1. Introduction
Olea europaea L. (Oleaceae) is a characteristic Mediterranean species used to produce olives and oils, representing key components of the traditional Mediterranean diet that includes a high intake of fruit, vegetables, fish, red wine, and extra virgin olive oil (EVOO). Such a diet is widely recognized to have an impact on good health, long life, prevention of chronic diseases (e.g., cardiovascular and neurodegenerative), and cancers in the Mediterranean population. In addition to EVOO, another phytochemical may be obtained from the olive plant: olive leaf extract (OLE), containing similar biologically active compounds, even at much higher concentrations and a wider variety. OLE is abundant in polyphenolic compounds, broadly categorized into secoiridoids including oleuropein, dimethyloleuropein, and oleuropein–aglycone, simple phenols such as hydroxytyrosol and tyrosol, and flavonoids (e.g., apigenin and luteolin). These polyphenols have been documented to exert anti-microbial [ 1 , 2 ], anti-viral [ 3 , 4 , 5 ], hypoglycemic [ 6 ], neuro-protective [ 7 , 8 ], anti-inflammatory [ 9 , 10 , 11 ] and anti-tumor activities [ 12 , 13 , 14 , 15 ]. The latter function was reported both in vitro and in vivo against a wide panel of human tumors, including breast, pancreatic, colorectal, and skin cancers, as well as melanoma, glioblastoma, neuroblastoma, and acute leukemias. In the context of prostate and breast cancers, oleuropein and hydrotyrosol were the main molecules endowed with anti-tumor properties; noteworthy, it was reported that they were able to discriminate between cancer and normal cells by inhibiting proliferation and inducing apoptosis only in the former [ 16 , 17 , 18 ]. In acute leukemias, the ability of OLE to counteract leukemia cell growth was evaluated exclusively against cell lines of myeloid origin (i.e., K562 and HL-60), but not in the B-acute lymphoblastic leukemias (ALL). In addition, B-cell lymphomas have not yet been investigated. The anti-tumor activity may be exerted through different and complementary mechanisms [ 15 ] mainly related to the (i) inhibition of genotoxicity and mutagenesis, (ii) induction of apoptosis, (iii) inhibition of cell proliferation, (iv) selective cytotoxicity, (v) regulation of oxidative stress through reactive oxygen species (ROS), and (vi) inhibition of angiogenesis, tumor cell migration and metastasis. Thus, olive polyphenols can target all phases of chemoprevention from primary prevention by inhibiting cancer initiation, to secondary prevention by inhibiting cancer promotion, and therapeutic intervention by inhibiting cancer progression. The broad beneficial effects of OLE are of particular relevance in the context of oncologic pediatric patients, especially those with poor prognoses, receiving debilitating treatments that affect the patients’ quality of life.
With this background, we investigated whether OLE may function as an anti-tumor agent against pediatric B-ALL, acute myeloid leukemias (AML), and human B-cell lymphomas, investigating the cellular pathways involved. B-ALL is the most common hematological cancer in children caused by an abnormal clonal proliferation of B-cell progenitors, thus manifesting as the accumulation of malignant and poorly differentiated lymphoid cells within the bone marrow, peripheral blood, and some extra-medullary sites. Despite the optimization of treatment protocols for pediatric B-ALL, approximately 15% of patients experience a relapse, and the overall survival of these patients is less than 10%. Moreover, other patients are refractory to the treatment and do not achieve remission. Hematopoietic stem cell transplantation is almost inevitably part of salvage treatment after relapse, but transplant-related mortality and long-term morbidity remain a pitfall. The prognosis of AML, which represents the second most common hematologic malignancy in children, remains less favorable compared to ALL, and approximately 30% of AML patients experience relapse and ultimately die from disease-related complications or treatment side effects. Finally, B-cell lymphoma is one of the more common cancers among children, teens, and young adults that starts in early forms of B lymphocytes. The overall 5-year relative survival rate is approximately 74% and patients, as for leukemias, undergo intensive chemotherapy and radiotherapy, surgery, hematopoietic stem cell transplantation, and immunotherapy.
Thus, novel therapeutic approaches that are less toxic, selectively target malignant cells, and potentially strengthen the immune responses of oncologic pediatric patients affected by leukemias or lymphomas are needed.
2. Materials and Methods
2.1. OLE Composition
Aqueous OLE, whose composition has been previously reported [ 12 ], was used throughout the study and was kindly provided by Evergreen Life, San Giovanni al Natisone, Italy. The manufacturer has implemented a patented method for the extraction at very low temperatures of different active compounds, mainly polyphenols, from olive leaves. The mean phytochemical compounds and their concentrations contained in OLE were the following: Oleuropein 2656 mg/L, Hydrohytyrosol 213 mg/L, Tyrosol 174 mg/L, Elenolic Acid 1393 mg/L, and Rutin 237 mg/L. Two different batches of OLE have been used throughout this study, with minimal variations in the composition, as disclosed by the manufacturer.
2.2. Cell Lines and Primary Leukemias from Pediatric Patients
The following human cell lines were used: NALM-6 and 697 B-ALL; K562, THP-1, and RPMI8866 myeloid leukemias; SUDHL-4, U937, and RAJI lymphomas (ATCC and Biobank at Ospedale San Martino, Genova, Italy). Cells were cultured in RPMI-1640 (Gibco/Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% of heat-inactivated fetal bovine serum (FBS, by Euroclone, Milano, Italy), 50 IU/mL penicillin, 50 g/mL streptomycin sulphate, and 2 mM L-glutamine (Euroclone, Milano, Italy). Fresh medium was replaced every three days.
Leukemia cells (five B-ALL and three AML) were derived from patients’ heparinized bone marrow aspirates (>90% blast cells) at diagnosis and, in patients experiencing disease recurrence, also at relapse, following written informed consent according to Helsinki Declaration. Leukemia cells were isolated by Ficoll-Hypaque Lympholyte ® (Cedarlane, Burlington, ON, Canada) density gradient centrifugation, then cryopreserved in FBS (Euroclone SPA, Milan, Italy), with 10% DMSO (AL.CHI.MI.A S.R.L., Padua, Italy) for later use. Characteristics of patients are summarized in Table 1 .
2.3. Cell Proliferation
Since oleuropein was the most effective and the most concentrated polyphenol in OLE, the concentration of OLE reported throughout the study was referred to the final concentration of oleuropein. Leukemia and lymphoma cell proliferation was assessed by flow cytometry using a CellTrace™ CFSE Cell Proliferation Kit (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, cell lines were resuspended in RPMI-1640 without serum, stained with Carboxyfluorescein diacetate N -succinimidyl ester (CFSE) at a final concentration of 2 μM, and incubated at 37 °C for 15 min. After washing in RPMI 10% FBS, cells were resuspended in RPMI 10% FBS, seeded in a 96-well plate (10 5 /well), and treated with OLE at the final concentrations of 50, 100, 200, and 400 µM. After 6 days of culture, cells were washed and analyzed for cell proliferation, as witnessed by CFSE dilution, by flow cytometry. Cells cultured for 6 days in the absence of FBS were used as negative controls. Cells were harvested, washed, and resuspended in 300 μL of MACS buffer (PBS supplemented with 2 μM EDTA and 0.5% BSA). Samples were then run on a Gallios ® flow cytometer (Beckman Coulter, Brea, CA, USA), acquiring at least 3 × 10 4 events, and data were analyzed using Kaluza ® analysis software version 2.0 (Beckman Coulter, Brea, CA, USA). The results were expressed as CFSE mean fluorescence intensity (MFI).
2.4. Leukemia and Lymphoma Cell Apoptosis
Leukemia and lymphoma cell lines, as well as primary samples from pediatric leukemia patients, were analyzed for induction of apoptosis driven by OLE. Cells were seeded at 105/well in 96-well plates, treated for 24, 48, and 72 hours (h) with OLE 50, 100, 200, and 400 µM, and tested for apoptosis by flow cytometry using the Annexin V-FITC/7AAD kit (Beckman Coulter, Brea, CA, USA), following the manufacturer’s instructions. Briefly, 10 5 cells were washed with PBS and resuspended in 100 μL of binding buffer 1X. Next, 7-AAD and FITC-conjugated Annexin V were added, and cells were incubated for 15 min on ice. Next, 300 μL of binding buffer 1X was added to each sample, and cells were run on a Gallios ® flow cytometer (Beckman Coulter, Brea, CA, USA), acquiring at least 3 × 10 4 events. Data were analyzed using Kaluza ® analysis software (Beckman Coulter, Brea, CA, USA). Annexin V + staining identified early apoptotic cells, 7AAD + /Annexin + the late apoptotic cells, and 7AAD + /Annexin − dead cells. B-ALL and AML primary samples were treated for 24 h with 100 and 200 µM OLE and tested for apoptosis.
