SEARCH

SEARCH BY CITATION

Keywords:

  • SH-7;
  • shikonin;
  • topoisomerase II inhibitor;
  • antitumor;
  • mitochondria

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enylfuran-2-caroxylate (SH-7), a new naphthoquinone compound, derived from shikonin, exhibited obvious inhibitory actions on topoisomerase II (Topo II) and topoisomerase I (Topo I), which were stronger than its mother compound shikonin. Notably, the SH-7's inhibitory potency on Topo II was much stronger than that on Topo I. In addition, SH-7 significantly stabilized Topo II-DNA cleavable complex and elevated the expression of phosphorylated-H2AX. The in vitro cell-based investigation demonstrated that SH-7 displayed wide cytotoxicity in diversified cancer cell lines with the mean IC50 value of 7.75 μM. One important finding is SH-7 displayed significant cytotoxicity in the 3 MDR cell lines, with an average IC50 value nearly equivalent to that of the corresponding parental cell lines. The average resistance factor (RF) of SH-7 was 1.74, which was much lower than those of reference drugs VP-16 (RF 145.92), ADR (RF 105.97) and VCR (RF 197.39). Further studies illustrated that SH-7 had the marked apoptosis-inducing function on leukemia HL-60 cells, which was validated to be of mitochondria-dependence. The in vivo experiments showed that SH-7 had inhibitory effects on S-180 sarcoma implanted to mice, SMMC-7721, BEL-7402 human hepatocellular carcinoma and PC-3 human prostate cancer implanted to nude mice. Taken together, these results suggest that SH-7 induces DSBs as a Topo II inhibitor, which was crucial to activate the apoptotic process, and subsequently accounts for its both in vitro and in vivo antitumor activities. The well-defined Topo II inhibitory activity, antitumor effects particularly with its obvious anti-MDR action, better solubility and less toxicity make SH-7 as a potential antitumor drug candidate for further research and development. © 2006 Wiley-Liss, Inc.

DNA topoisomerases I and II are nuclear enzymes that regulate DNA topology during DNA replication and recombination, DNA transcription, chromosome condensation-decondensation and segregation.1 They have been identified as important antitumor targets because of their essential physiological functions. Drugs that are targeted to DNA topoisomerases are divided into 2 categories: poisons and catalytic inhibitors. Classical topoisomerase poisons play a role in stabilizing the otherwise fleeting enzyme-DNA intermediates the so-called cleavable complexes,2, 3 and they are well known for their ability to efficiently induce DNA strand breaks.4

Shikonin is one of the active ingredients isolated from the roots of the traditional oriental medicinal herb Lithospermiumerythrorhizon.5 Many studies showed that shikonin exerted antitumor effects by inhibiting cancer cell growth,6 inducing apoptosis7 and inhibiting DNA topoisomerase I/II activity,8, 9, 10 antitelomerase activity11 and antiangiogenesis.12 Because of its poor solubility and toxicity, shikonin have little potential to be an anticancer drug. Over the past few years, many efforts have been made to synthesize new derivatives of this known structure, aiming at discovering the innovative antitumor drug candidate with more effective and less side effects. Ahn reported a series of acylated shikonin derivatives, some of which (such as acetylshikonin and benzoylshikonin) were more potent topoisomerase I poisons than shikonin.8 Couladouros also found that acetylshikonin and isovalylshikonin possessed more potent topoisomerase I inhibitory activity than shikonin.9 Encouraged by these findings, we synthesized a series of racemic versions of shikonin and its analogues,11 and shikonin derivatives with side chains of different heteroaromatic acyl moieties. Some of them showed good antitumor activities, and 1-(1,4-dihydro-5,8-dihydroxy-1,4- dioxonaphthalen-2-yl)-4-methylpent-3-enylfuran-2-caroxylate (SH-7) (Fig. 1) was one of the most potent compound. Thus, in this paper, we preferentially aim to address the possible mechanisms underlying its antitopoisomerase-associated events and the comprehensive anticancer activities of SH-7 both in vitro and in vivo.

thumbnail image

Figure 1. Chemical structure of Shikonin and SH-7.

Download figure to PowerPoint

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Compounds

Synthesis of SH-7
General

The melting point was recorded on a Fisher-John melting point apparatus and is uncorrected. 1H data were obtained on a Varian Unity 300 MHz NMR spectrometer. The chemical shifts are relative to the trace proton signal of the deuterated chloroform. Mass spectroscopic experiments were performed on MAT-711 and MAT-95 Mass Spectrometers. Flash column chromatography was performed on silica gel 60, 200–300 mesh. All of the starting materials were obtained from commercial sources.

1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enylfuran-2-caroxylate

Shikonin (144 mg, 0.5 mmol) was dissolved in methylene chloride (20 ml) and to the resulting mixture was added dicyclohexyl carbodiimide (113 mg, 0.55 mmol), 4-dimethylaminopyridine (3 mg, 0.025 mmol) and furan-2-carboxylic acid (30 mg, 0.5 mmol). The reaction mixture was stirred at room temperature, and reaction was completed in 3 hr. After completion of the reaction, petroleum (20 ml) was added to the reaction mixture. Precipitates was filtered off, and filtration was concentrated to dryness under reduced pressure, the remaining residue was subjected to flash column chromatography (silica gel, ethyl acetate:petroleum = 1:10), yielding a red oily gum, which was crystallized with a mixed solvent of petroleum and ethyl acetate to give a red powder (97 mg, 49%): mp 103-105°C; 1H NMR (CDCl3) δ (multiplet, m; singlet, s) 1.61 (s, 3H) 1.70 (s, 3H), 2.68 (m, 2H), 5.25 (m, 1H), 6.22 (m, 1H), 6.58 (m, 1H), 7.08 (s, 1H), 7.19 (s, 2H), 7.26 (m, 1H), 7.64 (s, 1H), 12.43 (s, 1H), 12.61 (s, 1H); EIMS (m/z) 382 (M+), 270 (100) 95; HRMS (EI) Calc. for C21H18O7 382.1053, Found 382.1045; Anal. (C21H18O7) C, H (Calc. C65.96 H4.74; Found C65.79 H4.87).

