The Antiproliferative and Pro-apoptotic Role of 2-aminoethyl dihydrogen Phosphate in Triple-negative Breast Tumor Cells

: Antineoplastic phospholipids are a new class of antitumor agents. These molecules interact with the plasma membrane, changing numerous pathways that induce cell death, with high selectivity for cancer cells. A representative of this class of antineoplastic agents is 2-aminoethyl dihydrogen phosphate (2-AEH 2 P). It is present in high intracellular concentrations in various tissues and organelles with antitumor, antiproliferative and pro-apoptotic action. Therefore, 4T1 triple-negative tumor cells were treated in different concentrations in order to assess the cytotoxic potential and its effects on the modulation of cell death pathways in association with the chemotherapeutic drug Paclitaxel. 2-AEH 2 P promoted cytotoxicity in tumor cells and significant morphological changes, however, it did not cause these effects in normal cells. There was positive regulation of proteins involved in the intrinsic pathway of cell death by apoptosis and regulation of the phases of cell cycle progression. Furthermore, structural and distribution changes in mitochondria, as well as decreased cell density and regression of the cytoskeleton were observed. The 2-AEH 2 P demonstrated a modulatory potential of pathways inducing cell death, being a new compound with antitumor properties.


Introduction 
Breast cancer occupies the second position in number of cases and, according to GLOBOCAN [1], for 2020, the global estimate is 2,179,457 and, for the year 2040, about 3,059,829 cases. In the female population, breast cancer is also commonly diagnosed (24.2%), being the most frequent in 154 countries and the leading cause of cancer death (15.0%) in 103 countries [2].
The most commonly diagnosed histological type is invasive ductal carcinoma (50-75%), followed by invasive lobular carcinoma (5-15%), and with mixed as well as studies of therapeutic strategies [7]. This tumor is highly tumorigenic and invasive, being able to spontaneously metastasize from the primary tumor to distant regions, as in lymph nodes, blood, liver, lung, brain and bone [8][9][10]. In addition, it has an easy implantation in the mammary gland and metastatic progression to lymph nodes and other organs similarly to triple negative human breast cancer, the human equivalent is MDA-MB-231 [11].
Breast cancer is known for its low response to traditional treatments adopted, such as chemotherapy and radiotherapy, therefore, the development of new therapeutic therapies is urgent. Thus, antineoplastic phospholipids and lipid precursors appear as a new class of antitumor agents. Without causing damage to DNA, these molecules interact with the plasma membrane, altering its update and inducing cell death, with high selectivity for cancer cells [12,13]. The phospholipid 2-aminoethyl dihydrogen phosphate (2-AEH 2 P), present in high intracellular concentrations in various tissues, is related to osmoregulation, neuromodulation and membrane stabilization [14,15]. In addition, ethanolamine-derived phospholipids are present in the cell membrane as an important structural component, performing regulatory functions in cell division, signaling, autophagy and phagocytosis [16].
Studies carried out by our group on murine B16-F10 melanoma cells with 2-AEH 2 P have demonstrated their antiproliferative effects and cell cycle block in S or G2/M phases [17]. The treatment caused antiproliferative effects reducing the Cyclin D1 mRNA as well as the transcription of the vascular endothelial growth factor receptor 1 (VEGFR1) gene and the expression of the VEGFR1 receptor [18]. In acute promyelocytic leukemia cell lines, after treatment with 2-AEH 2 P, there was a decrease in the expression CD177 + and Gr-17+ in immature myeloid cells bone marrow, spleen and liver [19]. The action of 2-AEH 2 P in the mitochondrial intrinsic pathway, promoted apoptotic effects in MCF-7 breast cancer cells, leading to an interruption followed by the release of cytochrome c. The 2-AEH 2 P induced apoptosis independent of Caspase-3 and stopped the cell cycle in phase G1 and decreased expression of cyclin D1 [20].
In the global scenario, speaking of the incidence of breast cancer, as well as resistance and low responses to therapies and non-specific treatments, it is necessary to search for new therapeutic strategies. In the study, therefore, we sought to evaluate the cytotoxicity of 2-AEH 2 P and its therapeutic effects, as well as the modulation of the pro-apoptotic pathway in murine 4T1 breast adenocarcinoma cells.

