Received date: March 11, 2014; Accepted date: April 26, 2014; Published date: May 07, 2014
Citation: Ranchal I, de Castro ML, Muntané J (2014) Olive Leaves and Mill Waste Exert Anti-Tumoral Properties in Hepatoma Cell Line. J Carcinog Mutagen 5:170. doi: 10.4172/2157-2518.1000170
Copyright: © 2014 Ranchal I, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Different phenolic compounds such as oleuropein and hydroxytyrosol have demonstrated antitumoral activity in cancer cell lines. Olive derivatives have demonstrated beneficial properties in cardiovascular diseases. The study was addressed to show the potential beneficial properties of olive leaves and olive oil mill waste extracts in hepatoma cell lines. Aflatoxin B1 (AFB1) was administered to hepatoma cell line (HepG2). Different parameters related to cell death, cell proliferation and DNA damage were determined. The expression of p53 and c-Src (activated and inhibitory phosphorylated state) was assessed by Western blot analysis in HepG2. The olive leaves and olive oil mill waste extracts contained oleuropein and hydroxytyrosol, respectively. AFB1 induced cell proliferation and death, associated to a rise on p53 and c-Src expression in HepG2 cells. Oleuropein and hydroxytyrosol reduced cell necrosis and DNA damage in HepG2 cells. However, the administration of olive leaves and olive oil mill waste extracts increased cell necrosis and DNA damage. These effects of natural extracts were associated with a reduction of activated c-Src expression and cell proliferation in AFB1-treated HepG2 cells. These results support that olive leaves extract and olive oil mill waste extracts, but not oleuropein and hydroxytyrosol, may exert antitumoral effect against a hepatoma cancer cell line.
HCC: Hepatocellular Carcinoma; AFB1: Aflatoxin B1; CDK: Cyclin-Dependent Protein Kinases; HepG2: Human Hepatoma Cell Line; DMSO: Dimethyl Sufoxide; EDTA: Ethylene Diamine Tetra Acetic Acid; FBS: Fetal Bovine Serum; PBS: Phosphate-Buffered Saline; PMSF: Phenylmethyl Sulfonylfluoride; RNase: Ribonuclease; SDS–PAGE: Sodium Dodecyl Sulfate–Polyacrylamide Gel; TBS: Tris Buffered Saline
Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths worldwide, and accounts for an overall incidence of 560,000 new cases per year [1,2]. In the era of molecularly targeted, rationally designed cancer therapeutics, natural environment, and especially plants continue to provide a rich source of anticancer agents.
The Mediterranean diet, characterized by an abundance of fruits and vegetables, contributes to the prevention of various chronic degenerative diseases such as cardiovascular diseases and cancer . Case-control, cohort, and prospective epidemiological studies have generated conflicting results regarding a protective effect of an olive oil-rich Mediterranean diet against several malignancies, especially breast cancer [4-8]. Olive tree (Olea Europaea) products are essential elements of Mediterranean diet. Olive oil is the major fat source of this diet and is considered an important component responsible, at least in part, for the low incidence of cardiovascular diseases in the Mediterranean area . The beneficial health effects of olive oil have been mainly attributed to the high content of the mono-unsaturated fatty acid oleic acid, while less attention has been given to other minor constituents such as phenolic antioxidant  present in appreciable amounts in crude olive oil (50-900 mg/kg) [11-13]. The phenolic compounds of olive oil and leaf are a complex mixture of compounds that include 3,4-dihydroxyphenylethanol (hydroxytyrosol), 4-hydroxyphenylethanol (tyrosol), 4-hydroxyphenylacetic acid, protocatechuic acid, caffeic acid and p-coumaric acid, among others [14,15]. The concentration of the phenolic fraction is several times higher in olive leaf than in oil. Apart from studies focused in olive oil composition, other studies have considered the constituents present in olive leaf extracts and olive oil mill waste (named “alperujo”) extracts mainly because of the availability and low cost of raw material in the region of Mediterranean.
