Selective Cytotoxic Effects of Annona Squamosa Leaves against Breast Cancer Cells via Apoptotic Signaling Proteins

Annona squamosa L. is an important medicinal plant used in traditional medicine for the treatment of various diseases. Different parts of A. squamosa L. have various therapeutic effects; however, the anticancer activity of the leaves has not yet been identified. In vitro, MTT, nuclear staining, and LDH assays were used to evaluate cell survival and proliferation in cells exposed to the extracts. The effect of the extracts on cell migration was investigated using a monolayer wound repair assay, and the apoptotic effects were evaluated using flow cytometry. A breast cancer model was used to study the effect of the extract on the tumor size, and the expression of different proliferative and apoptotic markers was evaluated by immunohistochemical analysis. At a concentration of 100 μg/mL, A. squamosa leaf extracts exerted strong antiproliferative and cytotoxic effects against various cell lines. The extracts reduced wound closure and strongly induced apoptosis. In vivo study, rats were sacrificed 24 h after the last injection, and tumor size, as well as the expression of proliferative and apoptotic markers, were observed to be greatly affected by treatment with the extracts. Therefore, A. squamosa leaf extract may be developed as a potential novel drug to treat breast cancer in the future.


Introduction
Breast cancer is the second most common cancer and the most common cause of death in women worldwide [1]. Nevertheless, surgery, chemotherapy, and radiotherapy remain the cornerstone of breast cancer treatment [2]. Chemotherapeutic agents have several disadvantages and significant side effects, such as the inability to differentiate between cancer and normal cells and drug resistance associated with repeated treatment. Hence, there is an urgent need for new drugs. It is necessary to identify natural products that target multiple signaling pathways and cause growth inhibitory effects on cancer cells with fewer harmful effects on healthy cells and the biological environment [3]. Plant medicines have gained recognition over the last 40 years, and have been widely used as alternative or complementary medicines in many countries and ethnic groups worldwide [4]. Even today, plant-derived compounds play a major role in primary healthcare as natural remedies in most countries, owing to their safety, effectiveness, quality, and availability [5]. It is reported that 49% of the 175 small molecules, which were approved for cancer treatment, were natural products or modified natural products. Additionally, it is estimated that almost 80% of people use plant medicines to treat inflammatory diseases [6]. Plants with a long history of use in ethnomedicine can be considered a vast resource of bioactive compounds, providing medicinal and health benefits against different diseases [7,8]. Therefore, in the field of phytoscience, researchers are working to elucidate the side effects, calculate appropriate dosages, and find the best method to extract and identify bioactive components. The bioactive compounds are secondary plant metabolites that elicit pharmacological or toxicological effects in humans and animals; however, one of the most critical challenges that researchers have faced is that a single plant may contain many bioactive compounds [9]. Currently, there is a global interest in medicinal plants as natural products that may yield the next generation of semi-synthetic derivatives, despite the decrease in the popularity of this approach in the 20 th century [10].
Annona squamosa L. is a small group of an edible plant of the genus Annona and the family Annonaceae, commonly known as a sugar apple or custard apple [11]. A. squamosa L. is a small tree that grows to 3-8 m in height and has conical fruits. The leaves are brilliant green on the top and bluish green on the bottom, with petioles of 0.7-1.5 cm ilong. The shape of leaves is oblong-elliptical; they measure 5 to 17 cm in length and 2 to 7 cm in width, with an obtuse or acuminate apex. The blade has 15 to 17 pairs of veins [12]. Different parts of A. squamosa L., such as the bark, root, seed, fruits, flowers, and leaves have been used in traditional medicine to treat various diseases [13].
A. squamosa L. plant parts are known to have various biological activities, although there are fewer studies of the leaves. To provide a clear understanding of the mechanism action of A. squamosa L leaf extracts, screening for the anticancer activity should confirm its use as an alternative to chemotherapeutic agents for breast cancer therapy. This study's objective was to investigate the in vitro and in vivo anti-breast cancer activity of A. squamosa leaf extracts.

