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Teroxirone inhibited growth of human non-small cell lung cancer cells by activating p53
Toxicology and Applied Pharmacology 273 (2013) 110–120
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Teroxirone inhibited growth of human non-small cell lung cancer cells by activating p53 Jing-Ping Wang a,1, Kai-Han Lin a,1, Chun-Yen Liu a, Ya-Chu Yu a, Pei-Tsun Wu a, Chien-Chih Chiu b, Chun-Li Su c, Kwun-Min Chen d, Kang Fang a,⁎ a
Department of Life Science, National Taiwan Normal University, Taipei, Taiwan Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan Department of Human Development and Family Studies, National Taiwan Normal University, Taipei, Taiwan d Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan b c
a r t i c l e
i n f o
Article history: Received 10 June 2013 Revised 26 July 2013 Accepted 7 August 2013 Available online 15 August 2013 Keywords: Teroxirone Human non-small-cell-lung-cancer cells Apoptosis p53
a b s t r a c t In this work, we demonstrated that the growth of human non-small-cell-lung-cancer cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutant p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells. © 2013 Elsevier Inc. All rights reserved.
Introduction Compounds with epoxy groups have attracted a great attention as perspective cancer therapy targets (Atassi et al., 1980, 1984; Fischer et al., 1984). Among them, the triepoxide derivative teroxirone (1,3,5triazine-2,4,6(1H,3H,5H)-tri-one-1,3,5-tri-(oxiranylmethyl), Fig. 1A) has been known active against a variety of tumors. The effectiveness of teroxirone was demonstrated in P388 and L1210 leukemia cell lines that are resistant to cyclophosphamide treatment (Atassi et al., 1980). As an alkylator, teroxirone has reportedly underdone phase I and II evaluation of clinical studies (Neidhart et al., 1984; Nicaise et al., 1986; Rubin et al., 1987). The antineoplastic effectiveness and the broad spectrum of antitumor activity together with its novel structural characteristics could be of potential clinical value (Spreafico et al., 1980). As a chemotherapeutic agent effective in eradicating tumors in different models, the drug has also been known helping patients recovering from leukemia and lymphomas (Atassi et al., 1980; Dombernowsky et al., 1983; Piccart et al., 1981; Spreafico et al., 1980). In spite of the distinct effectiveness in patients and in animal models, teroxirone remains poorly understood due to shortage of systematic and detailed biological study associated with the drug activity.
⁎ Corresponding author. Fax: +886 2 26749172. E-mail address: [email protected] (K. Fang). 1 The two authors contributed equally to this work. 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.08.007
Besides, the underlying mechanisms associated with it remain largely unexplored. In this work, we showed that low concentrations of teroxirone suppressed the growth of human non-small-cell-lung-cancer (NSCLC) cells. The induced apoptotic cell death can be reverted by caspase-3 inhibitor DEVD-CHO. The reduced cell viability is closely related to p53-activated apoptosis. Furthermore, the drug inhibited the growth of tumors in nude mice model. Teroxirone provides a good candidate for lung cancer treatment by suppressing proliferation in cell culture and animal models. Materials and methods Chemicals. The available synthetic teroxirone is of more than 98% purity. A stock solution of 10 mM in dimethyl sulfoxide (DMSO) was stored at −20 °C, and freshly dissolved in media. The DMEM medium, penicillin and streptomycin antibiotic mixture, sodium pyruvate and glutamine supplements were obtained from Sigma (St. Louis, MO). Fetal bovine serum was acquired from Invitrogen (Grands Islands, NY). Cell culture. The human lung cell carcinoma cell lines, H460, H1299 and A549 were obtained from ATCC. The acquired cells were thawed, grown and maintained in DMEM. All cells were supplemented with L-glutamine, sodium pyruvate, and supplemented with 10% heatinactivated FBS in the humidified atmosphere with 5% CO2 at 37 °C. All cell lines were periodically examined and found free of mycoplasma
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Fig. 1. (A) The chemical structure of the teroxirone (1,3,5-triazine-2,4,6(1H,3H,5H)-tri-one-1,3,5-tri-(oxiranylmethyl)). (B) Growth curves of H460, A549 and H1299 cells in media containing teroxirone. A total of 1.5 × 103 cells were cultured in 1% serum-supplemented DMEM. Twenty-four hours later, the cells were incubated with 2, 5 or 20 μM teroxirone for 48 h. The cells were then stained with MTT and converted into viability as specified in Materials and methods. Results are indicated as cell viability with teroxirone against controls, as determined from three independent experiments. Data are represented as the mean values ± standard deviation (SD). The asterisks (***) indicated a significant difference compared with the vehicle control DMSO (P b 0.005). (C) Colony formation assay. The trypsinized cells were plated in 12-well dishes of different densities (1000 cells of H460/well, 500 cells of A549/well and 200 cells of H1299/well). The attached cells were treated with 0, 0.5 and 1 μM of teroxirone, respectively, for 48 h. The media were replaced with fresh media and incubated at 37 °C. Eleven days later, the cells were fixed and stained with 10% methylene blue in 70% ethanol. (D) Statistical analysis of colony formation. The numbers of colonies, defined as more than 50 cells/ colony were counted and the remaining fractions were converted as the ratio of the numbers of colonies in the samples with treatment vs. those with vehicle control DMSO. Triplicate wells were set up for each condition. (E) Comet assay analysis. Cells were treated for 12 h with teroxirone at the concentrations as specified. DNA tail formation was determined in H460, H1299 cells and A549 cells after teroxirone treatment according to the protocol as specified in Materials and methods. Teroxirone caused DNA damage in all cell lines studied. (F) Relative scores of DNA damage in teroxirone-treated cells. Mean values of the average comet tail moment (percentage of DNA in tail × tail length) for the three fields were calculated. The relative scores of average comet tail were obtained by converting tail moment lengths of DNA tail formation in all cell lines of teroxirone treatment at different concentrations and compared those with vehicle control. Data represented three independent experiments of the mean values ± standard deviation (SD).
contamination using a MycoTect kit (Invitrogen, Grands Islands, NY). The selected stable H1299 clones transfected with cytomegalovirus promoter-driven pcDNA-p53 that encodes full-length wild type p53 (H1299/p53) or mutant p53R267L (H1299/p53R267L) were maintained in 10% serum-supplemented DMEM. Cell viability determination. Cell cytotoxicity was measured using the MTT assay (Crespo et al., 2011). For the assay, 1.5 × 103 cells per well were seeded on 96-well plates and cultured in 1% serumsupplemented DMEM. The suspension was incubated at 37 °C for 24 h to allow cell attachment. The mononuclear cells were incubated with different concentrations of teroxirone for the specified time. DMSO was added to the cell culture used as control at final concentration of 0.1%. After the specific exposure time, the medium was removed and then MTT assays were performed. Cells from each well were incubated with 10 μl of MTT (5 mg/ml) in PBS at 37 °C for 3 h. After this, the MTT was removed from wells and 100 μl DMSO was added into each
well. The amount of formazan formed was determined by measuring the absorbance at 570 nm using a 96-well microplate reader (Thermo Fisher Scientific, USA). The viability assays were performed in triplicate in three separate experiments. Cell viability was expressed as percentage of the vehicle controls. The results were presented as mean ± standard deviation. Statistical differences between two groups were determined by a two-tailed unpaired Student's t-test. P b 0.05 was considered significantly different. Comet assay. The comet assay was performed according to the method of the published work with minor modifications (Gualtieri et al., 2005; Tice et al., 2000). Briefly, conventional slides were covered with a layer of 70 μl 0.5% normal agarose and 0.5% low melting point agarose (GIBCO-BRL). An amount of 70 μl of low melting point agarose (0.5%, w/v) (GIBCO-BRL) was mixed with approximately 2 × 104 cells suspended in 15 μl; the mixture was then layered onto the slides, and immediately overlaid with coverslips. After
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Fig. 2. Apoptosis assay by annexin V-FITC and PI staining. Cells cultured in 1% serum-supplemented DMEM were incubated with different concentrations of teroxirone for 36 h before double staining with annexin-V and PI and the percentage of each quadrant determined by WinDM. The numbers on top right quadrant meant percentage distributions of late apoptosis; while those on the bottom right correspond to those of early apoptosis (top). The results on the right represented mean values ± SD of three individual experiments. All experiments were done independently in triplicate per experimental point, and representative results shown indicating early and late apoptosis, respectively, of different cells (bottom).
agarose solidification at 25 °C for 30 min, the coverslips were removed and the slides were immersed 60 min at 4 °C in fresh lysing solution (2.5 M NaCl; 100 mM Na2EDTA, 10 mM Tris, pH 10 containing 1% Triton X-100). The slides were equilibrated in alkaline solution (1 mM Na2EDTA, 300 mM NaOH) for 20 min and placed in alkaline electrophoresis solution (200 mM NaOH, 1 mM EDTA). The slides were neutralized by immersing in Tris buffer (0.4 M, pH 7.5) followed by distilled water and then soaked in methanol for 5 min. The air-dry slides were then stained with 5 μg/ml propidium iodide (PI) and viewed under an epifluorescence microscope (Nikon, Japan) at 460 nm for visual scoring. Visual image analyses of DNA damage were carried out in accordance with previously reported protocols (Lim et al., 2011). A total of at least 20 non-overlapping comet images per gel were visually assigned a score on an arbitrary scale of 0 (round and intact without discernible tail) to 4 (almost all DNA migrated
towards the tail without apparent head) based on the perceived comet tail length migration and relative proportion of DNA in the comet tail. A mean DNA damage score for each slide was obtained by dividing the total damage score gained with the total number of comets analyzed by CometScore™ software. Flow cytometry and determinations for cell cycle analysis and apoptosis assay. Cells in the early and late phases of apoptosis were quantitated using annexin V-FITC/PI Apoptosis Detection Kit (BD, Mansfield, MA). Briefly, 2 × 105 cells cultured in 1% serum-supplemented DMEM were incubated with different concentrations of teroxirone for 36 h before double staining. The collected cells were washed twice with PBS. After centrifugation at 1200 rpm for 5 min, cell pellet was stained with 0.5 μl PI (50 μg/ml, BD) plus 0.5 μl annexin-V FITC (20 μg/ml, BectonDickinson) in annexin-V binding buffer for 30 min at room temperature
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Fig. 3. (A) Western blot analysis in cells treated with different concentrations of teroxirone. Cells were analyzed by western blot to determine gene expression at each concentration ranging 0, 1, 2 and 5 μM after 24 h of treatment. Equal amounts of cell lysate protein were separated by SDS-PAGE separating gel and electro-blotted. The blots were then incubated in fresh blocking solution and probed for 1 h with 1:3000 dilution of p21, PARP, p53 or Bcl-2 antibody, followed by incubation with a 1:4000 dilution of horseradish peroxidase-conjugated secondary antibody and then developed by ECL detection system. (B) Western blot analysis in cells treated with teroxirone for different time points. Cells were analyzed by western blot to determine gene expression at time points (0, 12, 18 and 24 h) with 2 μM of teroxirone. (C) Mitochondrial release of cytochrome c. The induced release of cytochrome c from mitochondria in cells treated with 2 or 5 μM teroxirone or vehicle control DMSO for 24 h. Cells were fixed, permeabilized and stained with anti-cytochrome c antibody at 4 °C for 18 h. After washing, cells were stained with Mitotracker Green (mitochondrial staining), DAPI (nuclear staining) and secondary antibody conjugated with TRITC for cytochrome c following the description in Materials and methods. The pointed arrow signified the co-localization of red color cytochrome c and green color mitochondria, while blue color stood for nucleus.
