Amaryllidaceae-Type Alkaloids from Pancratium maritimum: Apoptosis-Inducing Effect and Cell Cycle Arrest on Triple-Negative Breast Cancer Cells

Aiming to find Amaryllidaceae alkaloids against breast cancer, including the highly aggressive triple-negative breast cancer, the phytochemical study of Pancratium maritimum was carried out. Several Amaryllidaceae-type alkaloids, bearing scaffolds of the haemanthamine-, homolycorine-, lycorine-, galanthamine-, and tazettine-type were isolated (3–11), along with one alkamide (2) and a phenolic compound (1). The antiproliferative effect of compounds (1–11) was evaluated by the sulforhodamine B assay against triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, breast cancer cells MCF-7, and the non-malignant fibroblast (HFF-1) and breast (MCF12A) cell lines. The alkaloids 3, 5, 7, and 11 showed significant growth inhibitory effects against all breast cancer cell lines, with IC50 (half-maximal inhibitory concentration) values ranging from 0.73 to 16.3 µM. The homolycorine-type alkaloid 7 was selected for further investigation in MDA-MB-231 cells. In the annexin-V assay, compound 7 increased cell death by apoptosis, which was substantiated, in western blot analyses, by the increased expression of the pro-apoptotic protein Bax, and the decreased expression of the anti-apoptotic protein Bcl-xL. Consistently, it further stimulated mitochondrial reactive oxygen species (ROS) generation. The antiproliferative effect of compound 7 was also associated with G2/M cell cycle arrest, which was supported by an increase in the p21 protein expression levels. In MDA-MB-231 cells, compound 7 also exhibited synergistic effects with conventional chemotherapeutic drugs such as etoposide.


Introduction
Cancer is a major health concern worldwide, mainly attributed to drug resistance. Breast cancer is the most frequent cancer diagnosed among women [1]. Triple-negative breast cancer, which is well known for its therapeutic resistance, is an aggressive subtype of breast cancer with defective expression of estrogen and progesterone receptors and a lack of human epidermal growth factor receptor 2. Triple-negative breast cancer is generally associated with hereditary conditions, representing 10-15% of the newly diagnosed breast cancer [2,3]. The treatment of this type of breast cancer is mainly based on standard chemotherapy, including platinum drugs, and more recently on poly (ADP-ribose) polymerase (PARP) inhibitors. However, the use of these anticancer drugs has been limited by the occurrence of drug resistance, related to the complexity of the tumor microenvironment and heterogeneity, which is characterized by the interaction of multiple factors and signaling pathways [4,5]. Despite all efforts that have been developed in the discovery of new chemotherapeutic agents, targeting triple-negative breast cancer is still a great challenge, and there is a need for new anticancer compounds to overcome its high drug resistance [6].
To overcome multidrug resistance (MDR) in cancer, several approaches have been proposed, mainly focused on the development of molecules that can act on MDR mechanisms. The deregulation of apoptosis and cell cycle pathways are among the most frequent alterations in cancer cells responsible for drug resistance and cancer development [7]. Apoptosis is characterized by a series of biochemical events that play a key role in the regulation of many physiological and pathophysiological processes. In drug-resistant cancer cells, the mechanisms of apoptosis are suppressed, causing an imbalance between cell death and proliferation [8,9]. Moreover, changes in the cell cycle regulatory signaling pathways are commonly altered in cancer cells, which often lose their checkpoint mechanisms, contributing to cell proliferation. Therefore, targeting deficient cell checkpoints and apoptosis are considered promising strategies for improving anticancer drug development and overcoming MDR [7,10].
Natural products have been an important source for the discovery of new bioactive compounds with anticancer properties. The most known clinically used chemotherapeutic agents for cancer treatment are from natural sources [11,12].
Plants from the Amaryllidaceae family are characterized by containing a wide range of unique and structurally diverse bioactive Amaryllidaceae-type alkaloids. Some of them have shown great anticancer activity, mainly lycorine [13,14].
In our search for new anticancer compounds targeting multidrug resistance in cancer, e.g., [15][16][17][18][19][20][21][22][23], recently, our group has reported a derivative of a monoterpene indole alkaloid isolated from the African medicinal plant Tabernaemontana elegans [24] that is shown to be highly effective against breast and ovarian cancer cells [25]. Aiming at finding new anticancer compounds against breast cancer cells, this study was conducted to obtain bioactive alkaloids from Pancratium maritimum L. (Amaryllidaceae family), which led to the isolation of nine Amaryllidaceae-type alkaloids, along with two other compounds.