2.5. OLE Molecular Pathways in Leukemia Cells
To investigate the molecular pathways involved in the apoptosis induced by OLE in leukemia cells, we selected the NALM-6 cells, due to their high response to OLE in terms of inhibition of cell proliferation and induction of apoptosis, and the K562 cell line for its low response to OLE treatment. Cells were treated in the presence or absence of 200 µM OLE for 24 h, and protein extracts were obtained following the protocol reported in each human proteome profiler kit. Proteins were quantified using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad, Hercules, CA, USA) and Tecan Infinite 200 Pro Bioreader (Tecan, Männedorf, Switzerland) (absorbance at 555 nm). A total mass of 200 µg of each protein lysate were assessed on 4 different human proteome profiler kits following the manufacturer’s instructions (Bio-Techne, Minneapolis, MN, USA): (i) apoptosis array detecting the relative levels of expression of 35 apoptosis-related proteins, (ii) NFkB pathway array analyzing 41 different proteins and 4 serine or tyrosine phosphorylation sites antibodies, (iii) phospho-kinase array with 37 different kinases and 2 related total proteins, and (iv) cell stress array analyzing 26 different stress-related proteins. The relative amount of each individual protein (corresponding to each spot tested in duplicate) was calculated using Image Lab software (Bio-Rad, version 6), upon normalization to internal controls. The fold increase was calculated for each protein as the ratio between values obtained in treated versus untreated cells. A fold increase of 1 indicated no modulation, whereas positive values indicated an up-regulation and negative values indicated a down-regulation. An arbitrary cut-off value for fold increase (1.8) was established, and only proteins with a fold increase >1.8 or >−1.8 are shown.
2.6. Combined Effect of OLE and Chemotherapeutic Agents on B-ALL Cell Lines
NALM-6 and 697 B-ALL cell lines were treated for 24 and 48 h with a combination of 50 or 100 µM OLE and cytarabine (Accord, 0.5 and 1 µM) or cyclophosphamide (Endoxan, Baxter, 0.5 and 1 µg/mL), obtained as a spare aliquot after therapeutic use. The percentage of live cells (7AAD − /Annexin V − ) was analyzed, as described above. The combined effect of OLE with the other drugs was analyzed using ZIP and Bliss models for additive effects and HSA and Loewe models for synergistic effects. These analyses were carried out using the SynergyFinder+ web application ( https://synergyfinder.org ) [ 19 , 20 , 21 , 22 , 23 ].
2.7. Statistical Analysis
Statistical analysis was carried out using Prism 5.03 software (GraphPad Inc., Boston, MA, USA). Data distribution was analyzed using the KS normality test and the D’Agostino and Pearson omnibus normality test. Comparison of data sets was analyzed using a t -test or Mann–Whitney test, depending on data distribution. p -values < 0.05 were considered statistically significant.
3. Results
3.1. OLE Inhibits the Proliferation of Leukemia/Lymphoma Cell Lines
First, we tested whether OLE could affect the proliferation of leukemia/lymphoma cells. To this end, we analyzed a panel of cell lines, including B-ALL (697 and NALM-6), Burkitt’s lymphoma (RAJI), myeloid leukemia (K562, RPMI8866, and THP-1), and histiocytic lymphoma (SU-DHL-4 and U937), treated for 48 h with different concentrations of OLE. As shown in Figure 1 , the proliferation of B-ALL cell lines was significantly inhibited by OLE at different concentrations, as witnessed by the increase in MFI CFSE. In detail, the proliferation of the 697 cell line (CFSE MFI mean ± SE 7.88 ± 1.29) was significantly reduced by OLE at 50 μM (11.87 ± 1.95, p = 0.03), 100 μM (13.98 ± 1.12, p = 0.0076), 200 μM (14.79 ± 1.22, p = 0.0043), and 400 μM (16.94 ± 0.79, p = 0.0011). Similarly, the proliferation of the NALM-6 cell line (11.36 ± 2.08) was significantly inhibited by OLE at 100 μM (19.38 ± 3.44, p = 0.01), 200 μM (22.58 ± 2.97, p = 0.01), and 400 μM (22.64 ± 2.87, p = 0.0011), but not at 50 μM (16.67 ± 3.82).
The effect of OLE on the proliferation of other cell lines was more limited at the same time point and concentrations. In particular, RAJI cell line proliferation (26.84 ± 3.67) was found to be significantly reduced by OLE only at 200 μM (61.46 ± 10.9, p = 0.0079) and 400 μM (57.74 ± 7.4, p = 0.004). Other OLE concentrations proved less effective. Similar results were obtained in histiocytic lymphoma cell lines SU-DHL-4 (CTR 16.63 ± 1.47; OLE 200 μM 31.1 ± 3.41, p = 0.0076; OLE 400 μM 36.62 ± 3.35, p = 0.0011), and U937 (CTR 10.78 ± 1.34; OLE 400 μM 24.32 ± 3.69, p = 0.01) cell lines.
Myeloid leukemia cells lines resulted the most resistant to OLE treatment, in particular, proliferation of K562 and THP-1 cell lines was inhibited only at the highest dose of OLE (K562: CTR 9.1 ± 0.5; OLE 400 μM 12.13 ± 0.98, p = 0.01; THP-1: CTR 31.59 ± 5.91; OLE 400 μM 46.38 ± 5.82, p = 0.04), whereas no modulation of cell proliferation was observed in RPMI8866 cell at any OLE concentration tested.
3.2. OLE-Induced Apoptosis in Leukemia/Lymphoma Cell Lines
Next, we investigated the ability of OLE to induce apoptosis of leukemia/lymphoma cells, at the same concentrations used for proliferation studies, after 24, 48, and 72 h of treatment. OLE-induced cell apoptosis at any time point tested, and data from 48 h treatment (i.e., intermediate time) are reported in Figure 2 . On the basis of the results obtained on cell proliferation, we first analyzed the B-ALL cell lines. The percentage of 697 cells in early apoptosis after 48 h of culture (% of Annexin V + /7AAD − cells, mean ± SE 0.68 ± 0.32) was significantly increased by OLE at 100 μM (3.86 ± 1.75, p = 0.05) and 200 μM (4.95 ± 1.66, p = 0.05). In addition, the percentage of 697 cells in late apoptosis (Annexin V + /7AAD + cells, 1.88 ± 0.49) was significantly increased in the presence of OLE at the concentration of 50 μM (16.72 ± 9, p = 0.05), 100 μM (45.88 ± 16.44, p = 0.05), 200 μM (75.97 ± 7.61, p = 0.05), and 400 μM (90.36 ± 4.28, p = 0.05). The percentage of dead cells (Annexin V − /7AAD + cells, 0.98 ± 0.07) was also significantly increased by OLE at 50 μM (2.1 ± 0.73, p = 0.05), 100 μM (4.52 ± 0.89, p = 0.05), 200 μM (4.36 ± 0.44, p = 0.05), and 400 μM (2.8 ± 0.59, p = 0.05). Similar results were obtained by analyzing the NALM-6 cell line, where the percentage of cells in early apoptosis after 48 h of culture (2.32 ± 0.72) was significantly increased by OLE at 100 μM (5.1 ± 0.45, p = 0.05) and 200 μM (8.12 ± 2.15, p = 0.05). In addition, the percentage of cells in late apoptosis (1.69 ± 0.43) was significantly increased by OLE at 50 μM (8.79 ± 4.52, p = 0.05), 100 μM (20.6 ± 10.6, p = 0.05), 200 μM (47.99 ± 14.51, p = 0.05), and 400 μM (86.83 ± 4.66, p = 0.05), as well as of dead cells (0.21 ± 0.12) that was significantly increased by OLE at 100 μM (3.9 ± 3.14, p = 0.05), 200 μM (4.47 ± 3.2, p = 0.05), and 400 μM (1.32 ± 0.35, p = 0.05).
Similarly to that reported in cell proliferation studies, limited effects were observed in terms of induction of apoptosis in RAJI and SU-DHL-4 lymphoma cell lines, where we observed only an increased percentage of cells in late apoptosis (0.88 ± 0.4 and 3.59 ± 1.62, respectively). Such an increase was observed in RAJI cells in the presence of OLE at 200 μM (14.32 ± 7.62, p = 0.05) and 400 μM (81.31 ± 6.45, p = 0.05) and in the SU-DHL-4 cell line at 400 μM OLE (22.94 ± 7.3, p = 0.05). We obtained similar results also in RPMI8866 and U937 cells.
Finally, we analyzed the myeloid leukemia cell lines. As expected, we did not observe any induction of early apoptosis in the K562 cell line, but only a slight increase of cells in late apoptosis (0.64 ± 0.13) and of dead cells (0.07 ± 0.01) in the presence of 400 μM OLE (1.55 ± 0.06 and 0.58 ± 0.18, respectively, p = 0.05). Superimposable results were obtained by analyzing the THP-1 cell line, with an increase in cells in late apoptosis (5.32 ± 3.16) and dead cells (0.76 ± 1.95) with 400 μM OLE (39.23 ± 7.53 and 4.44 ± 2.78, respectively, p = 0.05).
3.3. OLE-Induced Apoptosis in Primary Blasts from Leukemia Patients
To validate the results obtained with cell lines, we analyzed apoptosis of leukemic blasts obtained from patients with B-ALL and AML after 24 h of culture, in the presence or absence of OLE (200 and 400 μM). As shown in Figure 3 , the percentage of leukemic blasts from patients with B-ALL at diagnosis in late apoptosis (% of Annexin V + /7AAD − cells, mean ± SE 23.81 ± 6.99) was significantly increased by OLE at 200 μM (72 ± 6.53, p = 0.05) and 400 μM (81.91 ± 0.56, p = 0.05). The percentage of dead cells (% 7AAD + cells, mean ± SE 0.1 ± 0.1) was also significantly increased by OLE at 200 μM (3.08 ± 0.98, p = 0.05) and 400 μM (1.89 ± 0.75, p = 0.05). We obtained superimposable results from the same B-ALL patients at relapse, with a significantly higher percentage of both apoptotic cells (11.47 ± 1.69) and dead cells (0 ± 0.1) in the presence of OLE at 200 μM (49.74 ± 2.84, p = 0.05 and 12.2 ± 1.8, p = 0.05, respectively) and 400 μM (64.94 ± 4.14, p = 0.05 and 12.47 ± 2.87, p = 0.05, respectively). Regarding the leukemic blasts from patients who maintained disease remission, the percentage of blasts in late apoptosis (19.03 ± 3.42) was significantly increased by OLE at 200 μM (59.38 ± 1.2, p = 0.05) and 400 μM (74.3 ± 3.2, p = 0.05), as well as the percentage of dead cells (ctr: 0.04 ± 0.04; OLE 200 μM: 6.9 ± 1.7, p = 0.05; OLE 400 μM: 9.74 ± 2.74, p = 0.05).
