Introduction

The relationship between food consumption and cancer has been extensively studied. Specifically, the consumption of fruits and vegetables was found to be protective against different types of cancers¹ specially mammary cancer ². By virtue of dietary fiber ³ and antioxidants, fruits and vegetables are thought to improve immunity ⁴ and inhibit cancerous cell growth ⁴.

The date palm (Phoenix dactilyfera L.) is a monocotyledon of Palmae family growing annually ⁵. Worldwide, date palm fruit production has been steadily increasing over the last 30 years. Most of the worldwide production of the fruit is in the Arab countries ⁶. The last decade showed rapid increase in Jordanian areas cultivated with this plant because it tolerates desert climatic conditions of the Jordanian areas where it is cultivated ⁷.

Historically, the plant was used for medicinal purposes such as fever, throat soars, toothaches, liver, abdominal ⁸, eye, and respiratory problems ⁹. Additionally, it has been mentioned that the fruit contains stimulants that strengthen uterus muscles ¹⁰, and is recommended for breast milk production ^9,10. Recently, various fruit extracts have been used functionally as antioxidants ^11–13, and anticancer agents ¹⁴.

The consumption of date palm fruit is related to Islamic values. The fruit consumption reaches the highest levels during the month of Ramadan since eating this fruit is a custom favored by Prophet Mohammad (peace be upon him) ¹⁵. Date palm fruit has hundreds of varieties sharing three maturity stages: Khalal, Rutab, and Tamr.

Cancer is a risk factor for mortality; the World Health Organization (WHO) reported 9.5 million deaths from cancer in 2018. Mammary cancer caused 1.5% deaths in 2018. Deaths from cancer worldwide are expected to rise to 11 million in 2030. Breast, and liver cancers cause the most cancer deaths each year ¹⁶. Breast cancer is the second risk factor for cancer deaths in Jordan accounting for 11.3 % of total deaths of cancer in 2018 ¹⁷. MCF-7 mammary cancer cell line represents the in vitro model for estrogen receptor-positive breast cancer ¹⁸.

Jordanian date palm fruit of Barhi variety at the Rutab and Tamr maturity stages was found to reduce chemically-induced mammary cancer tumor multiplicity, size, and increase tumor latency ¹⁹ by inducing the hormone 17-β-estradiol ²⁰ and modulating the glutathione-S-transferase enzyme activity ²¹. In addition, the water extracts of the fruit were found to be rich in polyphenols and flavonoids and a potential for antioxidant activity ¹².

In the current study, we attempted to evaluate the effect of several concentrations of water extracts of three varieties of date palm fruit grown in Jordan (Barhi, Belle Huwaimil, and Medjool) at two maturity stages (Rutab and Tamr) on the viability of MCF-7 breast cancer cell line.

Materials and Methods

Fruit and Extract Preparation

Locally grown date palm fruits were provided thankfully by Wardat ElRaml shop. In this study, three fruit varieties were tested: Barhi, Belle Huwaimil, and Medjool. The tested fruits were at the two maturity stages Rutab (Barhi) and Tamr (Barhi, Belle Huwaimil, and Medjool varieties). Then, the water extracts were prepared with deionized water (1:10). Subsequently, serial decimal dilution series of 200, 100, 50, 25, 12.5, 6.25, 3.125 and 1.563 mg/ml were performed using Dulbecco’s minimal essential medium (DMEM) enriched with 10% fetal bovine serum (FBS).

Cell Line and Culture Conditions

The human breast adenocarcinoma cell line (MCF7) was purchased from American Type Culture Collection (MCF7 ATCC® HTB-22). The MCF7 cells were maintained DMEM, DMEM High Glucose (4.5 g/l), with L-Glutamine supplemented with 10% FBS, 1% penicillin-streptomycin and 0.25 mg/ml amphotericin B (Capricorn-scientific, Germany) at 37°C in a humidified atmosphere of 95% air and 5% CO₂ ¹⁸. The cell line that has been used in this assay was sourced from the ATCC and all the STR profile and data are available on the ATCC website. The authors would like to mention that cell lines in our labs are regularly checked for any microbial contamination and strict rules has been followed to discard any cell line or flask with possible contamination.

