Olive oil varieties cultivated in Morocco reduce reactive oxygen species and cell viability of human cervical cancer cells

Abstract

BACKGROUND:

The Moroccan diet incorporates olive oil as the primary source of fat and may reduce cancer risk. However, different olive oil varieties often have varying levels of anti-cancer polyphenols and thus have unique biologic effects.

OBJECTIVE:

The anti-cancer activity of five varieties of extra virgin Moroccan-cultivated olive oil on human cervical cancer cells was assessed in vitro.

METHODS:

The presence of phenolic compounds in five olive oil varieties cultivated in Morocco was analyzed using HPLC. Human cervical cancer cell lines (HeLa, SKG-II, and HCS-2) were incubated with the olive oils and cell viability was measured by MTT assay, reactive oxygen species were measured using the CellRox assay, and gene expression was measured by RT-PCR.

RESULTS:

Each of the five Moroccan-cultivated olive oil varieties had a unique composition of phenolic compounds. Incubation with the olive oils reduced cell viability and reactive oxygen species in human cervical cancer cells. The expression of genes involved in cervical cancer carcinogenesis and cell cycle were also altered. All five olive oil varieties decreased expression of E6, E7, p16, p63, and NRP2 and increased expression of IVL and miR 331-3p.

CONCLUSIONS:

Use of Moroccan-cultivated olive oils could be a promising anti-cancer agent for cervical cancer.

Keywords

Antioxidant cervical cancer gene expression olive oil

1 Introduction

Cervical cancer is the fourth most frequent cancer in women with an estimated 570,000 new cases reported in 2018. Approximately 90% of deaths from cervical cancer occur in low- and middle-income countries and cervical cancer is the second most common cancer affecting Moroccan women [1]. Before 2010, two thirds of the cervical cancer cases in Morocco were diagnosed and treated at a very late stage. However, in 2010, a systematic screening program was implemented throughout Morocco and this program has greatly contributed to reducing the dramatic consequences often caused by late detection and treatment.

Natural medicine has been used in cancer care for centuries and many of our modern chemotherapies are derived from plants and food. Many studies have shown that dietary manipulations can modulate physiological activities and prevent neoplastic disease [2, 3]. The Mediterranean diet is primarily based on the higher intake of olive oil. Beneficial effects of olive oil have been demonstrated in several studies and olive oil may be useful for the prevention or possible treatment of cancer [3 –6].

The anti-cancer, anti-proliferative, and pro-apoptotic activities of olive oil are attributed to many factors including the high content of oleic acid and other monounsaturated free fatty acids, squalene, phytosterols, and phenols [5 , 7–17]. In particular, many polyphenolic compounds present in extra-virgin olive oil, including tyrosol, hydroxytyrosol, oleocanthal, and oleuropein, induce apoptosis in multiple human cancer cell lines including cervical cancer [5 , 15–18]. Phenolic extracts of olive oil can also induce a significant decrease in cell viability and act as powerful antioxidants in breast and cervical cancer cells [19 –21]. In addition, the reduction of oxidative stress by phenolic compounds of olive oil may reduce oxidative DNA damage and slow the initiation steps of carcinogenesis in vivo [22]. However, different olive oil varieties have unique mixtures of polyphenols and may induce varying anti-cancer responses. The specific anti-cancer effects of olive oil varieties cultivated in Morocco on reactive oxygen species, gene expression, and viability of cervical cancer cells is not well-established. Therefore, the aim of this research is to evaluate the in vitro anti-cancer activity of five extra virgin Moroccan-cultivated olive oils against human cervical cancer cell lines.

2 Materials and methods

2.1 Moroccan olive oil source

Samples were collected from ‘Agropole Olivier’ Meknes in Morocco. The varieties of olive oil studied were 1 = Moroccan Picholine; 2 = Koroneiki; 3 = Arbequine; 4 = Picual; 5 = Arbosana. All varieties were Extra Virgin on the Physicochemical and Sensory Plan.