Cell Culture
The 4T1 line obtained from the American Type Culture Collection (ATCC® CRL-2539 ™, Rockville, MD, USA) and FN1 human fibroblast cells obtained from patients undergoing blepharoplasty normal dermatological surgery procedures, which were part of the FMUSP project 921/06, was grown in RPMI medium (LGC Biotecnologia, Cotia, SP, Brazil). The medium was supplemented with 2 mM l-glutamine (Cultilab, Campinas, SP, Brazil), 10 mM HEPES (Cultilab, Campinas, SP, Brazil), 24 mM sodium bicarbonate, 0.01% antibiotics and 10% of fetal bovine serum (Cultilab, Campinas, SP, Brazil). The cells were grown in 5% ambient CO 2 at 37 °C as monolayer cultures. The cells were checked for viability using the trypan blue exclusion test.

Cytotoxicity Assay by the MTT Method
Tumor cells 4T1 (10 5 cells/well) were seeded in 6-well plates and incubated for 24 and 48 h and were treated with concentrations of 2-AEH 2 P ranging from The Antiproliferative and Pro-apoptotic Role of 2-aminoethyl dihydrogen Phosphate in Triple-negative  Breast Tumor Cells   162 10 to 100 mM, Paclitaxel in concentrations from 0.5 to 8, 5 μM and Paclitaxel in combination with 2-AEH 2 P in concentration at 40 mM. The absorbance quantification was performed in an ELISA reader, and the inhibitory concentration of 50% (IC 50% ) of 2-AEH 2 P was determined from the dose-response curve.

Analysis of the Potential of Mitochondrial Membrane (ΔѰm)
The 4T1 tumor cells (10 5 cells/well) were seeded in 6-well plates and incubated for 24 h and treated with concentrations of 20, 40, 60 mM of 2-AEH 2 P. After 24 h, the cells were harvested and washed once with phosphate-buffered saline (PBS) and stained with Rhodamine-123 (Rho 123) for 15 min at 37 º C. Then, the cells were washed once with PBS and maintained at 4 °C during measurements. The mitochondrial membrane potential (ΔѰm) was measured by flow cytometry with FACScalibur (Becton Dickinson). A total of 10,000 cells/samples were analyzed and the average fluorescence intensity recorded.

Evaluation of Morphological Changes in the Cytoskeleton by Laser Confocal Microscopy
The 4T1 tumor cells were plated in 24 wells (10 5 cells/well) at a concentration of 20, 40 and 60 mM of 2-AEH 2 P, for 24 h. After the treatment period, the supernatant was removed and the cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Then, the cells were washed with PBS 3 times for 5 min each. The cell membrane was permeabilized with 0.1% Triton X-100 at room temperature for 15 min. The non-specific binding sites were blocked with PBS+1% BSA + 0.1% (100 µg/mL) at 37 °C for 40 min. After blocking, the cells were incubated with the combined FITC-Phalloidin (2 µg/mL) (Sigma Aldrich Code P5282) for 1 h. The cells were then washed with PBS 3 times for 5 minutes each and incubated with propidium iodide (PI) (20 µg/ml) for 5 min. Following the same cell culture above and the same concentrations, the cells were marked by rhodamine-123 (Molecular Probes, USA), for 15 min at 37 °C. After incubation, excess rhodamine-123 was removed and the cells washed in culture medium. After labeling the fluorochrome, the cells were washed 3 times with PBS for 5 min each under a confocal laser microscope (Carl Zeiss LSM 700; Leica, Mannheim, Germany).

Analysis of the Cell Cycle Phases
The 4T1 tumor cells were treated with 20, 40 and 60 mM of 2-AEH 2 P at a density of 1 x 10 6 per well for 24 h. The cultured cells were collected and fixed with 70% ethanol/20 mg/mL RNase (Sigma), and stored at -20 º C for 24 h. The samples were centrifuged and resuspended in 200 µL of Fac"s buffer, 20 µL of Triton X-100 (Sigma-Aldrich), and 50 µg/mL of propidium iodide (Sigma-Aldrich) maintained for 30 min at room temperature. The DNA content of each sample (10,000 events) was quantified for by flow cytometry, the data were analyzed by CellQuest software (Becton Dickinson) and the data were analyzed by ModFit program.

