Phytochemicals from Ajwa dates pulp extract induce apoptosis in human triple-negative breast cancer by inhibiting AKT/mTOR pathway and modulating Bcl-2 family proteins

Ajwa dates (Phoenix dactylifera L.) have been described in traditional and alternative medicine to provide several health benefits, but their mechanism of apoptosis induction against human triple-negative breast cancer MDA-MB-231 cells remains to be investigated. In this study, we analyzed the phytoconstituents in ethanolic Ajwa Dates Pulp Extract (ADPE) by liquid chromatography-mass spectrometry (LC–MS) and investigated anticancer effects against MDA-MB-231 cells. LC–MS analysis revealed that ADPE contained phytocomponents belonging to classes such as carbohydrates, phenolics, flavonoids and terpenoids. MTT assay demonstrated statistically significant dose- and time-dependent inhibition of MDA-MB-231 cells with IC50 values of 17.45 and 16.67 mg/mL at 24 and 48 h, respectively. Hoechst 33342 dye and DNA fragmentation data showed apoptotic cell death while AO/PI and Annexin V-FITC data revealed cells in late apoptosis at higher doses of ADPE. More importantly, ADPE prompted reactive oxygen species (ROS) induced alterations in mitochondrial membrane potential (MMP) in ADPE treated MDA-MB-231 cells. Cell cycle analysis demonstrated that ADPE induced cell arrest in S and G2/M checkpoints. ADPE upregulated the p53, Bax and cleaved caspase-3, thereby leading to the downregulation of Bcl-2 and AKT/mTOR pathway. ADPE did not show any significant toxicity on normal human peripheral blood mononuclear cells which suggests its safe application to biological systems under study. Thus, ADPE has the potential to be used as an adjunct to the mainline of treatment against breast cancer.

Scientific Reports | (2021) 11:10322 | https://doi.org/10.1038/s41598-021-89420-z www.nature.com/scientificreports/ (150 × 2.1, 2.6 μm) and elution was done for 30 min, using mobile phase solvent acetonitrile/water (5:95, v/v), acetonitrile, methanol and 5 mM ammonium acetate (95:5 H 2 O:Acetonitrile, pH 6.5). The mobile phase was kept at a flow ramp rate of 0.45 mL min -1 and the sample injection volume was 5 μL. Waters Acquity PDA detector type-UPLC eLambda 800 nm was used at wavelength range 210-800 nm and resolution 1.2 nm. The spectrometer was operated in positive and negative modes. The source temperature was 120 °C, the desolvation temperature was 350 °C and the cone voltage was set at 30 V. MS data was recorded in the mass range, m/z 200-2000 from 0-30 min under ionization mode of ES + and ES -. All eluted peaks from HPLC were recorded at different retention times. The fractions were then characterized by mass spectrometry. This analysis was carried out at the Sophisticated Analytical Instrumentation Facility (SAIF), CSIR-Central Drug Research Institute (CDRI), Lucknow, India.
MTT assay on breast cancer cells. The cell viability of ADPE against breast cancer cells was evaluated by MTT reduction assay following a previously published protocol 15 . MDA-MB-231 cells were seeded at a density of 1 × 10 4 cells/well in a 96-well microtiter culture plate and incubated overnight. ADPE was diluted in culture media and treated in triplicate with varying concentrations (10,12,15,18,20,22 and 25 mg/mL) of ADPE for 24 and 48 h. The absorbance values were read in an ELISA plate reader (BIORAD-PW41, USA) at 550 nm with a reference wavelength of 630 nm. The cellular morphological changes were observed under an inverted phasecontrast microscope (Nikon Eclipse TS100, Japan).
Cytotoxicity against PBMCs. Human PBMCs were isolated from heparinized blood using Histopaque-1077 density gradient (Sigma Aldrich, USA) following a previous protocol with slight modification 16 . Briefly, fresh blood was diluted with equal volume of PBS (1X) and lightly poured over equal volume of Histopaque 1077 in 15 ml falcon tube followed by centrifugation at 1500 rpm for 15 min at 20 °C in a hanging rotor centrifuge. The middle buffy-coat containing mononuclear cells was collected in a fresh tube and washed twice with PBS. For toxicity assay, 1 × 10 5 PBMCs/well were seeded into nutrient media in a 96-well culture plate. After 2 h, different concentrations of ADPE were added in each well in triplicate except the control wells and incubated for 24 h. Absorbance and percentage of cytotoxicity were determined as mentioned above using ELISA plate reader (BIORAD-PW41, USA).
Nuclear condensation assay. Based  www.nature.com/scientificreports/ with Rh123 at a final concentration of 10 µM for 30 min in the dark. After washing with PBS twice, cells were resuspended in 500 µl PBS and analyzed using flow cytometry.
Analysis of cellular DNA content. Cells were seeded at density 1 × 10 6 cells/mL into 6-well plate and treated with ADPE (15, 18 and 20 mg/mL) for 48 h. Different phases of the cell cycle with cellular DNA contents were analyzed using flow cytometry as described previously 15 .