2.2 Extraction and analysis of phenolic compounds from olive oils

All standard compounds were purchased from Sigma Aldrich. The standards used for comparison to the olive oils are shown in Table 1. The phenolic compounds for this study were selected based on previously published literature and availability of standards. Stock solutions were prepared using a solvent of 50 % v/v HPLC grade methanol (Acros) and 16.7 MΩ doubly deionized water (in-house system). Calibration standards were prepared ranging from 0.03 – 800 mg/L to determine the linear range for each compound and the appropriate calibration levels for the olive oil extracts. The final ranges selected for quantification are in Table 1.

Table 1
Summary of phenolic standards analyzed

Name Stock conc. mg/L Calibration range, mg/L Ret. Time, min Detector

Tyrosol 6920 0.6 – 13 5 DAD @ 280 nm

Oleuropin 770 0.7 – 4 11.5 FL (ex. 280, em. 320)

Vanillic acid 8420 0.08 – 17 6.5 FL (ex. 280, em. 320)

Luteolin 344 0.3 – 17 12.2 DAD @ 330 nm

Name	Stock conc. mg/L	Calibration range, mg/L	Ret. Time, min	Detector
Tyrosol	6920	0.6 – 13	5	DAD @ 280 nm
Oleuropin	770	0.7 – 4	11.5	FL (ex. 280, em. 320)
Vanillic acid	8420	0.08 – 17	6.5	FL (ex. 280, em. 320)
Luteolin	344	0.3 – 17	12.2	DAD @ 330 nm

The phenolic compounds were extracted from the olive oils based on a procedure that was previously published [23]. 0.6 g of oil was weighed,and mixed with 0.6 mL of dimethyl formamide (Acros). The tube was vortexed briefly and centrifuged for 3 minutes. The DMF layer was removed and this extraction was repeated three times. The pooled dimethyl formamide (DMF) extracts were washed with 2 mL of hexane (vortexed and centrifuged 3 minutes). The hexane layer was discarded and the DMF extract was diluted to a final volume of 2 mL with DMF and filtered through a 0.45μm nylon syringe filter into an autosampler vial for HPLC analysis.

The HPLC analysis was done on a Shimadzu Prominence HPLC system outfitted with a diode array detector (DAD) and a fluorescence detector (FL). The mobile phase consisted of (A) 50 mM phosphoric acid (prepared with 16.7 MΩ doubly deionized water and HPLC grade phosphoric acid, Fisher) and (B) HPLC grade acetonitrile (Fisher). The mobile phase gradient was as follows: initial solvent 10 % B, hold 10 % B for 2 min, increase to 90 % B at 18 min, hold 90 % B for 2 min (total analysis time 22 minutes). The column was a C18 stationary phase, 150 mm x 4.6 mm, 5μm particle size (Shimadzu), and the column oven was maintained at 40 °C. The injection volume was 10μL. Detection and retention times for each compound is shown in Table 1.

2.3 Cell lines

Human cervical cancer cell lines HeLa, SKG-II, and HCS-2, and MCF7 and T47D breast cancer cell lines were used. SKG-II cells were cultured in Ham’s F12 media supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100μg/ml streptomycin, 1 mM sodium pyruvate, 10 mM Hepes, and 0.1 mM non-essential amino acids. HeLa, HCS-2, MCF7, and T47D cells were cultured in Dulbecco’s Modified Eagle Medium media and supplemented as described above.

2.4 Cell viability assay

Human cervical and breast cancer cell lines (10⁴) were incubated in 96-well flat bottom plates in the presence of olive oil or media for 48 hours. To measure cell viability, the Cell Titer 96-well Non-radioactive Cell Viability Assay (MTT) (Promega, Madison, WI, USA) was used according to manufacturer’s instructions.

2.5 Flow cytometry for detection of reactive oxygen species

Human cervical cancer cell lines (10⁴) were incubated in 96-well flat bottom plates in the presence of olive oil or media for 48 hours. Cells were analyzed for reactive oxygen species using the CellROX Green flow cytometry assay kit according to manufacturer’s instructions (ThermoFisher Scientific). Cells were analyzed on an Accuri C6 Flow Cytometer using Accuri Analysis software (BD BioSciences).

