Synthetic phosphoethanolamine: the state of the art of scientific production

Cancer is a multifactorial disease and a serious public health problem. Currently, alternative drug treatments for cancer are actively being sought, which is the case of synthetic phosphoethanolamine (PHOS-S), a compound that could possibly have anticarcinogenic effects. To analyze the available scientific evidence to evaluate the anticarcinogenic effects of in vivo and in vitro PHOS-S. A systematic literature review of scientific articles aimed at evaluating the anticarcinogenic potential of PHOS-S, in vivo and in vitro, using the databases PubMed, ScienceDirect, SciElo, CAPES Portal and LILACS. The selected papers suggest a possible anticarcinogenic effect of PHOS-S by inhibiting tumor growth by inducing apoptosis and cell cycle blockade as well as cytotoxic potential against leukemia cells. However, a possible stimulatory effect of tumor growth was also observed. Although some of the evaluated studies indicated a possible anticarcinogenic effect of PHOS-S, the limitations of these studies must be evaluated. Most were performed by the same research group, and in the scientific literature, we identified only preclinical studies (in cells or in animals). No human study has been published. Thus, more studies are needed to confirm the anticarcinogenic capacity of PHOS-S.


INTRODUCTION
Cancer has become a serious public health problem in both developed and developing countries.Cancer is the second leading cause of death globally and accounted for 8.8 million deaths in 2015 (WHO, 2017).Current data and statistical projections indicate that, in 2016, 1,685,210 new cancer cases and 595,690 cancer deaths will occur in the United States alone (Siegel, Miller, Jemal, 2016).
Cancer must be understood as a multifactorial disease caused by a confluence of both endogenous and environmental factors.Diet and food, life habits and the environment in which a person lives may contribute to an increase in the likelihood of being affected by cancer.For example, the increased consumption of foods rich in food additives, alcohol consumption or smoking induce the emergence of cancer.Therefore, the incidence of cancer has been increasing steadily, and an increasing number of individuals are receiving cancer treatments (Boyle, Levin, 2008;Doll, Peto, 1981;Anastácio et al., 2016).
Currently, the standard cancer treatment is based on surgery, chemotherapy and radiation therapy, depending on the type and characteristics of the tumor (WHO, 2002;Weinberg, 2014).However, this type of conservative treatment can generate various types of side effects that decrease the quality of life of patients and may also hamper treatment.Furthermore, many cancers remain uncured even after therapy (WHO, 2002;Sikora et al., 1999;Macquart-Moulin, 1997).
Such outcomes have inspired modern medicine to search for safer and more effective chemotherapy drugs that act only at the specific local site of cancer cells (Gullotti, Yeo, 2010) or new therapeutic alternatives for the treatment of cancer (Vidal, Carvalho, Bispo, 2013).These alternative treatments may be based, for example, on changing the eating habits of the patient (Yu et al., 2009) or adopting new, non-conventional drugs for clinical treatment.An example of the latter is synthetic phosphoethanolamine (PHOS-S), a compound that has been gaining attention as a possible anticarcinogenic agent (Pondé, Ades, Azambuja, 2016;Ferreira, 2013;Meneguelo, 2007).
Phosphoethanolamine was first isolated in 1936 by Outhouse (Outhouse, 1936).The phospholipid ethanolamine plays diverse cellular roles, including in cell division and apoptosis, and phosphoethanolamine is a primary amine that makes up the cell membranes of many tissues (Brasil, 2013;Bakovic, Fullerton, Michel, 2007).This compound has been studied as a possible anticancer agent in response to evidence that it can suppress tumor growth (Ferreira, 2013;Meneguelo, 2007;Veronez, 2012).
However, this compound has not yet received approval for use as an anticancer drug.PHOS-S has just begun its clinical trials in humans, so, the analyses of its anticancer proprieties are based solely on laboratory models, resulting in uncertainties about the use of PHOS-S (Sarraf et al., 2016;Pivetta 2016).
Therefore, this systematic review aimed to analyze in detail the available scientific evidence to evaluate the anticarcinogenic effects of PHOS-S both in animals and in vitro.