Western blot analysis.
Western blotting was carried out as per a previously standardized protocol 20 .
Briefly, MDA-MB-231 cells at a density of 1 × 10 6 in T-25 cm 2 flask were treated with two effective doses viz. 15 and 20 mg/mL of ADPE. Based on the IC 50 value (16.67 mg/mL) of ADPE after 48 h, these two effective doses, one below IC 50 and the other above IC 50 , were selected to show their differential effects on selected apoptotic markers in MDA-MB-231 cells. The whole cell lysates were prepared by scrapping the cells in ice-cold RIPA lysis buffer supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, USA). The lysates from each sample were centrifuged at 13,000×g for 20 min and the protein concentration in the supernatant was determined with a BCA protein assay kit (Thermo Fisher Scientific, USA) as per manual instruction. Equal amounts (30 μg) from each sample of protein lysate were subjected to SDS-PAGE on a 6% gel for mTOR/p-MTOR and a 12% gel for AKT/p-AKT, p53, Bax, Bcl-2, cleaved caspase-3. Mini Trans-Blot Module (BIORAD) was used to electrotransfer separated proteins to a PVDF membrane and thereafter the blot was blocked with blocking buffer containing 5% BSA in Tris Buffer Saline Tween 20 (TBST) solution, pH 7.4 under constant agitation for 1 h at 4 °C. Membranes were then incubated with primary antibodies overnight at 4 °C. Following washing with TBST thrice, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT with gentle shaking. After washing, bands were visualized by ECL Western Blotting Substrate Kit (Thermo Fischer Scientific, USA) according to the manufacturer's instructions. The relative abundance of each band was quantified using Image J software (version 1.43, NIH, USA) and normalized to β-actin as a loading control.

Statistical analysis.
Cell viability data were expressed as the mean ± SEM from three independent experiments. Statistical evaluation was determined by one-way ANOVA followed by Dunnett's Multiple Comparison Test using GraphPad Prism software (Version 5.01). A p-value of less than 0.05 was considered statistically significant.  Table 1. The compounds having the highest peak were identified as maltose, catechin, myricetin, quercetin, β-sitosterol, digalacturonic acid, chlorogenic acid and β-carotene.

Effect of ADPE on cell viability of MDA-MB-231 cells and human PBMCs. MDA-MB-231 cells
were treated with increasing concentrations of ADPE and photographed at 24 and 48 h of ADPE exposure. As shown in Fig. 2a 2d). ADPE was also used to check the cytotoxic effect on normal human PBMCs. Interestingly; ADPE did not exhibit any significant toxicity against human PBMCs (Fig. 2f). The morphological features of PBMCs at different concentrations of ADPE also validated the data of cytotoxic assay, where no significant variations were found in number and morphology of normal cells (Fig. 2e). These results suggest that ADPE is not toxic to normal human cells in the range tested.

Stimulation of chromatin condensation and induction of apoptosis by ADPE.
As is evident from the photomicrograph (Fig. 3a), ADPE increased the chromatin condensation in MDA-MB-231 cells stained with Hoechst 33342 depending upon dose. Doses 15 and 18 mg/mL showed less condensation of nucleus whereas 20 mg/mL of ADPE exhibited maximum nuclear condensation. As shown in AO/EtBr staining, control cells appeared live and healthy having uniformly green-colored nucleus, while treated cells appeared to be either in early apoptosis (green-colored with condensed nuclei) or in late apoptosis stage (orange-red colored cells with condensed nuclei). Higher doses of ADPE increased the late apoptotic features (Fig. 3b) (Fig. 4b). The fluorescent microscopy analysis of ROS intensity was found to be consistent with flow cytometry data (Fig. 4a).

Decrease of MMP by ADPE.
As is evident from photomicrograph (Fig. 4c), the red fluorescent intensity of dye Rh123 was inversely proportional to increasing doses of ADPE due to loss of MMP in treated cells. Flow cytometry analysis depicted the percent level of MMP at various doses of ADPE (Fig. 4d) Figure S1. These results indicate that ADPE inhibits AKT/mTOR signaling pathway important in regulating cell growth and proliferation of breast cancer cells.

