Full Length Research Paper
ABSTRACT
Attallea speciosa Mart. (babassu) fruit contains a mesocarp that is rich in carbohydrates with immunomodulatory effects. The induction of the tolerogenic response is a tumor escape mechanism, and immunomodulator adjuvants have been studied to reestablish host immunogenicity. This study evaluates the adjuvant potential of babassu mesocarp carbohydrates in a tumor model. The babassu mesocarp extraction (BME) yield was 75.54%, and the total sugar concentration was 29.79 mg ml-1 containing monosaccharides, reducing sugars, polysaccharides and 0.506 mg ml-1 total protein. Chromatography analysis identified glucose, sucrose and fructose. Sensitization increased the spleen weight in the tumor group compared with the control, and a comparatively lower frequency of T helper and higher frequency of B-lymphocytes was also observed. The tumor+BME group had more cytotoxic T lymphocytes compared with the control. After co-culture with cancer cells, the tumor splenocytes showed lower proliferation, lower frequency of T helper cells and higher concentrations of interleukin (IL)-2, IL-6 and IL-10. However, the tumor+BME splenocytes presented results similar to the control, suggesting a reduction in the regulatory response of the tumor group. These results demonstrated that BME sensitization with cancer cells modulated an immune response in Balb/c animals, indicating an immunogenic effect.
Key words: Arecaceae, Attallea speciosa Mart., adjuvant, carbohydrate, antitumor.
INTRODUCTION
The palm tree Attallea speciosa Mart., known as babassu, is a common species in Northeastern Brazil. The mesocarp of babassu fruit predominantly consists of carbohydrates (Silva and Parente, 2001) and is popularly used as a health food. Previous studies have demonstrated that the babassu mesocarp has been traditionally used to treat inflammatory diseases (Agra et al., 2007), and the native communities have reported the use of babassu mesocarp to treat gastritis, leukorrhea and rounds (Souza et al., 2011). The biological effects of babassu mesocarp have been reported, including anti-inflammatory effects (Silva and Parente, 2001; Baldez, 2006; Nascimento et al., 2010; Silva et al., 2015). However, previous studies have shown that aqueous babassu mesocarp extract induces both in vitro and in vivo nitric oxide (NO) and tumor necrosis factor α (TNF-α) production in peritoneal macrophages (Nascimento et al., 2006). In addition, immunomodulatory (Guerra et al., 2011; Pessoa et al., 2014) and antitumoral effects (Rennó et al., 2008; de Souza et al., 2011; de Sousa et al., 2013) have been reported.
Herbal immunoadjuvants are substances that stimulate the immune response (Khyati, 2012), and carbohydrates are promising adjuvants because of their low cytotoxicity, good biocompatibility, and strong immune enhancement (Li and Wang, 2015).
Immune responses against tumors involve both innate and adaptive immunity (Lehrnbechter et al., 2008; Loose and Van de Wiele, 2009; Achyut and Arbab, 2016). Innate immunity includes natural killer (NK) cells and macrophages, while adaptive immunity involves cytotoxic T lymphocyte (CTL) cells, which are responsible for a more specific response against tumor antigens and the establishment of an immunologic memory (Klebanoff et al., 2006; Coulie et al., 2014; Dimeloe et al., 2016). Therefore, drugs with broad-spectrum activities, combining antitumoral and immunomodulatory effects with the capacity to mobilize the host immune system towards immunogenicity, are of great interest.
Considering its immunomodulatory activity and composition that is rich in carbohydrates, we hypothesized that the carbohydrates extracted from babassu mesocarp could act as adjuvants in sensitization against tumor cells. This study shows the ex vivo phenotypic, functional and cytokine production characteristics of splenocytes obtained from Balb/c mice sensitization using the extract of babassu mesocarp and tumor cells.