in the dark. Analysis was performed with FACSCalibur system (BD). The cell distributions were analyzed by Modfit software (Becton-Dickinson, Mansfield, MA). To determine cell cycle distribution, the cells were analyzed using FACS Calibur™ (BD Biosciences). A total of 2 × 106 cells were plated in different culture conditions. For sample preparation, both medium and trypsinized cells were centrifuged and then supernatant was removed. The collected cells were washed twice with PBS and then treated with 70% alcohol containing PBS for 24 h at −20 °C. Right before analysis, the sample cells were treated with 10 μg/ml PI (Sigma; St. Louis, MO), 10 μg/ml RNase A (ICN Pharmaceutical; Costa Mesa, CA) containing PBS
for 30 min in darkness. Data were analyzed by Modfit LT (Ver 2.0, Becton-Dickinson; Mountain View, CA). Analysis of cytochrome c release. Cells (1 × 105) were harvested and treated with 100 μl digitonin (50 μg/ml in PBS supplemented with 100 mM KCl and 1 mM EDTA) for 5 min on ice until more than 95% were permeabilized as assessed by trypan blue exclusion. Cells were fixed in 3.7% formaldehyde in PBS for 20 min at room temperature, washed thrice in PBS, and incubated in blocking buffer (3% bovine serum albumin, 0.05% saponin in PBS) for 1 h. The cells were incubated overnight at 4 °C with anti-cytochrome c mouse monoclonal antibody
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(BD PharMingen) diluted 1:200 in blocking buffer, washed thrice, and incubated for 1 h at room temperature with TRITC-conjugated goat anti-mouse (Santa Cruz) diluted 1:200 in blocking buffer. The cells were then counterstained with Mitotracker Green (Invitrogen Life Technologies). The samples were observed using a Leica TCS SP5 Confocal Spectral Microscope.
teroxirone for 36 h resulted in about 8.3% of H460 and 17.0% of A549 cells that entered early apoptotic phase and 27% and 6.0% late apoptosis (P b 0.05). As the drug concentrations were increased to 5 μM, both early and late apoptotic cells in H460 and A549 were elevated, respectively (P b 0.01), but not H1299 cells (Fig. 2). Taken together, the results suggested that teroxirone does trigger apoptotic cell death in NSCLC cells.
Western blot analysis. Cells treated with teroxirone were washed with PBS and scraped in lysate buffer containing 1% triton X-100, 150 mM NaCl, 5 mM EDTA, 1% aprotinin, 5 mM PMSF and 10 μg/ml leupeptin in 20 mM sodium phosphate. Protein concentrations were determined by the BCA assay (Pierce Biotechnology, Rockford, IL) and 20 μg of total protein was performed for western blot analysis. Protein samples were electrophoresed on SDS-PAGE gels, transferred to nitrocellulose filters, and immunoblotted with the antisera indicated. The immuno-active bands were visualized using horseradish peroxidaseconjugated secondary antiserum and enhanced chemiluminescence system ECL system (Amersham, Arlington Heights, IL). The blots are then incubated in fresh blocking solution and probed for 1 h with 1:3000 dilutions of p21Waf1/Cip1, PARP, GAPDH, caspase-3 and human p53 antibodies, respectively. Blots are washed twice in PBS-T and then incubated with a 1:4000 dilution of peroxidase-conjugated secondary antibody (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) in PBS-T for 1 h at 22 °C. Blots are again washed twice for 10 min in PBS-T and then developed by ECL detection system (Amersham).
The inducible PARP cleavage and expression mitosis regulators, p53 and p21Waf1/Cip1 (p21), accounted for the apoptotic cell death in H460 and A549 cells
Tumor xenograft study. The three- or four-week old female nu/nu mice were obtained from National Laboratory Animal Center (Tainan, Taiwan) and housed at the animal center (Kaohsiung Medical University, Kaohsiung, Taiwan) following the protocol of institutional animal care ethics. A total of 1 × 106 exponentially growing A549 cells in 0.2 ml (1:1 mixture ratio of PBS and 100 μl Matrixgel™ Basement Membrane Matrix (BD Biosciences, San Jose, CA)) were subcutaneously injected into the dorsal area of nude mice. Each group consisted of five mice. When the xenograft tumors reached 50 mm3 in size following cell inoculation, the mice were given an intraperitoneal injection of 50 mg/kg of teroxirone or vehicle control every other day to each group of mice for four days in a row. The volumes of tumors that formed were measured by a caliper and calculated according to the equation: volume in mm3 = (A × B2) / 2, where values of A and B corresponded to the largest and the smallest diameters of the implanted tumor, respectively. One-way ANOVA test was used for statistical comparisons between different groups.
The levels of tumor suppressor p53, its corresponding downstream regulator p21 and the cleaved PARP were elevated by teroxirone in both H460 and A549 cell lines and the enhanced intensities associated with the increased concentrations of the drug. No detectable p53, p21, active PARP fragment and cleaved caspase-3 were observed in H1299 cells. The maximal intensities of p53 and p21 were reached by 2 μM of teroxirone after 18 h of treatment (Figs. 3A and B). Being required to activate intrinsic apoptosis pathway, the cytotoxic mediator poly(ADPribose)polymerase (PARP) has been known to play a key role in base excision repair following DNA damage and in the maintenance of genome integrity (Chiu et al., 2005; Simbulan-Rosenthal et al., 1999). On the other hand, the appearance of 89-kDa PARP fragment in western blot means the commitment of apoptosis and the onset of cell death. The increased proteolytic cleavage of the precursor PARP implied the ultimate commitment of apoptotic cell death after drug-induced DNA damage.
Results Teroxirone-damaged DNA suppressed cell proliferation in A549 and H460 cells, but not in H1299 cells Human NSCLC cells exhibited different sensitivities against teroxirone. The cell growth of both A549 and H460 cells was inhibited in dose-dependent manners when treated with different concentrations of teroxirone, while the growth rates of H1299 cells were unaffected at all concentrations (Fig. 1B). The sensitive growth inhibition in A549 and H460 cells was also shown in colony formation assay (Figs. 1C and D). However, discrete DNA damages by teroxirone were detected in all cell lines (Fig. 1E) and the excluded lengths of DNA trail dose-dependent (Fig. 1F). The results proved that DNA lesions assisted in suppressing cell proliferation in H460 and A549 cells; while damaged DNA did not affect growth of H1299 cells. The increased annexin V and PI-positive cells by teroxirone in both H460 and A549 cells The induced apoptosis by teroxirone was quantified and confirmed by FACS analysis after staining cells with annexin-V/PI. Exposure to 2 μM
Fig. 4. (A) Cell cycle analysis by flow cytometry. Both H460 and A549 cells cultured in 1% serum-supplemented media were incubated with 10 μM of DEVD-CHO for 24 h before being treated with 5 μM of teroxirone for 36 h. The cells were collected and stained according to the description in Materials and methods for phase distribution analysis. Western blot analysis of (B) H460 and (C) A549 cells. The proteins were analyzed by western blotting by incubating with PARP, p53, cyclin B1, caspase-3 or GAPDH antibodies using lysates collected from cells with (+) or without (−) pretreatment of 10 μM DEVD-CHO before exposing to 1, 2 and 5 μM of teroxirone for 24 h. Protein levels were monitored based on a densitometer and expressed as a fold-change relative to protein level in untreated cells, in which changes of the cleaved PARP were labeled underneath. Data are representative of two independent experiments.
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Fig. 4 (continued).
Thus, elevated p53, p21 and cleaved PARP signaled the onset of apoptotic cell death in H460 and A549 cells that was absent in H1299 throughout the time course studied and various concentrations of teroxirone used. Release of cytochrome c in H460 and A549 cells when treated with teroxirone We next examined the effects of the drug on the release of cytochrome c from mitochondria by immunocytochemistry (Fig. 3C). In control cells, cytochrome c remained predominantly in mitochondria as evidenced by green color mitochondria. By treating 2 μM of teroxirone for 24 h, both H460 and A549 cells started to release of cytochrome c from mitochondria as shown by the appearance of yellow color of the coalesced green color of mitochondria and the diffused red color of cytochrome.
Caspase-3 inhibitor blocked teroxirone-mediated apoptosis Caspase-3 has been reported to be an effective downstream regulator of the signaling pathway during apoptosis. The synchronized cells under minimal serum supplementation were first treated with caspase-3 inhibitor. The appearance of sub-G1 cells after teroxirone treatment was offset by 24 h pretreatment with DEVD-CHO. In H460 and A549 cells, pretreatment with the peptide inhibitor prior to exposing cells to teroxirone for 36 h blocked the development of sub-G1 and G2/M population cells in exchange of G0/G1 cell accumulations (Fig. 4A). DEVD-CHO markedly attenuated the formation of cleaved caspase-3 and PARP that differed from those without pretreatment. The results indicated that p53 activation triggered formation of active caspase-3 fragment that repressed cell viabilities in both H460 (Fig. 4B) and A549 (Fig 4C) cells. Activation of cyclin B1 with DEVD-CHO pretreatment
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Fig. 5. (A) Growth curves. H1299 cells with stable expression of ectopic p53 (H1299/p53) and p53R267L (H1299/p53R267L) were treated with different concentrations (0, 1, 2 and 5 μM) of teroxirone for 48 h and the viabilities determined by MTT assay. (B) Cell cycle analysis. Both H1299/p53 and H1299/p53R267L cells were incubated with different concentrations (0, 1, 2 and 5 μM) of teroxirone for 36 h for cell cycle determination after being stained with PI. (C) Apoptosis assay. Both H1299/p53 and H1299/p53R267L cells were incubated with 0 and 5 μM of teroxirone for 36 h. Cells were double-stained with annexin V-FITC and PI, and then analyzed by flow cytometry (left). All experiments were done independently in triplicate per experimental point, and representative results are shown indicating early and late apoptosis, respectively (right). (D) Western blot analysis. Protein lysates of H1299/p53 and H1299/p53R267L cells treated with different concentrations (0, 1, 2 and 5 μM) of teroxirone for 24 h were analyzed by western blot using different antibodies with GAPDH as loading control. (E) Western blot analysis. Both H1299/p53 and H1299/p53R267L cells were treated with 2 μM of teroxirone for the time indicated and proteins analyzed.
