Betulinic Acid, a Catalytic Inhibitor of Topoisomerase I, Inhibits Reactive Oxygen Species–Mediated Apoptotic Topoisomerase I–DNA Cleavable Complex Formation in Prostate Cancer Cells but Does Not Affect the Process of Cell Death

DNA topoisomerase I is an ubiquitious and essential enzyme that relaxes DNA supercoiling inside cells during progress of several vital cellular processes, like replication, recombination, and transcription. The mechanism by which this enzyme alters the DNA topology involves three major steps: (a) nucleophilic attack by the hydroxyl group of the active site tyrosine on the scissile phosphate resulting in covalent attachment of enzyme to the 3′ end of the broken strand, (b) a topoisomerization step involving strand passage or free rotation, and (c) religation of the DNA strand and release of the enzyme (1, 2).

Under normal conditions, the topoisomerase I–DNA covalent complexes are transient and are found in very low levels because the religation rate is faster than the cleavage step. Stabilization of this complex (called cleavable complex) generates DNA lesions, thereby initiating cellular responses that induce cell cycle arrest and apoptosis (3).

Stabilization of topoisomerase I–DNA cleavable complexes can occur by two distinct mechanisms (4). First, these transient complexes can be trapped by specific inhibitors, like camptothecin, and its derivatives, which can bind specifically with the topoisomerase I–DNA intermediate complexes and prevent the religation step (5–7). The second mechanism does not involve any inhibitor directly, rather it is related to the oxidative DNA lesions and frequent DNA modifications. These modifications include oxidized bases (e.g., 8-oxoguanosine), abasic sites, mismatches, and strand breaks (8). Whereas the first mechanism is highly specific for the inhibitors like camptothecin, the second process involves different cellular events induced by agents which causes ROS generation and thereby subsequent DNA damage.

Recently, stabilization of topoisomerase I–DNA cleavable complexes was observed in various human cells, including leukemia and carcinoma cells undergoing apoptosis. Pommier and coworkers have established that topoisomerase I–DNA cleavable complexes are formed in cells exposed to staurosporine (9), arsenic trioxide (10), etoposide (11), and vinblastin (11), which are mechanistically different inducers of apoptosis. All these compounds are inactive on purified topoisomerase I in vitro. Whereas staurosporine is a protein kinase C inhibitor, vinblastin is an inhibitor of tubulin. Etoposide specifically inhibits topoisomerase II and has no direct effects on topoisomerase I. All these compounds results in generation of reactive oxygen species (ROS) inside cells that induce oxidative DNA damages, which in turn favors topoisomerase I–DNA cleavable complex stabilization. Thus, formation of cellular topoisomerase I–DNA cleavable complexes by these compounds do not result from their direct interaction with the topoisomerase I–DNA intermediates. Recently, Sen et al. also reported conservation of the apoptotic topoisomerase I–DNA cleavable complex formation by a protein kinase inhibitor, withaferin A, in parasite Leishmania donovani (12). Thus, topoisomerase I–DNA cleavable complexes are implicated directly or indirectly in apoptosis, irrespective of the initiating mechanism or the agent.

Betulinic acid, a pentacyclic triterpenoid (Fig. 1A) from Bacopa monniera has been shown to be a potent inhibitor of eukaryotic topoisomerase I and topoisomerase II (13). The compound is a catalytic inhibitor of the enzymes. Betulinic acid was reported to inhibit human topoisomerase I and rat liver topoisomerase I by interacting with the enzyme directly (14). The drug is shown to bind with the enzyme and thereby preclude the interaction with substrate DNA. Being a catalytic inhibitor, betulinic acid does not induce topoisomerase I–DNA cleavable complexes in vitro and in vivo. Most interestingly, the drug was found to abrogate camptothecin-mediated cleavable complex formation in mouse splenocytes. Dihydrobetulinic acid, a derivative of betulinic acid, was found to have similar effects on the enzyme from parasite L. donovani (15).

In the present study, we have analyzed the effects of betulinic acid on various types of topoisomerase I–DNA cleavable complexes that are formed inside cells during apoptosis induced by different agents. Betulinic acid is found to inhibit topoisomerase I–DNA cleavable complex formation both in vitro and in vivo. In this paper, we show that this inhibition is extendable for any type of topoisomerase I–DNA cleavable complex, either induced by camptothecin or by ROS-mediated DNA damage induced by agents like staurosporine. Betulinic acid has no effect on preformed cleavable complexes as evidenced by DNA cleavage assays. But pretreatment with betulinic acid abolishes all types of topoisomerase I–DNA cleavable complex formation. Interestingly, betulinic acid treatment in DU145 prostate cancer cells induces ROS formation and subsequent DNA damages but precludes formation of topoisomerase I–DNA cleavable complex. Cleavage reactions with oligonucleotide containing 8-oxoguanosine modifications provide evidence that betulinic acid precludes topoisomerase I from interacting with oxidatively damaged DNA. Moreover, down-regulation of topoisomerse I in DU145 cancer cells cannot substantially reduce betulinic acid–induced apoptotic process. Thus, our study proposes that, betulinic acid, although it targets topoisomerase I, sequesters the enzyme in the nucleoplasm so that the enzyme cannot be recruited for the formation of apoptotic topoisomerase I–DNA cleavable complexes.

