106   Original article  The in-vitro and in-vivo inhibitory activity of biflorin  in melanoma  Marne C. Vasconcellosa, Dan...
Melanoma growth inhibition by biflorin Vasconcellos et al. 107 Materials and methods                                      ...
108   Melanoma Research 2011, Vol 21 No 2  structure (Fig. 1a) were confirmed by spectroscopic analysis,   Pennsylvania, U...
Melanoma growth inhibition by biflorin Vasconcellos et al. 109 Development and survival of mice bearing B16 tumor         ...
110 Melanoma Research 2011, Vol 21 No 2  However, biflorin did not induce any significant increase                    were...
Melanoma growth inhibition by biflorin Vasconcellos et al. 111 condensation, and fragmentation of the nuclei. In addition,...
112 Melanoma Research 2011, Vol 21 No 2  mitochondria-dependent cell death. The mitochondrial                             ...
Melanoma growth inhibition by biflorin Vasconcellos et al. 113 the early failure of the membrane integrity [23]. Cells    ...
114 Melanoma Research 2011, Vol 21 No 2   4   Cragg GM, Newman DJ. Plants as a source of anti-cancer agents.              ...
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The in vitro and in-vivo inhibitory activity of biflorin

  1. 1. 106 Original article The in-vitro and in-vivo inhibitory activity of biflorin in melanoma Marne C. Vasconcellosa, Daniel P. Bezerrad, Aluısio M. Fonsecac, ´ Ana Jersia Araujo , Claudia Pessoa , Telma L.G. Lemosc, ´ ´ b ´ b Letıcia V. Costa-Lotufob, Manoel Odorico de Moraesb ´ and Raquel C. Montenegroe Biflorin, an ortho-naphthoquinone, is an active compound growth, and also increased the mean survival rate from found in the roots of Capraria biflora L. It has been reported 33.3 ± 0.9 days (control) to 44.5 ± 3.4 days (treated). Our that biflorin presents anticancer activity, inhibiting both findings suggest that biflorin may be considered as tumor cell line growth in culture and tumor development a promising lead compound for designing new drugs in mice. The aim of this study was to examine the to be used in the treatment of melanoma. Melanoma Res effectiveness of biflorin treatment using both in-vitro and 21:106–114 2011 Wolters Kluwer Health | Lippincott c in-vivo melanoma models. Biflorin displayed considerable Williams Wilkins. cytotoxicity against all tested cell lines, with half maximal Melanoma Research 2011, 21:106–114 inhibitory concentration values ranging from 0.58 lg/ml in NCI H23 (human lung adenocarcinoma) to 14.61 lg/ml in Keywords: apoptosis, biflorin, cytotoxic activity, melanoma MDA-MB-231 (human breast cancer) cell lines. In a second a ˆ ˆ Faculdade de Ciencias Farmaceuticas, Universidade Federal do Amazonas, set of experiments using B16 melanoma cells as a model, Manaus, Amazonas, bDepartamento de Fisiologia e Farmacologia, Faculdade de c ´ ˆ ˆ Medicina, Departamento de Quımica Organica e Inorganica, Universidade biflorin reduced cell viability but did not cause significant Federal do Ceara, Fortaleza, Ceara, dDepartamento de Fisiologia, Universidade ´ ´ e increase in the number of nonviable cells. In addition, the ´ ˆ ´ Federal de Sergipe, Sergipe, Aracaju and Instituto de Ciencias Biologicas, ´ ´ ´ Faculdade de Biotecnologia, Universidade Federal do Para, Belem, Para, Brazil DNA synthesis was significantly inhibited. Flow cytometry analysis showed that biflorin may lead to an apoptotic ˆ ´ Correspondence to Raquel C. Montenegro, PhD, Instituto de Ciencias Biologicas ˆ ´ ICB, Avenida Augusto Correa 01, CEP 66025-110, Belem, PA, Brazil death in melanoma cells, inducing DNA fragmentation and Tel: + 55 91 3201 8425; fax: + 55 91 3201 7601; mitochondria depolarization, without affecting membrane e-mail: rcm.montenegro@gmail.com integrity. In B16 melanoma-bearing mice, administration Received 23 March 2009 Accepted 16 December 2010 of biflorin (25mg/day) for 10 days inhibited tumor Introduction against autoxidation of oleic acid in a water/alcohol sys- Cutaneous melanoma is the most serious skin cancer tem [8]. Furthermore, its in-vivo anticancer effects seemed worldwide and its occurrence keeps rising every year to be associated with immunomodulatory properties [9]. especially in tropical countries [1]. Currently, the systemic chemotherapy is unsatisfactory because only a small mino- In a recent study on cytotoxic chemotherapy for treat- rity of patients respond in an acceptable manner [2]. In ment of metastatic melanomas, Gogas et al. [2] discussed this context, detection at early-stages is a key element in the aspects of single drug and combined chemotherapy, as the disease’s evolution, but the prognosis of melanoma well as chemotherapy combined with immunomodulators remains poor despite treatment advances, thus showing or with target-directed agents. They showed that fewer the importance of exploring additional ways to prevent than 20% of patients respond to a single drug treatment, and/or to treat this kind of cancer [3]. but there is a significant increase in the