MFTZ-1, an actinomycetes subspecies–derived antitumor macrolide, functions as a novel topoisomerase II poison Free

DNA topoisomerase (Topo) II is an essential enzyme that plays an important role in DNA replication, repair, transcription, and chromosome segregation (1). In addition to its critical functions, Topo II has been identified as an important antitumor target (2). Classic Topo II poisons stabilize DNA–Topo II complexes by blocking DNA religation. The accumulation of this covalent complex in rapidly dividing cancer cells results in the formation of multiple DNA strand breaks, and these genotoxic damages activate the cell death machinery (3, 4). Topo II poisons have shown a major clinical utility including mitoxantrone, etoposide (VP16), and Adriamycin. However, they often induce dose-limiting toxicities and multidrug resistance (MDR), resulting in treatment failure after initial effective therapy. As such, efforts to develop novel potent poisons have met a limited success, and there has been continuous interest in studying and developing new anti–Topo II agents.

Nonactic and homononactic acids are plant growth stimulators and exhibit specific insecticidal effects (5, 6). Nonactin, a macrotetrolide, derived from nonactic and homononactic acids, was shown to possess antitumor activity against both mammalian cell lines in vitro and Sarcoma 180 in vivo in mice (7, 8). 14-Ethyl-2,5,11-trimethyl-4,13,19,20-tetraoxa-tricyclo[14.2.1.1^7,10]eicosane-3,12-dione (MFTZ-1; Fig. 1A), a new derivative of nonactic and homononactic acids isolated from an endophyte Streptomyces sp. ls9131 of Magnolia hookeri, characterized to be a dimeric dinactin (9), stood out as a novel Topo II poison structurally distinct from all Topo II inhibitors known thus far. In the present study, we aimed to investigate the antitumor activities of MFTZ-1 both in vitro and in vivo and figure out the related possible mechanisms of action. Results show that MFTZ-1 functions as a novel Topo II poison to trigger the Topo II–dependent DNA double-strand breaks (DSBs) and apoptosis. These findings should help elucidate the precise mechanisms involved in the antitumor activities of MFTZ-1 and enable the rational design of novel Topo II–targeted drugs.

MFTZ-1 was isolated from an endophyte Streptomyces sp. ls9131 of M. hookeri as previously reported (9), and its purity is >95%. The compound was dissolved in DMSO to the concentration of 0.1 mol/L (in vitro) or in ethanol (in vivo) before each assay and then diluted with normal saline. The final DMSO or ethanol concentration did not exceed 0.1% (v/v) or 1% (v/v), respectively.

Adriamycin, VP16, camptothecin, and vincristine were prepared as 10 mmol/L stock solution in DMSO. Aclarubicin (5 mmol/L) was prepared with DMSO as a stock solution. All these agents were purchased from Sigma. Pan-caspase inhibitor Z-VAD-FMK and caspase-9 inhibitor Z-LEHD-FMK (Calbiochem) were dissolved at a concentration of 0.1 mol/L in DMSO. All stock solutions were stored at −20°C, thawed, and diluted with normal saline before each experiment.

BALB/Ca nu/nu female mice ages 4 to 5 weeks were bred in Shanghai Institute of Materia Medica. The animals were housed in sterile cages under laminar airflow hoods in a specific pathogen-free room with a 12-h light and 12-h dark schedule, and fed autoclaved chow and water ad libitum. All experiments were done according to institutional ethical guidelines on animal care.