Cell culture and drugs. DU145 prostate cancer cells were cultured in RPMI medium (Sigma) supplemented with 10% fetal bovine serum (Life Technologies) in 37°C CO₂ incubator. The cells were received as gift from Prof. Yves Pommier (National Cancer Institute, NIH).

Betulinic acid was isolated from leaves of B. monniera after the procedure described (14), characterized by spectral data (UV, ¹H nuclear magnetic resonance (NMR), ¹³C NMR, mass spectrometry), and compared with authentic samples.

Cell survival assay. DU145 cells were seeded in 96-well plates and treated with respective drugs. After 48-h treatment, cell survival was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, cells were washed and treated with MTT for 4 h at 37°C, and reactions were stopped with stop solution containing isopropanol and HCl. Plates were analyzed on Thermo MULTISKAN EX plate reader at 575 nm.

Purification of recombinant human topoisomerase I. The wild-type human topoisomerase I (91 kDa) was purified from Sf-9 insect cells infected with the recombinant baculovirus (a kind gift from Prof. J.J. Champoux). Approximately, 1 × 10⁹ Sf-9 cells were infected with the recombinant virus, and cells were harvested after 48-h infection. The cells were lysed and enzyme was purified as described (16).

DNA cleavage activity. Approximately 0.5 pmol of 5′ end labeled 25-mer or the 22-mer (containing 8-oxoguanosine) duplex oligonucleotides were incubated with ∼20 ng of recombinant human topoisomerase I or nuclear extracts in presence or absence of camptothecin, staurosporine, or betulinic acid as mentioned. The 22 mer oligonucleotide containing 8-oxoguanosine was received as a gift from Prof. Y. Pommier (NIH). Nuclear extracts from DU145 cells were prepared as described (17). The reactions containing 10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L KCl, 5 mmol/L MgCl₂, 0.1 mmol/L EDTA, and 15 μg/mL bovine serum albumin were incubated at 30°C for 30 min. Reactions were stopped with 0.5% SDS and loading buffer [80% formamide, 1 nmol/L sodium EDTA, 10 mmol/L sodium hydroxide, and 0.1% bromophenol blue (pH 8.0)]. Samples were denatured by heat-chill and separated by 20% denaturing PAGE (containing 7 mol/L urea) in 1× Tris-borate EDTA buffer and visualized by autoradiography.

In vivo cleavage activity. The in vivo formation of topoisomerase I–DNA cleavable complex was quantitated using the SDS precipitation assay as described (14, 18). Briefly, DNA in DU145 cells (2 × 107 cells per milliliter) was labeled by adding [methyl-³H]thymidine (specific activity, 83 Ci/mmol) into the medium to a final concentration of 5 μCi/mL for 18 h. Cells were pelleted and washed twice with PBS (137 mmol/L NaCl, 2.6 mmol/L KCl, 8.0 mmol/L Na₂HPO₄, 1.4 mmol/L KH₂PO₄), resuspended in complete medium, and distributed into 96-well plates. After incubation at 37°C for 2 h, cells were treated with betulinic acid and/or staurosporine, etoposide, or camptothecin for varying time points. Cells were pelleted and lysed by the addition of 200 μL of a prewarmed (65°C) lysis solution [1.25% SDS, 5 mmol/L EDTA (pH 8), calf-thymus DNA (0.4 mg/mL)]. Samples were cooled in ice for 10 min and centrifuged. The pellets were resuspended in 500 μL wash solution [10 mmol/L Tris-HCl (pH 8), 100 mmol/L KCl, 1 mmol/L EDTA, calf-thymus DNA (0.1 mg/mL)] and warmed at 65°C for 10 min with occasional shaking. The suspensions were cooled in ice for 10 min and recentrifuged. The pellets were washed again before resuspending in 200 μL H₂O prewarmed at 65°C. The suspensions were then mixed with 4 mL scintillation liquifluor, and radioactivity was determined in liquid scintillation counter.

