Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor

Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor

Chemico-Biological Interactions 186 (2010) 43–52 Contents lists available at ScienceDirect Chemico-Biological Interactions journal homepage: www.els...

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Chemico-Biological Interactions 186 (2010) 43–52

Contents lists available at ScienceDirect

Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint

Growth inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor Natália Bromberg a , Juliana L. Dreyfuss b , Caio V. Regatieri b , Marcelly V. Palladino b , Nelson Durán a , Helena B. Nader b , Marcela Haun d , Giselle Z. Justo b,c,d,∗ a

Biological Chemistry Laboratory, Instituto de Química, Universidade Estadual de Campinas (UNICAMP), C.P. 6154, CEP 13083-970, Campinas, SP, Brazil Departamento de Bioquímica (Campus São Paulo), Universidade Federal de São Paulo (UNIFESP), CEP 04044-020, São Paulo, SP, Brazil Departamento de Ciências Biológicas (Campus Diadema), Universidade Federal de São Paulo (UNIFESP), CEP 09972-270, Diadema, SP, Brazil d Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), C.P. 6109, CEP 13083-970, Campinas, SP, Brazil b c

a r t i c l e

i n f o

Article history: Received 17 December 2009 Received in revised form 8 April 2010 Accepted 14 April 2010 Available online 21 April 2010 Keywords: Ehrlich ascites tumor Violacein Antitumoral Apoptosis Chromobacterium violaceum Toxicology

a b s t r a c t The continuing threat to biodiversity lends urgency to the need of identification of sustainable source of natural products. This is not so much trouble if there is a microbial source of the compound. Herein, violacein, a natural indolic pigment extracted from Chromobacterium violaceum, was evaluated for its antitumoral potential against the Ehrlich ascites tumor (EAT) in vivo and in vitro. Evaluation of violacein cytotoxicity using different endpoints indicated that EAT cells were twofold (IC50 = 5.0 ␮M) more sensitive to the compound than normal human peripheral blood lymphocytes. In vitro studies indicated that violacein cytotoxicity to EAT cells is mediated by a rapid (8–12 h) production of reactive oxygen species (ROS) and a decrease in intracellular GSH levels, probably due to oxidative stress. Additionally, apoptosis was primarily induced, as demonstrated by an increase in Annexin-V positive cells, concurrently with increased levels of DNA fragmentation and increased caspase-2, caspase-9 and caspase-3 activities up to 4.5-, 6.0- and 5.5-fold, respectively, after 72 h of treatment. Moreover, doses of 0.1 and 1.0 ␮g kg−1 violacein, administered intraperitoneally (i.p.) to EAT-bearing mice throughout the lifespan of the animals significantly inhibited tumor growth and increased survival of mice. In view of these results, a 35-day toxicity study was conducted in vivo. Complete hematology, biochemistry (ALT, AST and creatinine levels) and histopathological analysis of liver and kidney indicated that daily doses of violacein up to 1000 ␮g kg−1 for 35 days are well tolerated and did not cause hematotoxicity nor renal or hepatotoxicity when administered i.p. to mice. Altogether, these results indicate that violacein causes oxidative stress and an imbalance in the antioxidant defense machinery of cells culminating in apoptotic cell death. Furthermore, this is the first report of its antitumor activity in vivo, which occurs in the absence of toxicity to major organs. © 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Abbreviations: EAT, Ehrlich ascites tumor; IC50 , 50% inhibitory concentration; ROS, reactive oxygen species; i.p., intraperitoneally; RPMI, Roswell Park Memorial Institute medium; FCS, fetal calf serum; HEPES, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid; NMR, nuclear magnetic resonance; UV–Vis, ultra violet–visible; PBS, phosphate balanced salt solution; DMSO, dimethyl sulfoxide; MTT, 3-(4,5-dimethiazol-zyl)-2-5-diphenyltetrazolium bromide; EDTA, ethylene diamine tetraacetic acid; GSH, reduced glutathione; DTNB, 5,5 dithio-bis-2-nitrobenzoic; DPA, diphenylamine; TCA, trichloroacetic acid; pNA, p-nitroanilide; GSSG, oxidized glutathione; DCFH, dichlorofluorescein; DCFH-DA, 2 ,7 -dichlorodihydrofluorescein diacetate; FITC, fluorescein isothiocyanate; PI, propidium iodide; AST, aspartate aminotransferase; ALT, alanine aminotransferase. ∗ Corresponding author at: Departamento de Bioquímica, Universidade Federal de São Paulo (UNIFESP), CEP 04044-020, São Paulo, SP, Brazil. Tel.: +55 11 5576 4444; fax: +55 11 5573 6407. E-mail address: [email protected] (G.Z. Justo). 0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2010.04.016

Natural products are invaluable as tools for identification of exploitable molecular targets and as platforms for developing front-line drugs. The literature indicates that many natural products are available as chemoprotective agents against commonly occurring cancers worldwide [1–3]. However, most of these chemotherapeutic agents exhibit severe normal toxicity, and cause undesirable side effects. Thus, a need is felt to find alternative drugs by screening newer molecules or plant products [1,3–5]. Violacein (Fig. 1), the main purple-colored pigment produced by the bacterial species of Chromobacterium violaceum found in the Rio Negro in the Amazon region of Brazil, has a dimeric structure composed of 5-hydroxyindole, oxindole and 2-pyrrolidone subunits. This secondary metabolite has been suggested to display several pharmacological properties [6,7]. Of most importance were the studies on its antitumoral activities, which focused on the ability

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were of analytical reagent grade purchased from commercial sources. 2.2. Violacein

Fig. 1. Chemical structure of violacein.