response of the conventional treatment combined with an immuno- In the field of anticancer therapy, plants, a prime source modulator agent [2,10,11]. of effective drugs, and natural products continue to play a highly significant role in the drug discovery and deve- Considering that biflorin seemed to present both anti- lopment process, thus providing leads for the develop- cancer and immunomodulator activities, the aim of this ment of potential novel agents [4,5]. Biflorin, a natural study was to determine the effectiveness of biflorin ortho-naphthoquinone, is an active compound found in treatment using in-vitro and in-vivo melanoma models. the roots of Capraria biflora L. (Scrophulariaceae). It pre- First, the antiproliferative activity of biflorin against seve- sents antifungal, antimicrobial, in-vitro (HL-60, CEM, ral tumor cell lines was determined. In a second set of MCF-7, and HCT-8 cell lines) and in-vivo (Sarcoma 180 experiments, the mechanisms involved in biflorin cyto- and Erlich Carcinoma) anticancer properties [6–9]. Apart toxic activity were investigated using B16 melanoma cells from the common pro-oxidant properties observed for as the model. Finally, the in-vivo effects of biflorin in quinones, biflorin showed potent antioxidant activity mice bearing B16 melanoma cells were also evaluated. 0960-8931 2011 Wolters Kluwer Health | Lippincott Williams Wilkins c DOI: 10.1097/CMR.0b013e328343ecc4Copyright © Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.
  2. 2. Melanoma growth inhibition by biflorin Vasconcellos et al. 107 Materials and methods light–dark cycle (lights on at 06:00 h) at 221C. Animals Cells were treated according to the ethical principles of animal The cytotoxicity of biflorin was tested against B16 ´ experimentation of Colegio Brasileiro de Experimentac˜o¸a (murine melanoma), UACC-62 (human melanoma), Animal, Brazil. The Animal Studies Committee of UACC-257 (human melanoma), MDA-MB-435 (human ´ Universidade Federal do Ceara approved the experimen- melanoma), M14 (human melanoma), K562 (human tal protocols. myeloblastoid leukemia), MDA-MB-231 (human breast carcinoma), MX 1 (human breast carcinoma), NCI H266 Isolation of biflorin (human lung carcinoma), NCI H23 (human lung adeno- Capraria biflora was collected from a plantation located in carcinoma), PC-3 (human prostate carcinoma), and SF- ´ Fortaleza (Ceara, Brazil) in April 2005 and identified by 295 (glioblastoma) cancer cell lines, all obtained from the Dr Edson Nunes (Botany Professor). A voucher specimen National Cancer Institute, Bethesda, Maryland, USA. (number: 30848) was deposited in the Herbarium Prisco Cells were grown in Roswell Park Memorial Institute- Bezerra of the Departamento de Biologia of Universidade 1640 medium supplemented with 10% fetal bovine ´ Federal do Ceara. serum, 2 mmol/l glutamine, 100 mg/ml streptomycin, and Air-dried powdered roots (6 kg) were extracted with light 100 U/ml penicillin, and incubated at 371C with a 5% petroleum (4 l) for 2 days. The extract was partially CO2 atmosphere. evaporated at room temperature until the formation of a solid material. The latter was filtered under vacuum and Animals yielded a purple solid (2 g). The purple solid material was A total of 30 C57BL/6 mice (female, 25–30 g), obtained chromatographed on silica gel and isocratic elution using a from the animal house of the Departamento de Fisiologia binary mixture of light petroleum/ethyl acetate 9 : 1 (v/v). ´ e Farmacologia, Universidade Federal do Ceara, Brazil, Fractions were pooled together according to thin layer were used. Animals were housed in cages with free access chromatographic analysis. Combined fractions having the to food and water. All animals were kept under a 12:12 h purple solid yielded 1.5 g of pure biflorin. The purity and Fig. 1 (a) (b) 25 O 20 (×104 cells/ml) Cell number ∗ O 15 ∗ 10 ∗ ∗ 5 O 0 C D 1.5 3.0 6.0 Biflorin (μg/ml) (c) 60 (d) 90 ∗ 50 80 ∗ ∗ Cell number (%) 70 ∗ BrdU-positive 40 ∗ ∗ 60 cells (%) ∗ 50 30 40 ∗ 20 30 ∗ ∗ 20 10 ∗ ∗ 10 0 0 C D 1.5 3.0 6.0 C D 1.5 3.0 6.0 Biflorin (μg/ml) Biflorin (μg/ml) Viable cells Apoptotic cells Necrotic cells (a) Chemical structure of biflorin (6,9-dimethyl-3-(4-methyl-3-pentenyl)naphtha[1,8–bc]-pyran-7,8-dione). Effect of biflorin on B16 melanoma cells after 24 h of incubation. (b) Cell viability determined by trypan blue staining, (c) Inhibition of 5-bromo-20 -deoxyuridine (BrdU) incorporation. (d) Cells viability determined by acridine orange and ethidium bromide staining. Negative control (c) was treated with the vehicle (0.1% dimethyl sulfoxide) used for diluting the tested substance. Doxorubicin (0.3 mg/ml) was used as positive control (d). Data are presented as mean values ± standard error of the mean from two independent experiments performed in duplicate (n = 4). *P 0.05 compared with control by one-way analysis of variance and followed by Student–Newman–Keuls multiple range test.Copyright © Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.
