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Keywords:

  • 2′-nitroflavone;
  • antitumor action;
  • apoptosis: caspases;
  • murine mammary adenocarcinoma

Abstract

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

We explored the in vitro and in vivo mechanism of antitumor action of the synthetic flavonoid 2′-nitroflavone on LM3 murine mammary adenocarcinoma cells. In vitro assays showed that 2′-nitroflavone increased the population of LM3 hypodiploid cells and produced a typical ladder of DNA fragmentation. Apoptotic cell death was also characterized by the activation of caspase-8, -9 and -3, by an increment in the expression levels of the proapoptotic protein Bax and by the release of cytochrome c to cytosol. The in vivo effect of 2′-nitroflavone on tumor growth was studied in BALB/c mice injected subcutaneously with LM3 cells. Results showed that tumor volume and weight were significantly reduced at doses of 10 and 40 mg/kg of 2′-nitroflavone, respectively. Apoptotic cells were identified by TUNEL assay in tumor slices from mice treated with 10 mg/kg of 2′-nitroflavone. Western blot analysis of tumor lysate supernatants from treated mice revealed an upregulation of the total levels of Bax and Fas receptor. In addition, administration of 40 mg/kg of 2′-nitroflavone to nontumor-bearing mice showed no histopathological effects on different organ tissues. This is the first report of the in vivo growth inhibitory effect of 2′-nitroflavone as an apoptotic agent likely useful for mammary adenocarcinoma treatment. © 2009 UICC

Flavonoids represent a group of polyphenolic compounds found in fruits, vegetables and different beverages.1, 2 These bioactive molecules possess anticarcinogenic effects and are considered potential chemopreventive candidates for cancer treatment.3–11 Different biological properties could explain the anticancer effects of flavonoids, including antiproliferation, induction of cell-cycle arrest and apoptosis, prevention of oxidation and induction of detoxification enzymes.3, 4, 7, 11

Carcinogenesis is a multistep process reflecting a succession of genetic changes that drive the progressive conversion of normal cells into malignant cells.12 Tumor growth represents a balance between the rate of cell proliferation and the rate of cell destruction and being resistant toward apoptosis, a feature that supports tumor development. Apoptosis is a regulated program of cell death that occurs under a variety of physiological and pathological conditions and is characterized by the activation of a complex intracellular pathway leading to a series of typical morphological and biochemical changes.13, 14 The inhibition of apoptosis may play a role in the pathogenesis of proliferative diseases, such as autoinmmune disorders or cancer.14 In this sense, many chemotherapeutic drugs used for the treatment of tumors induce tumor cell death by apoptosis.15, 16 In addition, the ability of several flavonoids to induce apoptosis has also been considered a relevant mechanism for the elimination of cancer cells.3, 7 Despite this, it has been reported that some flavonoids under pro-oxidant conditions restore cell viability and inhibit apoptosis of human vascular endothelial cells.17 Thus, the anticarcinogenic activity of these polyphenolic compounds might be attributed to a combination of their cytoprotective effect on normal cells and their cytotoxic effect on neoplastic cells.18

We have previously demonstrated that a synthetic flavonoid prepared in our laboratory, 2′-nitroflavone (Fig. 1), exerts a strong antimitogenic activity in various human and murine tumor cell lines, without disturbing the proliferation of nontumor epithelial cells.19 We have further studied the molecular pathways involved in the apoptotic cell death induced by 2′-nitroflavone in the human cervix HeLa adenocarcinoma cell line.20 To explore the in vivo action of 2′-nitroflavone, in this article we first examined the in vitro ability of this derivative to induce apoptosis in a cell line derived from a murine mammary tumor, and then investigated its in vivo growth inhibitory effects in a syngeneic BALB/c mouse model.

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Figure 1. Molecular structure of 2′-nitroflavone.

