Potential bioactivity of Phoenix dactylifera fruits, leaves, and seeds against prostate and pancreatic cancer cells

The use of functional foods’ phytochemicals in the chemoprevention of different cancer diseases has become one of the hot scientific areas in the clinical nutrition field. For instance, the Khalas palm cultivar (KPC; Phoenix dactylifera) is one of the natural sustainable resources that have high bioactivity and functionality. This study aimed to investigate the antiproliferative activity and mode of action of KPC’s different parts on prostate (Pc3) and pancreatic (panc1) cancer cells at a molecular level. In the methods, KPC’s leaves, seeds, and fruits’ chemical composition and phytochemical analysis were analyzed. Also, the cytotoxic effects of each extract were assessed against pc3 and panc1 cell lines. Besides, induction of apoptosis, cell cycle analysis, and gene expression of both Cap3 and Cap9 were studied. The obtained results indicated that KPC leaves extract exhibited the highest significant (P < 0.01) anti-proliferation activity against the utilized cancer cell lines compared to fruits and seeds extracts. Also, there were significant (P < 0.05) differences in the phenolic contents, flavonoid of compounds, and antioxidant power of the leaves when compared to the seeds and fruits. Additionally, the highest cytotoxic effect (lowest IC50) was recorded with leave extract than seeds and fruits. Meanwhile, the seeds extract induced (P < 0.05) the apoptosis and arrested cells in the G2/M phase as well as up-regulated the gene expression of the apoptotic-related genes (Casp3 and Casp9) compared to the control group. In conclusion, this study showed that the presence of bioactive components in the KPC different parts extracts have the significant ability to induce the apoptotic pathway that could down-regulate the proliferation of prostate (pc3) and pancreatic (panc1) cancer cells. The pathway mechanism of action was induced by the phytol molecule presented in its leaves extract.


Introduction
Cancer is one of the significant causes of global mortality and morbidity and remains a compelling challenge for public health (1,2). For instance, prostate cancer is the most common cancer in men after lung cancer (3). Rawla (4) reported that 1,276,106 new patients were recorded with prostate cancer with a 3.8% death count in men during the year 2018. Prostate cancer rates are increasing rapidly in most countries (5). Meanwhile, pancreatic cancer is considered an uncontrollable malignant tumor. Such cancer cells are the major cause of global cancer fatalities in developed and developing countries alike, including the United States, Egypt, and Saudi Arabia (4).
There are several treatment kinds for cancer diseases (2,6). These treatments include surgery, chemotherapy, radiotherapy, and immunotherapy. Such treatments have many different failure conditions due to adverse reactions, drug resistance, side effects, and drug specificity (7,8). Also, it is noteworthy that cancer prevention and therapy had been improved significantly by using phytochemical compounds, such as phenolics and flavonoid compounds (9)(10)(11). These organic compounds that represent above 50% of all modern clinical drugs have been discovered from plants and microorganisms (12). Moreover, functional foods including some of the commonly consumed herbs [plant leaves (13)] phytochemicals have significant antioxidant capacities that could act against cancer cell proliferation and differentiation (14-16). Rodríguez et al. (17) stated that natural origin therapeutics, which are rich in phenolics and flavonoid contents considered to be potent preventive drugs against cancer cells by their anti-apoptotic regulating functional groups.
The date palm tree (Phoenix dactylifera L.) belongs to the Arecaceae family and is an important crop in many regions of the world due to its overall nutritional and pharmacological effects, which have valuable components (18). There are several palm cultivators in the Middle East and North African regions such as Khalas (KPC), Riziz, Shish, Barhee, Ajwa, Anbra, and Mejdol palm date. From these varieties, the total number of KPC is about (760,476,7) trees (19,20). Additionally, Khalas date is mainly grown and manufactured in significant amounts in Saudi Arabia's eastern provinces, such as Al-Hassa, Al Kharj, and Qaseem (21). It has several secondary metabolites that act as high antioxidant, anticancer (22), hepatoprotective (23), neuroprotective, nephroprotective (24), gastrointestinal protective (25), antidiabetic (26), antihyperlipidemic (27), sexual improvement (28), and antimicrobial potentials (29,30). Moreover, In vitro trials were done to evaluate the anticancer activity of KPC extracts toward different cancer cell lines such as human epithelial colorectal adenocarcinoma (Caco-2) (31), the human melanoma-derived cell line (IGR-39) (32), ehrlich ascites carcinoma (33), and hepatocellular carcinoma (34). For instance, Rahmani et al. (35) found that KPC fruit extract reduced prostate cancer development. Additionally, the fruits contain the least amount of moisture and maintain a delicate texture and sweet taste in the last stage of ripe dates, characterized by good storage capacity. Mineral content in dates has been measured in a variety of studies. According to a recent study, the concentration ranges for Mn, Zn, Cu, and Se (percent mg) in dates from Bangladesh were 0.85-1.1, 0.69-0.72, 0.32-0.36, and 0.22-0.31, respectively (36). In prior research of five date cultivars (Ajwa, Beid, Burni, Rabia, and Safawi), potassium was shown to be the most prevalent macroelement (0.185-1.51%) in all of the dates studied. The concentrations of Zn and Cu were found to be between 27.5 and 72.70 ppm and 4.95 and 6.25 ppm, respectively (37). Thus, it can work as an effective anticancer agent. However, the actual preventive mechanism of its components is still not evident. Therefore, several reports have recommended further in-depth studies to understand the KPC biological, therapeutic, and nutritive roles of at the cellular and molecular levels (15,22,38).
The present study aimed to evaluate the antiproliferative impact of KPC's leaves, seeds, and fruit extracts against pancreatic and prostate cancer cell lines. Thus, to provide evidence of the applicability of KPC extracts as bioactive anticancer agents based on their unique chemical structures for its potential application in the clinical nutrition field.