Human gastric adenocarcinoma cell line SGC-7901, hepatocellular carcinoma cell line BEL-7402, and ovarian epithelioid carcinoma cell line HO-8910 were obtained from the cell bank of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Human premyelocytic leukemia cell line HL-60, HL-60/MX2 (Topo II deficient), chronic myelogenous leukemia K-562, lymphoblastic leukemia cell line MOLT-4, oral epidermoid carcinoma KB, cervical carcinoma cell line HeLa, colorectal adenocarcinoma HT-29, HCT-116, lung adenocarcinoma A549, NCI-H23, breast carcinoma cell line MCF-7, MDA-MB-231, and MDA-MB-468 were purchased from American Type Culture Collection. Human breast carcinoma cell line MDA-MB-435 and gastric adenocarcinoma cell line MKN-28 were from Japanese foundation of Cancer Research. Both Adriamycin-selected MDR cell sublines K-562/A02 (10) and MCF-7/Adriamycin (11, 12) were purchased from the Institute of Hematology, Chinese Academy of Medical Sciences. The vincristine-selected MDR subline KB/vincristine (13) was obtained from Zhongshan University of Medical Sciences. All these cell lines except MCF-7 were maintained in RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies), l-glutamine (2 mmol/L), penicillin (100 IU/mL), streptomycin (100 μg/mL), and HEPES [10 mmol/L; MCF-7 with additional 1 mmol/L sodium pyruvate and supplemented with 0.01mg/mL bovine insulin (pH 7.4)] in a humidified atmosphere of 95% air plus 5% CO₂ at 37°C.

The Saccharomyces cerevisiae strains used were the generous gift of Dr. Neil Osheroff (The School of Medicine, Vanderbilt University, Nashville, TN). The parent yeast strain used in this study was S. cerevisiae JN394. Two gene-modified strains from JN394 were used: JN394top1⁻, which had a chromosomal deletion of Topo I gene (14); JN394top2-4, in which the wild-type Topo II gene was replaced with the temperature-sensitive top2-4 mutant allele2-4 (15). Yeasts were grown in YPDA liquid medium (1% yeast extract, 2% bacto peptone, 2% glucose, and 40 μg/mL adenine) at 25°C or 30°C.

The inhibitory effect of MFTZ-1 on tumor cell line growth was assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) dye absorbance as described previously (16). The concentration required for IC₅₀ of tumor cells treated with MFTZ-1 for 72 h was determined from the dose-response curve.

DNA DSBs were evaluated using the neutral single-cell gel electrophoresis assay as previously described (17), with minor modifications (18). A fluorescence microscope (Olympus BX51) was used to capture the images. Quantization was achieved by analyzing ≥50 randomly selected comets per slide with the Komet 5.5 software (Kinetic Imaging, Ltd.) using the tail moment.

HL-60 cells (5 × 10⁵/mL) were treated with various concentrations of MFTZ-1 for 24 h. Cells were harvested and washed with cold PBS. Then, cells were fixed by 70% ethanol on ice overnight, and washed twice in PBS. Staining was done in PBS containing 50 μg/mL RNase and 10 μg/mL propidium iodide at room temperature in dark for 30 min. For each sample, ≥1 × 10⁴ cells were analyzed by flow cytometry (FACSCalibur).

HL-60 cells (5 × 10⁵/mL) were treated with various concentrations of MFTZ-1 for 24 h. The samples were left untreated or pretreated with aclarubicin for 0.5 h before MFTZ-1 addition. DNA fragmentation was extracted using the method on a previous report (19). The fragmentation of DNA was electrophoresed in 1.5% agarose gel, stained with ethidium bromide, and photographed through a ChemiGenius 2 Gel Documentation System (Syngene).

Terminal deoxyribonucleotide transferase–mediated nick-end labeling (TUNEL) assay was done according to the manufacturer's instructions (Roche). HL-60 cells (5 × 10⁵/mL) were incubated with MFTZ-1 for 24 h in indicated concentrations and stained for TUNEL. Cells were washed in PBS twice, fixed in 4% paraformaldehyde, and permeabilized in 0.1% Triton X-100 on ice. For the labeling reaction, the cells were stained for 1 h at 37°C. All cells were stained with 4′,6-diamidino-2-phenylindole (0.5 μg/mL). Cells were photographed using a fluorescence microscope.

DNA relaxation assays were according to the procedure as in previous studies (20). pBR322 DNA (0.25 μg) was incubated with 0.75 unit of Topo IIα (TopoGEN) at 37°C for 30 min in relaxation buffer [50 mmol/L Tris (pH 7.8), 50 mmol/L KCl, 50 mmol/L NaCl, 5 mmol/L MgCl₂, 0.1 mmol/L EDTA, 15 μg/mL of bovine serum albumin, and 1 mmol/L ATP]. Reactions were terminated by adding 3 μL of stop solution (100 mmol/L EDTA, 0.5% SDS, 50% glycerol, and 0.05% bromophenol blue). Electrophoresis was carried out in a 1% agarose gel in Tris-borate EDTA at 4 V/cm for 2 h. Gel was stained with ethidium bromide and photographed under UV light.