  3. 3. 108 Melanoma Research 2011, Vol 21 No 2 structure (Fig. 1a) were confirmed by spectroscopic analysis, Pennsylvania, USA), and allowed to dry for 2 h at room including one-dimensional and two-dimensional nuclear temperature. Cells that had incorporated BrdU were magnetic resonance spectral data, infrared, physical proper- labeled by direct peroxidase immunocytochemistry using ties, and comparison with data from the literature [12]. the chromogen diaminobenzidine. Slides were counter- stained with hematoxylin, mounted, and coverslipped. Inhibition of tumor cell proliferation Evaluation of BrdU-positivity was made by light micro- The tumor cell growth was quantified by the ability of scopy (Olympus, Tokyo, Japan). Two hundred cells were living cells to reduce the yellow dye 3-(4,5-dimethyl-2- counted per sample to determine the percentage of thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) positive cells. to a purple formazan product [13]. For all experiments, cells were seeded in 96-well plates (105 cells/well in Morphological analysis with hematoxylin–eosin staining 100 ml of medium). After 24 h, biflorin (0.39–25.0 mg/ml), dissolved in dimethyl sulfoxide (DMSO), was added to Untreated or biflorin-treated B16 melanoma cells were each well (using the HTS-high-throughput screening- examined for morphological changes by light microscopy Biomek 3000-Beckman Coulter Inc., Fullerton, California, (Olympus). To evaluate nuclear morphology, cells from USA) and incubated for 72 h. Doxorubicin (Sigma– cultures (after 24 h incubation) were harvested, trans- Aldrich Co., St. Louis, Missouri, USA) was used as a ferred to cytospin slides, fixed with methanol for 30 s, and positive control. At the end of the incubation, the plates stained with hematoxylin–eosin. were centrifuged and the medium was replaced by fresh medium (150 ml) containing 0.5 mg/ml MTT. Three hours Morphological analysis using fluorescence microscope later, the formazan product was dissolved in 150 ml DMSO B16 melanoma cells were treated with biflorin for 24 h. and the absorbance was measured using a multiplate Then, cells were pelleted and resuspended in 25 ml reader (DTX 880 Multimode Detector). The drug effect phosphate buffered saline (PBS). Thereafter, 1 ml of was quantified as the percentage of control absorbance of aqueous solution of acridine orange/ethidium bromide reduced dye at 595 nm. (AO/EB, 100 mg/ml) was added and the cells types were observed under a fluorescence microscope (Olympus). Analysis of mechanisms involved in the cytotoxic Three hundred cells were counted per sample and were activity classified as follow: viable cells, apoptotic cells, and The following experiments were performed to elucidate necrotic cells. the mechanisms involved in cytotoxic action of biflorin after 24 h drug exposure using B16 melanoma cells (0.3 Â 105 cells/ml). Biflorin was dissolved in DMSO at Cell cycle distribution and internucleosomal DNA the concentration of 5 mg/ml, and added to cell culture fragmentation analysis for final concentrations of 1.5, 3.0, and 6.0 mg/ml, based on B16 melanoma cells were harvested in a lysis solution its half maximal inhibitory concentration (IC50) value containing 0.1% citrate, 0.1% Triton X-100, and 50 mg/ml after 24 h incubation (2.95 mg/ml). The trypan blue propidium iodide. Cell fluorescence was then determined exclusion test was also carried out before each experi- by flow cytometry in a Guava EasyCyte Mini System ment described below to assess cell viability. Doxorubicin cytometer using the CytoSoft 4.1 software (Guava (0.3 mg/ml) was used as a positive control. Negative Technologies, Hayward, California, USA). Five thousand control was treated with the vehicle (0.1% DMSO) used events were evaluated per experiment and cellular debris for diluting the tested substance. was omitted from the analysis. Trypan blue exclusion Cell membrane integrity Cell viability was determined by the trypan blue dye The cell membrane integrity was evaluated by the ex- exclusion test. After the incubation period (24 h), cells clusion of propidium iodide (2 mg/ml). Cell fluorescence were