of violacein to induce apoptosis and on the molecular pathways involved in such effect [8–12]. Although neoplasia involves many other processes that also present targets for cancer therapy, in almost all instances, deregulated cell proliferation and suppressed cell death together provide the optimal conditions for neoplastic progression. Apoptosis is impaired in many human tumors, and its induction is frequently considered a more causal therapeutic approach in the treatment of malignant diseases [13–17]. There are several natural compounds that have been shown to induce apoptosis in Ehrlich tumor cells in vivo and in vitro [14,18,19]. In clinical situations, formation of ascites is often observed in patients with advanced cancer, especially ovarian cancer. Once accumulation of ascites occurs, involving intraperitoneal dissemination, the prognosis is poor. Relatively few animal tumors can grow in the abdominal cavity as ascites tumors. Ehrlich ascites tumor (EAT) is a spontaneous mammary carcinoma tumor, with a very aggressive behavior, and has been successfully transplanted in nearly all known mouse strains. After intraperitoneal (i.p.) inoculation, EAT readily grows in ascitic form [20]. The survival time after i.p. injection of EAT cells in untreated mice is said to be directly related to the number of tumor cells injected [21], hence the widespread use of this tumor in the screening of potential anticancer drugs. From these viewpoints and in continuation of our interest searching for violacein antitumoral mechanisms of action, the present study was carried out to evaluate the antitumor activity and apoptotic effects of violacein against EAT cells. Since there are no previous results focusing on the effect of violacein against tumor development in vivo, in this work, its potential to inhibit tumor growth in vivo was also investigated in EAT-bearing mice. Furthermore, in light of the aforementioned biological properties of violacein, toxicity studies were also performed in cultures of normal human peripheral blood lymphocytes and in vivo using hematological, biochemical and histopathological parameters. 2. Materials and methods 2.1. Reagents RPMI 1640 culture medium and fetal bovine serum (FBS) were purchased from GIBCO/Invitrogen (Grand Island, NY, USA). Trypan blue, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), sodium bicarbonate and 2-mercaptoethanol were from Sigma Chemical Co. (St. Louis, MO, USA), and HEPES and l-glutamine were from Life Technologies (Grand Island, NY, USA). Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) were from BD Biosciences (San Jose, CA, USA), and 2 ,7 -dichlorodihydrofluorescein diacetate (H2 -DCFDA) was from Molecular Probes/Invitrogen (Eugene, OR, USA). Colorimetric kits for the determination of caspases-2, caspase-9 and caspase-3 activities were purchased from R&D Systems (Minneapolis, MN, USA). All the other chemicals and reagents used in this study

Violacein (3-(1,2-dihydro-5-(5-hydroxy-1H-indol-3-yl)-2-oxo3H-pyrrol-3-ilydene)-1,3-dihydro-2H-indol-2-one), an indole derivative, was obtained from C. violaceum (CCT 3496) which was cultivated on cotton, in modified 1 l Roux bottles on a surface tray bioreactor. The pigment was extracted by a Soxhlet apparatus, purified and characterized by proton and carbon-13 nuclear magnetic resonance (NMR) spectroscopies, mass spectrometry, ultra violet–visible (UV–vis) spectroscopy and infrared spectroscopy as previously described [22]. 2.3. Animals and mouse tumor model The mice used in this study were bred at Centro de Bioterismo da UNICAMP (CEMIB) and raised under specific pathogen-free conditions. Male BALB/c mice, 6–8 weeks old, were housed in polycarbonate cages in a room with a 12 h day–night cycle and temperature at 22 ± 2 ◦ C. During the experimental period, the animals were fed with a balanced commercial diet (Labina-Purina, Brazil) and water ad libitum. Animal experiments were approved by the UNICAMP Institutional Animal Care and Use Commitee that follows the recommendations of the Canadian Council on Animal Care [23]. Ehrlich ascites tumor (EAT) was kindly provided by Prof. F.A. Dawood (Departamento de Microbiologia e Imunologia, FCM, UNICAMP, Campinas, SP, Brazil) and maintained in BALB/c mice in ascites form by successive transplantations. Ascitic tumor cell counts were done in a Neubauer hemocytometer using the trypan blue dye exclusion method. Cell viability was always found to be 95% or more. Tumor cell suspensions were prepared in phosphate balanced salt solution (PBS) at pH 7.4 to final concentrations of 6 × 107 viable cells ml−1 . Mice were inoculated intraperitoneally (i.p.) with 6 × 106 viable tumor cells per mouse in a volume of 0.2 ml [20]. 2.4. Antitumor evaluation The antitumor activity of violacein was evaluated by measuring survival time and tumor growth inhibition. Male BALB/c mice (7 per cage) were inoculated i.p. with 6 × 106 EAT cells. After 24 h, violacein (0.1 and 1.0 ␮g kg−1 doses) was administered throughout the lifespan of the animals by i.p. injections of 0.2 ml per mouse. Endpoint of experiments was determined by spontaneous death of animals. The ascitic fluid from the peritoneal cavity of tumorbearing mice was quantitatively isolated by peritoneal lavage after death. The total number of tumor cells was counted by the trypan blue exclusion method. Violacein solutions were prepared in PBS, pH 7.4, containing 10% Tween 80 (vehicle). Control mice received the vehicle for the same time period. 2.5. In vivo toxicological studies A 35-day intraperitoneal toxicity study of violacein was conducted in male BALB/c mice with daily doses of 0.1, 1.0 and 1000 ␮g kg−1 using hematological, biochemical and histopathological parameters. Twenty-four male mice, 7 weeks of age, were divided into four groups of six animals each and had free access to water and chow. Three groups were given 0.1, 1.0 and 1000 ␮g kg−1 violacein suspended in PBS, pH 7.4, containing 10% Tween 80 (vehicle), by i.p. injections of 0.2 ml per mouse, once daily for 35 days. One group was served as control and was given vehicle

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for the same time period. Clinical signs of gross toxicity, behavioral changes and mortality were observed once daily throughout the period of exposure. Individual animals from each group were weighed before and at the end of experiment and body weight was recorded. After the administration of the last dose, the animals were given rest overnight and then on the next day, they were anesthetized with ketamine hydrochloride (83 mg g−1 ) and xylazine hydrochloride (13 mg g−1 ) administered intraperitoneally. Whole blood was then sampled from the retro-orbital sinus through an uncoated microhematocrit tube (Becton Dickinson, USA) directly in Microtainer Brand Tubes (Becton Dickinson, USA) with EDTA (for hematology) or preservative-free Microtainer Brand Serum Separator Tubes (for biochemical analysis). The total volume for hematology was 150 ␮l and the remaining volume was used for serum biochemistry. The blood for serum biochemistry was allowed to clot at room temperature and was centrifuged at 3000 rpm for 10 min for serum separation. After blood collection, all mice were killed by cervical dislocation and liver and kidneys were removed, washed in PBS, individually fixed with 10% formalin and stored for histopathological examination.

reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) [24] and the phosphatase activity [25].