Immunoband depletion assay. DU145 cells were cultured in six-well plates with or without appropriate drugs. Nuclear extracts were prepared as described (12, 19). Briefly, cells were suspended in hypotonic buffer [10 mmol/L Tris-HCl (pH 7.5), 0.1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine hydrochloride, and 5 mmol/L DTT] and homogenized. The homogenate was centrifuged at 10,000×g for 10 min. The pellets were washed and were used as the source of nuclei. It was then lysed by 1% SDS, samples were subjected to SDS-PAGE (8%), and proteins that entered the gel were transferred to nitrocellulose membranes and immunoblotted with polyclonal antibody against topoisomerase I or topoisomerase II (Santa Cruz Biotech).

DNA fragmentation assay for detection of apoptosis. Cells were cultured in 24-well plates and treated with drugs for different times at different drug concentrations. Samples were collected at requisite time points and subjected to measurement of DNA fragmentations by detecting the cytoplasmic histone-associated DNA fragments (mononucleosome and oligonucleosomes) formed during apoptosis using a cell death detection ELISA kit (Roche Biochemicals) according to the manufacturer's protocol. DNA fragmentation was detected by spectrophotometric measurement of microtiter plates in a Thermo MULTISKAN EX plate reader at 405 nm, and relative percentages (with respect to samples treated with micrococcal nuclease and normalized to percentage values) were plotted as functions of time or drug concentrations.

Caspase activity assay. DU145 cells were treated with drugs for different time points, and caspase-3 activity was quantitated using ApoAlert colorimetric assay kit according to manufacturer's protocol. Briefly, cells were centrifuged at 400×g for 5 min, resuspended in lysis buffer, and incubated on ice for another 10 min. Cells were recentrifuged at 15,000×g for 10 min at 4°C, and supernatants were used to test caspase-3 cleavage activity. DEVD-pNA was used as a substrate, and pNA release was measured on a Thermo MULTISKAN EX plate reader at 405 nm.

Detection of ROS. Intracellular ROS was measured in drug-treated and untreated cells as described (20). Briefly, cells were washed and resuspended in 500 μL of 1× PBS and were loaded with 2 μg/mL of H₂DCFDA (Molecular Probes) for 30 min, and green fluorescence of 2,7-dichlorofluorescein was measured at 515 nm by spectroflorimeter.

Detection of oxidative DNA damage. After drug treatment, DU145 cells were permeabilized in hypotonic buffer [10 mmol/L Tris-HCl (pH 7.8), 70 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L DTT] containing 0.05% Triton X-100 at 4°C for 15 min by gentle mixing. formamidopyridine DNA glycosylase (Fpg; 1 mg/mL) was added and further incubated for 30 min at 25°C. DNA breaks were analyzed by alkaline comet assay with some modifications (12, 21). Briefly 5,000 to 10,000 cells were mixed with 100 mL of 0.75% low-melting agarose and kept at 37°C. The agarose cell suspension was spread on polylysine-coated coverslips.

The preparations were left on a chilled plate for 5 min before lysis (0.03 mol/L NaOH, 1 mol/L NaCl, 2 mmol/L EDTA, 0.5% N-lauryl sarkosyl) for 1.5 h and thereafter equilibrated (0.03 mol/L NaOH, 2 mmol/L EDTA) for 1 h. Electrophoresis of the agarose-imbedded cells was run at 0.67 V/cm for 20 min in the same solution. The agarose gel was neutralized in 0.4 mol/L Tris-HCl (pH 7.5). Cells were then stained with EtBr. Analysis of the DNA that migrated from the nuclei, the tail moment, was carried out using an Olympus fluorescence microscope, and data was analyzed using comet IV software.

TUNEL assay. To assess the extent of apoptotic DNA damage, treated and untreated DU145 cells were fixed with 2% paraformaldehyde and incubated with 0.2% Triton X-100 for 5 min for permeabilization and layered with TdT reaction mixture containing FITC-labeled dUTP for 1 h at 37°C according to the manufacturer's protocol (Apo Alert DNA fragmentation assay kit). Cells were stained with propidium iodide and visualized with Leica DMI 4000 B fluorescence microscope.

Small interfering RNA transfection. Gene silencing was achieved by transfecting small interfering RNA (siRNA) into DU145 cells. Control siRNA and siRNA for topoisomerase I were purchased from Santa Cruz Biotech. Transfections were carried out using Lipofectamine 2000 following manufacturer's protocol. Gene silencing was confirmed by Western blot analysis.