The effect of A. squamosa extract on cell survival
Treatment with A. squamosa leaf extracts did not induce clear changes in the morphology of either breast cancer cell line; however, the treatment affected the number of viable cells ( Figure 1). Therefore, the antiproliferative activity of various concentrations of A. squamosa extracts was determined against MCF-7 and MDA-MB-231 breast cancer cell lines using MTT assay. The methanolic extract resulted in a highly significant decrease in MCF-7 cell viability at 1 µ g/mL, 10 µ g/mL, and 100 µ g/mL ( Fig. 2-a). The 10 µ g/mL of the methanolic and acetonic extracts resulted in a highly significant decrease in the viability of MDA-MB-321 cells ( Fig. 2-b). However, it was found that all three extracts of A. squamosa leaf at 100 µ g/mL resulted in a highly significant decrease in MCF-7 and MDA-MB-321 cell viability, with cell proliferation inhibition of approximately 60%. Furthermore, to determine whether the antiproliferative activities of A. squamosa extracts were related to the induction of apoptosis, the morphological changes of cells were investigated using a nuclear staining assay. In both cell lines, the number of cells decreased with an increase in the extract concentration applied. Besides, nuclear damage occurred in a dose-dependent manner (Fig. 2-c). These results clearly showed the antiproliferative activity of A. squamosa extracts in human breast cancer ER+ and ER-cells.

The cytotoxic effects of A. squamosa extracts
The cytotoxicity of A. squamosa extracts was determined through the measurement of LDH leakage from degraded cells, which resulted from the cytotoxic effects of the extract. As shown in Figure 3, the extracts exerted cytotoxicity against MCF-7 and MDA-MB-231 in a dose-dependent manner.
Overall, the three different extracts showed highly significant cytotoxic effects in ER+ cells, MCF-7, compared with the ER-cells, MDA-MB-231. At the highest concentration tested (100 µ g/mL), the methanolic and acetonic extracts induced 100% cell death in MCF-7 cells, and the aqueous extract induced approximately 60% cell death. A. squamosa extracts resulted in approximately 70%-80% cell death of MDA-MB-231 cell lines at the highest concentration tested (100 µ g/mL).

The effect of A. squamosa extracts on cell migration
To clarify the potential of A. squamosa extracts as anticancer agents, the effect of the extracts on the migration of cancer cells was tested using a monolayer wound healing assay ( Figure 4). After 6 h, the cells treated with extracts had not migrated as far as the untreated cells. Furthermore, MCF-7 cells treated with the three different extracts showed 30%-33% less closure after 6 h than the DMSOtreated control cells (p < 0.005). It appears that the healing of ER-cells occurred much more rapidly than ER+ cells; therefore the migration of MDA-MB-231 treated cells was not greatly affected. In other words, the decrease in cell migration resulting from A. squamosa extract treatment was more considerable in ER+ cells.

Figure 4. The migration of breast cancer cell lines (MCF-7 and MDA-MB-231) after treatment with different A. squamosa extracts.
A scratch was made on each monolayer, the culture medium was refreshed, and 50 µ g/mL of extract was added. The cultures were maintained under the appropriate conditions, observed, and photographed at 0 and 24 h (n = 3, * p< 0.05, and ** p< 0.005).

The apoptotic effect of A. squamosa extracts
The methanolic, acetonic, and aqueous extracts of A. squamosa leaves on apoptosis induction in the breast cancer cell lines were measured using Annexin V/PI staining after 24 h of treatment at 50 µ g/mL (

Figure 5. Survival of breast cancer cells after treatment with A. squamosa extracts for 24 h. MCF-7
and MDA-MB-231 cells were seeded at 4 ×10 5 cells/well and treated with 50 µ g/mL methanol, acetone, or aqueous extract in DMEM medium with 10% FBS for 24 h. The apoptotic effect was measured using Annexin V-PI staining kit and flow cytometry.

The effect of A. squamosa extracts on anti-apoptotic/pro-apoptotic gene expression
To detect the apoptotic effects of the methanolic, acetonic, and aqueous extracts, the expression of anti-apoptotic (Bcl-2) and pro-apoptotic (Bax) genes in breast cancer cells were measured using qPCR. The exposure of MCF-7 cells to the extracts caused downregulation of the Bcl-2 mRNA; this change was significant after treatment with the methanolic and acetonic extracts. All three extracts upregulated the expression of Bax; however, the change was only significant after treatment with the aqueous extract ( Figure 6-a). In MDA-MB-231 cells, the expression of Bcl-2 was downregulated, and that of Bax was upregulated ( Figure 6-b). Therefore, these findings showed that A. squamosa extracts induced apoptosis through the suppression of Bcl-2 and the activation of Bax. Consequently, the Bcl-2/Bax ratios showed a highly significant decrease (P < 0.005).