METHODOLOGY
This systematic review was guided by the recommendations established by the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) (Moher et al., 2009).Five databases were used: PubMed, ScienceDirect, CAPES Portal, SciElo and LILACS.The databases were searched for articles evaluating the anticarcinogenic potential of PHOS-S.
The following terms were used as descriptors (previously consulted MeSH -Medical Subject Headings): neoplasm and cancer.Since the term synthetic phosphoethanolamine is not a descriptor, it was used as a keyword in the search.To perform the search, the authors used the descriptor "synthetic phosphoethanolamine" with the Boolean operator AND, followed by one of the two descriptors, 'neoplasm' or 'cancer'.According to the guiding question was established "PICOS": "P" (problem): the anticarcinogenic effects of PHOS-S; "I" (intervention): PHOS-S; "C" (Comparison): control (untreated); "O" (outcomes): tumor regression; "S" (study design): Pre-clinical trials (in cells or in animals), because there is no clinical studies of PHOS-S published in scientific articles.
Thirty-six articles were found.Duplicate articles with a later date of publication were excluded, and the authors applied eligibility criteria (inclusion and exclusion criteria) to the remaining articles to determine which publications to use in this review by analyzing the type of article (original and review -selected only original articles) and the abstract.These criteria were checked independently in a blinded manner by two authors of the present review who were provided general information and abstracts of the articles.After the selection of the articles, the following variables were collected: author, year of publication, country of study, animals used in in vivo tests, cell lines used for in vitro studies, sample size and doses (in vivo studies), objective of the study and main result.

RESULTS
Eight papers were selected as the basis for this review, as is presented in the flow diagram, shown in Figure 1, that summarizes the methodology of the article.After analysis, the authors concluded that all eight articles met the requirements for inclusion.
Eight articles were analyzed to obtain the results of the presented review.The general information of the selected articles is presented in Tables I and II.

Alterations in the cells of the immune response
The ability of PHOS-S to modulate the metabolic activity of macrophages, components of the immune system, has been evaluated.These experiments were performed to assess the ability of the compound to inhibit tumor growth by altering the activity of these cells (Arruda et al., 2011).
In laboratory tests, Ehrlich ascites tumor cell lines were inoculated in mice fed diets containing 800 mg/ kg PHOS-S.The researchers surgically removed the inoculated tumors (after a period of growth) and washed the abdominal cavity to remove peritoneal macrophages.The macrophages were then subjected to the following assays: spreading test and calculation of H 2 O 2 and NO (substances involved in tumor progression and dispersion) (Arruda et al., 2011).
These analyses indicated that PHOS-S affects the metabolism of macrophages; cells obtained from mice fed PHOS-S showed less dispersion in the spreading test and released minor amounts of H 2 O 2 and NO.Moreover, the histopathology of the excised tumors showed greater cell cohesion of tumor cells retrieved from mice treated with PHOS-S (Arruda et al., 2011).
The antitumor capacity of PHOS-S was evaluated in Ehrlich ascites tumor cells and other tumor cell lines (human breast adenocarcinoma [MCF-7], human malignant melanoma [SKMEL-28 and MeWo] murine malignant melanoma [B16-F10] and human mucoepidermoid pulmonary carcinoma [H292]).The researchers confirmed by MTT colorimetric assay (0.5 to 10 mg/mL) and Hoechst 33342/PI fluorescence assay (2.30 mg/mL) that PHOS-S has in vitro cytotoxic activity against tumor cells while preserving healthy cells and can cause morphological alterations in tumor lines.PHOS-S also directly inhibited tumor growth in in vivo models (at doses of 35 and 70 mg/kg/day).Finally, PHOS-S also decreased tumor proliferation and altered the cell cycle in these cells (reducing the proportion of cells in G2/M phase and increasing the number of apoptotic sub-G1 cells) (Ferreira et al., 2012a).
Other studies analyzed the ability of PHOS-S to inhibit the growth of melanoma in murine models (Ferreira et al., 2011;2012b).In one of these studies, groups of mice with melanomas (originating from B16F10 melanoma strain cells) received PHOS-S solutions (7 mg/kg and 14 mg/kg) via injection into the respective tumors.This particular study also studied the antitumor capacity of PHOS-S in tumor cells and healthy cells (in vitro) exposed to various concentrations of PHOS-S (0.005 to 6.0 mg/mL) (Ferreira et al., 2011).
In both experiments, PHOS-S exhibited antitumor effects.In the in vivo models, PHOS-S attained 86% tumor reduction, along with an increase in the apoptosis rate in the cells of tumors treated with PHOS-S.In the in vitro models, PHOS-S exhibited cytotoxic activity in tumor cells and decreased the mitotic index of this cell population (Ferreira et al., 2011).
Another study analyzed the antiproliferative capacity of PHOS-S in melanoma B16F10 tumor cells in both in vivo and in vitro models.When examined in vitro, PHOS-S showed antiproliferative capacity in an MTT colorimetric assay (12.5 to 100 mM without inducing morphological changes of the tumor cells.Additionally, in in vivo models, PHOS-S (50 and 100 mg/kg) showed antiproliferative capacity, decreasing the tumors size (by 60% and 47%, respectively), delaying tumor growth and significantly enhancing mouse survival (Ferreira et al., 2012b).
The ability of PHOS-S to inhibit tumor growth of MCF-7 breast cancer cell lines has also been reported.In this study, researchers ascertained that the compound had cytotoxic capacity based on the results of MTT colorimetric assays (from 20 mM) and reduced tumor cell metabolism (Ferreira et al., 2013a).
Finally, PHOS-S exhibited cytotoxic effects on murine renal carcinoma cells.Based on the results of MTT colorimetric assays, the researchers concluded that PHOS-S has concentration-dependent cytotoxic activity against tumor cells.In addition, the IC50 value (half the maximum inhibitory concentration) of PHOS-S in these cells was 90 mM (Ferreira et al., 2013b).