Apoptosis and Necrosis Detection Test
For the detection and evaluation of apoptosis, cells were seeded in 6-well culture plates and treated with 20, 40 and 60 mM 2-AEH 2 P within 24 h. Cells were collected, washed and resuspended in 100 μL PBS, followed by incubation. Then, the cells were stained with the apoptosis detection kit Anexina V-FITC/PI (BD Bioscience) and incubated in the dark at room temperature for 15 min. After incubation, 400 µL binding buffer was added and the cells were analyzed by flow cytometry (FACScalibur, Becton Dickinson) using the CellQuest software to determine the percentage of apoptotic cells with at least 10,000 events per sample.

Expression of Markers by Flow Cytometry
The 4T1 tumor cells treated with 2-AEH 2 P and the The Antiproliferative and Pro-apoptotic Role of 2-aminoethyl dihydrogen Phosphate in Triple-negative Breast Tumor Cells 163 control groups were incubated with 1 µg of the specific antibodies involved in cell death for 1 h at 4 °C, using cell cycle progression regulators (cytocrome-c, Bax, Bad, Bcl-2, p53, Ki-67, caspase-3, caspase-8 and TNF-DR4). After this period, the cells were centrifuged at 1500 rpm and washed twice with ice-cold PBS and 0.2% BSA. The supernatant was discarded and the pellet was resuspended in 200 µl of Facs buffer containing 0.1% paraformaldehyde. Read and analysis the expression of receptors on the surface of tumor cells was performed in a flow cytometer FACSCalibur (BD) at FL-1 intensity, and histograms were obtained and analyzed in Cell-Quest (BD). A least 10,000 events were purchased for each sample.

Synergy Finder 2.0 Analysis of Multiple Drug Combinations
The 4T1 adenocarcinoma cells were treated with phospholipid 2-AEH 2 P and chemotherapy paclitaxel at 24 h and 48 h, respectively, to determine the synergistic effect between the drugs. SynergyFinder 2.0 software quantified the degree of synergy of a single drug"s doubling effect as if they were acting independently (Bliss). The following higher-order formulations are used to quantify the multi-drug combination effect between 2 drugs:

2-AEH 2 P Has Cytotoxic Activity in 4T1 Triple-Negative Breast Tumor Cells
In order to investigate the cytotoxic effects of 2-aminoethyl dihydrogen phosphate (2-AEH 2 P) in 4T1 tumor cells, a MTT colorimetric assay was performed in the periods of 24 and 48 h. In 4T1 tumor cells, 2-AEH 2 P produces cytotoxic effects, with IC 50% values of 17.  Table 1).
After 24 h treatment, 4T1 tumor cells showed significant morphological changes with the presence of cells in abnormal cell division containing aneuploidy. After the treatment of 2-AEH 2 P associated with Paclitaxel, groupings of dead and detached cells from the substrate and spherical morphology were observed, as well as the formation of apoptotic bodies. Thus, the cytotoxic effect of 2-AEH 2 P on 4T1 tumor cells has been proven and, in association with Paclitaxel, have a marked cytotoxic effect ( Figure 1A).

Evaluation of Mitochondrial Electrical Potential and Cytoskeleton Rearrangement
The changes in the mitochondrial membrane

The Interference of 2-AEH 2 P in the Cell Cycle in 4T1 Triple-negative Breast Tumor Cells
It was possible to observe significant changes in the phases of the cell cycle, when compared to the control group, in 12 h and 24 h, after the treatment of 2-AEH 2 P. In the 12 h treatment period, the lowest concentration caused a reduction in the cell population in all phases of the cell cycle and, in 24 h, an abrupt decrease in the G2/M phase, in the highest concentration. There was also a notable increase in DNA fragmentation after treatment with 2-AEH 2 P, which was potentiated in the treatment of 40 mM, which indicates a higher concentration of dead cells, due to necrosis and apoptosis ( Figure 3A and 3B).