2.5.1. Hematological and biochemical analyses Whole blood was immediately analyzed for complete blood count with differential and platelet count using the fully automated CELL-DYN Ruby Hematology System Analyzer (Abbott, Germany). Serum samples were sent to the contract laboratory at the University Hospital (Laboratório Central do Hospital São Paulo, SPDM, São Paulo, SP, Brazil) for analysis on ice packs. Within the limitation of available serum volume, serum biochemistry determinations included aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, and creatinine levels by standard colorimetric assays.

2.9. Glutathione assay

2.5.2. Histopathological analysis Routine histological processes were employed for paraffin inclusion, sectioning and hematoxylin–eosin staining of liver and kidney from mice treated with 0.1, 1.0 and 1000 ␮g kg−1 violacein and vehicle. A histopathologist performed a complete examination of the tissues.

2.8. Leukocyte culture and evaluation of violacein cytotoxicity Peripheral human blood was obtained by venipuncture from healthy adult donors, diluted with an equal volume of RPMI 1640 medium, then layered over Ficoll-Hypaque density gradient separation solution (1.077 g ml−1 ), and centrifuged at 400 × g for 20 min at room temperature. The mononuclear cell layer was removed, washed twice in RPMI 1640 medium and resuspended in RPMI 1640 medium supplemented with 2 mM glutamine, antibiotics and 10% FBS. Leukocytes at a density of 1 × 106 plating cells ml−1 were cultured with 5 ␮g ml−1 of phytohemagglutinin in 96-well microtiter plates, in the absence and presence of violacein (0.25–12 ␮M), for 72 h at 37 ◦ C in a 100% humidity atmosphere containing 5% of CO2 . Control samples were treated with the corresponding volume of culture medium containing less than 0.1% DMSO. After treatment, cell proliferation was determined using the MTT reduction assay [24].

EAT cells (5 × 106 ), previously treated with violacein at different concentrations (2–5 ␮M) for 72 h, were washed with physiological solution and lysed with 2 ml of water. A volume of 3 ml of precipitant solution (1.67 g of glacial metaphosphoric acid, 0.2 g of EDTA and 30 g of NaCl in 100 ml of MilliQ water) was added to the lysate. After 5 min, the mixture was centrifuged. The GSH concentration was determined according to Torsoni et al. [26] with some modifications, by the reaction of 1.1 ml of the supernatant with 5,5 -dithio-bis-2-nitrobenzoic acid (DTNB) in the presence of PBS (0.2 M), pH 8.0. After 5 min of reaction, absorbance was measured at 412 nm and the GSH content calculated in relation to the control (ε = 13.6 mol−1 cm−1 ). 2.10. Reactive oxygen species (ROS) measurement

Ten days after inoculation of EAT cells in the abdominal cavity of mice, the cells were isolated by needle aspiration, washed in saline, and the erythrocytes were removed with a lysing solution. Cells were cultured in RPMI 1640 supplemented with HEPES (25 mM), l-glutamine (2 mM), sodium bicarbonate (25 mM), 10% FBS, 2mercaptoethanol (50 ␮M) and antibiotics (100 U ml−1 penicillin and 100 ␮g ml−1 streptomycin). Cells were incubated in culture flasks at 37 ◦ C in a humidity atmosphere containing 5% of CO2 . Viability and cell density were determined by the trypan blue dye exclusion test.

After EAT cells treatment with 5 ␮M violacein for 8, 12 and 24 h in a 96-well plate, analysis of intracellular ROS was performed using the oxidation-sensitive fluorescent probe 2 ,7 -dichlorofluorescein diacetate (DCFH-DA). The diacetate form of DCF (H2 -DCFDA) is taken up by cells and hydrolyzed to membrane-impermeant dichlorofluorescein (DCFH), which is oxidized in the presence of H2 O2 to form the highly fluorescent dichlorofluorescein (DCF). Briefly, EAT cells were loaded with 5 ␮M DCFH-DA for the last 30 min of violacein treatment and the fluorescence of the generated DCF was measured in a Fluoroskan Ascent FL fluorimeter plate reader (Labsystems, Finland) at 490 nm excitation and 538 nm emission. The readings were corrected according to the cell number estimated by the trypan blue assay and the amount of ROS formed was expressed relative to the control [11,27].

2.7. Evaluation of violacein cytotoxicity in EAT cells

2.11. Quantification of DNA fragmentation

EAT cells (3 × 105 ml−1 ) were seeded in quadruplicates into 96-well flat microtiter plates (Corning, USA) in enriched RPMI 1640 medium supplemented with 10% FBS. Violacein was dissolved in dimethyl sulfoxide (DMSO) which final concentration was adjusted to less than 0.1% (v/v) of the solvent in culture medium. The cells were treated with violacein (0.01–12 ␮M) while control samples were treated with the corresponding volume of culture medium containing DMSO. All samples were incubated for 72 h at 37 ◦ C in a 100% humidity atmosphere containing 5% of CO2 . After treatment, cell proliferation was determined using the standard

The amount of fragmented DNA was measured by the diphenylamine (DPA) method as described previously [8] with some modifications. Briefly, 5 × 106 EAT cells were collected after treatment with violacein (2.0–5.0 ␮M) for 72 h and incubated in lysis buffer (10 mM Tris/HCl, 10 mM EDTA, pH 8.0, 0.5% Triton X-100) with vigorous stirring. After the separation of the fragmented DNA from the intact chromatin, the supernatant was transferred into new test tube. The pellet was dissolved in TE buffer (10 mM Tris/HCl, 10 mM EDTA, pH 8.0) and 25% trichloroacetic acid (TCA) was added to all tubes. Following incubation overnight at 4 ◦ C,

2.6. In vitro EAT cell culture

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the precipitated DNA was collected by centrifugation for 10 min at 20,000 × g at 4 ◦ C. The supernatants were discarded and the DNA hydrolyzed by adding 5% TCA to the pellets followed by 15 min heating at 90 ◦ C. A blank with 5% TCA alone was also prepared. After the addition of freshly prepared DPA solution (15 mg of DPA ml−1 , 15 ␮l of conc. sulfuric acid ml−1 , and 90 (g of acetaldehyde ml−1 ), the mixture was incubated for 4 h at 37 ◦ C and the resulting absorbance was measured at 600 nm. The percentage of fragmented DNA was calculated as: % fragmented DNA =