Next, we analyzed leukemic blasts from patients with AML. In contrast to ALL, but consistent with that reported for AML cell lines, OLE did not induce a significant increase in apoptotic cells, but only a slight increase in dead cells (1.35 ± 1.15) either at 200 μM (6.7 ± 0.9, p = 0.05) and 400 μM (10.6 ± 0.62, p = 0.05).
These results were in accordance with those obtained with B-ALL and AML cell lines and confirm that B-ALL is the most sensitive acute leukemia to OLE treatment.
3.4. OLE Differentially Modulated Cellular Pathways in B-ALL and AML Cell Lines
To determine which cellular pathways may be affected in response to OLE treatment, we performed antibody array analyses on cellular lysates from the NALM-6 and K562 cell lines, the most treatment-sensitive and resistant, respectively. To this end, cells were cultured in the presence of OLE 200 μM for 24 h. The arrays selected included 139 molecules involved in cellular stress and apoptosis.
First, we investigated the expression of different proteins involved in cellular stress. As shown in Figure 4 A, the expression of different proteins was down-modulated in the NALM-6 cell line after OLE treatment, including Carbonic Anhydrase (CA-9, −33.3), Heat Shock Protein (HSP)-60 (−12.2), p27/kip-1 (−11.06), Superoxide Dismutase (SOD)2 (−7.01), cytochrome C (−2.23), HSP-70 (−2.17), and Tioredoxin-1 (−2.01). On the contrary, a limited effect of OLE was observed in K562 cells, with a moderate increase of cytochrome C (fold increase: 2.41) and phospho-HSP-27 (2.46) proteins.
The analysis of the NFkB pathway revealed that OLE-induced up-regulation of a few molecules, including TRAF2 (1.94) and Apoptosis-associated Speck-like protein containing CARD (ASC/CARD5, 1.86) in the NALM-6 cell line ( Figure 4 B), whereas a higher number of molecules were modulated by OLE in K562 cells. In particular, the expression of IkB epsilon was increased (1.91), and that of Tumor Necrosis Factor Receptor Associated Factors (TRAF)2 (−2.85), TNF-Related Apoptosis-Inducing Ligand Receptor (TRAIL R)1 (−2.22), CD40 (−1.91), and Ikkg/NEMO (−1.94) was down-regulated.
Next, we analyzed the apoptotic pathway. As shown in Figure 5 A, the expression of multiple proteins was up-regulated by OLE in NALM-6 cells, including TRAIL R1 (28.43), Heme Oxygenases (HO)-1/HSP-32 (6.77), clapsin (4.72), phospho-p53 S15 (2.22), Fas Associated via Death Domain (FADD, 2.27), and Bad (1.89), whereas in K562 cell line we observed only a slight down-modulation of pro-caspase 3 (−1.81). Finally, we analyzed the phospho-kinase pathway and, again, the expression of different protein was affected by OLE in NALM-6, with an increased phosphorylation of Akt 1-2-3 (10.22), CHK-2 (5.62), WNK1 (2.32), Glycogen Synthase Kinase (GSK)-3 α/β (2.55), c-jun (3.97), and phospho-p53 s46 (2.36) and decreased phosphorylation of phospho-p53 s392 (−15.51), STAT1 (−1.82), PYK2 (−1.92), HSP-27 (−1.8), and cAMP Response Element-binding protein (CREB, 1.97). In contrast, the phosphorylation of only a few kinases was decreased by OLE in K562 cells, including RSK1/2 (−2.33), WNK1 (−2.88), PLC-γ1 (−3.32), and STAT5 α/β (−2.3).
3.5. Induction of Apoptosis in B-ALL Cells by OLE in Combination with Chemotherapeutic Drugs
On the basis of previous results, we focused on B-ALL cells to assess the potential additive and/or synergistic pro-apoptotic activity of OLE in combination with chemotherapeutic drugs commonly used to treat leukemia patients. To this end, we selected cytarabine and cyclophosphamide/endoxan as chemotherapeutic agents that, in our in vitro experiments, were used at concentrations of 0.5 and 1 μM and of 0.5 and 1 μg/mL, respectively. At the same time, NALM-6 and 697 cells were treated alone or in combination with OLE at 50 or 100 μM. The percentage of 7AAD-/Annexin V-alive cells was evaluated by flow cytometry for each experimental condition, and data were analyzed using the SynergyFinder+ web application. The additive and synergistic effects were analyzed by the ZIP and HSA models, respectively.
As shown in Figure 6 A, the HSA model highlighted a synergistic effect of OLE in combination with cytarabine on NALM-6 cells (left panel, mean score: 8.26, p = 1.35 × 10 −5 ). A significant additive effect was also observed using the ZIP model (right panel, score: 3.45, p = 0.03). Conversely, experiments performed with OLE and cyclophosphamide/endoxan revealed no synergistic effects ( Figure 6 B) using the HSA model (left panel, score 0.022, p = 0.99), whereas the ZIP model suggested an antagonistic activity (right panel, score −3.05, p = 0.016).
As shown in Figure 7 A, similar results were obtained with the 697 cells, since a synergic effect emerged with a combined treatment of OLE and cytarabine (left panel, HSA model mean score: 14.38, p = 1.83 × 10 −9 ) and no significant additive activity (right panel, ZIP model, score: −3.04, p = 0.55). No synergistic or additive effect emerged from the combined treatment with OLE and cyclophosphamide/endoxan ( Figure 7 B; HSA model score 2.61, p = 0.45 on the left; ZIP model score −4.31, p = 0.11).
Similar results were obtained using Bliss and Loewe models to analyze synergistic or additive effects, respectively ( Supplementary Figures S1 and S2 ), as shown in the heat maps reported in Supplementary Figures S3–S6 .
4. Discussion
OLE has attracted much interest in recent years, since different studies demonstrated its efficacy in anti-cancer treatment [ 12 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. Indeed, OLE contains a high concentration of several molecules with a direct activity against solid and hematological tumors, and/or indirect anti-tumor effects via an increase in chemotherapy efficacy, such as oleuropein [ 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 ], rutin [ 43 , 44 , 45 , 46 , 47 , 48 , 49 ], hydrohytyrosol [ 50 , 51 , 52 , 53 , 54 ], and tyrosol [ 55 , 56 ]. Among them, oleuropein represents the main polyphenol and the most effective molecule against tumor cells [ 31 ]. The concentration of these compounds in OLE used in this study is reported in Section 2 , and the variability of these concentrations is very low among different batches. However, the standardization of the extract is crucial for future preclinical and clinical studies. So far, only a few studies have investigated the effects of OLE or oleuropein in the context of human leukemias [ 57 , 58 , 59 , 60 , 61 ]. Here, we reported that OLE has anti-proliferative and pro-apoptotic effects in vitro on B-ALL cell lines, whereas such activities were limited in AML and lymphoma cell lines. Furthermore, the induction of apoptosis and cell death in both B-ALL and AML cell lines was dose-dependent. More importantly, similar pro-apoptotic effects were also observed in primary leukemic blasts from patients with B-ALL but not in those from AML patients.
To clarify the molecular mechanisms underlying the anti-proliferative and pro-apoptotic effects of OLE against acute leukemias, we investigated the involvement of different cellular pathways using a panel of antibody arrays assessing the modulation of 102 molecules involved in the cellular stress, NFkB pathway, and apoptosis, as well as 37 phosphorylated kinases. In all of these pathways, we found a different OLE-mediated modulation between B-ALL and AML cell lines, which may explain the different and selective effects observed on proliferation and apoptosis. Indeed, B-ALL cells, more than AML cells, responded to OLE and showed greater inhibition of cell proliferation and induction of apoptosis. Such biological effects were paralleled by an increase in 6/35 and a decrease in 1/35 molecules involved in apoptotic processes in the B-ALL NALM-6 and in the AML K562 cells, respectively. In particular, the strongest effect observed in NALM-6 may be ascribed to the reduction of proteins known to be associated with the inhibition of proliferation and induction of apoptosis of cancer cells [ 62 , 63 ]. CA-9 is one of the most important proteins promoting different phases of cancer development, which is overexpressed in response to tumor hypoxia in many cancers. It plays a critical role in hypoxia-associated tumor acidosis and development of the metastatic phenotype, thus representing an attractive and promising target for systemic anticancer therapy [ 64 ]. Interestingly, it has recently been reported that a close relationship exists between CA-9, GSK3β, and AKT, which we found all modulated in the B-ALL NALM-6 cells [ 65 ]. The authors investigated the mechanism of action of ibuprofen on hypoxic cancer cells and whether ibuprofen may influence CA-9. They demonstrated that the drug down-regulated CA-9 and that such an effect was a consequence of the increased phosphorylation of AKT that led to increased inhibitory phosphorylation of GSK3β at Serine 9 (pS9-GSK3β) [ 65 ]. Since these modulations have also been observed in our study, we may speculate that the same pathway occurred in the induction of apoptosis and cell death of B-ALL cells upon treatment with OLE. Nonetheless, it is conceivable that additional molecules participated in the final anti-tumor effects driven by OLE. Indeed, four additional proteins related to cellular stress were down-regulated by OLE, namely HSP-60 and -70, SOD2, and Tioredoxin-1. HSP-60 and -70 are abundantly present in cancer, providing malignant cells a selective advantage through the suppression of apoptotic pathways, by regulating necrosis, interfering with tumor immunity, promoting angiogenesis, and supporting metastasis [ 66 , 67 ]. HSP-60 inhibits apoptosis mainly through a mitochondrial signal peptide [ 68 ], whereas HSP-70 prevents the activation of caspase [ 69 ]. Furthermore, the loss of these HSPs in normal cells, or their down-modulation in tumor cells, increases p53 expression and activates p53-dependent apoptosis, and finally, cell death [ 67 , 70 ]. Notably, in our study, we found that OLE modulated the phosphorylation of p53 (see below).
