DNA synthesis by blocking the movement of nascent DNA away from replication sites on
the nuclear matrix 27 . It is well established that topo
poisons mediated stabilized cleavable complexes collide with replication fork leading to the formation of
lethal double strand breaks and hence causes cytotoxi-
651
city28.30. On the other hand, collision of topo poisons
mediated stabilized cleavable complexes with transcription bubble leads to the generation of gapped
DNA fragments and hence the formation of single
strand breaks 3!,
Earlier studies have suggested the stabilization of
cleavable complexes as a prerequisite for cytotoxicity
of topoisomerase poisons with a direct correlation
between cleavable complexes and cytotoxicity, as
well as indirect evidences relating the rate of repair
and the formation of cleavable complexes in the damage response 32 . Many studies have revealed that the
damage by topo poisons is processed similar to frank
double strand breaks; but there are also evidences to
believe that DNA-Erotein cross-links are probably
handled differently 3. An unequivocal damage response pathway to topo poison induced cleavable
complexes has not emerged so far34 ,
DNA damage by UV irradiation and photoproducts
formation has shown an increase in the levels of to po
I-DNA complexes and p53 simultaneousll5. Although, the role of p53 in response to DNA damage
has been established, its interaction with endogenous
topo I has also been implicated in repair of damaged
genome. Recent studies have shown that although
MCF-7 cells with wild type p53 showed an elevation
in topo I-DNA complexes, p53 mutant SK-BR-3 cells
and null HL60 cells do not show such induction. A
physical interaction between p53 and topo I has also
been investigated and recruitment of topo I by p53 has
been proposed to cause an additional damage to genome, thereby committing cells to apoptosis 36.
Cellular responses to topo poisons-There are
enough evidences to believe that, while the stabilized
cleavable complexes can be repaired to a certain extent, moderate levels of the complex can mediate
other cellular responses like cell cycle arrest, induction of apoptosis, mitotic cell death and senescence
(Fig.2f' 8, 32. However, necrosis and non-specific cytotoxicity are observed at higher doses of topo poisons37 . Indirect evidences have shown that cleavable
complexes can be repaired by non-homologous end
joining (NHEJ) repair pathway38, while the role of
homologous recombination is equivocal. Cells deficient in NHEJ repair pathway (Ku deficient) are more
sensitive to topo poisons, which is correlated with
higher amount of cleavable complexes. Cells deficient
in double strand break (DSB) repair (ATM deficient)
also sensitize them to topo poisons39 . Inhibition of
patch strand repair also increases the cytotoxicity of
INDIAN J EXP BIOL, JULY 2004
652
topo poisons. The induced expression of DNA damage responsive genes DIN3 and RNR3 in TOP 1+
camptothecin treated yeast cells40 and deletion of
RAD52 gene, which is required for recombinational
repair of DSB, enhances cell sensitivity to camptothecin. The protein linked DSB doubled when incubated
with 3-aminobenzamide (inhibitor of poly ADP ribose
polymerase) in addition to camptothecin41 , shows that
protein concealed strand breaks can be lethal lesions
and intracellular activity of topoisomerase I and II
may be regulated coordinately through poly ADP ribosylation.
Ubiquitin (El enzyme)/26S proteasome-dependent
degradation of cleavable complexes has been
suggested to be a unique repair process for the repair
of topo I mediated DNA damage leading to its down
regulation 42 . Yong Mao et al. have shown that
treatment of mammalian or yeast cells expressing
human DNA topo I with camptothecin induces
covalent modification of topo I by SUMO-lISmt3p, a
ubiquitin like protein. Since, Ubc9 mutant yeast cells
expressing hu~an DNA topo I are hypersensitive to
CPT, it is suggested that UBC9/SUMOl may be
involved in the repair of topo I mediated DNA
damage 35 . The trimeric topo I-CPT-DNA cleavable
complex seems to be the signal for SUMO I
conjugation with topo I, which is supported by the
observation that majority of SUMO-l conjugated topo
1 is covalently linked to DNA 42 and mutant cell lines
(CPT-K5 and U-937/CR) are defective in CPT
induced SUMO-l conjugation to topo 143. Further, a
ubiquitin mutant with substitution at Lys-63 appears
Cleavable complex
~
~----{
??