Apoptosis induction effects
Based on the notion that phospholipids may be related to the induction of apoptosis (Meneguelo, 2007;Veronez, 2012;Elbayoumi et al., 2016), the ability of PHOS-S to induce apoptosis in cancer cells has been examined.
Studies in B16-F10 melanoma and MCF-7 breast cancer cell lines indicated that PHOS-S can induce tumor cell apoptosis regardless of blocking of the Bcl-2 protein.Loss of Bcl-2 expression was observed in tumor cells treated with the compound (Ferreira et al., 2011(Ferreira et al., , 2013a)).
Several studies have observed that PHOS-S can change the mitochondrial membrane potential, based on assays with Rhodamine-123 (Rho123).In these studies, PHOS-S reduced the membrane potential of mitochondria and caused dysfunction of these organelles in tumor cells (B16-F10 melanoma, Ehrlich ascites tumor, breast cancer MCF-7) pretreated with PHOS-S.These changes lead to an increased rate of apoptosis (Ferreira et al., 2012a(Ferreira et al., .b, 2012b(Ferreira et al., ., 2013a)).
The influence of PHOS-S on caspase-3 activity has also been studied by flow cytometry in tumor cells (B16-F10 melanoma, Ehrlich ascites tumor, MCF-7 breast The mice were divided into different groups (n=5): 40 mg/kg PHOS-S; 80 mg/kg PHOS-S; 1 mg/kg alltrans retinoic acid (ATRA) and 10 mg/kg daunorubicin (DA) (the last two were positive controls).In addition, mice were treated with 100 mL of PBS (vehicle) to induce the same stress conditions of treatment in the control animals

Ferreira et al. (2013b)
The mice were divided into different groups (n=5): 50 mg/kg/day PHOS-S, 100 mg/kg/day PHOS-S and 10 mg/kg/day sunitinib PHOS-S presented antiangiogenic and antimetastatic capacity cancer) treated with PHOS-S.An increase in caspase-3 activity was observed, confirming that PHOS-S can control tumor development by inducing apoptosis via the caspase pathway (Ferreira et al., 2011;2012a,b;2013a,b).
Other proteins analyzed in studies of PHOS-S include cyclin D1 and p53.Flow cytometry revealed that PHOS-S increased p53 expression and suppressed the expression of cyclin D1.Therefore, the authors hypothesized that PHOS-S may trigger apoptosis and have antitumor potential (Ferreira et al., 2012b(Ferreira et al., , 2013b)).
Finally, the included studies also analyzed the potential of PHOS-S to induce apoptosis by Annexin V/PI double-staining and flow cytometry.The authors concluded that PHOS-S can induce tumor cell apoptosis, as tumors treated with PHOS-S had greater numbers of cells at the early and late stages of apoptosis.PHOS-S also induced morphological changes in the tumor cells.Based on these findings, the authors hypothesized a role of PHOS-S in inducing apoptosis (Ferreira et al., 2011(Ferreira et al., , 2012a)).