allowed H460 and A549 cells to pass G2/M phase without apoptosis that attenuated the effectiveness of the drug. Teroxirone-induced apoptosis is dependent on p53 status To learn how the critical DNA binding site Arg267 of p53 affects cell sensitivity to teroxirone (Petty et al., 2011; Riley et al., 2008), we
constructed H1299 cell lines with stably expressing p53 or mutant p53R267L. Both H1299 cell clones with stable expression of ectopic p53 (H1299/p53) and p53R267L (H1299/p53R267L) were tested with teroxirone. The viabilities of H1299/p53 cells were decreased in dosedependent manner; whereas those of H1299/p53R267L cells were unaffected by teroxirone (Fig. 5A). The decreased viability in H1299/p53 was caused by apoptosis as shown in flow cytometry analysis (Fig. 5B)
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affect the growth of tumors of H1299 cells (Fig. 7A). The sizes of the dissected tumors of A549 cells by teroxirone were less than those by DMSO control (Figs. 7B and C). The liver, spleen and lung were removed from the mice following the experiment and examined. No detectable wound and lesion were observed. The molecular changes were analyzed by western blot in tumors of A549 cells between the control and teroxirone-treated groups. The levels of pro-survival genes, Akt, Bcl-2 and PCNA, were suppressed significantly in teroxirone-treated group as compared with those of the control group (Fig. 7D).
Discussion
Fig. 6. Effects of p53-specific siRNA on teroxirone sensitivity in both A549 and H460 cells (A) A total of 1.5 × 103 cells were either transfected withp53 siRNA or scrambled control (SO) for 18 h and then treated with 2 μM teroxirone for 48 h. The cells were then stained with MTT and converted into viability as specified in Materials and methods. Results are indicated as cell viability against DMSO vehicle controls in SO as determined from three independent experiments. Data are represented as the mean values ± SD. *P b 0.05, compared with drug alone in SO-transfected cells. (B) Expression of p53 in SO and p53 siRNA transfected cells after drug treatment as monitored by western blot using p53 antibody with β-actin expression as a loading control.
and annexin-V and PI staining (Fig. 5C). In western blot analysis, the p53 levels were first elevated and dropped later in H1299/p53 clone upon teroxirone treatment, and the turnover of p53 in H1299/p53R267L cells remained undetected (Fig. 5D). On the other hand, the increased 89kDa PARP fragment caused by teroxirone in H1299/p53 cells was timeand dose-dependent; the effect was not noticeable in H1299/p53R267L cells. The increased proteolytic cleavage of caspase-3 in H1299/p53 cells indicated that the intrinsic apoptotic activity by teroxirone is dependent on p53 status (Fig. 5E). Down-regulation of p53 proteins attenuated the onset of teroxironeinduced cell death in NSCLC cells To further verify p53 was a necessary determinant in the inducing cell death, a complemented experiment using siRNA against p53 along with scrambled control (SO). In teroxirone-treated H460 and A549 cells, transfection of p53 siRNA reduced drug sensitivity that differed from that of SO control (Fig. 6A). Western blot analysis showed that cells with or without SO induced expression p53 by teroxirone, while significant down-regulated p53 in cells transfected with p53 siRNA (Fig. 6B). The results altogether suggested that p53 status elicits drug effectiveness. The suppressed growth of xenograft tumors in teroxirone-treated A549 cells The xenograft tumors by subcutaneously inoculating A549 and H1299 cells in immunodeficient mice were established. A concentration of 50 mg/kg of teroxirone per mice was inoculated intraperitoneally for a total of four times. The growth of the tumors of A549 cells by teroxirone was suppressed effectively during the time intervals of the study as compared with that by vehicle control. Teroxirone did not
The work here showed that NSCLC cells and xenograft tumors with wild-type p53 are sensitive to teroxirone treatment (Figs. 1B and 6A). Previous reported work using human tumor cell line rhabdomyosarcoma indicated that 20 μM of teroxirone is needed to achieve 50% colony inhibition (Ames et al., 1984). The work here showed that the decreased viable cells by low concentrations of teroxirone in H460 and A549 cells can be attributed to apoptosis. Transient induction of p53, activation of p21 and caspase-3 fragment accounted for the final cell death by the drug. Tumor suppressor p53 can be activated transiently in response to a variety of cellular stresses, including DNA damage (Toledo and Wahl, 2006). Being regulated by p53 and regarded as an inherited property of cells, the development of cell death by treating with chemotherapeutic reagents can be characterized by DNA damage and apoptotic cell death (Boehme et al., 2008). The distinct apoptosis is an intrinsic biological feature to maintain tissue homeostasis (Christophorou, 2005; Christophorou et al., 2006). Cells normally respond to exogenous stress by cell cycle arrest and/or programmed cell death (Siddiqui-Jain et al., 2012). DNA damage in NSCLC cells and the subsequent p53-dependent apoptosis are closely associated with tumor eradication. While the potent anticancer drug activity may involve different intracellular targets, there is ample evidence that the decreased cell viability is mediated by apoptosis following DNA damage. Cells carrying wild-type p53 are more sensitive to genotoxic injury by anticancer agents than those carrying mutant p53 (Meek, 2009). Teroxirone caused DNA damage that began at 12 h in all NSCLC cell lines studied (Figs. 1E and F). The onset of apoptosis by teroxirone appeared in cells carrying wild-type p53 alleles only. The p53 pathway is stimulated by a very small number of DNA strand breaks or single-stranded gaps that trigger signals in eliminating DNA lesions in tumors (Ling and Lin, 2011). Once activated, p53 regulates the expression of an array of downstream effector genes, including those relating to DNA repair, growth arrest or apoptosis (Riley et al., 2008). By inducing DNA repair that permits the continuation of the cell cycle, p53 may mediate an exit from the cell cycle by inducing growth arrest or apoptosis (Offer et al., 2002). In the work as described, the induced p53 after 24 h drug treatment returned to their pretreatment levels and the characteristic checkpoint markers p21 varied accordingly (Figs. 3A and B). Serving as effective cyclin-dependent kinase (cdk) inhibitors by coordinating with p53, it is likely that activated p21 participated in mitotic arrest following drug induction (Dash and El-Deiry, 2005). As a p53 transcription target, p21 is implicated a potent inhibitor of the key cdk and has been regarded as the main intermediate of p53-dependent cell cycle arrest (Hsu et al., 2004). Since many therapeutic agents during management of cancer treatment ultimately target damaged DNA, the inhibitor p21 exerts vital role in preventing cell cycle exit and mitosis. The work here showed that, under stress from teroxirone, both H460 and A549 cells reversed their mitotic progress that is likely the result of p21 activation and the suppressed pathway prevented cell proliferation. To prevent further cycling on the damaged DNA template, the checkpoint blockage by p21 is regarded as a form of cellular resistance to proliferation by chemotherapeutic regimen that allows the DNA repair mechanisms to be activated and reduces the potential for aberrant mitosis (An et al., 2012).
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Fig. 7. (A) Teroxirone inhibits growth of xenograft tumors. A total of 1 × 106 of A549 or H1299 cells were subcutaneously injected into athymic nude mice. When the size of tumors reached approximately 50 mm3, the animals were administered with teroxirone (50 mg/kg) intraperitoneally every other day for four days. The mean tumor volumes were measured at the time intervals as specified and plotted following drug administration. Animals that received teroxirone were compared with those that received vehicle control (DMSO) alone in tumor growth rate. All tumor volume values plotted represented mean values ± SD of five mice in each group. (B) Comparison of the dissected tumors of the treated animals with those of control DMSO treatment. (C) Statistics of the collected tumor sample sizes. Significant reduction of tumor sample sizes in teroxirone-treated animals as compared with those of the control treatment. (D) Western blot analysis. Mice were euthanized after experiments and the extracted proteins of tumor samples subjected to western blot analysis. The blots were analyzed to quantify levels of Akt, Bcl-2 and PCNA against those of β-actin. *P b 0.05 compared with protein levels of the DMSO control group.
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Some evidence of DNA damage induced cell death, such as senescent fibroblasts that underwent extended cell arrest before undergoing apoptosis (Simbulan-Rosenthal et al., 1999). In human colon cancer cells of wild-type p53, treatment with low concentrations of camptothecin induced cell arrest that lead to immediate apoptosis (Oliver et al., 2008). On the other hand, human glioblastoma cells responded to DNA-methylating agents were arrested at G2/M phase and the cell fate depends on the status of p53 (Khan et al., 2012). While no significant change of cell viabilities in H1299 cells, the resulted apoptotic cell death in H460 and A549 cells was characterized by gradual cleavage of PARP. Having an active site and DNA-binding domain, the intense 89-kDa PARP fragment plus p53 and p21 activation committed cells to apoptotic death by teroxirone that corroborates with the attenuation of prosurvival signal Bcl-2. The absence of PARP cleavage and the intact Bcl-2 levels in H1299 cells signified the role of p53 in teroxirone-mediated apoptotic cell death. The cleavage of procaspase-3 after 24 h drug treatment suggested that activated caspase-3 participated in apoptotic cascade (Figs. 4B and C). The apoptotic cell death was offset in cultured cells following incubation with caspase-3 inhibitor, DEVD-CHO, by suppressing the accumulated G2/M phase cells. Inhibition of caspase activation in cancer cells is a potentially efficient mechanism to promote cell survival that contributes to chemo-resistance. The accumulated G0/G1 phase cells helped in reversing final apoptosis (Fig. 4A) and the inhibited caspase3 activities by DEVD-CHO blocked the final apoptotic cell death. The reduced procaspase-3 cleavage, suppressed p53 activation and inhibited PARP cleavage by the inhibitor indicated that teroxironeinduced apoptosis in NSCLC cells is caspase-3-dependent (Figs. 4B and C). Inclusion of caspase-3 inhibitors blocked the effects of a diverse range of cytotoxic drugs that reflected the essential role of the specific caspase in mediating their anticancer activity. Similar results were reported, in which paclitaxel-induced apoptotic morphological changes were reverted by the caspase-3 inhibitor, DEVD-CHO that accompanied with attenuated p53 turnover and increasing viable cells (Weigel et al., 2000). Our work here showed that caspase-3 activation is directly associated with p53-mediated apoptosis following DNA damage. In human NSCLC cells with wild-type p53, down-regulated cyclin B1 is associated with G2/M phase arrest and apoptosis of NSCLC cells (Yan et al., 2010). We have demonstrated that, by up-regulating cyclin B1, caspase-3 inhibitor arrested cells at G0/G1 phase and thereby attenuated drug sensitivities. In H460 and A549 cells, the coordination of different regulators prevented cell cycle exit and PARP cleavage marked for final cell apoptosis. Absence of molecular determinants such as p53 arrested cells without committing to apoptotic death after teroxirone treatment in H1299 cells. To underscore the importance of p53, H1299 cells with stable ectopic expression p53 became sensitive to teroxirone. The accompanied caspase-3 activation and PARP cleavage accounted for the final apoptotic cell death. Activation of downstream regulator caspase-3 prevented cell cycle exit and the enhanced PARP cleavage signaled final apoptosis in H1299/p53 cells. It is known that Arg267 contributes transcriptional activity by stabilizing DNA binding domain of p53 (Riley et al., 2008). Thus, the attenuated sensitivities in H1299 cells expressing ectopic mutant p53R267L accounted for the importance of DNA binding domain of p53 during teroxirone-mediated apoptosis. The results also corroborated with the findings that teroxirone sensitivity was offset by knocking down p53 (Figs. 6A and B). The observation was accentuated in xenograft mice in which teroxirone-treated A549 cell tumors were smaller in sizes (Figs. 7A, B and C) with decreased expression of prosurvival signals including Bcl-2 (Fig. 7D), while no such effect was detected in H1299 cells (data not shown). In summary, we showed that teroxirone caused DNA damage in NSCLC cells after 12 h treatment. Activation of p53 and p21 beginning at 18 h and the subsequent cytochrome c release accompanied with procaspase-3 and PARP cleavage committed cells to final apoptotic cell death. We identified that the toxic effect can also be attained in H1299
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cells with ectopic p53, but not those with ectopic mutant p53. Our findings asserted that regulation of cell proliferation by teroxirone in human lung cancer cells depends on the status of p53. The observed antitumor effect strongly suggested that teroxirone provides a feasible and alternative treatment strategy for cancer prevention.