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Material and methods

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

Chemicals

Synthesis of 2′-nitroflavone was performed as described previously.21, 22 The nitroflavone derivative was dissolved in dimethyl sulfoxide (DMSO) as 100 mM stock solution and stored at −70°C. For in vitro assays, stock solution was diluted 1:10 in ethanol and added at a 20-μM concentration to the culture medium. Caspase substrates Ac-DEVD-AMC (caspase-3), Ac-IETD-AMC (caspase-8) and Ac-LEHD-AMC (caspase-9) were obtained from Peptide Institute, Osaka, Japan. Caspase-8 inhibitor (Z-IETD-FMK), caspase-9 inhibitor (Z-LEHD-FMK), monoclonal anti-Bax and anti-Fas antibodies, and polyclonal anti-Bcl-2 and anti-Bcl-XL antibodies were from Santa Cruz Biotechnology, Santa Cruz, CA. Monoclonal anti-cytochrome c antibody was from BD Biosciences Pharmingen, CA.

Cell culture

LM3 murine mammary adenocarcinoma cell line,23 kindly provided by the Institute of Oncology Angel H. Roffo (Buenos Aires, Argentina), was grown at 37°C under 5% CO2 atmosphere in Dulbecco's Modified Eagle's Medium (DMEM)-F12 (Gibco, NY) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 0.6% HEPES, 50 U/mL penicillin and 50 μg/mL streptomycin. For harvesting, cells were treated with 0.05% trypsin/EDTA using standard procedures.

Flow cytometry DNA analysis

To evaluate the proportion of hypodiploid cells, LM3 cells were incubated for different times in the presence or absence of 20 μM 2′-nitroflavone. In some experiments, cells were preincubated for 1 hr with 20 μM caspase-8 inhibitor (Z-IETD-FMK) or 20 μM caspase-9 inhibitor (Z-LEHD-FMK). After harvesting and washing the cells with cold phosphate-buffer saline (PBS), 1 × 106 cells were fixed overnight with 1 mL of 70% cold ethanol and kept at 4°C. Then, cells were washed twice with PBS and resuspended in 500 μL of 0.1% sodium citrate buffer, pH 8.4, 0.1% Triton X-100, 50 μg/mL propidium iodide. After incubating overnight at 4°C, the hypodiploid DNA content was analyzed in a FACScan flow cytometer (Becton Dickinson, CA). Because debris and residuals of necrotic cells were eliminated during the gating procedure, the percentage of hypodiploid cells was considered to represent the amount of apoptotic cells.

DNA ladder fragmentation

The apoptotic response was also evaluated by measuring DNA fragmentation. After treating LM3 cells in the presence or absence of 20 μM 2′-nitroflavone for 48 hr, cells were detached with trypsin/EDTA, washed twice with PBS and lysed for 3 hr at 56°C with agitation in lysis buffer (50 mM Tris/HCl, pH 8.0, 10 mM EDTA, 0.5% SDS, 250 μg/mL proteinase K). Subsequently, RNAse A (0.5 mg/mL) was added and incubated for one more hour at the same conditions. DNA was extracted by adding 400 μL of phenol:chloroform:isoamilic alcohol (25:24:1) followed by centrifugation for 10 min at 14,500 rpm, 4°C. The aqueous phase was then washed with 200 μL of chlorophorm:isoamilic alcohol (24:1). DNA was precipitated by adding 40 μL of sodium acetate 3 M, pH 5.3, and 1 mL of absolute ethanol to the aqueous phase and kept overnight at −20°C. Precipitated DNA was washed with 70% cold ethanol and resuspended with 20 μL of TE buffer (10 mM Tris/HCl, 1 mM EDTA) at 50°C for 30 min with agitation. After electrophoresis in 2% agarose gels, DNA laddering was visualized by ethidium bromide staining.

Caspase activity assays

LM3 cells were incubated for different times in the presence or absence of 20 μM 2′-nitroflavone and after treatment cells were detached with trypsin/EDTA and washed twice with cold PBS. Then, 1 × 106 cells were lysed for 30 min at 4°C in 50 μL of lysis buffer [10 mM HEPES, pH 7.4, 50 mM NaCl, 2 mM MgCl2, 5 mM EGTA, 1 mM phenylmethanesulfonyl fluoride (PMSF), 2 μg/mL leupeptin, 2 μg/mL aprotinin] followed by three cycles of rapid freezing and thawing. Cell lysates were centrifuged at 17,000g for 15 min, and total protein concentration was determined using Bradford reagent (Bio-Rad, Hercules, CA). Aliquots containing 100 μg of protein were diluted in assay buffer [20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM K2HPO4, pH 7.4, 20% glycerol, 5 mM dithiothreitol (DTT)], and incubated for 2 hr at 37°C with 50 μM of the corresponding fluorogenic substrates (Ac-DEVD-AMC: caspase-3, Ac-IETD-AMC: caspase-8, Ac-LEHD-AMC: caspase-9). Cleavage of substrates was monitored by AMC liberation in a SFM25 Konton Fluorometer at 355 nm excitation and 460 nm emission wavelengths. Results were expressed as the change in fluorescence units (per μg of protein) relative to control.

