Cytostatic and Anti-tumor Potential of Ajwa Date Pulp against Human Hepatocellular Carcinoma HepG2 Cells

Ajwa dates (Phoenix dactylifera L.) are used by traditional therapeutic practitioners for several health benefits but most remain to be scientifically validated. In this study, we evaluated the apoptosis-inducing effect of ethanolic extract of Ajwa date pulp (ADP) on human hepatocellular carcinoma (HCC) HepG2 cells. High performance liquid chromatography analysis revealed the presence of polysaccharide β-D-glucan in ADP extract. Treated HCC cells revealed morphological characteristics of apoptosis under phase contrast microscopy. MTT assay demonstrated significant (p < 0.05) dose- and time-dependent inhibition of HCC cell growth. HCC cells were found to be in late apoptotic stage on treatment with higher doses of ADP extract as depicted by acridine orange/ethidium bromide and Annexin V-FITC/PI double stain. Importantly, ADP extract increased the reactive oxygen species level and decreased the mitochondrial membrane potential in treated HCC cells. Flow cytometry analysis demonstrated that ADP extract induced elevation of S and G2/M phases of cell cycle. Moreover, ADP extract induced apoptosis in HCC cells independent of tumor suppressor genes viz. CHEK2, ATM and TP53. Interestingly, ADP extract did not display any significant effect on normal cell line Vero. This study provides validation that ADP extract can be considered as a safe and natural potential drug candidate against human liver cancer.

HPLC analysis of ADP extract. ADP extract was characterized using HPLC on a Waters 515 HPLC Pump system (Milford, USA) equipped with a Waters 2998 PDA detector, a Waters column temperature controller, a pump control module coupled with an empower chromatography workstation. For chromatographic analysis, an XBridge C 18 Reverse Phase column (4.6 × 250 mm, 5 μm), with gradient elution as the mobile phase, was adopted. The mobile phase consisted of a mixture of water (Solvent A) and methanol-acetonitrile (Solvent B) which was applied as a gradient for 40 min. The standard β-D-glucan and ADP extract were dissolved in HPLC grade water and filtered through a 0.45 μm membrane filter and the injection volume was 10 μL. The flow rate was 1.0 mL/min and the column temperature was set at 30 °C. The gradient of mobile phase was as follows: 80% A, 20% B for 0-6 min; 70% A, 30% B for 6-12 min, 30% A, 70% B for 12-18 min; 20% A, 80% B for 18-25 min; 20% A, 80% B for 25-30 min; 80% A, 20% B for 30-35 min and 90% A, 10% B for 35-40 min. HPLC was monitored at 254 nm to provide real-time chromatograms of both standard and ADP extract.
Cell lines and culture. Human HCC HepG2 and normal kidney epithelial Vero cell lines were obtained from the cell repository of National Centre for Cell Sciences, Pune, India. Cells were grown in Dulbecco's Modified Eagle Medium: F12 (1:1) supplemented with 10% heat-inactivated Fetal Bovine Serum, 2 mM L-glutamine, 1% penicillin and streptomycin solution. Cells were cultured in cell culture flasks and were kept in an incubator (Thermo Scientific, USA) at 37 °C and 5% CO 2 .
MTT assay. The antiproliferative activity of ADP extract was evaluated by MTT reduction assay following a previously published protocol 20 . HepG2 and Vero cells were seeded at a density 1 × 10 4 cells/mL in 96-well microtiter culture plates and incubated overnight. ADP extract was diluted in culture media and treated in triplicate with different concentrations (10,15,20,25 and 30 mg/mL) of ADP extract 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 phase contrast microscope (Nikon Eclipse TS100, Japan).
Nuclear condensation assay. Based on the cell viability assay, the apoptosis-inducing effect of ADP extract was evaluated at two effective doses viz. 15 and 25 mg/mL. DNA condensation was measured using Hoechst 33258 staining as per a previously published method 21 . To assess nuclear morphology, stained cells were captured under an inverted fluorescence phase contrast microscope (Zeiss AxioVert 135, USA).
Acridine orange-ethidium bromide (AO/EtBr) assay. The mechanism of cytotoxicity of ADP extract on HepG2 cells was evaluated as reported previously 22   Analysis of cellular DNA content. Cells were seeded at density 1 × 10 6 cells/mL into 6-well plates and treated with ADP extract (15 and 25 mg/mL) for 48 h. Different phases of the cell cycle with cellular DNA contents were analyzed using flow cytometry as described previously 20 .