MATERIALS AND METHODS
Extract preparation and carbohydrate analysis
To prepare the babassu mesocarp extract (BME), the powder of babassu mesocarp supplied by the Cooperative of Coconuts breakers of Maranhão, located in Esperantinópolis-MA (Latitude: 04° 52' 00" S, Longitude: 44 42' 30" W) was used. The powder showed similarity regarding all of the botanical and phytochemical aspects compared with the mesocarp flour prepared in the laboratory (Nascimento et al., 2006). The botanical identifications
were obtained from the Herbario Ático Seabra, State of Maranhão,
Brazil (authenticated voucher specimen number 1135).
The babassu mesocarp powder was macerated in water at a concentration of 20 mg/ml for 24 h (Fortes et al., 2009), and subsequently, an aqueous babassu mesocarp extract was obtained (BME). The quantitative carbohydrate analysis was performed using a phenol-sulfuric acid method, with glucose as a standard (Dubois et al., 1956). The extract sample was deionized with a cationic-exchange column (Dowex 50W X 8 - 100) and anionic-exchange column (Dowex 1 X 8 -100). Next, the sample was filtered through 0.45-µm membrane filters. Samples with equivalent glucose concentrations (400 ug ml-1) were analyzed by anion exchange chromatography coupled with pulsed amperometric detection (HPAEC/PAD) using a DIONEX ICS3000 chromatograph and CarboPac PA-1 (2×250 mm). The carbohydrate separation was performed using a multi-step gradient obtained after mixing eluent A (water) and eluent B (250 mM sodium hydroxide): 0 to 15 min, 100 mM; 15.1 to 20 min, 200 mM; and 20.1 to 25.5 min, 100 mM. The applied PAD potentials for E1 (0 to 0.4 s), E2 (0.41 to 0.42 s), E3 (0.43 s) and E4 (0.44 to 1.00 s) were 0.1, 2.0, 0.6 and 0.1, respectively, and the flow rate through the column was 0.25 ml-1 min. The peaks were identified through comparison with authentic standards of Mio-inositol, Glucose, Fructose, Sacarose, Raffinose and Estaquiose, with 99% purity (Sigma) (Figure 1). The protein concentration was determined using the Bradford test (Bradford, 1976).
Animals
Balb/c male mice (25 to 30 g, 30 days old) were obtained from the Biotery at the University of São Paulo. The study was approved by the Ethics Committee on the Use of Animals of UFMA (Application number: 8608/2011-00).
MCF-7 cancer cells
The MCF-7 breast cancer cell line was obtained from the Cell Bank of Rio de Janeiro. The cells were cultured in flasks containing Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS) and maintained in a humidified incubator at 37°C under 5% CO2. When confluent, the cells were detached by addition of 3 ml of 0.025% trypsin-ethylene diamine tetraacetic acid and incubated at 37°C in a humidified incubator under 5% CO2 for 5 min. Subsequently, the cell suspension was transferred into a 15 ml polyethylene tube containing 12 ml of RPMI medium supplemented with 10% FBS, centrifuged at 800 g for 10 min at 18°C, and washed again. The cells were adjusted to a concentration of 1×105 cells and resuspended in 100 µl of enzyme assay buffer (EAB; at a concentration of 10 mg/kg) or saline solution (0.9%) 2 h prior to the sensitization assay.
Sensitization protocol
The animals were randomly divided into 4 groups (n=6/group): thecontrol group in which the animals were sensitized with 100 µl sterile saline solution; the BME group in which the animals were inoculated with 100 µl BME (66,6 mg/kg); the tumor group in which the animals were inoculated with 100 µl MCF-7 cancer cell suspension at a concentration of 1×106 ml-1 in saline solution; and the tumor+BME group in which the animals were sensitized with a 100 µl MCF-7 cancer cell suspension at a concentration of 1×106 ml-1 in BME (66.6 mg/kg). Sensitization was subcutaneously performed 3 times on the dorsum, at an interval of 5 days. On the 15th day, the animals were euthanized through intraperitoneal access with an excess of anesthetics, including 2% xylazine hydrochloride (20 mg/kg), and 5% ketamine hydrochloride (25 mg/kg). Blood, bone marrow cells, and splenocytes were collected.
