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 Mathurl•

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 ac­tivitlA (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, E-mail-bsd@inllias.org

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 cytotoxicity7.8. 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 remodeling9

• Topoi-

650 INDIAN J EXP BIOL, JULY 2004

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) 1 I. 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 Topo I I Topo-II

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 cells5

,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

.

TopoD

3 Topo-I "'110,,11"'" ,cA_I~'[".·?·.·.]

, .......................... ·41 • ...... ~~.~::.:~.~ .. ]~

~ ---------~~

Fig. I-Action of topoisomerase I and II and their inhibition

DW ARAKANATH et al.: TOPOISOMERASE POISONS AS ANTICANCER AGENTS 651

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 DNA25, 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 matrix27. 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-

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 breaks3

!,

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 response32. 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 apoptosis36.

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

652 INDIAN J EXP BIOL, JULY 2004

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 regulation42

. 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 damage35

. 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

~ ??

~----{ Frank DNA breaks

Ubiquitination SUMO

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 complexes45

• 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 apop­tosis48

.

Topo poisons can also mediate senescence, which correlates to a cytostatic but not quiescent cell popu­lation resting in cell cycle blocks8

. 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 fragments49

, 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 p5352 interact with sequences within the topo II a promoter.

DWARAKANATH et at. : TOPOISOMERASE POISONS AS ANTICANCER AGENTS 653

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 ob­served53

. 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 colon54

, 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 tumors56

. 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 tissues57

-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 tumors61

• A com­posite single agent response rate of greater than 20% has been observed in some of these tumors62

. 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 responses63

. Similarly, 86-95% responses have been observed in lung cancer in combination with cisplatin64

.

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

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 sites66

, 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 reported67

.

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 drugs69

. 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 regimens2

.

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 cluster regions71 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 Limitations Approaches

Enhance drug retention Drug retention and localization Drug target interaction Cleavable complex formation Repair of cleavable complex Initiation of cellular response: Cell cycle perturbations. Mitotic death. Apoptosis

Pgp mediated drug efflux Chromatin structure Inadequate enzyme levels Efficient repair systems Mutation in functional genes

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 malignancies72.

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 investigations76

.

Combinations of to po inhibitors with NF-kB inhibi­tors or GM-CSF to reduce myelo-suppression and related toxicities are currently under investigations77

.

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 tumors79

. 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 trials8o. Etoposide, ifosamide and cisplatin combination therapy has been in clinical use for refractory childhood solid tumors81

.

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 & U-87) 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 conditions82

.

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 A TP dependent, these observations suggest that 2-DG, induced fall in en­ergy status (A TP), 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 reported83

. 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 2-DG, 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 655

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 suggested35. 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

a]

~ Core diameter ().tm)

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 points47 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-

l-----~ Halo diameter (11m)

[b] ; ~ ~-='c '~~t~~-I- --=-:::~";; ;E:t ~ E ~2 ~iJ I 30 J .

fJ'J I o 25 -' ~ ...c:: 20 4-< o 15 o Z 10

5 - , , 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.

656 INDIAN J EXP BIOL, JULY 2004

'Q) U :::J C o o ~

25 a

Etoposide H-342 Camptothccin

60r-------------r=======~

~ 50

(;' c 40 III :::J CT & 30

en .~ 20

0. o 10 ~

b

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 efflux65

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 657

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