Antiproliferative Effect Evaluation
The antiproliferative effect of the isolated compounds (1-11) was investigated by the sulforhodamine B assay against human triple-negative breast cancer cell lines (MDA-MB-468 and MDA-MB-231) and breast ductal carcinoma cells (MCF-7) and the non-tumorigenic fibroblast (HFF-1) and breast cell lines (MCF12A). The IC 50 values were determined after 48 h of treatment (Table 1).
Lycorine ( Similarly, a pronounced antiproliferative activity was also found for compound 7 against MDA-MB-231 cells (IC 50 of 8.02 µM) and MCF-7 (IC 50 of 6.8 µM), whereas a higher IC 50 value of 16. A total of 3 µM was obtained in MDA-MB-468 cells. Interestingly, compound 7 displayed much higher activity than its structurally related compounds, 4 (IC 50 of 24.70 µM) and 9 (IC 50 > 40), suggesting that the higher number of free hydroxyl groups found in compound 7 may explain this different activity between the three compounds.
In addition, the antiproliferative effect of the compounds was evaluated on the nonmalignant fibroblast (HFF-1) and breast (MCF12A) cell lines. When comparing the IC 50 values obtained in HFF-1 cells with those obtained in cancer cell lines, a slight selectivity (SI) was found for compound 7 in MDA-MB-231 (SI of 1.65) and MCF-7 (SI of 2.37) cancer cells. Conversely, compounds 5 and 11 exhibited slight selectivity in all breast cancer cell lines in relation to MCF12A cells, particularly compound 5 against MDA-MB-231 cells (SI of 3.26) and both 5 and 11 in MCF-7 cells (SI of over 4).
In previous studies, compounds 3, 5, and 11 have shown antiproliferative activity against MDA-MB-231 breast cancer cells, which is in agreement with our results [30].     . Data are mean ± SEM of four to six independent experiments. b SI: Selectivity index; SI < 1 values denote lack of selectivity, 1 < SI < 3 mean slight selectivity and are marked with bold and italics; 3 < SI < 6 values indicate moderate selectivity and are highlighted in bold, whereas values of SI > 6 indicate that compounds are strongly selective [31]. Etoposide was used as a positive control.

Analysis of Cell Cycle, Apoptosis, and Mitochondrial ROS Generation
Compound 7, the antiproliferative effect of which has not been investigated yet, was selected for further studies in the triple-negative breast cancer cell line MDA-MB-231.
To explore the ability of compound 7 to regulate cell cycle progression, the percentage of cells in different phases was analyzed after 24 h of exposure. As shown in Figure 2B, a two-fold IC 50 concentration of compound 7 (16 µM) induced a significant accumulation of cells in the G2/M phase compared to the vehicle control cells. Importantly, at 16 µM, compound 7 did not significantly interfere with cell cycle, cell death, and mitochondrial ROS generation in HFF-1 cells. Immunoblots are representative of three independent experiments. GAPDH was used as a loading control. (F) The graph represents the quantification of protein expression levels relative to DMSO; data are mean ± SD of three independent experiments. The ability of compound 7 to induce apoptosis in this cell line was also assessed by cytometry, using the Annexin V-FITC assay after 48 h of treatment. Apoptosis is characterized by distinct morphologic features. In the early stages of apoptosis, the membrane phospholipid phosphatidylserine is translocated from the inner to the outer plasma membrane leaflet. Annexin V is a phospholipid-binding protein with a high affinity to phosphatidylserine, which is able to detect apoptotic cells with the externalization of phosphatidylserine [32]. The results showed a significant increase in the percentage of apoptotic cells in MDA-MB-231 cells compared to the vehicle control after treatment with 16 µM of compound 7 ( Figure 2C). In line with this, it was further observed that 16 µM of compound 7 increased ROS production in MDA-MB-231 cells ( Figure 2D). Immunoblots are representative of three independent experiments. GAPDH was used as a loading control. (F) The graph represents the quantification of protein expression levels relative to DMSO; data are mean ± SD of three independent experiments. The ability of compound 7 to induce apoptosis in this cell line was also assessed by cytometry, using the Annexin V-FITC assay after 48 h of treatment. Apoptosis is characterized by distinct morphologic features. In the early stages of apoptosis, the membrane phospholipid phosphatidylserine is translocated from the inner to the outer plasma membrane leaflet. Annexin V is a phospholipid-binding protein with a high affinity to phosphatidylserine, which is able to detect apoptotic cells with the externalization of phosphatidylserine [32]. The results showed a significant increase in the percentage of apoptotic cells in MDA-MB-231 cells compared to the vehicle control after treatment with 16 µM of compound 7 ( Figure 2C). In line with this, it was further observed that 16 µM of compound 7 increased ROS production in MDA-MB-231 cells ( Figure 2D).

