Anticancer potential, molecular mechanisms and toxicity of Euterpe oleracea extract (açaí): A systematic review

Cancer is an increasingly frequent malignancy worldwide, and despite the advances in drug development, it is still necessary to develop new plant-derived medicines. Euterpe oleracea (açaí) is abundant in South and Central America and has health benefits due to its high levels of phytochemicals, including lignans and polyphenols. The aim of this review was to systematically describe the safety and antitumor effects of açaí in preclinical models using rodents to provide a more comprehensive assessment of açaí for both therapeutic uses and the development of future clinical studies in cancer. Eligible studies were identified using four international databases (PubMed, Medline, Lilacs and SciELO) from their inception date through December 2017. The included studies were analyzed with methodological rigor (QATRS) to enable better quality control for these experimental studies. Sixty publications were identified in the databases, but only 9 articles were eligible: 6 evaluated the pharmacological effects of açaí in animal models of cancer (1 model each of esophageal cancer, urothelial cancer, melanoma and Walker-256 tumor and 2 models of colon cancer), and 3 were toxicological assays using preclinical models with rodents. Overall, 747 animals were analyzed. On a QATRS score scale of 0–20, the quality of the studies ranged from 16 to 20 points. Pulp was the main fraction of açaí administered, and an oral administration route was most common. The açaí dosage administered by gavage ranged from 30 mg/kg to 40,000 mg/kg, and açaí fed in the diet accounted for 2.5% to 5% of the diet. The anticarcinogenic and chemopreventive activities of açaí were observed in all experimental models of cancer and reduced the incidence, tumor cell proliferation, multiplicity and size of the tumors due to the antiinflammatory, antiproliferative and proapoptotic properties of açaí. No genotoxic effects were observed after açaí administration. The results of this review suggest that açaí is safe and can be used as a chemoprotective agent against cancer development. Açaí therapy may be a novel strategy for treating cancer.


Introduction
The use of natural products as medicines accounts for approximately 30% of the currently available drugs [1], and in some therapeutic areas, the amount of plant-derived medicines reaches 60% [2,3].Brazil has the greatest amount of biodiversity in the world and plays an important role in the area of natural bioactive compounds by contributing natural products to design new clinical medicines [1,4].Thus, there has been growing research aimed at establishing the therapeutic potential of natural products against several diseases.
Euterpe oleracea Mart. is a member of the family Arecaceae and is a typical palm of the rainforest in the Amazon region, in the states of the northern region of Brazil, including Guianas, Colombia, Ecuador, and Venezuela [5].The fruit, popularly known as "ac ¸aı ´", weighs approximately 2 g, and the color of the mature fruit is dark purple [6].Ac ¸aı ´is a traditional food in many regions of Brazil [7,8], and its consumption has increased significantly over the last several years, not only in Brazil but also in Europe and the USA, where the fruit gained popularity after being promoted as a "super fruit" [9].Currently, due to the health benefits and therapeutic potential of ac ¸aı ´, locally grown ac ¸aı ´are increasingly exported around the world as energy drinks [6,10], "functional foods" [7,8], cosmetics and pharmaceutical products [9].Ac ¸aı ´pulp is composed of approximately 48% lipids, 13% protein, 8% amino acids, 25% total sugars and minor compounds such as fiber and vitamins (A, B1, B2, B3, C and E) [8,11,12].Moreover, it is rich in several phytochemicals, including lignans, phenolic compounds (anthocyanins, proanthocyanidins and other flavonoids) and resveratrol, in low concentrations [8,11,12].The seeds of ac ¸aı ´possess the highest concentration of polyphenols (28.3%), followed by the whole fruit (25.5%) and the bark (15.7%) [13].
The aim of this review was to systematically describe the safety and antitumor effects of ac ¸aı ín preclinical models using rodents, to provide a comprehensive assessment of ac ¸aı ´for therapeutic use.Preclinical studies using rodents were evaluated to investigate whether the current knowledge supports cancer clinical trials with ac ¸aı ´.