Samples for biological experiments

SH-7 was dissolved in dimethylsulfoxide (DMSO) to the concentration of 0.1 M (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. DMSO (0.1%) and ethanol (1%) were used as a vehicle control throughout the study.

Camptothecin (CPT), doxorubicin (DOX), vincristine (VCR) and etoposide (VP-16) were purchased from Sigma (St. Louis, MO). 10 mM DOX, VCR and VP-16 were prepared with normal saline as stock solution. All stock solutions were stored at -20°C, thawed and diluted with complete medium prior to each experiment.

Animals

Seven-weeks-old specific pathogen free (SPF) female KM mice (weight, 18–22 g) were obtained from Shanghai Laboratory Animal Center, Chinese Academy of Sciences. BALB/cA nu/nu female mice aged 4–5 weeks were bred in the 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 performed according to institutional ethical guidelines on animal care.

Cell lines and culture

Human gastric adenocarcinoma cell line SGC-7901, hepatocellular carcinoma cell line BEL-7402, cervical carcinoma cell line Hela and ovarian epitheloid carcinoma cell line HO-8910 were obtained from the cell bank of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Human lung adenocarcinoma cell line A549 was from the National Cancer Institute (NCI, USA). Human hepatocellular carcinoma cell line SMMC-7721 was a gift from the Second Military Medical School. Human premyelocytic leukemia cell line HL-60, chronic myelogenous leukemia (CML) K562, hepatocellular carcinoma Hep-G2, oral epidermoid carcinoma KB, colorectal adenocarcinoma HT-29 and lung fibroblast WI-38 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). Human gastric adenocarcinoma cell line MKN-28, MKN-45, lymphoblastic leukemia cell line MOLT-4, colorectal carcinoma cell line HCT-116, breast carcinoma cell line MCF-7, MDA-MB-435 and ovarian carcinoma cell line OVCAR-5, SK-OV-3 were from Japanese foundation of Cancer Research (JFCR, Tokyo, Japan). Rhabdomyosarcoma (RMS) RH-30 cell line was a gift from St. Jude Children's Research Hospital (Memphis, TN). Both DOX-selected multidrug resistant (MDR) cell subline K562/A0213 and MCF-7/ADR14, 15 were purchased from the Institute of Hematology, Chinese Academy of Medical Sciences (Tianjin, China). The VCR-selected MDR KB/VCR16 subline was obtained from Zhongshan University of Medical Sciences (Guangzhou, China). All these cell lines except MCF-7 were maintained in RPMI-1640 medium (GIBCO, Grand Island, NE) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO), L-glutamine (2mM), penicillin (100 IU/ml), streptomycin (100 μg/ml) and HEPES (10 mM) (MCF-7 with additional 1 mM 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.

Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins by 0.1% type-I collagenase digestion at 37°C for 15 min and checked by immunofluorescence for von willebrand factor antibody (Sigma). HUVECs were cultured in M199 medium (GIBCO) supplemented with 20% heat-inactivated fetal bovine serum (GIBCO), 30 μg/ml ECGS (Beckon Dickinson Labware, MA) and 10 ng/ml EGF (Sigma). Cells at 3–7 passages were used in the experiments. Human microvascular endothelial cell line (HMEC-1) was obtained from American Type Culture Collection (ATCC) and propagated in MCDB131 (GIBCO) with 20% heat-inactivated fetal bovine serum (GIBCO), 30 μg/ml ECGS (BD), 10 ng/ml EGF (Sigma) and 1 μg/ml hydrocortisone (Sigma).

Topo I- and Topo II-mediated supercoiled PBR322 relaxation

DNA relaxation assays were based on the reported procedure.17 Reaction buffer contained 10 mM Tris–HCl (pH 7.9), 50 mM KCl, 50 mM NaCl, 5 mM MgCl2, 0.1 mM EDTA, 15 mg/ml of bovine serum albumin (BSA), 1 mM ATP (ATP was omitted in Topo I-mediated DNA relaxation), 12.5 μg/ml supercoiled pBR322 for Topo I assay (6.67 μg/ml for Topo II assay) and 0.01 unit/μl of Topo I (0.0467 units/μl Topo IIα) (TopoGEN, Columbus, OH). Relaxation was employed at 37°C for 15 min and was stopped by addition of 2 μl of 10% SDS. Electrophoresis was carried out in a 1% agarose gel in 1× TAE (40 mM Tris base, 40 mM acetate acid and 1 mM EDTA) at 4 V/cm for 1 hr. DNA bands were stained with 0.5 mg/ml of ethidium bromide (E.B.) solution and photographed through a ChemiGenius 2 Gel Documentation System (Syngene, Cambridge, UK).

kDNA decatenation assay

Topo II activity was measured by the ATP-dependent decatenation of kDNA.18 The standard reaction mixture consisted of 50 mM Tris–HCl (pH 7.7), 50 mM KCl, 5 mM MgCl2, 1 mM ATP, 0.5 mM dithiothreitol (DTT), 0.5 mM EDTA, 50 mg/ml of BSA, 20 μg/ml of kDNA and 1 unit of Topo IIαin a total volume of 15 μl. After incubation at 37°C for 15 min, the reaction was terminated by addition of 1 μl of 10% SDS. The DNA samples were subjected to electrophoresis under the same conditions as described earlier.

Trapped in agarose DNA immunostaining (TARDIS) assay

The experiment is operated strictly according to the previous report.19 Briefly, HL-60 cells (5 × 105 per each well) were incubated with SH-7 for 6 h in 6-well plates. Then, cells are embedded in agarose onto a microscope slide. Slides were immersed in lysis buffer (1% SDS (w/v), 80 mM Na2HPO4, 80 mM NaH2PO4, 10 mM EDTA, pH = 6.8, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 mM phenylmethysulfonyl fluoride, 1 mM dithiothreitol) to disrupt the cell membranes, and then were washed in 1 M NaCl. Only the proteins covalently bound to DNA remained. Slides were incubated with Topo IIα antibody (Santa cruz, CA), which binds to Topo II trapped in cleavable complexes on DNA. Finally, the complexes were made visible under microscope by using a secondary Alexa Fluro 488-conjugated antibody (Molecular Probes, Eugene, OR).