harvested and aliquots were removed from cultures was determined by flow cytometry as described above. and cells that excluded trypan blue were counted in a Newbauer chamber (LO, Laboroptik GmbH, Bad Hom- burg, Hessen, Germany). Measurement of mitochondrial transmembrane potential Mitochondrial transmembrane potential was determined Inhibition of DNA synthesis by the retention of the dye rhodamine 123 in B16 Twenty microliters of 5-bromo-20 -deoxyuridine (BrdU, melanoma cells. Cells were washed with PBS, incubated 10 mmol/l) was added to each well and incubated for 3 h with rhodamine 123 (5 mg/ml) at 371C for 15 min in the at 371C before completing the 24 h period of drug dark, and washed twice. The cells were then incubated exposure. To access the amount of BrdU incorporated again in PBS at 371C for 30 min in the dark and into DNA, cells were harvested, transferred to cytospin fluorescence was then measured by flow cytometry as slides (Shandon Southern Products Ltd., Sewickley, described above.Copyright © Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.
  4. 4. Melanoma growth inhibition by biflorin Vasconcellos et al. 109 Development and survival of mice bearing B16 tumor Table 1 Cytotoxic activity of biflorin on human cancer cells The effect of biflorin on tumor development and survival Biflorin IC50 Doxorubicin IC50 of mice with B16 melanoma tumor was evaluated. The Cell line mg/ml (mmol/l) mg/ml (mmol/l) tumor was injected subcutaneously in C57BL/6 female Melanoma cell lines mice (1.0 Â 106 cells/500 ml). Ten animals per group were M14 2.34 (7.02) 0.30 (0.55) 1.85–2.95 0.16–0.55 used. Treatment was performed by intraperitoneal route UACC-257 1.48 (4.44) 0.27 (0.50) starting 1 day after tumor injection. The chosen dose ND 0.23–0.32 (25 mg/kg/day) was based on earlier studies [9]. Dacar- UACC-62 1.67 (5.01) 0.09 (0.17) 1.55–1.8 0.0.8–0.99 bazine (25 mg/kg/day) was used as a positive control. MDA-MB-435 0.65 (2.10) 0.47 (0.86) 1.72–3.45 0.34–0.65 In the first experiments, animals were divided in two B16 (murino) 10.12 (16.86) 0.03 (0.05) groups: one treated intraperitoneally with biflorin at 6.98–14.67 0.02–0.04 Leukemic cell lines 25 mg/kg/day for a total of 10 days, and the other treated HL-60a 1.95 (6.30) 0.02 (0.03) intraperitoneally with 10% DMSO; the vehicle used to 1.26–2.69 0.01–0.02 dissolve biflorin. The radii of developing tumor were CEMa 1.02 (3.30) 0.02 (0.03) 1.24–1.96 0.01–0.02 measured using vernier calipers at 2-day intervals for K562 2.43 (7.86) 0.14 (0.26) 24 days, and tumor volume was calculated using the 1.41–2.37 0.09–0.22 following formula V = 0.4ab2, where ‘a’ and ‘b’ represent Breast cell lines MCF-7a 0.43 (1.30) 0.20 (0.37) the major and minor diameters, respectively. 1.28–1.77 0.17–0.24 MDA-MB-231 14.61–(47.26) 0.22 (0.40) In a second experiment, the effect of biflorin on the 11.54–18.50 0.18–0.26 survival of animals with B16 melanoma tumor was MX 1 1.11 (3.61) 0.076 (0.14) 0.98–1.29 0.05–0.10 evaluated. The animals were divided into four groups of Lung cell lines 10 animals each. Group 1: animals treated with the NCI H266 0.86 (2.80) 0.15 (0.28) vehicle, 10% DMSO. Group 2: animals treated with 0.67–1.12 0.13–0.18 NCI H23 0.58 (1.88) 0.06 (0.11) dacarbazine (25 mg/kg/day, intraperitoneally). Group 3: 0.51–0.65 0.05–0.07 animals treated with biflorin (25 mg/kg/day, intraperito- Other cell lines PC-3 (prostate) 6.09 (19.7) 0.45 (0.83) neally). Group 4: animals treated with biflorin (25 mg/kg/ 4.17–8.89 0.38–0.68 day, intraperitoneally) plus dacarbazine (25 mg/kg/day, SF-295 (CNS) 0.63 (2.02) 0.16 (0.29) intraperitoneally). The treatments started 1 day after 0.57–0.69 0.13–0.23 HCT-8 (colon)a 0.88 (2.80) 0.04 (0.07) tumor injection for a total of 10 days. The death pattern 4.73–12.5 0.03–0.05 of animals due to tumor burden was noted and the Doxorubicin was used as positive control. Data are presented as IC50 values and percentage of increase in life span was calculated using 95% confidence intervals from three independent experiments performed in the formula [(T–C)/C] Â 100, where ‘T’ and ‘C’ repre- duplicate. IC50, half maximal inhibitory