Cell Proliferation and Viability Assay

Different extract effects on MCF7 proliferation/inhibition was analyzed by MTT assay cell proliferation and viability after 24-hour incubation. Initially, 2×10⁴ cells/well were transferred into 96-well plates and let to attach overnight. Initially, 20000 cells/well were seeded into 96-well plates and let to attach overnight. After that, the cells were treated with different concentrations (100 mg/mL, 50 mg/mL, 25 mg/ml, 12.5 mg/mL, 6.25 mg/mL, 3.125 mg/mL or 1.56 mg/ml) of each fruit extract and incubated for 24 hours with 5% CO₂ at 37ºC in a humidified atmosphere. The wells with cells only was used as a positive control. After this treatment, MTT reagent (5 mg/ml) in 100 μl of fresh culture medium was added to each well for 4-hour incubation. The formazan crystals formed with MTT treatment were solubilized with acidic isopropanol and the optical density was -then- measured at 570 nm wavelength using a microplate reader (GloMax, promega). The assay controls were used to specify the cytotoxicity of the extract and to exclude the effect of the vehicle. In addition, water was used in different dilutions to exclude the effect of vehicle too. Further, DMSO was used as a positive control and the cell with media to mimic normal cell line growth. Percentage of inhibition was calculated by the following formula; (Average viability/ control) and inhibition (%)= 100 – viability. Furthermore, IC50 was estimated using Prism software ^22,23. The procedure was performed in triplicate.

Statistical Analysis        

To detect the interaction effect of date palm fruit variety (factor 1), maturity stage (factor 2), as well as concentration (factor 3), data of the duplicate readings of percentage inhibition as well as percentage of viable cells were analyzed by multivariate analysis of variance (MANOVA) using the statistical package for social sciences (SPSS) analysis system. Data in the figures was extracted from mean ± standard deviation values for the inhibition and viable cell percentages ²⁴ P<0.05 was set for significance.

Results

Results of inhibition of cell line are shown in Figure 1. All of date palm fruit concentrations showed inhibition effect on cell line (Figures 1 and 2). The statistical analysis of data showed that there is no interaction effect between date palm fruit variety, maturity stage, and extract concentration in terms of inhibition percentage, neither viable cell percentage (P>0.05). However, there are significant (P<0.01) differences between different extracts in terms of the interaction effect between date palm variety and extract concentration.

Figure 1: Inhibition Percentage of Different Concentrations of the Studied Dates Varieties. Click here to view Figure

Figure 2: Effect of the Date Palm Fruit Extracts on MCF-7 Cancer Cell Lines Viability at 24 Hours. Click here to view Figure

Results show that the inhibition percentage is dose-dependent. The effect of the tested concentrations (1.563 to 100 mg of dates extract (dry)/mL) on the viability of MCF-7 cells. There is insignificant (P>0.05) inhibition of the growth of cells observed at (1.563-100 mg/mL). However, results showed that the cytotoxic effect appeared at higher concentrations (50 and 100 mg/mL) where cell viability reduced by ~44% and 78%, respectively (Figure 2). MCF-7 cells growth inhibition as well as cytotoxicity were not significant until a concentration of 25 mg/mL (80% of cell viability). Interestingly, after 24 hours of treatment with date palm fruit extracts, MCF-7 cell viability decreased sharply and dose-dependently with IC50 values of 12 ± 0.02 mg/mL (P< 0.01) (Figure 2).

The most effective concentration was 100 mg/ml for all varieties. At this concentration, Tamr of Medjool variety exhibited the highest inhibition, followed by Belle Huwaimil variety at the Tamr stage, Barhi at the Rutab stage, and Barhi at the Tamr stage respectively (P=0.000^**) and results were similar at 50 mg/ml concentration extracts. At the medium concentrations of the extracts (12.5 and 25 mg/ml), the most effective fruit varieties were Belle Huwaimil and Barhi at the Rutab stage respectively. At these concentrations, Medjool and Belle Huwaimil at the Tamr stage were the least effective (P=0.000^**). At lower concentrations (3.125 and 6.25 mg/ml), Medjool at the Tamr stage and Belle Huwaimil respectively were the most effective extracts where Barhi at the Tamr and Rutab stages were the least effective (P=0.000^**).