Western blot analysis

LM3 cells were incubated for different times in the presence or absence of 20 μM 2′-nitroflavone, harvested and washed twice with cold PBS. Then, 1 × 106cells were lysed for 30 min at 4°C in 10 μL of lysis buffer (10% glycerol, 0.5% Triton X-100, 1 μg/mL aprotinin, 1 μg/mL trypsin inhibitor, 1 μg/mL leupeptin, 10 mM Na4P2O7, 10 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM PMSF, 150 mM NaCl, 50 mM Tris, pH 7.4). Clear cell lysate supernatants were prepared by centrifugation and aliquots containing 100 μg of protein were resuspended in 0.063 M Tris/HCl, pH 6.8, 2% SDS, 10% glycerol, 0.05% bromophenol blue, 5% 2-mercaptoethanol, submitted to 14% SDS-PAGE and then transferred onto nitrocellulose membranes (Amersham Biosciences, Piscataway, NY) for 1 hr at 100 V in 25 mM Tris, 195 mM glycine, 20% methanol, pH 8.2. After blocking with 10 mM Tris, 130 mM NaCl and 0.05% Tween 20, pH 7.4, (TBS-T), containing 3% bovine serum albumin, membranes were treated as the usual western blotting method. The applied secondary antibodies were anti-mouse IgG (horseradish peroxidase-conjugated goat IgG from Jackson ImmunoResearch Laboratories, West Grove, PA) or anti-rabbit IgG (horseradish peroxidase-conjugated goat IgG from Santa Cruz Biotechnology, CA). Immunoreactive proteins were visualized using the ECL detection system (Amersham Biosciences, Piscataway, NY) according to the manufacturer's instructions. For quantification of band intensity, Western blots were scanned using a densitometer (Gel Pro Analyzer). Equal protein loading was confirmed by reprobing membranes with a rabbit anti-actin antibody (Sigma-Aldrich, MO).

Immunodetection of cytosolic cytochrome c

After incubating LM3 cells for different times in the presence or absence of 20 μM 2′-nitroflavone, cells were detached with trypsin/EDTA and then, suspensions containing 1 × 106 cells were washed once with cold PBS, resuspended in 30 μL of sucrose buffer (250 mM sucrose, 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 PMSF, 1 μg/mL aprotinin, 1 μg/mL leupeptin) and incubated on ice for 15 min. Cells were then homogenized, cytosolic extracts were prepared by centrifugation and aliquots containing 20 μg of protein were loaded onto a 16% SDS-PAGE and then transferred onto nitrocellulose membranes as indicated above. Cytochrome c release was determined by Western blot and quantification of band intensity was performed by using a densitometer (Gel Pro Analyzer).

Toxicity studies and tumor growth

All experiments were carried out in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Female BALB/c mice (20–25 g), obtained from the Animal Care Facility of the School of Pharmacy and Biochemistry, University of Buenos Aires, were housed under controlled conditions. Food and water were administered ad libitum. We first explored if the administration of 2′-nitroflavone to a group of nontumor-bearing mice could induce some toxicity. To this end, nontumor-bearing mice were injected via i.p. twice a week for 3 weeks with doses of 40 or 80 mg/kg of 2′-nitroflavone or with vehicle. At the end of the treatment, different organs were excised and fixed in formol buffer 10% in PBS 0.1 M, pH 7.4, and then dehydrated and included in paraffin. Cuts of 5 μm were made in microtome (Leica RM 2125, Wetzlar, Germany) and mounted on 2% xylane-coated slides. Sections were then stained with hematoxylin–eosin for histological analysis. To study the effect of 2′-nitroflavone on tumor growth, LM3 cells (3 × 105) diluted in 200 μL of DMEM-F12 were injected subcutaneously in the right flank of each mouse. Seven days after cell inoculation, mice were divided into four groups (n = 12): group I (control) received 0.2 mL of vehicle (5% DMSO, 0.9% NaCl, 0.05% Tween-80); group II to IV received 1, 10 and 40 mg/kg 2′-nitroflavone in vehicle, respectively, via i.p. twice a week for 3 weeks. Animals were monitored daily and their body weights were recorded weekly throughout the study. Tumor sizes were measured with a caliper twice a week and tumor volumes were calculated using the formula: V= (D × d2)/2, where D is the larger diameter and d is the smaller. At the end of the study, mice were anesthetized via i.p. with 100 mg ketamine and 10 mg diazepam/kg of body weight, and tumors were excised, weighed and measured.

