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ARTICLE
Investigation of the Effectiveness of Oleuropein in a Three-Dimensional In Vitro Hepatocellular Tumor Sphere Model

Objectives: This study was conducted to examine the dose-related effects over time of oleuropein on the proliferation and area of tumor spheroids in hepatocellular carcinoma cells.
Materials and Methods: We examined the possible effects of 100 to 500 μM dose concentrations of oleuropein on HepG2 cell proliferation using a real-time cell analyzer. A 3-dimensional hepatocellular carcinoma tumor spheroid model was established by seeding HepG2 cells at a density of 160 cells/well in custom 96-well microplates with low attachment surfaces and culturing for 3 days. Tumor spheres were treated with increasing oleuropein doses for 72 hours, and images were captured every 24 hours. The dose-dependent effects of oleuropein on tumor sphere size were analyzed by measuring the area of tumor spheres with ImageJ software. We conducted oleuropein viability and cytotoxicity analyses using calcein acetoxymethyl ester-based and propidium iodide-based staining in the tumor model.
Results: Oleuropein inhibited cell proliferation; as the dose concentration of oleuropein increased, so did its capacity to inhibit cell proliferation (P < .001). The size of untreated tumor spheres increased at 72 hours (P < .001). However, treatment with 100 to 500 μM oleuropein reduced tumor size by 63.56% to 88.06% compared with untreated cells at the end of 72 hours (P < .001). With increasing concentrations, oleuropein inhibited the viability of tumor spheres, eliminating necrotic death caused by tumor hypoxia.
Conclusions: Overall, oleuropein reduced the size of tumors by inhibiting tumor proliferation and viability. In this context, oleuropein could be a candidate molecule for further extensive studies to reduce hepatocellular carcinoma tumors to meet Milan criteria for liver transplant.


Key words : Liver Transplantation, Real-time cell proliferation analysis, Reducing tumor size

Introduction

Hepatocellular carcinoma (HCC) is among the leading causes of cancer-related deaths worldwide and represents an important global health problem.1-3 Hepatocellular carcinoma is associated with a poor prognosis, resulting in approximately 905 677 new cases and 830 180 deaths yearly.2,4,5 Currently, a variety of treatment modalities are available for patients with HCC, including liver transplant (LT), surgical resection, percutaneous ablation, radiation, and transarterial and systemic therapies.1,6,7 Although surgical treatment options cannot be applied for patients with advanced HCC tumors, and thus only systemic chemotherapy can be used, it is emphasized that monotherapy using chemotherapy agents such as sorafenib is insufficient in terms of patient survival or improving patient quality of life.8-10 Patients with HCC also often develop severe chronic liver damage, including inflammatory, fibrotic, and cirrhotic lesions. Therefore, these patients frequently demonstrate poor systemic chemotherapeutic tolerance.5,11 Liver transplant is the best treatment option for HCC patients since it eliminates the tumor lesion and corrects the underlying liver failure, thus reducing the recurrence rate.12-14

Despite LT being an effective treatment for HCC, selecting and managing those patients appropriate for LT is a fundamental challenge due to the lack of donors.13-15 The most important factors determining survival in patients with HCC are tumor size, number of tumors, and extrahepatic spread. Although there are many criteria used in this context, the one most frequently used is Milan criteria (which classifies patients for LT with single HCC ≤5 cm or up to 3 HCCs ≤3 cm, without vascular invasion).16 Survival is lower in LT recipients when performed outside of Milan criteria.16

Reducing tumor size and the number of tumors in the preoperative period is also possible with various locoregional treatments. The aim of these treatments is to bridge patients until LT and to prevent dropouts on wait lists. Survival is acceptable with locoregional treatments that give a successful response, especially in selected patient groups.17 Although the response to these treatments is classified according to various criteria, some patients show no response.18 In addition, current strategies to treat HCCs are still lacking due to the continued growth of tumor cells and the complexity of molecular mec-hanisms that influence interactions between tumor cells and the tumor microenvironment surrounding them.5,19

The first step in developing effective chemot-herapeutic drugs is establishing an appropriate system to study complex tumor culture and microenvironment interactions. The 3-dimensional (3D) culture model is an important model that bridges the gap between in vivo and in vitro due to its ability to naturally mimic the complex in vivo tumor microenvironment compared with traditional 2-dimensional (2D) cultures.5,20