Ubiquitination
SUMO
Frank DNA breaks
Inhibition of apoptosis
Fig. 2-:-Schematic diagram showing cellular responses to
cleavable complexes stabilized by topo poisons
to be deficient in DNA repair RAD6 pathway and also
defective in its polymerization44 .
Brief exposure to topo poisons at moderate concentrations mediate a higher mitotic cell death in synchronized M phase cultures, leading to the formation
of micronuclei, which correlate well with the amount
of cleavable complexes 45 • In asynchronous cell culture most of the topoisomerase inhibitors arrest the
cell at G2/M cell cycle checkpoint, possibly facilitating the repair of cleavable complexes46 • G2/M cell
cycle arrest primarily arises because of alteration in
tyrosine dephosphorylation of p34cdc2 and cyclin B I
leading to inactivation of cyclin B/p34cdC2 complex47 .
Short exposure to topo poisons initiate ATM responsive p53 dependent, p21 and GAAD-45 mediated cell
cycle arrest48 . However, DNA synthesis is required at
the time of topo inhibitors treatment, since these cell
cycle dependent changes are not observed when incubated simultaneous with aphidicolin (DNA synthesis
inhibitor). Cleavable complex has also been found to
induce cellular proliferation through induction of excision repair enzymes or otherwise p53/p21 mediated
Bax synthesis and its translocation to the mitochondria resulting in membrane permeability transition
leading to cytochrome c release culminating in apoptosis 48 .
Topo poisons can also mediate senescence, which
correlates to a cytostatic but not quiescent cell population resting in cell cycle blocks 8 . Induction of p53
dependent expression of p21, which appears to be
necessary for cell cycle arrest, can also induce senescence8 • These studies further substantiated that the
loss of clonogenicity of cells does not necessarily result from loss of cell viability.
DNA degradation during etoposide induced apoptosis results in formation of high molecular weight
DNA fragments, but not oligonucleosomal DNA
fragments 49 , which is accompanied by downregulation of caspase-activated DNase (CAD). Further it is not affected by z-VAD-fmk (caspase inhibitor), suggesting that caspase is not involved in the
excision of DNA loop domains. It appears that topo II
is involved in caspase-independent excision of DNA
loop domains during apoptosis, and represents an alternative pathway of apoptotic DNA disintegration
different from CAD driven caspase-dependent oligonucleosomal DNA cleavage. Furthermore, there is
evidence that several oncogenes including ras50 ,
myb51 and p53 52 interact with sequences within the
topo II a promoter.
DWARAKANATH et at. : TOPOISOMERASE POISONS AS ANTICANCER AGENTS
Pre-clinical studies-Pre-clinical studies in different solid tumors have established etoposide as a primary treatment modality in lung tumors and leukemia
especially in Hodgkin's and non-Hodgkin's Lymphoma. Complete response in 12 % of small cell lung
tumors, significant response in acute amyloid leukemia and Hodgkins lymphoma has also been observed 53 . A overall response rate (14%) has been observed in combination with cisplatinum. Studies with
human xenografts have shown an unprecedented efficacy for 9-aminocamptothecin with complete remissions in colon 54, breast and non small cell lung carcinoma as well as in melanoma cell lines 55.
Clinical studies-A clear association between topo
II a and tumor cell proliferation has been established
in many of the human tumors 56 . Higher levels of expression qf to po I has been observed in colon, ovary,
prostate, lung and colorectal tumors as compared to
the levels in the corresponding normal tissues 57 -60 .
Clinical studies with topo II inhibitor, etoposide have
reported good responses primarily in patients with
Ewing's sarcoma, Hodgkin's disease, neuroblastoma,
rhabdomyosarcoma, small cell lung cancer, testicular
cancer, gestational choriocarcinoma, acute myelogenous leukemia and Kaposi's sarcoma, besides a spectrum of recurrent malignant solid tumors 61 • A composite single agent response rate of greater than 20%
has been observed in some of these tumors 62 . Topo
inhibitors have also been used as one of the primary
drugs among multi-drug therapy in lung, testicular
cancer and leukemias. Combination therapy with cyclophosphamide and vincristine in recurrent and refractory pediatric tumors have shown 95% responses
with 35% complete responses, 40% very good partial
responses and 20% partial responses 63 . Similarly,
86-95% responses have been observed in lung cancer
in combination with cisplatin 64 .