2.6 RNA isolation and real time polymerase chain reaction

Human cervical cancer cell lines (10⁴) were incubated in 96-well flat bottom plates in the presence of 10 ppm olive oil or media. After 8 hours, total RNA (including miRNA) was isolated using the SV Total RNA Isolation System (Promega Corporation) according to manufacturer’s instructions. For detection of mRNA, cDNA was synthesized using Random Hexamer primers and the Revert Aid First Strand cDNA Synthesis Kit (Promega). Gene expression was measured by Real Time Polymerase Chain Reaction (RT-PCR) using cDNA (5 ng) and Thermo Scientific Maxima SYBR Green qPCR Master Mix according to manufacturer’s instructions. Primer sequences (Integrated DNA Technologies) are listed in Table 2. β-Actin expression was used to normalize mRNA levels. For detection of miRNA331-3p, an ID3EAL miRNA qPCR Assay (BioVendor) was used according to manufacturer’s instructions.

Table 2
List of primer sequences for RT-PCR

Gene Sequence of sense and anti-sense primers

β-actin 5′-CTCTTCCAGCCTTCCTTCCT-3′

5′-AGCACTGTGTTGG CGTACAG-3′

E6 5′-CTCTGTGTATGGAGACACATTGGAA-3′

5′-GGCACCGCAGGCACCTTA-3′

E7 5′-TAGAAAGCTCAGCAGACGACCTT-3′

5′-GCACACCACGGACACACAAA-3′

p63 5′-GAAAGCAGCAAGTTTCGGAC-3′

5′-TTTCATAAGTCTCACGGCCC -3′

NRP2 5′-CTGGAAGTCAGCACTA ATGGAGAG-3′

5′-GCATCGTTGTTGGCTTGAAATACC -3′

IVL 5′-AGCCTTACTGTGAGTCTGGTTGA-3′

5′-GGGTATTGACTGGAGGAGGAACA -3′

p16 5′-CCAACGCACCGAATAGTTACG-3′

5′-GCGCTGCCCATCATCATG-3′

Gene	Sequence of sense and anti-sense primers
β-actin	5′-CTCTTCCAGCCTTCCTTCCT-3′
	5′-AGCACTGTGTTGG CGTACAG-3′
E6	5′-CTCTGTGTATGGAGACACATTGGAA-3′
	5′-GGCACCGCAGGCACCTTA-3′
E7	5′-TAGAAAGCTCAGCAGACGACCTT-3′
	5′-GCACACCACGGACACACAAA-3′
p63	5′-GAAAGCAGCAAGTTTCGGAC-3′
	5′-TTTCATAAGTCTCACGGCCC -3′
NRP2	5′-CTGGAAGTCAGCACTA ATGGAGAG-3′
	5′-GCATCGTTGTTGGCTTGAAATACC -3′
IVL	5′-AGCCTTACTGTGAGTCTGGTTGA-3′
	5′-GGGTATTGACTGGAGGAGGAACA -3′
p16	5′-CCAACGCACCGAATAGTTACG-3′
	5′-GCGCTGCCCATCATCATG-3′

2.7 Statistical analysis

Statistical analysis was run on each test using an unpaired two-tailed Student’s t-test or ANOVA with a post-hoc Tukey test when comparing multiple groups. The data was determined to be normally distributed using the Shapiro-Wilk test. Equality of variances was determined using Levene’s test. The program R was used for statistical analysis of the data. For all experiments, each sample was run in triplicate on at least three independent sets of tumor cells and p values < 0.05 were considered significant.

3 Results

3.1 Chemical composition of Moroccan-cultivated olive oil varieties

To analyze the anti-cancer effects of olive oil varieties cultivated in Morocco, samples of Extra Virgin olive oils were collected from ‘Agropole Olivier’ Meknes in Morocco. The varieties of olive oil studied were 1 = Moroccan Picholine; 2 = Koroneiki; 3 = Arbequine; 4 = Picual; 5 = Arbosana. Each variety of olive oil has unique properties. The Arbequina variety has been the most widely planted variety for several years in both high-density and super high-density systems in the Meknes region of Morocco. Koroneiki is one of the best-known varieties for super high-density systems and has the highest content of phenols and the best resistance to oxidation, followed by Arbosana’s olive oil. Comparatively, Arbequina and Picholine olive oils have low resistance to oxidation. Picual possesses high polyphenol content, oil stability, and high productivity.

