Gallotannin-rich Caesalpinia spinosa fraction decreases the primary tumor and factors associated with poor prognosis in a murine breast cancer model

  • Claudia Urueña1,

    Affiliated with

    • Juan Mancipe1,

      Affiliated with

      • John Hernandez1,

        Affiliated with

        • Diana Castañeda1,

          Affiliated with

          • Luis Pombo2,

            Affiliated with

            • Alejandra Gomez1,

              Affiliated with

              • Alexzander Asea3 and

                Affiliated with

                • Susana Fiorentino1Email author

                  Affiliated with

                  BMC Complementary and Alternative MedicineThe official journal of the International Society for Complementary Medicine Research (ISCMR)201313:74

                  DOI: 10.1186/1472-6882-13-74

                  Received: 24 July 2012

                  Accepted: 13 February 2013

                  Published: 3 April 2013

                  Abstract

                  Background

                  Several treatment alternatives are available for primary breast cancer, although those for metastatic disease or inflammation associated with tumor progression are ineffective. Therefore, there is a great need for new therapeutic alternatives capable of generating an immune response against residual tumor cells, thus contributing to eradication of micrometastases and cancer stem cells. The use of complex natural products is an excellent therapeutic alternative widely used by Chinese, Hindu, Egyptian, and ancestral Latin-American Indian populations.

                  Methods

                  The present study evaluated cytotoxic, antitumor, and tumor progression activities of a gallotannin-rich fraction derived from Caesalpinia spinosa (P2Et). The parameters evaluated in vitro were mitochondrial membrane depolarization, phosphatidylserine externalization, caspase 3 activation, DNA fragmentation, and clonogenic activity. The parameters evaluated in vivo were tumor growth, leukocyte number, metastatic cell number, and cytokine production by flow cytometry.

                  Results

                  The in vitro results showed that the P2Et fraction induced apoptosis with mitochondrial membrane potential loss, phosphatidylserine externalization, caspase 3 activation, DNA fragmentation, and decreased clonogenic capacity of 4T1 cells. In vivo, the P2Et fraction induced primary tumor reduction in terms of diameter and weight in BALB/c mice transplanted with 4T1 cells and decreased numbers of metastatic cells, mainly in the spleen. Furthermore, decreases in the number of peripheral blood leukocytes (leukemoid reaction) and interleukin 6 (IL-6) serum levels were found, which are events associated with a poor prognosis. The P2Et fraction exerts its activity on the primary tumor, reduces cell migration to distant organs, and decreases IL-6 serum levels, implying tumor microenvironment mechanisms.

                  Conclusions

                  Overall, the P2Et fraction lessens risk factors associated with tumor progression and diminishes primary tumor size, showing good potential for use as an adjuvant in breast cancer ER(+) treatment.

                  Keywords

                  Caesalpinia spinosa Breast cancer Apoptosis 4T1 IL-6 Metastasis

                  Background

                  One of the most common cancers in women worldwide is breast cancer. Its estimated incidence is 23% (1.38 million) new cases per year, and it comprised 14% (458,000) of all cancer deaths in 2008 [1]. According to the Colombian Cancer Institute, there were 551 new cases localized at the primary site in 2009, corresponding to 10% of all newly registered cancer cases [2]. There are several therapeutic alternatives for breast cancer, including surgery, hormone therapy, chemotherapy, and radiotherapy. Nonetheless, these therapies are ineffective for metastatic disease [3]. Therefore, there is a great need for new therapeutic alternatives capable of generating an immune response against residual tumor cells, thus contributing to eradication of micrometastases and cancer stem cells [4]. The use of complex natural products is an excellent therapeutic alternative widely used by Chinese, Hindu, Egyptian, and ancestral Latin-American Indian populations [5]. Biologically active compounds, such as hydrolyzable or condensed tannins [6], have high therapeutic potential in cancer treatment. Green tea-derived condensed tannins, for example, have been extensively studied. Their activities include tumor growth inhibition, decreased cell proliferation, increased apoptosis, and angiogenic suppression [7]. On the contrary, studies on the biological activity of hydrolyzable tannins are limited. Nevertheless, reports have shown that the tannin fraction derived from Eugenia jambos L. (Myrtaceae) is active against HL60 leukemia cells, inducing apoptosis and DNA fragmentation [8]. In addition, a gallotannin-rich fruit extract from Terminalia chebula induces apoptosis in the murine and human breast cancer cells S115 and MCF-7, respectively [9].

                  Soluble mediators may be exchanged between tumor cells and their microenvironment, affecting tumor growth and promoting metastases. Such mediators may be secreted as a result of the interaction between the innate and/or adaptive immune system with tumor cells, or produced by the tumor cell to modulate its surroundings for its own benefit [10]. Several reports indicate that high IL-6 serum levels in patients with breast carcinoma indicate a poor prognosis due to correlation with metastases in patients with untreated breast cancer [11].