Antileukemic effects
A study analyzed whether PHOS-S possesses antileukemic effects.Leukemia cell lines (KG-1, K562 To investigate the anticarcinogenic potential of PHO-S in melanoma B16-F10 cells both in vivo and in vitro C57BL/6 J Mice B16-F10 cell lines

Ferreira et al. (2013a)
To evaluate the anticarcinogenic activity of PHO-S in MCF-7 breast cancer cells MCF-7 cell lines Brazil

Ferreira et al. (2013c)
To investigate the antileukemic effects of PHO-S using acute promyelocytic leukemia (APL) as a model NOD/SCID Mice KG-1, K562 and Jurkat cell lines

Ferreira et al. (2013b)
To assess the anti-angiogenic and antimetastatic activity of PHO-S HUVEC -CRL 1730 cell lines Brazil and Jurkat) were exposed to PHOS-S in different types of experiments (Ferreira et al., 2013c).
The MTT colorimetric assay revealed cytotoxic effects of PHOS-S in the cell lines studied.PHOS-S also exhibited potent dose-dependent cytotoxic effects against KG-1, K562 and Jurkat cells, with IC50 values of 9, 6 and 12 mM, respectively (Ferreira et al., 2013c).
Flow cytometry also revealed that, in vitro, PHOS-S can decrease the mitochondrial membrane potential in the leukemic cell lines studied.Furthermore, the activity of caspase-3 in these cells was analyzed by the Annexin-V/PI double-staining apoptosis detection method.An increase in protein activity was observed in the studied cell lines, consistent with the evidence of the induction of apoptosis via a mitochondrial-dependent pathway by PHOS-S (Ferreira et al., 2013c).
The researchers also analyzed leukemic models in vivo (acute promyelocytic leukemia) obtained from leukemic induction in mice (mice NOD/SCID).In these leukemic mice, cyto-morphological analysis revealed a smaller number of immature cells in the bone marrow, peripheral blood, spleen and liver (tested at concentrations of 40 or 80 mg/kg PHOS-S) (Ferreira et al., 2013c).
Moreover, reductions of CD117 + and Gr-1 + cells (commonly observed in acute promyelocytic leukemia) were observed in the previously analyzed organs at the tested concentrations of the compound (40 or 80 mg/ kg).Treatment with 40 mg/kg induced an increase in the percentage of CD45 + cells, a decrease in hematopoietic progenitor cells from CD34 + and a decrease in CD34 + cells in the bone marrow of mice (Ferreira et al., 2013c).
Finally, researchers also evaluated the effects of PHOS-S on the induction of apoptosis by Annexin-V/ PI double-staining of CD117 + and Gr-1 + cells.The researchers observed an increase in apoptosis in the cells studied in the liver and spleen of guinea pigs pretreated with PHOS-S.These findings corroborate the possible antileukemic effects of synthetic phosphoethanolamine (Ferreira et al., 2013c).

Antiangiogenic activity
The ability of PHOS-S to influence the migration of endothelial cells was analyzed by wound healing assays.The researchers concluded that PHOS-S (10 mM) inhibited cell migration compared to the controls.In addition, human umbilical vein endothelial cells (HUVEC) treated with PHOS-S exhibited inhibition of the formation of capillaries in the tube formation assay, a three-dimensional model, confirming the antiangiogenic activity of PHOS-S (10 mM) (Ferreira et al., 2013b).
PHOS-S also inhibited the mRNA expression of cyclin D1 and VEGFR1 (vascular endothelial growth factor receptor 1).These factors are involved in angiogenesis modulation from cell proliferation and induction of the angiogenic mechanism and were suppressed by PHOS-S, consequently reducing angiogenesis.PHOS-S also inhibited oxidative stress in murine renal carcinoma cells, reducing the formation of malondialdehyde and H 2 O 2 (90 mM) and therefore exerting a protective effect (Ferreira et al., 2013b).