Search strategy
A careful literature search was performed to identify publications that studied the use of E. oleracea extract in experimental animal models of cancer and/or evaluated the safety/toxicity of ac ¸aı ín animal models.Studies were identified by searching the electronic databases: PubMed, Medline-Bireme, Lilacs and SciELO from their inception date through December 2017 (S1 Table ).The search terms were as follows: ("Euterpe oleracea" AND cancer treatment) OR ("Euterpe oleracea" AND cancer animal model) OR (Ac ¸aı ´AND cancer treatment) OR (Ac ¸aı ´AND cancer animal model) AND ("Euterpe oleracea" AND toxicity) OR (Ac ¸aı ´AND toxicity).The search was performed without restrictions on the language or year of publication.Two reviewers (KCRB and JA-P) selected the qualified studies independently by browsing the titles, abstracts or full texts based on the eligibility criteria.The duplicates were removed.The eligible articles were separated for analysis of the study methodology and results (S1 Table ).Any disagreements were resolved by discussion with two additional reviewers (DEM and JAP).

Inclusion and exclusion criteria
Articles were included if the following criteria were met: (1) evaluated the pharmacological effect of ac ¸aı ´in animal models of cancer and/or (2) performed toxicological analyzes after ac ¸aı ádministration in experimental animal models.Articles were excluded if the following criteria were met: (1) were reviews of literature; (2) did not analyze the use of ac ¸aı ´in vivo; (3) did not use the order Rodentia; (4) did not evaluate the toxicological effects of ac ¸aı ´administration in vivo; and (5) used only in vitro experimental models.

Data extraction
Three investigators (KCRB, JA-P and JAP) independently conducted the extraction of details from each study including the following: (1) basic information, including the publication year, the first author's name, the type of animals, the sex, the in vivo model and the experimental interventions; (2) basic information about the ac ¸aı ´treatment, including the fraction and origin of E. oleracea, dose, administration route, posology, diluents and treatment groups; and (3) outcome measures used to evaluate E. oleracea extract, therapeutic indications (pharmacodynamic), ac ¸aı ´signaling pathways and safety evaluations.When a single publication included studies with animals, posology or types of interventions that were different, these data were extracted and considered as independent experiments.Any disagreements regarding the extracted data were resolved by discussion with an additional reviewer (DEM).

Quality assessment
For assessment of quality, two independent reviewers (KCRB and JA-P) used a quality rating scale as an animal/tissue research scale (QATRS).The QATRS is a 20-point scaled evaluation chart that was designed based on randomization, blinding, the similarity of the animal/tissue model to human applications, standardization and the reliability of the measurement techniques, management of study withdrawals, and appropriateness of the statistical methods [23].Any disagreements were resolved by discussion with two additional reviewers (DEM and JAP).

Ac ¸aı ´information
Table 2 shows the basic information about the ac ¸aı ´extract used in the experimental models.The most commonly used ac ¸aı ´fraction was the pulp [40][41][42][43]46], followed by the juice [48], oil [44,47] and seeds [45].Seven studies mentioned the ac ¸aı ´origin, and all of the ac ¸aı ´extracts were from Brazil [40][41][42][43][45][46][47].The main administration route of ac ¸aı ´was oral; 4 studies administered ac ¸aı ´by gavage [45][46][47][48], and 4 studies administered ac ¸aı ´as part of the diet [40][41][42][43].The dosage ranged from 30 mg/kg to 40,000 mg/kg in studies that administered ac ¸aı ´by gavage and was administered as a single dose or as 1 daily dose for 90 consecutive days; in the studies that administered ac ¸aı ´as part of the diet 2.5% to 5% ac ¸aı ´supplementation was provided in the diet for 10 to 35 weeks (Table 2).In addition, Schauss and colleagues used oral and intraperitoneal administration of ac ¸aı ´at a dose of 0.1mg/0.15mL(daily dose during 7 consecutive days) to assess the possible genotoxic effects of ac ¸aı ´using BALB/c mice [48], and Monge-Fuentes and colleagues used 50 mg/mL of ac ¸aı ´administered intratumorally in an experimental model of melanoma [44].The results regarding the therapeutic indications, effects and safety of ac ¸aı ´in experimental models are summarized in Table 3.
Using a micronucleus test and a comet assay, Ribeiro and colleagues reported no differences between the control and ac ¸aı ´groups in bone marrow and peripheral blood cells polychromatic erythrocytes, and in liver and kidney cells, thus demonstrating the absence of genotoxic effects of ac ¸aı ´.In addition, ac ¸aı ´reduced DNA damage induced by doxorubicin  (DXR), suggesting a protective role in human health [46].In a study done by Schauss and colleagues, ac ¸aı ´did not cause mutagenic effects, as demonstrated by a bacterial reverse mutation assay, a chromosomal aberration assay, a mammalian cell mutation assay and an in vivo micronucleus study [48].In the same way, Marques and colleagues evaluated the genotoxic potential of ac ¸aı ´in rat cells.The authors used a comet assay and a micronucleus test and showed that on both cytogenetic tests, no significant genotoxic effects were observed at the three tested dosages of ac ¸aı ´ [47].

