All cell lines (HeLa, HepG2, A549, A431, MCF-7, PCS-600-010, MDA-MD-231, MCF-7, and SK-MEL-28) were purchased from American Type Culture Collection (ATCC, Manassas, VA). Dulbecco’s modified Eagles medium-high glucose (DMEM), penicillin/streptomycin, and L-glutamine were purchased from Mediatech, Inc. (Manassas, VA). Unless otherwise indicated, all antibodies were acquired from Cell Signaling Technology (Boston, MA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were obtained from Sigma-Aldrich (St. Louis, MO). All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). IGF-1 was purchased from Cell Signaling Technology (Beverly, MA). Inhibitors (i.e., Wortmannin, PF-3758309, FR-180204) were obtained from Cayman Chemical. ISIS2503 oligonucleotide inhibitor was prepared as previously described (Chen et al., 1996). Other chemicals were obtained from Sigma unless otherwise stated.

A. montana leaves were collected from the Redland region (Possum Trot Tropical Farm) in Miami, Florida, USA, by Dr. M.A. Barbieri and Mr. R. Barnum. Our botanist verified the taxonomic identity at the herbarium of the Department of Biological Sciences at Florida International University (Voucher: FTG182701, Fairchild Herbarium). The leaves of A. montana were separated from one individual plant and cleaned using running tap water. Then, the leaves were oven-dried in a convection oven at 60°C for 32 h. Dried leaves (2000 mg) were cut into small pieces and extracted with methanol (100 mL) in a 250 mL round bottom flask connected to a reflux condenser heated at 65°C for 30 min. The plant materials were filtered and washed twice with methanol (25 mL). All materials (extract and washes) were collected and pooled. Evaporation of the methanol extract by rotavapor/desiccation yielded a residue of 1180 mg for A. montana methanol extract (AmMe). Then each methanol extract was either dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 100 mg/mL plant extract or partitioned with hexane, ethyl acetate, and water, respectively, as described early (Chin et al., 2014). An aliquot (518 mg A. montana methanol extract) of the residue from the methanol extraction was partitioned between methanol and hexane. The hexane was removed from the methanolic phase and treated as previously described (Priestap et al., 2014). Finally, the residue was partitioned between water and ethyl acetate. All fractions collected (i.e., hexane-AmH-, ethyl acetate-Amea- and water-Amw-) were then individually evaporated and concentrated by rotorvapor/desiccation as previously described (Priestap et al., 2014). The hexane, ethyl acetate, and water extracts yielded 223, 102, and 95 mg of residue for A. montana, representing 82% of the initial methanol extract. A pool of several hexane fractions was collected (1000 mg), and was further fractionated using gravity column chromatography with hexane-ethyl acetate to increase polarity up to 90% ethyl acetate, then 100% ethyl acetate yielded twelve sub-fractions of increasing polarity (F1-F12). Each sub-fraction was dried, dissolved in DMSO, and tested for activity cell proliferation, cell cycle, invasion, colony formation, and Western blotting analysis.

GC/MS determinations were carried out in a Hewlett Packard model 6890 instrument coupled to a Q-Mass 910 quadrupole selective detector (Perkin-Elmer) as previously described (Chin et al., 2014; Priestap et al., 2014). GC/FID determinations were performed on a Trace GC Ultra apparatus (Thermo Electron Corporation), and the output was recorded using a ChromQuest version 4.1 data system. Analyses were performed on capillary columns DB-5MS at the abovementioned conditions (Adams, 2007; Araujo et al., 2007; Babushok et al., 2011; Yan et al., 2013).