Samples collection and extract preparation
The air-dried plant materials were cut into small pieces and dried in shade for 2 days until they were completely dried, then pulverized into fine powder. Next, 500 g of powdered materials were packed in the soxhlet apparatus extraction unit (Soxtherm, Gerhardt, Germany) and successive extraction was performed using ethanol solvent. The solution of the extract was filtered through Whatman filter paper no. 1 and concentrated using a rotary flash evaporator and dried under a vacuum condition.

Determination of total phenolic contents
The total phenolic contents were determined by the Folin-Ciocalteu method as described by Pieme et al. (40) with some modification at Food Technology Research Institute, Agriculture Research Centre, Giza, Egypt. In the mixture, 180 µL diluted extract, 750 µL diluted Folin Ciocalteu reagent, and 7.5% of 2 mL sodium carbonate were added together to make a substance. After mixing the substance, 7 mL of deionized water was added. After that, put the mixture in the dark at ambient conditions for 2 h so that the substance can finish its reaction. It should be noted that 765 nm absorbance was recorded. Moreover, Garlic acid was referred to as standard. The units were mg gallic acid (GAE; 5-100 µ g/mL).

Determination of total flavonoid contents
The total flavonoids were measured using the aluminum chloride colorimetric method according to Charoensin (41) at Food Technology Research Institute, Agriculture Research Centre, Giza, Egypt. One hundred µl were added together with 1.5 ml of 95% ethanol, 100 µL of 10% AlCl 3 , 100 µl of 1 M potassium acetate, and 2.8 ml of deionized water in each extract. It should be noted that the absorbance of the substance is found to be 415 nm. The unit of total flavonoid content was milligram quercetin equivalent per gram extract (mg QE/g extract).

Determination of antioxidant activity
The antioxidant activity of each extract was detected according to Bray et al. (42). Methanolic extracts in various concentrations (100-500 µL) were mixed separately with 2.5 mL of 0.2 mM PBS (pH 7.4) and 2.5 mL of potassium ferricyanide (1% w/v). Each mixture was incubated at 50 • C for 20 min. Then, 2.5 mL of trichloroacetic acid (10% w/v) was added and centrifuged at 3,500 rpm for 8 min, followed by 2.5 mL of distilled water and later 0.5 mL of ferrous chloride (0.1% w/v). The absorbance at 700 nm was estimated. As a positive reference standard, ascorbic acid was utilized.

Separation of the phytochemicals by GC/MS
Egypt. In brief, the mobile phase (helium; 99.99%) was used at a flow rate of 1 mL/min. The instrument was equipped with a capillary column DB-5MS (30 m length, 0.25 mm thickness, 0.25 m diameter). A 1 µL sample was injected into the split/splitless inlet in splitless mode at 260 • C. The temperature programming of the GC/MS method analysis starts with the oven temperature. 60 • C for 5 min, then increased by 10 • C/min to 240 • C for 15 min. the temperature of the interface was 220 • C and the ion source 200 • C. The range of scan mode (50-550 amu) was used for data acquisition. The relative amount of each constituent was calculated by measuring the corresponding peak area and represented as a percentage of the sum of areas of all peaks.