                  Our group recently reported that a Caesalpinia spinosa-derived fraction enriched in hydrolyzable tannins induces a cytotoxic effect on leukemia cells (K562), apoptosis with mitochondria membrane potential (MMP) loss, DNA fragmentation, and a decrease in clonogenic capacity [12]. Additional reports on the same plant have shown that hydrolyzable tannins such as tannic acid (TA) lower the biochemical marker levels associated with skin cancer and delay the development of tumors in mouse skin [13, 14]. However, reports on breast cancer have not been linked with these plant-derived tannins.

                  The murine breast adenocarcinoma cell line 4T1 is poorly immunogenic and highly invasive and expresses the estrogen receptor (ER+). When orthotopically transplanted into BALB/c mice, 4T1 cells are able to reproduce stage IV human breast cancer pathology [15]. Primary tumor metastasizes spontaneously to the lung, liver, brain, and bone marrow [16], making this an excellent model to study metastatic breast cancer pathophysiology and develop effective therapies.

                  We evaluated the effects of a C. spinosa plant fraction on 4T1 cells in vitro and in vivo. The fraction induced tumor cell apoptosis in vitro, an activity related to IL-6 secretion by the tumor cells. In vivo, it inhibited tumor growth, reduced metastatic cell numbers, and reduced peripheral blood leukocytes. Finally, IL-6 serum levels in fraction-treated mice were lower than those in untreated mice.

                  Methods

                  Plant material, extraction and purification

                  Caesalpinia spinosa pods were collected in Villa de Leyva, Boyacá, Colombia in March 2007 and identified by Luis Carlos Jiménez from the Colombian National Herbarium (voucher specimen number COL 523714). Authorization to collect the vegetal material was given by “Ministerio de Medio Ambiente y Desarrollo sostenible de Colombia” (Nº 5 from 10 may 2011). P2Et fraction was purified and characterized as previously described [12].

                  Cell culture conditions

                  The murine mammary tumor cell line 4T1 (a gift from Alexzander Asea of Texas A&M Health Science Center College of Medicine, Temple, TX, USA) was cultured in RPMI-1640 (Eurobio, Toulouse, France) supplemented with heat-inactivated fetal calf serum (10%) (Eurobio), 2mML-glutamine, 100 U/mL penicillin, 100μg/mL streptomycin, 0.01M Hepes buffer, and 1mM sodium pyruvate (Eurobio) and incubated in a humidified environment at 37°C and 5% CO2. Tumor cells were proven Mycoplasma-free with a MycoProbe Mycoplasma Detection Kit (R&D Systems, Minneapolis, MN) and maintained with ciprofloxacin (0.5μg/mL). Cells were grown until 75% confluence, removed with trypsin/EDTA 1X (Eurobio), washed with phosphate-buffered saline (PBS) (154mM, pH7.2), and suspended in supplemented RPMI-1640. Human peripheral blood mononuclear cells (PBMC) and fibroblasts obtained from normal healthy donors (with informed consent) were used as normal cells. Normal cells were maintained as previously reported [17]. This project was approved by the ethics committee (founded in 2002) of the Science Faculty at a meeting on August 21, 2007.

                  In vitro cytotoxicity assays

                  P2Et fraction and doxorubicin cytotoxic effects on normal and tumor cells were evaluated using trypan blue and methylthiazol tetrazolium (MTT) assays (Sigma-Aldrich, Saint Louis, MO) as previously reported [17]. Cell viability was assessed with a trypan blue dye exclusion test. The IC50 (50% inhibition of cell growth) value was calculated using Probit analysis (MINITAB Release 14.1; Minitab Inc. 2003 Statistical Software).

                  Evaluation of MMP

                  Cells (3 × 105) were treated with various concentrations of P2Et fraction (34.1, 17, and 8.5μg/mL), valinomycin (positive control, 0.1μg/mL) or ethanol (negative control, 0.02%) for 6, 12, 24, and 48h. MMP was measured using JC-1 (Sigma-Aldrich) dye as previously reported [17]. The cells were acquired on a FACSAria I (Becton, Dickinson and Company, Franklin Lakes, NJ) and analyzed with FlowJo software (Tree Star Inc., Ashland, OR), which calculated the red/green fluorescence ratios. Duplicate estimations were made, and the average was expressed as mean ± SEM in three independent experiments.

                  Annexin V assay

                  Phosphatidylserine (PS) externalization was assessed by flow cytometry using Annexin V-Alexa Fluor 488 (Molecular Probes, Invitrogen Corp., Carlsbad, CA) and propidium iodide (PI) (Sigma-Aldrich) as previously reported [12]. Briefly, 4T1 cells (3 × 105) were treated with P2Et fraction (34 and 17μg/mL), doxorubicin (positive control, 0.51 and 0.27μg/mL), or ethanol (negative control, 0.02%) for 24 and 48h. After treatment, the cells were dyed with Annexin V and PI, acquired on a FACSAria I (Becton Dickinson), and analyzed with FlowJo software (Tree Star Inc). Assays were performed in triplicate.

                  Caspase 3 activity assay

                  Caspase 3 activity was estimated using a caspase 3 colorimetric assay kit (Sigma-Aldrich). Briefly, cells (2 × 105 cells) were cultured at various concentrations of P2Et fraction, doxorubicin (positive control), or ethanol (negative control, 0.02%) for 48h. Caspase 3 activity was estimated following the manufacturer’s instructions. The increase in caspase 3 activity was calculated from a calibration curve prepared with pNA standards using the following formula: Activity, μmol pNA/min/mL = [(μmol pNA × d)/(t × v)], where d = dilution factor, t = reaction time in min, and v = volume of sample in milliliters.