Blood collection and leukocyte count
The blood was collected in a micro hematocrit capillary tube by puncturing the orbital plexus. The blood was collected after adding HEMSTAB EDTA anticoagulant and leukocyte (white blood cells, WBC) count was determined using the hematology automatic analyzer.
Collection and counting of bone marrow cells
To collect bone marrow cells, the femur was removed and perfused with 1 ml phosphate buffered saline (PBS) solution. To count the cells, 90 µl of the suspension was mixed with 10 µl of 0.1% crystal violet solution. Subsequently, the cells were counted under an optical (light) microscope in a Neubauer chamber.
Cell collection and splenocyte count
The spleen was surgically removed, weighed, and homogenized in 3 ml PBS. The suspension was stored on ice until counting. For quantification, 90 µl of cell suspension was mixed with 10 µl of crystal violet, and the cells were counted under an optical (light) microscope in a Neubauer chamber. The splenocytes were resuspended in RPMI medium supplemented with 10% FBS at a concentration of 1×106 ml-1 and stored in a Petri dish for 2 h in a CO2 incubator. After incubation, non-adherent cells were removed for phenotype characterization and co-culture experiments.
Phenotype characterization of splenocytes
The phenotypes of adherent and non-adherent cells, isolated from the spleen, were characterized with commercial monoclonal antibodies (BD Biosciences, San Jose, CA), according to the manufacturer’s instructions. Two panels of antibodies were used, one for adherent cells, including anti-CD14 (FITC), anti-IA/IE (PE), and anti-Ly6G (PE-Cy5), and the other for non-adherent cells, anti- CD3 (FITC), anti-CD19 (PE-Cy5), anti-CD4 (PE), and anti-CD8 (PE-Cy5). After the acquisition of 10,000 events in a FACSCalibur flow cytometer, the obtained data were analyzed using FlowJo software.
Establishment of a co-culture with MCF-7 cancer cells
For co-culture experiments, MCF-7 tumor cells (1×104/well) were seeded in 96-well microtiter plates (Corning Costar) in RPMI medium supplemented with 10% FBS. After 24 h, non-adherent splenocytes were seeded at a concentration of 3×105 cells/well in RPMI medium supplemented with 10% FBS. After three days, the splenocytes were used for proliferation assays and phenotype characterization, and the supernatant was used to determine cytokine production.
Lymphoproliferation assay
To evaluate cell proliferation, the splenocytes were labeled with carboxyfluorescein succinimidyl ester (CFSE) using the CellTrace CFSE cell proliferation kit according to the manufacturer’s instructions and subsequently added to co-culture plates containing MCF-7 cancer cells. After three days, 10,000 events were acquired using a FACSCalibur flow cytometer. The proliferation capacity was determined as the decay of the average fluorescence intensity of the population corresponding to splenocytes, according to the Side and Forward Scatter (SSC and FSC, respectively) obtained using FlowJo software. The proliferation index was calculated as the ratio between the mean fluorescence intensity (MFI) of CFSE obtained in control splenocytes and the MFI of CFSE obtained in the other splenocytes.
Phenotype characterization of splenocytes after co-culture
The phenotype of the non-adherent splenocytes in co-culture was characterized using the commercial monoclonal antibodies (BD Biosciences, San Jose, CA) anti-CD3 (FITC), anti-CD4 (PE), and anti-CD8 (PE-Cy5) according to the manufacturer’s instructions. After the acquisition of 10,000 events using a FACSCalibur flow cytometer, the obtained data were analyzed using FlowJo software.
Cytokine quantification by flow cytometry
The quantification of IL-2, IL-6, and IL-10 cytokines in the supernatant of the co-culture was performed using the Cytometric Beads Array (CBA) commercial kit according to the manufacturer’s instructions.
Statistical analysis
Statistical analyses were performed using Graph Pad Prism version 5.0 software with Student’s t-test and Newman-Keuls post hoc ANOVA test. Statistical significance was determined at a value of p<0.05. The data are expressed as the average±standard deviation of 6 animals per group.