Western Blot Analyses
Once compound 7 induced cancer cell death by apoptosis, we next explored the molecular pathways involved in its apoptotic effect. For that, some apoptotic-related proteins were checked in MDA-MB-231 cells treated with 16 µM of compound 7 for 48 h ( Figure 2E,F). The results showed increased levels of the pro-apoptotic protein Bax and reduced levels of the anti-apoptotic Bcl-xL, thus substantiating the data obtained in the Annexin V-FITC assay. Nonetheless, the compound did not interfere with the p53 protein expression levels (data not shown).
Moreover, in accordance with induction of a G2/M cycle arrest, compound 7 also increased the p21 protein levels.

Combination Therapy
To evaluate the ability of compound 7 of promoting the anticancer effect of conventional chemotherapeutic drugs, MDA-MB-231 cells were treated with a single concentration of compound 7 (at 1.5 µM; concentration with no significant effect on cancer cell growth) in combination with a DNA-damaging agent, such as etoposide. The results showed that 1.5 µM of compound 7 significantly increased the growth inhibitory effect of etoposide at the concentrations of 0.8, 1.5, and 3 µM (Figure 3). A multiple drug-effect analysis was carried out for each combination, with the calculation of the combination index (CI), which revealed synergistic effects between compound 7 and 0.8, 1.5, and 3 µM of etoposide (Table 2). Of note, for the same experimental conditions used in MDA-MB-231 cells, no synergistic effects were observed in HFF-1 cells (Table 2).

Western Blot Analyses
Once compound 7 induced cancer cell death by apoptosis, we next explored the molecular pathways involved in its apoptotic effect. For that, some apoptotic-related proteins were checked in MDA-MB-231 cells treated with 16 µM of compound 7 for 48 h ( Figure 2E,F). The results showed increased levels of the pro-apoptotic protein Bax and reduced levels of the anti-apoptotic Bcl-xL, thus substantiating the data obtained in the Annexin V-FITC assay. Nonetheless, the compound did not interfere with the p53 protein expression levels (data not shown).
Moreover, in accordance with induction of a G2/M cycle arrest, compound 7 also increased the p21 protein levels.

Combination Therapy
To evaluate the ability of compound 7 of promoting the anticancer effect of conventional chemotherapeutic drugs, MDA-MB-231 cells were treated with a single concentration of compound 7 (at 1.5 µM; concentration with no significant effect on cancer cell growth) in combination with a DNA-damaging agent, such as etoposide. The results showed that 1.5 µM of compound 7 significantly increased the growth inhibitory effect of etoposide at the concentrations of 0.8, 1.5, and 3 µM (Figure 3). A multiple drug-effect analysis was carried out for each combination, with the calculation of the combination index (CI), which revealed synergistic effects between compound 7 and 0.8, 1.5, and 3 µM of etoposide (Table 2). Of note, for the same experimental conditions used in MDA-MB-231 cells, no synergistic effects were observed in HFF-1 cells (Table 2).    The effect of 1.5 µM of compound 7 in combination with a range of concentrations of etoposide was evaluated after 48 h of treatment using CompuSyn software to calculate the combination index (CI) values for each combined treatment: CI < 1, synergy; 1 < CI < 1.1, additive effect; CI > 1.1, antagonism.

General Experimental Procedure
The NMR spectra were recorded on a Bruker 300 Ultra-Shield instrument ( 1 H 300 MHz, 13 C 75 MHz). 1 H and 13 C chemical shifts are expressed in δ (ppm) referenced to the solvent used and the proton coupling constants J are in hertz (Hz). Optical rotations were performed using a PerkinElmer 241 polarimeter, with quartz cells of 1 dm path length. The infrared spectra were collected on an Affinity-1 (Shimadzu, Kyoto, Japan) FTIR spectrophotometer. Low-resolution mass spectrometry was conducted with a Triple Quadrupole mass spectrometer Waters AcquityTM (Waters ® , Ireland). Column chromatography (CC) was performed on silica gel (Merck 9385, Darmstadt, Germany) or Combiflash system (teledyne-Isco; Lincolm, NE, USA), using SiO 2 or C 18 prepacked columns. Analytical thin-layer chromatography (TLC) was performed on pre-coated silica gel 60 F 254 and RP-18 F 254 plates (Merck 105,554 and 105,560, respectively), with visualization under UV light (λ 254 and 366 nm), and by spraying either with Dragendorff s reagent or a solution of H 2 SO 4 -MeOH (1:1), followed by heating.