Hydroxytyrosol (3,4-dihydroxyphenylethanol; DOPET) is the main ortho-diphenolic compound found in olive oil and responsible for its antioxidant properties [16,17]. Among the antioxidants in olive oil, hydroxytyrosol or 2-(3,4-dihydroxyphenyl)ethanol (DPE) has a remarkable protective effect against oxidative stress-related damage. It has been shown to prevent low-density lipoprotein oxidation  and platelet aggregation . Oleuropein is the most abundant of the phenolic compounds in olives . It actively scavenges reactive oxygen  and nitrogen species , with anti-angiogenic activity .
Both hydroxytyrosol and oleuropein have been shown to possess anti-inflammatory, bactericidal and bacteriostatic activities . Some in vivo studies on olive leaf have shown that its extract can decrease blood pressure and dilate the coronary arteries surrounding the heart . Moreover, hydroxytyrosol has been shown to have anti-cancer effect on human colon adenocarcinoma HT-29 cells and human promyelocytic leukemia HL-60 cells [26,27] have anti-melanogenesis activity, whereas oleuropein inhibited cell growth of LN-18, poorly differentiated glioblastoma; TF-1a, erythroleukemia; 786-O, renal cell adenocarcinoma; T-47D, infiltrating ductal carcinoma of the breastpleural effusion; RPMI-7951, malignant melanoma of the skin-lymph node metastasis; and LoVo, colorectal adenocarcinoma cells . Even though anticancer properties of oleuropein and hydroxytyrosol were confirmed in vitro with different cell lines, studies of their protective effect from liver cancer have not been demonstrated.
Aflatoxin B1 (AFB1) is naturally occurring mycotoxins elaborated by Aspergillus flavus and Aspergillus parasiticus that grow readily on foodstuffs stored in damp conditions like mouldy rice, hundredth wheat, barley, etc. The adverse health effects associated with AFB1 exposure range from acute liver toxicities to liver cancer in humans . AFB1-exo-8, 9-epoxide metabolized by hepatic cytochrome P450-family member, CYP1A2 and CYP3A4, which has been shown to bind and damage DNA . Different reports have showed that a G to T transversion of the third nucleotide in codon 249 of the p53 gene (249ser) [31-33], and Ras and Myc activation  are associated with AFB1-induced HCC.
Neoplastic progression of cancer cells is associated with chromosome damage that allows cells to escape from growth and proliferation controls and disables apoptosis. In these conditions, cell cycle progression is tightly regulated allowing arrest and repair of damaged DNA. The cell cycle is regulated through the sequential activation and inactivation of cyclin-dependent protein kinases (CDK) that control specific steps of the cycle progression, such as G1-S and G2-M transitions. CDK activation requires the binding to different cyclins (A, B, E, and D), which are timely expressed during the course of the cell cycle . CDK activity is also regulated by a diverse family of proteins termed CDK inhibitors (CKDi) that bind and inactivate CDK-cyclin complexes. There is a general agreement that the control of differentiation is tightly coupled to proliferation and some important molecules involved in the control of cell cycle progression, such as CIP/KIP family members named p21WAF1/Cip1 and p27Kip1, which are also implicated in the regulation of differentiation and apoptosis . The expression of p53 is important for cell proliferation and death [24,37]. The human SRC gene encodes pp60c-Src, a 60-kDa non-receptor, membrane-associated tyrosine kinase. C-Src was first identified based on homology to v-Src, the potent transforming oncogene encoded in the Rous Sarcoma Virus (RSV) genome . It is thought that c-Src cooperates with activated and/or overexpressed receptor tyrosine kinases (RTKs) in many human cancers where c-Src activation has been reported [39,40]. c-Src overexpression has been documented in upwards of 50% of human tumors derived from colon, liver, breast, lung, and pancreas [41-46].
The aim of the study was the identification of anticancer properties of olive leaves extract and olive oil mill waste extracts in control and pro-carcinogenic Aflatoxin B1-treated hepatoma cells. In addition, we evaluated if their most abundant compound such as oleuropein in olive leaves and hydroxytyrosol in olive mill waste extracts affect cell death in hepatoma cells. The study showed that the administration of natural extracts was to increase DNA damage, cell cycle arrest and apoptosis in hepatoma cell lines.