Discussion

The insignificant effect of maturity stage (P>0.05) is not surprising since all the fruit varieties were tested at the Tamr maturity stage and one variety only (i,e. Barhi) was tested at the Rutab maturity stage. Thus, probably, there was insufficient data for statistical comparison. On the other hand, the sole effect of variety as well as concentration on the inhibition ad cell viability percentages was significant (P=0.001).

Our results are concomitant with result found by ^15,25,26 This result suggests that the cell viability inhibition is probably related to the proliferation suppression neither to any cytotoxic nor cytolytic effects. This activity might be due to the presence of certain compounds within the fruit, such as quercetin (a flavonoids glucosides which known for anticancer effect) ²⁷. Previous studies revealed that polyphenol compounds reduce cancer cell growth by inducing apoptosis in many cell lines of mammary cancer ²⁸.

A study by Salomón-Torres et al. (2019) ²⁹ showed that water extracts of Medjool variety inhibited MCF-7 breast cancer cell line and the author mentioned that 100 µg/ml extracts was not effective and that is in disagreement with our study.

A previous study showed the 51% percentage of inhibition of cell line after incubation of MCF-7 with 100 mg/ml methanolic extracts ³⁰. The current study revealed that the percentage of inhibition of cell lines was between 14% to 93% after incubation for 24 hours with Medjool variety at Tamr stage. The variety of Medjool contains antioxidants such as ascorbic acid, phenolic compounds ¹², gallic acid ²⁹, p-hydroxybenzoic acid, vanillic, caffiec, synergic, and ferulic acids. Furthermore, this variety of date palm fruit exerted antioxidant activity ^14,31.

A study by El-sharkawy (2017) ²⁸reported that the inhibition of cell proliferation was 42% at 15 mg/ml concentration of extract. Inhibition values found in this research ranged from 9% to 92% and the results were close to those found by Odeh et al. (2014) ³⁰ who found inhibition value of 50 mg/ml extracts of Barhi variety at both maturity stages ³². Figure 3 shows summarization of the experimental procedure as well as suggested mechanisms and functional compounds that are thought to protect against mammary cancer.

Figure 3: (a) Summary of the Research Procedure and Key Findings and (b) Suggested Mechanisms by Which Date Palm Fruit Protects against Mammary Cancer. Click here to view Figure

The various compounds in the date palm fruit render it capable of having an antioxidant capacity. This property has been proved at the in vitro ^8,33–35 and the in vivo levels ^36–38. A previous study by Vayalil (2002) ¹⁴ showed the antioxidant and antimutagenic activity of the water extract of date palm fruit at the Tamr stage. The author found that the extract scavenged superoxide and hydroxyl radicals in a dose-dependent manner ¹⁴.

The compounds that are thought to add the (functional) antioxidant property to the date palm fruit are metals such as Se, Cu ³⁹, Zn, and Mn, phenolic acids ³⁸, enzymes such as: phytase, invertase ⁴⁰, polyphenols specially flavonoids ¹² such as catechins ³⁸ and rutin ³⁴, antioxidant vitamins such as the vitamins C and E ⁴¹.

Conclusion

This study revealed that the date palm fruit extract from Barhi and Belle Huwaimil varieties of Jordan provide a strong activity against MCF-7 cancer cell line proliferation. Given the folk claims of anticancer properties of Barhi and Belle Huwaimil date extract and our results on MCF-7 cells, the date fruits could be used as a supplemental food for chemoprotective effects against breast cancer and possibly other lesions. Results of this research supported the results of previous reports regarding Medjool variety. However, results of this study are limited by the type of extract, maturity stage, the indicators tested for anticarcinogenicity, and the lack of using control cell line like the MCF7-A10 to exclude general cytotoxicity effect on other cell line.

Funding Source

This research has been supported by the Deanship of Academic research at the University of Petra (Grant number 4/4/2015-2016).

Conflict of Interest

The author(s) declares no conflict of interest.

Acknowledgement

The authors would like to thank the Deanship of Academic Research at the University of Petra for financially supporting this research.

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