TUNEL assay

The apoptotic effect of 2′-nitroflavone was detected in tissue sections of tumors by TUNEL assay. To this end, tumors from control and 10 mg/kg treated mice were removed and fixed in formol buffer 10% in PBS 0.1 M, pH 7.4, for 48 hr. Soon, tissue sections were dehydrated and included in paraffin. Cuts of 5 μm were made in microtome (Leica RM 2125, Wetzlar, Germany) and mounted on 2% xylane-coated slides. DNA strand breaks were determined by the DeadEnd Colorimetric TUNEL System (Promega Corporation, WI) according to the manufacturer's instructions. The sections were observed by light microscopy (Nikon Eclipse 2000, NY).

Apoptosis-related protein expression in tumors

To evaluate the expression of apoptosis-related proteins, tumors were excised and lysed in a buffer solution containing 10% glycerol, 0.5% Triton X-100, 1 μg/mL aprotinin, 1 μg/mL trypsin inhibitor, 1 μg/mL leupeptin, 10 mM Na4P2O7, 10 mM NaF, 1 mM Na3VO4, 1 mM EDTA, 1 mM PMSF, 150 mM NaCl, 50 mM Tris, pH 7.4. Clear tumor lysate supernatants were prepared by centrifugation and aliquots containing 100 μg of protein were resuspended in 0.063 M Tris/HCl, pH 6.8, 2% SDS, 10% glycerol, 0.05% bromophenol blue, 5% 2-mercaptoethanol, loaded onto a 14% SDS-PAGE and then transferred onto nitrocellulose membranes as indicated above. Bax, Bcl-2, Bcl-XL and Fas were detected by Western blot and quantification of band intensity was performed by using a densitometer (Gel Pro Analyzer).

Statistical analysis

All values are expressed as mean ± SE. Statistical analysis of in vitro data was performed by using the Student's t test. Dunnett multiple comparison tests after ANOVA were used to establish the statistical significance of difference in tumor volumen and tumor weight between control and treated mice, and Newman–Keuls multiple comparison tests to establish the statistical significance between different treatments. All analyses were performed using the statistical software Graph Pad Prism (Prism 4.0).

Results

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

In vitro studies

We have previously demonstrated that 2′-nitroflavone exhibits a potent cytotoxic activity in murine mammary adenocarcinoma LM3 cells (IC50 4 ± 1 μM), without affecting the proliferation of NMuMG epithelial cells derived from normal mammary gland of mice or 3T3 fibroblastic cells from mouse embryo.19 To evaluate the in vitro apoptotic effect of 2′-nitroflavone, LM3 cells were incubated for 24, 48 and 72 hr in the presence or absence of a 20 μM concentration of the synthetic flavone and the population of hypodiploid cells was analyzed by flow cytometry. As shown in Figure 2a, the sub-G1 fraction of cells increased from 8 ± 1 (control) to 28 ± 7%, 9 ± 6 (control) to 41 ± 1%, and 12 ± 1 (control) to 51± 5% after 24, 48 and 72 hr of incubation, respectively. In addition, a typical DNA ladder fragmentation pattern was obtained in cells incubated for 48 hr with 20 μM 2′-nitroflavone, whereas no DNA internucleosomal fragmentation was observed in cells treated with vehicle (control) (Fig. 2b).

