Induction of apoptosis by phytochemicals of Ajwa dates extract in human triple-negative breast cancer cells by triggering cell cycle arrest, inhibiting Akt/mTOR signaling and modulating downstream Bcl-2 family of proteins


 Background:Ajwa date (Phoenix dactylifera L.) has been described in traditional and alternative medicine to provide several health benefits including anticholesteremic, antioxidant, hepatoprotective and anticancer effects, but most remain to be scientifically validated. In the present study, we evaluated the anticancer effects of the ethanolic extract of Ajwa Dates pulp on human triple-negative breast cancer MDA-MB-231 cells. MethodsAjwa Dates Pulp Extract (ADPE) was phytochemically characterized using high performance liquid chromatography coupled with mass spectrometry (LC-MS). The in vitro cytotoxicity of ADPE at various concentrations viz. 10, 12, 15, 18, 20, 22 and 25 mg/mL were evaluated against MDA-MB-231 and MCF-7 cells at 24 and 48 h. The apoptosis effect was examined by Hoechst 33342 and AO/PI-double-stain using fluorescence microscopy. The proportion of apoptotic cells, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP) and cell cycle phase distribution were estimated using flow cytometry. Apoptosis-related protein expression was determined using western blot analysis. ResultsLC-MS analysis showed that ADPE contained phytocomponents belonging to chemical classes such as carbohydrates, phenolics, flavonoids and terpenoids. Phase contrast microscopy analysis revealed various morphological variations in ADPE treated cells. MTT assay demonstrated statistically significant dose- and time-dependent inhibitions of MDA-MB-231 cells with a half-maximal inhibitory concentration 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, Ajwa date extract prompted ROS induced alterations in MMP in ADPE treated MDA-MB-231 cells. Cell cycle analysis demonstrated that ADPE induced cell arrest in S and G2/M checkpoints. In addition, ADPE upregulates p53, Bax and cleaved caspase-3, thereby leading to downregulation of bcl-2 protein and Akt/mTOR pathway. ConclusionsThe results of the present study indicate that ADPE exerts significant anticancer effects on MDA-MB-231 cells via the induction of apoptosis and suppression of AKT/mTOR signaling pathway. Therefore, ADPE has the potential to be used as an adjunct to the main line of treatment against breast cancer and can be further studied as a potential lead in breast cancer treatment.

The results of the present study indicate that ADPE exerts significant anticancer effects on MDA-MB-231 cells via the induction of apoptosis and suppression of AKT/mTOR signaling pathway. Therefore, ADPE has the potential to be used as an adjunct to the main line of treatment against breast cancer and can be further studied as a potential lead in breast cancer treatment.

Background
Breast cancer is an abnormal growth of cells lining the breast lobules or ducts. These cells grow uncontrollably and have the potential to spread to other parts of the body. Breast cancer impact 2.1 million women each year, and also causes the greatest number of cancer-related deaths among women. Though, the prevalence of breast cancer in Asia is lesser than that in Western countries, but in the modern time, the relative contribution to the global burden of breast cancer is rising rapidly in Asia [1]. In 2019, there was an estimation of 268,600 new cases of invasive breast cancer among female to be reported in the US and 41,760 estimated death due to breast cancer [2]. Breast cancer is the most common cancer of females in India, which accounts for 25-32% of female cancers in India.
One woman is diagnosed with breast cancer every four min and one women dies of breast cancer every thirteen min in India [3]. Breast cancer projection is expected to go as high as 17,97,900 in 2020 [4].
At the present time, various treatments including surgical resection, chemotherapy, radiation therapy, hormonal therapy and synthetic lethality have been developed. However, surgical resection at the metastasis stage often limits its effectiveness and chemotherapy and radiotherapy can have a negative effect on normal cells [5]. Therefore, treatment with insignificant adverse effects, specifically killing cancer cells with less toxicity to normal cells may present as a practical approach for improving breast cancer therapy. Molecularly targeted therapies that block tumor cell proliferation and are less toxic to normal cells, may contribute to improving survival and quality of life in breast cancer patients [6,7].
Phytonutrients like carotenoids, isothiocynates, phenolic compounds, flavanoids, organo-sulphides, isoflavones and indoles found in plant-based foods such as fruits, vegetables, beans and grains may help to prevent and fight breast cancer. Foods that are rich in fiber, such as whole grains, beans, and legumes may also help in fighting breast cancer. Ajwa date fruits being a rich source of antioxidants, flavanoids as well as fiber can serve as a good anticancer agent against breast cancer. Ajwa dates (Phoenix dactylifera L.), belonging to family Arecaceae contain polyphenolics and other bioactive compounds, which are used in traditional remedies for potential prevention of cell damage and cancer therapeutics [8]. Date fruits have been consumed in Arab and its neighboring areas since time unmemorable as part of the essential diet. Recent research has shown the anticancer potential of Ajwa dates extract in combination with 5-fluorouracil against human breast adenocarcinoma cell line MCF-7 [9] and apoptosis-inducing effect and cell cycle arrest in prostate cancer cells PC3 [10]. In addition, the ethnopharmacological significance of date palm is well-described in terms of its antioxidant, anti-inflammatory and antitumoral activities against breast carcinoma cells [11]. Other parts of the date palm have alos been found to exhibit potent anticancer activity viz. seed extract against azoxymethane-induced colon carcinoma in rats, root hair extract in combination with silver nanoparticles [12] and palm pollen extract in combination with silver and gold nanoparticles against breast adenocarcinoma cells [13] and leaf extract against human malignant melanoma cell line IGR-39 [14]. However, none of the studies have, so for, reported the mechanism of anticancer action of Ajwa dates pulp extract by modulation of Bcl-2 family proteins and downregulation of Akt/mTOR signaling pathway in breast carcinoma MDA-MB-231 cells.
In the present study, MDA-MB-231 cell line was treated with various concentrations of ADPE and was analyzed for the underlying regulatory mechanism(s) of apoptotic cell death. To identify major phytoconstituents in ADPE, phytochemical characterization of ADPE was done using LC-MS.

