The Effect of Dietary Intervention With High-Oleocanthal and Oleacein Olive Oil in Patients With Early-Stage Chronic Lymphocytic Leukemia: A Pilot Randomized Trial

Abstract

Aim: Oleocanthal and oleacein (OC/OL) have important in vitro and in vivo antitumor properties; however, there is no data about their anticancer activity in humans. The aim of this pilot study was to test if patients at early stage of chronic lymphocytic leukemia (CLL) could adhere to and tolerate an intervention with high OC/OL extra virgin olive oil (EVOO) and if this intervention could lead to any changes in markers related to the disease.

Methods: A pilot dietary intervention (DI) was made in patients with CLL in Rai stages 0-II who did not follow any treatment (NCT04215367). In the first intervention (DI1), 20 CLL patients were included in a blind randomized study and were separated into two groups. One group (A) of 10 patients consumed 40 ml/day of high OC/OL-EVOO (416 mg/Kg OC and 284 mg/kg OL) for 3 months. A second group (B) of 10 patients consumed 40 ml/day of low OC/OL (82 mg/kg OC and 33 mg/kg OL) for 3 months. After a washout period of 9-12 months, a second intervention (DI2) only with High OC/OL-EVOO for 6 months was performed with 22 randomly selected patients (16 from the DI1 (8 from each group) and 6 new). Hematological, biochemical, and apoptotic markers were analyzed in the serum of the patients. In addition, cellular proliferation and apoptosis markers were studied in isolated proteins from peripheral blood mononuclear cells.

Results: The results of the DI1 showed beneficial effects on hematological and apoptotic markers only with High OC/OL-EVOO. During the DI2, a decrease in the white blood cell and lymphocyte count was observed (p ≤0.05), comparing 3 months before the intervention and 6 months after it. After 3 and 6 months of DI2, an increase (p ≤0.05) was observed in the apoptotic markers ccK18 and Apo1-Fas, and also in the cell cycle negative regulator p21, and also a decrease in the antiapoptotic protein Survivin, and in the cellular proliferation marker Cyclin D.

Conclusions: This is the first clinical trial with High OC/OL-EVOO that indicates that it could be a promising dietary feature for the improvement of CLL inducing the apoptosis of their cancer cells and improving the metabolism of the patients.

Clinical trial registration: https://clinicaltrials.gov/ct2/show/NCT04215367, identifier: NCT04215367.

Keywords: Chronic Lymphocytic Leukemia (CLL); apoptosis; dietary intervention; extra virgin olive oil (EVOO); oleacein; oleocanthal; randomized trial.

Figure 1

Trial profile: This figure displays the trial profile, including the total number of screened and randomized subjects, the group distribution in both dietary interventions, and an overview of reasons for withdrawal.

Figure 2

(A) Flow chart of the Dietary Intervention protocol (DI1). (B) Flow chart of the Dietary intervention protocol (DI2).

Figure 3

Variation on the white blood cells during the DI2 with High OC/OL-EVOO correlating the differences WBC₍₋₆₎ − WBC₍₊₆₎ with WBC(−6), WBC₍₋₃₎ − WBC₍₊₆₎ with WBC(−3) and WBC₍₀₎ − WBC₍₊₆₎ with WBC₍₀₎. Linear regression was performed and R² is depicted.

Figure 4

Variation on the apoptotic marker ccK18 (A), Apo1-Fas (B) and antiapoptotic Survivin (C) during the dietary intervention DI2 with High OC/OL-EVOO. Linear regression was performed and R² is depicted.

Figure 5

TUNEL Assay (top) performed on isolated PMBC from patients with CLL during the dietary intervention DI2 with High OC/OL-EVOO. (A) Baseline. (B) 3 months of dietary intervention. (C) 6 months of dietary intervention. DAPI staining (middle) and overlay (bottom) are also illustrated. The data in the histograms are the mean ± SD of 22 different experiment subjects. Data were compared using the unpaired two-tailed t-test. Significant difference between the time points of the dietary intervention at p <0.05 (*0–3, **3–6 and 0–6) is indicated. For each subject each experiment was performed twice.

Figure 6

Effect of High OC/OL-EVOO on cyclin D, Survivin and p21 protein expression in PMBC cells of patients with CLL during DI2. The protein expression level was measured by Western blot analysis at baseline, at 3 and 6 months after dietary intervention in equal amount of cell lysate (40 lg/lane) from isolated PMBC cells of each participant. Cell lysates was subjected to electrophoresis and analyzed by Western blot. (A) Representative blots shown for each studied protein for selected patients (P2, 7, 10, 11, 17, 19). (B) The densitometry measurements are depicted in histograms. The data in the histograms are the mean ± SD of 22 different experiment subjects. The protein expression levels are normalized according to the measurement of β-actin in a semi quantitative analysis. Quantification of relative protein expression of the Western signals (complexed protein bands) was performed manually using the image analysis program, Image-J (IE 6.0, Microsoft Java). Data were compared using the unpaired two-tailed t-test. Significant difference between the time points of the dietary intervention at p <0.05 (*0–3, **3–6 and 0–6) is indicated.