2-AEH 2 P Induced 4T1 Triple-negative Breast Tumor Cells to Cell Death by Apoptosis
The 2-AEH 2 P monophosphoester induces a significant reduction in the number of viable cells in all treatment groups compared to control groups. After treatment, the cell population in both initial and late apoptosis increased significantly, at all concentrations. Treatment with 2-AEH 2 P increased the population of cells in late apoptosis and, in the concentration above 40 mM, there was a decrease in cell density,and increase in necrosis cells when compared to the control group (Figure 4).

The Reduction of Cell Proliferation by Modulation of Pro-apoptotic Proteins due to the Effects Caused by 2-AEH 2 P
We investigated whether caspase 3-8 and the death receptor (DR4) may be involved in monophosphoester-mediated apoptosis 2-AEH 2 P. Thus, it was possible to observe a significant increase in these proteins in 4T1 cells treated with 2-AEH 2 P and, mainly, in the concentration of 40 mM ( Figure  5A). The pro-apoptotic proteins of the Bcl-2, Bax and Bad family, which are also involved in the permeability of the mitochondrial membrane, were also evaluated. The pro-apoptotic caspases 3 and 8 had a significant increase, while the anti-apoptotic protein (Bcl-2) was suppressed, when compared to the control group, and even more accentuated in the concentration of 40 mM ( Figure 5B).
The expression of cytochrome c was also analyzed and it was evident that 2-AEH 2 P induced the release of cytochrome c, after 24 h of treatment, a mechanism that occurs during cell death by apoptosis by the intrinsic route. The expression of p53 was also analyzed, which showed an increase mainly in the 40 mM concentration of 2-AEH 2 P. When analyzing KI-67, it was evident that associative treatment decreased the proliferation of tumor cells ( Figure 5C).

The Association of 2-AEH 2 P with Paclitaxel Potentiated the Antiproliferative Effects
In order to evaluate the associative effects between