Conflict of interest statement None declared.
Acknowledgment This work is supported by grants from the National Science Council, Executive Yen, Taiwan and National Taiwan Normal University (99-D). Technical assistance of College of Life Science and Instrumentation Center, National Taiwan University with the confocal laser microscopy is appreciated.
References Ames, M.M., Kovach, J.S., Rubin, J., 1984. Pharmacological characterization of teroxirone, a triepoxide antitumor agent, in rats, rabbits, and humans. Cancer Res. 44, 4151–4156. An, J., Gao, Y., Wang, J., Zhu, Q., Ma, Y., Wu, J., Sun, J., Tang, Y., 2012. Flavokawain B induces apoptosis of non-small cell lung cancer H460 cells via Bax-initiated mitochondrial and JNK pathway. Biotechnol. Lett. 34, 1781–1788. Atassi, G., Spreafico, F., Dumont, P., Nayer, P., Klastersky, J., 1980. Antitumoral effect in mice of a new triepoxyde derivative: 1,3,5-triglycidyl-s-triazinetrione (NSC 296934). Eur. J. Cancer 16, 1561–1567. Atassi, G., Dumont, P., Fisher, U., Zeidler, M., Budnowski, M., 1984. Preclinical evaluation of the anti tumour activity of new epoxide derivatives. Cancer Treat. Rev. 11 (Suppl. A), 99–110. Boehme, K.A., Kulikov, R., Blattner, C., 2008. p53 stabilization in response to DNA damage requires Akt/PKB and DNA-PK. Proc. Natl. Acad. Sci. U. S. A. 105, 7785–7790. Chiu, C.C., Lin, C.H., Fang, K., 2005. Etoposide (VP-16) sensitizes p53-deficient human nonsmall cell lung cancer cells to caspase-7-mediated apoptosis. Apoptosis 10, 643–650. Christophorou, M.A., 2005. Temporal dissection of p53 function in vitro and in vivo. Nat. Genet. 37, 718–726. Christophorou, M.A., Ringshausen, I., Finch, A.J., Swigart, L.B., Evan, G.I., 2006. The pathological response to DNA damage does not contribute to p53-mediated tumour suppression. Nature 443, 214–217. Crespo, R., Villaverde, M.L., Girotti, J.R., Güerci, A., Juárez, M.P., de Bravo, M.G., 2011. Cytotoxic and genotoxic effects of defence secretion of Ulomoides dermestoides on A549 cells. J. Ethnopharmacol. 136, 204–209. Dash, B.C., El-Deiry, W.S., 2005. Phosphorylation of p21 in G2/M promotes cyclin B-Cdc2 kinase activity. Mol. Cell. Biol. 25, 3364–3387. Dombernowsky, P., Lund, B., Hansen, H.H., 1983. Phase-I study of alpha-1,3,5-triglycidyls-triazinetrione (NSC 296934). Cancer Chemother. Pharmacol. 11, 59–61. Fischer, H., Zeidler, U., Budnowski, M., Atassi, G., Dumont, P., Venditti, J., Yoder, O.C., 1984. Investigation of the antitumor activity of new epoxide derivatives. Part I: s-triazinetrione derivatives. Arzneim. Forsch. 34, 543–547. Gualtieri, M., Rigamonti, L., Galeotti, V., Camatini, M., 2005. Toxicity of tire debris extracts on human lung cell line A549. Toxicol. In Vitro 19, 1001–1008. Hsu, Y.L., Kuo, P.L., Lin, C.C., 2004. The proliferative inhibition and apoptotic mechanism of saikosaponin D in human non-small cell lung cancer A549 cells. Life Sci. 75, 1231–1242. Khan, M., Zheng, B., Yi, F., Rasul, A., Gu, Z., Li, T., Gao, H., Qazi, J.I., Yang, H., Ma, T., 2012. Pseudolaric acid B induces caspase-dependent and caspase-independent apoptosis in u87 glioblastoma cells. Evidence-based Compl. Alt. Med.: eCAM 2012. 957568. Lim, S.W., Ting, K.N., Bradshaw, T.D., Zeenathul, N.A., Wiart, C., Khoo, T.J., Lim, K.H., Loh, H.S., 2011. Acalypha wilkesiana extracts induce apoptosis by causing single strand and double strand DNA breaks. J. Ethnopharmacol. 138, 616–623. Ling, S., Lin, W.-C., 2011. EDD inhibits ATM-mediated phosphorylation of p53. J. Biol. Chem. 286, 14972–14982. Meek, D.W., 2009. Tumour suppression by p53: a role for the DNA damage response? Nat. Rev. Cancer 9, 714–723. Neidhart, J.A., Derocher, D., Grever, M.R., Kraut, E.H., Malspeis, L., 1984. Phase I trial of teroxirone. Cancer Treat. Rep. 68, 1115–1119. Nicaise, C., Rozencweig, M., Crespeigne, N., Dodion, P., Gerard, B., Lambert, M., Decoster, G., Kenis, Y., 1986. Phase I study of triglycidylurazol given on a 5-day i.v. schedule. Cancer Treat. Rep. 70, 599–603. Offer, H., Erez, N., Zurer, I., Tang, X., Milyavsky, M., Goldfinger, N., Rotter, V., 2002. The onset of p53-dependent DNA repair or apoptosis is determined by the level of accumulated damaged DNA. Carcinogenesis 23, 1025–1032. Oliver, P.G., LoBuglio, A.F., Zinn, K.R., Kim, H., Nan, L., Zhou, T., Wang, W., Buchsbaum, D.J., 2008. Treatment of human colon cancer xenografts with TRA-8 anti-death receptor 5 antibody alone or in combination with CPT-11. Clin. Cancer Res. 14, 2180–2189. Petty, T.J., Emamzadah, S., Costantino, L., Petkova, I., Stavridi, E.S., Saven, J.G., Vauthey, E., Halazonetis, T.D., 2011. An induced fit mechanism regulates p53 DNA binding kinetics to confer sequence specificity. EMBO J. 30, 2067–2076.
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Piccart, M., Rozencweig, M., Dodion, P., Cumps, E., Crespeigne, N., Makaroff, O., Atassi, G., Kisner, D., Kenis, Y., 1981. Phase I clinical trial with alpha 1,3,5-triglycidyl-striazinetrione (NSC-296934). Eur. J. Cancer Clin. Oncol. 17, 1263–1266. Riley, T., Sontag, E., Chen, P., Levine, A., 2008. Transcriptional control of human p53regulated genes. Nat. Rev. Mol. Cell Biol. 9, 402–412. Rubin, J., Kovach, J.S., Ames, M.M., Moertel, C.G., Creagan, E.T., O'Connell, M.J., 1987. Phase I study of two schedules of teroxirone. Cancer Treat. Rep. 71, 489–492. Siddiqui-Jain, A., Bliesath, J., Macalino, D., Omori, M., Huser, N., Streiner, N., Ho, C.B., Anderes, K., Proffitt, C., O'Brien, S.E., Lim, J.K.C., Von Hoff, D.D., Ryckman, D.M., Rice, W.G., Drygin, D., 2012. CK2 inhibitor CX-4945 suppresses DNA repair response triggered by DNA-targeted anticancer drugs and augments efficacy: mechanistic rationale for drug combination therapy. Mol. Cancer Ther. 11, 994–1005. Simbulan-Rosenthal, C.M., Rosenthal, D.S., Iyer, S., Boulares, H., Smulson, M.E., 1999. Involvement of PARP and poly(ADP-ribosyl)ation in the early stages of apoptosis and DNA replication. Mol. Cell. Biochem. 193, 137–148.
Spreafico, F., Atassi, G., Filippeschi, S., Malfiore, C., Noseda, S., Boschetti, D., 1980. A characterization of the activity of alpha-1,3,5-triglycidyl-s-triazinetrione, a novel antineoplastic compound. Cancer Chemother. Pharmacol. 5, 103–108. Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.C., Sasaki, Y.F., 2000. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206–221. Toledo, F., Wahl, G.M., 2006. Regulating the p53 pathway: in vitro hypotheses, in vivo veritas. Nat. Rev. Cancer 6, 909–923. Weigel, T.L., Lotze, M.T., Kim, P.K., Amoscato, A.A., Luketich, J.D., Odoux, C., 2000. Paclitaxel-induced apoptosis in non-small cell lung cancer cell lines is associated with increased caspase-3 activity. J. Thorac. Cardiovasc. Surg. 119, 795–803. Yan, K.H., Yao, C.J., Chang, H.Y., Lai, G.M., Cheng, A.L., Chuang, S.E., 2010. The synergistic anticancer effect of troglitazone combined with aspirin causes cell cycle arrest and apoptosis in human lung cancer cells. Mol. Carcinog. 49, 235–246.