Cancer cell lines and culture
Cells of pancreatic cancer (Panc1) and prostate cancer (Pc3) were suspended in a medium containing 10% fetal bovine serum (RPMI 1640) and incubated at 37 • C in atmospheric 5% CO 2 .

Detection of cell viability
The MTT cell viability assay was utilized to explore the activity of each extract against pancreatic (Panc1) and prostate (Pc3) cancer cell lines. MTT assay relies on the dehydrogenase enzyme of a living cell to split the MTT tetrazolium yellow rings to make an unsolvable dark blue formazan crystal. This crystal was impermeable to cell membranes that cause its growth in healthy cells. The solubility of formazan's dark blue color depends on the count of living cells. It was found that dwindling in MMT measured to be 570 nm approximately. The used cell lines were plated in serum-free culture media in a flat bottom 96 well microplate 0.5 × 10 5 cell/well and used with multiple concentrations ranging from 312.5 to 10,000 µg/mL of each extract in a mummified 5% CO 2 atmosphere at 37 • C for 48 h. After the incubation, the media was removed. Each time 40 µL of MTT solution/well was included. The plates were swung and then the solution kept at room temperature for 4 h. A microplate ELISA reader was used to calculate the absorbance at 570 nm. Three replicates were measured for each sample. Data were presented as a relative viability% as follows: Then, the IC 50 % was calculated from the dose-dependent curve.

Induction of apoptosis and cell cycle analysis
Apoptosis and necrosis induction was investigated by Annexin V-FITC/PI apoptosis assay according to Pumiputavon et al. (44). Cell lines at 1 × 10 5 cells/mL were fixed in 24well plates under IC 50 % methanolic concentration for 24 h in each extract. The population of apoptotic cells was calculated by Annexin V-FITC (fluorescein isothiocyanate)/P.I. (propidium iodide) co-staining assay. After 24 h of incubation, the cells were collected and then passed through the centrifugal process at 1,800 rpm for 8 min. The pellet was resuspended in a 50 µL binding buffer containing 0.5 µL Annexin V-FITC and then incubated at 4 • C for 30 min in the dark. Propidium iodide (PI) (Sigma-Aldrich, USA) (50 µg/mL) in 200 µL binding buffer was supplemented in each tube and then incubated for 5 min. Lastly, flow cytometry was used to further investigate the cell.
For cell cycle analysis, cells (1 × 10 5 cells/mL) were treated with the IC 50 % concentration of each extract for 24 h as previously described by Azadmehr et al. (45). Cells were centrifuged and fixed in 70% ethanol. After washing, cells were resuspended in 1 mL PBS containing 10 mg/mL RNase and 1 mg/mL PI (Sigma-Aldrich, USA). After that, these cells were incubated for 1 h at 37 • C in the dark. Lastly, the cells were examined on a FACScalibur flow cytometer (BD Bioscience Ltd. Co., Canada).

Expression analysis of apoptotic-related genes
The expression of apoptosis-related genes, caspase-3 (CASP3) and caspase-9 (CASP9) were analyzed using real-time PCR according to Fard et al. (22). The cell lines were treated with each extract and controlled at established IC 50 concentration. Thereafter, RNA was isolated using the Trizol reagent according to manufacturer instructions. The concentration and purity of the RNA samples were verified on an agarose gel and by the A260/A280 ratio using a NanoDrop Spectrophotometer. RT-qPCR was performed by the use of SYBR R Green dye (One-Step RT-PCR Kit, Bio-Rad) on a Rotor-Gene Q real-time PCR cycler. The sequences of used primers for amplification are as follows: Caspase3-F 5 -TTC ATT ATT CAG GCC TGC CGA GG-3 and Caspase3-R 5 -TTC TGA CAG GCC ATG TCA TCC TCA-3 , Caspase 9 F 5 -CGAACTAACAGGCAAGCAGC-3 , and Caspase 9 R 5 -ACCTCACCAAATCCTCCAGAAC-3 and GAPDH F 5 -GTCTCCTCTGACTTCAA-3 and GAPDH R 5 -ACCACCCTGTTGCTGTA-3 .
The PCR cycling conditions for caspases-9 and -3 were as follows: amplification at 95 • C for 1 min, denaturation for 30 cycles at 95 • C for 15 s, annealing at 56.6 • C, and 57 • C for GAPDH for 15 s, primer extension at 72 • C for 10 s, and final extension at 72 • C for 7 min. Additionally, the quantitative analysis measured the threshold cycle (CT) values in exponential phase amplification, and CT was calculated from the difference between CT values of Casp3, Caspase9, and GAPDH gene. Then, 2 − Ct was calculated from the fold changes of treated cells in correlation with untreated cells.
Where, CTE is the difference between the CT values of the Casp3, Cas9, and the CT is the value of the GAPDH gene of the treated cells.