To analyze the polyphenol composition of these five Moroccan-cultivated olive oil varieties, the levels of various compounds with known anti-cancer properties were quantified. A sample chromatogram of one of the olive oil samples is shown in Fig. 1. Of note, the compound apigenin was detected in each of the olive oils, but not quantified due to lack of a quantitative standard. Several other peaks were visible but not identifiable with the standards selected for this study. The compounds selected for this preliminary analysis and the concentration ranges used for calibration (see Table 1) were based on availability of analytical standards and the detection limits and dynamic range of the instrumentation. The quantitative data for each of the four compounds identified in the olive oils are shown in Table 3. These results indicate that tyrosol was the main contributor to the phenolic content of the Moroccan-cultivated olive oils and that there were significant variations in the relative amounts of the other compounds that were included in the study. The concentrations of vanillic acid, luteolin, and tyrosol calculated in the oils were comparable in magnitude to previously published work [24, 25]. Although the quantitative analysis of the phenolic compounds in these oils is currently limited by using HPLC with spectrophotometric detectors, this preliminary analysis indicates that the Moroccan-cultivated olive oils do have significant levels of important phenols found in common olive oil varieties and supports the findings of anti-cancer activity of the oils. Further analysis with mass spectrometric detection would allow lower detection limits and identification of additional compounds.

Fig. 1

Selected chromatogram of olive oil 3, Arbequine. Apigenen was detected but not quantified, and vanillic acid is not detectable with the absorbance detector (quantification for vanillic acid was on the FL detector as noted in Table 1).

Table 3

Summary of quantitative analysis of four phenolic compounds identified in Moroccan-cultivated olive oils. Average and±standard deviation is given for three replicate samples

	Luteolin	Oleuropein	Vanillic acid	Tyrosol
	Average conc. (mg/kg oil)	Average conc. (mg/kg oil)	Average conc. (mg/kg oil)	Average conc. (mg/kg oil)
Moroccan Picholine oil (1)	2.06±0.04	5.6±0.4	1.9±0.3	29.4±0.6
Koroneiki oil (2)	1.02±0.02	5.8±0.6	6±2	14±1
Arbequine oil (3)	4.1±0.1	3.9±0.2	1.5±0.3	22±3
Picual oil (4)	2±1	1.5±0.8	2±1	12±6
Arbosana Arbquina oil (5)	4.0±0.3	3.0±0.5	4.5±0.1	13.8±.04

3.2 Moroccan olive oil varieties reduced viability and reactive oxygen species in cervical cancer cells

Incubation with different olive oil varieties decreased cell viability of HeLa, SKG-II, and HCS-2 cervical cancer cells, and this decrease was dose dependent. The Moroccan-cultivated olive oils also decreased the viability of MCF7 and T47D breast cancer cells (Fig. 2), demonstrating the potential anti-cancer effects on cell types other than cervical cancer cells.

Fig. 2

Moroccan-cultivated olive oil varieties reduced cell viability of cervical cancer and breast cancer cell lines. (A) Human cervical cancer cell lines or (B) breast cancer cell lines were incubated with various concentrations of olive oil varieties (1 = Moroccan Picholine; 2 = Koroneiki; 3 = Arbequine; 4 = Picual; 5 = Arbosana) as indicated. After 48 hours, cell viability was measured using an MTT assay. Data are shown as average + standard deviation of triplicates and are representative of three separate experiments. Cell viability was significantly decreased when the cells were cultured with all concentrations of olive oils as indicated by * (p < 0.05 when compared to 0 ppm).

Next the effect of the Moroccan-cultivated olive oil varieties on reactive oxygen species was measured using the CellROX assay. Compared to untreated cells, incubation with different Moroccan olive oils significantly decreased reactive oxygen species in HeLa, HSC-2, and SKG-II cells (Fig. 3). There was no significant difference in the reduction in reactive oxygen species among the olive oil varieties. This indicates that exposure to Moroccan olive oil may reduce reactive oxygen species and contribute to a decrease in cervical cancer formation or growth.