Discussion
Antineoplastic phospholipids emerge as a new antitumor strategy that acts directly on the plasma membrane, altering its renewal and, consequently, inducing cell death [12,13]. Therefore, the monophosphoester 2-AEH 2 P was shown to be effective in the ability to inhibit the proliferation of  [19,20].
Due to the rapid cell division and high proliferation rate, tumor cells have a greater energy requirement than normal cells [21]. Thus, the tumor cells alter their metabolism and these changes try to supply the constant need for nutrients and energy [22]. Therefore, since mitochondria serve as the "potency" of cells, they can be successful targets for tumor cell therapies [22]. These organelles are dynamic and play a central role in the apoptotic process, therefore, the decrease in mitochondrial potential (ΔѰm) is known as a trigger for cell death due to apoptosis and this mechanism may be associated with the intrinsic apoptotic signaling pathway [23]. Analyzing the ΔѰm of Ehrlich tumor cells, it showed that the effects of 2-AEH 2 P are attributed to its ability to induce apoptosis by reducing ΔѰm [24]. These data corroborate the results obtained in this work, since 2-AEH 2 P induced a similar mechanism in 4T1 tumor cells, reducing ΔѰm, which may be a key event to trigger signals that will result in cell death due to apoptosis. The reduction in mitochondrial electrical potential and the increase in pro-apoptotic proteins, linked to membrane permeability are related to the release of cytochrome c by mitochondria [25]. It was possible to observe the increased expression of cytochrome c in 4T1, as well as in other tumor lines after treatment with 2-AEH 2 P, reinforcing its role in modulating the intrinsic pathway of apoptosis [19,20,[26][27][28].
Most of these apoptotic events are modulated by caspases, which are divided into initiators (8, 9 and 12) and executors (3, 6 and 7), the latter being responsible for morphological changes in the nucleus and cytoskeleton [29]. The 2-AEH 2 P increased the expression of caspases 3 and 8 in 4T tumor cells. The activation of different caspases can initiate or amplify the apoptosis processes, depending on the mitochondrial pathway or independent caspases [30]. Results also obtained in other studies corroborate that 2-AEH 2 P modulates the intrinsic pathway of apoptosis in response to the activation of caspase 8 [17,31].
The Bcl-2 protein exhibits an anti-apoptotic effect and Bax and Bad work in the opposite way, being able to induce cell death, therefore being pro-apoptotic proteins [25]. Our results show an increase in Bad and Bax expression and a reduction in Bcl-2 in 4T1 tumor cells after treatment with 2-AEH 2 P. These data obtained are convergent to those reported in the literature [32] where 2-AEH 2 P increased the expression of Bax and Bad and had a negative regulation of Bcl-2 in human hepatocarcinoma [33] and human breast adenocarcinoma MCF-7 [27].
The p53 protein regulates the S transition to the G2/M phase of the cell cycle, which involves the regulation of cell division cycle 2 (Cdc2 kinase), essential for cells to enter mitosis [32]. Thus, our results showed an increase in p53 expression in the treatment with 2-AEH 2 P and similar data were found in squamous cell tumor lines of the oral cavity squamous cell carcinoma (SCC9 and SCC25) [26] and in human breast tumor lines [27] when treated with 2-AEH 2 P.
We analyzed the percentage of distribution of tumor populations in the phases of the cell cycle after treatment with 2-AEH 2 P and the data show an increase in fragmented DNA and a significant decrease in all other phases of the cycle. The increase in the population of cells in the G2/M phase was also observed, when treated with 2-AEH 2 P, in murine melanoma cells B16-F10 and in breast cancer MCF-7 [17,20].
The 2-AEH 2 P induced cell death by apoptosis and not by necrosis, which was confirmed by the Annexin/PI assay. In this study, 2-AEH 2 P increased the percentage of cells in late apoptosis compared to the treated control group. This same compound was tested in other tumor cell lines and obtained results similar to those observed in this work [17,19,28]. Therefore, the data indicate that 2-AEH 2 P modulates the cell cycle of 4T1 tumor cells by altering the cell proliferation process that is so intense in these tumor cell types.
The cytotoxic effect of 2-AEH 2 P associated with the chemotherapy drug Paclitaxel was evidenced, an increase in the antiproliferative potential. The effect of this association was clearly evident in changes in cell morphology, which showed a marked cytoplasmic retraction and regression of the cytoskeleton. Paclitaxel inhibits cell growth and stabilizes the polymerization of microtubules, preventing the cell from replicating and remaining in the G2 phase of the cell cycle [34,35].
The association with Paclitaxel was considered a potent antineoplastic agent used in several types of tumors, such as pancreas, breast, stomach, ovary and lung cancer [36,37]. This chemotherapy promotes the formation of exceptionally stable microtubules, inhibiting the normal dynamics of the reorganization of the network formed by microtubules necessary for mitosis and cell proliferation [38]. In addition, Paclitaxel induces the accumulation of cells in the G2/M phases of the cell cycle and leads to apoptosis in sensitive cells [39,40]. It was observed, in MCF-7 breast cancer cells, treated with this drug, the presence of apoptotic bodies in 24 h of treatment and peaks in 48-72 h [41,42].

Conclusions
The 4T1 triple-negative breast tumor cell, after treatment with the 2-aminoethyl dihydrogen phosphate monophosphoester, showed selective cytotoxicity and significant morphological changes in cell death due to apoptosis. The association with the chemotherapy drug Paclitaxel, then, which had additive effects, may favor antitumor therapy and mitigate possible adverse effects. In addition, when the cells were treated with 2-AEH 2 P, there was positive regulation of proteins involved with the intrinsic pathway of cell death by apoptosis. There was an increase in populations of cells in late apoptosis, as well as structural and distribution changes in mitochondria and a decrease in cell density. In addition, 2-AEH 2 P decreased the proteins that regulate cell proliferation, increased fragmented DNA and altered the distribution of the cell cycle phases. Thus, the 2-AEH 2 P monophosphoester has been shown to have specificity in tumor cells and plays a modulatory role in apoptotic pathways, thus being a promising antitumor and antiproliferative agent.