concentration; ND, not determined. sent the number of days the treated and control animals a Published by Vasconcellos et al. [8]. survived, respectively. Statistical analysis values. The IC50 values ranged from 0.58 mg/ml in NCI Data are presented as IC50 values and their 95% H23 (human lung adenocarcinoma) to 14.61 mg/ml in confidence intervals obtained by nonlinear regression or MDA-MB-231 (human breast cancer) cell lines. Doxo- as mean ± standard error of the mean of n experiments. rubicin showed IC50 values from 0.03 mg/ml in B16 Differences between the experimental groups were (murine melanoma) to 0.47 mg/ml in MDA-MB-436 compared by one-way analysis of variance followed by (human breast carcinoma) cell lines. Student–Newman–Keuls multiple range test (P 0.05). Despite the high value of IC50 obtained, all subsequent For survival assay, Kaplan–Meier survival risk assessment, experiments were carried out in B16 melanoma cells, as it was used. Chi-squared test was used for curve compar- was possible to use the same cell line for in-vitro and ison. All analyses were carried out using the GRAPHPAD in-vivo evaluation. For mechanistical purposes, in-vitro program (Intuitive Software for Science, San Diego, experiments were conducted after 24 h incubation, at California, USA). concentrations corresponding to 1.5, 3.0, and 6.0 mg/ml of biflorin. These concentrations were chosen based on Results biflorin IC50 value after 24 h incubation (2.95 mg/ml). Biflorin displayed in-vitro cytotoxicity against different Furthermore, mice bearing B16 melanoma tumor were cell lines used to confirm the in-vivo effectiveness of biflorin. In Several tumor cell lines were treated with increasing addition, internucleosomal DNA fragmentation was concentrations of biflorin for 72 h and analyzed by the performed at 48 h drug exposure. MTT assay. A significant suppression of cell growth was detected in the presence of biflorin, which occurred in a Biflorin reduced the number of viable cells in a dose-dependent manner. Table 1 shows the obtained IC50 concentration-dependent manner (P 0.05) (Fig. 1b).Copyright © Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.
  5. 5. 110 Melanoma Research 2011, Vol 21 No 2 However, biflorin did not induce any significant increase were calculated. Green-stained cells (live), with normal in the number of nonviable cells in any tested concen- morphology, were seen in B16 melanoma cells control tration (P 0.05) (data not shown). groups reaching more than 90% of cells counted. After biflorin-treated B16 melanoma cells, an increasing To further understand the lack of proliferation, the rate of number of apoptotic cells were observed at concentra- DNA synthesizing cells was determined based on the tions of 3.0 and 6.0 mg/ml (P 0.05, Fig. 1d). Only at BrdU incorporation into B16 melanoma cells (Fig. 1c). At higher concentration were necrotic cells found. In all tested concentrations, the inhibition DNA synthesis addition, the doxorubicin-treated cells also showed was significantly decreased (P 0.05). Biflorin inhibited apoptotic characteristics. BrdU incorporation by 33.02, 29.95, and 43.16% at 1.5, 3.0, and 6.0 mg/ml, respectively. Doxorubicin inhibited Morphological examination of B16 melanoma cells 88.92%. showed severe drug-mediated changes (Fig. 2). B16 Morphological analysis of treated cells was also investi- melanoma cells treated with biflorin, at all tested gated using AO/EB staining for fluorescent microscopy concentrations, presented morphology consistent with and the percentage of viable, apoptotic, and necrotic cells apoptosis, including reduction in cell volume, chromatin Fig. 2 (a) (b) (c) (d) (e) (f) Microscopic analysis of hematoxylin/eosin stained B16 melanoma cells. Cells were untreated (a), treated with biflorin (1.5 mg/ml, c and d), biflorin (3.0 mg/ml, e), or biflorin (6.0 mg/ml, f) in B16 melanoma cells , and analyzed by using a light microscope ( Â 400). Doxorubicin (0.3 mg/ml) was used as a positive control (b). Black arrows: cells blocked in different phases of the cell cycle.Copyright © Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.