Betulinic acid inhibits camptothecin-induced topoisomerase I–DNA cleavable complex formation in vitro and in vivo. Betulinic acid, which is a catalytic inhibitor of topoisomerase I, has been shown to inhibit the formation of topoisomerase I–DNA cleavable complex in cleavage reactions with plasmid DNA in vitro. To confirm that betulinic acid directly inhibits the cleavage step of topoisomerase reaction, we have performed cleavage reactions with 25 mer duplex oligonucleotide in presence of betulinic acid and/or camptothecin. Each reaction contained 0.5 pmol of 5′ end–labeled substrate DNA, 20 ng of recombinant human topoisomerase I, and varying concentrations of drugs. In presence of camptothecin, there is substantial amount of cleavage product, which is inhibited to the extent of 90% in presence of betulinic acid. Moreover, pretreatment with betulinic acid before addition of camptothecin markedly reduced camptothecin-mediated cleavage (Fig. 1B , lanes 7 and 8). Thus, betulinic acid can interact with the enzyme and preclude the trapping of cleavable complex by camptothecin. But when camptothecin is added before betulinic acid in the reaction, betulinic acid cannot inhibit the formation of cleavable complex in vitro. This finding allows us to speculate that betulinic acid has no effect on preformed topoisomerase I–DNA cleavable complexes.

Camptothecin-mediated apoptosis invariably involves trapping of topoisomerase I–DNA cleavable complexes inside cells. The amount of this trapped cleavable complex can be measured by incorporation of [³H]thymidine and subsequent SDS-K⁺ precipitation of the trapped complex. This experiment provides direct and quantitative measurements of the cleavable complex formed in vitro. To understand whether the precipitated count is actually due to topoisomerase I–linked complexes, we performed immunoband depletion assay (12, 18). Nuclear extracts were prepared from untreated and treated cells and subjected to SDS-PAGE. If topoisomerase I can form a covalent complex with the genomic DNA inside the cells, the complex cannot enter the gel. On the other hand, if there is no complex formation, the free protein will enter the gel. The proteins were transferred to nitrocellulose membranes, and free topoisomerase in gel was detected by Western blotting with polyclonal topoisomerase I antibody as described in Materials and Methods. The IC₅₀ values for camptothecin and betulinic acid were 0.4 and 6 μmol/L, respectively, for 48 h of drug treatment as determined by MTT assay (Table 1). Respectively, 10 μmol/L and 20 μmol/L of camptothecin and betulinic acid were used to ensure accumulation of detectable amounts of topoisomerase I–DNA cleavable complexes and subsequent DNA fragmentation within 4 to 6 h of drug treatment. As shown in Fig. 1E and F, there is 74% DNA fragmentation for camptothecin at 10 μmol/L and 66.5% fragmentation for 20 μmol/L betulinic acid within 6 h of drug treatment. When DU145 prostate cancer cells were treated with betulinic acid, very little cleavable complex was detected. Camptothecin on the other hand exhibits extensive stabilization of the cleavable complex with increased time points (Fig. 1C), which is evident from the depleted band of topoisomerase I in Fig. 1D. But when cells were pretreated with betulinic acid for 2 h and subsequently treated with camptothecin, there was substantial decrease in the amount of cleavable complex formation, which is evident by the appearance of the immunoband of topoisomerase I (Fig. 1D). As evident from Fig. 1C, with increasing time, there is no concomitant increase in the amount of cleavable complex formed. Thus, the results indicate that betulinic acid precludes formation of camptothecin-mediated topoisomerase I–DNA cleavable complexes in vivo. In contrast, there is no inhibition of cleavable complex if camptothecin is added before addition of betulinic acid (Fig. 1D). This result support the notion that betulinic acid associates with topoisomerase I inside cells and precludes its further association with the substrate to form camptothecin-mediated cleavable complexes.

Betulinic acid inhibits formation of ROS-mediated topoisomerase I–DNA cleavable complex inside cells induced by protein kinase C inhibitor staurosporine and topoisomerase II inhibitor etoposide. Topoisomerase I–DNA cleavable complexes inside cells are trapped by two distinct mechanisms: (a) by the drugs that interact directly with the transient covalent complex and (b) by the formation of oxidative DNA lesions, allowing topoisomerase I to link covalently with the damaged DNA. Betulinic acid has been shown to inhibit the first type of cleavable complex formation induced by topoisomerase poisons like camptothecin. We therefore tested the effect of betulinic acid on the second type of cleavable complexes inside cells. Staurosporine, a protein kinase inhibitor, has been reported to induce ROS generation inside cells and, in turn, oxidative base damages, which subsequently leads to trapping of topoisomerase I–DNA cleavable complexes (9). Etoposide, although an inhibitor of topoisomerase II, is also found to induce stabilization of topoisomerase I–DNA cleavable complexes as a part of the apoptotic process. Both these drugs therefore lead to trapping of topoisomerase I–DNA cleavable complex (11) by an indirect mechanism distinct from that of camptothecin. Thus, we chose to see the effect of betulinic acid on the ability of these two drugs (staurosporine and etoposide) to induce topoisomerase I–DNA cleavable complexes.