Topo II activity was measured by the ATP-dependent decatenation of kinetoplast DNA (21). Briefly, 0.25 μg kinetoplast DNA was incubated 0.75 unit of Topo IIα at 37°C for 15 min in a 15 μL total reaction buffer [50 mmol/L Tris (pH 8.0), 120 mol/L KCl, 10 mmol/L MgCl₂, 0.5 mmol/L ATP, 0.5 mmol/L DTT, and 30 μg/mL bovine serum albumin]. The reaction was terminated by adding 2 μL 10% SDS. The DNA samples were subjected to electrophoresis under the same conditions as described above.

Trapped in agarose DNA immunostaining assay was done as previously reported (22). Briefly, untreated or treated HL-60 cells (5 × 10⁵/mL) were harvested and embedded in agarose onto a microscope slide. Slides were immersed in lysis buffer [1% SDS, 80 mmol/L phosphate buffer (pH 6.8), and 10 mmol/L EDTA] containing protease inhibitors (2 μg/mL pepstatin A, 2 μg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L DTT) to disrupt the cell membranes and then were washed in 1 mol/L NaCl. Only the proteins covalently bound to DNA remained. Slides were incubated with Topo IIα antibody (Santa Cruz Biotechnology, Inc.), which binds to Topo II trapped in cleavable complexes on DNA. Finally, the complexes became visible under microscope by using a secondary Alexa Fluor 488–conjugated antibody (Molecular Probes).

DNA-unwinding effects of MFTZ-1 were assayed according to the procedure as in previous studies (23). Plasmid pBR322 was linearized by EcoRI restriction endonuclease and recovered by phenol and ethanol precipitation. Linearized DNA and testing compounds (0.1 μg) were incubated with 2 units of T4 DNA ligase at 22°C for 30 min in T4 ligase buffer. The reaction was stopped by shifting the temperature to 60°C for 10 min. The compounds were removed from the reaction mixture by extraction with phenol. The DNA samples were subjected to electrophoresis under the same conditions as described above.

The binding reaction between MFTZ-1 and Topo II was carried out using Biacore 3000 instrument (Biacore). Human Topo IIα ATPase domain (HsATPase) was purified from a yeast overexpressing system (24). Briefly, human Topo IIα ATPase domain was immobilized onto CM5 sensor chip strictly following the ligand Thiol protocol described in BIAapplication Handbook. The unreacted moieties on the surface were blocked with ethanolamine. To correct for nonspecific binding and bulk refractive index change, a blank channel (FC2) without drugs was used as a control for each experiment. Sensorgrams for all binding interactions were recorded in real-time and analyzed after subtracting that from the blank channel. Changes in mass due to the binding response were recorded as resonance units. All binding experiments were done at 25°C with a constant flow rate of 20 μL/min HBS-EP. The system buffer was 20 mmol/L HEPES, 150 mmol/L NaCl, 5 mmol/L EDTA, and 0.05% P20. The equilibrium dissociation constants (K_D) evaluating the protein-ligand binding affinity were determined by the steady-state affinity fitting analysis of the results from Biacore data.

Variations of mitochondrial transmembrane potential (Ψ_m) were assessed as the retention of the fluorescent dye DiOC₆ (Calbiochem). After treatment with MFTZ-1 for an indicated time, 10⁶ cells in 2 mL of complete RPMI 1640 were loaded with the probe DiOC₆ (40 nmol/L) for 30 min as described previously (25) and analyzed by flow cytometry.