Statistical analysis
Experiments were conducted in three replicates and the mean of values ± standard deviation (SD) were used in the figures and table. One-way analysis of variance (ANOVA) by using SPSS 20.0 (Chicago, IL, USA) was used for the statistical measurements, and P < 0.05 ( * ) was considered statistically significant and P < 0.01 ( * * ) was considered highly significant. In which, Least significant differences (LSD) have been calculated to measure the signification among the tested groups. Also, IC 50 was calculated based on the regression equation.

Phoenix dactylifera (KPC) chemical composition
The chemical composition of the different parts of the date cultivar (KPC) was investigated to better explain the observed biological activity of these different parts of the date palm on human cancer cell lines. In Table 1, it has been observed that the KPC date cultivar was very low in sucrose content with a high percentage of fiber (2.33%) and protein contents (1.175%). The total contents of phenols and flavonoids of leaves, seeds, and fruits' ethanolic extracts are evaluated and illustrated in Figure 1. The highest concentration of total phenolic contents and flavonoids was observed in leaves extract followed by seeds extract, while the lowest concentration was noted in the fruit extract. Several previous studies showed the high carbohydrate content of dates (approximately 70-80%), making them a quick and bountiful energy source. Moreover, date fruits are low in starch and high in protein content ranging between (1.03 and 5.60%), dietary fiber (6.40-11.50%), and the amount of minerals (0.10-916 mg/100 g dry weight), and also contains a group of specific vitamins such as (C, B1, B2, B3, and A) (46,47). Dates vary in chemical composition based on many factors, including the type of variety and soil conditions of different moisture contents and salts. There is also a role for agricultural procedures and the stage of maturity (48)(49)(50). Many variations were identified, such as color, texture, and taste/flavor. The texture of unripe green dates (chimera stage) is firm, with the highest moisture content and tannin acid. Dates begin to lose moisture and produce large amounts of sugar when they reach complete, colorful ripeness (Besser) (51). In addition, moisture loss accelerates in the soft brown (wet) phase, the fruits become smoother in texture, and sucrose turns into inverted sugars. The wet stage of dates is the most attractive because it is the softest and sweetest. Meanwhile, the separation of the extracts by GC/MS showed that the leaves had more compounds than seeds extract with a very high percentage of phytol and octadecenoic acid (Figure 2). A higher percentage of the phytochemicals was achieved in the leaf parts of the KPC tree. Also, the high presence of phytol in the leaves was observed compared to the seed extract.

Cell viability
The cytotoxicity of Panc1 and Pc3 cells was significantly increased following incubation with the ethanolic extracts of leaves, seeds, and fruit parts. Inhibition of Panc1 and Pc3 cell proliferation by these extracts was dose-dependent (Figures 3, 4). The Pc3 IC 50 of leaves extract was the lowest (P < 0.01) with 475 µg/mL compared to both seeds with 950 µg/mL and fruits with 985 µg/mL. Meanwhile, the seeds Pnc1 IC 50 was significantly (P < 0.05) lower than fruits extract, respectively.