SOD2 is a member of the SOD family of antioxidants, which play an essential role in cellular protection against mitochondrial oxidative damage [ 71 ]; thus, its inhibition may impair the mitochondrial functions [ 72 ], increasing ROS and finally inducing apoptosis. In this context, HO-1/HSP32 and Bad, two molecules associated with oxidative stress and consequent apoptosis [ 73 ], were up-regulated by OLE, while not increasing the amount of cytochrome C. However, it is worth mentioning that we assessed the modulation of all these molecules in total cellular extracts; thus, we cannot exclude that the subcellular compartmentalization of cytochrome C changed upon OLE treatment and, in particular, its release from mitochondria. Tioredoxin-1 physiologically inhibits apoptosis by regulating the activities of caspases and procaspases through S-nitrosylation, and its inhibition was associated with the induction of apoptosis of tumor cells [ 74 ]. Controversial effects may be ascribed to p27/Kip1, down-modulated by OLE in NALM-6 cells, which was identified as a pivotal player during metabolic stress. Accordingly, p27 is known to be involved in pathways correlated with autophagy and apoptosis [ 75 ], although with dual effects depending on the cellular context. Notably, Russo et al. [ 76 ] investigated the mechanisms by which polyphenols interact with cellular processes by modulating the signal transduction pathways. They reported that the majority of polyphenols induced a p27 increase in a panel of tumor cell lines (e.g., promyelocytic leukemia, ovarian carcinoma, and multiple myeloma), but not in pulmonary mesothelioma and breast carcinoma. Nonetheless, the final result was the induction of apoptosis and cell cycle arrest in all cell lines.
Six out of the 35 proteins analyzed and associated with the induction of apoptosis have been found to be up-regulated in B-ALL upon OLE treatment, whereas only one was modulated in K562 cells. Among them, a strong up-regulation of TRAIL R1 was paralleled by the increase of FADD and TRAF2. The apoptosis induced by the TRAIL-R1 pathway needs the recruitment of the adaptor protein FADD to the cytoplasmic region of the receptor, followed by recruitment of pro-caspase-8 or pro-caspase-10. The formation of this death-inducing signaling complex triggers cleavage and activation of caspase-8 or caspase-10, which in turn activate downstream caspase-3, -6, and -7, and apoptosis [ 77 ]. Although we have not studied the modulation of caspases in the present work, we may hypothesize that this apoptotic pathway may also be utilized by OLE. In addition, the observed up-regulation of TRAIL R1 may render B cells sensitive to TRAIL-induced apoptosis [ 78 ].
The NFkB pathway was studied since we have previously reported that OLE-induced apoptosis in neuroblastoma cell lines by activating NFkB through phosphorylation of the p65 subunit [ 12 ]. In B-ALL cells, we did not observe this modulation, whereas we observed an increase in TRAF2 and ASC/CARD5. TRAF2, which is a key player in cancer cell survival, has been described as a tumor promoter, but may also act as a tumor suppressor [ 79 ]. Thus, it is conceivable that the regulation of TRAF2 induced by OLE may be implicated in the inhibition of proliferation and induction of apoptosis in our experimental setting. ASC/CARD5, which is an adaptor protein involved in the activation of apoptosis, was also up-regulated, as well as FADD, another crucial adaptor protein connecting death receptors to caspases [ 80 ]. Notably, upon OLE treatment of the K562 AML cell line, we highlighted a negative modulation of five different molecules, all involved in the TRAF2 signaling (i.e., TRAF2, TRAIL R1, IkB epsilon, CD40, and Ikkg/NEMO) [ 81 , 82 ]. These data are consistent with the opposite effects driven by OLE on B-ALL and AML cells.
Regarding the phosphorylation pattern, we detected a peculiar modulation of p53, a key regulator of apoptosis, affected by OLE in B-ALL, with enhanced phosphorylation at S15 and S46 and decreased phosphorylation at S392. It has been reported that the phosphorylation of the S46 (and also S15) residue is crucial for p53 activation and its binding to DNA and transcription of apoptotic target genes [ 83 , 84 , 85 ]. In contrast, phosphorylation of S392 is required for the mitochondrial localization and transcription-independent apoptotic function of p53 [ 86 ]. Thus, the down-modulation of the p53 phosphorylation at this site suggested that in our experimental setting, p53 predominantly acts in the nucleus through the activation of transcription of apoptosis-related genes. In addition, OLE induced the phosphorylation (and thus activation) of CHK2, which is a tumor suppressor involved in the induction of apoptosis [ 87 ]. Two additional factors that may lead either to apoptosis or survival of cancer cells have been found to increase, and are STAT1 [ 88 ] and WNK1 [ 89 ]. Conversely, the phosphorylation of CREB and HSP-27, which promote cell survival and inhibit apoptosis, was reduced by OLE [ 90 , 91 ].
We also found increased phosphorylation of c-jun and AKT upon OLE treatment. These results are difficult to explain, since the phosphorylation of c-jun and AKT usually promotes cell survival [ 92 , 93 ]. We may hypothesize that B-ALL cells reacted to OLE through the phosphorylation of these two factors in order to counterbalance the induction of apoptosis. Furthermore, the phosphorylation of PYK2, which mediated anti-apoptotic signaling of AKT, is decreased by OLE in B-ALL. Beyond the abovementioned relationship between CA-9, GSK3β, and AKT, it is to be taken into account that all these pathways underlying apoptosis and cell death are very complex, with an impressive number of molecules involved. Thus, it is conceivable that OLE triggers apoptosis as a result of a balance of several modulated molecules, which interact with each other. Here, we demonstrated for the first time to our knowledge that OLE, similarly to what we previously reported in neuroblastoma cells, acts as direct anti-tumor agent in B-ALL and with a lesser extent in AML cells, highlighting the potential molecular mechanisms involved, which are related to the induction of cellular stress and apoptosis, and are conceivably mediated by the extrinsic pathway, as witnessed by the highest modulation of TRAIL R1. Nonetheless, to unambiguously demonstrate such a conclusion, additional studies are required involving analysis of caspases and/or experiments focused on blocking death receptors such as TRAIL. The impact of OLE on these cellular pathways was modest in AML, resulting in a greater resistance to induction of apoptosis and inhibition of cell proliferation. Indeed, most of the proteins in AML were modulated in an opposite way compared to what was observed in B-ALL.
In summary, the effects of OLE on B-ALL cells are mainly related to (i) down-regulation of proteins involved in cancer progression, antioxidants, and heat-shock proteins, (ii) up-regulation of proteins related to apoptosis, and (iii) alteration of protein phosphorylation. The final effects based on these pathways are the inhibition of cell proliferation and induction of apoptosis. Thus, the resistance of AML cells to OLE is conceivably related to the minimal or absent alteration of these molecular pathways and, in addition, to a modulation of key proteins in the opposite way.
Interesting and promising data were obtained in experiments performed with OLE in combination with chemotherapeutic drugs. We selected two standard-of-care chemotherapeutic agents used for the management of acute leukemias and tested clinical-grade cytarabine and cyclophosphamide, as spare aliquots after therapeutic use in pediatric leukemic patients. In particular, cytarabine is currently used to treat patients with relapse/refractory disease [ 94 , 95 ], whereas cyclophosphamide represents a standard of care treatment for B-ALL patients [ 96 ]. Our data suggested that OLE may be used as a food supplement for these patients in combination with the drug to implement its anti-tumor efficacy. Notably, the presence or absence of synergistic and additive effects of OLE with cytarabine and cyclophosphamide, respectively, may conceivably be related to their different mechanisms of action. Indeed, cyclophosphamide acts as an alkylating agent, interfering with DNA replication and transcription of mRNA [ 97 ]. In contrast, cytarabine induces DNA damage through the incorporation of similar pyrimidine and purine nucleosides, leading to the activation of two kinase pathways, namely ataxia telangiectasia mutated and Rad3-related Checkpoint kinase 1 (ATR-CHK1) and ataxia telangiectasia mutated Checkpoint kinase 2 (ATM-CHK2), which may lead to DNA repair or apoptosis [ 98 , 99 ]. Due to the finding that OLE upregulated CHK2 in ALL (but not in AML) cell lines, we may speculate that the synergistic/additive effects observed with cytarabine may be related to the concomitant induction of CHK2 in target cells.