Whole-cell lysates and cytoplasmic extracts (26) were prepared, and Western blotting was done as previously described. Briefly, equal amounts of proteins were separated on SDS polyacrylamide gels. Then, proteins were electroblotted onto nitrocellulose membranes and identified with anti–phosphohistone H2AX (Ser¹³⁹), anti–Bcl-xL, anti-Bax, anti–Bcl-2, anti–cytochrome c, anti–caspase-3, and anti–caspase-7 antibodies (1:1,000; Cell Signaling); as well as anti–apoptosis-inducing factor, anti-poly(ADP)ribose polymerase (PARP), anti–caspase-8, anti–caspase-9, and anti–β-actin antibodies (1:1,000; Santa Cruz Biotechnology). Detection was done using horseradish peroxidase–conjugated secondary antibody and SuperSignal West Pico Chemiluminescent Substrate (Pierce, Inc.) according to the manufacturer's instructions.

Human ovarian carcinoma HO-8910 xenografts were established by inoculating 2 × 10⁷ cells s.c. in nude mice. The experiments began when the xenografts had three passages in nude mice. Under a sterilization condition, well-grown tumors were cut into 1-mm³ fragments, and the fragments were transplanted s.c. into the right flank by trocar in nude mice. When tumor reached a volume of 100 to 200 mm³, the mice were randomized to control and treated groups and received vehicle, mitomycin C, or MFTZ-1 at indicated doses by i.v. administration. The size of tumors were measured individually twice per week with microcalipers. Tumor volume (V) was calculated as V = (length × width²) / 2. The individual relative tumor volume (RTV) was calculated as follows: RTV = V_t / V₀, where V_t is the volume on each day of measurement, and V₀ is the volume on the day of initial treatment. Therapeutic effect of compound was expressed in terms of treated versus control percent, and the calculation formula is as follows: treated versus control (%) = mean RTV of the treated group / mean RTV of the control group × 100%.

Data were presented as X̄ ± SD, and significance was assessed with Student's t test. Differences were considered significant at P < 0.05.

MFTZ-1 Exhibits Antiproliferative Activity in Tumor Cells. We first determined the effects of MFTZ-1 on growth of the human tumor cell lines. MFTZ-1 displayed wide potent cytotoxicity in human cancer cell lines. The mean IC₅₀ of MFTZ-1 for all tested tumor cell lines was 0.905 μmol/L. Although MFTZ-1 exhibited various cytotoxic effects against different cell lines, no significant tissue specificity was observed (Fig. 1B).

MFTZ-1 Overcomes Drug Resistance. To determine whether MFTZ-1 was able to overcome drug resistance, we examined the effect of MFTZ-1 on three MDR sublines, K-562/A02, MCF-7/Adriamycin, and KB/vincristine. Drug-sensitive parental K-562, KB, MCF-7 cell lines, and three conventional anticancer drugs, VP16, Adriamycin, and vincristine were used as references. MFTZ-1 displayed significant cytotoxicity in the three MDR sublines examined, with an average IC₅₀ value of 1.08 μmol/L, which was close to that observed in the corresponding parental cell lines (average IC₅₀, 0.525 μmol/L). The average resistance factor (RF) of MFTZ-1 on MDR cells was 2.08 versus 101.95, 75.60, and 229.35 for VP16, Adriamycin, and vincristine, respectively (Table 1).

Because of the potent antitumor activity of MFTZ-1 in vitro, its antitumor properties were further examined in vivo. We tested the antitumor efficacy of MFTZ-1 in the human ovarian carcinoma HO-8910 xenograft as it was highly cytotoxic to HO-8910 cell lines in vitro. As indicated in Fig. 1C, MFTZ-1 displays an obvious antitumor activity at the dosage of 1 mg/kg after i.v. injection weekly for 3 weeks with the treated versus control values of 38.91% (at day 17), 46.6% (at day 21), and 48.5% (at day 24). Moreover, no body weight loss and other apparent toxicity were observed in MFTZ-1–treated nude mice.

Cytotoxic agents exert antitumor activity by damaging tumor-cell DNA, further inhibiting cell proliferation or inducing apoptosis. A broad range of DNA damage–inducing drugs have been developed for treatment of neoplastic disease. These drugs, although acting in many different ways, are all capable of activating the cellular responses that have evolved to respond to physiologically induced DNA damage (27).