3. Results 3.1. Effect of violacein on proliferation and viability of EAT cells and lymphocytes in vitro Cytotoxicity of violacein in EAT cells was assessed after 72 h of treatment using different endpoints: MTT reduction and phosphatase activity (Fig. 2). Exposure of EAT cells to various concentrations of violacein resulted in a significant decline in cell

Amount of fragmented DNA in supernatant × 100 Amount of fragmented DNA in the supernatant + amount of DNA in the pellet

2.12. Determination of caspases activities Caspase-2, caspase-9 and caspase-3 activities were measured using colorimetric protease kits following the manufacturer’s recommendations, after incubation of the EAT cells with violacein (3.0–5.0 ␮M) for 72 h. The assays were based on the spectrophotometric detection of the cromophore p-nitroanilide (pNA) after cleavage from the substrates X-pNA, where X stands for the amino acid sequences VDVAD, LEHD and DEVD recognized by caspase-2, caspase-9 and caspase-3, respectively. For the assay, 2 × 106 cells were pelleted by centrifugation and lysed on ice. The protein concentration in the lysate was measured using the Bio-Rad protein assay (Bio-Rad Laboratories Inc., USA). Two hundred micrograms of proteins were incubated with each X-pNA substrate (200 ␮M final concentration) at 37 ◦ C for 4 h in a microtiter plate. The absorbance of the samples was measured at 405 nm and the increase in the caspase activity of treated cells was determined by comparing the results with the untreated cells after background correction.

proliferation, with an IC50 of 5.0 ␮M, as seen for MTT reduction and phosphatase activity. In spite of the appearance of an acceleration of cell growth at low concentrations of violacein, this effect is not statistically significant (Fig. 2). We also verified if violacein was able to decrease EAT cell viability in vitro evaluating this effect by the trypan blue exclusion method. As presented in Fig. 3, exposure of cells to violacein for 72 h decreased cell viability in a dose-dependent manner. At the concentration of 5.0 ␮M violacein revealed 70% of EAT cell viability as long as the maximum decrease (15%) was seen after violacein treatment with 10.0 ␮M. These results suggest that the compound potently inhibits the proliferation and viability of EAT cells. Even though violacein was able to inhibit the proliferation of human lymphocytes, these cells were less sensitive to the compound, presenting an IC50 value almost twofold higher (9.0 ␮M) than for EAT cells, as verified by the MTT assay after 72 h of incubation with violacein in the same range of concentrations (Fig. 2). These data together with previous studies of violacein cytotoxicity in normal fibroblasts (V79 cells) [28,30] and in human erythrocytes [31] and mononuclear cells [9,29] suggest a lower sensitivity of normal cells to the compound.

2.13. Annexin-V/PI double-staining and analysis by flow cytometry

3.2. Changes in cellular glutathione and reactive oxygen species (ROS) levels induced by violacein in EAT cells

EAT cells were harvested after treatment with 2.0–5.0 ␮M violacein for 24 h. Cells were washed with cold PBS and resuspended in 1× binding buffer (0.01 M HEPES, pH 7.4, 0.14 M NaCl and 2.5 mM CaCl2 ) at a concentration of 1 × 106 cells ml−1 . The suspensions were transferred to 5-ml tubes and 5 ␮l Annexin-V-FITC and 25 ␮g ml−1 PI was added. The cells were incubated at room temperature for 20 min, after which 300 ml of 1× binding buffer was added and analysis (10,000 events were collected per sample) was performed in a FACSCalibur flow cytometer (Becton Dickinson, USA) using the CellQuest software.

ROS have been observed to take part in highly organized cellular functions like pathways of signal transduction and apoptosis [32]

2.14. Statistical analysis All in vitro experiments were performed at least in triplicate and the results shown in the graphs represent the means ± standard deviation (S.D.). To verify significant differences among groups, analysis of variance (ANOVA) was used. When significant difference was obtained, the Tukey test was used to evaluate the minimal differences among the groups. The survival of mice was demonstrated using the Kaplan–Meier method and the logrank (Cox–Mantel) statistical test was applied to compare the curves for non-parametric procedures. Differences were considered significant at P < 0.05, representing two-sided test of statistical significance. All experiments were repeated at least twice. A 0.01 level of significance was used for statistical evaluations of hematological and biochemical data. Equal variances were indicated by the Bartlett’s test and ANOVA was performed for statistical comparison among groups.

Fig. 2. Cytotoxicity of violacein in Ehrlich tumor cells (3 × 105 cells ml−1 ; solid line) and human lymphocytes (1 × 106 cells ml−1 ; dashed line) after 72 h of incubation. Leukocytes were collected from normal human donors, separated by density gradient and treated with violacein as described in Section 2. The curves show the effects of violacein on MTT reduction () and protein phosphatase activity (䊉). The inhibition was expressed relative to control cell viability (100%) and each point represents the mean ± S.D. of three experiments run in quadruplicate.

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Fig. 3. Effect of violacein on the viability of Ehrlich tumor cells (3 × 105 cells ml−1 ) determined by trypan blue exclusion test after 72 h of incubation. The results present the mean ± S.D. of three experiments run in quadruplicate.

and a role for oxidative signaling in the cytotoxicity of violacein in colon cancer cells has been previously shown [11]. Therefore, we investigate a possible role of oxidative stress in the alteration of cellular sensitivity to violacein. ROS levels were measured on violacein-treated EAT cells for 30 min after the addition of DCFH-DA. Figure 4 shows the time-course effect of violacein on intracellular peroxide levels in EAT cells. Significant increases in intracellular ROS production (P < 0.01) could be observed 8–24 h after incubation of tumor cells with 5.0 ␮M violacein as compared to control cells. Moreover, the increase in peroxides amounts generated by EAT cells was time-dependent, being significantly higher (P < 0.01) at the beginning of treatment (8–12 h) in comparison to late time (24 h). Actually, peroxides levels were approaching normal after 72 h exposure in EAT cells (data not shown). As we observed the existence of an oxidative cytotoxic mechanism, we next measured the level of glutathione (GSH), the major non-protein thiol in mammalian cells with chemoprotective action, particularly due to its role in antioxidant defense. Treatment with violacein reduced the GSH levels in EAT cells (Fig. 5). Moreover, this decrease was statistically significant at all concentrations (2.0, 3.0 and 5.0 ␮M, P < 0.01) of violacein when compared with the value obtained in the untreated cells.