Material, reagents and standards
The olive leaves were collected, washed, dried in open air, and stored in freeze. The humidity content of olive oil mill waste or “alperujo” was eliminated by drying at 35°C for 24 h and stored at -20°C. Oleuropein and hydroxytyrosol were obtained from Extrasynthese (Genay, France).
The olive phenols from leaves were submitted to microwave-assisted extraction (MAE) with ethanol-water according to previously described procedure [47,48]. The irradiation power was set at 200 W with 80:20 (v/v) ethanol–water during 8 minutes. The superheated liquid extraction has been demonstrated to provide quantitative extraction of the target phenols of “alperujo” [47,48]. Thus, a static–dynamic method using 80:20 (v/v) ethanol-water at 200°C was used for sample preparation. The extraction time was 12 min for the static step and 15 min for the dynamic one using 1 mL/min extracting flow-rate.
Cell Lines and culture conditions
Human hepatoma cell line (HepG2) was obtained from the ECACC (European Collection of Cell Cultures) and routinely maintained in MEM medium pH 7.4 supplemented with 10% fetal bovine serum, 2.2 g/L HCO3Na, 100 mM sodium pyruvate, 0.292 gr/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin and 0.25 µg/mL amphotericin in 5% CO2 in air at 37°C. The study was initiated 24 hours after seeding in fresh culture medium. It was carried out a kinetic study of citotoxicity with AFB1 (50 µg/mL) dissolved in DMSO. Oleuropein (0.5, 0.05, 0.005 mg/mL), olive leaf extract (at a 0.5, 0.05, 0.005 mg/mL in relation to oleuropein concentration), hydroxytyrosol (0.05, 0.005, 0.0005 mg/mL), and “alperujo” (0.05, 0.005, 0.0005 mg/mL in relation to hydroxytyrosol concentration). Compounds were co-administered with AFB1. Samples were collected 0, 12 and 24 h after the addition of compounds. The final concentration of DMSO (0.005 % v/v) had no effect on experimental parameters.
Preparation of cytoplasmic and nuclear extracts
Nuclear extracts from hepatocytes were prepared according to Schreiber et al. . Briefly, hepatocytes were recovered in 200 µl of lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 5 µg/ml aprotinin, 10 µg/ml leupeptin, 0.5 mM phenyl methyl sulfonyl fluoride, 1 mM DTT,0.6% Nonidet NP-40) allowing to swell for 10 min on ice. Afterwards, samples were homogenized and centrifuged at 15,000 x g for 1 min at 4 ºC. After removal the supernatant (cytoplasmic fraction), the nuclear pellet was resuspended in 25 µL of nuclear extraction buffer (20 mM HEPES pH 7.9, 0.4 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 µg/mL aprotinin, 10 µg/mL leupeptin, 0.5 mM phenyl methyl sulfonyl fluoride, and 1 mM DTT). The tube was incubated for 20 min on ice with continuous mixing and centrifuged at 15,000 x g for 5 min at 4°C. Aliquots of the supernatant (nuclear fraction) were stored at -80°C until use.
Measurement of lactate dehydrogenase release
Lactate dehydrogenase (LDH) in the culture medium was measured by modification of a colorimetric routine laboratory method . Briefly, a volume of medium or cytoplasmic fraction ranging from 5-125 µL was incubated with 0.2 mM ß-NADH and 0.4 mM pyruvic acid diluted in 100 mM PBS pH 7.4. LDH concentration in the sample was proportional to the linear decrease in the absorbance at 334 nm and calculated using a commercial standard. LDH release represents the percentage of LDH in culture medium in relation to the total LDH (culture medium and cell lysate).
Assay for caspase-3-associated activity
The cytoplasmic fractions obtained above were also used to measure caspase-3 activity in cultured hepatocytes. The enzymatic activity in the cell extract (15 µg) was measured using the corresponding peptide-based substrate (Ac-DEVD-AFC, 100 µM) in caspase-incubating buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 10% sucrose, 0.1% CHAPS, 1 mM EDTA and 5 mM DTT) up to 100 µL total volume. The increase in fluorescence of the sample of enzymatically released AFC measured at Ex 400 nm and Em 505 nm was recorded using a GENios Reader (TECAN, Salzburg, Austria).