Preparation of Ajwa dates extract
Fresh Ajwa dates were procured from Al-Madina Al-Munawwarah, Kingdom of Saudi Arabia. The pulp part of date fruit was manually separated, washed with double distilled water, sun-dried and coarsely powdered using pestle and mortar. The coarse powder contents were then extracted in 95% ethanol

MTT assay
The cell viability of ADPE against breast cancer cells was evaluated by MTT reduction assay following a previously published protocol [15]. MDA-MB-231 and MCF-7 cells were seeded at a density of 1 × 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
To know the expression pattern of apoptosis-inducing proteins, MDA-MB-231 cells at a density of 1 ×

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.  (Fig. 1). On comparison of the mass spectra of the constituents, eight phytocomponents were characterized and identified as shown in Table 1

ADPE stimulates chromatin condensation and induces apoptosis
As is evident from the photomicrograph (Fig. 3A), ADPE increased the chromatin condensation in MDA-MB-231 cells 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 aappeared 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). ADPE was further analyzed to determine DNA fragmentation in MDA-MB-231 cells. Intact undamaged DNA band was obtained in control well, while treated cells displayed progressive DNA damage and fragmentation depending upon dose (Fig. 3C). ADPE at 15 mg/mL showed less shearing of DNA, but it was found to be increased at 20 mg/mL of extract.

Intracellular ROS generation by ADPE
The flow cytometry analysis of ROS measurement revealed that the level of ROS in control cells was 5.5%. This small amount of ROS level shows the characteristic feature of normal healthy cells. ROS levels were enhanced by 8.5 and 14.7% at 15 and 18 mg/mL of ADPE as compared to control.
Moreover, ROS production in treated cells was increased enormously by 51.9% at 20 mg/mL of ADPE (Fig. 4B). The fluorescent microscopy analysis of ROS intensity was found to be consistent with flow cytometry data (Fig. 4A).

ADPE decreases the MMP
As is evidenced in 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) Moreover, the results demonstrated that both p-AKT and p-mTOR levels were reduced in MDA-MB-231 cells following 48 h of ADPE treatment (Fig. 6). These results indicate that ADPE inhibits AKT/mTOR signaling pathway important in regulating cell growth and proliferation in breast cancer cells.

Discussion
Among breast cancer, TNBC has increased the risk of cancer progression and also poorer prognosis due to the lack of targeted hormonal therapy. Herbal extracts and their isolated molecules have been found to be effective against various cancers [18]. In the present study, the unprecedented evaluation of mitochondrial-mediated apoptosis, cell cycle arrest and underlying intracellular signaling pathways of cell death were carried out using ADPE against human TNBC MDA-MB-231 cells.  [19]. Further, to confirm apoptosis cell death, MDA-MB-231 cells were investigated using Hoechst 33258 nuclear stain under a fluorescence 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. For quantitative analysis, treated cells were stained with Annexin-V/PI double stain and examined with flow cytometry. The result 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. 4). 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 [20]. 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 [21]. Therefore, the generation of ROS levels in treated cells was examined using DCFH-DA stain through fluorescence microscopy and flow cytometry study. Results revealed that ADPE induced the level of ROS generation in a dosedependent manner. 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 [22]. Oxidative stress can disrupt the balance between ROS production and radical-scavenging leading to loss of MMP and release of cytochrome c from the intracellular space of the mitochondria and resulting in cellular apoptosis [23]. Thus, ADPE has been found to decrease MMP in MDA-MB-231 cells in a dose-dependent manner. Moreover, excessive ROS production can activate various signaling molecules in cancer cells which initiate cell cycle arrest and apoptosis [24]. Eventually, the modulation of the Bcl-2 family proteins initiates caspases cascade reaction and activates caspase-3 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 [28]. 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 [29]. 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 [30]. 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

Conclusions
In conclusion, the present study has revealed the potent growth-inhibitory effect of Ajwa dates pulp against human TNBC MDA-MB-231 cells. 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 anticancer effect of ADPE against MDA-MB-231 cells. It is evident from the present study that ADPE has the potential for being developed into a novel and potent anticancer drug against human breast cancer in future, and/or as a powerful adjunct to the mainline of breast cancer therapy; albeit with further clinical studies to validate the therapeutic basis of drug development.

Ethics approval and consent to participate
Not applicable.

Consent for publication
Not applicable.

Availability of data and material
All data generated or analyzed during this study are included in this manuscript.
for providing technical assistance in flow cytometry analysis and Mr. R.K. Purshottam, Senior       Schematic diagram summarizing the mechanism of ADPE action on MDA-MB-231 cells.
Bioactive components from ADPE arrest G2/M and S phase checkpoints and induce apoptosis by upregulation of p53, Bax and cleaved caspase-3 thereby leading to downregulation of bcl-2 protein and Akt/mTOR pathway.

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