Cell proliferation assay

Cell proliferation was evaluated by SRB assay.20 Cells were seeded into 96-well plates and cultured over night. The cells were treated with SH-7 for 72 hr (VP-16 as a positive control). Medium was dumped immediately after the drug treatment (suspended cells were spin at 3,000 rpm for 10 min and medium was sip up carefully), and the cells were then treated with 100 μl of 10% precooled TCA in each well. After that, cells were fixed for 1 hr at 4°C. The plate was washed 5 times with distilled water and dried. 100 μl of 4 mg/ml SRB (Sigma) in 1% acetic acid was added to each well for 15 min. Then plate was washed 5 times with 1% acetic acid and dried. SRB in the cells was dissolved in 150 μl of 10 mM Tris–HCl and was measured at 515 nm using a multiwell spectrophotometer (VERSAmax, Molecular Devices, USA). The inhibition rate on cell proliferation was calculated as (1 − (A515 treated/A515 control)) × 100%. IC50 value was obtained by Logit method and was determined from the results of at least 3 independent tests. The resistance factor (RF) to each drug was calculated as the ratio of the IC50 value of resistant cells to that of parental cells.21

AnnexinV–propidium iodide binding assay

Quantification of early apoptosis by measuring phosphatidylserine (PS) exposure was carried out by annexin V (Sigma) staining. Briefly, following 2 hr treatment of agents, cells were resuspended with the cold binding buffer. Subsequently, 5 μl of annexin-V-FITC and 5 μl of propidium iodide (PI) were added and the cells were incubated for 10 min in dark at room temperature. Flow cytometry analysis was performed using a FACS-Calibur cytometer (Becton Dickinson, San Jose, CA). The annexin-V+/PI- cells were defined as early apoptotic cells.22

PI staining for flow cytometry

HL-60 cells (5×105 each well) were seeded into 6-well plates and treated with SH-7 for 6 hr. Cells were harvested and washed once with cold phosphate buffer saline (PBS). Then cells were fixed in 70% ethanol on ice for 15 min. Staining went along in PBS containing 40 μg/ml RNase and 10 μg/ml PI at room temperature in dark for 30 min. Cells were then analyzed using an FACS-Calibur cytometer (Becton Dickinson, San Jose, CA). The cells undergoing apoptosis was obtained from the distinct subdiploid region of the DNA distribution histograms. At least 10,000 events were counted for each sample.

Terminal transferase dUTP nick endlabeling (TUNEL) assay

TUNEL assay was performed according to the manufacturer's instructions (Roche, Basel, Switzerland). Briefly, HL-60 cells (5 × 105) were incubated for 6 hr with SH-7 in indicated concentrations and vehicle control and stained for terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end labeling (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 hr at 37°C. Samples were washed with PBS and observed in a fluorescent microscope (Olympus BX51, Tokyo, Japan).

Agarose electrophoresis of DNA fragmentation

HL-60 cells (0.8 to 1.0 × 106 per each well) were seeded into a six well plate. SH-7 was added at the indicated concentrations for 6 hr. DNA fragmentation was extracted using the method on a previous report.23 Briefly, harvested cells were lysed by equal volume of 1.2% SDS. By adding 7/10 volume of precipitation solution (3 M CsCl, 1 M potassium acetate, 0.67 M acetic acid) and spinning for 15 min at 14,000g, DNA fragmentation was kept in the supernatant, which was then absorbed by a miniprep spin column. Finally, DNA was eluted with 50 μl TE buffer (pH = 8.0). This production can be loaded into an agarose gel and run at 80 V for about 50 min.

Measurement of mitochondrial transmembrane potential (Ψm)

Variations of mitochondrial transmembrane potential (Ψm) were assessed in cells plated and treated with SH-7 and vehicle under the same conditions described for AnnexinV-propidium iodide binding assay. Then cells were stained with the fluorochrome DiOC6(3) (40 nM) (Calbiochem, San Diego, CA) as described previously24 and analyzed by flow cytometry.

Western Blotting Analysis

The cytosol fraction was prepared using the method previous described25 and modified slightly. In brief, after treated with SH-7 for 6 hr, HL-60 cells were harvested. And then the cell pellets were resuspended at 5 × 107 cells/ml in extraction buffer (20 mM HEPES, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM phenyl-methyl sulfonyl fluoride (PMSF) and 250 mM sucrose) and homogenized using a microhomogenizer. The homogenates were centrifuged at 750g for 10 min at 4°C. The supernatants were centrifuged at 10,000g for 15 min at 4°C, the remained supernatant was designated as the cytosol fraction. The whole cell lysate was prepared by resuspending cell pellets in lysis buffer (0.15 M NaCl, 50 mM Tris–HCl (pH 8.0), 0.5% deoxycholic acid (Sodium Salt), 0.02% Sodium azide, 1% NP-40, 2.0 μg/ml aprotinin, 100 mM PMSF) on ice for 1h and then centrifuged at 10,000g for 10 min.

Equal amounts of proteins were separated on 12% (Cytochrome c, p-H2AX (Ser139), caspase-3) or 8% (PARP) Tris-glycine-SDS polyacrylamide gels and electroblotted onto nitrocellulose membranes. Proteins were identified with rabbit-antiphospho-histone H2A.X (Ser139) polyclonal antibody (1:1,000) (Cell signaling, Beverly, MA), rabbit-anticytochrome c polyclonal antibody (1:1,000) (Cell signaling), rabbit-anti-PARP polyclonal antibody (1:1,000) (Santa Cruz), mouse-anticaspase-3 polyclonal antibody (1:1,000) (Santa Cruz) and goat-anti-β-actin polyclonal antibody (1:1,000) (Santa cruz). Detection was performed using horseradish peroxidase-conjugated secondary antibody and SuperSignal West Pico Chemiluminescent Substrate (Pierce Inc, Rockford, IL) according to the manufacturer's instructions.