MFTZ-1 Triggers DNA DSBs. The neutral signal cell electrophoresis was used to measure chromosomal DNA DSBs in HL-60 cells exposed to MFTZ-1. Upon treatment with MFTZ-1 for 1 h, chromosomal DNA DSBs, which were measured using the comet tail moment, were increased in a dose-dependent manner in HL-60 cells (Fig. 2A).

MFTZ-1 Induces Tumor Cell Apoptosis. Cells can respond to DNA damage either by undergoing cell cycle arrest, to facilitate DNA repair, or by undergoing cell suicide. It seems that in some types of tumors, the propensity to undergo apoptosis is a critical determinant of their sensitivity to anticancer therapy. Accordingly, we investigated the ability of MFTZ-1 to induce apoptosis in HL-60 cells. MFTZ-1 significantly increased sub–G₀-G₁ phase in a dose-dependent manner by flow cytometry analysis (Fig. 2B). TUNEL assay showed that HL-60 cells incubated with various concentrations of MFTZ-1 for 24 h underwent apoptosis (Fig. 2C). In addition, MFTZ-1 dose dependently induced internucleosomal DNA fragmentation (Fig. 2D).

Caffeine Antagonizes MFTZ-1–Induced Apoptosis. We also tested whether MFTZ-1–induced apoptosis involves nuclear kinases ATM/ATR, which is known to mediate apoptosis induced by many DNA-damaging agents (27, 28). Caffeine, an inhibitor of ATM/ATR, was preferentially used. As shown in Fig. 2E, PARP cleavage and caspase-3 activation induced by MFTZ-1 were greatly reduced after pretreatment with caffeine, suggesting that MFTZ-1–induced apoptosis in HL-60 cells requires the ATM/ATR. The involvement of ATM/ATR in MFTZ-1–induced apoptosis indicated the possible involvement of DNA damage.

A number of DNA-damaging agents currently used for the treatment of human cancers are targeted to DNA topoisomerases. A yeast (S. cerevisiae) genetic system that allowed manipulation of topoisomerase activity and drug sensitivity is commonly used to determine whether topoisomerases are the primary cytotoxic target of DNA-damaging agents (14). Here, we used this system to investigate whether topoisomerases are involved in MFTZ-1–mediated tumor cell death.

MFTZ-1 Fails to Target Topo I. Initial experiments addressed whether Topo I accounts for MFTZ-1–induced cytotoxicity. This was accomplished by using a JN394top1⁻ strain that is devoid of Topo I activity (14). As a result, top1⁻ strains are refractory to Topo I poison camptothecin (Fig. 3A). In contrast, no resistance was observed when JN394top1⁻ cells were treated with MFTZ-1, and MFTZ-1 displayed significant cytotoxicity in both JN394 and JN394top1⁻ cells (Fig. 3B). This finding suggests that Topo I was not the cytotoxic target for MFTZ-1 in yeast.

MFTZ-1 Targets Topo II in Yeast. Because Topo I was excluded to be the target of MFTZ-1, we are then interested in addressing whether Topo II plays responsive roles for the cytotoxicity of MFTZ-1. The yeast strain JN394top2-4 contains a temperature-sensitive mutation that alters Topo II activity in the yeast strain according to the culture temperature (15). At the semipermissive temperature of 30°C, Topo II activity is reduced to about 5% to 10% of that the permissive temperature of 25°C. Our result showed that MFTZ-1, such as VP16, was a potent toxic agent toward JN394top2-4 at 25°C. Conversely, VP16 or MFTZ-1 had no obvious influence on JN394top2-4 at the semipermissive temperature of 30°C, even up to the concentrations of 200 μmol/L (Fig. 3C). The failure of MFTZ-1 to induce cytotoxicity to the cells lacking Topo II activity strongly suggests that Topo II is the primary cellular target for MFTZ-1.

MFTZ-1 Inhibits the Catalytic Activity of Topo II. We further confirm the effect of MFTZ-1 on Topo II activity next. First, we detected the effect of MFTZ-1 on the catalytic activity of Topo II by evaluating the enzyme-mediated, negatively supercoiled pBR322 relaxation. As shown in Fig. 4A, MFTZ-1 significantly inhibited the relaxation of supercoiled pBR322 by the enzyme in a dose-dependent manner. MFTZ-1 (200 μmol/L) made most of pBR322 in a supercoiled state. To further confirm the inhibitory effect of MFTZ-1 on Topo II, one more specific Topo II–mediated kinetoplast DNA decatenation was carried out. As shown in Fig. 4B, MFTZ-1 displayed significant inhibition of this reaction in a concentration-dependent manner.