Analysis of c-Src Total, Phospho-Src (Tyr527), Phospo-Src (Tyr416), p21, p27, Wild and Mutated p53 Expression by Western-Blot in HepG2
The cytoplasmic and nuclear fractions obtained above were used to measure total c-Src, phospho-Src (Tyr527) and phospo-Src (Tyr416), as well as p21Waf1/Cip, p27Kip1, native and mutated p53 protein expression in cultured HepG2 cells, respectively. Proteins (20-100 µg) were separated by 10-14% SDS-PAGE electrophoresis and transferred to nitrocellulose for Western-blot analysis using anti-c-Src (2109, Cell Signalling Technology, Inc), phospho-Src (Tyr527) (2105, Cell Signalling Technology, Inc), phospho-Src (Tyr416) (2101, Cell Signaling Technology, Inc), p27Kip1 (sc-528, Santa Cruz Biotechnology), p21Cip1 (sc-397, Santa Cruz Biotechnology), wild type p53 (sc-6243, Santa Cruz Biotechnology) and mutated p53 (sc-91, Santa Cruz Biotechnology) primary antibodies. Samples were incubated with the corresponding secondary antibodies. The sample was incubated with horseradish peroxidase.
Cell proliferation was determined by BrdU (5-bromo-2'-deoxyuridine)-ELISA based assay (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions. Briefly, BrdU (10 µM) was added 4 h before collecting samples, washed with 10% SFB containing medium, and fixed with 200 µL of precooled commercial fixative solution during 30 min at -20°C. Cells were treated with nuclease for 30 min at 37°C, and incubated with anti-BrdU-POD for 30 min at 37°C. The BrdU incorporation was measured at 405/492 nm (Tecan, Spectrofluor, Salburg Astria).
Detection of 8-Hydroxydeoxiguanosine formation in hepatocytes
Cells were cultured onto glass bottom culture dishes coated with collagen (MatTek Corporation, Ashland, MA, USA). Cells were fixed with cold 70% ethanol for 10 minutes, and incubated with 100 µg/mL RNAse diluted in 10 mM Tris-HCl, 1 mM EDTA and 0.4 mM NaCl pH 7.5 at 37°C for 1 hour. After cell washing with PBS, DNA was denatured by soaking the slides in 50 mM Tris-base at room temperature for 5 minutes, and reduced unspecific binding with 10% SBF in 10 mM Tris-HCl pH 7.5 and at 37°C for 1 hour. Samples were incubated with goat polyclonal anti-8-hydroxydeoxiguanosine antibodies (Chemicon International) (1:300) in 10% fetal bovine serum at 4 ºC overnight. Biotin anti-goat Ig G (H+L) polyclonal antibodies (Jackson Immuno-Research Laboratories, West Grove, PA, USA) diluted (1:500) in 10 % fetal bovine serum and 10 mM Tris-HCl pH 7.5 at 37°C for 30 minutes. At this point, slides were treated with H2O2 (3%) in methanol at room temperature for 30 minutes in order to avoid DNA damage before immunological detection. Samples were incubated with streptavidin-horseradish peroxidase for 30 min, washed twice with 1% Triton X-100, and incubated with in DAB and H2O2 (0.001%) for 10 min. Slides were washed with deionised water and counterstained with 1% methyl green for 2 min. The samples were washed, dehydrated in 95% and 100% ethanol, clarified by xylene and mounted with Eukkit® mounting media. Apoptotic index was determined by counting the percentage of positive cells in 200 cells.
Data were expressed as Mean ± SE of five experiments. Data were compared using ANOVA with the Least Significant Difference (LSD) test as post-hoc multiple comparison analysis. The statistical differences were set at p = 0.05. The groups with “a” were significantly different vs. the corresponding control group. The groups with “b” were significantly different vs. the corresponding group without AFB1. The images or blots are representative of five independent experiments.