Antitumor activity assay in vivo

Female KM mice were inoculated subcutaneously into right armpit with 2 × 106 S-180 sarcoma cells. After 24 hr, daily treatment with SH-7 or vehicle was administrated by i.v. injection for 7 days. Subsequently, mice were killed by cervical dissociation, and the tumors were excised and weighed. The inhibition rate was calculated as (Average tumor weight of NS group − Average tumor weight of test group)/Average tumor weight of NS group × 100%.

Human hepatocellular carcinoma SMMC-7721, BEL-7402 and prostate cancer PC-3 xenograftes were established by 5 × 106 cells subcutaneously inoculated in nude mice. The experiments began when the xenografts had 3 passages in nude mice. Under a sterilization condition, well-grown tumors were cut into 1 mm3 fragments, and the fragments were subcutaneously transplanted by trocar into the right flank in nude mice. When tumor reached a volume of 50–150 mm3, the mice were randomized to control and treated groups, and received vehicle or SH-7 at indicated doses by i.v. administration twice a week for 3 weeks. The size of tumors were measured individually twice per week with microcalipers. Tumor volume (V) was calculated as V = (length × width)2/2. The individual relative tumor volume (RTV) was calculated as follows: RTV= Vt/V0, where Vt 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 T/C % and the calculation formula is T/C (%) = mean RTV of the treated group/mean RTV of the control group × 100%.

Statistical analysis

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

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Inhibition of topoisomerases' activity

First, we detected SH-7's effect on the catalytic activity of Topo II by evaluating the enzyme-mediated negatively supercoiled pBR322 relaxation. As shown in Figure 2a, SH-7 displayed significant inhibition of this reaction in a concentration-dependent manner. 2.5 μM of SH-7 began to inhibit the activity of Topo II, and as the concentration is up to 10 μM, SH-7 made most of pBR322 in supercoiled sate. Likewise, 5 μM of shikonin intrigued the inhibition on the Topo II activity, and 10 μM of shikonin produced similar potency in suppressing Topo II's relaxation activity as SH-7 did. However, 10 μM of VP-16 failed to influence the activity of Topo II (data not shown), and it only exerted its inhibition at the concentration of 100 μM (Fig. 2a). It suggested that SH-7 exerted a slightly stronger inhibition efficiency on Topo II's relaxation activity than shikonin, and much stronger than VP-16.

thumbnail image

Figure 2. Effects of SH-7 and shikonin on Topoisomerase. (a) Supercoiled PBR322 relaxation assay for TOPO II. (b) kDNA decatenation assay for TOPO II. (c) Effect of SH-7 and shikonin on Topoisomerase I. The results are typical of those obtained in three independent experiments that gave similar results.

Download figure to PowerPoint

To further corroborate the SH-7's effect on Topo II, one more specific Topo II-mediated kDNA decatenation was carried out. Similarly, 20 μM of SH-7 pushed all the minicircles back to gel aperture, indicating that SH-7 completely restrained Topo II activity (Fig. 2b).

We also investigated the effects of SH-7 on Topo I-mediated supercoiled PBR322 relaxation. Results revealed that 100 μM of SH-7 inhibited almost all the relaxation activity of Topo I, paralleling to that of 100 μM CPT. However, 100 μM of shikonin failed to show any effect on Topo I. (Fig. 2c).

Stablization of cleavable complexes

Tardis assay is typically used to detect Topo II drug-stabilized cleavable complexes, which is crucial for understanding the key mechanism of topo II poison.26 The covalently bound topo II can be detected by immunofluorescence by staining with an antitopo IIα primary antibody followed by an Alexa Fluro 488-conjugated secondary antibody. There was no visible immunofluorescence associated with the DNA in untreated HL-60 cells (Fig. 3). When cells treated with SH-7, the staining intensity was increased dose-dependently. Green immunofluorescence was just visible in HL-60 cells treated with 20 μM SH-7, and the fluorescence was easily detectable in cells exposed to 40 μM SH-7. VP-16 also yielded very high levels of fluorescence at the concentration of 100 μM. However, little staining was observed in cells treated with 40 μM shikonin (Fig. 3).

thumbnail image

Figure 3. Fluorescence of SH-7 treated HL-60 cells. Cells were treated with SH-7 for 2 hr at indicated concentrations before embedding and staining. Upper images showed the DAPI-stained nuclei (blue) whereas lower ones showed corresponding Alexa Fluro 488-stained (green) immunofluorescence. These images were captured under 200× or 400× fluorescent microscope and are typical of those seen in replicate experiments.

Download figure to PowerPoint

Aggravation of DNA double strand breaks

Recently, an immunocytochemical assay capable of specifically recognizing rH2AX has become the gold standard for the detection of DNA double strand breaks (DSBs). This assay is currently accepted as being an extremely sensitive and specific indicator for the existence of only one DSB; specifically, one rH2AX focus correlates to one DSB.27, 28 Since topoisomerase II poison is well known to efficiently cause DSBs, we further examined whether SH-7 could induce DSBs. As illustrated in Figure 5, the level of phosphorylated-H2AX was significantly elevated in SH-7-treated cells when compared with that in the control. It began to increase upon exposure to 0.25 μM of SH-7, and remained at a high level in a dose-dependent manner (Fig. 4).

thumbnail image

Figure 4. Increase of phosphorylation of H2AX caused by SH-7. Whole cell lysate was prepared from HL-60 cells treated with SH-7 for 2 hr at indicated concentrations. The level of phosphorylation of H2A.X was examined by Western Blotting analysis. The result is typical of results obtained in three independent experiments that gave similar results.

Download figure to PowerPoint

thumbnail image

Figure 5. Inhibitory effects of SH-7 on cells proliferation. (Mean IC50 values were calculated from at least three independent experiments).