MFTZ-1 Induces Stable DNA-Topo II Complexes. Trapped in agarose DNA immunostaining assay is typically used to detect Topo II drug-stabilized cleavable complexes, which is crucial for understanding the key mechanism of Topo II poison (29, 30). We further test whether MFTZ-1 produced DNA–Topo II complexes using this method. In our study, there was no visible green immunofluorescence associated with DNA in untreated cells. When cells were treated with MFTZ-1, the staining intensity was increased dose dependently and the fluorescence was easily detectable in cells exposed to 25 μmol/L MFTZ-1. VP16 also yielded high levels of fluorescence at the concentration of 100 μmol/L (Fig. 4C). All these results indicate that MFTZ-1 is a novel Topo II poison.

Clinically relevant Topo II poisons fall into two general classes. One class intercalates DNA, and apparently impedes the Topo II–dependent religation step by altering DNA structure (31). The other class has weak affinity for DNA and is thought to prevent the DNA religation step by directly binding to the enzyme (32). Our aforementioned data release strong evidence that MFTZ-1 functions as Topo II poison. We next intended to learn whether MFTZ-1 accomplished its Topo II poisoning activity via intercalating into DNA or via directly binding to Topo II.

MFTZ-1 Does Not Intercalate into DNA. To investigate whether MFTZ-1 intercalated into DNA, an unwinding assay was done using linearized pBR322 and T4 DNA ligase. As shown in Fig. 4D, an obvious inhibition of the activity of T4 DNA ligase by Adriamycin was observed, as reported previously (33). However, MFTZ-1, such as VP16, had no influence on T4 DNA ligase activity even up to the concentrations of 200 μmol/L.

MFTZ-1 Binds to the ATP Domain of Human Topo IIα. To further test whether MFTZ-1, such as VP16, directly bound to the enzyme in the absence of DNA, we assessed MFTZ-1–Topo II interaction using surface plasmon resonance assay. With the ATPase domain of human Topo IIα immobilized on sensor chip, the different concentrations of MFTZ-1 or VP16 run over. Results indicated that both MFTZ-1 and VP16 bound to human Topo IIα ATPase domain in a concentration-dependent manner, yielding a dissociation constant (K_D) of 11.1 and 41.6 μmol/L, respectively (Fig. 4E).

Topo II poisons, such as VP16 and Adriamycin, are cytotoxic as a result of their ability to stabilize the cleavable complex, thereby decreasing the ability of the enzyme to religate the transient DNA DSBs. Therefore, we next investigated the role of Topo II in MFTZ-1–induced DNA DSBs, apoptosis, and cytotoxicity.

MFTZ-1–Induced Damage, Apoptosis, and Cytotoxicity Are Reduced in Topo II–Deficient Cells. To test the involvement of Topo II in MFTZ-1–induced DNA DSBs, we used HL-60 and HL-60/MX2 (Topo II deficient) cells. DNA damage was monitored by Ser¹³⁹-phosphorylated H2AX (γ-H2AX), an established marker of DSBs (34). As shown in Fig. 5A, γ-H2AX was obviously elevated after treatment with MFTZ-1 or VP16 in HL-60 cells but not in Topo II–deficient HL-60/MX2 cells. These results suggested that MFTZ-1, such as VP16, induces Topo II–mediated DNA damage.

Because DNA DSBs caused by MFTZ-1 may be responsible for the apoptosis of MFTZ-1, we then examined whether MFTZ-1–induced apoptosis involved the formation of Topo II cleavage complexes. Apoptosis was monitored by specific proteolytic cleavage of PARP. As shown in Fig. 5B, PARP cleavage induced by MFTZ-1 was greatly reduced in HL-60/MX2 compared with HL-60 cells. In addition, we found that HL-60/MX2 cells exhibited a reduced sensitivity to MFTZ-1, with the RF value of 7.65. RF for novantrone, VP16, and Adriamycin were 28.19, 13.38, and 6.88, respectively. However, there was no significant difference in the IC₅₀ values of camptothecin between HL-60 and HL-60/MX2 (Fig. 5C).

