Leaf extract from european olive (Olea europaea L.) post-transcriptionally suppresses the epithelial-mesenchymal transition and sensitizes gastric cancer cells to chemotherapy

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Abstract

The overall survival of patients with the advanced and recurrent gastric cancer (GC) remains unfavorable. In particular, this is due to cancer spreading and resistance to chemotherapy associated with the epithelial-mesenchymal transition (EMT) of tumor cells. EMT can be identified by the transcriptome profiling of GC for EMT markers. Indeed, analysis of the TCGA and GTEx databases (n = 408) and a cohort of GC patients (n = 43) revealed that expression of the CDH2 gene was significantly decreased in the tumors vs. non-tumor tissues and correlated with the overall survival of GC patients. Expression of the EMT-promoting transcription factors SNAIL and ZEB1 was significantly increased in GC. These data suggest that targeting the EMT might be an attractive therapeutic approach for patients with GC. Previously, we demonstrated a potent anti-cancer activity of the olive leaf extract (OLE). However, its effect on the EMT regulation in GC remained unknown. Here, we showed that OLE efficiently potentiated the inhibitory effect of the chemotherapeutic agents 5-fluorouracil (5-FU) and cisplatin (Cis) on the EMT and their pro-apoptotic activity, as was demonstrated by changes in the expression of the EMT markers (E- and N-cadherins, vimentin, claudin-1) in GC cells treated with the aforementioned chemotherapeutic agents in the presence of OLE. Thus, culturing GC cells with 5-FU + OLE or Cis + OLE attenuated the invasive properties of cancer cells. Importantly, upregulation of expression of the apoptotic markers (PARP cleaved form) and increase in the number of cells undergoing apoptosis (Annexin V-positive) were observed for GC cells treated with a combination of OLE and 5-FU or Cis. Collectively, our data illustrate that OLE efficiently interferes with the EMT in GC cells and potentiates the pro-apoptotic activity of certain chemotherapeutic agents used for GC therapy.

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About the authors

Cagla Tekin

Bursa Uludag University

Email: btunca@uludag.edu.tr

Department of Medical Biology, Faculty of Medicine

Turkey, 16059 Bursa

Melis Ercelik

Bursa Uludag University

Email: btunca@uludag.edu.tr

Department of Medical Biology, Faculty of Medicine

Turkey, 16059 Bursa

Pavel Dunaev

Kazan State Medical University

Email: boichuksergei@mail.ru

Department of Pathology

Russian Federation, 420012 Kazan

Aigul Galembikova

Kazan State Medical University

Email: boichuksergei@mail.ru

Department of Pathology

Russian Federation, 420012 Kazan

Gulcin Tezcan

Bursa Uludag University

Email: btunca@uludag.edu.tr

Department of Fundamental Sciences, Faculty of Dentistry

Turkey, 16059 Bursa

Secil Ak Aksoy

Bursa Uludag University

Email: btunca@uludag.edu.tr

Experimental Animal Breeding and Research Unit, Faculty of Medicine, Inegol Vocation School, Bursa Uludag University

Turkey, 16059 Bursa; 16059 Bursa

Ferah Budak

Bursa Uludag University

Email: btunca@uludag.edu.tr

Department of Immunology, Medical Faculty

Turkey, 16059 Bursa

Ozgen Isık

Bursa Uludag University

Email: btunca@uludag.edu.tr

Department of General Surgery, Medical Faculty

Turkey, 16059 Bursa

Nesrin Ugras

Bursa Uludag University

Email: btunca@uludag.edu.tr

Department of Pathology, Medical Faculty

Turkey, 16059 Bursa

Sergei Boichuk

Kazan State Medical University; Russian Medical Academy of Continuous Professional Education; Kazan Federal University

Email: boichuksergei@mail.ru

Department of Pathology, Department of Radiotherapy and Radiology, “Biomarker” Research Laboratory, Institute of Fundamental Medicine and Biology