Discussion
Amongt breast cancer, TNBC is the most dangerous with increased risk of cancer progression and poorer prognosis due to lack of targeted hormonal therapy. MDA-MB-231 is a TNBC cell line as it does not express estrogen, progesterone and HER2 receptors. Herbal extracts and their isolated molecules have been found to  21 . In the present study, the evaluation of mitochondrial-mediated apoptosis, cell cycle arrest and underlying intracellular signaling pathways of cell death by ADPE against human TNBC MDA-MB-231 cells has been reported for the first time.
The anti-proliferative effect of ADPE on MDA-MB-231 cell line has been shown in Fig. 1. The MTT results revealed that exposure of MDA-MB-231 cells to ADPE resulted in growth inhibition of cells in a dose-and timedependent manner. ADPE showed IC 50 values of 17.45 and 16.67 mg/mL at 24 and 48 h, respectively. ADPE was also tested on normal human PBMCs to ascertain its toxicity. Interestingly, ADPE was found to be non-toxic without any associated morphological effects on PBMCs (Fig. 2e and f), which suggests that ADPE is non-toxic to normal human cells. As majority of the phytocomponents present in the pulp of Ajwa dates were characterized as polysaccharides in LC-MS, therefore, larger proportions are needed to elicit biological efficacy of ADPE. This study suggests that consumption of large portions of Ajwa dates may be conducive to lead a healthy and cancerfree life. The morphological data under inverted phase-contrast microscopy revealed the natural and fibroblastic morphology of untreated TNBC cells. ADPE caused a decrease in the number of cells by an alteration in their shape and adherence. These events represent the classical features of early apoptosis 22 . Further, to confirm apoptotic cell death, MDA-MB-231 cells were investigated using Hoechst 33342 nuclear stain under a fluorescence www.nature.com/scientificreports/ microscope. The treated cells displayed cell shrinkage, blebbing of plasma membrane without loss of integrity, nuclear condensation and formation of pyknotic bodies (Fig. 3). Further, AO/EtBr double stain was used to analyze the early and late apoptosis in ADPE treated cells. In early apoptosis, AO binds within the fragmented DNA of the cells emanating bright green fluorescence, while in late apoptosis; PI binds to denatured DNA displaying reddish-orange color. This study suggests that lower doses of ADPE lead to early apoptosis while higher doses lead to the late stages of apoptosis. The preliminary assays such as nuclear condensation assay, AO/EtBr assay and DNA fragmentation assay are the initial confirmation of apoptotic/necrotic cell death. Many previous studies have tested these preliminary assays to validate the induction of cell death via apoptosis or necrosis [23][24][25] . Therefore, based on these preliminary tests, Annexin V-FITC& PI double staining assay was carried out to quantify the percent of early and late apoptosis and necrotic cells. Flow cytometry analysis of Annexin-V/FITC & PI double stain depicted that the percentage of viable cells decreased with a concomitant increase in the percentage of cells undergoing early and late apoptosis. At a low dose, ADPE treatment resulted in early apoptotic cells while late apoptotic stages were found at higher doses (Fig. 3). This data suggests that ADPE treatment pushes the cancer cells into late apoptosis stage. A previous study has also reported that methanolic extract of Ajwa dates induces apoptosis in breast cancer MCF-7 cells by increasing the percentage of cells in late apoptosis 26 . DNA fragmentation data also confirmed the apoptosis-inducing efficacy of ADPE against MDA-MB-231 cells.
The regulation of intracellular ROS levels is crucial in maintaining cellular homeostasis and thus, different ROS levels can cause diverse biological responses. At low levels, ROS act as signaling molecules while at high levels they induce cell damage and death 27 . Therefore, the generation of ROS levels in treated cells was examined using DCFH-DA stain through fluorescence microscopy and flow cytometry. Results revealed that ADPE increased the level of ROS generation in a dose-dependent manner (Fig. 3). Excess cellular levels of ROS are responsible for damaging the various biomolecules such as proteins, lipids, nucleic acids, cell membranes and organelles which may result in progressive cell dysfunctions and cellular apoptosis 27 . Oxidative stress can disrupt the balance between ROS production and radical-scavenging leading to loss of MMP and release of cytochrome c from the inner mitochondrial membrane resulting in cellular apoptosis 28 . Thus, ADPE has been found to decrease MMP in MDA-MB-231 cells