All cell lines were cultured according to standard mammalian tissue culture protocols and sterile technique as indicated by ATCC. Cell lines were cultured for less than 2 months, splitting them twice a week in a humidified incubator with 5% CO₂ at 37°C. Hela, SK-MEL-28, A431, A549, HepG2, MDA-MB-231, and MFC-7 cells were grown in DMEM (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin, and 50 μg/mL streptomycin. MCF-10A cells were grown in DMEM supplemented with 10% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, 10 ng/mL human epidermal growth factor (hEGF), 100 ng/mL cholera toxins, 5 μg/mL bovine insulin and 500 ng/mL hydrocortisone. PCS-600-010 cells were grown in mammary epithelial cell basal medium supplemented with 10% FBS, 50 U/mL penicillin, 50 μg/mL streptomycin, 100 ng/mL recombinant human transforming growth factor-alpha (rhTGF-α), 5 μg/mL Apotransferrin, 1 μM epinephrine, 5 μg/mL recombinant human insulin (rhI) and 100 ng/mL hydrocortisone. Cells were harvested at approximately 80% confluence and then plated for subsequent passage and treatments.

Cell cycle progression was determined by flow cytometry analysis (Alvarez et al., 2010). Cells were incubated in the absence or presence of A. montana extract for 24 h. A suspension of cells was fixed with methanol (70%) at 4°C, washed, and stained with propidium iodide (PI). Cells were analyzed with an Acurri-C6 Flow Cytometer (BD Biosciences, San Diego, CA), and cell cycle progression was measured with the Modfit LT program (Verity Software House, Topsham, ME).

The Annexin V/fluorescein isothiocyanate (FITC) assay was performed using Annexin V Kit (BD Pharmigen, USA) to analyze the potential of A. montana extract in causing apoptosis. (The Annexin V signal indicates cellular apoptosis, while PI staining indicates necrotic or late apoptosis) (Elmore et al., 2016). The cells were seeded in a 6-well plate at a concentration of 2.4 × 10⁵ cells/mL and incubated overnight. The next day, the seeded cells were treated with A. montana extract and incubated for 24, 48, and 72 h. The cells were harvested at a time point, and the resulting pellets were resuspended in a binding buffer. To stain the cell suspension, 25 μL of FITC Annexin V and 5 μL of PI were added and allowed to stand in a dark place at room temperature for 15 min. Afterward, the stained cells were analyzed by an Acurri-C6 Flow Cytometer (BD Biosciences, San Diego, CA).

Cells were seeded onto 6-well plates at a density of 1 × 10⁶ cells per well, incubated for one day, and then treated with A. montana extract for 24 h. The cells were collected and washed with 1 mL phosphate buffer saline (PBS). Cells were fixed with paraformaldehyde for 30 min at room temperature, washed, and then permeabilized by 0.2% Triton X-100 for 15 min. After incubation, cells were washed and resuspended in 1 mL of PBS containing TUNEL solution (de Felice et al., 2019) and incubated for 1 h at 37°C in the dark. Afterward, the stained cells were analyzed by an Acurri-C6 Flow Cytometer (BD Biosciences, San Diego, CA).

Cells (3.5 × 10⁵) were seeded in a 6-well plate and incubated overnight. To evaluate migration (non-directional migration) the next day, a straight wound line was drawn across the 100% confluent attached cell layer with pipette tips as previously described (Cappiello et al., 2018). Plates were washed to remove unattached cells with PBS and replaced with a fresh DMEM medium. A. montana extract was added to the wells, and images of the closure of the wound were recorded at 24 h using the inverted microscope equipped with a camera.

Cells were seeded in 96-well plates at 2 × 10³ cells/well density in 100 μL medium and incubated overnight. The next day, the seeded cells were treated with A. montana extract or subfraction and incubated for 8 h (for extract) or 24, 48, and 72 h (for subfraction). After the incubation, the MTT solution (5 mg/mL) was added to the plates, and the cells were incubated at 37°C for 6 h. The formazan, derived from MTT by living cells, was dissolved in 10% SDS (150 μL per well), and the absorbance was measured at OD₅₉₅ nm as described (Mosmann, 1983). All MTT experiments were performed in duplicate and repeated at least three times.