Apoptosis and cell cycle analysis
The apoptotic effect of the examined extracts was investigated by Annexin-FITC/propidium iodide double staining flow cytometric assay. Obtained results showed that Frontiers in Nutrition 05 frontiersin.org Total phenolic contents, flavonoid, and antioxidants of leaf, seed, and fruit ethanolic extracts. The highest antioxidant potential was observed with leaf extract followed by seed and fruit extracts. *P-value was considered statistically significant (P < 0.05) and **P-value was considered highly significant (P < 0.01). all used cancer cell populations tend to shift from viable to apoptotic in comparison with the control group. Moreover, the late apoptosis events were increased than early apoptotic events in all treatments with all cancer cell lines, as shown in Figures 5-10. The apoptotic populations increased significantly in the Pc3 cell line due to the treatment with the extracts when compared with the control cells. But the apoptosis percent was increased significantly than necrosis in all treatments of utilized cancer cells, as shown in Figures 5, 6. Also, the morphological images of Pc3 have shown that all 1,250-10,000 µg/mL concentrations of the leaves extract had very high cytotoxic effects compared to the 1,250 and 2,500 µg/mL fruit extract with lower cytotoxic effects (Figure 7). Figures 8, 9 show the apoptotic effect of the different extracts on Panc1 compared to the control group. It has been observed that the leaves extract was significantly highest (P < 0.01) compared to the control group. On the other hand, the necrosis had no significant changes among the different Cytotoxic effect of leaf, seed, and fruit extract on prostate cancer cells (Pc3). * * P-value was considered highly significant (P < 0.01). Cytotoxic effect of leaf, seed, and fruit extract on pancreatic cancer cells (Panc1). *P-value was considered statistically significant (P < 0.05) and **P-value was considered highly significant (P < 0.01). Percentages of apoptotic and necrotic cells in and Pc3 cell population after treatment with leaf, seed, and fruit extract separately. *P-value was considered statistically significant (P < 0.05) and **P-value was considered highly significant (P < 0.01).
groups. Additionally, the concentration of 5,000 µg/mL from seed extract showed high cytotoxic effects on the Panc1 cell lines (Figure 10).
Moreover, the effect of each extract on the cell cycle progression of both Pc3 and Panc1 cancer cell lines showed that the treatments caused cell growth arrest at the G2/M phase Induction of apoptosis after treatment of Pc3 cells with leaf, seed, and fruit extracts separately in comparison with control. Pc3 cytotoxic morphology after the treatment with leaf, seed, and fruit extracts separately in comparison with control. (Figures 11, 12). The highest effects were presented by leaf extracts, followed by seed and fruit extracts.

Expression analysis of related apoptotic genes
Treatment of Panc1 and Pc3 IC 50 of each extract exhibited a significant increase in casp3 and casp9 mRNA levels as compared to untreated controls (Figures 13, 14). Also, the effect of leaf extract was more prominent on the utilized cell lines than on seeds and fruit extracts.