                  Analysis of DNA fragmentation

                  Cells (3 × 105) were treated with different concentrations of P2Et fraction (34.1 and 17μg/mL), doxorubicin (positive control, 0.27μg/mL) or ethanol (negative control, 0.02%) for 48h. DAPI (4',6-diamidino-2-phenylindole; Sigma-Aldrich)-stained cells were monitored under a confocal microscope as previously reported [17]. Slides were mounted using a ProLong Antifade Kit (Molecular Probes), and cells were analyzed under a fluorescence confocal microscope (FluoView 1000; Olympus, Japan).

                  Clonogenic assays

                  The clonogenic assays were performed as previously described [17]. Briefly, 4T1 cells (2.5 × 105 cells) were plated (96-well plates) and treated with P2Et fraction (34.1, 17, and 3.4μg/mL), doxorubicin (0.51, 0.25, and 0.17μg/mL), or ethanol (negative control, 0.02%) and incubated for 6h in a humidified environment at 37°C and 5% CO2. After treatment, cells were re-plated onto 0.5% agar dishes (60mm; 20,000 cells/dish), incubated for 14 d (37°C and 5% CO2), and stained with violet crystal (0.4% in ethanol). Cell colonies with >50 cells were counted. Treatments were performed in triplicate, and results are expressed as mean ± SEM.

                  Cytokine secretion assessment

                  Cytokine secretion was assessed by flow cytometry using a BD Cytometric Bead Array (CBA) Mouse Inflammation Kit (Becton Dickinson). Briefly, 4T1 cells (2 × 106 cells) were cultured with different concentrations of P2Et fraction (34, 17, and 3.4μg/mL), doxorubicin (0.5, 0.27, and 0.05μg/mL), or ethanol (negative control, 0.02%) for 6, 12, 24, and 48h. Secreted cytokines were acquired on a FACSAria II (Becton Dickinson) and analyzed with FCAP Array software (Becton Dickinson). Treatments were performed in triplicate, and results are expressed as mean ± SEM.

                  Mice

                  Female BALB/c mice, 6 to 12weeks old and purchased from Charles Rivers Laboratories International, Inc. (Wilmington, MA), were housed in our animal research facility following the established protocols of the Ethics Committee of the Science Faculty and National and International Legislation for Live Animal Experimentation (Colombia Republic, Law 84, 1987; Colombia Republic, Resolution 08430, 1993; National Academy of Sciences, 2010). Mice were housed in polyethylene cages with food and water ad libitum, controlled temperature, and a 12-h light/dark cycle. Before treatments, the mice were acclimated for 1week under standard conditions.

                  Acute toxicity evaluation

                  Female BALB/c mice (6 to 12weeks of age) weighing approximately 25g, were divided into groups of five and inoculated intraperitoneally (IP) with 1, 2, 4, and 8mg/mL of P2Et fraction corresponding to 40, 80, 160 and 320mg/kg. After 72h, deceased animals were counted and the lethal dose, 50% (LD50) was calculated with Probit version 14 (Minitab Inc.). To ensure no toxicity animals were treated with two different doses of P2Et fraction (18.7 and 9.3mg/Kg) which corresponds to 4 times lower doses than LD50.

                  Tumor model, treatment protocol, and cytokine detection

                  4T1 cells (1 × 104) suspended in 100 μL of saline phosphate-buffer were injected into the right mammary fat pad (subcutaneously [SC]) on day 0. After 5days of inoculation, the mice (7–8 mice/group) were treated with IP injection of P2Et fraction (9.3 or 18.7mg/kg) or control vehicle twice a week. The leukocyte count was determined weekly using a hemocytometer (ABX SAS Micros 60; Horiba, Madrid, Spain). Blood was obtained from a small incision made to a lateral tail vein and added directly into an EDTA containing vial and then diluted 1:1 with PBS and counted immediately using a Calibrated Cell Counter. Additionally, differential counts were performed using Wright staining. Tumors were measured with vernier calipers twice a week, and tumor volume was calculated using the following formula: tumor volume (mm3) = [(width)2 × length]/2 [18]. Mice were sacrificed when the primary tumor reached 2000mm3, and lung, spleen, liver, brain, primary tumor, and bone samples were collected for histopathology studies. Tumor weight was determined in a high-sensitivity balance. Serum was collected by cardiac puncture, and cytokine evaluation was performed using a BD CBA Mouse Inflammation Kit (Becton Dickinson). Experiments were performed twice, and the results are expressed as the mean of two independent experiments.