Cells (~70% confluent) were serum-starved for 24 h. Then, cells were seeded (2 × 10⁵ cells/mL) in the insert chamber coated with solidified Matrigel (BD Biosciences) for the invasion assay, whereas, for the migration assay (directional migration), the Matrigel basement membrane did not coat the chamber. In the lower compartment of the chamber, 2 mL of DMEM supplemented with 10% FBS and the desired concentration of A. montana extract were added. The inserts were incubated in a 37°C CO₂ incubator for 24 h. The inserts were removed afterward, and the inner side of the inserts was swabbed to remove the non-invaded cells. The outer side of the inserts bearing the migrated/invaded cells was then fixed in methanol for 30 min before being stained with 0.01% of crystal violet. Images of the membranes were acquired or counted under an inverted microscope with a camera (×100). The dye was extracted, and the absorbance reading was obtained using a spectrophotometer (Hulkower and Herber, 2011).

For measurement of anchorage-independent growth, cells were trypsinized and resuspended in a culture medium containing 0.9% methylcellulose (Fluka, Saint Louis, MO) in DMEM containing 5% FBS. Aliquots of 1 mL containing 2 × 10³ cells were plated on 35-mm bacteriological dishes in the absence or presence of A. montana extract containing 5% FBS. As previously described, colonies were counted under an inverted microscope after 14–16 days (Borowicz et al., 2014).

Cells were grown in a 48-well culture plate in DMEM with 10% FBS containing antibiotics and then incubated with either DMSO vehicle or A. montana extract for 24 h. After incubation, the released lactate dehydrogenase (LDH) into the culture supernatant was measured by the colorimetric LDH Cytotoxicity Assay (BioVision) according to the manufacturer’s instructions (Smith et al., 2011).

To prepare cell lysates, cell monolayers were washed with ice-cold lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM Na2 EDTA, 1% NP40, and 0.1% Na deoxycholate) containing both protease and phosphatase inhibitors. Lysates were collected by centrifugation, and protein concentrations were estimated by using the BCA protein assay (Thermo Fisher Scientific, Inc., Pittsburgh, PA) following the manufacturer’s instructions. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes, which were blocked, probed with the specific antibodies, and then visualized by western blotting analysis. Relative levels of MMP-2 and MMP-9 proteins were determined by densitometry using the ratio of MMP-2 and MMP-9 to GAPDH. Relative levels of PI3K, AKT, SGK1, JNK, ERK, mTOR, p38, and RAF-1 were determined using the ratio of p-PI3K, p-AKT, p-SGK1, p-JNK, p-ERK, p-mTOR, p-p38, p-RAF-1 to t-PI3K, t-AKT, t-SGK1, t-JNK, t-ERK, t-mTOR, t-p38, and t-RAF-1, respectively. Relative levels of survivin, IAP-1, IAP-2, Cyclin-D1, COX-2, VEGF, Caspase, PARP, p53, IGF-1R, and p21 proteins were determined by densitometry using the ratio of these proteins to GAPDH as previously described (Tall et al., 2001).

Cells were lysed using a buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM dithiothreitol, 5 mM MgCl₂, 5% (v/v) glycerol, and 1% (v/v) Triton-X-100 supplemented with 2 mM phenylmethylsulfonyl fluoride (lysis buffer). Lysates (1 mL) were then incubated with 100 μL of glutathione beads containing 10 μg of GST-RAF-RBD at 4°C while rocking for 1 h. After incubation, the beads were washed three times using Hank’s balanced salt solution (HBSS) with 5 mg/mL bovine serum albumin (BSA) (HBSS-BSA). The pull-downs were subjected to SDS-PAGE and analyzed by western blotting using an anti-Ras antibody as previously described (Camalier et al., 2010).

All experiments were done in duplicates, and they were repeated at least three times. Values are represented as the standard error of the mean of triplicates (SEM), and the statistical significance was analyzed by two-way ANOVA or two-tail student’s t-test. Results with *p < 0.05, **p < 0.01, and ***p < 0.001 were statistically significant.