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 less than 0.05 was considered as statistically significant.

Results
HPLC characterization of ADP extract. The chromatograms of standard β-D-glucan and ADP extract are presented in Fig. 1. The peak area and percentages of different components with a specific retention time (R t ) in HPLC chromatograms are shown in Table S1. The HPLC chromatographic analysis using a reverse phase column and water (A) and methanol-acetonitrile (B) solvents as mobile phase provided fine separation of β-D-glucan with R t value of 24.831 min at 254 nm in chromatograms ( Fig. 1a and Table S1). The corresponding peak of ADP extract in HPLC analysis was found at a R t value of 24.87 min under similar conditions (Fig. 1b). This study revealed the presence of β-D-glucan as an active component in ethanolic extract of ADP.  ADP extract stimulated chromatin condensation and induced apoptosis. As is evident from photomicrograph (Fig. 3a), ADP extract at 15 mg/mL increased the chromatin condensation in HCC cells as compared to control. However, 25 mg/mL of ADP extract exhibited maximum nuclear condensation. Furthermore, the AO/EtBr double stain revealed that control cells displayed uniformly stained green-colored nuclei indicating live and healthy cells. Treated cells appeared either green-colored with condensed nuclei indicative of early apoptosis, or orange-red colored cells with condensed nuclei indicative of late apoptosis. HCC cells displayed early apoptotic features at low doses whereas late apoptotic features were observed at higher doses of the ADP extract (Fig. 3b). Moreover, ADP extract was tested to ascertain DNA fragmentation in HCC cells. The results obtained (Fig. 3c) showed undamaged DNA with the intact band in control well, whereas treated cells displayed progressive DNA fragmentation in a dose-dependent manner. Little DNA shearing was observed at 15 mg/mL of extract which was found to increase at 20 mg/mL of ADP extract. . Untreated cells showed 88.4% viability and were typed as being alive and healthy whereas 15 mg/mL ADP extract increased the cell death by inducing 8.8% early apoptotic and 10.5% late apoptotic cells (Fig. 4). Moreover, 25 mg/mL ADP extract caused induction of a remarkable 32.8% early apoptotic and 46.7% late apoptotic cells as compared to control group.
ADP extract induced intracellular ROS generation. As revealed in Fig. 5a, HCC cells treated with ADP extract showed a significant increase in ROS intensity in a dose-dependent manner as compared to untreated cells. The results of the flow cytometry measurement of ROS generation showed that control cells displayed 3.6% ROS which is a characteristic of normal healthy cells that generate little amount of ROS intensity. However, 15 mg/mL of ADP extract enhanced ROS level by 10.5% as compared to control. Moreover, ROS production was found to increase enormously by 25% at 25 mg/mL of ADP extract (Fig. 5b).
ADP extract decreased the MMP. Loss of MMP was indicated by the decrease of red fluorescence of the fluorescent dye Rh 123 as revealed in the photomicrograph (Fig. 6a). Figure 6b depicts the percent MMP activity analyzed by flow cytometry. Results suggested that treatment of HepG2 cells with ADP extract resulted in a

ADP extract regulated the apoptosis-related genes in HCC cells. ADP extract was further used
to investigate the expression of tumor suppressor genes of TP53 pathway and to ascertain whether these were involved in the apoptosis-inducing mechanism of cell death or not. As is clear from the qRT-PCR analysis, ADP extract modulated the expression of tumor suppressor genes such as CHEK2, ATM and TP53 in treated HCC cells. As shown in Fig. 8, ADP extract decreased the expression of CHEK2 by 0.77-fold and 0.67-fold at 15 and

Discussion
The present study undertook the unprecedented investigation of cytotoxicity and underlying mechanism of apoptotic cell death induced by ADP extract on human HCC HepG2 cells. This study also examined the active component of ADP extract, which may have a potential role in causing cytotoxicity. The result of the HPLC study revealed the presence of β-D-glucan as an active component in ethanolic extract of ADP. A previous study has reported that β-glucan, a phytochemical component of Libyan Ajwa dates, demonstrated antitumor activity against solid tumor in mice, which was found to be related to (1 → 3)-β-D-glucan linkages 24 . Likewise, a study has examined the anticancer activity of the low molecular weight β-glucan from oats against skin melanoma cell line Me45 and skin epidermoid carcinoma cell line A431, which significantly decreased cancer cell viability in a time-and