Approaches for overcoming limitations-Therapeutic efficacy of topoisomerase inhibitors is limited
primarily by the resistance of tumors at lower doses
and toxicity to the normal tissues at moderate to
653
higher doses. Among several factors that contribute to
the resistance of human tumors is decrease in intracellular drug availability due to multiple drug resistance (MDR) gene product, P-glycoprotein (Pgp) mediated drug efflux process appears to be common to
many drugs, although camptothecin, a topo I inhibitor
seems to be an exception65 . Besides drug efflux, alterations or mutations in drug' s cellular target or
binding sites 66 , distribution of cleavage sites, increase
in efficient repair of drug-induced damage (cleavable
complexes) and cross resistance to other structurally
and mechanistically related drugs also contribute to
the drug resistance. Lower levels of topoisomerases in
slow growing tumors and reduction of its expression
due to hypermethylation have been reported 67 .
Ubiquitin mediated repair and mutations in critical
proapoptotic and antiapoptotic regulatory proteins like
p53 and Bch family or alterations in cell cycle machinery, which activate checkpoints and prevent initiation of apoptosis can also be the cause of resistance68 . Coordinately regulated detoxifying systems,
such as DNA repair, glutathione S-transferase and
cytochrome P450 mixed-function oxidases can also
confer resistance to drugs 69 . Resistance can also result
from defective apoptotic pathways. A brief summary
of the factors that limit topo inhibitors as effective
anti-cancer drugs and suggested approaches to overcome them is presented in Table 1.
Some of the topo II poisons induce acute and delayed toxicities in normal tissue that include bone
marrow suppression, mild nausea or vomiting and
hair loss with myelo-suppression being the dose limiting toxicity. Mucosities, liver toxicity, fever and
chills are also observed with high dose regimens 2 .
Although, most of the side effects occur within 1-10
davs of drug administration, delayed induction of secondary tumors mainly in the form of leukemias have
been observed70 • De novo translocations leading to
fusion proteins involving topo I and MLL break point
71
cluster regions and transcriptional activation of short
interspersed elements (SINES, like Alu elements) by
Table I-Factors limiting the efficacy of topoisomerase poisons and approaches to overcome them.
Factors
Drug retention and localization
Drug target interaction
Cleavable complex formation
Repair of cleavable complex
Initiation of cellular response:
Cell cycle perturbations. Mitotic death.
Apoptosis
Limitations
Pgp mediated drug efflux
Chromatin structure
Inadequate enzyme levels
Efficient repair systems
Mutation in functional genes
Approaches
Enhance drug retention
Induction of alteration in chromatin condensation
Over- expression of topoisomerase genes
Inhibition of repair
Modifiers of cellular responses:
Cell cycle progression. Apoptosis
654
INDIAN J EXP BIOL, JULY 2004
topo II poisons have recently been implicated for induction of secondary malignancies 72.
There has been a constant effort for the development of newer class of drugs with higher sequence
specificit/ 3 . Many different analogues of camptothecin CPT-II have been evaluated in pre-clinical studies and are under clinical trials 74. Strategies using
MDR inhibitors to enhance intracellular drug retention 75 and agents that leads to induction of topo II levels ego Hoechst-33342 etc are under investigations 76 .
Combinations of to po inhibitors with NF-kB inhibitors or GM-CSF to reduce myelo-suppression and
related toxicities are currently under investigations 77 .
Combinations of cyclophosphamide with etoposide
and vincristine for pediatric solid tumors have been in
phase IIII clinical trial 78 • Use of topo poisons also potentiates the radioimmunotherapy of tumors 79 . Combination of 'proteasome inhibitors PS-341 with topo
poisons has also enhanced the efficacy of these drugs
with little or no normal tissue toxicity in vitro are currently under clinical trials 8o . Etoposide, ifosamide and
cisplatin combination therapy has been in clinical use
for refractory childhood solid tumors 81.
Energy-linked modification of cellular responses to
topo poisons using the glycolytic inhibitor 2-deoxy-Dglucose-The repair of cleavable complex, the primary lesions responsible for cytotoxicity as well as
the drug retention linked to Pgp pump mediated efflux
process are both known to be energy dependent processes. Since cancer cells derive a major portion of the
energy (A TP) through glycolysis, it has been suggested that inhibitors of glycolysis, like 2-deoxy-Dglucose (2-DG) may selectively enhance the cytotoxicity of topo poison in cancer cells.