In vivo effect of A. squamosa extract on tumor growth
The therapeutic effects of A. squamosa extract in a rat model of breast cancer were assessed after treatment, via local injection, with the aqueous extract for one month. As shown in Figure 7, the tumor size in the treatment group was significantly reduced compared with that of the control (corn oiltreated) group, 1083 mm 3 , and 6583 mm 3 , respectively. These data suggested that the local treatment Hematoxylin and eosin staining was used for the histopathological examination, whereas antibodies for Ki67 and p53 were used for immunohistochemical staining. Magnification 400×.

Discussion
From ancient times, plants have been used for the treatment of diseases by humans. They are used as complementary medicine or to synthesize chemical compounds. According to the World Health Organization, more than 80% of the world's population depends on nature-derived traditional alternative medicine for their primary healthcare needs [16]. One of these plants with extensive traditional use is A. squamosa; however, biologically, it is less well characterized. Few studies have illustrated the anticancer activity of A. squamosa leaf extracts. Therefore, this study aimed to investigate the anticancer effect of A. squamosa leaf extract on breast cancer cells.
In the current study, the anticancer activity of A. squamosa leaf extracts was investigated in vitro against two breast cancer cell lines; MCF-7 and MDA-MB-231. Our results revealed that the different A. squamosa leaf extracts did not induce any changes in the morphology of the cell lines ( Figure 1). However, different extracts were found to induce cytotoxicity and inhibit the proliferation of MCF-7 and MDA-MB-231 cells in a dose-dependent manner (Figures 2 and 3). In this study, the antiproliferative and cytotoxicity activities of the extracts were confirmed by nuclear staining, which revealed damaged nuclei (Figure 2-c). These appear to be consistent with the results of a recent study on the isolation of different acetogenin compounds from A. squamosa seeds; the compounds were able to suppress the proliferation of multi-drug resistant MCF-7 cells via cell cycle arrest in the G1 phase [8]. Indeed, it is well known that the leading cause of death among patients with cancer is the ability of cancer cells to metastasize and invade [17], and our results present the first demonstration that different A. squamosa leaf extracts partially inhibited breast cancer migration ( Figure 4). Furthermore, the current study showed that different extracts induced late apoptosis and necrosis in both MCF-7 and MDA-MB-231 cells ( Figure 5). These results were confirmed by the study of the gene expression of pro-apoptotic and anti-apoptotic markers, and different extracts were found to induce the expression of Bax and suppress the expression of Bcl-2 ( Figure 6). These results agreed with those of Pardhasaradhi et al. [18], which showed that treatment of MCF-7 with A. squamosa seed extracts resulted in nuclear condensation, DNA fragmentation, ROS generation, and apoptosis induction via downregulation of the ratio of Bcl-2/Bax.
An in vivo study can offer conclusive insights about the nature of the biological effect of A. squamosa leaf extracts; therefore, a breast cancer model developed in our laboratory was used. It was interesting to note that aqueous extracts significantly decrease tumor size. To better understand the mode of action of the extracts, immunohistochemistry was used to evaluate the expression of proliferative and tumor suppressor genes, Ki67, and p53. Stronger immunostaining intense for p53 was found in the treated tissue than the control one, and less intense staining Ki67 was observed in the treated tissue. This finding was consistent with those of Chen et al. [19], which showed that A. squamosa seeds extracts inhibited the growth of H22 hepatoma cells by 69.55% in mice without any side effects. According to these results, we can infer that p53 directly or indirectly targeted either the apoptotic or non-apoptotic cell death pathway. For example, Caspase-3 activation drives the expression of different microRNA species that are known to target the Bcl-2 or the expression of different genes that are involved in cell cycle arrest [20].
In summary, A. squamosa leaf extracts demonstrated potential anticancer activity against breast cancer in both in vitro and in vivo experiments; therefore, further investigation may contribute to the development of a potential new anticancer therapy for both ER+ and ER-breast cancer. Further research is needed to fully understand the mechanism of action of A. squamosa leaf extracts and its role in cell autophagy, and to identify the pathway targeted by these extracts.

Cell lines and culture medium
MCF-7 and MDA-MB-231 breast cancer cell lines were obtained from American Type Culture Collection (ATCC, USA) and cultured in Dulbecco's Modified Eagle's medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Sigma) and 2×10 -3 v/v penicillin-streptomycin (composed of 31 g/L penicillin and 50 g/L streptomycin) in a humidified atmosphere of 5% CO 2 at 37 °C throughout the assays.