The analysis of olive leaves extract and olive oil mill waste showed that oleuropein hydroxytyrosol (3,4 DHPEAEDA) were the most abundant compound, respectively. The concentrations of polyphenols (mg/L) detected in olive leaf and oil mill wastes are shown in Table 1.
|Olive oil mil waste||Hydroxytyrosol||15.2|
Table 1: Phenolic compounds present in olive leaves and olive oil mill waste extracts.
Effects of extracts in cell death, proliferation and DNA damage in AFB1-treated HepG2
AFB1 increased caspase-3 activity (Figure 1A) and LDH (Figure 1B) in HepG2 cells (p=0.05). Oleuropein and Hydroxityrosol reduced cell necrosis (Figure 2A and 2C) and DNA damage (Figure 2B and 2D) in control and AFB1-treated hepatocytes (p = 0.05). However, the corresponding containing extracts such as olive leaves and olive oil mill waste extracts increased LDH release (Figures 3A and 3B, respectively) and DNA damage (Figure 4A and 4B, respectively) in hepatoma cells. Interestingly, this effect of olive leaves and olive oil mill waste extracts was related to arrest of cell proliferation at 24 hours in control and AFB1-treated HepG2 cells (Figures 5A and 5B) (p = 0.05).
Figure 1: Effects of Aflatoxin B1 (AFB1) on apoptosis (A) and necrosis (B) in hepatoma cancer cells. Apoptosis and necrosis were measured by caspase-3 activity and LDH release in HepG2 cells, respectively. The groups with “a” were significantly different vs. the corresponding control group. The groups with “b” were significantly different vs. the corresponding group without AFB1.
Figure 2: Effects of oleuropein and hydroxytyrosol on cell necrosis (A and C,respectively) and DNA damage (B and D, respectively) in hepatoma cancer cells. Cell necrosis and DNA damage were measured by LDH release and 8-hydroxydeoxiguanosine, respectively. The groups with “a” were significantly different vs. the corresponding control group. The groups with “b” were significantly different vs. the corresponding group without AFB1(Image magnification x100).
Figure 3: Effects of olive leaves (A) and olive oil mill waste (B) extracts on cell necrosis in Aflatoxin B1 (AFB1)-treated HepG2 cells. Cell necrosis was assessed by LDH release in HepG2. The groups with “a” were significantly different vs. the corresponding control group. The groups with “b” were significantly different vs. the corresponding group without AFB1
Figure 4: Effects of olive leaves (A) and olive oil mill waste (B) extracts on cell proliferation in Aflatoxin B1 (AFB1)-treated HepG2 cells. Cell proliferation was determined by BrdU incorporation in HepG2 cells. Samples were collected at 24 hours. The groups with “a” were significantly different vs. the corresponding control group. The groups with “b” were significantly different vs. the corresponding group without AFB1.
Effects of natural extracts on the expression of p53 and c-Src oncoprotein in HepG2
The administration of AFB1 increased the expression of p53 in nuclear fraction from HepG2 cells (Figure 6). AFB1-containing samples did not show p21 and p27 expression in HepG2 cells (data not shown). The administration of olive leaves (Figure 6A) and olive oil mill waste (Figure 6B) extracts reduced specially at their highest concentration p53 expression in control and AFB-treated HepG2 cells. c-Src is an oncoprotein with tyrosine kinase activity which exerts a relevant role in cell proliferation. The phosphorylation in Tyr416 and Tyr527 residues acts as a negative and positive regulator of the kinase activity. Tyr416 phosphorylation of c-Src was not detected in control and AFB1-treated HepG2 cells (data not shown). AFB1 increased the expression of total c-Src with a remarkable reduction of Tyr527 phosphorylation at 12 hours in HepG2 cells (Figure 7). The administration of natural extracts counteracted the effect of AFB1 on c-Src total levels and increased its Tyr527 phosphorylation (Figure 7).