Studies carried out in human glioma (BMG-l & U87) and squamous carcinoma (4197 & 4451) cell lines
have shown that the cytotoxicity of etoposide, a topo
II inhibitor, camptothecin, a topo I inhibitor and bisbenzimidazole derivative, Hoechst-33342 (H-342), an
AT specific minor groove binding DNA ligand that
inhibits both topo I and topo II can be significantly
enhanced by 2-DG under certain conditions 82 .
Presence of 2-DG (5 mM, equimolar to glucose
concentration in the media) for 2-4 hr following exposure to the topo inhibitors enhanced the cell death by
a factor of nearly 2 with all the three drugs (Fig.3),
implying that 2-DG (in fact the metabolic stress created by 2-DG) facilitates certain common processes
that are responsible for cytotoxicity of topo inhibitors.
These include enhanced drug retention, inhibition of
repair of cleavable complexes, manifestation of cytogenetic damage and up-regulation of damage dependent apoptosis as wen as alterations in damage related
cell cycle delays.
Interestingly, when 2-DG has been added along
with the topo inhibitors, a small but significant decrease in the cell death is noticed in case of etoposide,
while no effect is observed with camptothecin (Fig.3).
Since functioning of topo II is ATP dependent, these
observations suggest that 2-DG, induced fall in energy status (ATP), may reduce the formation of
cleavable complexes if the two are added simultaneously or 2-DG before etoposide. Reduced etoposide
toxicity in glucose regulated protein (GRP) induced
cells following long exposure to 2-DG (4-6 hr) has
also been reported 83 . Irrespective of the mechanisms
involved in reduced toxicity of etoposide by 2-DG,
our results as well as earlier findings suggest that the
efficacy of topo II inhibitors can be enhanced by 2DG, only when 2-DG is added (administered) after
the cells are treated with the inhibitor, i.e. when significant amounts of cleavable complexes have been
formed.
Analysis of the DNA damage using the nuclear
halo assal4 revealed that under these conditions the
presence of 2-DG significantly enhanced the halo diameters of BMG-l cells implying a higher level of
DNA strand breaks under these conditions (Fig.4).
Although, the mechanisms involved in the repair of
cleavable complex are not completely understood,
cells defective in double strand break repair are found
to be more sensitive to topo II inhibitors 85,80.
c:: 0.8
o·
.~
<b 0.6
gp
.;; 0.4
.~
::s
r.n 0.2
None
EI (10 uM)
H-342 (5 uM)
Ca (0.1 uM)
Treatment
Fig. 3--Effects of 2-DG (5mM) on the cytotoxicity of topoisomerase inhibitors etoposide, camptothecin and Hoechst-33342
in exponentially growing a human glioma cell line (BMG-l).
DWARAKANATH ef at.: TOPOISOMERASE POISONS AS ANTICANCER AGENTS
More recently, ubiquitination mediated protein (topoisomerases) degradation resulting in the formation
of frank DNA strand breaks, facilitating the completion of repair by strand break repair pathways has
been suggested 35. 43. 45. Therefore, it appears that 2-DG
does not interfere in the conversion of cleavable complex into frank strand breaks, while it clearly inhibits
the repair of strand breaks. Inhibition of DNA double
strand break repair and excision repair following UV
as well as ionizing radiation by 2-DG has been reported earlier87-92 •
. Studies carried out to investigate the role of cytogenetic damage clearly show that, 2-DG enhances the
topo inhibitor induced micronuclei formation by 40%
and 60% respectively with etoposide and H-342,
while a 20% increase has been observed with camptothecin (Fig.S b,d). These observations are similar to
the earlier results on the enhancement of radiationinduced micronuclei formation in tumor cell lines as
well as short-term organ cultures of tumors88.89.92-95.
Modifications of the topo inhibitor induced cell death
and micronuclei formation by 2-DG observed here
(Fig.Sd) imply that enhanced mitotic death is partly
655
responsible for chemosensitization by 2-DG in these
cells. Interestingly, it has also been found that 2-DG
enhances the topo inhibitor induced delayed apoptosis, particularly in case of topo II inhibitor etoposide
(Fig.S c,e). However, no significant induction of early
apoptosis can be observed under these conditions,
implying thereby that the enhanced cytotoxicity is
primarily due to an increase in the mitotic death.