Antitumoral effects of ac ¸aı The
anticarcinogenic and chemopreventive activities of ac ¸aı ´, as evidenced by reductions in the incidence of tumors, tumor cell proliferation, and multiplicity and size of tumors, were observed in all the experimental models of cancer [40][41][42][43][44][45] (Table 3).
Stoner and colleagues reported that ac ¸aı ´was effective at inhibiting the progression of esophageal tumorigenesis, reducing the levels of the serum cytokines (IL-5 and IL-8), and increasing serum antioxidant capacity and interferon-gamma (IFNγ) levels [40].By contrast, the esophageal tumor size and serum levels of IL-1β, IL-4, IL-13 and tumor necrosis factor-alpha (TNFα) were not significantly affected by adding ac ¸aı ´to the diet for 35 weeks [40].
Fragoso and colleagues reported that ac ¸aı ´was effective at inhibiting urinary bladder carcinogenesis, reducing DNA damage, and reducing the expression of p63 and proliferating cell nuclear antigen (PCNA) [41].However, altered cytoplasmatic and nuclear β-catenin were not significantly affected by adding ac ¸aı ´to the diet for 10 weeks [41].Two studies reported that ac ¸aı ´was effective at inhibiting colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) in Wistar rats [42] and azoxymethane (AOM) with dextran sulfate sodium (DSS) in ICR mice [43].Nevertheless, the opposite results were observed with regard to cleaved caspase-3 expression after supplementation with 2.5% and 5% of ac ¸aı ´in the diet for 10 [42], 14 [43] or 20 weeks [42].Despite the discrepancies between these studies, the quality evaluation of the results of the articles showed good quality QATRS (16/20 and 20/20, respectively) [42,43].Moreover, Choi and colleagues reported that ac ¸aı ´treatment down-regulated myeloperoxidase (MPO) and proinflammatory cytokines (TNF-α, IL-1β and IL-6), inhibited cyclooxygenase 2 (COX-2), PCNA and Bcl-2, and increased Bad and cleaved caspase-3 expression in an experimental model of cancer colon [43].
Monge-Fuentes and colleagues reported that ac ¸aı ´was an effective photosensitizer because it reduced melanoma carcinogenesis by increaseing the necrotic tissue per tumor area after 5 applications of intratumoral ac ¸aı ´during a period of 15 days [44].
Nascimento and colleagues reported an anticarcinogenic effect (tumor diameter and weight) of ac ¸aı ´in anorexia-cachexia syndrome induced by Walker-256 tumors due to the antioxidant activity of ac ¸aı ´after 1 daily dose of ac ¸aı ´over 14 consecutive days [45].
Finally, based on the results of this review study, we created a schematic representation of the effects of ac ¸aı ´in tumor cells (Fig 2).Ac ¸aı ´showed antitumoral functions due to its antiinflammatory, antiproliferative and proapoptotic properties.