Cell viability assay
The antiproliferative activity of the extracts was determined by the 3-(4.5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay following the method of Semlali et al. [21]. The extracts were dissolved in DMSO and then diluted in a culture medium. Briefly, the cells were seeded in 6-well plates (4 × 10 5 cells/well) and incubated at 37 °C for overnight growth. When cells reached 80% confluence, the culture medium was removed and replaced with 1 mL of fresh culture medium, and the cells were treated with various concentrations of the leaf extract (1 µ g/mL, 10 µ g/mL, and 100 µ g/mL) for 24 h. The untreated cells were assayed as a negative control. After that, cells were incubated with 100 µ L MTT (5 mg/mL; Sigma) in fresh medium for 3 h. The colored MTT-formazan crystals were dissolved by the addition of 0.04 N HCl in isopropanol (500 µ L) to each well. Finally, 100 µ L of the reaction mixture was transferred to a 96-well plate, and the maximum absorbance at 550 nm was detected using an ELISA plate reader (X-Mark Microplate Spectrophotometer, Bio-Rad, USA). The experiment was conducted in triplicate, and the percentage of viable cells was calculated using the following formula: Cell viability (%) = (absorbance of test sample/absorbance of negative control) × 100

Nuclear staining
The morphology of the treated cells was assessed using a Hoechst 33342 staining assay (H42), as previously described by Semlali et al. [21]. The cells were seeded in 6-well tissue culture plates (3 × 10 5 cells/well). After overnight incubation, the medium was removed and replaced with fresh medium. Different concentrations of the extracts were added to the medium, and the cells were incubated for 24 h. The supernatant was then removed, and the cells were washed twice with PBS and fixed in cold methanol for 15 min. After fixing, the cells were washed twice with PBS, and then stained with 2 µ g/mL Hoechst 33342 (Thermo Fisher Scientific, DE) for 15 min. The stained nuclei were washed twice with PBS and observed and photographed using Leica DM2500 & DM2500 LED optical microscopes (LEICA Microsystems, DE).

Cytotoxicity assay
Breast cancer cell lines were cultured for 24 h in 6-well plates (4×10 5 cells/well). After overnight growth, the culture medium was refreshed, and cells were treated with various concentrations of the leaf extract (1 µ g/mL, 10 µ g/mL, and 100 µ g/mL) for 24 h. Cell injury was assessed by the measurement of the release of lactate dehydrogenase (LDH) [22]. 10 µ L of the culture supernatant was transferred to a 96-well plate, and the enzyme reaction was conducted in accordance with the manufacturer's instructions (LDH cytotoxicity colorimetric assay kit II, BioVision, USA). Briefly, LDH reaction mixture (100 µ L) was added to each well and the plate was incubated for 30 min at 18-21 °C (room temperature) in the dark; thereafter, 10 µ L of stop solution was added to terminate the reaction. The absorbance was measured at 450 nm using an X-Mark microplate spectrophotometer (Bio-Rad). Negative, positive, and background controls for total LDH activity were included in the experiment. The cytotoxicity was calculated as a percentage from the following formula: Cytotoxicity (%) = (test sample -negative control) / (positive control -negative control) × 100

Monolayer wound repair assay
Breast cancer cell lines were seeded in 6-well plates and grown to confluence. A 10 µ L pipette tip was used to made scratch wounds. The detached cells were removed by washing with PBS; thereafter, the cells were treated with 50 µ g/mL of the methanolic, acetonic, or aqueous extracts. The migration of cells into the wounded region was observed using a LEICA DFC450 C digital camera (LEICA Microsystems, DE) at 0 and 24 h Semlali et al. [23]. The percentage of wound closure was calculated following the formula: Wound closure (%) = (initial scratch size -size of the scratch after an identified culture period) / (initial scratch size) × 100

Annexin V/PI apoptosis assay:
To discriminate the type of cell death induced by A. squamosa leaf extracts, APC Annexin V Apoptosis Detection Kit with PI (BioLegend, USA) was used. This assay is based on the detection of morphological features associated with apoptosis or necrosis. First, the cells were treated with methanolic, acetonic, or aqueous extracts (50 µ g/mL) for 24 h; then, adherent cells were collected, rinsed twice with cold PBS, and resuspended in 100 µ L of annexin-binding buffer. This was followed by the addition of 5 µ L APC Annexin V and 10 µ L PI solution, and incubation at room temperature for 15 min. Subsequently, 400 µ L of annexin-binding buffer was added to the reaction mixture, and the cells were immediately analyzed using BD FACSCalibur TM flow cytometer (Becton Dickinson, USA).