Cancer cells must continuously refurbish cellular metabolism to fulfill the demands of growth and proliferation (Fares et al., 2020). Cell proliferation was determined by comparing the survival of cells for 24 h in the untreated cells, which was normalized to 100%. Cells were treated once with different concentrations of three types of A. montana extracts (i.e., A. montana methanol, hexane, ethyl acetate, and water). Fig. 1A shows that the A. montana methanol extract inhibited the proliferation of human tumorigenic MCF-7 and metastatic MDA-MB-231 breast cancer cell lines. Specifically, the A. montana methanol extract shows an IC₅₀ of 315 ± 15 μg/mL for MDA-MB-231 cells and an IC₅₀ of 245 ± 8 μg/mL for MCF-7 cells, respectively. Fig. 1A also shows the effect of the A. montana methanol extract on the proliferation of MCF-10A, which was considerably lower. It only inhibited 19.5 ± 2.2% at 600 μg/mL. Consistent with this observation, we found that this A. montana methanol extract was unable to affect the viability of PCS-600-010 primary normal mammary epithelial cells (compare the viability of non-treated cells (92 ± 8%) with cells treated with 600 μg/mL of A. montana methanol extract (90 ± 6% cell viability) as evidenced by the quantification of LDH as described in Material and Methods section. Furthermore, adding 0.01% DMSO did not affect the cell viability of any of the treated cell lines (Figs. 1A and 1B). As a positive control, we used wortmannin, a potent inhibitor of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) activity, which also inhibited the proliferation of MDA-MB-231 and MCF-7 cells with 43 ± 9% and 57 ± 5% of inhibition, respectively. Furthermore, the addition of wortmannin (100 μg/mL; 0.233 μM) did not significantly affect the viability of MCF-10A cells (96 ± 4% cell viability) as compared with control cells (100 ± 3% cell viability), and it is consistent with our previous observations (Chin et al., 2014). These results suggest a strong activity in inhibiting the proliferation of the A. montana methanol extract on these cancer cells. To obtain additional information about the nature of the active compound in A. montana leaves, the methanol extract was subsequently fractionated as described in the Materials and Methods section. Three fractions (named hexane, ethyl acetate, and water fractions) were obtained. The A. montana hexane fraction exhibited the most potent inhibitory effect (IC₅₀ 164 ± 4 μg/mL) on cell proliferation of MDA-MB-231 cells as compared with the A. montana ethyl acetate fraction, which showed inhibition of cell proliferation with IC₅₀ 225 ± 5 μg/mL (Fig. 2A). Interestingly, the aqueous fraction did not show any effect (Fig. 2A), and more importantly, these A. montana fractions showed a limited inhibitory effect on the proliferation of MCF-10A cells (Fig. 2B). As expected, these fractions did not show a significant effect on the viability of MDA-MB-231 cells (compare the viability of non-treated cells (94 ± 6% cell viability) and 0.01% DMSO-treated cells (93 ± 5% cell viability) with cells treated with 300 µg/mL of A. montana hexane (94 ± 5% cell viability), ethyl acetate (95±3% cell viability) and water extracts (98 ± 2% cell viability) as evidenced by the quantification of LDH as described in Material and Methods (Fig. 2C). We also fractionated the hexane fraction with a combination of Hexane: Ethyl acetate in order of increasing polarity. Each sub-fraction was tested for its activity on cell proliferation. As shown in Table 1, we observed that A. montana hexane subfraction V, and in less extent, fraction VII, strongly decreased the proliferation of MBA-MD-231 cells. On the other hand, the IC₅₀ results for the anti-proliferative effect of the A. montana hexane sub-fraction V on several types of cancer cell lines (i.e., HeLa, PC-3, HepG2, A549, A431, SK-MEL-28, MDA-MB-231, and MCF-7) are shown in Table 2. The proliferation of these cancer cell lines was susceptible to A. montana hexane subfraction V. A549, and SK-MEL-28 cell lines were the least affected by adding A. montana hexane subfraction V (i.e., high IC₅₀). In contrast, the proliferation of HeLa and MCF-7 cell lines showed high activity inhibiting cell proliferation as they have lower IC50 values. Interestingly, HeLa and MCF-7 cell lines were the most affected after 72 h of treatment with A. montana hexane subfraction V. We also considered the possibility that the extent of inhibition is time-dependent. Thus, we then determine the effect of adding A. montana hexane subfraction V on the proliferation of MDA-MB-231 cells for up to 72 h. Specifically, MDA-MB-231 cells were incubated with 0.01% DMSO, 20 μg/mL, or 40 μg/mL of A. montana hexane extract for three different time intervals: 24, 48, and 72 h, respectively. After the last incubation, the cell proliferation was analyzed using an MTT assay described in Material and Methods. As shown in Fig. 3, 40 μg/mL of A. montana hexane subfraction V significantly suppressed cell proliferation of MDA-MB-231 cells in a time-dependent matter with potent inhibition of cell proliferation (>90% inhibition) at 72 h of treatment.