Fig. 3

Moroccan-cultivated olive oil varieties reduced reactive oxygen species in cervical cancer cell lines. Human cervical cancer cell lines were incubated with various concentrations of olive oils (1 = Moroccan Picholine; 2 = Koroneiki; 3 = Arbequine; 4 = Picual; 5 = Arbosana) as indicated. After 48 hours, reactive oxygen species were measured using a CellRox Green flow cytometry assay. Data are shown as average + standard deviation of triplicates and are representative of three separate experiments. Reactive oxygen species were significantly decreased when the cells were cultured with all concentrations of olive oils as indicated by * (p < 0.05 when compared to 0 ppm).

3.3 Moroccan-cultivated olive oil varieties altered gene expression in cervical cancer cells

Many studies indicate that cervical cancer develops through evolution of preinvasive lesions to invasive cancer [26]. Among the different components of the HPV genome, the most commonly expressed viral proteins associated with cervical cancer development are the E6 and E7 oncoproteins [27]. The E6 and E7 viral proteins contribute to cancer development through many mechanisms including binding to and degrading the host tumor suppressor proteins, p53 and Rb, respectively. Thus, these viral proteins override the functions of cell cycle checkpoints and cause cellular transformation. Other genes that are altered during transformation of cervical cancer cells includes p63 which is a member of the p53 transcription factor family, the cell cycle regulator cyclin dependent kinase inhibitor 2A (p16), neuropilin 2 (NRP2) which is a high-affinity receptor for VEGF isoforms, involucrin (IVL) which is part of the cell envelope of the stratified squamous epithelia and is an important keratinocyte differentiation marker, and miR-331-3p, which has a principal role in the regulation of E6 and E7 expression in HPV-infected squamous cell carcinoma cell lines [28 –31]. MiR-331-3p also down-regulates p63 and NRP2 and up-regulates involucrin (IVL) and overexpression of miR-331-3p decreases cell proliferation [21].

Therefore, the effect of Moroccan-cultivated olive oils on the expression of these aforementioned genes that play a role in cervical cancer development (E6, E7, p16, p63, NRP2, IVL, and miR-331-3p) was measured by RT-PCR. Cells were incubated with media or 10 ppm of each olive oil variety for 8 hours. Five genes (E6, E7, p16, p63, NRP2) showed decreased expression in the three cervical cancer cell lines compared to cells cultured in media (Fig. 4). Strikingly, the expression of E6 was decreased the most in cells incubated with olive oil variety 1 (Picholine) whereas expression of E7 and p63 was lowest in samples incubated with olive oil variety 2 (Koroneiki) and expression of p16 was lowest in samples incubated with olive oil variety 4 (Picual). The expression of IVL and miR-331-3p increased in all three cervical cancer cell lines by 4 to 15-fold compared to the media samples (Fig. 4). In particular, the expression of miR-331-3p was up-regulated the most with olive oil variety 1 (Picholine) and IVL was highest in samples incubated with olive oil variety 2 (Koroneiki) or 5 (Arbosana). In summary, these data demonstrate that these five Moroccan-cultivated olive oils have unique anti-cancer properties and may be used to prevent or treat cervical cancer.

Fig. 4

Moroccan-cultivated olive oil varieties altered gene expression in cervical cancer cells. Human cervical cancer cell lines were incubated with 10 ppm olive oil (1 = Moroccan Picholine; 2 = Koroneiki; 3 = Arbequine; 4 = Picual; 5 = Arbosana) or media as indicated. After 8 hours, gene expression of genes involved in cervical cancer development and growth was measured using RT-PCR. Data are shown as average + standard deviation of triplicates and are representative of three separate experiments. Gene expression was significantly altered when the cells were cultured with olive oils as indicated by * (p < 0.05 when compared to 0 ppm (media only)).

4 Discussion

These results showed that five Moroccan-cultivated olive oils varieties decreased cell viability and reactive oxygen species in human cervical cancer cell lines HeLa, SKG-II, and HCS-2. These olive oil varieties also decreased the proliferation of MCF7 and T47D breast cancer cells. Therefore, the anti-cancer effects of the different Moroccan-cultivated olive oil varieties may be a potential preventative measure for cervical cancer and possibly other cancer types.