Discussion
Research on the pharmacological effects of natural products for the treatment of several diseases has significantly increased in the last decades.In this sense, ac ¸aı ´has been marketed as a dietary food supplement because of its health benefits due to its high levels of phytochemicals, including lignans and polyphenols.Studies have demonstrated that ac ¸aı ´has biological effects, such as antioxidant, antiinflammatory, antiproliferative, antinociceptive and antitumorigenic activities [13,16,17,19,[49][50][51][52][53][54][55].
As far as we know, the present work is the first review to focus on the antitumorigenic and toxicological effects of ac ¸aı ´in preclinical trials using rodents.Overall, nine studies were included in this review [40][41][42][43][44][45][46][47][48].Although we conducted a thorough literature search, using four international databases, one limitation is that our conclusions may be narrow due to the lack of availability of published articles and because all of the included studies were published in English.In spite of the small number of studies found, we assessed them with a range of methodological rigor in accordance with the QATRS, which encompasses various aspects that enable better quality control for these experimental studies [23].A strong point of our review is that all the included studies had good quality as assessed by the QATRS score (all had a score greater than or equal to 16/ 20).A total of 747 animals of the order Rodentia were analyzed.The results indicated that ac ¸aı has a chemopreventive effect (anticancer) by inhibiting tumor growth and leads to a reduction in tumor size, suggesting antiproliferative, pro-apoptotic and anti-inflammatory activity [40][41][42][43][44][45].In addition, the toxicological studies showed that ac ¸aı ´did not cause DNA damage or genotoxic or mutagenic effects in the evaluated animals, suggesting that it is safe for clinical testing [46][47][48].
Most of the studies found that ac ¸aı ´significantly decreased tumor incidence or tumorigenic inhibition and prevented DNA damage without causing genotoxic effects when it was administered orally (in the diet or by gavage).These results suggest that the oral route is a good choice for evaluation of the effects of ac ¸aı ´in humans clinical studies since this is an easy and safe route of administration.It should be noted that the significant results found with the oral administration of ac ¸aı ´have also been described in other diseases, such as obesity and hepatic steatosis [67], endometriosis [55], renovascular hypertension [68] and neuropathic pain [53].The articles included in this review described different doses of ac ¸aı ´that were administered orally by gavage (range 30 mg/kg to 40,000 mg/kg) [45][46][47][48].However, Marques and colleagues observed that at an ac ¸aı ´dose of 300 mg/kg, a few animals showed signs of toxicity (diarrhea and bristling of the hair), which is why they did not test higher doses [47].Recently, our group reported that a dose of ac ¸aı ´of 200 mg/kg administered by gavage for 30 consecutive days had efficacy in suppressing endometriotic lesions in a Sprague-Dawley rat model without any signs of toxicity [55].Although considered a benign disease, endometriosis frequently presents with characteristics of malignancy [69].Therefore, we suggest that an ac ¸aı ´dose of 200 mg/kg is safe for preclinical testing and is a promising novel pharmacological treatment for cancer due to its anticarcinogenic and chemopreventive effects.
With regard to the ability of ac ¸aı ´to inhibit carcinogenesis, and the incidence and multiplicity of tumors in experimental models of cancer using rodents, in vitro studies also showed that ac ¸aı ´decreased cell viability, suppressed proliferation and induced apoptosis, suggesting the anticancer and antioxidant activity of ac ¸aı ´against C-6 rat brain glioma cells [49], MCF-7 breast cancer cells [13,38] and colon cancer cells [34].These results suggest that ac ¸aı ´contains phytochemicals that can be used as natural chemopreventive agents [13,40,42].