  6. 6. Melanoma growth inhibition by biflorin Vasconcellos et al. 111 condensation, and fragmentation of the nuclei. In addition, change was observed. However, at concentrations of 3.0 and biflorin caused an increase in the mitotic figures at 6.0 mg/ml there were fewer cells in the G2/M phase (13.15 different stages of mitotic process. Doxorubicin also indu- and 11.96% against 20.36% of the control). In addition, ced cell shrinkage, chromatin condensation, and nuclear biflorin caused significant internucleosomal DNA fragmen- fragmentation, morphology consistent with apoptosis. tation at the higher concentration (P 0.05, Fig. 3c) after 24 and 48 h. Biflorin also induced mitochondrial depolar- The reduction of cell number due to biflorin treatment ization in B16 melanoma cells, as measured by incorpora- was confirmed by the flow cytometric analysis (Fig. 3a). tion of rhodamine-123 using flow cytometry, suggesting Cell membrane integrity was also accessed, but biflorin did not disrupt membrane at any tested concentration (P 0.05, Fig. 3b). Furthermore, biflorin caused cell Table 2 Effect of biflorin in cell cycle distribution in B16 cells shrinkage, at concentrations of 3.0 and 6.0 mg/ml, as determined by flow cytometry using propidium iodide, triton X-100 observed by the decrease in forward light scatter and and citrate after 24 h incubation nuclear condensation, as observed by a transient increase Phase of the cell cycle (%) in side scatter, both morphological modifications were Concentration compatible with apoptotic cells (data not shown). Drug (mg/ml) G0/G1 S G2/M Control — 60.69 ± 2.42 14.31 ± 1.45 20.36 ± 1.82 The effect of biflorin on the cell cycle progression was Doxorubicin 0.3 38.02 ± 2.27* 13.88 ± 1.35 10.19 ± 2.12* also examined using flow cytometry. All DNA subdiploid Biflorin 1.5 59.12 ± 2.53 13.17 ± 1.33 18.14 ± 1.64 3.0 61.33 ± 2.17 11.43 ± 1.31 13.15 ± 1.16* in size (sub-G1) were considered as internucleosomal 6.0 60.21 ± 2.55 10.42 ± 1.00 11.96 ± 1.22* DNA fragmentation. Table 2 shows the obtained cell Negative control was treated with the vehicle used for diluting the tested cycle distribution. On control, the percentage of cells substance. Doxorubicin (0.3 mg/ml) was used as positive control. Data are corresponding to G0/G1 was 60.69%, to S phase was presented as mean values ± standard error of the mean from three independent 14.31%, and to G2/M was 20.36%. Cells with inter- experiments performed in triplicate. Five thousand events were analyzed in each experiment. nucleosomal DNA fragmentation (sub-G1) corresponded *P 0.05 compared with control by analysis of variance followed by Student– to 5.17%. At the concentration of 1.5 mg/ml, no significant Newman–Keuls multiple range test. Fig. 3 (a) (b) 0.6 100 Cell membrane integrity (%) Number of cells (×106) 0.5 80 0.4 ∗ ∗ 60 0.3 ∗ 0.2 40 0.1 20 0.0 C D 1.5 3.0 6.0 0 C D 1.5 3.0 6.0 Biflorin (μg/ml) Biflorin (μg/ml) (c) (d) 50 DNA fragmentation (%) 80 ∗ ∗ 24 h 48 h despolarization (%) 40 Internucleosomal Mitochondrial 60 #,∗ 30 ∗ #,∗ #,∗ 40 ∗ ∗ ∗ ∗ 20 20 # 10 0 0 C D 1.5 3.0 6.0 C D 1.5 3.0 6.0 Biflorin (μg/ml) Biflorin (μg/ml) Effect of biflorin on B16 melanoma cell number (a), membrane integrity (b), internucleosomal DNA fragmentation (c), and mitochondrial transmembrane potential (d) determined by flow cytometry. Negative control, C, was treated with the vehicle (0.1% dimethyl sulfoxide ) used for diluting the tested substance. Doxorubicin (0.3 mg/ml) was used as a positive control (d). Data are presented as mean values ± standard error of the mean from three independent experiments performed in triplicate. Five thousand events were analyzed in each experiment. *P 0.05 compared with control by analysis of variance followed by Student–Newman–Keuls multiple range test.Copyright © Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.