Plant Material
The whole plant of Pancratium maritimum was collected in August 2018, during the flowering period, at Cabo Raso, Cascais, Portugal. The plant was identified by Dr. Teresa Vasconcelos (plant taxonomist) of Instituto Superior de Agronomia, Technical University of Lisbon, Portugal. A voucher specimen (no. LISI044123) has been deposited at the herbarium João de Carvalho Vasconcellos of Instituto Superior de Agronomia.

Extraction and Isolation
The fresh bulbs (14.8 kg) of P. maritimum were sliced, dried at 40 • C for 48 h, and then minced. The resulting powder (8.19 kg) was extracted with MeOH (11 × 30 L). Afterward, the crude methanol extract (1.034 kg) was suspended in a solution of HCl 2% (v/v; 2.0 L), and the neutral material was removed with Et 2 O (3 × 1 L). The pH of the aqueous phase was adjusted to 5 by the addition of dilute NH 4 OH and successively extracted with CH 2 Cl 2 (3 × 500 mL), EtOAc (4 × 1 L), and n-butanol (4 × 1 L), yielding, after drying over anhydrous Na 2 SO 4 and evaporating the solvents under reduced pressure, the CH 2 Cl 2pH5 (13.9 g) and EtOAc pH5 (20 g) and the n-butanol pH5 (109 g)-soluble fractions. Then, the pH of the aqueous phase was adjusted to 9, by adding NH 4 OH, and extracted again with EtOAc (3 × 1 L) and n-butanol (4 × 1 L), originating two new alkaloid fractions after the above procedure (EtOAc pH9 10.3 g; and n-butanol pH9 32.2 g) (see Figure S1 and Table S1, Supplementary Materials).
The residue of the powdered bulbs was further extracted with MeOH using a Soxhlet apparatus (500 g each dose for 10 h). The resulting MeOH extract (168 g) was submitted to acid-base extraction, as described above, yielding the EtOAc pH9 (3.5 g) and n-butanol pH9 (4.5 g)-soluble fractions (see Table S1, Supplementary Materials).

Human Cell Lines and Growth Conditions
Human breast adenocarcinoma MDA-MB-231, MDA-MB-468, and MCF-7 cell lines and non-tumorigenic foreskin fibroblast (HFF-1) and breast (MCF12A) cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). All cancer cell lines, except MCF12A, were routinely cultured in RPMI-1640 medium with glutamine from Corning (VWR, Carnaxide, Portugal) supplemented with 10% fetal bovine serum (FBS) from Biowest (Labclinics, Barcelona, Spain). MCF12A cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium, 20 ng/mL human epidermal growth factor, 100 ng/mL cholera toxin, 0.01 mg/mL bovine insulin, and 500 ng/mL hydrocortisone and supplemented with 10% FBS. All cells were maintained in a humidified incubator at 37 • C with 5% CO 2 . All cells were routinely tested for mycoplasma contamination using the MycoAlertTM PLUS detection kit (Lonza, Basel, Switzerland).

Sulforhodamine B Assay
Human cell lines were seeded in 96-well plates at a density of 5.0 × 10 3 (MDA-MB-231, MCF-7, HFF-1, and MCF12A) and 7.5 × 10 3 (MDA-MB-468) cells/well and allowed to adhere for 24 h. Cells were treated with serial dilutions of compounds (ranging from 0.1 to 40 µM) for an additional 48 h. Effect on cell proliferation was measured by sulforhodamine B assay, as described in [33], and the half-maximal inhibitory concentration (IC 50 ) was then determined for each cell line using the GraphPad Prism software version 7.0 (La Jolla, CA, USA).