The olive tree (Olea europaea), a member of the Oleaceae, is a plant native to the Mediterranean climate. Extracts from the leaves of Olea europaea (OLE) contain high levels of bioactive components such as secoiridoid, triperthene, and flavonoids. Oleuropein is a coumarin-like compound called secoiridoids, the most detectable (17%-23%) in OLE.21 In addition to its antioxidant and anti-inflammatory effects, oleuropein has been reported to treat oxidant and inflammatory-related diseases such as cardiovascular disease, hepatic disorders, obesity, and diabetes.21,22 In our previous studies, we reported that OLE and oleuropein have effects on suppressing the aggressiveness of tumors with features such as inducing apoptosis, reducing tumor size, and suppressing epithelial mesenchymal transformation and cancer stem cell characteristics in glioblastoma and gastric cancer types.23-26 Here, we investigated the dose- and time-dependent antitu-moral effects of oleuropein in HCC cells in a 3D model of HCC that is well representative of the tumor microenvironment to reveal the potential for tumor size reduction in HCC cells.

Materials and Methods

All data and materials are available from the corresponding author. This article does not contain any studies with human participants or animals.

Cell culture and reagents
The human HCC cell line HepG2 was provided by Assoc. Dr. Zeynep Güne? Özünal (Maltepe University, Istanbul, Turkey). HepG2 cells were grown in Dulbecco’s modified eagle’s medium, high glucose medium (Thermo Fisher Scientific) containing L-glutamine, 10% fetal bovine serum (Biochrome), 1 mM sodium pyruvate, 100 μg/mL of streptomycin, and 100 μg/mL penicillin and incubated in a 5% CO2 humidified incubator at 37 °C. Oleuropein (no 12247; Sigma-Aldrich) was dissolved in dimethyl sulfoxide according to the manufacturer’s instructions to form a solution at a concentration of 10 μM.

Real-time cell analyzer system
The cell proliferation assay was measured with the real-time cell analyzer system on the xCELLigence (ACEA Biosciences) instrument according to the manufacturer’s instructions. Briefly, HepG2 cells were seeded into an E-plate 16 (ACEA Biosciences) at 20 × 103/100 μL of medium per well. After incubation for 24 hours, cells were treated with doses of oleuropein at concentrations from 100 to 500 μM. The xCELLigence real-time cell analyzer system measured the impedance caused by cells to determine cell proliferation every 30 minutes for 96 hours using interactive microelectrode sensors placed under the E-plate 16 inserts.27 The cell index of untreated HepG2 cells was used as the control group.

Three-dimensional tumor sphere assay
The 3D in vitro HCC tumor sphere model was constructed using Perkin-Elmer cell carrier sphere ULA 96-well polystyrene microplates (Perkin-Elmer) according to the manufacturer’s recommended protocol. HepG2 cells were grown in each well of the plate to 160 cells/100 μL medium to form tumor spheres and incubated for 3 days during which sphere formation was observed.23,24 Tumor spheres were then treated with doses of 100 to 500 μM oleuropein for 72 hours, and changes in sphere size were recorded every 24 hours by viewing them with an inverted microscope. The area of tumor spheres was measured by ImageJ software. Untreated HepG2 tumor spheres were used as the control group.

Calcein acetoxymethyl ester and propidium iodide staining assays
Cell cytotoxicity or viability of 3D HepG2 tumor spheres was measured using calcein acetoxymethyl ester (calcein-AM; Abcam) and propidium iodide (PI; Thermo Fisher Scientific) staining. At 72 hours of treatment of the formed tumor spheres with oleuropein, a dye combination of 2 μM calcein-AM and 4.5 μM PI were added to the wells and incubated for 60 minutes at 5% CO2 and 37 °C.28 Tumor spheroids stained with calcein-AM were visualized at excitation and emission wavelengths of 488 and 520 nm, and tumor spheroids stained with PI were visualized at cleavage and emission wavelengths of 535 and 615 nm by a fluorescence microscope (EVOS M5000 Imaging System; Thermo Fisher Scientific). The viable or proliferative cells showed calcein-AM staining, dead cells showed PI staining, and quiescent cells appeared weakly stained with calcein-AM but unstained with PI.28

Statistical analyses
We evaluated the statistical significance of the dose-dependent effects of oleuropein on cell proliferation, tumor sphere size, and aggressiveness by 1-way and 2-way analysis of variance using SPSS version 23 (IBM SPSS Inc). Data are shown as numbers, percentages, and mean. We plotted graphs using GraphPad Prism 8 (GraphPad Software Inc). P < 0.05 was considered significant in all experiments with 95% confidence interval (CI).