in a dose-dependent manner (Fig. 4). Excessive ROS production can activate various signaling molecules in cancer cells which initiate cell cycle arrest and apoptosis 29 . The cell cycle data revealed that ADPE treatment of MDA-MB-231 cells resulted in arrest of cells in S phase and sparingly in the G2/M phase with an accompanying decrease in the G0/G1 phase (Fig. 5). This study confirmed that ADPE interferes the initiation of DNA replication and thus arrests MDA-MB-231 cells at the S phase, while cells arrested in G2/M phase are restricted by ADPE to repair damaged DNA before entering mitosis. A previous www.nature.com/scientificreports/ study has also shown that Allium atroviolaceum flower extract induces S and G2/M phase arrest in MCF-7 and S phase arrest in MDA-MB-231 cells 30 . ROS cause cell cycle arrest and induce apoptosis by activating various signaling cascades and signal-regulating kinase pathways 27,31 . Apoptosis also requires permeabilization of the outer mitochondrial membrane which is controlled by the Bcl-2 family proteins. Therefore, the present study also attempted validation of our hypothesis on the modulation of the Bcl-2 family proteins and signal-regulating AKT/mTOR pathway in the ADPE-extract mediated apoptosis against MDA-MB-231 cells. For this, protein expression of p53, Bax, Bcl-2 and cleaved caspase-3 was analyzed along with the expression of p-AKT and p-mTOR in ADPE-treated MDA-MB-231 cells. Protein expression data revealed that ADPE increased the expression of tumor suppressor p53, pro-apoptotic Bax, and effector cleaved caspase-3 and reduced the expression of anti-apoptotic Bcl-2 protein (Fig. 6). Tumor suppressor p53 gene, also called guardian of the genome, plays an important role in cell growth inhibition and induction of apoptosis after DNA damage. After DNA damage, p53 gene is activated and it promotes high expression of pro-apoptotic regulator Bax and low expression of anti-apoptotic gene Bcl-2. Eventually, the modulation of the Bcl-2 family proteins initiates cascade reaction of caspases leading to caspase-3 activation and resulting in nuclear apoptosis 22,27 . Thus, our results clearly show that ADPE exhibited apoptosis via intrinsic pathway.
Growth factors can suppress apoptosis and regulate cell growth and cell survival in a transcription independent manner by activating the serine/threonine specific protein kinase, AKT 32 . Activated AKT, also known as Protein kinase B (PKB), translocates to the cytoplasm and nucleus and activates a number of downstream targets following activation of mTOR 33 . The mammalian target of rapamycin (mTOR) functions as a serine/threonine protein kinase, which can affect gene transcription and translation by regulating cell growth, cell proliferation and cell survival 34 . AKT promotes cell survival by inhibiting apoptosis, and thus it can be said that AKT is downregulated during apoptosis processes. Therefore, due to suppression of the AKT/mTOR pathway, cells lose their survival and proliferation capability; which may trigger programmed cell death, including apoptosis, autophagy and necroptosis. Interestingly, our protein expression data revealed that the expression level of p-AKT and p-mTOR underwent a down regulation after 48 h of ADPE treatment in MDA-MB-231 cells (Fig. 6). A previous study has also reported that Murraya koenigii leaves extract induced mitochondrial apoptosis in DLD-1 colon

Conclusions
In conclusion, the present study revealed the potent growth-inhibitory effect of Ajwa dates pulp against human TNBC MDA-MB-231 cells with no significant toxic effect on normal human PBMCs. The growth-inhibitory effect was found to be associated with ROS generation, MMP depletion, cell cycle arrest, upregulation of tumor suppressor p53, pro-apoptotic Bax, and effector cleaved caspase-3 and downregulation of anti-apoptotic Bcl-2 protein, p-AKT and p-mTOR signaling molecules. Figure 7 summarizes the proposed mechanism of action underlying The current research has focused on a single TNBC cell line and not all TNBC cell lines or triple-negative breast cancer cases. Furthermore, the cytotoxicity test was carried out on normal human PBMCs rather than a normal mammary cell line. As a result, further research into the mechanism of ADPE is needed on all TNBC cell lines and normal mammary cells. Based on the findings of this study, ADPE has the potential to be developed into a novel and effective anticancer drug against human breast cancer in future, and/or as a potent adjunct to the mainline of breast cancer therapy. However, further clinical trials are required to confirm the therapeutic basis of drug development. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.