Discussion
There are multiple natural phytochemicals that could help in protecting the individual from different cancer diseases FIGURE 8 Percentages of apoptotic and necrotic cells in and Panc1 cell population after treatment with leaf, seed, and fruits extract separately. *P-value was considered statistically significant (P < 0.05) and **P-value was considered highly significant (P < 0.01). Induction of apoptosis after treatment of panc1 cells with leaf, seed, and fruit extracts separately in comparison with control. (52,53). In addition, functional foods, such as date fruits, have the potential to regulate cancer cell proliferation and differentiation (12). Zhang et al. (54) elaborated those different medicated plants have multiple natural substances such as taxol, vincristine, and vinblastine. Such products have characteristics to compete against cancer cells with the lowest side effects.
In this study, the ethanolic extracts of different KPC parts for Panc1 and Pc3 were evaluated to find the antiproliferative activity in leaves, seeds, and fruits. The results showed that the extracts of that date palm parts gave a defense line to counter cancer cells. It reveals major findings of the tested extracts that might be assorted, like the antiproliferative effect, the apoptotic Frontiers in Nutrition 09 frontiersin.org Panc1 cytotoxic morphology after the treatment with leaf, seed, and fruit extracts separately in comparison with control.
effect, and the influence on Casp3 and casp9 gene expression. The leaf extract showed the highest antiproliferative activity, higher than seed and fruit extracts. A higher percentage of the phytochemicals was achieved by the KPC leaves extract compared to the other parts. Also, the high presence of phytol in the leaves could explain its high activity against cancer cells. de Alencar et al. (55) mentioned that phytol phytochemical has a very high anticarcinogenic and antitumoral impacts on the different cancer types. This could be explained through the mechanism of action of the phytol prosthetic group presented in KPC leaves. In which, phytol molecule has a high ability to induce ligand-dependent transcription factor called retinoid X receptor (RXR) that functions as a key regulator of cell growth, differentiation, and metabolism of cancer cells (56,57). Besides, it has been suggested that phytol metabolites have functional impacts on cellular metabolism through activating the uncoupling protein-1 gene transcription (58). This sheds new light on the consequences which might involve excessive activation of phytol targets that have been presented in the leaf extract of KPC. Furthermore, the results showed that the highest phenolics, flavonoid contents, and antioxidant activity were found in leaf extract, followed by seed and eventually by fruit extracts. It is noteworthy that the leaf extract had improved phenolic content more than the other parts (P < 0.01). Significantly, the outcomes found in this study were aligned with Qadoos et al.
(59) study. Also, these results showed that different P. dactylifera extracts had multiple TPC and TFC stages. Mehraban et al. (28) described that the different concentrations of TPC and TFC are contributing to the used parts of KPC. Also, the presence of large amounts of phenolic compounds in the ethanolic extract showed a high antioxidant agent (60)(61)(62). Earlier studies found that palm date has antioxidant potential to suppress free radicals based on its TPC and TFC (63,64). Also, Zineb et al. (65) reported that dates constitute considered a good source of antioxidants. Al-Farsi et al. (66) investigated the antioxidant activities in Fard, Khasab, and KPC. Their study concluded that KPC had the most activity as compared to other types of dates. Besides, Ajwa and KPC date extracts showed higher polyphenol contents than Sukkari date extract (63).
As a precise colorimetric approach, MTT assay is a quick technique that shows cell growth and cytotoxicity. It also examines the phytochemcials cytotoxicity and could evaluate their impacts on the cancer cells' proliferation and viability (67). MTT assay stated that extracts could be used as an antiproliferative agent which attacks cancer cells with an appropriate dose. The cytotoxic effect of leaf extract was the highest one, followed by seed and fruit extract. This impact was through the functional groups of phytol that is the part of the chlorophyll dye presented in the leaves extracts. Thakor et al. (56) mentioned that the mechanism of action of phytol cytotoxicity came from its significant impact on the intrinsic pathway enzyme caspase 9 as well as the common executioner caspase 3. Moreover, the cytotoxic effect of Ajwa date pulp FIGURE 11 Cell cycle analysis after treatment of Pc3 cells with leaf, seed, and fruit extracts separately in comparison with control.
against hepatocellular carcinoma (hepG2) was attributed to β-D-glucan as reported by Siddiqui et al. (68). Also, the dates quercetin and kaempferol showed high cytotoxic activity against pc3 cells (69,70). Another in vitro study was conducted by Al-Sayyed et al. (71) and reported that Barhi date palm fruit prevents 7,12-Dimethylbenzathracene (DMBA)-induced mammary cancer. Its outcomes can be correlated with the other two drugs, i.e., tamoxifen and 17-β-estradiol. This effect is caused due to its high phenolic and flavonoid contents.
Also, the high cytotoxic effect of the allocated in the phenolic compounds found in the KPC's extracts of leaves, seeds, and fruit could be from the pro-oxidant and modification of the redox balance in the Pc3 and Panc1 cancer cells (72, 73). Mirza et al. (70) reported that cancer cells can be controlled through the cell cycle and induction of apoptosis. Additionally, Stewart (74) briefed that the apoptotic morphological changes could explain the cytotoxic effect of date phytochemicals. In which, Panc1 and Pc3 cells that reacted with the selected extracts showed the characteristics of apoptosis. Regarding induction of apoptosis and cell cycle arrest. Flow cytometry results using Annexin V-FITC and PI were presented as a percentage of 786 Annexin V+/PI− or Annexin V+/PI+ to indicate the status of apoptosis and necrosis induced by the 787 test compounds.
In our study, the seed extract caused Pc3 cells to move toward the late apoptosis stage and then induced cancer cell death. Khan et al. (75) reported that Ajwa dates extract tended to induce apoptosis in breast cancer MCF-7 cells by damaging the cancer cells' DNA. Also, Vessoni et al. (76) reported that if the cell is severely damaged, it can be forwarded to apoptosis to stop the proliferation of cancer cells. This study identified that cell cycle analysis shows a high level of cells found in preG1 and G2/M phases to control cells. The outcomes of this study are validated by the earlier studies which discussed anticancer drugs, i.e., paclitaxel. Such a drug inhibited human tenon's fibroblast cell proliferation through cell cycle arrest at G2/M phases (77). Also, these findings are in line with a recently published report by Siddiqui (68) on Ajwa dates. Its extracts are helpful to counter HepG2 cells. In addition, their findings FIGURE 12 Cell cycle analysis after treatment of Panc1 cells with leaf, seed, and fruit extracts separately in comparison with control.