                  To evaluate the effectiveness of oral treatment with P2Et fraction, animals were randomly assigned in control or treatment groups (7 mice per group). One week prior to tumor cell inoculation (day 0), the treatment group started P2Et fraction consumption (0.306mg/Kg daily equivalent to dose schedule used in IP administration experiment plus an adjustment for bioavailability) administered in water. 4T1 cells (1 × 104) suspended in 100 μL of PBS were injected into the right mammary fat pad SC on day 7. All animals were weighed once a week and food and water consumption was recorded once a week. To identify and quantitate surface lung metastatic nodules, at the end of the experiment, animals were euthanized and the chest cavity and trachea was exposed through a midline chest incision. The trachea was cannulated with a 30-gauge syringe, and the lungs were slowly insufflated with India Ink (1:15 PBS). The lungs were excised en bloc and immersed in Fekete’s solution (EtOH 70%, formaldehyde 10%, glacial acetic acid at a 60:22:1 ratio) to destain the pulmonary nodules. White tumor nodules against a blue lung background were counted by two independent observers.

                  Histopathologic analysis

                  After treatment with P2Et fraction, primary tumors, lung, liver, spleen, brain, and bone tissues were dissected and fixed in 10% formaldehyde (Sigma-Aldrich), embedded in paraffin, cut into 2-μm sections, and stained with hematoxylin and eosin (H&E). Metastatic tumor infiltrations were evaluated for each tissue and counted under a microscope using a micrometric grid and counted in 5 different fields (magnification power, 10× or 40×).

                  Statistical analysis

                  Values are given as mean ± SEM. Data were analyzed by one- and two-way ANOVA, and differences between the control and treated groups were determined using the Bonferroni test. Differences were considered significant at p < 0.05 and determined with GraphPad Prism 5.0 software (La Jolla, CA, USA).

                  Results

                  P2Et fraction is cytotoxic to 4T1 tumor cells in vitro

                  The cytotoxicity of P2Et fraction on 4T1 cells was evaluated by MTT assay. Tumor cells were treated with P2Et fraction (at different concentrations), gallic acid, and doxorubicin for 48h. The fraction reduced 4T1 cell viability in a dose-dependent manner with an IC50 of 34.1μg/mL. Notably, one of the compounds present in P2Et fraction is gallic acid, and gallic acid by itself exhibits less cytotoxicity (96.2μg/mL) than does the full P2Et fraction. This suggests that compounds other than gallic acid exert this cytotoxic activity [19]. Doxorubicin was used as positive control with an IC50 of 0.5μg/mL, as expected (Table1, Additional file 1: Figure S1). To evaluate whether the fraction activity is selective enough for tumor cells, normal fibroblasts and human mononuclear cells were treated with P2Et fraction. The outcome was that P2Et fraction exerted a slight cytotoxic effect on normal cells at high doses where the IC50 was more than 125μg/mL, as shown in Table1.
                  Table 1

                  IC 50 values of P2Et fraction, gallic acid, and doxorubicin in 4T1 and normal cells (gingival fibroblasts and PBMC) estimated using the MTT assay

                   

                  IC50 values after treatment with the following compounds

                  Cell Type

                  P2Et fraction*

                  Gallic acid*

                  Doxorubicin*

                  4T1

                  34.1 ± 1.6

                  96.2 ± 0.0

                  0.5 ± 0.1

                  PBMC + PHA

                  > 125 ± 1.0

                  ND

                  2023 ± 0.2

                  Fibroblasts

                  > 125 ± 1.0

                  ND

                  0.4 ± 0.1

                  *Concentration (μg/mL) ± SEM.

                  ND = not done.

                  P2Et fraction induces apoptosis through the mitochondrial pathway

                  Defining the type of tumor cell death induced by cytotoxic or cytostatic agents may give information about the patient’s response. To assess the cell death type induced by P2Et fraction, we estimated loss of MMP, externalization of PS, activation of effector caspases, and DNA fragmentation [20]. 4T1 cells treated with P2Et fraction at different concentrations for 6, 12, 24, and 48h showed MMP loss in a dose-dependent manner starting at 6h after treatment until 48h (Figure1a). Similarly, PS externalization was evidenced by Annexin V-Alexa Fluor 488 dye, where Annexin V+/PI- cells were early apoptotic cells (Figure1b). In addition, the late appearance of Annexin V+/PI + cells at 48h after treatment showed the cell death was apoptotic, not necrotic. Doxorubicin showed a typical apoptotic pattern after treatment for 24 and 48h. On the other hand, caspase 3 activation was higher with P2Et fraction than with doxorubicin after treatment for 48h. Caspase 3 activation was estimated by the cleavage of Ac-DEVD-pNA substrate compared with ethanol treatment (Figure1c). Finally, DNA fragmentation was assessed and apoptotic body formation was detected after DAPI staining, a distinctive cell death characteristic after DNA fragmentation (Figure2a). These results confirm that P2Et fraction induces apoptosis of tumor cells through the mitochondrial pathway.
                  http://static-content.springer.com/image/art%3A10.1186%2F1472-6882-13-74/MediaObjects/12906_2012_1323_Fig1_HTML.jpg
                  Figure 1