Pharmacokinetics and/or bioavailability of the different compounds present in OLE must be taken into account in view of clinical perspectives. Previous studies have addressed this issue, demonstrating that oleuropein and hydroxytyrosol are stable after digestion and reach their peak in the plasma of subjects consuming OLE within 20–75 min, and are still present 2–3 h after ingestion [ 100 , 101 ].
5. Conclusions
We here showed that exclusively the combination of OLE with cytarabine led to induction of apoptosis with effects ranging from synergy to addiction, whereas no synergic nor additive effects were observed when OLE was tested in combination with cyclophosphamide. These findings suggest that OLE not only has autonomous anti-tumor potential but can also increase the effectiveness of existing treatments in vitro. Although these data represent a proof-of-concept, we believe that a deep mechanistic insight aimed at validating the data raised from this study should be addressed in the future. Furthermore, our results highlighted a potential use of OLE in the clinical setting and management of pediatric B-ALL patients. Notably, recent technologies delineate areas for new interactions between food engineering and the medical field to aid in the best efficacy of cancer drugs, and to ensure more attractive and proper nutrition for cancer patients. In this view, it is to be taken into mind that OLE is an aqueous nutraceutical that may be easily administered as a food integrator without any side effects. Thus, we may envisage clinical settings with the use of OLE as an adjuvant compound during chemotherapy with cytarabine in pediatric oncology, taking advantage of its anti-proliferative and pro-apoptotic effects on B-ALL cells described here and of the already known anti-inflammatory properties. However, this hypothesis needs to be validated in future clinical studies to confirm the potential effectiveness of OLE as a food supplement in combination with existing therapies for B-ALL patients.
Acknowledgments
We thank the Scientific Committee of Evergreen Life Products S.r.l. (Valentina Brovedani and Claudia Pesle) for their support. The graphical abstract was created in BioRender. Morandi, F. (2025) https://BioRender.com/ky80g8d (accessed on 11 December 2025).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/nu18010015/s1 , Figure S1: OLE displays synergistic effects with cytarabine; Figure S2: OLE did not show any synergy or additive effects with cyclophosphamide; Figure S3: Heat maps of OLE/cytarabine in NALM-6 cells; Figure S4: Heat maps of OLE/cyclophosphamide synergistic effects in NALM-6 cells; Figure S5: Heat maps of OLE/cytarabine synergistic effects in 697 cells; Figure S6: Heat maps of OLE/cyclophosphamide synergistic effects in 697 cells.
Author Contributions
Conceptualization: F.M.; Data curation: I.A., L.C. and F.M.; Formal analysis: I.A., L.C. and F.M.; Funding acquisition: I.A. and F.M.; Investigation: I.A., L.C. and F.M.; Methodology: I.A., F.M., C.B. and E.C.; Project administration: F.M.: Resources: F.M., D.M. and I.A.; Software: F.M., I.A. and C.B.; Supervision: F.M.; Validation: F.M. and I.A.; Writing—original draft: I.A., C.B., D.M. and F.M.; Writing review and editing: I.A., C.B., D.M., L.C., E.C. and F.M. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The original contributions presented in this study are included in the article/ Supplementary Materials . Further inquiries can be directed to the corresponding author.
Conflicts of Interest
The authors declare no conflicts of interest.
Abbreviations
The following abbreviations are used in this manuscript: OLE Olive leaf extract B-ALL B-acute lymphoblastic leukemias ROS Reactive oxygen species AML Acute myeloid leukemias CFSE Carboxyfluorescein diacetate N-succinimidyl ester
References
1. Pereira A.P. Ferreira I.C. Marcelino F. Valentão P. Andrade P.B. Seabra R. Estevinho L. Bento A. Pereira J.A. Phenolic compounds and antimicrobial activity of olive ( Olea europaea L. Cv. Cobrançosa) leaves Molecules 2007 12 1153 1162 10.3390/12051153 17873849 PMC6149345
2. Baysal G. Kasapbaşı E.E. Yavuz N. Hür Z. Genç K. Genç M. Determination of theoretical calculations by DFT method and investigation of antioxidant, antimicrobial properties of olive leaf extracts from different regions J. Food Sci. Technol. 2021 58 1909 1917 10.1007/s13197-020-04702-0 33897027 PMC8021670
3. Micol V. Caturla N. Pérez-Fons L. Más V. Pérez L. Estepa A. The olive leaf extract exhibits antiviral activity against viral haemorrhagic septicaemia rhabdovirus (VHSV) Antivir. Res. 2005 66 129 136 10.1016/j.antiviral.2005.02.005 15869811
4. Ben-Amor I. Gargouri B. Attia H. Tlili K. Kallel I. Musarra-Pizzo M. Sciortino M.T. Pennisi R. In Vitro Anti-Epstein Barr Virus Activity of Olea europaea L. Leaf Extracts Plants 2021 10 2445 10.3390/plants10112445 34834807 PMC8622071
5. Ben-Amor I. Musarra-Pizzo M. Smeriglio A. D’Arrigo M. Pennisi R. Attia H. Gargouri B. Trombetta D. Mandalari G. Sciortino M.T. Phytochemical Characterization of Olea europea Leaf Extracts and Assessment of Their Anti-Microbial and Anti-HSV-1 Activity Viruses 2021 13 1085 10.3390/v13061085 34200316 PMC8229950
6. Hadrich F. Mahmoudi A. Chamkha M. Isoda H. Sayadi S. Olive Leaves Extract and Oleuropein Improve Insulin Sensitivity in 3T3-L1 Cells and in High-Fat Diet-Treated Rats via PI3K/AkT Signaling Pathway Oxidative Med. Cell. Longev. 2023 2023 6828230 10.1155/2023/6828230 PMC9840553 36647430
7. Hadrich F. Chamkha M. Sayadi S. Protective effect of olive leaves phenolic compounds against neurodegenerative disorders: Promising alternative for Alzheimer and Parkinson diseases modulation Food Chem. Toxicol. 2022 159 112752 10.1016/j.fct.2021.112752 34871668
8. Romero-Márquez J.M. Navarro-Hortal M.D. Forbes-Hernández T.Y. Varela-López A. Puentes J.G. Pino-García R.D. Sánchez-González C. Elio I. Battino M. García R. Exploring the Antioxidant, Neuroprotective, and Anti-Inflammatory Potential of Olive Leaf Extracts from Spain, Portugal, Greece, and Italy Antioxidants 2023 12 1538 10.3390/antiox12081538 37627533 PMC10451848
9. Somma F. Romano B. Maresca D.C. Maisto M. Tenore G. Ianaro A. Ercolano G. Olive leaf extract (OLE) reduces mast cell-mediated allergic inflammation Biomed. Pharmacother. 2025 182 117784 10.1016/j.biopha.2024.117784 39719741
10. Fayez N. Khalil W. Abdel-Sattar E. Abdel-Fattah A.M. In vitro and in vivo assessment of the anti-inflammatory activity of olive leaf extract in rats Inflammopharmacology 2023 31 1529 1538 10.1007/s10787-023-01208-x 37029328 PMC10229708
11. De Cicco P. Maisto M. Tenore G.C. Ianaro A. Olive Leaf Extract, from Olea europaea L., Reduces Palmitate-Induced Inflammation via Regulation of Murine Macrophages Polarization Nutrients 2020 12 3663 10.3390/nu12123663 33260769 PMC7761141
12. Morandi F. Bensa V. Calarco E. Pastorino F. Perri P. Corrias M.V. Ponzoni M. Brignole C. The Olive Leaves Extract Has Anti-Tumor Effects against Neuroblastoma through Inhibition of Cell Proliferation and Induction of Apoptosis Nutrients 2021 13 2178 10.3390/nu13072178 34202787 PMC8308225
13. Nath P. Modak S. Aktar T. Maiti S. Ghosh A. Singh R. Debnath M. Saha B. Maiti D. Olive leaves extract alleviates inflammation and modifies the intrinsic apoptotic signal in the leukemic bone marrow Front. Immunol. 2022 13 1054186 10.3389/fimmu.2022.1054186 36741365 PMC9894250
14. Pessoa H.R. Zago L. Difonzo G. Pasqualone A. Caponio F. Ferraz da Costa D.C. Olive Leaves as a Source of Anticancer Compounds: In Vitro Evidence and Mechanisms Molecules 2024 29 4249 10.3390/molecules29174249 39275097 PMC11397062