Results

Oleuropein inhibited HepG2 cell proliferation with increasing dose concentrations
The cytotoxic dose-dependent effects of oleuropein in HepG2 cells were measured by the xCELLigence system. The real-time cell analysis system determined the cell index of HepG2 cells, where the cell index decreased with increasing oleuropein dose concentration at up to 96 hours of treatment (Figure 1A). Accordingly, treatment with 100 to 500 μM oleuropein reduced HepG2 cell proliferation at 24 hours by 82.48%, 81.45%, 31.23%, and 18.45%, respectively (Figure 1B; P < .05 and P < .001). Based on the capacity of oleuropein treatment at different concentrations at 24 hours to reduce cell proliferation, the 50% inhibitory concentration was 186.1 μM. Treatment with 100 to 500 μM oleuropein inhibited the proliferation of HepG2 cells depending on the increased dose concentration compared with untrea-ted HepG2 cells (Figure 1B). We also determined that oleuropein in nontumor fibroblast cells had no cytotoxic effects on cells at a dose concentration of 267 μM at 24 hours (data not shown).

Oleuropein dose-dependently decreased the size of tumor spheroids
The possible effects of increasing dose concentration of oleuropein on tumor size and aggressiveness were analyzed by a 3D in vitro HCC tumor spheroid model. Tumor spheres derived from HepG2 cells were treated with increasing dose concentrations of oleuropein (100-500 μM) for 72 hours (Figure 1C). The analysis showed 301.49% enhancement in the size of untreated tumor spheroids at 72 hours compared with at 0 hours (P < .001). The size of the tumor spheroids were reduced by 63.56%, 73.38%, 80.14%, and 88.06% after treatment with 100 to 500 μM of oleuropein, respectively, compared with untreated tumor spheres at 72 hours. We observed that 100 μM oleuropein treatment resulted in a 2.75-fold (P < .05), 200 μM oleuropein treatment resulted in a 3.75-fold (P < .05), 300 μM oleuropein treatment resulted in a 5.03-fold (P < .001), and 500 μM oleuropein treatment resulted in an 8.38-fold (P < .001) decrease in size of tumor spheres compared with untreated tumor spheres (Figure 1D). Oleuropein treatment with increasing dose concentrations inhibited cell proliferation, reducing the size of tumor spheroids. The morphological appearance of tumor spheres at 24, 48, and 72 hours with 100 to 500 μM of oleuropein is shown in Figure 1, C and D.

Increased concentrations of oleuropein inhibited the viability of tumor spheres
We analyzed the effects of oleuropein on the viability and cytotoxicity of tumor spheres using calcein-AM/PI staining. We found that, after 72 hours, tumor spheroids showed consistent results in response to different concentrations of oleuropein (Figure 1E). Findings indicated more proliferative cells with more intense calcein-AM staining of the outer wall of untreated HepG2 tumor spheres and quiescent cells with calcein-AM staining weakly bound between the core region and the wall. In untreated tumor spheres, the core region showed a hypoxia-induced necrotic area with PI-stained cells, representing dead cells. With increasing dose concentrations from 100 to 500 μM of oleuropein, calcein-AM-stained cells in the walls of the tumor spheres gradually decreased compared with untreated tumor spheres. This result indicated that oleuropein treatment inhibited cell proliferation and created a dose-dependent response depending on the increa-sing dose concentration. In addition, as oleuropein dose concentrations increased, the PI-stained region in the core region decreased compared with untreated tumor spheres. Furthermore, increased oleuropein concentrations reduced the occurrence of tumor hypoxia-induced necrosis in the tumor core region compared with untreated tumor spheres.

Discussion

Liver transplant remains the best treatment option for HCC. In this context, the Milan criteria (which specifies single HCC ≤5 cm or up to 3 HCCs ≤3 cm, without vascular invasion) remains the most frequently used criteria with good results shown in terms of survival analyses.29 Milan criteria also depends on UCSF criteria according to size and number criteria (solitary HCC ≤6.5 cm or ≤3 nodules and largest tumor ≤4.5 cm, and total diameter ≤8 cm.30 Patients with MC have better survival after transplant and patients without MC.31 In addition, the use of specific selection criteria, including the use of specified tumor size and number in LT, reduced recurrence rates to 8% at 4 years and resulted in survival rates similar to patients who received a transplant for nonmalignant disease.29,32 Various downstaging treatments have been described in the literature (such as radiofrequency ablation, microwave ablation, and transarterial chemoembolization); however, these methods require invasive interventions and may be ineffective. In a review that included 13 studies, the success rate was found to be between 11% and 77%.33 Therefore, new downstaging methods are needed. Thus, we aimed to examine a new therapeutic target aimed to reduce tumor size so that it can be used in patient groups with HCC who are waiting for LT but who are outside the Milan criteria due to tumor size. Accordingly, we found that oleuropein dose-dependently and time-dependently reduced the proliferation of HCC cells and reduced tumor size and the area of 3D tumor spheroids, a system that mimics the tumor microenvironment in vitro.