                  MMP loss, PS externalization, and activation of caspase 3 in 4T1 cells induced by P2Et fraction. (a) 4T1 cells treated with P2Et fraction for 6, 12, 24, or 48h, stained with JC-1, and analyzed by flow cytometry. Ethanol (negative control) and valinomycin (positive control) (***p < 0.001). (b) PS externalization in 4T1 cells by flow cytometry after treatment for 24h and 48h with P2Et fraction, doxorubicin (positive control), or ethanol (negative control). Top panel is a representative dot plot showing the various treatments; bottom is a representative histogram showing percent (%) PS/PI cells after 24 and 48h of treatment. (c) Caspase 3 activity by enzyme-linked immunosorbent assay (ELISA) after cell treatment with P2Et fraction or doxorubicin (positive control) for 48h (***p <0.001).

                  http://static-content.springer.com/image/art%3A10.1186%2F1472-6882-13-74/MediaObjects/12906_2012_1323_Fig2_HTML.jpg
                  Figure 2

                  P2Et fraction decreases 4T1 cell nuclear segmentation and clonogenic activity. (a) 4T1 cells treated with P2Et fraction for 48h and stained with DAPI. White arrows show apoptotic cells or fragmented nuclei observed under a fluorescence microscope (40×). (b) 4T1 cells treated with P2Et fraction, ethanol (negative control), and doxorubicin (positive control). Data represent the mean number of colonies ± SEM of three independent experiments (***p < 0.001) versus control.

                  4T1 tumor cell line clonogenic capacity is decreased by P2Et fraction

                  Clonogenic assays are regarded as the gold standard technique with which to determine antitumor activity of fractions and/or isolated compounds [21]. Doxorubicin and P2Et fraction significantly decreased 4T1 cells’ clonogenic ability compared with the negative control (Figure2b). We may expect that if P2Et fraction has such an effect in vitro, the fraction effect in vivo could be foreseen to reduce metastasis.

                  P2Et fraction induces 4T1 cells to secrete IL-6 and monocyte chemoattractant protein-1 in vitro

                  It is well known that multiple cell components and the tumor microenvironment influence breast cancer progression. Cytokines such as monocyte chemoattractant protein-1 (MCP-1), IL-6, and matrix metalloproteinases have been suggested to promote cancer progression [22]. We evaluated the secretion of IL-6, MCP-1, IL-10, IFN-γ, IL-12p70, and TNF-α by 4T1 cells after treatment with P2Et fraction, doxorubicin, or ethanol (negative control) for 6, 12, 24, and 48h. Cell culture supernatants were used to determined cytokine secretion by flow cytometry. In basal conditions or after treatment, no significant changes were found in IL-10, IFN-γ, IL-12p70, or TNF-α levels (data not shown). However, IL-6 levels were detected in cells treated with P2Et fraction compared with the control (ethanol) after 6h of treatment. After 12h of treatment, IL-6 levels peaked followed by a decrease, and low levels were maintained until 48h at all P2Et fraction concentrations. Doxorubicin-treated cells showed no significant increase in IL-6 levels at 6 to 24h of treatment, but the secretion levels peaked at 48h at a concentration corresponding with the IC50 (Figure3a, 3b). P2Et fraction and doxorubicin can induce a slight increase in MCP-1 secretion by 4T1 cells in a dose-dependent manner with a peak at 48h (Figure3c, 3d). In addition to having a cytotoxic effect on 4T1 cells, P2Et fraction and doxorubicin may also positively modulate IL-6 and MCP-1 secretion, but with different kinetics. Doxorubicin increased IL-6 secretion at the IC50 concentration; while P2Et fraction increased IL-6 secretion at all tested concentrations.
                  http://static-content.springer.com/image/art%3A10.1186%2F1472-6882-13-74/MediaObjects/12906_2012_1323_Fig3_HTML.jpg
                  Figure 3

                  IL-6 and MCP-1 production by 4T1 cells after treatment with P2Et fraction in vitro. 4T1 cells were treated with P2Et fraction and doxorubicin for 6, 12, 24, or 48h, and IL-6 (a, b) or MCP-1 (c, d) Secretion was determined by flow cytometry (***p < 0.001) versus control.

                  P2Et fraction inhibits 4T1 cell growth and metastasis in BALB/c mice

                  To assess the chemotherapeutic effect of P2Et fraction under physiological conditions, a murine model of metastatic breast cancer was used after transferring 4T1 cells into mouse mammary fat pad. The P2Et therapeutic dose was determined following LD50 estimation as described in the Materials and Methods. The LD50 corresponded to 69.6mg/kg, and the therapeutic dose was 4-fold less to ensure low toxicity (Additional file 2: Figure S2). Female BALB/c mice in groups of 7 to 8 were inoculated with SC injection of 1 × 104 4T1 cells. After 5days, the tumors were palpable, and the mice were treated with 18.7 and 9.3mg/kg of P2Et fraction or vehicle (PBS) twice a week. Figure4a shows that P2Et fraction significantly reduced tumor growth compared with the control, becoming increasingly marked from day 23 until 30. Similarly, there was a significant decrease in primary tumor weight at the two tested concentrations (Figure4b).
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                  Figure 4

                  Tumor growth inhibition and decrease in leukemoid reaction in BALB/C mice by P2Et fraction. BALB/c mice implanted SC with 1 × 104 4T1 cells for 5days and randomly divided into three groups. Group 1 was treated with PBS (vehicle), group 2 was treated with 18.7mg/kg of P2Et fraction, and group 3 was treated with 9.3mg/kg of P2Et fraction. Leukocyte count was determined weekly using a hemocytometer (ABX Micros 60). After 31days, all animals were sacrificed. (a) Tumor volume. The graph represents the mean of each group with 7–8 animals per group. (b) Tumor weight. The graph represents the total animals by each group. For these experiments we used 7–8 animals per group and each experiment was performed independently two times. (c) Leukocyte count. The graph represents the total number animals used per group. For these experiments we used 7–8 animals per group and each experiment was performed independently two times.