15. Melo Ferreira D. Oliveira M. Alves R.C. A Comprehensive Review of the Antitumor Activity of Olive Compounds: The Case of Olive Oil, Pomace, and Leaf Extracts, Phenolic Alcohols, Secoiridoids, and Triterpenes Antioxidants 2025 14 237 10.3390/antiox14020237 40002421 PMC11852221
16. Acquaviva R. Di Giacomo C. Sorrenti V. Galvano F. Santangelo R. Cardile V. Gangia S. D’Orazio N. Abraham N.G. Vanella L. Antiproliferative effect of oleuropein in prostate cell lines Int. J. Oncol. 2012 41 31 38 10.3892/ijo.2012.1428 22484302
17. Sirianni R. Chimento A. De Luca A. Casaburi I. Rizza P. Onofrio A. Iacopetta D. Puoci F. Andò S. Maggiolini M. Oleuropein and hydroxytyrosol inhibit MCF-7 breast cancer cell proliferation interfering with ERK1/2 activation Mol. Nutr. Food Res. 2010 54 833 840 10.1002/mnfr.200900111 20013881
18. Luo C. Li Y. Wang H. Cui Y. Feng Z. Li H. Li Y. Wang Y. Wurtz K. Weber P. Hydroxytyrosol promotes superoxide production and defects in autophagy leading to anti-proliferation and apoptosis on human prostate cancer cells Curr. Cancer Drug Targets 2013 13 625 639 10.2174/15680096113139990035 23597197
19. Tang J. Informatics Approaches for Predicting, Understanding, and Testing Cancer Drug Combinations Kinase Signaling Networks Methods in Molecular Biology Humana Press New York, NY, USA 2017 1636 485 506 10.1007/978-1-4939-7154-1_30 PMC6322649 28730498
20. Yadav B. Wennerberg K. Aittokallio T. Tang J. Searching for Drug Synergy in Complex Dose–Response Landscapes Using an Interaction Potency Model Comput. Struct. Biotechnol. J. 2015 13 504 513 10.1016/j.csbj.2015.09.001 26949479 PMC4759128
21. Zagidullin B. Aldahdooh J. Zheng S. Wang W. Wang Y. Saad J. Malyutina A. Jafari M. Tanoli Z. Pessia A. DrugComb: An integrative cancer drug combination data portal Nucleic Acids Res. 2019 47 W43 W51 10.1093/nar/gkz337 31066443 PMC6602441
22. Zheng S. Aldahdooh J. Shadbahr T. Wang Y. Aldahdooh D. Bao J. Wang W. Tang J. DrugComb update: A more comprehensive drug sensitivity data repository and analysis portal Nucleic Acids Res. 2021 49 W174 W184 10.1093/nar/gkab438 34060634 PMC8218202
23. Zheng S. Wang W. Aldahdooh J. Malyutina A. Shadbahr T. Tanoli Z. Pessia A. Tang J. SynergyFinder Plus: Toward Better Interpretation and Annotation of Drug Combination Screening Datasets Genom. Proteom. Bioinform. 2022 20 587 596 10.1016/j.gpb.2022.01.004 35085776 PMC9801064
24. Milanizadeh S. Reza Bigdeli M. Pro-Apoptotic and Anti-Angiogenesis Effects of Olive Leaf Extract on Spontaneous Mouse Mammary Tumor Model by Balancing Vascular Endothelial Growth Factor and Endostatin Levels Nutr. Cancer 2019 71 1374 1381 10.1080/01635581.2019.1609054 31074644
25. Vizza D. Lupinacci S. Toteda G. Puoci F. Ortensia I.P. De Bartolo A. Lofaro D. Scrivano L. Bonofiglio R. La Russa A. An Olive Leaf Extract Rich in Polyphenols Promotes Apoptosis in Cervical Cancer Cells by Upregulating p21(Cip/WAF1) Gene Expression Nutr. Cancer 2019 71 320 333 10.1080/01635581.2018.1559934 30661406
26. Barrajón-Catalán E. Taamalli A. Quirantes-Piné R. Roldan-Segura C. Arráez-Román D. Segura-Carretero A. Micol V. Zarrouk M. Differential metabolomic analysis of the potential antiproliferative mechanism of olive leaf extract on the JIMT-1 breast cancer cell line J. Pharm. Biomed. Anal. 2015 105 156 162 10.1016/j.jpba.2014.11.048 25560707
27. Ruzzolini J. Peppicelli S. Bianchini F. Andreucci E. Urciuoli S. Romani A. Tortora K. Caderni G. Nediani C. Calorini L. Cancer Glycolytic Dependence as a New Target of Olive Leaf Extract Cancers 2020 12 317 10.3390/cancers12020317 32013090 PMC7072393
28. Mijatovic S.A. Timotijevic G.S. Miljkovic D.M. Radovic J.M. Maksimovic-Ivanic D.D. Dekanski D.P. Stosic-Grujicic S.D. Multiple antimelanoma potential of dry olive leaf extract Int. J. Cancer 2011 128 1955 1965 10.1002/ijc.25526 20568104
29. Albogami S. Hassan A.M. Assessment of the Efficacy of Olive Leaf ( Olea europaea L.) Extracts in the Treatment of Colorectal Cancer and Prostate Cancer Using In Vitro Cell Models Molecules 2021 26 4069 10.3390/molecules26134069 34279409 PMC8272070
30. Tezcan G. Taskapilioglu M.O. Tunca B. Bekar A. Demirci H. Kocaeli H. Aksoy S.A. Egeli U. Cecener G. Tolunay S. Olea europaea leaf extract and bevacizumab synergistically exhibit beneficial efficacy upon human glioblastoma cancer stem cells through reducing angiogenesis and invasion in vitro Biomed. Pharmacother. 2017 90 713 723 10.1016/j.biopha.2017.04.022 28419967
31. Ruzzolini J. Peppicelli S. Andreucci E. Bianchini F. Scardigli A. Romani A. la Marca G. Nediani C. Calorini L. Oleuropein, the Main Polyphenol of Olea europaea Leaf Extract, Has an Anti-Cancer Effect on Human BRAF Melanoma Cells and Potentiates the Cytotoxicity of Current Chemotherapies Nutrients 2018 10 1950 10.3390/nu10121950 30544808 PMC6316801
32. Leto G. Flandina C. Crescimanno M. Giammanco M. Sepporta M.V. Effects of oleuropein on tumor cell growth and bone remodelling: Potential clinical implications for the prevention and treatment of malignant bone diseases Life Sci. 2021 264 118694 10.1016/j.lfs.2020.118694 33130080
33. Sherif I.O. Al-Gayyar M.M.H. Oleuropein potentiates anti-tumor activity of cisplatin against HepG2 through affecting proNGF/NGF balance Life Sci. 2018 198 87 93 10.1016/j.lfs.2018.02.027 29476769
34. Park S.M. Kim D.Y. Lee K.H. Shin Y.I. Han S.C. Kwon S.M. Anti-Tumor Efficacy of Oleuropein-Loaded ZnO/Au Mesoporous Silica Nanoparticle in 5-FU-Resistant Colorectal Cancer Cells Int. J. Nanomed. 2024 19 2675 2690 10.2147/IJN.S439392 38505168 PMC10948330
35. Tekin C. Ercelik M. Ak Aksoy S. Camlibel M. Ferah S. Gurbuz M. Aksoy F. Kaya E. Tunca B. Investigation of the Effectiveness of Oleuropein in a Three-Dimensional In Vitro Hepatocellular Tumor Sphere Model Exp. Clin. Transplant. 2025 23 207 213 10.6002/ect.2023.0020 37503799
36. Blanco E. Silva-Pilipich N. Bocanegra A. Chocarro L. Procopio A. Ausín K. Fernandez-Irigoyen J. Fernández L. Razquin N. Igea A. Oleuropein-driven reprogramming of the myeloid cell compartment to sensitise tumours to PD-1/PD-L1 blockade strategies Br. J. Cancer 2024 130 869 879 10.1038/s41416-023-02561-y 38195888 PMC10912768
37. Yılmaz G. Özdemir F. Novel Anti-tumor Strategy for Breast Cancer: Synergistic Role of Oleuropein with Paclitaxel Therapeutic in MCF-7 Cells Anti-Cancer Agents Med. Chem. 2024 24 224 234 10.2174/0118715206284107231120063630 PMC10909830 38629155
38. Nassir A.M. Ibrahim I.A.A. Md S. Waris M. Tanuja Ain M.R. Ahmad I. Shahzad N. Surface functionalized folate targeted oleuropein nano-liposomes for prostate tumor targeting: In vitro and in vivo activity Life Sci. 2019 220 136 146 10.1016/j.lfs.2019.01.053 30710640
39. Asghariazar V. Makaremi S. Zare E. Danesh H. Matin S. Fouladi N. Safarzadeh E. Oleuropein induces apoptosis in gastric cancer cell lines by regulating mir-34a, mir-21, and related genes: An experimental and bioinformatic study Int. J. Biol. Macromol. 2024 265 130527 10.1016/j.ijbiomac.2024.130527 38431007
40. Türkdoğan M.K. Koçyiğit A. Güler E.M. Özer Ö.F. Demir K. Uğur H. Oleuropein exhibits anticarcinogen effects against gastric cancer cell lines Mol. Biol. Rep. 2023 50 9099 9105 10.1007/s11033-023-08773-y 37747682
41. Bossio S. Perri A. Malivindi R. Giordano F. Rago V. Mirabelli M. Salatino A. Brunetti A. Greco E.A. Aversa A. Oleuropein Counteracts Both the Proliferation and Migration of Intra- and Extragonadal Seminoma Cells Nutrients 2022 14 2323 10.3390/nu14112323 35684123 PMC9182631