  7. 7. 112 Melanoma Research 2011, Vol 21 No 2 mitochondria-dependent cell death. The mitochondrial after the tumor inoculation, while the dacarbazine- depolarization occurred at all tested concentrations treated animals survived for 31.9 ± 2.0 days. Animals (Fig. 3d). treated with biflorin survived for 44.5 ± 3.4 days (P 0.05), while those treated in association with dacarbazine survived for 35.7± 2.7 days. Biflorin (25 mg/kg/day) treat- Biflorin also displayed in-vivo antitumor effects ment significantly increased the life span by 33.7%, but The effects of biflorin on mice bearing B16 melanoma cells the association of biflorin and dacarbazine was inactive. were also examined. There was a significant reduction of tumor volume in biflorin-treated animals (Fig. 4a). Tumor volume of control mice was 0.94 ± 0.13 cc3 on day 20, while Discussion that of biflorin-treated animals was only 0.33 ± 0.08 cc3, on The search for new metabolites or drugs that effectively the same day. In addition, the life span of mice-bearing intervene in neoplastic processes has a great value tumors was significantly increased by biflorin treatment especially in melanoma therapy. Many drugs, which are (Fig. 4b). The control animals survived for 33.3 ± 0.9 days clinically used in the therapy of solid tumors, contain a quinone moiety. This moiety is well known to possess anticancer properties, although its exact mechanism is Fig. 4 still unclear [14]. The ortho-naphthoquinones constitute promising groups of anticancer compounds [15]. In this (a) 3.0 way, this study reported the mechanisms involved in the Control cytotoxic activity of biflorin, an ortho-naphthoquinone 2.5 Biflorin isolated from C. biflora, in cultured cancer cell lines, as Tumor volume (cc3) 2.0 well as in mice bearing B16 melanoma tumor. Vasconcellos et al. [8] had shown earlier that biflorin 1.5 ∗ inhibits the proliferation of cancer cell lines in culture. 1.0 Herein, the tested panel included 13 cell lines from ∗ different tissues, including human skin (melanoma), ∗ 0.5 ∗ breast, lung, prostate, colon, central nervous system, ∗ and also leukemia cells. Biflorin exhibited a strong 0.0 cytotoxicity for all tested cell lines, showing the ability 0 5 10 15 20 25 30 to suppress the cell growth in a concentration-dependent Days after tumor implant manner despite the tissue origin. (b) 120 Such antiproliferative effects were further investigated to show the mechanism of cytotoxic action exhibited by 100 biflorin in the B16 melanoma cell line, and also to confirm its in-vivo effectiveness. The B16 melanoma cell is a well- 80 established model for the study of experimental cancer Survival (%) 60 therapies [16,17]. To evaluate whether this activity was related to DNA synthesis, the BrdU incorporation test Biflorin 40 was carried out. The analysis showed that biflorin decreased the number of cell divisions at all concentra- 20 tions (P 0.05) and corroborated with trypan blue Control Dac Bif + Dac exclusion where biflorin reduced viable cell numbers 0 without increasing the number of nonviable cells. 0 10 20 30 40 50 60 70 Days Drugs that induce cell death by apoptosis in tumor cells can be useful for cancer chemotherapy [18–20]. Apopto- Control Biflorin Dacarbazine sis, or programmed cell death, has an essential role in Biflorin + dacarbazine controlling cell number in many developmental and physiological settings and in chemotherapy-induced Effect of biflorin on melanoma tumor development (a) and on the life span (b) of tumor bearing mice. Mice (C57BL/6) were injected with B16 cells tumor cell killing [21]. Cells undergoing apoptosis show (1.0 Â 106 cells/500 ml subcutaneously). Biflorin was administered by typical, well-defined morphological changes, including intraperitoneally at a dose of 25 mg/kg, starting 1 day after tumor implant, for 10 days. Control mice received vehicle (10% dimethyl sulfoxide). plasma membrane blebbing, chromatin condensation with Dacarbazine was used as a positive control (25 mg/kg). Data are margination of chromatin to the nuclear membrane, representative of 10 animals. *P 0.05 compared with control by karyorhexis (nuclear fragmentation), and formation of analysis of variance followed by Student–Newman–Keuls multiple range test. The survival rates were analyzed by Kaplan–Meier and apoptotic bodies [22]. During the initial stages of apop- compared by w2-test for controls and treated groups. tosis, the cell shrinks whereas the membrane remains intact. During necrosis, cell swelling occurs as a result ofCopyright © Lippincott Williams Wilkins. Unauthorized reproduction of this article is prohibited.