                  Inoculation of 4T1 cells into mouse mammary fat pad induces extramedular hematopoiesis, generating the so-called leukemoid reaction [23], a condition also observed in advanced human cancers. Leukocytosis of >50,000 cells/μL is associated with an increase in tumor invasive capacity and size, which is probably caused by tumor cell secretion of GM-CSF [24]. Because the leukemoid reaction in several human cancers is associated with a poor prognosis [25], we evaluated whether P2Et fraction therapy influences the leukocyte number in the peripheral blood of mice. The decrease in leukocyte number, seen since day 22, was considered significant on day 30 (Figure4c). The latter results suggest that P2Et fraction activity may decrease mediators triggering the leukemoid reaction in this model, which can be beneficial for the disease outcome.

                  Since the experimental model developed spontaneous metastasis, histopathological analysis to evaluate the number of tumor cells present in lung, liver, spleen, and primary tumor tissues was carried out. Treated mice tissues had fewer tumor cells than did untreated mice tissues (Figure5a, Additional file 3: Figure S3), with significant differences in the primary tumor and spleen (Figure5b). Similarly, no tumor cells were found in the bone marrow or brain tissues of either group (data not shown). P2Et fraction exerted activity on the primary tumor as evidenced by the decrease in size and weight of the tumor and the decrease in metastases in the spleen and liver.
                  http://static-content.springer.com/image/art%3A10.1186%2F1472-6882-13-74/MediaObjects/12906_2012_1323_Fig5_HTML.jpg
                  Figure 5

                  Reduction of spleen metastasis and decreased IL-6 serum levels in BALB/c mice treated with P2Et fraction. After treatment, primary tumors and lung, liver, and spleen tissues were dissected and fixed in 10% formaldehyde, embedded in paraffin, and stained with H&E. Metastatic infiltrations were evaluated in each tissue and counted using a microscope with a micrometric grid (magnification power, 10×). (a) Tissue Images. White arrows show metastatic tumor infiltration, white dotted arrows show alveolar septum, black arrows show adipocytes, and black dotted arrows show necrotic areas. (b) Graphic shows tissue cell number in each (***p < 0.001) versus control. The graphs represents mean of each group with 7–8 animals by group and each experiment was performed independently two times. (c) After treatment, mice serum was collected by cardiac puncture, and cytokine estimation was performed using a CBA mouse inflammation kit. (c) IL-6 secretion. (d) MCP-1 secretion (***p < 0.001) versus control. The graphs represents mean of each group with 7–8 animals by group and each experiment was performed independently two times.

                  P2Et fraction reduces IL-6 serum levels in BALB/c mice

                  Microenvironmental factors such as proinflammatory cytokines and adhesion molecules may support tumor progression toward a malignant phenotype by favoring a suitable environment and interactions with tumor cells [26]. As mentioned above, cytokines such as MCP-1 and IL-6 may promote breast cancer progression [22]. There are also reports associating IL-6 with growth and progression of ovarian and myeloma cancers [27, 28]. Thus, we quantified IL-6, MCP-1, and the proinflammatory cytokines IL-10, IFN-γ, IL-12p70, and TNF-α in treated and control mice serum. Treated mice had significantly lower IL-6 serum levels compared with the controls (Figure5c). We also observed a decreasing trend in MCP-1, although it was not significant (Figure5d). The other tested cytokines showed no differences or were not detected in the serum of mice (data not shown). These results suggest that P2Et fraction diminishes the host inflammatory response, apparently indirectly, and that this change is associated with a decrease in tumor diameter and metastasis control, confirming its potential antitumor function.

                  To determine its biological activity and bioavailability, the P2Et fraction was orally administrated to female BALB/c transplanted with 4T1 cells as described in detail in the Material and Methods section. We demonstrated that mice receiving the P2Et fraction had significantly reduced tumor volumes as compared to control treated mice (Figure6a). In addition, we demonstrated that mice receiving the P2Et fraction exhibited significantly less lung macro-metastasis as judged by India ink staining (Figure6b).
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                  Figure 6

                  Tumor growth and lung metastases formation is decreased after treatment with P2Et fraction. BALB/c mice were randomly assigned in control or treatment groups (7 mice per group). One week prior to tumor cell inoculation (day 0), the treatment group started P2Et fraction consumption administered in water. 4T1 cells (1 × 104) were injected into the right mammary fat pad SC on day 7. After 33days, all animals were sacrificed. (a) Tumor volume. The graph represents the mean of each group with 7 animals per group. (b) Lung metastatic number. White tumor nodules against a blue lung background were counted by two independent observers. The graph represents the total animals by each group. For these experiments we used 5 animals per group.