A large number of studies have shown the importance of chronic exposure to proinflammatory cytokines in tumorigenesis [70][71][72].The results of this review show that ac ¸aı ´acts in the inflammatory processes involved in induced-cancer in animals by decreasing the levels of IL-1β, IL-5, IL-6, IL-8, COX-2, TNF-α and MPO and increasing the levels of IFN-γ [40,43].An in vitro study of polymorphonuclear cells showed a reduction in the IL-8 levels that was associated with the decreasing inflammatory conditions after ac ¸aı ´treatment [64].Xie and colleagues evaluated flavonoids isolated from ac ¸aı ´pulp and observed a reduction in serum levels, gene expression and protein levels of both the cytokines TNF-α and IL-6 in the resident macrophages cells [73].Ac ¸aı ´was also able to prevent increases in the levels of IL-1β and TNF-α in the brain tissues of a CCI4 experimental model [27].In addition, ac ¸aı ´reduced the COX-2 expression and PGE 2 levels in an experimental model of endometriosis [55] and reduced the MPO levels in a rat renal ischemia/reperfusion model [74].As a result of this review, it was possible to identify the antiproliferative pathways by which ac ¸aı ´acts by reducing PCNA, Ki-67 and p63 [41][42][43].These proteins are involved in tumor development, survival and metastasis of different tumors [75][76][77].In addition, the anti-apoptotic proteins Bcl-2 was also reduced after ac ¸aı ´treatment in animals with induced-cancer [43], in agreement with a study of human colon cancer cells in which the proapoptotic activities of polyphenolics from ac ¸aı ´were described [34].Polyphenolics may regulate distinct steps of the apoptotic process and/or the expression of regulatory proteins, such as the downregulation of Bcl-2 and cleavage of caspase-3 [78,79].Ac ¸aı ´polyphenolics were previously described to have proapoptotic and antiproliferative activities in leukemia cancer cells through caspase-3 activation [80].Surprisingly, as a result of this review, it was possible to identify the discrepancies in the levels of cleaved caspase-3 in colon carcinogenesis induced after ac ¸aı ´treatment [41,43].Choi and colleagues observed that ac ¸aı ´increased the cleaved caspase-3 levels in the supernatants of colon strips [43], but Fragoso and colleagues described the opposite results using immunohistochemical techniques in colon tumor tissues [41].
Another specie from Brazilian Euterpe, o Euterpe edulis, has been studied because has important nutritional properties for human health.E. edulis Mart., commonly known as juc ¸ara or jussara and ac ¸aı ´-do-sol, is a native tree of the Atlantic Forest and has similar nutritional properties of ac ¸aı ´ [81], however ac ¸aı ´has twice of the polyphenols concentration [82].Recently, a review described 25 articles about the phytochemical characterization and biological activities of juc ¸ara [81].Nevertheless, none of these studies evaluated the effect of E. edulis in the cancer treatment and two studies described the safety evaluation of E. edulis, however with controversial results.Barros Freitas et al, 2017 showed juc ¸ara prevent the oxidative damage resulting from the cafeteria diet and no evidenced signs of lipid peroxidation in renal or in cardiac tissue in Wistar rats [82].On the other hand, Felzenszwald et al., 2013, demonstreted E. edulis was able to induce mutagenicity and clastogenic/aneugenic effects in Wistar rats [25].
Toxicity data are decisive for evaluating the safety of natural products for clinical treatment because these data investigate the potential for mutagenicity, genotoxicity, clastogenicity and aneugenicity [83].The toxicological studies included in this review showed that ac ¸aı ´is nontoxic [42,44,[46][47][48], has no genotoxic or mutagenic effects, and has a protective effect on DNA damage caused by antitumoral agents [46][47][48].Similarly, Santos and colleagues showed that antioxidant compounds prevented the induction of DNA damage induced by DXR [84].On the other hand, ac ¸aı ´showed mutagenic effects when assayed in high concentrations in eukaryotic cells of Saccharomyces cerevisiae yeast; however, there is a low mutagenic risk for humans because the tested concentrations were significantly elevated [31].Since only 3 studies investigated the genetic toxicity of ac ¸aı ´in preclinical trials of rodents [46][47][48], future research is needed to better understand the efficacy of ac ¸aı ´because its antimutagenic and antioxidant activities may prevent DNA damage and thus improve human health.

Conclusions
The results of this review suggest that ac ¸aı ´is safe and can be used as a chemoprotective agent against cancer by exhibiting antiinflammatory, antioxidant, antiproliferative, and proapoptotic properties.Further studies on the functional relevance of ac ¸aı ´are necessary to build a database that can be used in future clinical investigations aimed at discovering antitumor agents.

Table 2 . Basic information regarding the E. oleracea extract used in the in vivo experimental models. Fraction Origin of ac ¸aı ´Dosing Diluent and placebo Administration Posology Reference Juice b
Juice of MonaVie Active1 = In addition to ac ¸aı ´, contains lesser amounts of 19 fruits and berries.
AIN = American Institute of Nutrition; IP = intraperitoneal; SP = São Paulo; PBS = Phosphate buffered saline.a A reference can have different methods of administration of ac ¸aı ´. b c A cereal-based commercial diet for mice formulated by the Orient Bio Group (Seongnam, Korea).