Cell Cycle and Apoptosis Analysis
The analyses were performed basically as described in [33]. Briefly, MDA-MB-231 cells were seeded in 6-well plates at a density of 1.5× 10 5 cells/well for 24 h, followed by treatment with 16 µM of compound 7 for an additional 48 h. For cell cycle analysis, cells were stained with propidium iodide (PI; Sigma-Aldrich, St. Louis, MO, USA). The cells were then analyzed by flow cytometry, and cell cycle phases were identified and quantified using the FlowJo × 10.0.7 Software (Treestar, Ashland, OR, USA). For apoptosis, cells were stained using the Annexin V-FITC Apoptosis Detection Kit I from BD Biosciences (Enzifarma, Porto, Portugal), according to the manufacturer's instructions. The AccuriTM C6 flow cytometer and the BD Accuri C6 software (BD Biosciences) were used.

Mitochondrial Reactive Oxygen Species (ROS) Generation
For mitochondrial ROS generation assessment, MDA-MB-231 cells were seeded in 6-well plates at a density of 1.5 × 10 5 cells/well for 24 h, followed by treatment with 16 µM of compound 7 for an additional 24 h. Thereafter, cells were incubated with 3 µM MitoSOX (Thermo Fisher Scientific, Invitrogen, Portugal) for 30 min at 37 • C and analyzed by flow cytometry.

Western Blot Analysis
MDA-MB-231 cells were seeded in 6-well plates at a density of 1.5 × 10 5 cells/well for 24 h, followed by treatment with 16 µM of compound 7. Protein extracts were quantified using the Bradford reagent (Sigma-Aldrich). Proteins were run in SDS-PAGE and transferred to a Whatman nitrocellulose membrane from Protan (VWR, Carnaxide, Portugal). After blocking, proteins were identified using specific primary antibodies: rabbit polyclonal anti-p21 (Santa Cruz Biotechnology, Dallas, DX, USA), rabbit polyclonal anti-Bax, and rabbit monoclonal anti-Bcl-xL (Invitrogen, Waltham, MA, USA), followed by the respective anti-mouse/anti-rabbit HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). GAPDH was used as a loading control. The signal was detected with the ECL Amersham kit from GE Healthcare (VWR, Carnaxide, Portugal). Two detection methods were used: the Kodak GBX developer and fixer (Sigma-Aldrich) or the ChemiDoc™ XRS Imaging System from Bio-Rad Laboratories (Amadora, Portugal).

Combination Therapy Assays
To assess the synergistic effect of compound 7 with the conventional chemotherapeutic drug etoposide, MDA-MB-231 cells were treated with compound 7 at 1.5 µM (concentration that did not interfere with the growth of cancer cells) and/or increasing concentrations of etoposide, for 48 h. The effect of combined treatments on cell proliferation was analyzed by SRB assay. For each combination, the combination index (CI) values were calculated using the CompuSyn Software version 1.0; CI values < 1, 1 < CI < 1.1, and >1.1 indicate synergistic, additive, and antagonistic effects, respectively [34].

Statistical Analysis
The data were analyzed statistically using the GraphPad Prism (La Jolla, CA, USA; version 8.0.2) software for Microsoft Windows 10. For the comparison of the two groups, the unpaired Student's t-test was used. For the comparison of multiple groups, the two-way ANOVA followed by Sidak's test was used. Statistical significance was set as * p < 0.05.

Conclusions
To summarize, among the nine Amaryllidaceae-type alkaloids isolated from P. maritimum, bearing different scaffolds, the most significant growth inhibitory activity against different human breast cancer cell lines was found for compounds 3, 5, 7, and 11. When comparing the IC 50 values obtained for compound 7 with those of 4 and 9, sharing the same homolycorine-type scaffold, the higher number of hydroxyl groups in compound 7 seems to be associated with its higher activity. Further investigation of the mechanism of action of compound 7 against the triple-negative human breast cancer MDA-MB-231 cell line revealed that its antiproliferative effect was associated with the induction of apoptosis and cell cycle arrest through a p53-independent pathway. Interestingly, compound 7 seems to interfere with the expression levels of proteins of the Bcl-2 family, namely increasing Bax and reducing Bcl-xL, which may indicate a potential involvement of the mitochondrial pathway in its antitumor activity. In fact, an increase in mitochondrial ROS production was also observed upon compound 7 treatment. The results obtained in this work also revealed that triple-negative breast cancer therapy might greatly benefit from the combination of etoposide with compound 7.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27185759/s1, Figure S1: Extraction and fractionation of the methanol extract of P. maritium and the isolated compounds (1-11); Table S1: Fractions obtained from acid-base extraction; Figure S2- Figure S17: spectroscopic data of the isolated compounds; NMR data of some compounds.