Our results indicate that betulinic acid substantially inhibits the topoisomerase I–DNA cleavable complex formation induced by staurosporine and etoposide. As shown in Fig. 2A, the amount of cleavable complex formation with time decreases in cells pretreated with betulinic acid. This is also evident from the immunoband depletion experiment, wherein reappearance of topoisomerase I band is observed when cells were pretreated with betulinic acid before addition of staurosporine or etoposide. The IC₅₀ values for staurosporine and etoposide were 1.5 and 2.6 μmol/L for 48 h of drug treatment as determined by MTT assay (Table 1). The drug concentrations (10 μmol/L) for etoposide and staurosporine used have been optimized on the basis of percentage of DNA fragmentation induced at 6 h of drug treatment. As shown in Fig. 2D, there is 70% DNA fragmentation for staurosporine and 64% for etoposide within 6 h of drug treatment.

Etoposide, which is a topoisomerase II inhibitor, stabilizes topoisomerase II–DNA covalent complexes in vitro and in vivo. Moreover, betulinic acid is a catalytic inhibitor of topoisomerases I and II. Hence, we checked for topoisomerase II–DNA cleavable complexes after etoposide treatment and its subsequent inhibition by betulinic acid. Results indicate that, along with topoisomerase I–DNA cleavable complexes, topoisomerase II covalent complexes are also formed after etoposide treatment. These topoisomerase II–DNA covalent complexes leads to ROS generation, which subsequently leads to apoptotic topoisomerase I–DNA cleavable complexes (11). Pretreatment with betulinic acid can reduce topoisomerase II–DNA covalent complexes as evidenced in immunoband depletion assay (Fig. 2C). Although the effect of betulinic acid on topoisomerase I–DNA complexes are partly due to the inhibition of topoisomerase II–DNA complexes, it, however, does not affect our conclusion significantly. It has been shown that it is the topoisomerase I cleavable complexes and not the topoisomerase II complexes which are responsible for mediating apoptotic cell death by etoposide (11). To check whether staurosporine also have any effect on topoisomerase II–DNA cleavable complexes, we performed immunoband depletion assays after staurosporine treatment (Fig. 2C). There was no depletion of topoisomerase II observed after staurosporine treatment. Hence, the cleavable complexes observed in case of staurosporine are solely due to topoisomerase I. Thus, from the above results, we can conclude that betulinic acid inhibits apoptotic topoisomerase I–DNA cleavable complexes irrespective of the mechanism of formation of such complexes.

Betulinic acid induces ROS generation, caspase cleavage, and subsequent DNA damage inside cells. The indirect mechanism of trapping of topoisomerase I–DNA cleavable complex involves generation of ROS inside cells due to mitochondrial dysfunction and cleavage of caspases. Both staurosporine and etoposide induce ROS formation and subsequent oxidative base damages which in turn is responsible for trapping of the complex. Therefore, the most obvious question that needs to be addressed is, whether betulinic acid, like staurosporine and etoposide, also induce generation of ROS and subsequent DNA base damage inside cells.

DU145 prostate cancer cells were treated with betulinic acid, and ROS were measured by conversion of H₂DCFDA to 2,7-dichloroflurescein. Betulinic acid treatment leads to 4-fold increase of ROS formation inside cells compared with control cells (Fig. 3A). Treatment with staurosporine also exhibits high level of ROS as expected (9). When cells were treated with NAC before the treatment with betulinic acid, the level of ROS generation was reduced by 2-fold to 3-fold, both in betulinic acid–treated and staurosporine-treated cells (Fig. 3A). Because mitochondrial factors and cleavage of caspases also contribute to generation of ROS inside the cells, we checked for the extent of caspase cleavage in betulinic acid–treated cells. As evident from Fig. 3B, there is substantial amount of caspase cleavage at 6 and 12 h of betulinic acid treatment.

With these evidences in hand, we next proceeded to check for the extent of oxidative DNA damage and modification, which is responsible for trapping of topoisomerase I–DNA cleavable complexes during the apoptotic process. For the detection of oxidative DNA lesions, we performed alkaline comet assay.

Generation of ROS inside cells causes oxidative DNA lesions, such as formation of oxidized bases, abasic sites, and strand breaks. Here, we investigated the generation of such lesions by betulinic acid. DU145 cells treated with betulinic acid or staurosporine were permeabilized and exposed to Fpg, an enzyme that converts oxidized purines (e.g., 8-oxoguanosine) into DNA single-strand breaks. Cells were visualized by fluorescence microscope and the comet tail moments were measured from the images using the software Comet IV.

By using the alkaline comet assay, we observed that Fpg-induced single-strand breaks increased substantially in betulinic acid–treated cells (Fig. 3C) compared with the control cells. The comet moments were calculated and plotted to get a comparative picture of DNA damage. Betulinic acid exhibits substantial DNA damage similar to staurosporine. When cells were treated with NAC before betulinic acid treatment, there was substantial reduction in DNA damage, indicating that the base damages in DNA were due to betulinic-induced ROS generation inside cells. Treatment with caspase inhibitor z-VAD-fmk reduced the ROS generation and also inhibited betulinic acid–induced apoptotic DNA fragmentation (Fig. 3D), indicating the role of mitochondrial ROS in betulinic acid–induced cell death.