Although many previous studies on Olea europaea L. have identified this extract as a traditional medicine to treat various ailments, such as fever, recent studies have reported its cancer-preventing potential.34-36 Because the antioxidant and anticancer activity of Olea europaea L. was assumed to be directly related to its polyphenol content, our focus was on oleuropein as the major secondary metabolite of the extract.37

Current research has reported that oleuropein inhibits cell proliferation and induces apoptosis of cancer cell lines by affecting different mechanisms. Our work over the past decade have provided evidence of the tumor-inhibitory effects of oleuropein on glioblastoma cell lines when used with the chemotherapy drug.24,26,38 Barbaro and colleagues39 suggested that the antitumor activity of olive leaf and oleuropein in different types of cancer may be related to its reactive oxygen species suppressive, antiproliferative, apoptosis directing, and anti-invasive effects. Cardeno and colleagues40 reported that oleuropein inhibited cell growth and induced apoptosis in HT29 cells via a p53-dependent pathway.

With the recent study showing that oleuropein gradually inhibits cell growth with increasing dose and time, we wondered whether oleuropein had any effects on 3D tumor spheroids. Although monolayer culture systems (2D) have been the best models for anticancer agents for decades, results have been inconsistent when switching to in vivo models after these analyses. Tumor spheroids have better results in mimicking the architectural features and tumor microenvironment of solid tumors compared with 2D systems.

Tumor spheroids are characterized by a complex structure that summarizes the complexity of cell-cell interaction, cell-extracellular matrix interconnection, and the structure’s differential access to oxygen and nutrients.41 All of these features have led to the emergence of 3D tumor spheroid models as a viable models in cancer biology and drug screening. Morandi and colleagues reported that OLE reduces tumor cell viability in a time- and dose-dependent manner with cytotoxic experiments performed on tumor spheroids, supporting the results obtained in 2D cultures.42 Our findings showed that tumor sphere size decreased in the 3D HCC tumor model through inhibition of cell proliferation as the oleuropein dose concentration increased. In this context, for patients with HCC who are on transplant wait lists according to Milan criteria, its use for reduction of tumor size is promising. In addition, cell viability was reduced due to increasing dose concentration of oleuropein in tumor spheres by prevention of tumor-induced hypoxia-induced necrotic death. In our previous study, we reported that oleuropein did not affect cytotoxicity in nontumor fibroblast cells (unpublished observations). In this regard, the effect of oleuropein in reducing tumor size by inhibiting cell proliferation supported that it could be a candidate molecule to support chemotherapy in patients who are waiting for organ transplant.

Conclusions

The properties shown with oleuropein indicate it may be a novel anticancer compound targeting different steps in cancer progression, as shown by our 3D spheroid tumor model in which oleuropein inhibited HCC tumor proliferation. Oleuropein, as an anticancer agent, can prevent tumor progression and directly inhibit cancer cells, leading to tumor regres-sion. Although more extensive preclinical studies are required, the in vitro results shown here support the possibility of using oleuropein in the clinical setting to reduce tumor size in patients waiting for LT.


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DOI : 10.6002/ect.2023.0020


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From the 1Department of Medical Biology, Faculty of Medicine, Bursa Uludag University; the 2Inegol Vocation School, Bursa Uludag University; and the 3Organ Transplantation Center, Faculty of Medicine, Bursa Uludag University, Bursa, Turkey
Acknowledgements: Melis Ercelik was supported by grant 100/2000 from The Council of Higher Education as a PhD Scholar in Molecular Biology and Genetics (Gene Therapy and Genome Studies). This study was supported by a grant from the Scientific Research Projects Foundation of the Bursa Uludag University, Bursa, Turkey (Project No. TGA-2020-174). The authors have no declarations of potential conflicts of interest.
Corresponding author: Berrin Tunca, Bursa Uludag University, Faculty of Medicine Department of Medical Biology, Gorukle, Bursa, Turkey
Phone: +902242954161
E-mail: tuncaberrin@gmail.com