Fig. 4. Time-course effect of violacein on ROS generation in Ehrlich ascites cells. The fluorescence intensity of DCF was monitored at 538 nm, with excitation wavelength set at 490 nm, and used to indicate the level of intracellular peroxides formation. Changes in DCF fluorescence in tumor cells were measured at 8, 12 and 24 h after treatment with 5 ␮M of violacein. *P < 0.01 compared to control. **P < 0.01 compared to control and to 8 and 12 h-time points (ANOVA, Tukey test). The results express the mean ± S.D. of three independent experiments run in duplicate. C denotes control.

Fig. 5. Alteration on GSH levels of Ehrlich ascites cells (3 × 105 cells ml−1 ) treated with violacein for 72 h. *P < 0.01 compared to control cells (ANOVA, Tukey test). The results present the mean ± S.D. of three experiments run in duplicate. C denotes control.

3.3. Apoptosis induction in violacein-treated EAT cells To investigate whether apoptosis is involved in the cytotoxicity of violacein to EAT cells, the nuclear DNA fragmentation, a classic hallmark of apoptotic cells, was assayed. As shown in Fig. 6, the extent of DNA fragmentation in EAT cells increased dose-dependently with violacein treatment. The untreated cells presented 11% of fragmentation, while cells treated with 2.0, 3.0, 4.0 and 5.0 ␮M of violacein for 72 h presented 22, 26, 28 and 42% of DNA fragmentation, respectively (P < 0.05). These results suggest that violacein induces EAT killing via apoptosis. For precise differentiation between cells undergoing necrosis or apoptosis in the violacein-mediated cell death, EAT cells were treated with increasing concentrations of violacein (2.0, 4.0 and 5.0 ␮M) for 72 h and cells were analyzed by flow cytometry using PI and fluorochrome-conjugated Annexin-V labeling. Changes in membrane phospholipid bilayers, such as externalization of the phosphatidylserine, which can be stained with Annexin-V-FITC, are characteristic of cells undergoing apoptosis. In contrast, loss

Fig. 6. Percentage data of DNA fragmentation obtained by DPA method in Ehrlich tumor cells (3 × 105 cells ml−1 ) treated with different concentrations of violacein for 72 h. *P < 0.05 compared to control cells. **P < 0.05 compared to control cells and to 2.0 and 5.0 ␮M treatments (ANOVA, Tukey test). The results express the mean ± S.D. of three experiments run in duplicate. C denotes control.

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Fig. 7. Effect of violacein on apoptosis induction. Ehrlich ascites cells were treated for 72 h with 2, 4 and 5 ␮M violacein for 72 h and harvested for quantification of Annexin-V-positive, PI-positive, and Annexin-V/PI-positive cells by flow cytometry. *P < 0.05 compared to control. **P < 0.05 compared to control and to 2.0 and 4.0 ␮M treatments. ***P < 0.001 compared to control and to 2.0 and 4.0 ␮M treatments (ANOVA, Tukey test). The results represent the mean ± S.D. of two independent experiments run in duplicate.

of membrane integrity, shown by PI staining, has been associated with necrosis. The results shown in Fig. 7 for analysis by flow cytometry of violacein-treated cells stained with AnnexinV-FITC indicated that apoptosis is predominant, as detected by significant increases in Annexin-V-FITC positive populations after 72 h of exposure to 4.0 ␮M (P < 0.05) and 5.0 ␮M (P < 0.001) violacein. Moreover, a substantial increase in Annexin-V-FITC staining of 5.0 ␮M over 4.0 ␮M-treated samples was observed (Fig. 7). These results corroborated with the higher DNA fragmentation levels determined in 5.0 ␮M violacein-treated cells (Fig. 6). In addition, small, but statistically significant (P < 0.05), populations of cells were Annexin-V-FITC/PI double stained after treatment with 4.0 and 5.0 ␮M, while only at the highest dose of violacein a significant (P < 0.05) PI-positive population could be determined (Fig. 7), reflecting cell death by necrosis, which might be related to the longer period of incubation with the compound. Because of the significance of caspases in apoptosis, the role of caspase-2, caspase-9 and caspase-3 in the violacein-induced EAT cell death was evaluated. After 72 h of incubation with violacein, cells treated with 3.0 ␮M of the compound presented a significant increase of 2.5-fold for all caspases activities when compared to the control cells (P < 0.01) (Fig. 8). Treatment of cells with 5.0 ␮M violacein resulted in 4.5-, 6.0- and 5.5-fold increases of caspase2, caspase-9 and caspase-3 activities, respectively (P < 0.01). Taken together, these biochemical features, as high DNA fragmentation, low membrane rupture, high phosphatidylserine externalization and activation of effector caspases are most likely indicative of activation of an apoptotic death pathway by violacein in EAT cells.

Fig. 8. Alterations on caspase-2, caspase-9 and caspase-3 activities after incubation of Ehrlich tumor cells (3 × 105 cells ml−1 ) with different concentrations of violacein for 72 h. *P < 0.01 compared to control cells. **P < 0.01 compared to control cells and to 3.0 and 4.0 ␮M treatment (ANOVA, Tukey test). The results express the mean ± S.D. of two experiments run in triplicate. C denotes control.

the 18th day. In contrast, survival of violacein-treated EAT mice was 100% on the 16th day and 14.3% in the 30th day, with no animal alive beyond day 31. Both doses of the pigment tested in this experiment (0.1 and 1.0 ␮g kg−1 ) significantly enhanced the rate of mice survival (P < 0.05). No significant statistical difference was observed between mice treated with 0.1 and 1.0 ␮g kg−1 of violacein. Higher doses of violacein, 5.0 and 7.5 mg kg−1 , did not significantly affect survival (data not shown) while a single dose of 10 mg kg−1 produced toxic side effects, such as ruffling of hair and sluggishness leading to animal death. The administration of 0.1 and 1.0 ␮g kg−1 of violacein after tumor inoculation resulted in a significant inhibition of tumor growth (P < 0.05), as evident from a 70% reduction in intraperitoneal tumor cell burden on the day of death. Mice treated with 0.1 and 1.0 ␮g kg−1 violacein presented 3.3 ± 2.2 × 107 and 3.7 ± 2.4 × 107 viable ascites cells, respectively, while the control group presented 11.7 ± 3.6 × 107 .