MFTZ-1–Induced DNA Damage and Apoptosis Are Antagonized by a Topo II Inhibitor, Aclarubicin. To further test the possibility of Topo II involvement, aclarubicin, a Topo II catalytic inhibitor, was used to antagonize the formation of Topo II cleavage complexes. Unlike Topo II poisons, aclarubicin specifically inhibits the catalytic activity of Topo II without significantly elevating the level of cleavage complexes (35). Therefore, aclarubicin was commonly used to evaluate whether Topo II poison–induced DNA DSBs are due to the formation of cleavage complexes (36, 37). In the study, we found that aclarubicin significantly antagonized MFTZ-1–induced DNA DSBs in HL-60 cells (Fig. 5D). Furthermore, aclarubicin effectively reversed the level of γ-H2AX induced by MFTZ-1 (Fig. 5E). In addition, aclarubicin also counteracted MFTZ-1–triggered apoptosis in HL-60 cells (Fig. 5F). Taken together, these results indicate that Topo II cleavage complex may contribute to MFTZ-1–induced DNA DSBs, apoptosis, and cytotoxicity.

The aforementioned result suggested that MFTZ-1 induced DNA damage–mediated apoptosis, which involved Topo II. Given that genotoxic DNA-damaging agents may activate both membrane death receptors and mitochondria-dependent apoptotic pathway (28, 38, 39), we are thus interested in how MFTZ-1 is involved in the activation of apoptosis.

The mitochondrial apoptotic pathway is controlled by Bcl-2 family proteins, including the proapoptotic Bax and antiapoptotic Bcl-2 and Bcl-xl (40, 41). In our study, we found that MFTZ-1 increased the expression of Bax and decreased the expression Bcl-2 and Bcl-xl, accompanied by activation of caspase-3, caspase-7, caspase-9, and cleavage of PARP in HL-60 cells (Fig. 6A). Consistent with this, MFTZ-1 induced a dose-dependent drop in mitochondrial membrane potential (Fig. 6B) then caused the release of cytochrome c and apoptosis-inducing factor into cytosol (Fig. 6C). All these findings implicated that mitochondrial pathway were involved in MFTZ-1–driven apoptosis.

Activation of the apoptotic pathway may occur via Fas-L/Fas-R and activation of the downstream caspase-8 (42, 43). However, treatment of HL-60 cells with MFTZ-1 failed to up-regulate the expression of Fas-L/Fas-R, and the receptor proximal caspase-8 was not activated either (data not shown).

To examine whether inhibition of caspase cleavage blocks MFTZ-1–induced apoptosis, caspase inhibitors were preferentially used. Pan-caspase inhibitor (Z-VAD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) significantly inhibited procaspase-3, procaspase-7, procaspase-9, and PARP cleavage triggered by MFTZ-1 treatment (Fig. 6D). These data help clarify that MFTZ-1–induced apoptosis was partly mitochondria dependent but Fas-R/Fas ligand independent.

Macrotetrolide antibiotics, including nonactin, monactin, dinactin, trinactin, and tetranactin, isolated from a variety of streptomyces species are cyclotetralactones derived from nonactic acid and homononactic acid (5). Nonactin is the parent compound of this group of macrotetrolide antibiotics. It has been shown to possess antitumor activity both in vitro and in vivo (7). In our study, we found that these two monomer, nonactic and homononactic acids, did not show obvious cytotoxicity to human tumor cells (data not shown). However, MFTZ-1, which was determined to be dimeric dinactin, has appreciable antiproliferative activities against various human tumor cell lines and possesses potent anti-MDR activities, highlighted by its encouragingly marked tumor growth arrest against human ovarian carcinoma xenograft. Nuclear magnetic resonance data of MFTZ-1 was very similar to those of dimeric nonactic acid. However, MFTZ-1 was cyclized to form a dimacrolide, and the substituent at C-8′ was ethyl in this compound but methyl in dimeric nonactic acid. Dimeric nonactic acid did not show obvious antifungal or antibacterial activity; however, the dimeric dinactin showed strong antibacterial activity (9). Overall, it seems that the lactone ring may play an important role in these activities.