RESULTS
The babassu mesocarp aqueous extract was rich in carbohydrates
The babassu mesocarp aqueous extract showed a yield of 75.54%. The aqueous extract showed a total sugar concentration of 29.79 mg/ml, with positive results for the presence of monosaccharides, reducing sugars, aldoses and ketoses, confirming the presence of polysaccharides. HPAEC analysis identified glucose, sucrose and fructose (Figure 2). The protein concentration in the aqueous extract was 0.506 mg/ml.
Sensitization with tumor cells altered the balance between T and B lymphocytes of the spleen
After the sensitization of Balb/c mice, the numbers of cells in the blood, spleen, and bone marrow were determined. The data showed that sensitization did not alter the number of circulating leukocytes and bone marrow cells in the different groups. However, the animals in the Tumor group showed an increase in spleen weight compared with the control group (Table 1).
Phenotyping of the cellular population of the spleen revealed that the frequency of B cells was higher in the tumor group compared with the other groups (Figure 3A). In contrast, the frequency of T cells was lower in the tumor group compared with the other groups (Figure 3B). The decreased frequency of T cells in the tumor group was followed by a reduction in the frequency of CD4+ T cells (Figure 3C). However, the frequency of CD8+ T cells in the tumor group was similar to that in the other groups (Figure 3D). No changes were observed in the frequency of CD14+, IA/IE+, and Ly6G+ splenocytes in any of the groups analyzed (data not shown).
Lymphoproliferative activity of splenocytes co-cultured with MCF-7 cells
To evaluate whether sensitization interferes with the proliferative capacity of splenocytes, non-adherent cells, and primarily represented by lymphocytes, were co-cultured with MCF-7 cancer cells. The data showed that splenocytes obtained from the BME group presented 3 times higher proliferative activity compared with the control group. No significant difference was observed in the tumor+BME group compared with the control group; however, significantly higher cell proliferation was observed compared with the tumor group (Figures 4A and B).
Phenotype characterization of splenocytes after co-culture with MCF-7 cancer cells
Splenocytes and MCF-7 tumor cells were co-cultured and the phenotypic analysis of T cells was performed. The results showed that the percentage of CD4+ T lymphocytes was lower in the tumor group compared with the other groups (Figure 5A). The results obtained in the tumor+BME group were similar to those obtained in the control group. However, no changes in cytotoxic T cells were observed (Figure 5B).
Cytokine production and release in the co-culture supernatant of splenocytes and MCF-7 cancer cells
The supernatant of the co-culture was removed to determine cytokine production. The data revealed that the concentrations of IL-6 and IL-10 were lower in the BME group compared with the control group. However, the tumor group showed significantly higher levels of IL-2, IL-6 and IL-10 than the control and BME groups. The splenocytes obtained from animals in the tumor+BME group did not show a significant difference in cytokine production compared with the other groups, but there was a decrease in the IL-6 and IL-10 concentrations compared with the tumor group (Table 2).
DISCUSSION
In the present study, Balb/c mice were subcutaneously sensitized with MCF-7 cancer cells, which are ideal for antigen recognition by resident immune cells and subsequent migration and presentation to secondary lymphoid organs. In the context of immunization with tumor cells, the immune system might experience two types of naive responses: the low capacity of the host to trigger local inflammation that might compromise tumor antigen presentation, and poor immunogenicity in response to the tumor (Edelman and Jefford, 1968). Balb/c mice induced an adequate response after immunization (Crowther and Wasgstaff, 1983; Ochsenbein et al., 2001; Ochesenbein, 2002). Although these cells do not proliferate in mice, upon sensitization, human MCF7 cancer breast cancer cells induce the proliferation of splenocytes and after fusion, form hybridomas that produce specific monoclonal antibodies (Ochsenbein et al., 2001; Ochsenbein, 2002; Schunck and Macallum, 2005).