3.4. Antitumor evaluation Despite of the antitumor potential of violacein evidenced in several in vitro studies, there are no previous results reporting this property in vivo. Therefore, in this study, the effect of violacein on the survival time of EAT-bearing mice was evaluated as shown in Fig. 9. EAT cells when injected intraperitoneally to mice grow as ascites tumor with accumulation of large volume of ascitic fluid in the peritoneal cavity. Survival of the control group halved on the 16th day after tumor inoculation and no animal survived beyond

Fig. 9. Survival of EAT-bearing animals treated with violacein. BALB/c mice were inoculated i.p. with 6 × 106 cells and after 24 h, they were treated i.p. with daily 0.2 ml injections of 0.1 ␮g kg−1 () and 1.0 ␮g kg−1 violacein () throughout their lifespan. Mice from the control group were treated with 0.2 ml of vehicle, PBS containing 10% Tween 80 (䊉). Curves were represented by the method described by Kaplan–Meier and differences among groups were analyzed by Logrank (Cox–Mantel) test for non-parametric procedures (*P < 0.05 compared with vehicle-treated tumor group and n = 7/group).

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Table 1 Body weights (g) of control and violacein-treated BALB/c mice during the period of the studya . Day

P

Control Mean ± S.D.

0.1 ␮g kg−1 violacein Mean ± S.D.

1.0 ␮g kg−1 violacein Mean ± S.D.

1000 ␮g kg−1 violacein Mean ± S.D.

0 (before treatment) 36

0.9784 0.5347

21.4 ± 1.14 26.4 ± 1.14

21.6 ± 0.894 26.4 ± 1.15

21.6 ± 1.14 25.6 ± 1.14

21.4 ± 0.894 26.6 ± 1.16

a Mice were treated i.p. with daily 0.2 ml injections of violacein (0.1, 1.0 and 1000 ␮g kg−1 doses) for 35 days and, on the next day after the last injections, mice were weighed and body weight was recorded. Control mice received the vehicle (PBS containing 10% Tween 80) for the same time period. The results express the mean ± S.D. (n = 6/group). ANOVA was performed for statistical comparison among groups and no differences were observed (P value shown in table). Equal variances were indicated by the Bartlett’s test (P < 0.01).

Table 2 Hematological parameters measured in BALB/c mice treated with different doses of violacein for 35 daysa . Parameter (unit)

P value

0.1 ␮g kg−1 violacein

Control Mean ± S.D.

−1

RBC (10 ␮l ) HGB (g dl−1 ) HCT (%) Platelet (105 ␮l−1 ) MCV (fL) MCH (pg) MCHC (g dl−1 ) WBC (103 ␮l−1 ) PMN (%) Lymphocytes (%) Monocytes (%) Eosinophils (%) Basophils (%) 6

0.6286 0.7532 0.8342 0.8851 0.2479 0.5729 0.9658 0.8473 0.9125 0.6761 0.7391 0.4020 0.1413

9.76 14.2 41.2 5.77 46.2 15.3 32.4 4.88 14.4 77.3 6.1 1.3 0.9

± ± ± ± ± ± ± ± ± ± ± ± ±

0.996 1.79 3.83 1.89 1.10 0.576 3.29 1.22 6.08 7.58 1.5 1.0 0.5

Min

Max

Mean ± S.D.

8.50 11.9 35.2 3.44 45.9 14.9 27.0 3.61 5.00 75.7 4.0 0 0

11.1 16.8 45.8 8.07 48.0 16.3 36.0 6.69 20.7 89.0 8.0 3.0 1.5

9.15 13.3 39.5 7.03 47.0 16.1 33.2 5.48 14.0 80.2 5.1 0.5 0.3

± ± ± ± ± ± ± ± ± ± ± ± ±

1.70 1.29 5.20 2.83 1.26 1.52 3.15 1.17 6.45 6.68 1.5 0.3 0.2

1.0 ␮g kg−1 violacein

Min

Max

Mean ± S.D.

7.29 12.1 35.5 3.83 45.0 14.3 29.2 4.23 5.70 71.4 3.1 0 0

11.1 15.4 47.8 11.0 48.0 17.8 36.8 6.90 23.2 90.2 7.0 1.0 1.0

8.98 13.7 41.7 5.57 46.2 15.3 33.0 4.91 15.1 82.2 5.6 1.3 0.8

± ± ± ± ± ± ± ± ± ± ± ± ±

1.04 1.50 3.56 2.16 0.792 1.08 2.41 1.15 5.09 10.8 1.7 0.9 0.4

1000 ␮g kg−1 violacein

Min

Max

Mean ± S.D.

7.74 11.2 36.1 3.35 45.0 14.2 29.5 3.94 7.30 73.8 4.0 0 0

10.2 15.2 46.0 8.94 47.0 16.9 36.0 6.4 20.2 89.4 8.4 2.6 1.0

8.76 13.4 40.0 6.47 45.7 15.4 32.6 5.07 16.5 76.7 5.4 1.1 0.3

± ± ± ± ± ± ± ± ± ± ± ± ±

1.13 1.17 4.01 2.50 0.412 0.618 2.90 1.23 5.90 6.13 1.4 0.6 0.2

Min

Max

7.90 11.9 35.3 3.50 45.0 14.8 28.1 3.94 7.60 70.2 4.0 0.2 0

10.6 14.7 44.1 9.84 46.0 16.3 35.9 7.0 22.0 84.9 7.6 2.0 1.2

a Mice were treated i.p. with daily 0.2 ml injections of violacein (0.1, 1.0 and 1000 ␮g kg−1 doses) for 35 days and, on the next day after the last injections, 150 ␮l of whole blood per animal were collected from the retro-orbital sinus in microtainer tubes with EDTA for hematological analysis. Control mice received the vehicle (PBS containing 10% Tween 80) for the same time period. The results express the mean ± S.D. (n = 6/group) and ranges (minimum and maximum values). ANOVA was performed for statistical comparison among groups and no differences were observed (P value shown in table). Equal variances were indicated by the Bartlett’s test (P < 0.01). RBC: red blood cell count; HGB: hemoglobin; HCT: hematocrit; MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration; WBC: white blood cell count; PMN: polymorphonuclear neutrophils.