To further define the inhibitory effect of A. montana hexane sub-fraction V, we investigate the effect of this extract on the cell cycle by flow cytometry as described in Material and Methods. In Figs. 4A and 4B, we show that after incubation of cells with 20 μg/mL A. montana hexane sub-fraction V for 48 h, a significant increase in the SubG1 peak (i.e., untreated cells: 3 ± 0.2; treated cells: 27 ± 2.1). Also, the percentage of cell population in the Go/G1, S, and G2+M phases was significantly lower than in untreated cells. Taken together, our data indicate that treatment of cells with A. montana hexane sub-fraction V was effective and led to the suppression of cell proliferation and induced apoptosis, as evidenced by the increased accumulation of cells in the Sub-G1 phase of the cell cycle. Based on our observations that continued the exposure of A. montana hexane sub-fraction V to MDA-MB-231 cells strongly diminished cell proliferation with an increase in the Sub-G1 peak, we decided to investigate the effect of this extract on apoptosis. We stained MDA-MB-231 cells with Annexin V-FITC after treating them with 20 μg/mL of A. montana hexane subfraction V for 24 h. Figs. 5A and 5B shows that cells stained with Annexin V antibodies (regarded as early apoptosis) increase compared to non-treated cells (Control). We then examined for late apoptosis using the TUNEL assay. Again, apoptotic cells were significantly increased on treatment with A. montana hexane subfraction V for 24 h, as observed by flow cytometry (compare Figs. 5C and 5D). In Table 3, we also show more specific results of Annexin V FITC and PI staining of MDA-MB-231 cells treated with 20 μg/mL of A. montana hexane subfraction V at two different times intervals in addition to 24 h intervals (i.e., 48 and 72 h). A significant increase in the population of early apoptotic cells from 1.14% (untreated cells) to 21.70% (treated cells) when cells were incubated with 20 μg/mL A. montana hexane subfraction V for 48 h. Moreover, a further increase in the population of early apoptotic MDA-MB-231 cells after 72 h of incubation (i.e., untreated cells: 3.09; treated cells: 37.01). Similarly, we also observed an increase in the population of late apoptosis at 48 h (i.e., untreated cells: 1.33; treated cells: 12.21) and 72 h (i.e., untreated cells: 1.39; treated cells: 15.31). After treatment, dead cells were also increased with 20 μg/mL A. montana hexane subfraction V (Table 3). These results suggest that A. montana hexane subfraction V arrested the cell cycle at the Sub-G0/G1 phase and induced apoptosis in vitro in a time-dependent manner.