In the present study, the results showed no significant differences in the leukocyte number and cellularity of the lymphoid organs. However, increased spleen weight and a lower frequency of T helper cells were observed in animals sensitized with MCF-7 cells. These data contradict the expectation, as the inoculation of human cells in mice should lead to a xenograft reaction involving cell rejection and T lymphocyte stimulation (Mandal-Ghosh et al., 2007; Menard et al., 1983). However, studies with athymic mice showed that the overexpression of transforming growth factor beta (TGF-β) induces immunosuppression in the MCF-7 cancer cell line (Sun et al., 1992; Arteaga et al., 1993; Sachs, 1995; Schunk and Macallum, 2005; Koch et al., 2013). Although, the thymus of the animals was maintained in the present study, we suggest the existence of potential mechanisms that might be involved in the steps occurring between xenograft rejection and tumor cell escape, presumably affecting the T helper cell response.
However, the frequency of CD3+CD4+ cells in the group sensitized with MCF-7 cancer cells and BME was similar to that in the control group. Although the difference was not significant, sensitization with babassu mesocarp, with or without MCF-7 cancer cells, increased the frequency of IA/IE+ cells, demonstrating the occurrence of improved antigen presentation, the modulation of the resident cells’ pro-inflammation response (Azevedo et al., 2003), or the generation of complexes with tumor proteins that increase immunogenicity (Richmond and Su, 2008; Ju et al., 2001). Nascimento et al. (2006) showed that in animals intraperitoneally treated with babassu mesocarp, the migration of cells into the peritoneum increased, primarily through the activation of macrophages that release hydrogen peroxide (H2O2) and nitric oxide (NO). This finding indicates that babassu mesocarp promotes an M1 type response. M1 macrophages are activated through classical pathways and play an important role in eliminating tumor cells via the production of high levels of pro-inflammatory cytokines that lead to a Th1 response (Koch et al., 2013; Schunk and Macallun, 2005; Richmond and Su, 2008; Sachs, 1995; Arteaga et al., 1993; Sun et al., 1992). Using isogenic C57Bl/6 and Balb/c mice, Fortes et al. (2009) showed antitumor activity and improved survival of animals sensitized with solid and ascitic Ehrlich tumors. In the present study, in addition to the recovery of T helper cells, sensitization with BME and tumor cells increased the frequency of cytotoxic T cells and reduced the frequency of B cells compared with animals sensitized with the tumor alone. These data reinforce the idea that the carbohydrates of babassu mesocarp might exhibit immunomodulatory activity through the induction of a cytotoxic response.
In the proliferation assay, the group sensitized with MCF-7 cancer cells did not present any differences compared with the control group, and the population of T helper cells remained lower after co-culture. These data reinforce the hypothesis that the presence of a tumor might activate immunosuppressive mechanisms that could affect effector lymphocyte ability. However, the proliferation rates of the splenocytes obtained from the BME and tumor+BME groups were higher, suggesting that BME treatment induced the immune response against tumor-associated immunosuppression.
The data show that increased levels of IL-2, IL-6, and IL-10 were observed in the supernatant of the splenocyte co-culture obtained from animals of the tumor group, thus indicating a regulatory response. The splenocytes obtained from the animals of the tumor+BME group showed a decrease in all cytokines, similar to the control group. The data reinforce a Th1 adjuvant role for the carbohydrates extracted from babassu mesocarp, as the entire response induced in Balb/c mice stimulated with MCF-7 cancer cells was reversed when the tumor stimulus was associated with BME.