Anti-metastatic activity
The anti-metastatic activity of PHOS-S has also been evaluated.Murine renal carcinoma cells were injected into mice (BALB).After 15 days of treatment (intraperitoneal administration of PHOS-S, 50 or 100 mg/ kg/day), metastatic nodules were examined in the lungs and other organs of the animals (Ferreira et al., 2013b).
The number of metastases in the lungs and spreading to other organs was lower in mice that received prior administration of PHOS-S.Furthermore, the cell density of metastatic nodules in treated mice was lower than that in the control mice in the experiment, suggesting antimetastatic properties of PHOS-S (Ferreira et al., 2013b).
In another study, mice with melanoma (B16-F10) tumors were treated with different doses of PHOS-S (7 and 14 mg/kg).The control mice exhibited a higher number of metastases compared with the treated mice, again confirming the anti-metastatic potential of PHOS-S (Ferreira et al., 2011).

Effects on the promotion of tumor cell proliferation
Researchers evaluated the possible role of PHOS-S as an inducer of tumor proliferation in a murine mammary carcinoma line (64-24 cell line isolated from hormonedependent mammary carcinoma -MCCLX).Previous evidence suggested that an unknown phospholipid of pituitary origin could stimulate the proliferation and growth rate of these tumor cells.To test this hypothesis, the authors exposed the tumor cells at different concentrations of compound (0.2 to 50 nmol/mL).The tests indicated that PHOS-S induces tumor growth in a dose-dependent manner (Kano-Sueoka et al., 1979).

DISCUSSION
A subset of the analyzed studies converged to a hypothesis that PHOS-S has anticarcinogenic potential.Assessments of the influence of this compound on the activity of mononuclear phagocyte system cells (macrophages), which are important for neoplastic development (Laoui et al., 2014), indicated that PHOS-S has inhibitory activity in these cells.
Moreover, in some of the analyzed studies, cytotoxic and inhibitory effects of PHOS-S were observed in tumor cells.These results theoretically confirm evidence that phospholipids have anticarcinogenic activities (Koufaki et al., 1996;Hochhuth et al., 1990).Finally, the study by Ferreira and colleagues (2013c) of the antileukemic properties of PHOS-S also corroborates findings by Dhakshinamoorthy et al. (2015) of a possible role of PHOS-S in the inhibition of leukemic cells.
However, most of the studies of the possible anticarcinogenic effects of PHOS-S that were analyzed in this review were from the same research group.In addition, the in vivo and in vitro results are controversial when compared with other sources of information (Ferreira et al., 2013c;Brasil, 2013;Brasil, 2016a;Brasil, 2016b).
Reports commissioned by the Brazilian's Ministry of Science, Technology and Innovation showed contrary implications of the anticarcinogenic effects of this substance.The experiments carried out in these reports (even using extremely high concentrations of the compound) did not support PHOS-S as an inhibitor of cancer (Brasil, 2013(Brasil, , 2016a,b),b).Finally, conflicting results were obtained by Kano-Sueoka and colleagues in a study published in 1979 and conducted in the USA (the only study outside Brazil).
It is also important to analyze the limitations of the studies published to date.As discussed at the beginning of this article, the analysis of the properties of PHOS-S is still based exclusively on preclinical animal or cellular models.Clinical studies have started recently, and they are still in a reduced scale (Pivetta, 2016).The use of a drug based exclusively on preclinical studies should be avoided.This was not the case with PHO-S, which began to be used in humans even without further clinical studies (Escobar, 2016).

CONCLUSION
A portion of the analyzed studies suggest possible anticarcinogenic potential of PHOS-S.However, the limitations of the published studies must be considered.
In the literature, we identified only preclinical studies (in cells or in animals).No clinical study in humans has been published in the literature until the present moment.
The lack of more complex studies in humans does not allow a more in-depth and qualitative analysis of the actual toxicological implications of the compound.
Even though PHOS-S did indeed have anticarcinogenic properties, there was insufficient evidence that the drug would not negatively affect its users.Thus, it's certain to assert that an analysis of the actual implication of using this compound as an anticancer drug will still take time.
Therefore, it is necessary to evaluate the real implications of PHOS-S as a possible drug for use in cancer treatments.Further studies are needed on a broader scale to assert the legitimate anticarcinogenic capability of this compound.

FIGURE 1 -
FIGURE 1 -Flow diagram of search procedure and screening process through the different phases of the systematic review.

TABLE I -
Description of the articles included in the systematic review of the synthetic phosphoethanolamine and its anticarcinogenic effects

TABLE II -
Description of the articles included in the systematic review of the synthetic phosphoethanolamine and its anticarcinogenic effects, according to the main goals