Cell culture conditions for RNA extraction
The breast cancer cell lines were cultured for 24 h in a T25 flask (1 × 10 6 cells/flask). The culture medium was replaced with medium containing 1% FBS for 24 h, and the cells were then treated with 50 µ g/mL of the leaf extracts in 2 mL fresh medium containing 1% FBS, or 0.25% DMSO as the control. The cells were allowed to grow overnight. After washing with PBS, adherent cells were detached by the application of trypsin (Gibco, USA).

Reverse-Transcription PCR (RT-PCR)
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, DE). The RNA quantity, purity, and quality were determined using a Nanodrop 8000 spectrophotometer (Thermo Fisher Scientific, USA). One microgram of RNA from each sample was reverse-transcribed into cDNA using the Hyperscript Kit (GeneAll, KR) and random hexamers (GeneAll, KR), as described in the manufacturer's protocol.

Quantitative real-times PCR
The mRNA expression of Bcl-2 and Bax was measured using a QuantStudio 7Flex Detection System (Applied Biosystems, USA). The reactions were performed using iTaq TM Universal SYBER Green Supermix (Bio-Rad). Primers (Table 1) were added to the reaction mix at a final concentration of 10 pM. Five microliters of each cDNA sample were added to a 20 µ L PCR mixture containing 12.5 µ L of 2 × iTaq TM Universal SYBER Green Supermix, 0.5 µ L of primers for Bcl-2, Bax, GAPDH (Macrogen, KR), and 7 µ L RNase/DNase-free water (Qiagen, DE). The thermal cycling conditions for Bcl-2 and Bax were established as 5 min at 95 °C, followed by 40 cycles of 30 s at 95 °C and 30 s at 60 °C, and final 10 s at 95°C. The specificity of each primer pair was verified by the presence of a single melting temperature peak. The Applied Biosystems software automatically determined the threshold cycle. The expression of GAPDH, a housekeeping gene, was used as an endogenous control for this study.
Table1. Description of primers used for qPCR.

Gene
Primer sequence (

Animal model
To confirm the in vitro results, twelve female Wistar albino rats (Rattus norvegicus), aged 5-8 weeks of age and with a bodyweight of 110 ± 20 g were used. The rats were divided into three groups, with four animals in each group. The rats were obtained from the breeding facility at the College of Pharmacy at King Saud University, Riyadh, and the written informed consent to use the animals in our study was obtained from this facility. The animals were housed in plastic cages at room temperature (25 ± 2 °C) with a constant relative humidity (45% ± 5%) under a 12 h light/dark cycle. Mammary tumors were induced in two groups of rats (groups I and II) by a single subcutaneous injection in the pectoral area with the potent carcinogen, dimethylbenzanthracene (DMBA), as described previously [24]. After tumor palpation (8 weeks post-injection), animals in group I received corn oil as vehicle control, whereas animals in group II were injected with A. squamosa aqueous extract dissolved in corn oil at a dose of 300 mg/kg directly into the tumor. A single dose was injected for 4 weeks every 48 h, following a previously described protocol [25,26]. After treatment, the rats were euthanized by administering a ketamine (125 mg/kg), and xylazine (10 mg/kg) overdose and mammary gland tissues were collected from all sacrificed rats. All animal procedure was reviewed and approved in accordance with ICH GCP guidelines by the Institutional Review Board at King Fahd Medical City, Riyadh, KSA (IRB log number: 18-586, registration number with KACST, KSU: H-01-12-012 and registration number with OHRP/NIH, USA: IRB00010471).

Immunohistochemistry (IHC) analysis
Polyclonal antibodies were used to assess the expression of different tumor markers, including the genes involved in the proliferation signaling pathway, such as Ki67 and p53. Briefly, IHC was performed on 3-μm slices of deparaffinized and rehydrated breast tissue by a Ventana Benchmark XT automated staining (Ventana Medical Systems Inc., Tucson, AZ) for the assessment of Ki67 and p53 (Santa Cruz Biotechnology, CA, USA), as previously described by Semlali et al. [27]. The immunostained sections were analyzed by an Olympus BX51 light microscope and DP72 Olympus digital camera with powers 400X (Olympus America Inc, Center Valley, PA, USA).

Statistical analysis
Data are presented as an arithmetical mean ± standard deviation (±SD) using Statistical Package for the Social Sciences (SPSS v.21, Chicago, IL, USA). All statistical differences were evaluated with one-way analysis of variance (ANOVA) followed by student's t-test. p ≤0.05 was considered as statistically significant, while value ≤0.005 was taken as statistically highly significant compared to relevant control.