The mesocarp of the babassu fruit is rich in carbohydrates and in particular, a mucopolysaccharide, MP1, which was shown to induce phagocytic activity (Silva and Parente, 2001). Other studies demonstrated that treatment with mesocarp exerts immunoregulatory effects. Nascimento et al. (2006) showed that intraperitoneal injections of BME increased cellular migration to the peritoneal cavity, promoted the activation of peritoneal macrophages both in vitro and in vivo, and increased the expression of major histocompatibility complex (MHC) class II and the recruitment of immune cells (Nascimento et al., 2006). Fortes et al. (2009) showed that BME exerted an immunomodulatory effect, resulting in increased survival of the animals and a decreased size of the solid Ehrlich tumors when this tumor cell line was pretreated with BME. Guerra et al. (2011) demonstrated that immunization with promastigote forms of Leishmania amazonensis and the addition of BME induced a Th1-dependent immune response in Balb/c mice, thus showing promising effects compared with other adjuvants. Despite their poor immunogenicity, the ability of carbohydrates to exert an immunomodulatory effect through the formation of complexes with tumor proteins potentiates immunospecific responses. This is the case for the oligosaccharides in Lewis Y-containing glycoproteins, which are used in mice to form complexes that induce the production of antibodies that exert cytotoxic effects against MCF-7 cells through the activation of macrophages (Kudryashov et al., 1998; Silva and Parente, 2001; Mantovani et al., 2004) by the binding of these molecules to membrane receptors (Um et al., 2002; Moretão et al., 2003; Ma et al., 2010) or the increase in the host immune response against tumors, which enhances leukocyte action and cytokine production (Mosser, 2003). The results showed that BME induces cell proliferation, increases T helper cells and reduces IL-6 and IL-10 levels, thereby reversing tumor-associated immunosuppression.
Therefore, this study showed that BME modulates the frequency of T helper cells and B-lymphocytes in the spleen of Balb/c mice after sensitization with human MCF-7 cancer cells. Moreover, in vitro experiments demonstrated that BME improved the lymphoproliferative activity and modulation of cytokines in the presence of MCF-7 cancer cells. These data reinforce the immunomodulatory effect of the carbohydrates extracted from babassu mesocarp, thus demonstrating an adjuvant role in the development of antitumor vaccines.
CONFLICT OF INTERESTS
The authors have not declared any conflicts of interests.
ACKNOWLEDGEMENTS
The authors would like to thank the Brazilian funding agencies CAPES, CNPq, and FAPEMA for the ï¬nancial support and Ana Karlla S. Sousa and Juliana Lucena for the technical support.
REFERENCES
Agra MF, Freitas PF, Barbosa-Filho JM (2007). Synopsis of the plants known as medicinal and poisonous in Northeast of Brazil. Rev. Bras. Farmacogn. 17(1):114-140. |
|
Achyut BR, Arbab AS (2016). Myeloid cell signatures in tumor microenvironment predicts therapeutic response in cancer. Onco. Targets Ther. 9:1047-1055. |
|
Arteaga CL, Carty-Dugger T, Moses HL, Hurd SD, Pietenpol JA (1993). Transforming growth factor beta 1 can induce estrogen-independent tumorigenicity of human breast cancer cells in athymic mice. Cell Growth Differ. 4(3):193-201. |
|
Azevedo AP, Ferreira SCP, Guerra RMN, Nascimento FRF (2003). Efeito do tratamento com mesocarpo de Babassu sobre o edema de pata e mediadores da inflamação. Rev. Ciênc. Saude 5:21-28. |
|
Baldez RN (2006). Análise da cicatrização do cólon com uso do extrato aquoso da Orbignya phalerata (Babaçu) em ratos. Acta Cir. Bras. 21(2):31-38. |
