Dietary Intervention With High Phenolic EVOO in CLL

Extra virgin olive oil (EVOO) is the major component of the Mediterranean diet. More than 200 different chemical compounds have been detected in olive oil, including fatty acids, sterols, carotenoids, terpenoids, flavonoids, tocopherols and olive polyphenols. Virgin olive oil contains a variety of important phenolic components, mainly corresponding to the compounds tyrosol, hydroxytyrosol, oleocanthal (OC) ,oleacin (OL) ,ligstroside and oleuropein aglycones .Among these olive phenols, oleocanthal , a dialdehydic form of dicarboxymethyl ligstroside aglycone which has been isolated from EVOO, presents a wide range of biological effects including antioxidant, anti-inflammatory, anticancer, neuroprotective and antidiabetic activities and for this reason has attracted more scientific attention.

Chronic Lymphocytic Leukemia (CLL), the most commonly diagnosed adult leukemia in Western countries, is responsible for about 1 in 4 cases of all leukemias. It is characterized by the accumulation of monoclonal B-cells in bone marrow, peripheral blood, lymphatic tissues and spleen, while it is often asymptomatic and slow in its development. Patients with CLL do not always require immediate therapy. However, patients who appear symptoms have an approximate median survival range from 18 months to 6 years depending on the clinical stage. Criteria for the diagnosis of CLL are monoclonal B lymphocytes ≥5000 lymphocytes/ml in the peripheral blood for at least 3 months, prolymphocytes ≤55%, co-expression of claster of diferentiation (CD)5 and B-cell surface antigens CD 19, CD 20, and CD 23, low levels of CD 20, CD 79b and low expression of surface immunoglobulins (sIg), and kappa or lambda light chain restriction. The standard clinical procedures to estimate prognosis are the clinical staging systems developed by Rai et al and Binet et al.

The level of apoptosis in hematopoiesis may provide information about the mechanism which is responsible for the regulation of progenitor's and stem cell's proliferation. The dysfunction of apoptosis has found to be associated with leukemia. An increase in the expression of Apo-1/fas (CD 95) apoptotic marker in myeloid progenitor cells in CLL patients and a corresponding decrease in the apoptotic B-cell lymphoma 2 (BCL-2) inhibitor has been described. Recent studies showed also that the increase in CLL patients' lymphocytes may be due to the disturbed balance between the proliferation and apoptosis of CLL cells while the changes in apoptosis-related protein expression are recognized during all the stages of Binet in CLL. The p38 levels are reduced (resistance to apoptosis) and Induced myeloid leukemia cell differentiation protein (Mcl-1) and Survivin levels are elevated, so as to enhance the survival of B lymphocytes.

Survivin is a protein inhibitor of apoptosis which inhibits caspases and blocks cell death. It is highly expressed in most cancers, including hematological, and is associated with a poor clinical outcome. The apoptotic marker CD 95 (Apo-1/fas) and its ligand, CD 95 is a death receptor ligand that mediates apoptosis induction in order to maintain immune homeostasis and has the capacity to mediate apoptosis induction in cancer cells, generating an 18-kilodalton fragment termed caspase-cleaved cytokeratin-18 (cCK-18).

Oleocanthal has been shown to induce cytotoxicity and apoptosis in vitro in human acute promyelocytic leukemia and in myeloma cells. It may also cause primary necrosis and cellular apoptosis by inducing lysosomal membrane permeability in different cancer cell lines.

The aim of this study taking into account the strong apoptotic properties of oleocanthal in vitro, was to investigate its ability, under real clinical settings, to affect the hematological and biochemical profile; also the cellular apoptotic markers Survivin, cCK18- (caspase activity marker) and Apo1fas/CD 95 in CLL patients after taking into consideration the progression of their disease. For this reason was conducted a dietary intervention study comparing the antineoplastic effects of an oleocanthal-rich EVOO in CLL patients.

Material and Methods

In the current study, a screening of 300 commercial samples (selected among a database of >3000 samples), obtained from olives (Olea europaea L.) harvested in 2017-2018 season, was performed by the Quantitative NMR spectroscopy (qNMR) method in order to identify oils which could fulfill the following criteria: The extravirgin oil would have high oleocanthal and oleacin content (D1 index) and low or even no content in other secoiridoid conjugated phenolics (3-8) or free tyrosol and hydroxytyrosol Specifically Oil rich in Oleocanthal was obtained from Monovarietal oil of Lianolia Kerkyras variety provided by "The Governor" Company from Agios Matthaios, Corfu, Greece. This oil contained: Oleocanthal 416±7 mg/Kg, oleacin 284±10 mg/Kg, Tyrosol < 10 mg/Kg, Hydroxytyrosol < 10 mg/Kg (D1 = 700 mg/Kg). It should be mentioned that apart the qNMR analysis, the hydroxytyrosol and tyrosol content was also measured by the standard IOC method for measurement of olive oil biophenols. Selected EVOO immediately after their analysis and until the beginning of the study period were stored at 4 celsious degree in order to minimize possible alterations in chemical composition.

Subjects Twenty one patients from the hematologic clinic of the General hospital of Lakonia (Greece), were enrolled to participate in the dietary intervention. CLL diagnosis was confirmed by standard criteria. Patients were untreated, in Rai stage 0 to II.

Dietary Intervention At the begin of the pilot study medical exams of each patient`s history were recorded for third and sixth month before dietary the intervention. The clinical record before the dietary intervention concerns the hematological profile, biochemical exams including, lipidemic profile, fasting glucose, Liver and kidney function markers, as well as data concerning blood marrow biopsy, neoplastic markers and immunophenotype. Drug treatment or comorbidities were also recorded.

At the begin of the pilot dietary intervention a group of 21 patients with CLL consumed before their meals, 40 ml/day of EVOO rich in olecanthal and oleacin (OC/OL) for 6 months, in order to explore the effect of the dietary consumption of the A-EVOO on the studied hematological, biochemical and molecular markers. The amount of the consumed EVOO OC/OL was chosen according to a previous study. (15) Patients were advised to continue their regular diet; their adherence to the study protocol was assessed by two 24-h telephone recalls every two weeks.

Peripheral blood samples from the 21 patients with CLL were gathered at the begin, after 3 months and after 6 months from the begin of the dietary intervention. Whole blood count and complete biochemical tests were performed for fasting glucose, blood lipid profile, and markers of liver and kidney function. Serum analyses were conducted using the same procedure and at the same lab with the use of a biochemical analyzer (Olympus AU-600, Olympus, Tokyo, Japan).

A commercially available enzyme-linked immunosorbent assay (ELISA) was used according to the instructions of the supplier to assay the apoptotic markers M30 Apoptosense-cCK18 (VLVbio Hästholmsvägen, Sweden), Apo-1/Fas/CD 95 (Novus Biologicals, Colorado USA) and the antiapoptotic marker Survivin (OriGene Technologies, Inc USA) into plasma samples of all the patients at all the studied time points.

Statistical analysis was performed using SPSS v.24 (SPSS Inc., Chicago, IL. USA) and the significance level was set at 0.05. Initially a descriptive analysis of the sample was performed in terms of age, sex, clinical, biochemical and molecular markers. The markers were found to be skewed at p < 0.05 significance level when checked by using the Kolmogorov-Smirnov test. Therefore non-parametric Mann-Whitney U test were used to find out the differences between the intervention and the control group.

The differences between the time intervals of the intervention was assessed with Friedman non‐parametric test followed by Dunn's test for multiple comparison Finally, the correlations of the molecular markers with the clinical parameters included in the study and were checked with Spearman Rho.