3.5. In vivo toxicity studies In view of the positive effect of violacein in preventing cancer progression in vivo, we evaluated possible side effects of the i.p. administration of daily doses of 0.1, 1.0 and 1000 ␮g kg−1 violacein for 35 days in healthy BALB/c mice. Toxicity was evaluated by clinical signs of gross toxicity, behavioral changes and mortality, as well as by hematological, biochemical and histopathological parameters. No animal died in any of the groups during the experimental period (35 days). There were no abnormal clinical signs or behavioral changes in any of the groups, and changes in body weights of the violacein-treated groups were not significantly different from those of the control group after a 35-day treatment period (Table 1). There were no significant changes in hematological parameters in the violacein-treated groups (Table 2). Similarly, no significant differences were found between the violacein-treated groups and

the controls for the three blood chemical parameters evaluated (Fig. 10), ALT, AST and creatinine, which were within the physiological range of values expected for the method of blood collection [33]. These data indicate that daily intraperitoneal injections of violacein at doses up to 1000 ␮g kg−1 for 35 days did not cause hematotoxicity nor poses risks of renal or hepatotoxicity. At necropsy, no visible pathological changes were noted in the livers and kidneys of mice administered violacein at 0.1, 1.0 and 1000 ␮g kg−1 doses. Histological analysis of formaldehyde-fixed, paraffin embedded liver and kidney sections stained with hematoxylin and eosin showed normal architecture in all experimental groups. Livers of animals treated with different doses of violacein showed no sign of necrosis, fatty degeneration, or inflammation (Fig. 11A). Similarly, glomerulus structures, and proximal and distal tubules in kidneys showed normal architecture (Fig. 11B), pointing out that violacein did not cause toxicity to these organs.

Fig. 10. (A) Aspartate aminotransferase (AST) and (B) alanine aminotransferase (ALT) activities, and (C) creatinine levels in serum from mice treated with different doses of violacein for 35 days. BALB/c mice were treated i.p. with daily 0.2 ml injections of 0.1, 1.0 and 1000 ␮g kg−1 violacein for 35 days and, on the next day after the last injections, whole blood was collected from the retro-orbital sinus in preservative-free microtainer tubes for serum biochemical analysis using standard colorimetric assays. Mice from the control group (denoted as C) were treated with 0.2 ml of vehicle, PBS containing 10% Tween 80. The results express the mean ± S.D. (n = 6/group). ANOVA was performed for statistical comparison among groups and no differences were observed (P value shown in figures).

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N. Bromberg et al. / Chemico-Biological Interactions 186 (2010) 43–52

Fig. 11. Histological findings in liver and kidney of mice treated with different doses of violacein for 35 days. BALB/c mice were treated i.p. with daily 0.2 ml injections of 0.1, 1.0 and 1000 ␮g kg−1 violacein for 35 days and, on the next day after the last injections, liver and kidney were submitted to routine histological processes. Mice from the control group (denoted as Ctr) were treated with 0.2 ml of vehicle, PBS containing 10% Tween 80. (A) Normal liver histological findings in control and 0.1, 1.0 and 1000 ␮g kg−1 violacein-treated groups. (B) Normal kidney histological findings in control and 0.1, 1.0 and 1000 ␮g kg−1 violacein-treated groups.

4. Discussion Much of nature remains to be explored, particularly microbial environments, and the interplay of this source with new technologies that can be used to enhance the discovery process, leads to the optimism that examining new natural products will continue to turn up even useful drugs to treat cancer. Violacein has attracted attention as a possible candidate for cancer treatment due to its high toxicity to transformed cells and the ability to induce apoptosis [8–12], in contrast to the low toxic effect in relevant concentrations in untransformed cells [9,28–31]. In view of these facts, in this study we attempted to investigate the in vivo and in vitro tumoricidal activity of violacein against EAT. In addition, cultures of normal human peripheral blood lymphocytes and a 35-day toxicity study in mice were conducted to determine its possible toxic effects. Violacein cytotoxicity, considered primarily as the potential of the compound to induce cell death, was demonstrated by MTT assay and phosphatase activity determination. Reduction of MTT in isolated cells is regarded as an indicator of cell redox activity and the reaction is attributed mainly to mitochondrial enzymes and electron carriers [34]. Phosphatase activity determination has been

successfully applied to natural products as a tool to cytotoxicity studies and a parameter for studying cellular adaptation to apoptosis and oxidative stress [29,35]. Both methods revealed the same IC50 value and this cytotoxicity probably reflects the cell response to particular kinds of damage, in this case, mitochondria insult and/or oxidative stress. Furthermore, the trypan blue exclusion assay confirmed that the reduction on cell viability and cell number was due to the cytotoxic action of violacein to EAT cells. Interestingly, violacein was less effective against normal human peripheral blood lymphocytes in the same experimental conditions, exhibiting an IC50 value twofold higher than that obtained for EAT cells, as evaluated by the MTT assay. This finding is in agreement with previous results reported by Bromberg et al. [29], in which an IC50 value of 10 ␮M was determined by protein phosphatase activities and MTT reduction assay in human lymphocytes exposed to violacein for 24 h. Normal V79 fibroblasts, a model cell system for standard cytotoxicity studies, were also more resistant to violacein activity, presenting 70% of viability after 72 h of treatment with a concentration of 5 ␮M, as evaluated by the nucleic acid content [28]. It is worth mentioning that reduced cytotoxic effects of violacein on normal lymphocytes and monocytes relative to leukemia cells (IC50 = 1.0 ␮M) were also described [9], reinforcing the lower vio-