dose-dependent manner, while for the normal keratinocyte cell line HaCaT, it was reported to be non-toxic 25 . Interestingly, the present study also demonstrated that ADP extract mediated both dose-and time-dependent anti-proliferative effects against HCC cells, while having no toxicity against the normal Vero cell line. A previous study has reported that the date pulp also contains phenolics like quercetin and kaempferol 15 , which possess anticancer activity against HCC cells 26,27 . Thus, it can be postulated that the anticancer effect of ADP extract against HCC cells might be due to the synergistic or combined effect of the potential bioactive components of Ajwa dates. Previously, various plant extracts have been evaluated for their anticancer activity against HCC cell line. The IC 50 values of Chinese medicine Fanbaicao extract and aqueous extract of Chlorella vulgaris have been reported to be 2.03 and 1.6 mg/mL, respectively, at 24 h 28,29 , whereas 1.30 mg/mL for Sophora moorcroftiana seed extract at 48 h 30 . In the present study, ADP extract induced cell death of HCC cells with an IC 50 value of 20.03 and 16.78 mg/mL after 24 and 48 h exposure, respectively (Fig. 2d). In a previous study, the total sugar content of Ajwa date pulp was found to be 74.3 g/100 g dry weight 31 . Because of a large amount of polysaccharide in ADP, higher doses would be needed for the efficacy of ADP extract against HCC cells. Interestingly, Al-Bukhaari (5445) and Muslim (2047) have narrated from Sa' d ibn Abi Waqqaas that the Prophet (PBUH) said: "Whoever eats seven Ajwa dates in the morning, will not be harmed by any poison or witchcraft that day. " This is the reason why one should intake a large amount of Ajwa pulp to lead a healthy life.
Previous studies have also reported the growth inhibitory and cytotoxic effects of ADP extract against various cancer cells 18,19 . The cell viability data of the present study has indicated that ADP extract is equally effective on HCC cells. However, the underlying mechanism of ADP extract mediated anti-proliferative effect in HCC cells has not been studied so far.
The morphological data revealed that ADP extract treated HCC cells acquired a round shape, showed cluster shrinkage and detachment from the surface. In contrast, untreated cells remained intact with regularity in shape. This result showed the initial characteristic features of apoptotic cell death 32 . To confirm the efficacy of apoptosis, this study further investigated the major apoptotic events in ADP extract treated HCC cells under a fluorescence microscope. Nuclear condensation data revealed that exposed cells displayed typical apoptotic features viz. chromatin condensation, fragmented nuclei and extensive cytoplasmic vacuolization as compared to untreated cells (Fig. 3). The AO/EtBr double stain data depicted early and late apoptosis in ADP treated cells. The early apoptotic cells were detected via the binding of AO within the fragmented DNA displaying a bright green fluorescence at a low dose of ADP extract. However, higher dose of ADP extract led to the late stages of apoptosis as indicated by the presence of a reddish-orange color because of the binding of PI to denatured DNA. Moreover, to justify these results quantitatively, a flow cytometry analysis of Annexin-V/PI double stain was performed. The result indicated that the percentage of viable cells was decreased with a concomitant increase in the percentage of cells undergoing early and late apoptosis. A lower dose of the ADP extract led to early apoptotic cells while late apoptotic stages were found at a higher dose of the ADP extract (Fig. 4). This quantitative data suggested that ADP A previous study has also reported that methanolic extract of Ajwa dates induced apoptosis in breast cancer MCF-7 cells by increasing the percentage of cells in late apoptotic stage 18 . DNA fragmentation data also confirmed the apoptotic efficacy of ADP extract against HCC cells.
To confirm the apoptotic mechanism of cell death, intracellular ROS generation was evaluated in ADP treated HCC cells. Overproduction of ROS disrupts the plasma membrane and cytoskeleton and finally leads to chromosomal damage 33 . ROS has been regarded as an important regulator of both extrinsic and intrinsic pathways of cell survival and cell death 34 . Various natural agents that are used as anticancer compounds can lead to cell death of many cancer cells by causing overproduction of ROS 35 . Flow cytometry analysis of ROS generation confirmed that ADP extract stimulated ROS production in HCC cells by causing oxidative stress, destabilizing mitochondria and consequently induced apoptosis (Fig. 5).