A similar study showed that phenolic extracts of Moroccan olive cakes reduced proliferation and cell viability of the P815 murine mastocytoma cell line [24]. Compared to our study, phenolic extracts of olives had a dose-dependent cytotoxic effect starting at low concentrations (less than 0.05 ppm) with an IC50 range from 20 to 40 ppm [24]. These findings are similar to our results that demonstrated decreased cell viability starting at 0.1 ppm and increasing to 100 ppm (Fig. 2). Similarly, the cytotoxicity of olive oil on the HeLa cancer cell line has previously been tested in the range of 10–10,000 ppm for 72 hours, and the maximum cytotoxic effect occurred at 10,000 ppm [7]. Our data demonstrated that the maximum cytotoxic effect of the Moroccan-cultivated olive oils occurred at the highest concentration (100 ppm), suggesting that these olive oil varieties may have enhanced anti-cancer properties compared to other olive oils.

While the preventative effects of olive oil on cancer are not fully understood, polyphenols in olive oil has been shown to have effects on a variety of cell processes including oxidative stress and regulation of gene expression [32 –35]. In particular, oleuropein, tyrosol, luteolin, and vanillic acid have strong antioxidant effects [36 –39]. The Moroccan-cultivated olive oil varieties tested in this study had detectable levels of the antioxidants oleuropein, tyrosol, luteolin, and vanillic acid and significantly reduced reactive oxygen species in cervical cancer cell lines. In this study, Moroccan Picholine and Koroneiki had the highest content of oleuropein. Tyrosol was also highest in the Moroccan Picholine variety. Therefore, while the Moroccan-cultivated olive oil varieties all had significant levels of these polyphenols, each had a unique combination of compounds and may have distinct anti-cancer effects. In addition to the anti-cancer compounds detected in this study, there are many other molecules in olive oil with well-known anti-cancer effects, including hydroxytyrosol, oleic acid, oleocanthal, apigenin, quercetin, and many others [5 , 16]. To further study the anti-cancer mechanisms of Moroccan-cultivated olive oils, the content of these anti-cancer compounds should be determined in future studies.

Many studies have also shown a relationship between olive oil consumption and gene expression changes [40]. Olive oil and its functional components often affect the regulation of genes associated with the arrest of cell cycle during G0/G1 or G2/M transitions, which result in cell senescence and activation of apoptosis [41]. This study was the first to demonstrate that multiple genes involved in the formation and growth of cervical cancer were altered by Moroccan-cultivated olive oils. In particular, expression of E6, E7, p16, p63, and NRP2 were down-regulated by olive oil, while expression of IVL and miR-331-3p were up-regulated. MicroRNAs (miRNAs) are small non-coding RNAs that have an important role in regulation gene expression and in tumor formation [42, 43]. In cervical cancer cells, miR-331-3p directly regulates NRP2 and contributes to a down-regulation of E6, E7, and p63 mRNA and up-regulation of IVL mRNA leading to reducing proliferation and increased apoptosis [28 , 44]. The data presented in this study demonstrate that all Moroccan-cultivated olive oils tested reduced the gene expression of E6, E7, p16, p63, and NRP2, while the expression of IVL and miR-331-3p increased in the cervical cancer cell lines. Since overexpression of miR-331-3p can inhibit cell proliferation and induce apoptosis in SKG-II, HCS-2 and HeLa cells, our findings suggest that these Moroccan-cultivated olive oil varieties might have a beneficial effect in preventing or treating cervical cancer through altered gene expression.

In summary, we conclude that the varieties of Moroccan-cultivated olive oil tested reduced cell viability and reactive oxygen species in cervical cancer cells, and thus have demonstrated anti-cancer effects for cervical cancer. Furthermore, there may be anti-cancer applications in cancer types other than cervical cancer. The current research demonstrated that olive oil has beneficial effects on inhibiting cancer cells and its use in medical practices may help cancer patients. Further research is needed to continue investigating the effects of Moroccan-cultivated olive oil on the physiological and genetic characteristics of cancer cells and on the potential therapeutic application of olive oils.

Funding

The authors report no funding.

Conflict of interest

The authors have no conflict of interest to report.

Footnotes

Acknowledgments

This work was supported by in part by Longwood University’s Faculty Research Grants and the Department of Biological and Environmental Sciences. We are thankful to Dr. Nacer Bellaloui for reviewing and editing the manuscript.

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