To visualize the extent of apoptotic DNA damage in the betulinic acid–treated DU145 cells, we performed TUNEL assay. As evident from Fig. 3E, there is substantial number of TUNEL-positive cells after treatment with betulinic acid for 6 h. But pretreatment with NAC reduces the number of TUNEL-positive cells, indicating the involvement of ROS in betulinic acid–induced apoptosis.

Thus, from these experiments, we conclude that betulinic acid does not affect the events upstream to the apoptotic topoisomerase I–DNA cleavable complex formation. Most importantly, betulinic acid induces events like ROS and oxidative base damage favorable for trapping of topoisomerase I–DNA cleavable complex and yet inhibits the complex formation.

Betulinic acid renders cellular topoisomerase I less sensitive to cleavable complex formation and inhibits its interaction with DNA containing oxidative base modification. The fact that betulinic acid induces oxidative DNA damage and yet cannot induce the formation of topoisomerase I–DNA cleavable complexes prompted us to investigate whether the drug interacts with the cellular pool of topoisomerase I and precludes its interaction with the damaged DNA inside cells.

To check this possibility, we treated DU145 cells with betulinic acid for different time periods (0, 2, and 6 h), prepared nuclear extracts form these betulinic acid treated, and examined their ability to cleave the 25 mer duplex oligonucleotide in assays in presence and absence of camptothecin. In absence of camptothecin, no cleavage was observed with nuclear extracts from cells treated with betulinic acid for 0, 2, and 6 h (Fig. 4, lanes 2–7). In presence of camptothecin, cleavage was observed only with nuclear extracts from cells treated with betulinic acid for 0 h. Cleavage was found to diminish and finally abolished with extracts from cells treated with betulinic acid for 2 and 6 h (Fig. 4, lanes 6 and 7). This result strengthens our hypothesis that betulinic acid interacts with the cellular topoisomerase I and sequesters the enzyme in the nucleoplasm, thereby inhibiting its interaction with damaged DNA.

To confirm that betulinic acid interacts with topoisomerase I and prohibits its interaction with oxidatively damaged DNA, we performed in vitro cleavage reactions with DNA substrate containing 8-oxoguanosine, a frequently observed oxidative base modification. Cleavage reactions were performed under standard assay conditions with recombinant human topoisomerase I, as well as with nuclear extracts from betulinic acid–treated cells. Results of this experiment are shown in Fig. 4B. Presence of 8-oxoguanosine at +1 position of the siscile strand of DNA enhances cleavage by topoisomerase I by 5-fold (22). As evident from the figure, huge amounts of cleavage products accumulate when the DNA was incubated with topoisomerase I in absence and presence of camptothecin. But in samples pretreated with betulinic acid, there is substantial reduction of cleavage products indicating that betulinic acid interferes with the cleavage reaction by interacting with the enzyme. Similar reduction in cleavage was observed when DNA substrate was incubated with nuclear extracts from betulinic acid–treated DU145 cells (Fig. 4B , lanes 8 and 9). Thus the results of this experiment strongly support our hypothesis that betulinic acid prohibits topoisomerase I from interacting with oxidatively damaged DNA.

Down-regulation of topoisomerase I reduces but cannot abrogate betulinic acid–induced apoptosis. Topoisomerase I have been depicted to act as a general apoptotic nuclease in death processes induced by various agents in different cell types (3). Topoisomerase I–DNA cleavable complex formation has been shown to be a general event in many apoptotic processes. All the above experiments argue in favor of the fact that topoisomerase I–DNA cleavable complex formation is not a requisite event in apoptotic process induced by betulinic acid. This catalytic inhibitor is also found to intervene the process of topoisomerase I–DNA complex formation induced by other agents like camptothecin, staurosporine, or etoposide.

To establish this argument and also to delineate the role of topoisomerase I in betulinic acid–induced cell death, we chose to see the effect of down-regulation of topoisomerase I on betulinic acid–induced apoptosis. Topoisomerase I was transiently down-regulated by transfecting topoisomerase I siRNA in DU145 cells, and the effect of different drugs were examined. As expected, down-regulation of topoisomerase I led to marked difference in apoptotic DNA fragmentation in camptothecin-treated and staurosporine-treated cells. There were almost 66% and 50% reduction in apoptotic DNA fragmentation in camptothecin and staurosporine treated cells, respectively. On the contrary, there was only 27% reduction of DNA fragmentation in topoisomerase I depleted cells treated with betulinic acid compared with cells transfected with a control siRNA. PARP cleavage was also examined in control and siRNA-transfected cells after betulinic acid treatment. PARP cleavage was found to be unaffected in topoisomerase I depleted cells treated with betulinic acid (Fig. 5D). Thus, it can be concluded from this experiment that, unlike camptothecin and staurosporine, topoisomerase I–DNA cleavable complex formation is not a requisite event for apoptosis induced by betulinic acid.