Mitochondria play a vital role in both cell survival and cell death by sending the death signals to the cascades. When cells undergo apoptosis, the mitochondria lose their membrane integrity and release cytochrome c into the cytosol that ultimately leads to the formation of apoptosome and completes the intrinsic apoptotic pathway 36,37 . In the present study, both fluorescence microscopy and flow cytometry data showed the disruption of the mitochondrial membrane integrity and loss of MMP in ADP extract treated HCC cells (Fig. 6). Loss of fluorescence intensity of Rh 123 dye inside mitochondria due to loss of mitochondrial integrity revealed the comprehensible difference between the apoptotic and viable cells. This study suggested that ADP extract induced the apoptotic events through the intrinsic pathway. Cell-cycle arrest in response to stress is integral to the maintenance of genomic integrity. Cell cycle arrest provides sufficient time for the cells to repair damaged DNA. In case of severe damage, cells proceed to apoptosis, thus stopping the proliferation of cancer cells 38 . The cell cycle analysis in the present study revealed a higher percentage of cells in the S and G2/M phase whereas the percentage of cells in the G0/G1 phase was decreased as compared to control cells (Fig. 7). These findings are consistent with a previously published study in which paclitaxel, an anticancer drug, inhibited human tenon's fibroblast cell proliferation through cell cycle arrest at both S and G2/M phases 39 . These results indicated that ADP extract inhibited cell proliferation via S and G2/M phase arrest in a dose-dependent manner.
The present study has also attempted a validation of our hypothesis about TP53 implication in the ADP-extract mediated apoptosis of HCC cells. For this study, the gene expression of ATM, CHEK2 and TP53 were analyzed by qRT-PCR. The ATM is a key checkpoint molecule regulating cell cycle responses to DNA damage either by cell cycle arrest or apoptosis 40 . CHEK2, a tumor suppressor gene, is involved in DNA repair, cell cycle arrest or apoptosis in response to DNA damage 41 . Upon severe damage of cells, ATM along with ataxia telangiectasia and Rad3-related protein (ATR) phosphorylates and activates the protein kinase CHEK2 which results in the activation of tumor suppressor gene TP53 42 . Increased level of TP53 augments numerous target genes such as p21, Mdm2 and Bax that mediate cell cycle arrest and apoptotic cell death 43 . On the basis of cell cycle arrest, other possible mechanisms of cell death in HCC cells can be suggested. In a previous study, pemetrexed induced S-phase arrest and apoptosis in human non-small-cell lung cancer A549 and H1299 cell lines through serine/ threonine protein kinase (Akt) activation which stimulated Cdk2/Cyclin A-associated kinase activation and then promoted the movement of cells into the S phase 44 . Another recent study has shown that the down-regulation of serine-threonine kinase receptor-associated protein (STRAP), an important regulator of cell proliferation might be responsible for the anti-proliferation and S-phase arrest in HepG2 cells by blocking the DNA repair function of p53 30 . On the basis of G2/M phase arrest, another possible mechanism of apoptotic cell death may be implicated. A study has shown that inhibition of c-Jun N-terminal kinase (JNK) leads to a G2/M phase arrest in breast cancer cells independent of p53 function 45 . Moreover, a previous report showed that erianin induced apoptosis and G2/M-phase arrest in human osteosarcoma cells via the ROS/JNK signaling pathway 46 . Thus, it can be postulated that ADP extract induced apoptotic cell death in HCC might be mediated through the Akt pathway, downregulation of STRAP as well as through ROS/JNK signaling pathway. The present study, thus, confirmed that ADP extract mediated cell cycle arrest at both S and G2/M phase in HCC cells followed by apoptosis through a TP53-independent pathway. Interestingly, in independent studies, Artonin E, a ruthenium-xanthoxylin complex and a novel analog of varacin C have been reported to induce apoptosis in target cancer cells via a TP53-independent pathway [47][48][49] . It would be our endeavor in future to evaluate and assess the anticancer activity of ADP-extract via alternative pathways of apoptosis induction in vitro and in vivo.
In conclusion, the present study revealed the potent growth-inhibitory effect of ethanolic extract of ADP against human liver carcinoma HepG2 cells with little to no effect on normal Vero cells. The effect was found to be associated with ROS generation and MMP depletion in cancer cells. Moreover, ADP extract induced DNA damage in HCC cells leading to cell cycle arrest at S and G2/M phases, and followed by apoptosis through a TP53-independent pathway. This study also examined the presence of β-D-glucan in ADP extract, which has a potential role in apoptotic cell death. To conclude, ADP extract has the potential for development into a novel and potent anticancer drug against human liver cancer in future, albeit with further clinical studies to validate the therapeutic basis of drug development.