Cytotoxic drugs act in principle by damaging DNA and triggering apoptosis in tumor cells. In the present study, we found that MFTZ-1 significantly mediated apoptosis involving DNA damage in defined tumor cells. Caffeine, an ATM/ATR kinase inhibitor capable of abrogating the DNA damage–induced apoptosis and desensitizing cancer cells to Topo II poisons, counteracted MFTZ-1–mediated apoptosis involving DNA damage. Given that many active DNA-damaging drugs currently clinically available hold Topo II as their primary target, we thus used the well-accepted yeast genetic system and found that Topo II was the primary cellular target of MFTZ-1.

Topo II has two closely related isoforms in mammalian species, α and β. Although Topo IIα is dramatically up-regulated during periods of rapid cell proliferation, Topo IIβ levels are relatively constant across cell growth cycles (44, 45). Both isoforms seem to play a role in mediating the effects of Topo II poisons; however, Topo IIα is believed to be the more important drug target (46, 47). In the present studies, we found that MFTZ-1 significantly inhibited Topo IIα–mediated supercoiled pBR322 relaxation and kinetoplast DNA decatenation, accompanied by an obvious stabilization of the Topo II-DNA “cleavable complex.” All these favor that MFTZ-1 is a Topo IIα poison. In fact, clinically relevant Topo II poisons fall into two general classes. One intercalates into DNA, and apparently impedes the Topo II–dependent religation step by altering DNA structure (31). The other has weak affinity for DNA and is thought to prevent the DNA religation step by directly binding the enzyme (32). In current study, we found that MFTZ-1 rather than intercalating into DNA is capable of directly binding to human Topo II ATPase domain, suggesting that enzyme-drug interactions play an important role in the ternary Topo II drug–DNA complex. This notion helps clarify that MFTZ-1 function as a nonintercalating Topo IIα poison.

Topo IIα poisons generally result in a series of biochemical changes that culminate in cell death (48). In general, the stabilization of Topo IIα cleavage complexes plays critical roles for the fate of the drug-poisoned cells. However, it is not always true for those poisons to ensure tumor cell apoptosis in a sufficient manner. Sometimes, they activate the apoptosis cascades by passing the formation of Topo II cleavage complexes. In the present study, DNA DSBs induction, PARP cleavage, and cell killing ability of MFTZ-1 were dramatically reduced in Topo II–deficient HL-60/MX2 cells, encouraging us to disclose that the stable Topo II cleavable complexes account for MFTZ-1–mediated DNA DSBs and subsequent apoptosis. Another finding that the use of Topo II catalytic inhibitor aclarubicin has led to reduced DNA DSBs and apoptosis in HL-60 cells provides more convincing data for this theory.

Addressing the issue of apoptotic pathway that becomes activated in response to MFTZ-1 will offer an alternative view of the detailed antitumor machinery of MFTZ-1. In this study, we found that MFTZ-1 increased the expression of Bax and decreased the expression of Bcl-2 and Bcl-xl in HL-60 cells, accompanied by induction of cytochrome c release from the mitochondria into cytoplasm and activation of downstream caspases, including caspase-3, caspase-7, and caspase-9. These findings highlighted the decisive roles of mitochondria-mediated caspase activation in the MFTZ-1–driven events. Additional resultant data from caspase inhibitors further help understand this story. More interestingly, MFTZ-1–induced apoptosis in HL-60 cells in a FasL/FasR–independent manner standing in contrast to the well-known Topo II poisons, including VP16 and Adriamycin, aids in distinguishing MFTZ-1 from the conventional ones.

Here, we show for the first time that MFTZ-1 functions as a novel Topo II poison structurally distinct from all Topo II inhibitors known thus far. The consequently appreciable pharmacologic profiles of MFTZ-1, including well-defined antitumor activities, anti-MDR capacities, and less toxicity promise it as an attractive subject deserving further development.