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Toxicology and Applied Pharmacology journal homepage: www.elsevier.com/locate/ytaap
Teroxirone inhibited growth of human non-small cell lung cancer cells by activating p53 Jing-Ping Wang a,1, Kai-Han Lin a,1, Chun-Yen Liu a, Ya-Chu Yu a, Pei-Tsun Wu a, Chien-Chih Chiu b, Chun-Li Su c, Kwun-Min Chen d, Kang Fang a,⁎ a
Department of Life Science, National Taiwan Normal University, Taipei, Taiwan Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan Department of Human Development and Family Studies, National Taiwan Normal University, Taipei, Taiwan d Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan b c
a r t i c l e
i n f o
Article history: Received 10 June 2013 Revised 26 July 2013 Accepted 7 August 2013 Available online 15 August 2013 Keywords: Teroxirone Human non-small-cell-lung-cancer cells Apoptosis p53
a b s t r a c t In this work, we demonstrated that the growth of human non-small-cell-lung-cancer cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutant p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells. © 2013 Elsevier Inc. All rights reserved.
Introduction Compounds with epoxy groups have attracted a great attention as perspective cancer therapy targets (Atassi et al., 1980, 1984; Fischer et al., 1984). Among them, the triepoxide derivative teroxirone (1,3,5triazine-2,4,6(1H,3H,5H)-tri-one-1,3,5-tri-(oxiranylmethyl), Fig. 1A) has been known active against a variety of tumors. The effectiveness of teroxirone was demonstrated in P388 and L1210 leukemia cell lines that are resistant to cyclophosphamide treatment (Atassi et al., 1980). As an alkylator, teroxirone has reportedly underdone phase I and II evaluation of clinical studies (Neidhart et al., 1984; Nicaise et al., 1986; Rubin et al., 1987). The antineoplastic effectiveness and the broad spectrum of antitumor activity together with its novel structural characteristics could be of potential clinical value (Spreafico et al., 1980). As a chemotherapeutic agent effective in eradicating tumors in different models, the drug has also been known helping patients recovering from leukemia and lymphomas (Atassi et al., 1980; Dombernowsky et al., 1983; Piccart et al., 1981; Spreafico et al., 1980). In spite of the distinct effectiveness in patients and in animal models, teroxirone remains poorly understood due to shortage of systematic and detailed biological study associated with the drug activity.
⁎ Corresponding author. Fax: +886 2 26749172. E-mail address: [email protected] (K. Fang). 1 The two authors contributed equally to this work. 0041-008X/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.taap.2013.08.007
Besides, the underlying mechanisms associated with it remain largely unexplored. In this work, we showed that low concentrations of teroxirone suppressed the growth of human non-small-cell-lung-cancer (NSCLC) cells. The induced apoptotic cell death can be reverted by caspase-3 inhibitor DEVD-CHO. The reduced cell viability is closely related to p53-activated apoptosis. Furthermore, the drug inhibited the growth of tumors in nude mice model. Teroxirone provides a good candidate for lung cancer treatment by suppressing proliferation in cell culture and animal models. Materials and methods Chemicals. The available synthetic teroxirone is of more than 98% purity. A stock solution of 10 mM in dimethyl sulfoxide (DMSO) was stored at −20 °C, and freshly dissolved in media. The DMEM medium, penicillin and streptomycin antibiotic mixture, sodium pyruvate and glutamine supplements were obtained from Sigma (St. Louis, MO). Fetal bovine serum was acquired from Invitrogen (Grands Islands, NY). Cell culture. The human lung cell carcinoma cell lines, H460, H1299 and A549 were obtained from ATCC. The acquired cells were thawed, grown and maintained in DMEM. All cells were supplemented with L-glutamine, sodium pyruvate, and supplemented with 10% heatinactivated FBS in the humidified atmosphere with 5% CO2 at 37 °C. All cell lines were periodically examined and found free of mycoplasma
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Fig. 1. (A) The chemical structure of the teroxirone (1,3,5-triazine-2,4,6(1H,3H,5H)-tri-one-1,3,5-tri-(oxiranylmethyl)). (B) Growth curves of H460, A549 and H1299 cells in media containing teroxirone. A total of 1.5 × 103 cells were cultured in 1% serum-supplemented DMEM. Twenty-four hours later, the cells were incubated with 2, 5 or 20 μM teroxirone for 48 h. The cells were then stained with MTT and converted into viability as specified in Materials and methods. Results are indicated as cell viability with teroxirone against controls, as determined from three independent experiments. Data are represented as the mean values ± standard deviation (SD). The asterisks (***) indicated a significant difference compared with the vehicle control DMSO (P b 0.005). (C) Colony formation assay. The trypsinized cells were plated in 12-well dishes of different densities (1000 cells of H460/well, 500 cells of A549/well and 200 cells of H1299/well). The attached cells were treated with 0, 0.5 and 1 μM of teroxirone, respectively, for 48 h. The media were replaced with fresh media and incubated at 37 °C. Eleven days later, the cells were fixed and stained with 10% methylene blue in 70% ethanol. (D) Statistical analysis of colony formation. The numbers of colonies, defined as more than 50 cells/ colony were counted and the remaining fractions were converted as the ratio of the numbers of colonies in the samples with treatment vs. those with vehicle control DMSO. Triplicate wells were set up for each condition. (E) Comet assay analysis. Cells were treated for 12 h with teroxirone at the concentrations as specified. DNA tail formation was determined in H460, H1299 cells and A549 cells after teroxirone treatment according to the protocol as specified in Materials and methods. Teroxirone caused DNA damage in all cell lines studied. (F) Relative scores of DNA damage in teroxirone-treated cells. Mean values of the average comet tail moment (percentage of DNA in tail × tail length) for the three fields were calculated. The relative scores of average comet tail were obtained by converting tail moment lengths of DNA tail formation in all cell lines of teroxirone treatment at different concentrations and compared those with vehicle control. Data represented three independent experiments of the mean values ± standard deviation (SD).
contamination using a MycoTect kit (Invitrogen, Grands Islands, NY). The selected stable H1299 clones transfected with cytomegalovirus promoter-driven pcDNA-p53 that encodes full-length wild type p53 (H1299/p53) or mutant p53R267L (H1299/p53R267L) were maintained in 10% serum-supplemented DMEM. Cell viability determination. Cell cytotoxicity was measured using the MTT assay (Crespo et al., 2011). For the assay, 1.5 × 103 cells per well were seeded on 96-well plates and cultured in 1% serumsupplemented DMEM. The suspension was incubated at 37 °C for 24 h to allow cell attachment. The mononuclear cells were incubated with different concentrations of teroxirone for the specified time. DMSO was added to the cell culture used as control at final concentration of 0.1%. After the specific exposure time, the medium was removed and then MTT assays were performed. Cells from each well were incubated with 10 μl of MTT (5 mg/ml) in PBS at 37 °C for 3 h. After this, the MTT was removed from wells and 100 μl DMSO was added into each
well. The amount of formazan formed was determined by measuring the absorbance at 570 nm using a 96-well microplate reader (Thermo Fisher Scientific, USA). The viability assays were performed in triplicate in three separate experiments. Cell viability was expressed as percentage of the vehicle controls. The results were presented as mean ± standard deviation. Statistical differences between two groups were determined by a two-tailed unpaired Student's t-test. P b 0.05 was considered significantly different. Comet assay. The comet assay was performed according to the method of the published work with minor modifications (Gualtieri et al., 2005; Tice et al., 2000). Briefly, conventional slides were covered with a layer of 70 μl 0.5% normal agarose and 0.5% low melting point agarose (GIBCO-BRL). An amount of 70 μl of low melting point agarose (0.5%, w/v) (GIBCO-BRL) was mixed with approximately 2 × 104 cells suspended in 15 μl; the mixture was then layered onto the slides, and immediately overlaid with coverslips. After
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Fig. 2. Apoptosis assay by annexin V-FITC and PI staining. Cells cultured in 1% serum-supplemented DMEM were incubated with different concentrations of teroxirone for 36 h before double staining with annexin-V and PI and the percentage of each quadrant determined by WinDM. The numbers on top right quadrant meant percentage distributions of late apoptosis; while those on the bottom right correspond to those of early apoptosis (top). The results on the right represented mean values ± SD of three individual experiments. All experiments were done independently in triplicate per experimental point, and representative results shown indicating early and late apoptosis, respectively, of different cells (bottom).
agarose solidification at 25 °C for 30 min, the coverslips were removed and the slides were immersed 60 min at 4 °C in fresh lysing solution (2.5 M NaCl; 100 mM Na2EDTA, 10 mM Tris, pH 10 containing 1% Triton X-100). The slides were equilibrated in alkaline solution (1 mM Na2EDTA, 300 mM NaOH) for 20 min and placed in alkaline electrophoresis solution (200 mM NaOH, 1 mM EDTA). The slides were neutralized by immersing in Tris buffer (0.4 M, pH 7.5) followed by distilled water and then soaked in methanol for 5 min. The air-dry slides were then stained with 5 μg/ml propidium iodide (PI) and viewed under an epifluorescence microscope (Nikon, Japan) at 460 nm for visual scoring. Visual image analyses of DNA damage were carried out in accordance with previously reported protocols (Lim et al., 2011). A total of at least 20 non-overlapping comet images per gel were visually assigned a score on an arbitrary scale of 0 (round and intact without discernible tail) to 4 (almost all DNA migrated
towards the tail without apparent head) based on the perceived comet tail length migration and relative proportion of DNA in the comet tail. A mean DNA damage score for each slide was obtained by dividing the total damage score gained with the total number of comets analyzed by CometScore™ software. Flow cytometry and determinations for cell cycle analysis and apoptosis assay. Cells in the early and late phases of apoptosis were quantitated using annexin V-FITC/PI Apoptosis Detection Kit (BD, Mansfield, MA). Briefly, 2 × 105 cells cultured in 1% serum-supplemented DMEM were incubated with different concentrations of teroxirone for 36 h before double staining. The collected cells were washed twice with PBS. After centrifugation at 1200 rpm for 5 min, cell pellet was stained with 0.5 μl PI (50 μg/ml, BD) plus 0.5 μl annexin-V FITC (20 μg/ml, BectonDickinson) in annexin-V binding buffer for 30 min at room temperature
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Fig. 3. (A) Western blot analysis in cells treated with different concentrations of teroxirone. Cells were analyzed by western blot to determine gene expression at each concentration ranging 0, 1, 2 and 5 μM after 24 h of treatment. Equal amounts of cell lysate protein were separated by SDS-PAGE separating gel and electro-blotted. The blots were then incubated in fresh blocking solution and probed for 1 h with 1:3000 dilution of p21, PARP, p53 or Bcl-2 antibody, followed by incubation with a 1:4000 dilution of horseradish peroxidase-conjugated secondary antibody and then developed by ECL detection system. (B) Western blot analysis in cells treated with teroxirone for different time points. Cells were analyzed by western blot to determine gene expression at time points (0, 12, 18 and 24 h) with 2 μM of teroxirone. (C) Mitochondrial release of cytochrome c. The induced release of cytochrome c from mitochondria in cells treated with 2 or 5 μM teroxirone or vehicle control DMSO for 24 h. Cells were fixed, permeabilized and stained with anti-cytochrome c antibody at 4 °C for 18 h. After washing, cells were stained with Mitotracker Green (mitochondrial staining), DAPI (nuclear staining) and secondary antibody conjugated with TRITC for cytochrome c following the description in Materials and methods. The pointed arrow signified the co-localization of red color cytochrome c and green color mitochondria, while blue color stood for nucleus.