Download figure to PowerPoint

Inhibition of cell proliferation in vitro

The cytotoxicity of SH-7 was evaluated by SRB assay against a panel of human tumor cell lines including leukemia, lung cancer, oral epidermoid cancer, gastric cancer, hepatocellular cancer, colon cancer, breast cancer, cervical cancer, ovarian cancer and rhabdomyosarcoma. SH-7 displayed wide potent cytotoxicity in diversified cancer cell lines. The IC50 values varied from hundred nanomoles to dozen micromoles. For the sensitive cell lines such as lymphoblastic leukemia MOLT-4, lung adenocarcinoma A549 and rhabdomyosarcoma RH-30, the IC50 were 0.30, 0.46 and 0.47 μM, respectively. On the other hand, it exhibited weak inhibitory effects on chronic myelogenous leukemia K562, colorectal adenocarcinoma HT-29 and ovarian carcinoma OVCAR-5, SK-OV-3. The mean IC50 value for all the tested tumor cell lines was 7.75μM. This agent also had better activities on 2 endothelial cell lines HMEC and HUVEC with average IC50 of 1.95 μM. Though SH-7 exhibited various cytotoxic effects against different cell lines, it did not show significant tissue specificity (Fig. 5).

In addition, we investigated the effects of SH-7 on 3 MDR sublines, K562/A02,13 KB/VCR16 and MCF-7/ADR14, 15 tumor cells. Drug-sensitive parental K562, KB and MCF-7 cell lines and 3 conventional anticancer drugs (DOX, VCR and VP-16) were used as references. SH-7 displayed significant cytotoxicity in the 3 MDR sublines examined, with an average IC50 value of 17.02 μM, which was nearly equivalent to that observed in the corresponding parental cell lines (average IC50: 11.07) (Table I). The average resistance factor of SH-7 on MDR cells was 1.74, which was much lower than those of reference drugs VP-16 (RF 145.92), ADR (RF 105.97) and VCR (RF 197.39) (Table I).

Table I. Resistance Factors of SH-7 and Reference Drugs in MDR Sublines (Mean IC50 Values were Calculated from at Least Three Independent Experiments)
DrugIC50 (μM, X ± SD)Resistance factorAverage value of resistance factor
K562K562/A02KBKB/VCRMCF7MCF7/AdrK562/A02KB/VCRMCF7/ADR
  1. NT: not tested.

SH-716.2 ± 5.58.6 ± 2.611.5 ± 6.131.9 ± 7.65.5 ± 0.810.6 ± 4.60.52.81.91.7
VP-165.1 ± 0.9258.8 ± 84.50.5 ± 0.1163.0 ± 76.913.1 ± 2.2343.0 ± 84.950.6361.026.2145.9
ADR0.4 ± 0.246.6 ± 11.4NTNT1.6 ± 0.8150.3 ± 68.6117.0NT94.9106.0
VCRNTNT3.1 ± 1.60.6 ± 0.03NTNTNT197.4NT197.4

Apoptosis induced by SH-7

Topo II poisons play a role in stabilizing the otherwise fleeting enzyme-DNA intermediates, inducing DNA strand breaks, and then trigger the process of cell apoptosis. Accordingly, we investigated the ability of SH-7 to induce apoptosis in HL-60 cells using Annexin-V, TUNEL, internucleosomal DNA fragmentation and PI staining.

Annexin-V identifies the early stage of apoptosis while PI only stains late apoptotic cells and dead cells.29 Hereby, Annexin-V positive but PI negative cells were identified to be the early apoptotic cells, while both PI and Annexin-V positive cells were the late apoptotic and dead cells. In Figure 6A, we found that treatment of SH-7 at various concentrations for 2 h triggered HL-60 cells to enter into early apoptotic process, and 27.25% cells with well-characterized apoptosis were observed at the concentration of 1 μM.

thumbnail image

Figure 6. Effects of SH-7 and shikonin on inducing apoptosis of HL-60 cells. (a) AnnexinV-PI binding assay. Cells were treated with SH-7 for 2 hr at indicated concentrations. Cells were stained with Annexin V and PI before subjecting into FACScan for analysis. Lowerright part (annexin-V+/PI-) was considered as early apoptotic cells. (b) PI staining for flow cytometry. Cells were treated with SH-7 for 6 hr at 0.125, 0.25, 0.5 μM. FACS analyzed cells after they were fixed by 70% ethanol and stained with PI. Sub-G1 cells were collected as apoptotic cells. (c) TUNEL assay Cells were treated with SH-7 for 6 hr at indicated concentrations, and then were stained with dUTP-FITC by TUNEL method. Upper images showed the DAPI-stained nuclei (blue) whereas lower ones showed corresponding FITC (green) immunofluorescence. (d) Electrophoresis of DNA fragmentation (dose-dependent). Cells were treated with SH-7 for 6 hr at indicated concentrations, and fragmented DNA was extracted and separated in 1% agarose gel electrophoresis. (e) Electrophoresis of DNA fragmentation (time-dependent). Cells were treated with 2 μM of SH-7 for indicated times, and fragmented DNA was extracted and separated in 1% agarose gel electrophoresis. The results are typical of those obtained in three independent experiments that gave similar results.

Download figure to PowerPoint

Flow cytometry analysis, after PI staining, was another method to characterize the apoptosis process. About 46.6% HL-60 cells were detected to be apoptotic following the treatment of SH-7 at 0.25 μM for 6 hr. And over 50% of HL-60 cells underwent apoptosis when the concentration was up to 0.5 μM (Fig. 6b).

TUNEL assay is used to indicate late apoptotic cells. We found that HL-60 cells incubated with various concentrations of SH-7 for 6 hr underwent apoptosis, with obvious effects observed at the concentration of 0.25 μM, and severer apoptosis occurred at 0.5 μM (Fig. 6c).