Involvement of topoisomerase I–DNA cleavable complexes in the apoptotic process is not only restricted to topoisomerase inhibitors like camptothecin but can be extended to many other agents that have different cellular targets.

Recently, Pommier and coworkers have shown that several agents, like staurosporine, arsenic trioxide, etoposide, and tubulin inhibitors, induce formation of topoisomerase I–DNA complexes inside cells in spite of having different cellular targets (4). Most drugs that induce apoptosis generate ROS inside cells, which in turn leads to oxidative DNA damages. These oxidized bases are preferred sites for the formation of topoisomerase I–DNA cleavable complexes. Thus, topoisomerase I seems to play a pivotal role in the process of apoptosis.

In this study, we show that betulinic acid, which is a catalytic inhibitor of topoisomerases, inhibits the process of apoptotic topoisomerase I–DNA complex formation by interacting with the enzyme directly inside cells. This work establishes the effects of betulinic acid on the formation of topoisomerase I–DNA cleavable complexes inside cells induced by both direct and indirect mechanisms. We have reported earlier that betulinic acid inhibits camptothecin-mediated cleavage in vitro and in vivo in blasted mouse splenocytes (14). Recently, formation of topoisomerase I–DNA cleavable complexes has been depicted to be a general event in many programmed cell death processes (4). Therefore, we chose to investigate the effect of betulinic acid on this apoptotic process and its involvement in cell death.

As evidenced by 25 mer duplex oligo cleavage, betulinic acid inhibits camptothecin-mediated formation of cleavable complexes in vitro. In cells pretreated with betulinic acid, there was substantial reduction of camptothecin-mediated cleavable complex formation. Thus, betulinic acid can inhibit the direct trapping of cleavable complex induced by camptothecin. We then chose to see the effects of betulinic acid on the indirect mechanism of topoisomerase I–DNA cleavable complex formation by agents like staurosporine and etoposide. Although staurosporine and etoposide have different cellular targets and have no direct effect on topoisomerase I, both these compounds induce oxidative DNA damage through generation of ROS inside cells. Therefore, we chose to see the effect of betulinic acid on the ROS-mediated indirect mechanism of topoisomerase I–DNA cleavable complex formation. As evidenced by in vivo cleavage assay and immunoband depletion assay, topoisomerase I–DNA cleavable complexes induced by both staurosporine and etoposide were inhibited in cells pretreated with betulinic acid for 2 h. This clearly suggests that betulinic acid also inhibits the indirect mechanism of topoisomerase I–DNA cleavable complex formation.

Generation of ROS and subsequent DNA damage facilitate the process of topoisomerase I–DNA cleavable complex trapping in cells. This phenomenon is common to many apoptotic processes, which are initiated by generation of ROS or cleavage of caspases. Therefore, agents which induce generation of ROS inside cells are expected to form topoisomerase I–DNA complex as an indispensable event in the process of apoptosis. Because betulinic acid was found to intervene the cleavable complex formation by staurosporine and etoposide, we tested whether betulinic acid can induce generation of ROS and subsequent oxidative DNA lesions. Betulinic acid was found to generate ROS inside cells within 4 to 6 h of treatment. NAC inhibited this ROS generation and also inhibited apoptotic DNA fragmentation in betulinic acid–treated cells, indicating that ROS plays a vital role in betulinic acid–induced cell death. Caspase 3 is known to generate ROS inside cells by feedback mechanism. Recently, caspase-3 was shown to feed back on permeabilized mitochondria and cleave the 75 kDa subunit of complex I. This event leads to the disruption of mitochondrial membrane potential and subsequent production of ROS inside cells (23, 24). We therefore tested the extent of betulinic acid–induced caspase cleavage and effect of caspase inhibitor z-VAD-fmk on betulinic acid–induced ROS generation. Caspase-3 cleavage was observed in betulinic acid–treated cells as evidenced by the pNA release. Treatment with caspase inhibitor z-VAD-fmk reduced the ROS generation and also inhibited betulinic acid–induced apoptotic DNA fragmentation, indicating the role of mitochondrial ROS in betulinic acid–induced cell death. In case of staurosporine, arsenic trioxide, and other compounds, ROS mediate cell death through oxidative DNA lesions and subsequent trapping of topoisomerase I–DNA cleavable complexes. We therefore checked for oxidative DNA damages due to betulinic acid–induced ROS formation in DU145 cells. Alkaline comet assay results suggest that oxidative DNA lesions are formed in betulinic acid–treated cells, similar to that in staurosporine-treated cells. However, in spite of this ROS-induced DNA damage inside cells, there is no trapping of topoisomerase I–DNA cleavable complex in betulinic acid–treated cells. Thus, it can be concluded that although similar initiation events occur both in case of betulinic acid and staurosporine treatment, the death processes do not converge at the point of topoisomerase I–DNA cleavable complex formation.