in the dark. Analysis was performed with FACSCalibur system (BD). The cell distributions were analyzed by Modfit software (Becton-Dickinson, Mansfield, MA). To determine cell cycle distribution, the cells were analyzed using FACS Calibur™ (BD Biosciences). A total of 2 × 106 cells were plated in different culture conditions. For sample preparation, both medium and trypsinized cells were centrifuged and then supernatant was removed. The collected cells were washed twice with PBS and then treated with 70% alcohol containing PBS for 24 h at −20 °C. Right before analysis, the sample cells were treated with 10 μg/ml PI (Sigma; St. Louis, MO), 10 μg/ml RNase A (ICN Pharmaceutical; Costa Mesa, CA) containing PBS
for 30 min in darkness. Data were analyzed by Modfit LT (Ver 2.0, Becton-Dickinson; Mountain View, CA). Analysis of cytochrome c release. Cells (1 × 105) were harvested and treated with 100 μl digitonin (50 μg/ml in PBS supplemented with 100 mM KCl and 1 mM EDTA) for 5 min on ice until more than 95% were permeabilized as assessed by trypan blue exclusion. Cells were fixed in 3.7% formaldehyde in PBS for 20 min at room temperature, washed thrice in PBS, and incubated in blocking buffer (3% bovine serum albumin, 0.05% saponin in PBS) for 1 h. The cells were incubated overnight at 4 °C with anti-cytochrome c mouse monoclonal antibody
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(BD PharMingen) diluted 1:200 in blocking buffer, washed thrice, and incubated for 1 h at room temperature with TRITC-conjugated goat anti-mouse (Santa Cruz) diluted 1:200 in blocking buffer. The cells were then counterstained with Mitotracker Green (Invitrogen Life Technologies). The samples were observed using a Leica TCS SP5 Confocal Spectral Microscope.
teroxirone for 36 h resulted in about 8.3% of H460 and 17.0% of A549 cells that entered early apoptotic phase and 27% and 6.0% late apoptosis (P b 0.05). As the drug concentrations were increased to 5 μM, both early and late apoptotic cells in H460 and A549 were elevated, respectively (P b 0.01), but not H1299 cells (Fig. 2). Taken together, the results suggested that teroxirone does trigger apoptotic cell death in NSCLC cells.
Western blot analysis. Cells treated with teroxirone were washed with PBS and scraped in lysate buffer containing 1% triton X-100, 150 mM NaCl, 5 mM EDTA, 1% aprotinin, 5 mM PMSF and 10 μg/ml leupeptin in 20 mM sodium phosphate. Protein concentrations were determined by the BCA assay (Pierce Biotechnology, Rockford, IL) and 20 μg of total protein was performed for western blot analysis. Protein samples were electrophoresed on SDS-PAGE gels, transferred to nitrocellulose filters, and immunoblotted with the antisera indicated. The immuno-active bands were visualized using horseradish peroxidaseconjugated secondary antiserum and enhanced chemiluminescence system ECL system (Amersham, Arlington Heights, IL). The blots are then incubated in fresh blocking solution and probed for 1 h with 1:3000 dilutions of p21Waf1/Cip1, PARP, GAPDH, caspase-3 and human p53 antibodies, respectively. Blots are washed twice in PBS-T and then incubated with a 1:4000 dilution of peroxidase-conjugated secondary antibody (Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD) in PBS-T for 1 h at 22 °C. Blots are again washed twice for 10 min in PBS-T and then developed by ECL detection system (Amersham).
The inducible PARP cleavage and expression mitosis regulators, p53 and p21Waf1/Cip1 (p21), accounted for the apoptotic cell death in H460 and A549 cells
Tumor xenograft study. The three- or four-week old female nu/nu mice were obtained from National Laboratory Animal Center (Tainan, Taiwan) and housed at the animal center (Kaohsiung Medical University, Kaohsiung, Taiwan) following the protocol of institutional animal care ethics. A total of 1 × 106 exponentially growing A549 cells in 0.2 ml (1:1 mixture ratio of PBS and 100 μl Matrixgel™ Basement Membrane Matrix (BD Biosciences, San Jose, CA)) were subcutaneously injected into the dorsal area of nude mice. Each group consisted of five mice. When the xenograft tumors reached 50 mm3 in size following cell inoculation, the mice were given an intraperitoneal injection of 50 mg/kg of teroxirone or vehicle control every other day to each group of mice for four days in a row. The volumes of tumors that formed were measured by a caliper and calculated according to the equation: volume in mm3 = (A × B2) / 2, where values of A and B corresponded to the largest and the smallest diameters of the implanted tumor, respectively. One-way ANOVA test was used for statistical comparisons between different groups.
The levels of tumor suppressor p53, its corresponding downstream regulator p21 and the cleaved PARP were elevated by teroxirone in both H460 and A549 cell lines and the enhanced intensities associated with the increased concentrations of the drug. No detectable p53, p21, active PARP fragment and cleaved caspase-3 were observed in H1299 cells. The maximal intensities of p53 and p21 were reached by 2 μM of teroxirone after 18 h of treatment (Figs. 3A and B). Being required to activate intrinsic apoptosis pathway, the cytotoxic mediator poly(ADPribose)polymerase (PARP) has been known to play a key role in base excision repair following DNA damage and in the maintenance of genome integrity (Chiu et al., 2005; Simbulan-Rosenthal et al., 1999). On the other hand, the appearance of 89-kDa PARP fragment in western blot means the commitment of apoptosis and the onset of cell death. The increased proteolytic cleavage of the precursor PARP implied the ultimate commitment of apoptotic cell death after drug-induced DNA damage.
Results Teroxirone-damaged DNA suppressed cell proliferation in A549 and H460 cells, but not in H1299 cells Human NSCLC cells exhibited different sensitivities against teroxirone. The cell growth of both A549 and H460 cells was inhibited in dose-dependent manners when treated with different concentrations of teroxirone, while the growth rates of H1299 cells were unaffected at all concentrations (Fig. 1B). The sensitive growth inhibition in A549 and H460 cells was also shown in colony formation assay (Figs. 1C and D). However, discrete DNA damages by teroxirone were detected in all cell lines (Fig. 1E) and the excluded lengths of DNA trail dose-dependent (Fig. 1F). The results proved that DNA lesions assisted in suppressing cell proliferation in H460 and A549 cells; while damaged DNA did not affect growth of H1299 cells. The increased annexin V and PI-positive cells by teroxirone in both H460 and A549 cells The induced apoptosis by teroxirone was quantified and confirmed by FACS analysis after staining cells with annexin-V/PI. Exposure to 2 μM
Fig. 4. (A) Cell cycle analysis by flow cytometry. Both H460 and A549 cells cultured in 1% serum-supplemented media were incubated with 10 μM of DEVD-CHO for 24 h before being treated with 5 μM of teroxirone for 36 h. The cells were collected and stained according to the description in Materials and methods for phase distribution analysis. Western blot analysis of (B) H460 and (C) A549 cells. The proteins were analyzed by western blotting by incubating with PARP, p53, cyclin B1, caspase-3 or GAPDH antibodies using lysates collected from cells with (+) or without (−) pretreatment of 10 μM DEVD-CHO before exposing to 1, 2 and 5 μM of teroxirone for 24 h. Protein levels were monitored based on a densitometer and expressed as a fold-change relative to protein level in untreated cells, in which changes of the cleaved PARP were labeled underneath. Data are representative of two independent experiments.
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Fig. 4 (continued).
Thus, elevated p53, p21 and cleaved PARP signaled the onset of apoptotic cell death in H460 and A549 cells that was absent in H1299 throughout the time course studied and various concentrations of teroxirone used. Release of cytochrome c in H460 and A549 cells when treated with teroxirone We next examined the effects of the drug on the release of cytochrome c from mitochondria by immunocytochemistry (Fig. 3C). In control cells, cytochrome c remained predominantly in mitochondria as evidenced by green color mitochondria. By treating 2 μM of teroxirone for 24 h, both H460 and A549 cells started to release of cytochrome c from mitochondria as shown by the appearance of yellow color of the coalesced green color of mitochondria and the diffused red color of cytochrome.
Caspase-3 inhibitor blocked teroxirone-mediated apoptosis Caspase-3 has been reported to be an effective downstream regulator of the signaling pathway during apoptosis. The synchronized cells under minimal serum supplementation were first treated with caspase-3 inhibitor. The appearance of sub-G1 cells after teroxirone treatment was offset by 24 h pretreatment with DEVD-CHO. In H460 and A549 cells, pretreatment with the peptide inhibitor prior to exposing cells to teroxirone for 36 h blocked the development of sub-G1 and G2/M population cells in exchange of G0/G1 cell accumulations (Fig. 4A). DEVD-CHO markedly attenuated the formation of cleaved caspase-3 and PARP that differed from those without pretreatment. The results indicated that p53 activation triggered formation of active caspase-3 fragment that repressed cell viabilities in both H460 (Fig. 4B) and A549 (Fig 4C) cells. Activation of cyclin B1 with DEVD-CHO pretreatment
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Fig. 5. (A) Growth curves. H1299 cells with stable expression of ectopic p53 (H1299/p53) and p53R267L (H1299/p53R267L) were treated with different concentrations (0, 1, 2 and 5 μM) of teroxirone for 48 h and the viabilities determined by MTT assay. (B) Cell cycle analysis. Both H1299/p53 and H1299/p53R267L cells were incubated with different concentrations (0, 1, 2 and 5 μM) of teroxirone for 36 h for cell cycle determination after being stained with PI. (C) Apoptosis assay. Both H1299/p53 and H1299/p53R267L cells were incubated with 0 and 5 μM of teroxirone for 36 h. Cells were double-stained with annexin V-FITC and PI, and then analyzed by flow cytometry (left). All experiments were done independently in triplicate per experimental point, and representative results are shown indicating early and late apoptosis, respectively (right). (D) Western blot analysis. Protein lysates of H1299/p53 and H1299/p53R267L cells treated with different concentrations (0, 1, 2 and 5 μM) of teroxirone for 24 h were analyzed by western blot using different antibodies with GAPDH as loading control. (E) Western blot analysis. Both H1299/p53 and H1299/p53R267L cells were treated with 2 μM of teroxirone for the time indicated and proteins analyzed.
allowed H460 and A549 cells to pass G2/M phase without apoptosis that attenuated the effectiveness of the drug. Teroxirone-induced apoptosis is dependent on p53 status To learn how the critical DNA binding site Arg267 of p53 affects cell sensitivity to teroxirone (Petty et al., 2011; Riley et al., 2008), we
constructed H1299 cell lines with stably expressing p53 or mutant p53R267L. Both H1299 cell clones with stable expression of ectopic p53 (H1299/p53) and p53R267L (H1299/p53R267L) were tested with teroxirone. The viabilities of H1299/p53 cells were decreased in dosedependent manner; whereas those of H1299/p53R267L cells were unaffected by teroxirone (Fig. 5A). The decreased viability in H1299/p53 was caused by apoptosis as shown in flow cytometry analysis (Fig. 5B)
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affect the growth of tumors of H1299 cells (Fig. 7A). The sizes of the dissected tumors of A549 cells by teroxirone were less than those by DMSO control (Figs. 7B and C). The liver, spleen and lung were removed from the mice following the experiment and examined. No detectable wound and lesion were observed. The molecular changes were analyzed by western blot in tumors of A549 cells between the control and teroxirone-treated groups. The levels of pro-survival genes, Akt, Bcl-2 and PCNA, were suppressed significantly in teroxirone-treated group as compared with those of the control group (Fig. 7D).