We also examined the internucleosomal DNA fragmentation at various concentrationsof SH-7 and exposure times. Upon treatment with SH-7 for 6 hr, SH-7 (0.125–2 μM) dose-dependently induced internucleosomal DNA fragmentation (Fig. 6d). An SH-7 dose of 2 μM started to induce DNA ladder after 4 hr-treatment, and the fragmentation appeared more obvious accompanied with increase in time (Fig. 7e).

thumbnail image

Figure 7. Mitochondria dependent apoptosis induced by SH-7. (a) Loss of mitochondria membrane potential (MMP). After 2 h's treatment with SH-7, the MMP loss (characterized by left shifting of the peak) of HL-60 was detected by DiOC63 and analyzed by FACS. (b) Cytochrome c release. HL-60 cells were treated with SH-7 at the indicated concentrations for 6 hr. Cells were homogenized and lysate (cytosolic fraction) were assayed for cytochrome c levels by Western blotting analysis. The result is typical of those obtained in three independent experiments that gave similar results.

Download figure to PowerPoint

Apoptosis via mitochondria dependent pathway

The aforementioned result suggested that SH-7 exhibited apoptosis-inducing activity. Considering that the mitochondrial pathway is crucial for the induction of apoptosis, we thus hypothesize that the apoptosis-triggering impact of SH-7 might be related to the loss of mitochondrial membrane potential. To answer this issue, flow cytometry analysis after DiOC6(3) staining was used to test the membrane potential of mitochondria. The experimental results showed that SH-7 induced a dose-dependent drop in mitochondrial membrane potential (Fig. 7a), suggesting SH-7-indcued apoptosis might be mitochondria-dependent.

In light of this notion, we further tested whether SH-7 would trigger a release of cytochrome c from mitochondria. Treatment with SH-7 for 6 hr promoted cytochrome c release from mitochondria in a dose-dependent manner. As shown in Figure 7b, 1 μM of SH-7 induced a marked increase of cytochrome c in cytoplasm. Moreover, we evaluated the influence of SH-7 on specific proteolytic cleavage of PARP, a biochemical characteristic of apoptosis, and its upstream activator caspase-3. As a result, 0.25 μM of SH-7 began to make pro-caspase-3 and PARP cleaved, and 1 μM of SH-7 nearly make these proteins cleaved totally (Fig. 8). All these findings collectively verified that SH-7's apoptosis-inducing action is a mitochondria-targeting event.

thumbnail image

Figure 8. Effects of SH-7 on PARP and pro-caspase-3 in HL-60 cells. Cells were treated with SH-7 at the indicated concentrations for 6 hr. Whole cell lysate was prepared and assayed for PARP and pro-caspase-3 by Western blotting analysis. The results are typical of results obtained in three independent experiments that gave similar results.

Download figure to PowerPoint

In vivo antitumor activities

Because of the potent antitumor activity of SH-7 in vitro, its antitumor properties were examined further in vivo. SH-7 exerted an inhibitory effect of 51.1% at the dosage of 2 mg/kg on S-180 sarcoma after daily i.v. administration for 7 days (Table II). The antitumor efficacy of SH-7 was also tested in the human carcinoma BEL-7402, SMMC-7721 and PC-3 xenograft models in nude mice. As indicated in Table III, SH-7 began to show the BEL-7402 xenograft growth inhibition at the dosage of 2.5 mg/kg after i.v. injection twice a week for 3 weeks, and significant antitumor activity was observed at the dosage of 10 mg/kg with the T/C value of 47.4%. The similar tumor growth inhibitory effect of SH-7 was seen on SMMC-7721 xenograft (Table IV, Fig. 9). SH-7 exhibited more potent antitumor effect in human prostate PC-3 xenograft model. At the dosage of 10 mg/kg, the T/C value was 38.4% (Table V, Fig. 9).

thumbnail image

Figure 9. Tumor growth inhibition of SH-7 in nude mice. The animals were randomly divided into 4 groups and given iv injection of SH-7 or vehicle twice per week for a period of 3 weeks. Data are expressed as the mean ± SD of a typical experiment.

Download figure to PowerPoint

Table II. SH-7 Decreased Tumor Weight in Mice Implanted with S-180 Sarcoma
GroupDose (mg/kg)RoutesNumbersBWC (%)TW (g), mean ± SDTWI (%)
  • *

    p > 0.05.

  • **

    p < 0.01, compared with control group.

Control 2020+52.81.37 ± 0.86
SH-71i.v.1010+38.51.11 ± 0.76*19.0
2i.v.1010+36.20.67 ± 0.39**51.1
4i.v.1010+33.90.72 ± 0.34**47.4
Table III. Assessment of the Effects of SH-7 against Human Liver Tumor Xenocraft BEL-7402 in Nude Mice
GroupDose (mg/kg)RoutesNumberTV (mm3, mean ± SD)RTV (mean ± SD)T/C (%)
d0d29D0d29  
  • *

    p > 0.05, compared with control group.

Controli.v.101095 ± 282334 ± 121130.1 ± 15 
SH-710i.v.5597 ± 381271 ± 27514.2 ± 3.7*47.4
5i.v.5594 ± 501291 ± 65217.8 ± 9.859.1
2.5i.v.5597 ± 371652 ± 69217.6 ± 6.758.6
Table IV. Assessment of the Effects of SH-7 against Human Liver Tumor Xenocraft SMMC-7721 in Nude Mice
GroupDose (mg/kg)RoutesAnimalTV (mm3, mean ± SD)RTV (mean ± SD)T/C (%)
d0d29d0d29  
  • *

    p > 0.05, compared with control group.

Controli.v.1010144 ± 541971 ± 81316.5 ± 6.3 
SH-710i.v.55146 ± 511572 ± 10439.2 ± 4.7*55.9
5i.v.55145 ± 991241 ± 6939.3 ± 4.4*56.1
2.5i.v.55142 ± 1021341 ± 10489.8 ± 3.0*59.6
Table V. Assessment of the Effects of SH-7 against Human Prostate Tumor Xenograft PC-3 in Nude Mice
GroupDose (mg/kg)RoutesNumber of miceTV (mm3 mean ± SD)RTV (mean ± SD)T/C (%)
   d0d24d0d24  
  • *

    p > 0.05.