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Figure 2. In vitro apoptotic effect of 2′-nitroflavone on LM3 cells. (a) Cells (100,000 cells/mL) were incubated for 24, 48 and 72 hr in the absence (control) or presence of 20 μM 2′-nitroflavone and then hypodiploid DNA content was evaluated by flow cytometry after propidium iodide staining. The percentage of apoptotic cells ±SE of three different experiments is shown in each histogram. Statistical significance in comparison with the corresponding control values is indicated by *p < 0.005, **p < 0.001. (b) DNA fragmentation after exposure of LM3 cells to 2′-nitroflavone was evaluated after incubating cells (3 × 106 cells/lane) in the absence (control) or presence of 20 μM 2′-nitroflavone for 48 hr. Genomic DNA was extracted, electrophoresed on 2% agarose gel and then visualized by ethidium bromide staining.

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To examine the role of caspases in the apoptosis induced by 2′-nitroflavone, we measured the proteolytic activity of the executioner caspase-3 and the initiator caspases -8 and -9. As shown in Figure 3a, caspase-3 activity increased and reached a peak after 24 hr of exposure to 2′-nitroflavone, remaining this activity elevated for at least 48 hr. Activation of both caspases -8 and -9 was evident after 24 hr of incubation and persisted up to 48 hr of treatment.

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Figure 3. Caspases activation induced by 2′-nitroflavone. (a) LM3 cells (100,000 cells/mL) were incubated for different times in the absence (control) or presence of 20 μM 2′-nitroflavone and then caspase-3, -8 and -9 activities were determined as indicated in “Material and methods”. Enzymatic activities are expressed as the ratio of the fluorescence per μg of protein of the treated sample with respect to the fluorescence per μg of protein of the control and represent mean values ± SE of three different experiments. (b) LM3 cells were treated for 1 hr with 20 μM caspase-8 inhibitor (Z-IETD-FMK) or 20 μM caspase-9 inhibitor (Z-LEHD-FMK), and then incubated in the absence (control) or presence of 20 μM 2′-nitroflavone for 24 hr. After propidium iodide staining, the hypodiploid DNA content was evaluated by flow cytometry. The percentage of apoptotic cells ±SE of three different experiments is shown in each histogram. Statistical significance in comparison with the corresponding control values is indicated by *p < 0.05, **p < 0.005; #p < 0.01 with respect to 2′-nitroflavone treatment.

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To confirm the participation of caspases, 2′-nitroflavone-treated cells were preincubated in the presence of the specific inhibitors of caspase-8 (Z-IETD-FMK) and -9 (Z-LEHD-FMK), at concentrations that completely blocked the activation of the corresponding caspases (data not shown). As shown in Figure 3b, incubation with 2′-nitroflavone for 24 hr increased the sub-G1 population of LM3 cells from 14 ± 2 (control) to 30 ± 3%, whereas a lower percentage of hypodiploid cells was obtained in the presence of Z-IETD-FMK (21 ± 1) or Z-LEHD-FMK (20 ± 1).

We also examined if the apoptotic cell death induced by 2′-nitroflavone produced a change in the expression of Bcl-2 family proteins. Results obtained showed that the expression of Bcl-2 and Bcl-XL remained unchanged after 24 hr of exposure to 2′-nitroflavone, whereas the amount of the proapoptotic protein Bax increased after 6 and 12 hr of treatment (Fig. 4). Furthermore, a significant increase in the cytosolic level of cytochrome c was obtained after 12 and 24 hr of treatment with 2′-nitroflavone, indicating the release of this protein from mitochondria to cytosol (Fig. 4).

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Figure 4. Levels of Bcl-2 family proteins and cytosolic cytochrome c in cells treated with 2′-nitroflavone. (a) Cells were incubated for different times in the presence or absence of 20 μM 2′-nitroflavone. Total cell lysates (for Bcl-2 family proteins) and cytosolic fraction (for cytochrome c) were processed for Western blot analysis as described in “Material and methods”; equal loading was confirmed by stripping and reprobing each blot for actin. (b) Quantification by densitometric analysis was performed by using Gel Pro software. Results are expressed as mean ± SE of three different experiments (*p < 0.05).

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In vivo studies

To explore if the administration of 2′-nitroflavone to a group of nontumor-bearing mice could induce some toxicity, we injected either vehicle or doses of 40 or 80 mg/kg of 2′-nitroflavone via i.p. twice a week for 3 weeks. As shown in Figure 5a, examination of different tissues stained with hematoxylin–eosin showed similar histological characteristics for 40 mg/kg treated mice and nontreated mice. However, the administration of 80 mg/kg of 2′-nitroflavone revealed the presence of an hematic infiltrate in lung tissue, both surrounding and within the alveolus (Fig. 5b). Thus, further experiments were performed with doses ≤40 mg/kg.