Discussion
Fig. 6. Effects of p53-specific siRNA on teroxirone sensitivity in both A549 and H460 cells (A) A total of 1.5 × 103 cells were either transfected withp53 siRNA or scrambled control (SO) for 18 h and then treated with 2 μM teroxirone for 48 h. The cells were then stained with MTT and converted into viability as specified in Materials and methods. Results are indicated as cell viability against DMSO vehicle controls in SO as determined from three independent experiments. Data are represented as the mean values ± SD. *P b 0.05, compared with drug alone in SO-transfected cells. (B) Expression of p53 in SO and p53 siRNA transfected cells after drug treatment as monitored by western blot using p53 antibody with β-actin expression as a loading control.
and annexin-V and PI staining (Fig. 5C). In western blot analysis, the p53 levels were first elevated and dropped later in H1299/p53 clone upon teroxirone treatment, and the turnover of p53 in H1299/p53R267L cells remained undetected (Fig. 5D). On the other hand, the increased 89kDa PARP fragment caused by teroxirone in H1299/p53 cells was timeand dose-dependent; the effect was not noticeable in H1299/p53R267L cells. The increased proteolytic cleavage of caspase-3 in H1299/p53 cells indicated that the intrinsic apoptotic activity by teroxirone is dependent on p53 status (Fig. 5E). Down-regulation of p53 proteins attenuated the onset of teroxironeinduced cell death in NSCLC cells To further verify p53 was a necessary determinant in the inducing cell death, a complemented experiment using siRNA against p53 along with scrambled control (SO). In teroxirone-treated H460 and A549 cells, transfection of p53 siRNA reduced drug sensitivity that differed from that of SO control (Fig. 6A). Western blot analysis showed that cells with or without SO induced expression p53 by teroxirone, while significant down-regulated p53 in cells transfected with p53 siRNA (Fig. 6B). The results altogether suggested that p53 status elicits drug effectiveness. The suppressed growth of xenograft tumors in teroxirone-treated A549 cells The xenograft tumors by subcutaneously inoculating A549 and H1299 cells in immunodeficient mice were established. A concentration of 50 mg/kg of teroxirone per mice was inoculated intraperitoneally for a total of four times. The growth of the tumors of A549 cells by teroxirone was suppressed effectively during the time intervals of the study as compared with that by vehicle control. Teroxirone did not
The work here showed that NSCLC cells and xenograft tumors with wild-type p53 are sensitive to teroxirone treatment (Figs. 1B and 6A). Previous reported work using human tumor cell line rhabdomyosarcoma indicated that 20 μM of teroxirone is needed to achieve 50% colony inhibition (Ames et al., 1984). The work here showed that the decreased viable cells by low concentrations of teroxirone in H460 and A549 cells can be attributed to apoptosis. Transient induction of p53, activation of p21 and caspase-3 fragment accounted for the final cell death by the drug. Tumor suppressor p53 can be activated transiently in response to a variety of cellular stresses, including DNA damage (Toledo and Wahl, 2006). Being regulated by p53 and regarded as an inherited property of cells, the development of cell death by treating with chemotherapeutic reagents can be characterized by DNA damage and apoptotic cell death (Boehme et al., 2008). The distinct apoptosis is an intrinsic biological feature to maintain tissue homeostasis (Christophorou, 2005; Christophorou et al., 2006). Cells normally respond to exogenous stress by cell cycle arrest and/or programmed cell death (Siddiqui-Jain et al., 2012). DNA damage in NSCLC cells and the subsequent p53-dependent apoptosis are closely associated with tumor eradication. While the potent anticancer drug activity may involve different intracellular targets, there is ample evidence that the decreased cell viability is mediated by apoptosis following DNA damage. Cells carrying wild-type p53 are more sensitive to genotoxic injury by anticancer agents than those carrying mutant p53 (Meek, 2009). Teroxirone caused DNA damage that began at 12 h in all NSCLC cell lines studied (Figs. 1E and F). The onset of apoptosis by teroxirone appeared in cells carrying wild-type p53 alleles only. The p53 pathway is stimulated by a very small number of DNA strand breaks or single-stranded gaps that trigger signals in eliminating DNA lesions in tumors (Ling and Lin, 2011). Once activated, p53 regulates the expression of an array of downstream effector genes, including those relating to DNA repair, growth arrest or apoptosis (Riley et al., 2008). By inducing DNA repair that permits the continuation of the cell cycle, p53 may mediate an exit from the cell cycle by inducing growth arrest or apoptosis (Offer et al., 2002). In the work as described, the induced p53 after 24 h drug treatment returned to their pretreatment levels and the characteristic checkpoint markers p21 varied accordingly (Figs. 3A and B). Serving as effective cyclin-dependent kinase (cdk) inhibitors by coordinating with p53, it is likely that activated p21 participated in mitotic arrest following drug induction (Dash and El-Deiry, 2005). As a p53 transcription target, p21 is implicated a potent inhibitor of the key cdk and has been regarded as the main intermediate of p53-dependent cell cycle arrest (Hsu et al., 2004). Since many therapeutic agents during management of cancer treatment ultimately target damaged DNA, the inhibitor p21 exerts vital role in preventing cell cycle exit and mitosis. The work here showed that, under stress from teroxirone, both H460 and A549 cells reversed their mitotic progress that is likely the result of p21 activation and the suppressed pathway prevented cell proliferation. To prevent further cycling on the damaged DNA template, the checkpoint blockage by p21 is regarded as a form of cellular resistance to proliferation by chemotherapeutic regimen that allows the DNA repair mechanisms to be activated and reduces the potential for aberrant mitosis (An et al., 2012).
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Fig. 7. (A) Teroxirone inhibits growth of xenograft tumors. A total of 1 × 106 of A549 or H1299 cells were subcutaneously injected into athymic nude mice. When the size of tumors reached approximately 50 mm3, the animals were administered with teroxirone (50 mg/kg) intraperitoneally every other day for four days. The mean tumor volumes were measured at the time intervals as specified and plotted following drug administration. Animals that received teroxirone were compared with those that received vehicle control (DMSO) alone in tumor growth rate. All tumor volume values plotted represented mean values ± SD of five mice in each group. (B) Comparison of the dissected tumors of the treated animals with those of control DMSO treatment. (C) Statistics of the collected tumor sample sizes. Significant reduction of tumor sample sizes in teroxirone-treated animals as compared with those of the control treatment. (D) Western blot analysis. Mice were euthanized after experiments and the extracted proteins of tumor samples subjected to western blot analysis. The blots were analyzed to quantify levels of Akt, Bcl-2 and PCNA against those of β-actin. *P b 0.05 compared with protein levels of the DMSO control group.
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Some evidence of DNA damage induced cell death, such as senescent fibroblasts that underwent extended cell arrest before undergoing apoptosis (Simbulan-Rosenthal et al., 1999). In human colon cancer cells of wild-type p53, treatment with low concentrations of camptothecin induced cell arrest that lead to immediate apoptosis (Oliver et al., 2008). On the other hand, human glioblastoma cells responded to DNA-methylating agents were arrested at G2/M phase and the cell fate depends on the status of p53 (Khan et al., 2012). While no significant change of cell viabilities in H1299 cells, the resulted apoptotic cell death in H460 and A549 cells was characterized by gradual cleavage of PARP. Having an active site and DNA-binding domain, the intense 89-kDa PARP fragment plus p53 and p21 activation committed cells to apoptotic death by teroxirone that corroborates with the attenuation of prosurvival signal Bcl-2. The absence of PARP cleavage and the intact Bcl-2 levels in H1299 cells signified the role of p53 in teroxirone-mediated apoptotic cell death. The cleavage of procaspase-3 after 24 h drug treatment suggested that activated caspase-3 participated in apoptotic cascade (Figs. 4B and C). The apoptotic cell death was offset in cultured cells following incubation with caspase-3 inhibitor, DEVD-CHO, by suppressing the accumulated G2/M phase cells. Inhibition of caspase activation in cancer cells is a potentially efficient mechanism to promote cell survival that contributes to chemo-resistance. The accumulated G0/G1 phase cells helped in reversing final apoptosis (Fig. 4A) and the inhibited caspase3 activities by DEVD-CHO blocked the final apoptotic cell death. The reduced procaspase-3 cleavage, suppressed p53 activation and inhibited PARP cleavage by the inhibitor indicated that teroxironeinduced apoptosis in NSCLC cells is caspase-3-dependent (Figs. 4B and C). Inclusion of caspase-3 inhibitors blocked the effects of a diverse range of cytotoxic drugs that reflected the essential role of the specific caspase in mediating their anticancer activity. Similar results were reported, in which paclitaxel-induced apoptotic morphological changes were reverted by the caspase-3 inhibitor, DEVD-CHO that accompanied with attenuated p53 turnover and increasing viable cells (Weigel et al., 2000). Our work here showed that caspase-3 activation is directly associated with p53-mediated apoptosis following DNA damage. In human NSCLC cells with wild-type p53, down-regulated cyclin B1 is associated with G2/M phase arrest and apoptosis of NSCLC cells (Yan et al., 2010). We have demonstrated that, by up-regulating cyclin B1, caspase-3 inhibitor arrested cells at G0/G1 phase and thereby attenuated drug sensitivities. In H460 and A549 cells, the coordination of different regulators prevented cell cycle exit and PARP cleavage marked for final cell apoptosis. Absence of molecular determinants such as p53 arrested cells without committing to apoptotic death after teroxirone treatment in H1299 cells. To underscore the importance of p53, H1299 cells with stable ectopic expression p53 became sensitive to teroxirone. The accompanied caspase-3 activation and PARP cleavage accounted for the final apoptotic cell death. Activation of downstream regulator caspase-3 prevented cell cycle exit and the enhanced PARP cleavage signaled final apoptosis in H1299/p53 cells. It is known that Arg267 contributes transcriptional activity by stabilizing DNA binding domain of p53 (Riley et al., 2008). Thus, the attenuated sensitivities in H1299 cells expressing ectopic mutant p53R267L accounted for the importance of DNA binding domain of p53 during teroxirone-mediated apoptosis. The results also corroborated with the findings that teroxirone sensitivity was offset by knocking down p53 (Figs. 6A and B). The observation was accentuated in xenograft mice in which teroxirone-treated A549 cell tumors were smaller in sizes (Figs. 7A, B and C) with decreased expression of prosurvival signals including Bcl-2 (Fig. 7D), while no such effect was detected in H1299 cells (data not shown). In summary, we showed that teroxirone caused DNA damage in NSCLC cells after 12 h treatment. Activation of p53 and p21 beginning at 18 h and the subsequent cytochrome c release accompanied with procaspase-3 and PARP cleavage committed cells to final apoptotic cell death. We identified that the toxic effect can also be attained in H1299
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cells with ectopic p53, but not those with ectopic mutant p53. Our findings asserted that regulation of cell proliferation by teroxirone in human lung cancer cells depends on the status of p53. The observed antitumor effect strongly suggested that teroxirone provides a feasible and alternative treatment strategy for cancer prevention.