42. Liu L. Ahn K.S. Shanmugam M.K. Wang H. Shen H. Arfuso F. Chinnathambi A. Alharbi S.A. Chang Y. Sethi G. Oleuropein induces apoptosis via abrogating NF-κB activation cascade in estrogen receptor-negative breast cancer cells J. Cell. Biochem. 2019 120 4504 4513 10.1002/jcb.27738 30260018
43. Liu H. Xu Q. Wufuer H. Li Z. Sun R. Jiang Z. Dou X. Fu Q. Campisi J. Sun Y. Rutin is a potent senomorphic agent to target senescent cells and can improve chemotherapeutic efficacy Aging Cell 2024 23 e13921 10.1111/acel.13921 37475161 PMC10776113
44. Yılmaz S. Doğanyiğit Z. Oflamaz A.O. Ateş Ş. Söylemez E.S.A. Nisari M. Farooqı A.A. Determination of Rutin’s antitumoral effect on EAC solid tumor by AgNOR count and PI3K/AKT/mTOR signaling pathway Med. Oncol. 2023 40 131 10.1007/s12032-023-01999-7 36971893
45. Chen H. Miao Q. Geng M. Liu J. Hu Y. Tian L. Pan J. Yang Y. Anti-tumor effect of rutin on human neuroblastoma cell lines through inducing G2/M cell cycle arrest and promoting apoptosis Sci. World J. 2013 2013 269165 10.1155/2013/269165 PMC3891239 24459422
46. Huo M. Xia A. Cheng W. Zhou M. Wang J. Shi T. Cai C. Jin W. Zhou M. Liao Y. Rutin Promotes Pancreatic Cancer Cell Apoptosis by Upregulating miRNA-877-3p Expression Molecules 2022 27 2293 10.3390/molecules27072293 35408691 PMC9000526
47. Zhou M. Zhang G. Hu J. Zhu Y. Lan H. Shen X. Lv Y. Huang L. Rutin attenuates Sorafenib-induced Chemoresistance and Autophagy in Hepatocellular Carcinoma by regulating BANCR/miRNA-590-5P/OLR1 Axis Int. J. Biol. Sci. 2021 17 3595 3607 10.7150/ijbs.62471 34512168 PMC8416719
48. Zhang P. Sun S. Li N. Ho A.S.W. Kiang K.M.Y. Zhang X. Cheng Y.S. Poon M.W. Lee D. Pu J.K.S. Rutin increases the cytotoxicity of temozolomide in glioblastoma via autophagy inhibition J. Neuro-Oncol. 2017 132 393 400 10.1007/s11060-017-2387-y 28293765
49. Lin J.P. Yang J.S. Lin J.J. Lai K.C. Lu H.F. Ma C.Y. Sai-Chuen Wu R. Wu K.C. Chueh F.S. Gibson Wood W. Rutin inhibits human leukemia tumor growth in a murine xenograft model in vivo Environ. Toxicol. 2012 27 480 484 10.1002/tox.20662 21254320
50. Perta N. Torrieri Di Tullio L. Cugini E. Fattibene P. Rapanotti M.C. Borromeo I. Forni C. Malaspina P. Cacciamani T. Di Marino D. Hydroxytyrosol Counteracts Triple Negative Breast Cancer Cell Dissemination via Its Copper Complexing Properties Biology 2023 12 1437 10.3390/biology12111437 37998036 PMC10669715
51. Calahorra J. Blaya-Cánovas J.L. Castellini-Pérez O. Aparicio-Puerta E. Cives-Losada C. Marin J.J.G. Rementeria M. Cara F.E. López-Tejada A. Griñán-Lisón C. Unlocking the effective alliance of β-lapachone and hydroxytyrosol against triple-negative breast cancer cells Biomed. Pharmacother. 2024 174 116439 10.1016/j.biopha.2024.116439 38518601
52. El-Azem N. Pulido-Moran M. Ramirez-Tortosa C.L. Quiles J.L. Cara F.E. Sanchez-Rovira P. Granados-Principal S. Ramirez-Tortosa M. Modulation by hydroxytyrosol of oxidative stress and antitumor activities of paclitaxel in breast cancer Eur. J. Nutr. 2019 58 1203 1211 10.1007/s00394-018-1638-9 29468462
53. Costantini F. Di Sano C. Barbieri G. The Hydroxytyrosol Induces the Death for Apoptosis of Human Melanoma Cells Int. J. Mol. Sci. 2020 21 8074 10.3390/ijms21218074 33137997 PMC7662312
54. Todoerti K. Gallo Cantafio M.E. Oliverio M. Juli G. Rocca C. Citraro R. Tassone P. Procopio A. De Sarro G. Neri A. Oleil Hydroxytyrosol (HTOL) Exerts Anti-Myeloma Activity by Antagonizing Key Survival Pathways in Malignant Plasma Cells Int. J. Mol. Sci. 2021 22 11639 10.3390/ijms222111639 34769070 PMC8584245
55. Guo J. Meng F. Hu R. Chen L. Chang J. Zhao K. Ren H. Liu Z. Hu P. Wang G. Inhibition of the NF-κB/HIF-1α signaling pathway in colorectal cancer by tyrosol: A gut microbiota-derived metabolite J. Immunother. Cancer 2024 12 e008831 10.1136/jitc-2024-008831 39343509 PMC11440206
56. Quiles J.L. Farquharson A.J. Simpson D.K. Grant I. Wahle K.W. Olive oil phenolics: Effects on DNA oxidation and redox enzyme mRNA in prostate cells Br. J. Nutr. 2002 88 225 234 225–234; discussion 223–224 10.1079/BJN2002620 12207832
57. Nath P. Majumder D. Debnath R. Debnath M. Singh Sekhawat S. Maiti D. Immunotherapeutic potential of ethanolic olive leaves extract (EOLE) and IL-28B combination therapy in ENU induced animal model of leukemia Cytokine 2022 156 155913 10.1016/j.cyto.2022.155913 35640418
58. Abaza L. Talorete T.P. Yamada P. Kurita Y. Zarrouk M. Isoda H. Induction of growth inhibition and differentiation of human leukemia HL-60 cells by a Tunisian gerboui olive leaf extract Biosci. Biotechnol. Biochem. 2007 71 1306 1312 10.1271/bbb.60716 17485840
59. Samet I. Han J. Jlaiel L. Sayadi S. Isoda H. Olive ( Olea europaea ) leaf extract induces apoptosis and monocyte/macrophage differentiation in human chronic myelogenous leukemia K562 cells: Insight into the underlying mechanism Oxidative Med. Cell. Longev. 2014 2014 927619 10.1155/2014/927619 PMC3997986 24803988
60. Anter J. Fernández-Bedmar Z. Villatoro-Pulido M. Demyda-Peyras S. Moreno-Millán M. Alonso-Moraga A. Muñoz-Serrano A. Luque de Castro M.D. A pilot study on the DNA-protective, cytotoxic, and apoptosis-inducing properties of olive-leaf extracts Mutat. Res. 2011 723 165 170 10.1016/j.mrgentox.2011.05.005 21620995
61. Pieme C.A. Santosh G.K. Tekwu E.M. Askun T. Aydeniz H. Ngogang J.Y. Bhushan S. Saxena A.K. Fruits and barks extracts of Zanthozyllum heitzii a spice from Cameroon induce mitochondrial dependent apoptosis and Go/G1 phase arrest in human leukemia HL-60 cells Biol. Res. 2014 47 54 10.1186/0717-6287-47-54 25723957 PMC4335363
62. Cianchi F. Vinci M.C. Supuran C.T. Peruzzi B. De Giuli P. Fasolis G. Perigli G. Pastorekova S. Papucci L. Pini A. Selective Inhibition of Carbonic Anhydrase IX Decreases Cell Proliferation and Induces Ceramide-Mediated Apoptosis in Human Cancer Cells J. Pharmacol. Exp. Ther. 2010 334 710 719 10.1124/jpet.110.167270 20519553
63. Temiz E. Koyuncu I. Durgun M. Caglayan M. Gonel A. Güler E.M. Kocyigit A. Supuran C.T. Inhibition of Carbonic Anhydrase IX Promotes Apoptosis through Intracellular pH Level Alterations in Cervical Cancer Cells Int. J. Mol. Sci. 2021 22 6098 10.3390/ijms22116098 34198834 PMC8201173
64. Tülüce Y. Ahmed B.A. Koyuncu İ. Durgun M. The cytotoxic, apoptotic and oxidative effects of carbonic anhydrase IX inhibitor on colorectal cancer cells J. Bioenerg. Biomembr. 2018 50 107 116 10.1007/s10863-018-9749-9 29520697
65. Grossmannova K. Belvoncikova P. Puzderova B. Simko V. Csaderova L. Pastorek J. Barathova M. Carbonic anhydrase IX downregulation linked to disruption of HIF-1, NFκB and STAT3 pathways as a new mechanism of ibuprofen anti-cancer effect PLoS ONE 2025 20 e0323635 10.1371/journal.pone.0323635 40408503 PMC12101644
66. Singh M.K. Shin Y. Ju S. Han S. Choe W. Yoon K.S. Kim S.S. Kang I. Heat Shock Response and Heat Shock Proteins: Current Understanding and Future Opportunities in Human Diseases Int. J. Mol. Sci. 2024 25 4209 10.3390/ijms25084209 38673794 PMC11050489
67. Koo N. Sharma A.K. Narayan S. Therapeutics Targeting p53-MDM2 Interaction to Induce Cancer Cell Death Int. J. Mol. Sci. 2022 23 5005 10.3390/ijms23095005 35563397 PMC9103871
68. Cao S. Li Y. Chen L. Lei X. Feng X. Li Y. HSP60 inhibits DF-1 apoptosis through its mitochondrial signal peptide Poult. Sci. 2025 104 104571 10.1016/j.psj.2024.104571 39637657 PMC11664397
69. Beere H.M. Wolf B.B. Cain K. Mosser D.D. Mahboubi A. Kuwana T. Tailor P. Morimoto R.I. Cohen G.M. Green D.R. Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome Nat. Cell Biol. 2000 2 469 475 10.1038/35019501 10934466