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Figure 5. Toxicity studies. (a) Tissues from vehicle (control) or 40 mg/kg 2′-nitroflavone treated mice were fixed in formol buffer 10% in PBS and embedded in paraffin. Sections of 5 μm were then stained with hematoxylin and eosin (magnification ×400). (b) Histological examination of lung tissue from vehicle (control) or 80 mg/kg 2′-nitroflavone treated mice (magnification ×400). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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To examine the in vivo effects of 2′-nitroflavone on tumor growth, mice were inoculated with murine mammary carcinoma LM3 cells and treated 7 days after with vehicle or different doses of the synthetic flavonoid twice a week for 3 weeks. The administration of 2′-nitroflavone at doses of 10 and 40 mg/kg reduced tumor volume 70 and 43% (p < 0.01), respectively, and no significant reduction was observed at 1 mg/kg of flavonoid (Fig. 6a). Tumor weight at the end of the experiment diminished 50 and 40% (p < 0.01 and 0.05), respectively, at 10 and 40 mg/kg of 2′-nitroflavone (Fig. 6b). The effect of 2′-nitroflavone on induction of apoptosis in tumors from nontreated mice (control) and from 10 mg/kg 2′-nitroflavone treated mice was evaluated by TUNEL assay. As shown in Figure 7a, the presence of dark brown apoptotic nuclei was evident in tumor sections from treated mice. The expression of Bcl-2 family proteins was also evaluated in tumor lysates from mice treated with 10 mg/kg 2′-nitroflavone or vehicle (control). Results obtained by Western blot showed a significant increment in the expression levels of Bax and Fas proteins and no change in the amount of Bcl-XL and Bcl-2 (Fig. 7b). Thus, Bax and Fas levels increased approximately 2- and 3-fold, respectively, in tumor lysates from treated mice (Fig. 7b).

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Figure 6. Effect of 2′-nitroflavone treatment on tumor growth. LM3 cells (3 × 105) were injected subcutaneously in the right flank of each mouse, and 7 days after cell inoculation, mice received vehicle or different doses of 2′-nitroflavone via i.p. twice a week for 3 weeks as described in “Material and methods”. (a) Tumor sizes were measured with a caliper twice weekly. Tumor volume was calculated by the formula (D × d2)/2. (b) At the end of the treatment, tumors were excised and the wet weights were measured. Results represent mean values ± SE. Dunnett's multiple comparison test was applied after one way ANOVA, *p < 0.05, **p < 0.01 respect to control group. Newman–Keuls multiple comparison test after one-way ANOVA, #p < 0.05 respect to 40 mg/kg dose.

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Figure 7. In vivo induction of apoptosis. (a) Representative TUNEL results from nontreated tumors (control) and tumors from mice treated with 10 mg/kg of 2′-nitroflavone. Brown nuclei indicate apoptotic cells (magnification ×1,000). (b) Levels of Bcl-2 family proteins and Fas receptor in cells treated with 10 mg/kg of 2′-nitroflavone. Tumor lysates were processed for Western blot analysis as described in “Material and methods”; equal loading was confirmed by stripping and reprobing each blot for actin. Quantification by densitometric analysis was performed by using Gel Pro software. Results are expressed as mean ± SE of three different experiments (*p < 0.005). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Discussion

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

The screening of the in vitro antiproliferative activity of various natural and synthetic flavonoids on different human and murine tumor cell lines allowed us to identify 2′-nitroflavone as a potent and selective antitumor compound.19 To evaluate the in vivo effect of this derivative in a mouse mammary tumor model, in this study we first explored its mechanism of antitumor action in vitro on LM3 cells, a cell line derived from a murine mammary adenocarcinoma. When LM3 cells were incubated for different times in the presence of 2′-nitroflavone, a significant increment in the amount of hypodiploid cells was observed, suggesting that this derivative induced an apoptotic cell death. A typical ladder of DNA fragmentation, characteristic of an apoptotic response,24 was also obtained after 48 hr of treatment.