                  Discussion

                  P2Et is a C. spinosa-derived fraction mainly composed of hydrolyzable tannins, gallotannins, and minor concentrations of pentagalloylglucose (PGG), as previously stated [12]. Our study gives evidence of the fraction’s cytotoxic effects on 4T1 cells, inducing apoptosis by the mitochondrial pathway, a path selective for cells with high proliferative rates when compared with normal mononuclear cells or human fibroblasts. Apoptosis is a type of programmed cell death that can be activated by two pathways: extrinsic and intrinsic. The latter involves mitochondrial translocation of pro-apoptotic members of the Bcl-2 family, such as Bax, causing MMP loss followed by the release of several apoptosis mediators, including cytochrome c, into the cytosol [29], which binds Apaf-1 and, in association with other proteins, the apoptosome is formed. This in turn recruits and activates pro-caspase 9, leading to the activation of effector caspases such as caspase 3. Finally, DNA endonucleases are activated [30, 31]. This cell death pathway involving caspase activation produces typical morphological changes such as chromatin condensation, nucleosomal DNA fragmentation, nuclear membrane disruption, PS externalization, and formation of apoptotic bodies [29]. We herein demonstrated that P2Et fraction activity induces apoptosis with MMP loss, PS externalization, caspase 3 activation, and DNA fragmentation.

                  The activities of isolated compounds that are also present in P2Et fraction have been previously studied. Gallotannins or gallic acid derivatives, specifically alkyl gallates and gallamides, have proven cytotoxic effects on L1210 leukemia cells in a dose-dependent manner [32]. Eugenia jambos L. (Myrtaceae) plant extract exerts cytotoxic activity on HL60 leukemia cells. This extract contains hydrolyzable tannins (1-O-galloyl castalagin, casuarinin, and gallotannins) and induces apoptosis and DNA fragmentation [8]. Similarly, we previously reported that P2Et fraction has a cytotoxic effect on leukemia cells (K562), inducing apoptosis [12]. The compound’s broad activity range can also be seen in Terminalia chebula fruit extract, rich in gallotannins capable of inducing apoptosis in the murine and human breast cancer cells MCF-7 and S115, respectively, among other activities [9].

                  PGG is a compound also present in P2Et fraction, and it is capable of inducing G1-phase arrest [33], suppressing the phosphorylation and protein level of estrogen receptor alpha by promoting degradation in the lysosome and negatively modulating the ErbB/PI3K/Akt pathway in MCF-7 cells [34]. This causes the cells to become refractory to estrogenic stimulation. In our previous studies, we also observed a G1-phase arrest in MCF-7 cells [12], but not in 4T1 cells, suggesting a cell line-selective mechanism. We also showed that the activities of gallotannins and PGG are regulated by the same cell diversity.

                  Caesalpinia spinosa fruits are well known to be good tannins sources, yet evidence of their cytotoxic or antitumor activity is quite limited. Gali-Muhtasib’s group reported that C. spinosa hydrolyzable tannins such as TA lower skin cancer biochemical marker levels, delaying tumor development in mice [13, 14]. Similarly, epigallocatechin 3-gallate (EGG), a gallic acid condensed tannin present in green tea, is capable of inducing apoptosis through the mitochondrial pathway in murine breast cancer cells (4T1) [35]. In general, such compounds exhibit low toxicity to normal cells, as observed herein and previously demonstrated for 1-O-galloyl castalagin, casuarinin, and gallotannins from Eugenia jambos, which are slightly cytotoxic to human lymphocytes [6].

                  Our study evaluated the fraction’s ability to modulate tumor cell cytokine production. We found that both P2Et fraction and doxorubicin induced early IL-6 secretion from ER(+) 4T1 cells and in both treatments there was apoptosis induction, inferring that IL-6 favors their latter removal [36]. No significant changes in the levels of the other tested cytokines were found in tumor cell secretion. To control tumor growth in vivo, is necessary to induce direct death of tumor cells and regulate microenvironmental and tissue factors involved in primary tumor proliferation and migration. IL-6 is a mediator with dual activity in tumor growth and metastatic processes. Intrinsic production of IL-6 in the ER(+) breast cancer cell lines MCF-7, T47D, ZR-75-1, and SK-BR promotes cell death in response to drugs, inducing morphological changes, and apoptosis with DNA fragmentation [3739]. On the contrary, in the ER(-) breast cancer cell lines HS578T and MDA-MB-231, IL-6 is related to a drug-resistant phenotype with increased expression of MDR-1 [40]. In our system, IL-6 production by 4T1 cells favored apoptotic cell death.

                  On the other hand, when overexpressed in organs such as the liver, brain, or lung, IL-6 can promote metastasis by attracting and promoting tumor cells [41]. In patients with breast cancer, IL-6 is associated with a poor prognosis because the levels increase in advanced stages and are associated with a higher number of metastases [42]. We observed a decrease in circulating IL-6 that may have been correlated with the reduction in tumor volume and metastasis; however, it also may have been correlated with negative regulation of the inflammatory microenvironment, favoring the activation of mechanisms implied in tumor elimination.