  • **

    p < 0.01, compared with control group.

Controli.v.101055 ± 301268 ± 35426.2 ± 8.0 
SH-710i.v.5557 ± 31576 ± 21610.1 ± 3.8**38.4
5i.v.5555 ± 26656 ± 24614.0 ± 6.4*54.4
2.5i.v.5557 ± 181025 ± 40318.0 ± 6.069.6

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References

Topoisomerases inhibitor is a kind of most active and widely prescribed antineoplastic drugs. There has been continuous interest in studying and developing new antitopo agents and several potential compounds are currently in clinical trial. Of a series shikonin derivatives, a new naphthazarine analogue, SH-7, was favorably selected for further preclinical development owing to its potent antitoposiomerase feature at molecular levels, in our initial experiments. Using the convincing techniques, we found that this agent significantly inhibited Topo II-mediated supercoiled pBR322 relaxation and kDNA decatenation, whereas the inhibitory efficacy on Topo I was much lower than that of Topo II. In addition, SH-7 could obviously increase the level of p-H2A.X and may stabilize the Topo II-DNA “cleavable complex” in Tardis assay, resembling other known Topo II poisons. All these findings demonstrated that SH-7 is a new topoisomerase II inhibitor with the inhibitory potency on topoisomerases stronger than its mother compound shikonin.

Though chemical agents interfering with DNA topoisomerases are widespread in nature, yet only a few might highlight the clinical efficacy as promising antitumor drugs. SH-7 retained a superior cytotoxic effect on diversified tumor cell lines. The wide sensitivity on cell lines appears to be identical to those of other known clinic-used topoisomerases inhibitors. Encouragingly, a marked activity was observed in SH-7-treated group at doses as low as 2 mg/kg/day against sarcoma 180, and 5 mg/kg twice per week against human tumor xenocrafts. Comparatively, the inhibition rate of sarcoma 180 only reached 29.6% when treated with shikonin at 4 mg/kg/day (i.p.) for 7 days (data not shown), similar to the result got by Sankawa et al.30 It is worth noting that the mice in SH-7-treated group showed a rapid body weight rise after treatment when compared to the shikonin-treated mice. Moreover, another very important finding of SH-7 was that it possessed much stronger cytotoxic activity against MDR tumor cells than several clinically wideused anticancer durgs, including VP-16, ADR and VCR. MDR is a major clinical problem in treating human cancers with conventional chemotherapeutic drugs. The development of novel anticancer drugs insensitive to or bypassing MDR mechanisms is currently a major focus of research. The effectiveness activity in killing MDR tumor cells of SH-7 in vitro furtherly implies its value of anticancer therapy.

The chemotherapeutic efficacy of topoisomerases poisons is suggested to be due to stabilization of a topoisomerase-DNA-cleavable complex, leading to protein-linked DNA breaks and thus activating apoptosis cascade.31 SH-7's remarkable apoptosis-inducing potential was authenticated in HL-60 cells. Of the 2 primary caspase activation pathways (death receptor-mediated and mitochondrially mediated), the mitochondrial pathway is most commonly associated with apoptosis triggered by cytotoxic stress. In fact, a decrease in mitochondrial membrane potential (Ψm) due to the mitochondria permeability transition is an early event in apoptosis,32, 33, 34, 35 and the release of cytochrome c is another key event in the mitochondria-dependent cell apoptotic pathway.36, 37 In current studies, administration of SH-7 preferentially decreased the susceptibility of mitochondria, and subsequently allowed the translocation of cytochrome c from the mitochondrial intermembrane space into the cytosol. As a consequence, SH-7 eventually activates caspase-3 and PARP. All these putatively support that SH-7's inhibitory action on topoisomerase activities account for the mitochondria-targeted apoptosis-inducing action and thus its broad-spectra antitumor activities in vitro.

