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.
The anti-proliferative effect of ADPE on MDA-MB-231cell lines has been shown in Fig. 1. The MTT results show that exposure of MDA-MB-231 cells to ADPE resulted in growth inhibition of cells in a dose- and time-dependent manner. ADPE showed IC50 values of 17.45 and 16.67 mg/mL at 24 and 48 h, respectively. Since 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 cancer-free 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 classic features of early apoptosis [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 dose-dependent 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]. 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. 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 by ADPE restricts the cells to repair damaged DNA before entering mitosis. A previous 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 [25].
ROS cause cell cycle arrest and induce apoptosis by activating various signaling cascades and signal-regulating kinase pathways [22, 26]. 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, we analyzed protein expression of p53, Bax, Bcl-2 and cleaved caspase-3, along with the expression of p-AKT and p-mTOR in ADPE- treated MDA-MB-231 cells. Protein expression data has revealed that ADPE increases the expression of tumor suppressor p53, pro-apoptotic Bax, and effector cleaved caspase-3, while reduces the expression of anti-apoptotic Bcl-2 protein. 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 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 and p-mTOR undergoes a down regulation after 48 h in ADPE treated MDA-MB-231 cells. A previous study has also reported that Murraya koenigii leaves extract induced mitochondrial apoptosis in DLD-1 colon cancer cells by downregulating mTOR/AKT pathway [31]. LC-MS data revealed the presence of carbohydrates containing disaccharides and their derivative along with phenolics, flavonoids, phytosterols, and carotenoids in ADPE. The identified bioactive components such as maltose, catechin, myricetin, quercetin, β-sitosterol, digalacturonic acid, chlorogenic acid and β-carotene are known to exhibit various pharmacological activities such as antioxidant, anti-inflammatory, antimicrobial, hepatoprotective and anticancer [32, 33].