Many vegetable foods in Mediterranean diet contain substances possessing anticancer properties . Epidemiological studies performed in different countries (Spain, Greece and Italy) have shown that the consumption of olive oil reduces the estimated relative risk of breast cancer [52-54]. Epidemiological studies have reported the strong correlation between the intake of olive oil and colon  or prostate  cancer. However, few experimental reports have demonstrated the antitumoral mechanism of olive-derived compounds. It has been recently shown that olive oil prevents the development of azoxymethane-induced aberrant crypt foci and colon carcinomas in rats .
Several phenolic compounds from olive, such as oil, leaves, fruits and waste processing may play a role in cancer protection. In this sense, hydroxytyrosol [3,4-dyhydroxyphenylethanol (3,4-DHPEA)], an ortho-diphenol derived from the hydrolysis of oleuropein, recently received particular attention because it may inhibit both initiation and promotion steps of carcinogenesis in vitro. The present study has shown that olive leaves extracts and olive mill waste, that contain oleuropein and hydroxytyrosol respectively, are able to increase DNA damage, reduce cell proliferation and promote cell death of hepatoma cells.
Olive mill waste or “alperujo” used in the present study contains an important amount of hydroxytyrosol (15.2 mg/L). In the last years, different antitumoral and cardiovascular activities have been observed with hydroxytyrosol administration. Hydroxytyrosol inhibits platelet aggregation , alters eicosanoid metabolism , and inhibits low-density lipoprotein oxidation in vitro  and in vivo . Hydroxytyrosol has demonstrated antitumoral properties in different cell types (Rasione et al)  have reported that hydroxytyrosol increase mitochondrial cytochrome c release and cell death in HL60 cells. Hydroxytyrosol induce a cell cycle arrest and apoptosis in HL60  and colon cancer cells . However, this compound possesses a clear antioxidant activity protecting cells against oxidative damage [51,63,64]. These results suggest that hydroxytyrosol may prevent cancer by efficiently preventing the mutagenic activity caused by oxidative stress, or may interfere with other steps inducing cell cycle arrest, thereby reducing the growth and proliferation of cancerous cells and subsequently, inducing apoptosis and necrosis death. Other major components of the studied extracts such as oleuropein, hydroxytyrosol, hydroxytyrosol acetate, and the flavonoids luteolin, luteolin- 7-O-glucoside, and luteolin-49-O-glucoside also induce cell cycle arrest [65-69].
Several cell cycle regulators such as p27 and p53 have been identified as potential prognostic markers for the outcome of HCC [70-72]. AFB1 increased the expression of p53 in cells that were reduced by olive leaves and “alperujo” extracts. In many cases, the induction of p21 required the functional p53 tumor suppressor protein that is activated by DNA damage. In our conditions, we could not detect p21 and p27 in HepG2 cells (data not shown). The c-Src activity has strong association with advanced tumor stage and metastasis in different human carcinoma tissue [73,74]. c-Src affects cell proliferation and the metastatic properties of cancer cells [75,76]. In the present study we showed that the expression of c-Src is increased in AFB1-treated HepG2 cells. The activity of pp60c-Src is regulated through phosphorylation of different tyrosine residues. The phosphorylation of tyrosine residue 416 (Try416) has a positive regulatory effect on pp60c-Src kinase, whereas phosphorylation at the C-terminal tyrosine residue 527 (Tyr527) acts as a negative regulator of the kinase activity . The reduction of tyrosine 527 phosphorylation by AFB1 may exert an inducer effect on cell proliferation. In consequences, the reduction of c-Src and relative increase of Try527 c-Src phosphorylation by the administration of olive leaves and “alperujo” extracts may be related to a reduction of cell proliferation in HepG2 cells.
In conclusion, the administration of olive leaves and olive oil mill extracts (“alperujo”) is able to increase DNA damage, cell cycle arrest and apoptosis in hepatoma cell line. More studies should be done in order to identify if oleuropein, hydroxytyrosol or other components are responsible for this antitumoral effect.
This study has been supported in part by the Consejería de Innovación, Ciencia y Empresa (P06-FQM-01515) and Consejería de Salud (SAS 0103/2006) both from Junta de Andalucía (Spain). We thank Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd) founded by “Carlos III” Health Institute for its support.