It is clear from the above experimental evidences that betulinic acid cannot promote the formation of topoisomerase I–DNA cleavable complexes in spite of formation of oxidatively damaged DNA inside cells. One possible explanation for this can be that the drug interacts directly with the cellular topoisomerase I and prevents its interaction with the damaged DNA. In vitro cleavage assays with nuclear extracts from betulinic acid–treated cells strongly support this notion. Even in presence of camptothecin, nuclear extracts from betulinic acid–treated cells exhibit severely reduced trapping of topoisomerase I–DNA cleavable complexes. Thus, interaction of betulinic acid with the cellular topoisomerase I prevents the enzyme from binding with the DNA and refrains it from participating in the apoptotic process. Similar experiments using oligonucleotide substrate containing 8-oxoguanosine modification further strengthen this hypothesis. Cleavage was inhibited in the presence of betulinic acid. Thus, our results argue in favor of the notion that betulinic acid precludes the interaction between topoisomerase I and oxidatively damaged DNA inside cells.

Because topoisomerase I plays the role of an apoptotic nuclease in death processes initiated by staurosporine, etoposide, arsenic trioxide, or camptothecin, silencing of the topoisomerase I gene severely affects the apoptotic process in case of these drugs. On the contrary, similar role of topoisomerase I is not apparent in case of betulinic acid–induced cell death, although topoisomerase I is a major target for the drug. To address this issue, we silenced the topoisomerase I gene in DU145 cells and looked for its effect on the apoptotic process induced by betulinic acid. DNA fragmentation was reduced by 27% but still continued to be substantial at 6 h of treatment. PARP cleavage was observed to be almost similar as that of wild-type cells indicating that apoptotic process, although reduced, is not abrogated due to silencing of topoisomerase I. On the other hand, there was substantial reduction of apoptotic DNA fragmentation in apoptosis induced by staurosporine when topoisomerase I gene is silenced. Thus, it is evident that topoisomerase I, although it is an initial target for betulinic acid, is not the actual mediator of cell death process.

The formation of topoisomerase I–DNA cleavable complexes depends on caspase activation and generation of ROS inside cells. It is also well established that caspases activate endonucleases during apoptosis. The DNA breaks produced by apoptotic nucleases, such as CAD/DFF40 and endonuclease G, may also contribute to the trapping of topoisomerase I–DNA cleavable complexes. Moreover, there are reports of occurrence of topoisomerase I–DNA cleavable complexes during apoptosis induced by tumor necrosis factor–related apoptosis ligand, Fas ligand, and BH3 mimetics antimycin A (25, 26). Thus, apoptotic topoisomerase I–DNA cleavable complex formation has been shown to be involved in many of the cell death processes. We have summarized the effect of betulinic acid on topoisomerase I–DNA cleavable complex formation in Fig. 6. We have shown that betulinic acid interacts with the enzyme and inhibits all types of topoisomerase I–DNA cleavable complex formation irrespective of the process of initiation. Although, betulinic acid itself induce caspase cleavage, ROS generation, and oxidative DNA damage, the cell death process induced by the drug does not involve formation of topoisomerase I–DNA cleavable complex. Our study provides evidence for the first time that betulinic acid can inhibit topoisomerase I–DNA cleavable complexes inside cells, and this inference can be extrapolated for all the apoptotic processes discussed above. Because these cleavable complexes are common mediators of cell death, betulinic acid cannot be used to potentiate cell death in combination with most anticancer drugs which induce apoptosis through formation of topoisomerase I–DNA cleavable complexes. Taken together, this study will help in understanding the mechanism of synergistic drug action and development of newer strategies for combinatorial drug treatment in cancer chemotherapy.

Grant support: Network Project SMM 003 of Council of Scientific and Industrial Research (H.K. Majumder) and Senior Research Fellowship from Council of Scientific and Industrial Research, Government of India (A. Ganguly).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Prof. Yves Pommier (National Cancer Institute, NIH) for sending us DU145 cells and DNA substrate containing 8-oxoguanosine modification, Prof. J.J. Champoux (University of Washington) for providing us with the recombinant baculovirus containing wild-type human topoisomerase I, and Dr. S. Roy (Indian Institute of Chemical Biology) for his interest in this work.