Conflict of interest statement None declared.
Acknowledgment This work is supported by grants from the National Science Council, Executive Yen, Taiwan and National Taiwan Normal University (99-D). Technical assistance of College of Life Science and Instrumentation Center, National Taiwan University with the confocal laser microscopy is appreciated.
References Ames, M.M., Kovach, J.S., Rubin, J., 1984. Pharmacological characterization of teroxirone, a triepoxide antitumor agent, in rats, rabbits, and humans. Cancer Res. 44, 4151–4156. An, J., Gao, Y., Wang, J., Zhu, Q., Ma, Y., Wu, J., Sun, J., Tang, Y., 2012. Flavokawain B induces apoptosis of non-small cell lung cancer H460 cells via Bax-initiated mitochondrial and JNK pathway. Biotechnol. Lett. 34, 1781–1788. Atassi, G., Spreafico, F., Dumont, P., Nayer, P., Klastersky, J., 1980. Antitumoral effect in mice of a new triepoxyde derivative: 1,3,5-triglycidyl-s-triazinetrione (NSC 296934). Eur. J. Cancer 16, 1561–1567. Atassi, G., Dumont, P., Fisher, U., Zeidler, M., Budnowski, M., 1984. Preclinical evaluation of the anti tumour activity of new epoxide derivatives. Cancer Treat. Rev. 11 (Suppl. A), 99–110. Boehme, K.A., Kulikov, R., Blattner, C., 2008. p53 stabilization in response to DNA damage requires Akt/PKB and DNA-PK. Proc. Natl. Acad. Sci. U. S. A. 105, 7785–7790. Chiu, C.C., Lin, C.H., Fang, K., 2005. Etoposide (VP-16) sensitizes p53-deficient human nonsmall cell lung cancer cells to caspase-7-mediated apoptosis. Apoptosis 10, 643–650. Christophorou, M.A., 2005. Temporal dissection of p53 function in vitro and in vivo. Nat. Genet. 37, 718–726. Christophorou, M.A., Ringshausen, I., Finch, A.J., Swigart, L.B., Evan, G.I., 2006. The pathological response to DNA damage does not contribute to p53-mediated tumour suppression. Nature 443, 214–217. Crespo, R., Villaverde, M.L., Girotti, J.R., Güerci, A., Juárez, M.P., de Bravo, M.G., 2011. Cytotoxic and genotoxic effects of defence secretion of Ulomoides dermestoides on A549 cells. J. Ethnopharmacol. 136, 204–209. Dash, B.C., El-Deiry, W.S., 2005. Phosphorylation of p21 in G2/M promotes cyclin B-Cdc2 kinase activity. Mol. Cell. Biol. 25, 3364–3387. Dombernowsky, P., Lund, B., Hansen, H.H., 1983. Phase-I study of alpha-1,3,5-triglycidyls-triazinetrione (NSC 296934). Cancer Chemother. Pharmacol. 11, 59–61. Fischer, H., Zeidler, U., Budnowski, M., Atassi, G., Dumont, P., Venditti, J., Yoder, O.C., 1984. Investigation of the antitumor activity of new epoxide derivatives. Part I: s-triazinetrione derivatives. Arzneim. Forsch. 34, 543–547. Gualtieri, M., Rigamonti, L., Galeotti, V., Camatini, M., 2005. Toxicity of tire debris extracts on human lung cell line A549. Toxicol. In Vitro 19, 1001–1008. Hsu, Y.L., Kuo, P.L., Lin, C.C., 2004. The proliferative inhibition and apoptotic mechanism of saikosaponin D in human non-small cell lung cancer A549 cells. Life Sci. 75, 1231–1242. Khan, M., Zheng, B., Yi, F., Rasul, A., Gu, Z., Li, T., Gao, H., Qazi, J.I., Yang, H., Ma, T., 2012. Pseudolaric acid B induces caspase-dependent and caspase-independent apoptosis in u87 glioblastoma cells. Evidence-based Compl. Alt. Med.: eCAM 2012. 957568. Lim, S.W., Ting, K.N., Bradshaw, T.D., Zeenathul, N.A., Wiart, C., Khoo, T.J., Lim, K.H., Loh, H.S., 2011. Acalypha wilkesiana extracts induce apoptosis by causing single strand and double strand DNA breaks. J. Ethnopharmacol. 138, 616–623. Ling, S., Lin, W.-C., 2011. EDD inhibits ATM-mediated phosphorylation of p53. J. Biol. Chem. 286, 14972–14982. Meek, D.W., 2009. Tumour suppression by p53: a role for the DNA damage response? Nat. Rev. Cancer 9, 714–723. Neidhart, J.A., Derocher, D., Grever, M.R., Kraut, E.H., Malspeis, L., 1984. Phase I trial of teroxirone. Cancer Treat. Rep. 68, 1115–1119. Nicaise, C., Rozencweig, M., Crespeigne, N., Dodion, P., Gerard, B., Lambert, M., Decoster, G., Kenis, Y., 1986. Phase I study of triglycidylurazol given on a 5-day i.v. schedule. Cancer Treat. Rep. 70, 599–603. Offer, H., Erez, N., Zurer, I., Tang, X., Milyavsky, M., Goldfinger, N., Rotter, V., 2002. The onset of p53-dependent DNA repair or apoptosis is determined by the level of accumulated damaged DNA. Carcinogenesis 23, 1025–1032. Oliver, P.G., LoBuglio, A.F., Zinn, K.R., Kim, H., Nan, L., Zhou, T., Wang, W., Buchsbaum, D.J., 2008. Treatment of human colon cancer xenografts with TRA-8 anti-death receptor 5 antibody alone or in combination with CPT-11. Clin. Cancer Res. 14, 2180–2189. Petty, T.J., Emamzadah, S., Costantino, L., Petkova, I., Stavridi, E.S., Saven, J.G., Vauthey, E., Halazonetis, T.D., 2011. An induced fit mechanism regulates p53 DNA binding kinetics to confer sequence specificity. EMBO J. 30, 2067–2076.
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J.-P. Wang et al. / Toxicology and Applied Pharmacology 273 (2013) 110–120
Piccart, M., Rozencweig, M., Dodion, P., Cumps, E., Crespeigne, N., Makaroff, O., Atassi, G., Kisner, D., Kenis, Y., 1981. Phase I clinical trial with alpha 1,3,5-triglycidyl-striazinetrione (NSC-296934). Eur. J. Cancer Clin. Oncol. 17, 1263–1266. Riley, T., Sontag, E., Chen, P., Levine, A., 2008. Transcriptional control of human p53regulated genes. Nat. Rev. Mol. Cell Biol. 9, 402–412. Rubin, J., Kovach, J.S., Ames, M.M., Moertel, C.G., Creagan, E.T., O'Connell, M.J., 1987. Phase I study of two schedules of teroxirone. Cancer Treat. Rep. 71, 489–492. Siddiqui-Jain, A., Bliesath, J., Macalino, D., Omori, M., Huser, N., Streiner, N., Ho, C.B., Anderes, K., Proffitt, C., O'Brien, S.E., Lim, J.K.C., Von Hoff, D.D., Ryckman, D.M., Rice, W.G., Drygin, D., 2012. CK2 inhibitor CX-4945 suppresses DNA repair response triggered by DNA-targeted anticancer drugs and augments efficacy: mechanistic rationale for drug combination therapy. Mol. Cancer Ther. 11, 994–1005. Simbulan-Rosenthal, C.M., Rosenthal, D.S., Iyer, S., Boulares, H., Smulson, M.E., 1999. Involvement of PARP and poly(ADP-ribosyl)ation in the early stages of apoptosis and DNA replication. Mol. Cell. Biochem. 193, 137–148.
Spreafico, F., Atassi, G., Filippeschi, S., Malfiore, C., Noseda, S., Boschetti, D., 1980. A characterization of the activity of alpha-1,3,5-triglycidyl-s-triazinetrione, a novel antineoplastic compound. Cancer Chemother. Pharmacol. 5, 103–108. Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., Miyamae, Y., Rojas, E., Ryu, J.C., Sasaki, Y.F., 2000. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 35, 206–221. Toledo, F., Wahl, G.M., 2006. Regulating the p53 pathway: in vitro hypotheses, in vivo veritas. Nat. Rev. Cancer 6, 909–923. Weigel, T.L., Lotze, M.T., Kim, P.K., Amoscato, A.A., Luketich, J.D., Odoux, C., 2000. Paclitaxel-induced apoptosis in non-small cell lung cancer cell lines is associated with increased caspase-3 activity. J. Thorac. Cardiovasc. Surg. 119, 795–803. Yan, K.H., Yao, C.J., Chang, H.Y., Lai, G.M., Cheng, A.L., Chuang, S.E., 2010. The synergistic anticancer effect of troglitazone combined with aspirin causes cell cycle arrest and apoptosis in human lung cancer cells. Mol. Carcinog. 49, 235–246.