Further investigations have also shown that 2-DG
significantly enhanced the cell cycle delay induced by
all the three topo inhibitors. Since the extent of cell
cycle delay arising on account of the functioning
check points 47 is related to the level of DNA damage,
the additional delay observed also indicates the presence of a higher level of DNA damage in cells treated
with 2-DG following exposure to topo inhibitors.
Taken together, the available evidences so far indicate that the presence of 2-DG for a few hours following exposure to topoisomerase inhibitors can significantly enhance their cytotoxicity, by inhibiting the
reversibility of cleavable complexes, the primary lesions responsible for the cell death. A higher level of
DNA strand breaks accumulated under these condi-
a]
~
Core diameter ().tm)
l-----~
Halo diameter (11m)
; ~ ~-='c '~ ~t~~-I- --=-:::~";;;E:t ~ E ~2 ~iJ I
[b]
30 J
.
I
fJ'J
o 25 -'
~
20
o 15
...c::
4-<
o
Z
10
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o
,,
-t--fi-r--(1<"...,o-,r-n-,--G-r-IIFT-.......,.-III--r----T"I:l-r-o.. !
o
4
8
12 16 20 24 28 32 36 40
Halo diameter ().tM)
Fig. 4-Effect of 2-DG (SmM, 2hr) on etoposide induced DNA damage studied by nuclear halo assay in a human glioma cell line
(BMG-l). [a] Photo-micrographs of nuclear halo [b] Frequency distribution of halo diameters following various treatments.
INDIAN J EXP BIOL, JULY 2004
656
60r-------------r=======~
25
a
~
(;'
c
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b
50
40
:::J
CT
&
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30
en
U
.~
o
C
0.
~
~
:::J
o
o
Etoposide
H-342
Camptothccin
20
10
Etoposide
H-342
Carnptothcc in
Fig 5-Photomicrographs of DAPI stained BMG-l cells showing treatment-induced micronucleus (arrowhead) and apoptosis (arrow). [aj
Control; [bj Etoposide (10 /-lM); [c] Etoposide (10 /-lM) 2-DG (5 mM) in a human glioma cell line (BMG-J). Enhancement of etoposide
induced micronuclei formation [dj and apoptosis [e] by 2-DG observed at 48hr after treatment are also shown.
tions and the significant increase in micronuclei formation lends support to this proposition. However,
maintenance of higher intracellular levels of topo inhibitors, due to the inhibition of energy dependent,
Pgp mediated process responsible for the drug efflux 65
can also contribute to the enhanced cytotoxicity _ Preclinical studies in tumor bearing animals to investigate the effects of the combined treatment (topo inhibitors + 2-DG) on tumor control as well as systemic
toxicity responsible for the therapeutic efficacy are
warranted before contemplating clinical trials. Preliminary studies in Ehrlich ascites tumor bearing mice
have indeed shown that a combination of etoposide
and 2-DG could be more effective than etoposide
alone in achieving the tumor control96, 97.
Future perspectives
Considerable progress has been made in the last
decade towards the understanding of molecular and
cellular mechanisms underlying the cytotoxicity of
topoisomerase inhibitors, as well as natural and acquired tumor cell resistance to these drugs. These advancements should facilitate the design of new topo
poisons as well as adjuvant biological response modi-
fiers that selectively enhance the sensitivity of neoplastic cells. One of the challenges for the future is to
develop predictive assays and to individualize therapy
to benefit patients from the tumor specific therapeutic
regimens. Further, development of appropriate in vitro and in vivo models to predict major toxicity is expected to facilitate the development of agents and
strategies for ameliorating treatment induced toxicity
with topo poisons.
In conclusion, topoisomerase inhibitors form an
important class of anticancer drugs. They could be
expected to play a greater role in the future, when
limitations associated with their use are overcome by
some of the approaches outlined here.
Acknowledgement
We are grateful to Gen T Ravindranath, Director
INMAS for his keen interest and constant encouragement in this work. We thank Dr Seema Gupta and Dr
Shailja Singh for helpful discussions during the
prepration of this manuscript. These studies have been
supported by DRDO project (INM-280), Ministry of
Defence, Govt. of India_Mr. Rohit Mathur is thankful
to CSIR, New Delhi for awarding JRF.
DW ARAKANATH et al.: TOPOISOMERASE POISONS AS ANTICANCER AGENTS
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