|
Bradford MM (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7(72):248-254. |
|
Coulie PG, Van den Eynde BJ, Van der Bruggen P, Boon T (2014). Tumour antigens recognized by T lymphocytes: at the core of cancer immunotherapy. Nat. Rev. Cancer 14(2):135-146. |
|
Crowther D, Wagstaff J (1983). Lymphocyte migration in malignant disease. Clin. Exp. Immunol. 51:413-420. |
|
de Souza PA, Palumbo AJ, Alves LM, de Souza VP, Cabral LM, Fernandes PD, Takiya CM, Menezes FS, Nasciutti LE (2011). Effects of a nanocomposite containing Orbignya speciose lipophilic extract on Benign Prostatic Hyperplasia. J. Ethnopharmacol. 135(1):135-46. |
|
de Sousa VP, Crean J, de Almeida Borges VR, Rodrigues CR, Tajber L, Boylan F, Cabral LM (2013). Nanostructured systems containing babassu (Orbignya speciosa) oil as a potential alternative therapy for benign prostatic hyperplasia. Int. J. Nanomed. 8(1):3129-3139. |
|
Dimeloe S, Burgener AV, Graehlert J, Hess C (2016). T cell metabolism governing activation, proliferation and differentiation ;a modular view. Immunology 1:1-25. |
|
Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956). Colorimetric method for determination of sugars and related substances. Anal. Chem. 28(3):350-356. |
|
Edelman J, Jefford TG (1968). The mechanisim of fructosan metabolism in higher plants as exemplified in Helianthus tuberosus. New Phytol. 67(3): 517-531. |
|
Fortes TS, Fialho EMS, Reis AS, Assunção AKM, Azevedo APS, Barroquiero ESB, Guerra RNM, Nascimento FRF (2009). Desenvolvimento do tumor de Ehrlich em camundongos apoÌs tratamento in vitro com mesocarpo de Babassu Mart. Rev. CieÌ‚nc. SauÌde 11:101-105. |
|
Guerra RN, Silva VM, Aaragão-França LS, Oliviera PR, Feitosa R, Nascimento FR, Pontes-De-Carvalho LC (2011). Babassu aqueous extract (BAE) as an adjuvant for T helper (Th)1-dependent immune responses in mice of a Th2 immune response-prone strain. Immunology 12(1):1. |
|
Ju YH, Allred CD, Allred KF, Karko KL, Doerge DR, Helferich WG (2001). Physiological concentrations of dietary genistein dose-dependently stimulate growth of estrogen-dependent human breast cancer (MCF-7) tumors implanted in athymic nude mice. J. Nutr. 131(11):2957-2962. |
|
Khyati P (2012). A review on herbal immunoadjuvant. Int. J. Pharm. Life Sci. 3:1568-1576. |
|
Klebanoff CA, Gattinoni L, Restifo NP (2006). CD8+ T-cell memory in tumor immunology and immunotherapy. Immunol. Rev. 211(1): 214-224. |
|
Koch J, Hau J, Christensen JP, Jensen HE, Handen MB, Rieneck K (2013). Immune cells from SR/CR mice induce the regression of established tumors in BALB/c and C57BL/6 mice. Plos. One 8(3):e59995. |
|
Kudryashov V, Kim HM, Ragupathi G, Danishefsky SJ, Livingston PO, Lloyd KO (1998). Immunogenicity of synthetic conjugates of Lewis (y) oligosaccharide with proteins in mice: towards the design of anticancer vaccines. Cancer Immunol. Imunother. 45(6):281-286. |
|
Lehrnbechter T, Koehl U, Wittekindt B, Bochennek K, Tramsen L, Klingebiel T, Chanock SJ (2008). Changes in host defense induced by malignancies and antineoplastic treatment: implication for immunotherapeutic strategies. Lancet Oncol. 9(3):269-278. |
|
Li P, Wang F (2015). Polysaccharides: Candidates of promising vaccine adjuvants. Drug Discov. Ther. 9(2):88-93. |
|
Loose D, Van de Wiele C (2009). The immune system and cancer. Cancer Biother. Radiopharm. 24(3):369-376. |
|
Ma J, Liu L, Che G, Yu N, Dai F, You Z (2010). The M1 form of tumor-associated macrophages in non-small cell lung cancer is positively associated with survival time. BMC Cancer 10(1):1. |
|