                  Additional results suggest that P2Et fraction could modulate different mediators. When 4T1 cells are orthotopically transplanted to BALB/c mice, a leukemoid reaction is induced. This reaction is characterized by leukocytosis of >50,000 cells/μL with predominance of immature granulocytes Gr-1dim and splenomegaly due to massive granulocyte infiltration into the red pulp [23]. P2Et fraction-treated animals showed a reduced number of peripheral blood leukocytes at day 30. The leukemoid reaction has been associated with a poor prognosis in animal cancer models and human cancers [25]. The decrease in response by P2Et fraction therapy could improve the course of the disease.

                  The in vivo P2Et fraction activity showed that the fraction favors not only a decrease in tumor diameter and weight, but also a significant decrease in spleen and liver metastases. 4T1 tumor cells are highly aggressive and metastatic in BALB/c mice, causing death mostly by metastasis and not by the primary tumor [16, 43]. Polyphenols, particularly EGG, reportedly have a role in metastasis control in 4T1 models [35] and that their activity is linked to a decrease in proliferating cell nuclear antigen, resulting in a decreased cell proliferation rate.

                  Tumor progression into a malignant phenotype is favored by different signals generated by microenvironmental factors such as proinflammatory cytokines and adhesion molecules as well as conditions that support the tumor microenvironment by interacting with specific tumor cell receptors [26], promoting breast cancer progression [22]. Thus, high IL-6 serum levels are associated with lower survival, and at later stages, IL-6 can contribute to tumor growth progression [11]. We found that IL-6 serum levels in mice treated with P2Et fraction were significantly lower than those in untreated mice. Similarly, MCP-1 levels were reduced, although not significantly. The latter findings of the decrease in leukemoid reaction and direct activity over tumor cells allow us to conclude that P2Et fraction, besides reducing primary tumor diameter and weight, lessens the migration to distant organs by mechanisms involving the tumor microenvironment as shown by the decreased serum IL6. With regards to data demonstrating that treatment of tumor-bearing mice with P2Et fraction results in significant reduction in serum IL-6, we hypothesize that locally produced IL-6 may improve apoptosis mechanisms and are consumed immediately by tumor cells during its death process thus acting in an autocrine fashion as previously discussed. We concur that tumor reduction after P2Et treatment cannot simply explain IL-6 serum reduction and hypothesize that some other immune-regulatory mechanisms are mediated by the P2Et fraction cannot be excluded, including the inhibition of suppressor macrophages.

                  Overall, P2Et fraction decreases associated risk factors and primary tumor size, supporting its potential use as an adjuvant in ER(+) breast cancer treatment. Moreover, it is important to note that antitumor activity of the fraction is observed in immunocompetent mice; consequently, tumor microenvironment modulation together with antitumor immune response activation allows for control of distant metastases. We are currently evaluating this hypothesis in our laboratory.

                  Conclusions

                  P2Et fraction exerts its activity on the primary tumor, reduces cell migration to distant organs, and implies the involvement of tumor microenvironment-associated mechanisms in the drop in IL-6 serum levels. Overall, P2Et fraction lessens risk factors associated with tumor progression and diminishes primary tumor size, offering great potential for use as an adjuvant in ER(+) breast cancer treatment.

                  Abbreviations

                  CBA: 

                  Cytometric bead array

                  DAPI: 

                  4,´6-diamidino-2-phenylindole

                  DMSO: 

                  Dimethyl sulfoxide

                  EGG: 

                  Epigallocatechin 3-gallate

                  H&E: 

                  Hematoxylin and eosin

                  IC50: 

                  50% inhibition cell growth

                  IP: 

                  Intraperitoneally

                  IL-6: 

                  Interleuquin 6

                  LD50: 

                  Lethal dosis 50

                  MCP1: 

                  Monocyte chemoattractan protein-1

                  MMP: 

                  Mitochondria membrane potential

                  MTT: 

                  Methylthiazol tetrazolium

                  PGG: 

                  Pentagalloylglucose

                  PI: 

                  Propidium iodide

                  PMBC: 

                  Human peripheral blood mononuclear cells

                  PS: 

                  Phosphatidylserine

                  S.C: 

                  Subcutaneously

                  Declarations

                  Acknowledgments

                  The authors thank the Science Faculty of the Pontificia Universidad Javeriana, Fundación Universitaria Juan N. Corpas for their support and the Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnología “Francisco Jose de Caldas” (COLCIENCIAS, code 12010NK0101103), Bogotá, Colombia, for financial support. We thank Dra. Claudia Cifuentes (Pontificia Universidad Javeriana, Bogotá, Colombia) for critical reading of the manuscript.

                  Authors’ Affiliations

                  (1)
                  Grupo de Inmunobiología y Biología Celular. Departamento de Microbiología. Facultad de Ciencias, Pontificia Universidad Javeriana
                  (2)
                  Grupo de Farmacología Vegetal, Fundación Universitaria Juan N. Corpas
                  (3)
                  Department of Microbiology, Biochemistry & Immunology, Morehouse School of Medicine

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                  44. Pre-publication history

                    1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1472-6882/​13/​74/​prepub

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                  © Urueña et al.; licensee BioMed Central Ltd. 2013

                  This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.