  8. 8. Melanoma growth inhibition by biflorin Vasconcellos et al. 113 the early failure of the membrane integrity [23]. Cells the only cytotoxic chemotherapy agent approved by the treated with biflorin did not decrease the cell membrane Food and Drug Administration (the American regulatory integrity but showed chromatin condensation, apoptotic agent for medication) for metastatic melanoma. However, bodies, and blebs, suggesting that treated cells under- it has a limited result for patients inducing a 15–25% go apoptosis. These findings were confirmed by the fluo- response, in a mean time of 5–6 months. Long-term rescence microscopy using AO/EB staining. dacarbazine therapy shows that fewer than 2% of patients have a survival rate above 6 years [2]. As mentioned The mitochondria has been suggested to be fundamental earlier, melanoma tumors are quite refractory to systemic for the biochemistry of apoptosis because it might form the chemotherapy [33]. nidus where the decision of life and death is actually being made [24,25]. In this way, apoptosis has been character- In this way, investigation of cytotoxic agents in combination ized by several biochemical criteria, including changes in with immunomodulators and with minimal efficacy has mitochondrial membrane permeability [26]. Biflorin in- been carried out and similar results between the associa- duced mitochondrial depolarization in B16 melanoma cells, tions of dacarbazine with tamoxifen or interferon-a were suggesting apoptosis by an intrinsic pathway. observed. A 28% survival rate with a 41-week survival mean Measurements of DNA content made it possible to time was observed with a dacarbazine + tamoxifen combi- recognize the cell cycle phase specificity of the apoptotic nation treatment, while dacarbazine alone induced a 12% process [27]. DNA fragmentation and formation of survival rate. When dacarbazine was associated with high molecular weight ( 50 kbp) and nucleosome-sized interferon-a, 12 complete responses and four partial (200 bp) DNA fragments [28,29] are commonly observed responses were observed on a 30 patient study, while in apoptotic cells. DNA fragmentation can be analyzed by dacarbazine alone produced only two complete responses flow cytometry by detection of DNA hypoploidy, after and four partial ones. These results emphasize the need adding propidium iodide to the dying cells and permea- for a greater efficacy of single drugs, rather than alone or bilizing them by a detergent. Propidium iodide inter- in combination, to reduce tumor resistance. Moreover, it calates in the DNA and the size of DNA fragments reflects the need for new agents with lesser side effects, appears as a hypoploid DNA histogram. This technique and this might have the most significant impact on the allows discrimination between apoptotic and necrotic cell treatment of cancers [3]. Herein, the association of death [30]. In addition, biflorin effect on cell cycle biflorin and dacarbazine was not efficient in the treat- progression was determined by flow cytometry. DNA ment of mice bearing melanoma tumor. fragmentation observed during biflorin treatment was accompanied with membrane integrity, thus suggesting In conclusion, these data show that biflorin possesses in- an apoptotic cell death pattern. The cell population in vitro and in-vivo anticancer activities in melanoma models. the G2/M phase was shifted to the sub-G1 population In cultured cells, biflorin was able to preferentially lead the (internucleosomal DNA fragmentation) after biflorin cells of the G2/M phases to mitochondrial-dependent treatment (3.0 and 6.0 mg/ml) and no significant change apoptosis in B16 melanoma cell line. In the in-vivo of cell population in the G0/G1 or S phase was observed antitumor assay, biflorin was found to be associated with a (data not shown). This implies that biflorin might decrease in tumor volume and a significantly increase in life preferentially lead the cells of the G2/M phases to span. Further studies must be carried out to understand the apoptosis. underlying mechanism involved in biflorin activities. Herein, we reported the antitumor activity of biflorin in mice bearing B16 melanoma tumor. A reliable criterion for judging the value of any anticancer agent is the increase Acknowledgements in life span of the animals [31,32]. A decrease in tumor The authors are grateful to the Brazilian Agencies FINEP, volume as mentioned above finally reduced the tumor CNPq, BNB/FUNDECI, PRONEX, and CAPES for burden and enhanced the life span of mice bearing fellowships and financial support. They also thank Silvana tumors. Earlier studies showed that biflorin exhibits Franca dos Santos and Luciana Franca for providing ¸ ¸ antitumor effects on experimental tumors (sarcoma 180 excellent technical assistance. and Ehrlich carcinoma) without an expressive toxicity. This activity seemed to be related to its immunostimu- There is no conflict of interest to declare. lating and cytotoxic properties [8,9]. References In this study, biflorin (25 mg/kg/day) improved the life 1 Molenkampa BG, Van Leeuwena PAM, Van den Eertweghb AJM, Sluijter BJ, Scheper RJ, Meijer S, et al. 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