70. Dahiya V. Agam G. Lawatscheck J. Rutz D.A. Lamb D.C. Buchner J. Coordinated Conformational Processing of the Tumor Suppressor Protein p53 by the Hsp70 and Hsp90 Chaperone Machineries Mol. Cell 2019 74 816 830.e817 10.1016/j.molcel.2019.03.026 31027879
71. Ibrahim N.K. Schreek S. Cinar B. Stasche A.S. Lee S.H. Zeug A. Dolgner T. Niessen J. Ponimaskin E. Shcherbata H. SOD2 is a regulator of proteasomal degradation promoting an adaptive cellular starvation response Cell Rep. 2025 44 115434 10.1016/j.celrep.2025.115434 40131931 PMC12094083
72. Rath-Deschner B. Nogueira A.V.B. Memmert S. Nokhbehsaim M. Augusto Cirelli J. Eick S. Miosge N. Kirschneck C. Kesting M. Deschner J. Regulation of Anti-Apoptotic SOD2 and BIRC3 in Periodontal Cells and Tissues Int. J. Mol. Sci. 2021 22 591 10.3390/ijms22020591 33435582 PMC7827060
73. Chiang S.K. Chen S.E. Chang L.C. The Role of HO-1 and Its Crosstalk with Oxidative Stress in Cancer Cell Survival Cells 2021 10 2401 10.3390/cells10092401 34572050 PMC8471703
74. Raninga P.V. Trapani G.D. Vuckovic S. Bhatia M. Tonissen K.F. Inhibition of thioredoxin 1 leads to apoptosis in drug-resistant multiple myeloma Oncotarget 2015 6 15410 10.18632/oncotarget.3795 25945832 PMC4558160
75. Liang J. Shao S.H. Xu Z.X. Hennessy B. Ding Z. Larrea M. Kondo S. Dumont D.J. Gutterman J.U. Walker C.L. The energy sensing LKB1-AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis Nat. Cell Biol. 2007 9 218 224 10.1038/ncb1537 17237771
76. Russo G.L. Stampone E. Cervellera C. Borriello A. Regulation of p27(Kip1) and p57(Kip2) Functions by Natural Polyphenols Biomolecules 2020 10 1316 10.3390/biom10091316 32933137 PMC7564754
77. Oh Y.T. Sun S.Y. Regulation of Cancer Metastasis by TRAIL/Death Receptor Signaling Biomolecules 2021 11 499 10.3390/biom11040499 33810241 PMC8065657
78. Staniek J. Lorenzetti R. Heller B. Janowska I. Schneider P. Unger S. Warnatz K. Seidl M. Venhoff N. Thiel J. TRAIL-R1 and TRAIL-R2 Mediate TRAIL-Dependent Apoptosis in Activated Primary Human B Lymphocytes Front. Immunol. 2019 10 951 10.3389/fimmu.2019.00951 31114586 PMC6503035
79. Albini A. Di Paola L. Mei G. Baci D. Fusco N. Corso G. Noonan D. Inflammation and cancer cell survival: TRAF2 as a key player Cell Death Dis. 2025 16 292 10.1038/s41419-025-07609-w 40229245 PMC11997178
80. Gupta S. Kim C. Yel L. Gollapudi S. A role of fas-associated death domain (FADD) in increased apoptosis in aged humans J. Clin. Immunol. 2004 24 24 29 10.1023/B:JOCI.0000018059.56924.99 14997030
81. Borghi A. Verstrepen L. Beyaert R. TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death Biochem. Pharmacol. 2016 116 1 10 10.1016/j.bcp.2016.03.009 26993379
82. Kim C.M. Park H.H. Comparison of Target Recognition by TRAF1 and TRAF2 Int. J. Mol. Sci. 2020 21 2895 10.3390/ijms21082895 32326186 PMC7215387
83. Sun X. Dyson H.J. Wright P.E. A phosphorylation-dependent switch in the disordered p53 transactivation domain regulates DNA binding Proc. Natl. Acad. Sci. USA 2021 118 e2021456118 10.1073/pnas.2021456118 33443163 PMC7817127
84. Smeenk L. van Heeringen S.J. Koeppel M. Gilbert B. Janssen-Megens E. Stunnenberg H.G. Lohrum M. Role of p53 serine 46 in p53 target gene regulation PLoS ONE 2011 6 e17574 10.1371/journal.pone.0017574 21394211 PMC3048874
85. Yogosawa S. Yoshida K. Tumor suppressive role for kinases phosphorylating p53 in DNA damage-induced apoptosis Cancer Sci. 2018 109 3376 3382 10.1111/cas.13792 30191640 PMC6215896
86. Castrogiovanni C. Waterschoot B. De Backer O. Dumont P. Serine 392 phosphorylation modulates p53 mitochondrial translocation and transcription-independent apoptosis Cell Death Differ. 2018 25 190 203 10.1038/cdd.2017.143 28937686 PMC5729520
87. Hirao A. Cheung A. Duncan G. Girard P.M. Elia A.J. Wakeham A. Okada H. Sarkissian T. Wong J.A. Sakai T. Chk2 is a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia mutated (ATM)-dependent and an ATM-independent manner Mol. Cell. Biol. 2002 22 6521 6532 10.1128/MCB.22.18.6521-6532.2002 12192050 PMC135625
88. Stephanou A. Latchman D.S. STAT-1: A novel regulator of apoptosis Int. J. Exp. Pathol. 2003 84 239 244 10.1111/j.0959-9673.2003.00363.x 14748743 PMC2517573
89. Xiu M. Li L. Li Y. Gao Y. An update regarding the role of WNK kinases in cancer Cell Death Dis. 2022 13 795 10.1038/s41419-022-05249-y 36123332 PMC9485243
90. Trevisan R. Daprai L. Acquasaliente L. Ciminale V. Chieco-Bianchi L. Saggioro D. Relevance of CREB phosphorylation in the anti-apoptotic function of human T-lymphotropic virus type 1 tax protein in serum-deprived murine fibroblasts Exp. Cell Res. 2004 299 57 67 10.1016/j.yexcr.2004.05.024 15302573
91. Katsogiannou M. Andrieu C. Rocchi P. Heat shock protein 27 phosphorylation state is associated with cancer progression Front. Genet. 2014 5 346 10.3389/fgene.2014.00346 25339975 PMC4186339
92. Hettinger K. Vikhanskaya F. Poh M.K. Lee M.K. de Belle I. Zhang J.T. Reddy S.A.G. Sabapathy K. c-Jun promotes cellular survival by suppression of PTEN Cell Death Differ. 2007 14 218 229 10.1038/sj.cdd.4401946 16676006
93. Kim A.H. Khursigara G. Sun X. Franke T.F. Chao M.V. Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1 Mol. Cell. Biol. 2001 21 893 901 10.1128/MCB.21.3.893-901.2001 11154276 PMC86680
94. Bradstock K.F. Morley A. Byth K. Szer J. Prosser I. Cannell P. Irving I. Seymour J.F. Effects of intensive induction and consolidation chemotherapy with idarubicin and high dose cytarabine on minimal residual disease levels in newly diagnosed adult precursor-B acute lymphoblastic leukemia Contemp. Clin. Trials Commun. 2016 4 9 13 10.1016/j.conctc.2016.06.004 29736466 PMC5935861
95. Li Y.-Y. Zhou H.-X. Huang S.-M. Wan C.-L. Xu M.-Z. Gong W.-J. Qian C.-S. Wu D.-P. Dai H. Xue S.-L. Venetoclax, Cladribine and Cytarabine for the Treatment of Relapse/Refractory Acute Lymphoblastic Leukemia: Interim Analysis of a Phase 2 Trial Blood 2023 142 1506 10.1182/blood-2023-185312
96. Zhao Y.R. Song H.M. Ni L. Cyclophosphamide for the treatment of acute lymphoblastic leukemia: A protocol for systematic review Medicine 2019 98 e14293 10.1097/MD.0000000000014293 30702599 PMC6380740
97. Dabbish E. Scoditti S. Shehata M.N.I. Ritacco I. Ibrahim M.A.A. Shoeib T. Sicilia E. Insights on cyclophosphamide metabolism and anticancer mechanism of action: A computational study J. Comput. Chem. 2024 45 663 670 10.1002/jcc.27280 38088485
98. Shabashvili D.E. Feng Y. Kaur P. Venugopal K. Guryanova O.A. Combination strategies to promote sensitivity to cytarabine-induced replication stress in acute myeloid leukemia with and without DNMT3A mutations Exp. Hematol. 2022 110 20 27 10.1016/j.exphem.2022.03.008 35306047 PMC9133110
99. Takagaki K. Katsuma S. Kaminishi Y. Horio T. Tanaka T. Ohgi T. Yano J. Role of Chk1 and Chk2 in Ara-C-induced differentiation of human leukemia K562 cells Genes Cells 2005 10 97 106 10.1111/j.1365-2443.2005.00821.x 15676021
100. de Bock M. Thorstensen E.B. Derraik J.G. Henderson H.V. Hofman P.L. Cutfield W.S. Human absorption and metabolism of oleuropein and hydroxytyrosol ingested as olive ( Olea europaea L.) leaf extract Mol. Nutr. Food Res. 2013 57 2079 2085 10.1002/mnfr.201200795 23766098
101. Soldo B. Bilušić T. Giacometti J. Ljubenkov I. Čikeš Čulić V. Bratanić A. Bošković P. Šola I. Ilić K. A Comparative Study of Oleuropein Extraction from Wild Olive Leaves ( Olea europea subsp. oleaster, Hoffmanns. & Link), Its Gastrointestinal Stability, and Biological Potential Appl. Sci. 2024 14 869 10.3390/app14020869