We further investigated the molecular pathways involved in the apoptosis. The execution of an apoptotic cell death is dependent on the activity of caspases, cysteine proteases involved in the activation of the death receptor and the mitochondrial pathways.25–30 The interaction of members of the death receptors family (Fas, TNF or TRAIL) with their corresponding ligands in the cell surface leads to the formation of a death-inducing signaling complex that activates the initiator caspase-8. The mitochondrial pathway involves the release of multiple polypeptides from mitochondria, including cytochrome c, which induces the formation of the apoptosome and the activation of caspase-9. The initiator caspases can proteolytically activate the effector caspase-3, which induce the cleavage of cellular substrates that are finally responsible for the execution of cell death.25–29 We herein demonstrated that 2′-nitroflavone induced the activation of initiator caspases -8 and -9, both leading to the activation of the effector caspase-3. Thus, both death receptor and mitochondrial pathways would be involved in the apoptotic response. These pathways would be independently activated, as the presence of caspase-8 or caspase-9 specific inhibitors partially prevented 2′-nitroflavone-induced apoptosis. The significant higher levels of cytosolic cytochrome c found in 2′-nitroflavone treated cells indicated the release of cytochrome c from mitochondria. We also studied the expression of Bcl-2 family proteins which are involved in the mitochondria-mediated cell death.29, 31 We found that although the levels of the antiapoptotic proteins Bcl-2 and Bcl-XL did not change after 24 hr of incubation, the expression of the proapoptotic protein Bax increased in a time-dependent manner.

Different studies have reported the in vivo chemopreventive effect of various flavonoids.5, 11 The in vivo antitumor activity of natural flavonoids,32–34 synthetic flavonoids as flavopiridol,35 and cinnamic acid derivatives, such as caffeic acid phenethyl ester36 have been described in animal tumor models. However, the potential of 2′-nitroflavone as an effective in vivo antitumor agent has not yet been explored. Because the in vitro effectiveness of a compound may not be directly related to the in vivo action, we decided to evaluate whether 2′-nitroflavone could inhibit LM3 tumor growth in BALB/c mice. We demonstrated that 2′-nitroflavone caused a significant inhibition of tumor volume at doses of 10 and 40 mg/kg, although the response seemed to exhibit an inverted U-shaped profile, because volume reduction was significantly higher in mice treated with 10 mg/kg in comparison with mice receiving 40 mg/kg of 2′-nitroflavone.37 No loss in body weight was observed during the whole treatment. Furthermore, 2′-nitroflavone did not seem to exhibit a potential toxicity on other tissues, because any histological alteration was found in different organs from tumor-bearing mice (data not shown).

We also explored if the administration of 2′-nitroflavone to a group of nontumor-bearing mice could induce some toxicity. To this end, nontumor-bearing mice were injected via i.p. twice a week for 3 weeks with vehicle or doses of 40 or 80 mg/kg of 2′-nitroflavone. At the end of the treatment, histological examination of different organs, such as spleen, pancreas, kidney, liver, brain, lung and colon showed no differences between treated and nontreated mice. Thus, neither inflammatory infiltrates nor congestive and hemorrhagic areas were observed in these tissues. However, the infiltration of red blood cells was evident in lung tissue after the administration of 80 mg/kg, suggesting a toxic effect of the nitroflavone derivative at this dose.

Apoptotic cells were identified by TUNEL assay in tumors from mice treated with 10 mg/kg of 2′-nitroflavone. In addition, Western blot analysis of tumor lysates showed a significant increment in the amounts of Bax and Fas receptor proteins, indicating that 2′-nitroflavone also induced an apoptotic response in vivo. Based on in vitro and in vivo experiments, our results suggested that both death receptor and mitochondria-dependent pathways are involved in the apoptotic cell death triggered by 2′-nitroflavone in tumor LM3 cells.

In summary, this is the first report of the in vivo action of 2′-nitroflavone as an agent capable of inhibiting murine mammary tumor growth in mice by inducing apoptosis. This synthetic nitroflavone derivative may be, therefore, considered a potential candidate for cancer treatment.

Acknowledgements

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

The authors are indebted to Dr. Viviana Blank and Dr. Julieta Marino (IQUIFIB, Buenos Aires, Argentina) for helpful discussion and critical revision of the manuscript.

References

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