The anti-metastatic abilities of the A. montana hexane subfraction V were evaluated in MDA-MB-231 cells by using several in vitro assays (i.e., wound healing analysis, migration, invasion, and clonogenic assay). In Fig. 6A, we show a significant decrease in the percentage of wound closure in the A. montana hexane subfraction V extract-treated MDA-MB-231 cells compared with untreated cells (compare 67 ± 4% of closure of treated cells vs. 100% closure for untreated-control cells) after 48 h incubation. We also observed that cells treated with A. montana hexane sub-fraction V inhibited migration (83 ± 7% inhibition) through the Transwell membrane compared to untreated cells (Fig. 6B). Then, we evaluated the invasive capacity of MDA-MB-231 cells in an in vitro system. From the Matrigel invasion assay, as shown in Fig. 6C, the ability of MDA-MB-231 cells to invade this loaded membrane was significantly compromised (72 ± 5% inhibition invasion) in the A. montana hexane sub-fraction V-treated cells relative to the control cells. We also investigated the effect of the A. montana hexane sub-fraction V on the ability of MDA-MB-231 breast cancer cells to form colonies to understand better the inhibitory effect of A. montana hexane sub-fraction V on MDA-MB-231 breast cancer cells. From the clonogenic assay, as shown in Fig. 6D, the ability of MDA-MB-231 cells to form colonies was significantly reduced (64 ± 5% inhibition of invasion) in the A. montana hexane sub-fraction V-treated cells relative to the control cells. In addition, the inhibitory effect of A. montana hexane subfraction V on all these assays was time and concentration-dependent (data not shown). Furthermore, we observed that the protein expressions of MMP-2 and MMP-9 were analyzed to evaluate the effect of A. montana hexane sub-fraction V on the motility of MDA-MB-231 cells. It showed that A. montana hexane sub-fraction V significantly suppressed the protein expressions of MMP-2 (84 ± 5% inhibition of protein expression) and MMP-9 (54 ± 3% inhibition of protein expression) (Fig. 6E). Thus, these results indicated that A. montana hexane sub-fraction V significantly inhibited the migration, invasion, and colony formation of MDA-MB-231 breast cancer cells in vitro. These observations are consistent with the fact that MMP-2 and MMP-9 proteins are closely related to the migration and invasion of various types of cancer cells (Brown and Murray, 2015; Folgueras et al., 2004).

The Ras small GTPases control signaling pathways that are key regulators of several aspects of the uncontrol cell growth (Downward, 2003). The downstream of Ras proteins is the RAF/MEK/ERK kinase cascade (Kolch et al., 1991). Also, the active Ras turn on the PI3K/AKT/mTOR/S6K1 pathway regulates different cellular functions (i.e., anti-apoptotic pathways) (Yuan et al., 2020). We first investigated whether A. montana hexane sub-fraction V extract affects the PI3K/AKT/mTOR/S6K1 activation in MDA-MB-231 breast cancer cells. As shown in Figs. 7A–7D, PI3K and Akt activation was suppressed by 20 μg/mL of A. montana hexane subfraction V. In addition, the activation of S6K1 and mTOR was also decreased upon A. montana hexane extract treatment in MDA-MB-231 breast cancer cells. Our data indicate that inhibiting PI3K/AKT/mTOR/S6K1 signaling cascade by A. montana hexane sub-fraction V leads to the suppression of cell proliferation in MDA-MB-231 breast cancer cells. We next determine whether A. montana hexane subfraction V could alter the activation of MAPK cascades, including JNK, ERK1/2, and p38 MAPK in MDA-MB-231 breast cancer cells. As shown in Figs. 7E–7H, A. montana hexane subfraction V substantially decreased the phosphorylation of Raf-1 (84 ± 5% inhibition) and Erk1/2 (56 ± 3% inhibition) at a concentration of 20 μg/mL and slightly downregulated the phosphorylation of JNK (30 ± 4% inhibition) and p38 MAPK (19 ± 5% inhibition) in MDA-MB-231 breast cancer cells.

Our observations indicate that AKT and ERK1/2 activities are affected by the A. montana hexane subfraction V. We next examined the effects of A. montana hexane subfraction V on the expression of survivin, IAP-1, and IAP-2 proteins. They have been implicated in apoptosis and act downstream to PI3K/Akt/mTOR/S6K1 signaling cascade (Hassan et al., 2013; Paplomata and O’Regan, 2014). We found that A. montana hexane subfraction V suppressed the expression of anti-apoptotic proteins (Figs. 8A–8C). Also, A. montana hexane sub-fraction V decreased the expression of Cyclin D1, COX-2, and VEGF (Figs. 8D–8F). In addition, we determined the effect of A. montana hexane sub-fraction V on the expression and cleaved procaspase-3 and PARP. Procaspase-3 is an excellent marker to monitor activated apoptotic and triggers cleavage of PARP, which is involved in repairing DNA damage (Kitazumi and Tsukahara, 2011). In Figs. 8G and 8H, we showed that A. montana hexane subfraction V induced the cleavage of procaspase-3 and PARP, as observed by the disappearance of the main band and the appearance of its cleaved forms. The tumor-suppressor proteins p53 and p21 are significant regulators of the cell–cycle checkpoint (Kulaberoglu et al., 2016). The p53 protein is a tumor suppressor in preventing cancer development (Luo et al., 1995). The cyclin-dependent kinase inhibitor p21 is regulated by p53 and negatively modulates cell cycle progression by inhibiting the activities of cyclin E/CDK2 and cyclin D/CDK4 complexes (Pandey et al., 2019). We found that A. montana hexane subfraction V induced the expression of both p53 and p21 in MDA-MB-231 breast cancer cells (Figs. 8I and 8J). These results demonstrate that A. montana hexane subfraction V induces apoptosis via the down-regulation of anti-apoptotic, proliferative, metastatic, and angiogenetic proteins via caspase-3 activation in MDA-MB-231 breast cancer cells.