Mandal-Ghosh I, Chattopadhyay U, Baral R (2007). Neem leaf preparation enhances Th1 type immune response and anti-tumor immunity against breast tumor associated antigen. Cancer Immun. 7(1):8. |
|
Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M (2004). The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol. 25(12):677-686. |
|
Menard S, Tagliabue E, Canevari S, Fossati G, Colnaghi MI (1983). Generation of monoclonal antibodies reacting with normal and cancer cells of human breast. Cancer Res. 43(3):1295-1300. |
|
Moretão MP, Buchi DF, Gorin PA, Iacomini M, Oliveria MBM (2003). Effect of an acidic heteropolysaccharide (ARAGAL) from the gum of Anadenanthera colurbina (Angico branco) on peritoneal macrophage functions. Immunol. Lett. 89(2):175-185. |
|
Mosser DM (2003). The many faces of macrophage activation. J. Leukoc. Biol. 73(2):209-212. |
|
Nascimento FR, Barroquiero ES, Azevedo APS, Lopes AS, Ferreira SC, Silva LA, Maciel MCG, Rodriguez D, Guerra RN (2006). Macrophage activation induced by Orbignya phalerata Mart. J. Ethnopharmacol. 103(1):53-58. |
|
Nascimento FR, Barroquiero ES, Azevedo APS, Maciel MCG, Periera WS, Ferreira SC, Farias JC, Phinheiro MT, Silva LA, Guerra RN (2010). Differential effects of subcronic and chronic oral treatments with Orbignya phalerata Mart. mesocarp on the inflammatory response. Comp. Bioact. Nat. Prod. 5:267-281. |
|
Ochsenbein AF, Sierro S, Odermatt B, Pericin M, Karrer U (2001). Roles of tumour localization, second signals and cross priming in cytotoxic T-cell induction. Nature 411(6841):1058-1064. |
|
Ochsenbein AF (2002). Principles of tumor immunosurveillance and implications for immunotherapy. Nature 9(12):1043-1055. |
|
Pessoa RS, França EL, Ribeiro EB, Lanes PK, Chaud NG, Moraes LC, Honorio-França AC (2014). Microemulsion of babassu oil as a natural product to improve human immune system function. Drug Des. Dev. Ther. 16(9):21-31. |
|
RennoÌ MN, Barbosa GM, Zancan P, Veiga VF, Alviano CS, Sola-Penna M, Menezes FS, Holandino C (2008). Crude ethanol extract from babassu (Orbignya speciosa): cytotoxicity on tumoral and non-tumoral cell lines. An. Acad. Bras. Cienc. 80(3):467-476. |
|
Richmond A, Su Y (2008). Mouse xenograft models vs GEM models for human cancer therapeutics. Dis. Model. Mech. 1(2):78-82. |
|
Sachs DH (1995). The immunologic response to xenografts. ILAR J. 37(1):16-22. |
|
Schunk MK, Macallum GE (2005). Applications and optimization of immunization procedures. ILAR J. 46(3):241-257. |
|
Silva BP, Parente JP (2001). An anti-inflammatory and immunomodulatory polysaccharide from Orbignya phalerata. Fitoterapia 72(8):887-893. |
|
Silva CE, Santos OJ, Ribas-Filho JM, Tabushi FI, Kume MH, Jukonis LB, Cella IF (2015). Effect of Carapa guianensis Aublet (Andiroba) and Orbignya phalerata (Babassu) in colonic healing in rats. Rev. Col. Bras. Cir. 42(6):399-406. |
|
Souza MH, Monteiro CA, Figueredo PM, Nascimento FR, Guerra RN (2011). Ethnopharmacological use of babassu (Orbignya phalerata Mart) in communities of babassu nut breakers in Maranhão, Brazil. J. Ethnopharmacol. 133(1):1-5. |
|
Sun XB, Matsumoto M, Yamada H (1992). Anti-ulcer activity and mode of action of the polysaccharide fraction from the leaves of Panax ginseng. Plant. Med. 58(5):432-435. |
|
Um SH, Rhee DK, Pyo S (2002). Involvement of protein kinase C and tyrosin kinase in tumoricidal activation of macrophage induced by Streptococcus penumoniae type II capsular polysaccharide. Int. Immunopharmacol. 2(1):129-137. |
Copyright © 2024 Author(s) retain the copyright of this article.
This article is published under the terms of the Creative Commons Attribution License 4.0