FIGURE 13
Effect of leaf, seed, and fruit extracts on Casp3 and Casp9 gene expression in the pancreatic cancer cell (Pc3). *P-value was considered statistically significant (P < 0.05) and **P-value was considered highly significant (P < 0.01).
Frontiers in Nutrition 12 frontiersin.org Effect of leaf, seed, and fruit extracts on Casp3 and Casp9 gene expression in the pancreatic cancer cell (Panc1). *P-value was considered statistically significant (P < 0.05) and **P-value was considered highly significant (P < 0.01).
showed that extract of KPC date palm inhibited cell proliferation via G2/M phase arrest. Yamashita and Kawanishi (78) and Marvibaigi et al. (79) reported that phenolic and flavonoids contents helped apoptosis and induced the cell cycle arrest through the inhibition of DNA replication. The effect of KPC date extracts of leaves, seeds, and fruits on the expression levels of apoptosis-related genes (Casp3 and Casp9) was observed among Pnc1 and Pc3 cells. The cysteine protease activation, particularly caspases, is a crucial intracellular regulator of cell apoptosis (80). CASP 3 and CASP 9 genes belong to the cysteine-aspartic acid protease family and play a central role in inducing apoptosis (81). Alenzi et al. (82) stated that caspase 3 has a substantial arbitrator of apoptosis. Hünten et al. (80) reported that it activates caspase 9 through a wide range of activators. Also, apoptosis is activated from two signaling pathways, i.e., extrinsic and intrinsic pathways. The extrinsic pathway is arbitrated through the death receptor. However, the intrinsic pathways are arbitrated to the mitochondrion. Su et al. (83) stated that the caspase gene has a vital role in mitochondrionmediated apoptosis. Zhang et al. (54) indicated that casp-3 has an activated protease in mammalian cell apoptosis. It came through activation, proteolysis, and hydrolysis of particular substrates, i.e., DNA-dependent protein kinases. Badgujar et al.
The expression of Casp3 and Casp9 was tested to analyze the mechanism underlying the induction of apoptosis due to treatment with KPC extracts of leaves, seeds, and fruits. Casp3 and Casp9 expression were significantly up-regulated, and the mRNA level was increased among treated cells compared to untreated cells in all treatments with the two utilized cancerous cell lines. This result indicates that the induction of cell death of these lines was in the apoptosis way (85). The upregulation of Casp3 and Casp9 gene expression suggested that the treatment of Panc1 and Pc3 cells with the tested extracts induces mitochondrial dysfunction by discharging cytochrome c and inducing the intrinsic pathway apoptosis of cancer cells caspases 3 and 9 activations (86). Casp3 and Casp9 gene expression up-regulation might be due to the phytochemical contents in examined plant extracts (87)(88)(89). Leaves extract showed the highest effect on the gene expression change, followed by seeds extract and eventually by fruit extract. This result might be due to the different phytochemical contents in different plant parts (90).

Conclusion
This study concluded that KDC leaves, seeds, and fruit extracts have significant impacts on the Pc3 and Panc1 through their significant impacts on Casp3 and Casp9 gene expressions. The leaves extract showed a high significant (P < 0.01) effect in down-regulating Pc3 proliferation as compared to fruits and seeds extracts. Also, Panc1 had the least Casp3 and Casp9 expressions. After treating Panc1 and Pc3 cells with leaves extract, the cell cycle analysis was maximum in G0-G1%. Moreover, the least%PreG1 was found to be 1.39% in the control group of Pc3. Additionally, the percentage of apoptotic and necrotic cells in Panc1 after the treatment with leaf extract was found to be 16.11% in total apoptosis of leaves/Panc1, which was the highest compared to seeds (12.07%), and fruits (5.61%). The IC 50 of seed extracts on Pc3 had a significantly (P < 0.05) lower concentration than Fruit extracts with cytotoxicity of 1.13% compared to the control with 88.63%. Thus, the present results indicated that KPC extracts have antiproliferation activity by inducing apoptosis in the caspase-3 independent pathway and G2/M phases in Panc1 and Pc3 cells. This study is opening the way toward using the phytochemicals of KPC through new technologies like micro and nanoencapsulation for establishing functional bioactive food that could be used for the prevention Frontiers in Nutrition 13 frontiersin.org of prostate and pancreatic cancers. In which, the molecular pathway was confirmed for the related expressed genes. Further in vivo studies could help in tracking the changes in Casp3 and Casp9 in the vital body system.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.