In summary, we have demonstrated for the first time that SH-7, a favorable Topo II inhibitor and thus apoptosis-inducer, possesses potent inhibitory effects on the growth of tumor both in vitro and in vivo. The well-defined antitumor activities of SH-7, particularly with its obvious anti-MDR action, better solubility and less toxicity overwhelming over its parent compound shinkonin, may be an attractive aspect upon further development. Of significance is its characterized mechanisms with its unique attributes will alternatively provide insights into the rationale optimization of shinkonin-like structures to develop novel anticancer drugs.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. References
  • 1
    Lock RB, Ross WE. DNA topoisomerases in cancer therapy. Anticancer Drug Des 1987; 2: 15164.
  • 2
    Pommier Y, Pourquier P, Fan Y, Strumberg D. Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme. Biochim Biophys Acta 1998; 1400: 83105.
  • 3
    Liu LF, Liu CC, Alberts BM. Type II DNA topoisomerases: enzymes that can unknot a topologically knotted DNA molecule via a reversible double-strand break. Cell 1980; 19: 697707.
  • 4
    Liu LF. DNA topoisomerase poisons as antitumor drugs. Annu Rev Biochem 1989; 58: 35175.
  • 5
    Fu SL, Shang TM, Xiao PG. Analysis of naphthaquinone pigments in Chinese medicinal “zicao”, Arnebia euchroma. Yao Xue Xue Bao 1984; 19: 9215.
  • 6
    Guo XP, Zhang XY, Zhang SD. Clinical trial on the effects of shikonin mixture on later stage lung cancer. Zhong Xi Yi Jie He Za Zhi 1991; 11: 5989.
  • 7
    Yoon Y, Kim YO, Lim NY, Jeon WK, Sung HJ. Shikonin, an ingredient of Lithospermum erythrorhizon induced apoptosis in HL60 human premyelocytic leukemia cell line. Planta Med 1999; 65: 5325.
  • 8
    Ahn BZ, Baik KU, Kweon GR, Lim K, Hwang BD. Acylshikonin analogues: synthesis and inhibition of DNA topoisomerase-I. J Med Chem 1995; 38: 10447.
  • 9
    Plyta ZF, Li T, Papageorgiou VP, Mellidis AS, Assimopoulou AN, Pitsinos EN, Couladouros EA. Inhibition of topoisomerase I by naphthoquinone derivatives. Bioorg Med Chem Lett 1998; 8: 338590.
  • 10
    Fujii N, Yamashita Y, Arima Y, Nagashima M, Nakano H. Induction of topoisomerase II-mediated DNA cleavage by the plant naphthoquinones plumbagin and shikonin. Antimicrob Agents Chemother 1992; 36: 25892594.
  • 11
    Lu Q, Liu W, Ding J, Cai J, Duan W. Shikonin derivatives: synthesis and inhibition of human telomerase. Bioorg Med Chem Lett 2002; 12: 13758.
  • 12
    Hisa T, Kimura Y, Takada K, Suzuki F, Takigawa M. Shikonin, an ingredient of Lithospermum erythrorhizon, inhibits angiogenesis in vivo and in vitro. Anticancer Res 1998; 18: 78390.
  • 13
    Yang CZ, Luan FJ, Xiong DS, Liu BR, Xu YF, Gu KS. Multidrug resistance in leukemic cell line K562/A02 induced by doxorubicin. Zhongguo Yao Li Xue Bao 1995; 16: 3337.
  • 14
    Fairchild CR, Moscow JA, O'Brien EE, Cowan KH. Multidrug resistance in cells transfected with human genes encoding a variant P-glycoprotein and glutathione S-transferase-pi. Mol Pharmacol 1990; 37: 8019.
  • 15
    Cowan KH, Batist G, Tulpule A, Sinha BK, Myers CE. Similar biochemical changes associated with multidrug resistance in human breast cancer cells and carcinogen-induced resistance to xenobiotics in rats. Proc Natl Acad Sci USA 1986; 83: 932832.
  • 16
    Zhang XH, Zhang FY, Ji XJ, Li ZY. Vincristine-resistant human KB cell line and mechanism of multidrug resistance. Yao Xue Xue Bao 1994; 29: 24651.
  • 17
    Osheroff N, Shelton ER, Brutlag DL. DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA. J Biol Chem 1983; 258: 953643.
  • 18
    Tanabe K, Ikegami Y, Ishida R, Andoh T. Inhibition of topoisomerase II by antitumor agents bis(2,6-dioxopiperazine) derivatives. Cancer Res 1991; 51: 49038.
  • 19
    Willmore E, Frank AJ, Padget K, Tilby MJ, Austin CA. Etoposide targets topoisomerase IIα and IIβ in leukemic cells: isoform-specific cleavable complexes visualized and quantified in situ by a novel immunofluorescence technique. Mol Pharmacol 1998; 54: 7885.
  • 20
    Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 1990; 82: 110712.
  • 21
    Miao ZH, Tang T, Zhang YX, Zhang JS, Ding J. Cytotoxicity, apoptosis induction and downregulation of MDR-1 expression by the anti-topoisomerase II agent, salvicine, in multidrug-resistant tumor cells. Int J Cancer 2003; 106: 10815.
  • 22
    Li L, Krajewski S, Reed JC, Choi YS. The apoptosis and proliferation of SAC-activated B cells by IL-10 are associated with changes in Bcl-2, Bcl-xL, and Mcl-1 expression. Cell Immunol 1997; 178: 3341.
  • 23
    Steinfelder HJ, Quentin I, Ritz V. A fast and sensitive technique to study the kinetics and the concentration dependencies of DNA fragmentation during drug-induced apoptosis. J Pharmacol Toxicol Methods 2000; 43: 7984.
  • 24
    Amarante-Mendes GP, Finucane DM, Martin SJ, Cotter TG, Salvesen GS, Green DR. Anti-apoptotic oncogenes prevent caspase-dependent and independent commitment for cell death. Cell Death Differ 1998; 5: 298306.
  • 25
    Miura K, Aminova L, Murayama Y. Fusarenon-X induced apoptosis in HL-60 cells depends on caspase activation and cytochrome c release. Toxicology 2002; 172: 10312.
  • 26
    Osheroff N. Effect of antineoplastic agents on the DNA cleavage/religation reaction of eukaryotic topoisomerase II: inhibition of DNA religation by etoposide. Biochemistry 1989; 28: 615760.
  • 27
    Sedelnikova OA, Rogakou EP, Panyutin IG, Bonner WM. Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody. Radiat Res 2002; 158: 48692.
  • 28
    Takahashi A, Ohnishi T. Does γH2AX foci formation depend on the presence of DNA double strand breaks? Cancer letters 2005; 229: 1719.
  • 29
    van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 1998; 31: 19.
  • 30
    Sankawa U, Ebizuka Y, Miyazaki T, Isomura Y, Otsuka H. Antitumor activity of shikonin and its derivatives. Chem Pharm Bull (Tokyo) 1977; 25: 23925.
  • 31
    Pommier Y, Leteurtre F, Fesen MR, Fujimori A, Bertrand R, Solary E, Kohlhagen G, Kohn KW. Cellular determinants of sensitivity and resistance to DNA topoisomerase inhibitors. Cancer Invest 1994; 12: 53042.
  • 32
    Zoratti M, Szabo I. The mitochondrial permeability transition. Biochim Biophys Acta 1995; 1241: 13976.
  • 33
    Zamzami N, Marchetti P, Castedo M, Zanin C, Vayssiere JL, Petit PX, Kroemer G. Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. J Exp Med 1995; 181: 166172.
  • 34
    Zamzami N, Marchetti P, Castedo M, Decaudin D, Macho A, Hirsch T, Susin SA, Petit PX, Mignotte B, Kroemer G. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 1995; 182: 36777.
  • 35
    Brennan J, Mahon G, Mager DL, Jefferies WA, Takei F. Recognition of class I major histocompatibility complex molecules by Ly-49: specificities and domain interactions. J Exp Med 1996; 183: 15539.
  • 36
    Green DR. Apoptotic pathways: the roads to ruin. Cell 1998; 94: 6958.
  • 37
    Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 2001; 15: 292233.