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lacein cytotoxicity for non-tumor cells than for tumor ones and suggesting violacein as a promising agent for cancer therapy. GSH is the major non-protein thiol in mammalian cells with chemoprotective action. The maintenance of optimal reduced glutathione (GSH):oxidized glutathione (GSSG) ratios by the cell is critical to survival; a deficiency of GSH puts the cell at risk for oxidative damage, since this ubiquitous cellular tripeptide plays a vital role in protecting cells against oxidative damage by free radicals. It is not surprising that an imbalance in GSH levels is observed in a wide range of pathologies, including cancer [13,36]. Distinct responses to chemotherapeutic drugs have prompted cellular GSH modulation as target for cancer chemotherapy. An increase in the GSH level of a cell makes it more resistant to certain antitumor agents, radiation and oxidative effects. On the other hand, therapy that decreases cellular GSH levels usually promotes sensitivity to certain drugs, radiation and oxygen [5,37]. Many authors have considered its participation in primary cellular processes such as signal transduction, gene expression, cell proliferation and apoptosis [38]. The results obtained in the present study demonstrate that violacein exhibits a dose-dependent cytotoxicity to Ehrlich ascites tumor cells concurrent with diminished levels of GSH for all concentrations of violacein. Significant decreases in GSH levels were also reported in EAT cells after treatment with green tea polyphenols [39], 1 -acetoxychavicol acetate [18], ninhydrin [40] and medicinal plants [5]. In addition, Jagetia and Rao [4] have observed that the depletion of GSH levels was accompanied by a drastic elevation in lipid peroxidation when EAT-bearing animals were treated with a guduchi extract. Considering the vast literature that describes the chemoprotective action of GSH and its decreased levels when processes such as lipid peroxidation and oxidative stress take place, we, therefore, hypothesized that in this experiment, the reduction on GSH levels observed in EAT cells treated with violacein has occurred due to the oxidative stress tempted by the addition of the indolic compound. Indeed, the present study demonstrates that intracellular peroxides level was rapidly increased after 8–12 h exposure, corroborating previous experiments in colon cancer cells which indicated that violacein is capable of entailing production of ROS and triggering of apoptosis [11]. If we accept that the efficacy of chemotherapy is associated with a reduction of GSH content, it is reasonable to suppose that the excessive levels of ROS generated in violacein-treated EAT cells might have a detrimental effect in the pool of GSH available. Thus, the oxidative stress-induced cell death by violacein could result in perturbation of growth or death related factors and in reduction of intracellular GSH, which, in turn, conferred weak antioxidant system. In addition to these findings, increase in intracellular ROS and depletion of intracellular GSH has been reported to occur with the onset of apoptosis in a number of studies. The support in favor of the suggested hypothesis initially came from the results of the morphological alterations such as ruffling, blebbing, condensation of the plasma membrane, and the aggregation of nuclear chromatin observed in EAT cells treated with violacein. And, finally, the involvement of ROS production in EAT and Caco-2 cells death induced by violacein demonstrated in the present study and by Carvalho et al. [11] and the nuclear fragmentation investigated as proof of violacein-induced apoptosis in HL60 cells [8,9]. Most drugs currently used in anticancer therapy kill target cells by induction of apoptosis, both by receptor-mediated and mitochondrial-mediated pathways. Disruption of the mitochondrial membrane potential, cytochrome c release and activation of different caspases have already been described following treatment of EAT cells with different natural agents [14,19]. DNA fragmentation is observed in several cases of apoptosis and is considered to occur at an early stage of apoptosis [41]. In this work, apoptosis was also demonstrated in EAT cells after incubation with violacein by the increase

51

in the percentage of fragmented DNA quantified by the diphenylamine method, which occurs concurrently with an increase in Annexin-V-FITC positive cells. Further, we also found that violacein treatment of EAT cells enhanced caspase-2, caspase-9 and caspase3 activities. Caspases play a significant role in apoptosis induction and it is known that the increase of ROS may affect the mitochondrial membrane potential, leading to the release of pro-apoptotic factors, including those involved in caspases cascade activation [42]. Activation of the upstream caspases by pro-apoptotic signals leads to proteolytic activation of effector caspases, such as caspase3, that cleave a set of vital proteins and thus, initiate and execute the apoptotic degradation phase including DNA degradation and the typical morphologic features. Apoptosis occurrence was also found in Caco-2 cells after treatment with violacein, which was correlated with the elevation of ROS and calcium levels, the impairment of mitochondrial function and the activation of caspase-3 [11]. Since depletion of glutathione has been reported to enhance cell death and apoptosis of tumor cells along with loss of essential sulfhydryl groups that result in an altered calcium homeostasis and eventually loss of cell viability, the reduced GSH contents induced as a result of ROS generation by violacein treatment may have contributed to the killing of EAT cells. These reported results illustrated that violacein exhibited antiproliferative and apoptotic activities against EAT cells in vitro and could therefore be candidate for further stages of screening in vivo. We describe for the first time, to the best of our knowledge, the in vivo antitumoral activity of violacein. In EAT-bearing mice, a regular rapid increase in ascites tumor volume was noted. Ascitic fluid is the direct nutritional source for tumor cells and a rapid increase in ascitic fluid with tumor growth would be a means to meet the nutritional requirement of tumor cells. Treatment with low doses of violacein inhibited the tumor volume, the number of viable tumor cells and increased survival rate of EAT-bearing mice, differing from other works with natural products in the literature which spend high doses of the compounds. Even though the exact mechanism of action of violacein in vivo is still unknown, changes in ROS production, GSH status and activation of apoptotic process might contribute to its anticancer activity. Certainly, the increase in EAT-bearing mice survival time after treatment with violacein is the first in vivo result that deserves further studies. Additionally, this is probably the first consistent toxicity study of violacein in vivo, in which complete hematology was described, and the liver and kidney functions were investigated by biochemical determination of ALT, AST and creatinine levels and histopathological examination of these tissues in mice given daily i.p. doses of 0.1, 1.0 and 1000 ␮g kg−1 violacein for 35 days. The results revealed no abnormal clinical signs of gross toxicity or behavioral changes and no animal died in any of the groups. Also, violacein treatment does not affect body weight gain when compared the control group. More importantly, the data indicate that daily doses of violacein up to 1000 ␮g kg−1 for 35 days are well tolerated and did not cause hematotoxicity, renal and hepatotoxicity when administered intraperitoneally to BALB/c mice. These results further indicate that the same effects observed for the doses of 0.1 and 1.0 ␮g kg−1 violacein on survival and tumor burden of EAT-bearing mice were not associated with detrimental effects caused by the higher dose. It is worth mentioning at this point that previous studies from our group indicated that a single oral dose of 10 mg kg−1 violacein was not toxic to rats [43], as well as i.p. administration of daily doses up to 7.5 mg kg−1 violacein to mice infected with Plasmodium chabaudi chabaudi for 11 days did not cause significant toxicity [31], implying that violacein toxicity must dependent on the route and the treatment schedule. Nevertheless, these results are particularly important, since most traditional chemotherapeutic drugs exhibit severe normal toxicity and cause undesirable side effects, thus limiting their application in clinical sets.

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