To further understand how A. montana hexane subfraction V may modulate various signaling molecules downstream of Ras pathways, we decided to treat MDA-MB-231 breast cancer cells with the following inhibitors (i.e., PF-3758309, FR180204, Wortmannin, and ISIS2503). We then investigated whether the A. montana hexane subfraction V was able to enhance its inhibitory effect by combination with the pharmacological inhibitors mentioned above. As shown in Figs. 9A–9D, treatment of cells with either A. montana hexane subfraction V or wortmannin inhibited AKT activation but had a weak inhibitory effect on mTOR and S6K1 activation. Interestingly, mTOR and S6K1 activation were blocked by combining A. montana hexane subfraction V alone or with wortmannin. In Figs. 9E–9G, we showed that the PF-3758309, a PAK1 inhibitor, decreased the phosphorylation of RAF. Moreover, it also affected the activation of ERK1/2. However, the addition of FR180204, an ERK1/2 inhibitor, strongly diminished the activation of ERK1/2 without affecting the activity of RAF. Interestingly, we also observed inhibition of the phosphorylation of RAF and ERK1/2 when A. montana hexane subfraction V was combined individually with each inhibitor. We also observed that the addition of A. montana hexane subfraction V was unable to affect the expression of Ras when compared with the ISIS2503 inhibitor alone in MDA-MB-231 breast cancer cells. As expected, the combination of A. montana hexane subfraction V with ISIS2503 inhibitor did not potentiate the inhibitory effect of the ISIS2503 inhibitor alone (Figs. 9H and 9I). We next examined the effects of A. montana hexane subfraction V extract on the activation of AKT and ERK1/2 activities upon IGF-1 stimulation. Fig. 10 shows that AKT and ERK1/2 activities were affected by the A. montana hexane subfraction V upon IGF-1 stimulation. However, neither IGF-1 receptor phosphorylation nor activation of Ras was affected. These data imply that A. montana hexane subfraction V can act downstream of the Ras protein through modulation of multiple signal transduction pathways to induce apoptosis in tumor cells.

Chemical studies with species of the Annona genus have reported on the essential oils from fruits because many fruits from the Annonaceae family are edible (Nugraha et al., 2019; Al Kazman et al., 2022). On the contrary, only some studies have been performed on the leaves. Thus, we conducted a GC-MS analysis as described in Material and Methods section to gain more information about the composition of the constituents, specifically in the A. montana hexane sub-fraction V. Our results on A. montana hexane sub-fraction V indicated that ten compounds out of a total of fifteen compounds were identified, (Table 4 and Suppl. Table S1) in a complex mixture of phytosterols (i.e., campesterol, γ-sitosterol, β-sitosterol, and γ-stigmasterol), γ-tocopherol, 1,2-epxyoctadecane, methyl-elaidolinolenate, 2-clhloroethyllinoleate, solanesol, and palmitic acid. Interestingly, some of these compounds have been reported to affect cell proliferation and apoptosis cellular mechanisms in cancer cells (Awad and Fink, 2000; Nugraha et al., 2019; Al Kazman et al., 2022; Cioccoloni et al., 2022).