Russian Federation, 420012 Kazan; 125445 Moscow; 420012 Kazan

Berrin Tunca

Bursa Uludag University

Author for correspondence.
Email: btunca@uludag.edu.tr

Department of Medical Biology, Faculty of Medicine

Turkey, 16059 Bursa

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2. Fig. 1. Regulation of epithelial-mesenchymal transition (EMT) gene expression in gastric cancer (GC). a – CDH1 gene expression in GC tissue and non-tumor tissues (GEPIA database); b – CDH1 gene expression in GC samples obtained from patients participating in this study; c – the effect of CDH1 gene expression impairment on overall survival of GC patients (GEPIA database); d – CDH2 gene expression in GC tissue (GEPIA database); d – CDH2 gene expression in GC samples obtained from patients participating in this study; e – the effect of CDH2 gene expression impairment on overall survival of GC patients (GEPIA database); g–i – expression of SNAI1, ZEB1, and TWIST genes encoding transcription factors associated with EMT in GC (GEPIA database). k – expression of non-coding RNA MALAT1 in GC (GEPIA database); l – expression of non-coding RNA MALAT1 in GC samples obtained from patients participating in this study; m – relationship between expression of non-coding RNA MALAT1 and overall survival of patients with GC (GEPIA database); n – interaction of non-coding RNA MALAT1 and the CDH2 gene (LncRRIsearch database); o and p – expression of miR-23b-3p and miR-200c in GC; p – binding of miR-23b-3p to the site of non-coding RNA MALAT1 (lncRNASNP database); c – binding of miR-200c to the site of the ZEB1 gene (DIANA-TarBase database). The level of statistical significance (p) was calculated using Student's t-test (for a, b, d, e, g–l, o and n) and the Kaplan–Meier test (for c, f and m); * p < 0.05; TPM – number of transcripts per million mapped reads

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3. Fig. 2. Effect of OLE, 5-FU and Cis on the viability of AGS cells. a – Expression of CDH2, SNAI1 and ZEB1 genes in different gastric cancer cells (HPA database); b – Signaling pathways in AGS tumor cells (HPA database); c – Viability of AGS cells at different concentrations of OLE (incubation for 24, 48 and 72 h); d – Effect of half inhibitory concentration (IC50) of OLE on AGS cell proliferation; d and g – Effect of half inhibitory concentration (IC50) of 5-FU on AGS cell proliferation; f and h – Effect of half inhibitory concentration (IC50) of Cis on AGS cell proliferation. i – study of the ability of OLE, 5-FU and Cis to induce apoptosis of AGS cells by fluorescence microscopy (staining with AO/PI dye containing acridine orange – AO and propidium iodide – PI); k – study of the ability of OLE, 5-FU and Cis to induce apoptosis of AGS cells by flow cytometry. Tumor cells were stained with Annexin V and propidium iodide (PI) dyes. OLE – Olea europaea leaf extract; 5-FU – 5-fluorouracil; Cis – cisplatin. The level of statistical significance (p) was calculated by one-way analysis of variance (ANOVA) and using Tukey’s post hoc test; * p < 0.05; n = 3

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4. Fig. 3. Effect of combinations of OLE (1.5 mg/ml) with 5-FU (25 μM) and Cis (20 μM) on the viability of AGS cells. a – Viability of AGS cells when cultured with OLE alone and under conditions of combinations of OLE with chemotherapy drugs (OLE + 5-FU, OLE + Cis); n = 3. b–d – Study of AGS cell proliferation when cultured with OLE, 5-FU, Cis and their combinations; d – Study of the interaction of OLE with chemotherapy drugs (5-FU, Cis) using the Bliss synergy scale; e – synergistic effect of the interaction of OLE and Cis on AGS cells (data from Synergy Finder software); g – additive effect of the interaction of OLE and 5-FU (data from Synergy Finder software). OLE – Olea europaea leaf extract; 5-fluorouracil – 5-FU; cisplatin – Cis. The level of statistical significance (p) was calculated by one-way analysis of variance (ANOVA) and using Tukey's post hoc test; * p < 0.05

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5. Fig. 4. Study of the ability of OLE (1.5 mg/ml), 5-FU (25 μM), Cis (20 μM) and their combinations to induce AGS cell death via apoptosis. a–c – Study of the ability of combinations of OLE + 5-FU, OLE + Cis, OLE + 5-FU + Cis to induce AGS cell apoptosis using flow cytometry. Tumor cells were stained with Annexin V and propidium iodide (PI); g and e – study of the ability of OLE, 5-FU, Cis and combinations of OLE + 5-FU, OLE + Cis to induce AGS cell apoptosis using Western blotting. Apoptosis markers – cleaved forms of PARP1 and caspase-3. β-Actin reflects the protein level in the samples. The graphs show the expression of cleaved forms of PARP1 and caspase-3 proteins, expressed in pixels. OLE – Olea europaea leaf extract; 5-fluorouracil – 5-FU; cisplatin – Cis. The level of statistical significance (p) was calculated by one-way analysis of variance (ANOVA) and using Tukey's post hoc test; * p < 0.05; n = 3

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6. Fig. 5. Study of the ability of OLE (1.5 mg/ml), 5-FU (25 μM), Cis (20 μM) and their combinations to inhibit the epithelial-mesenchymal transition (EMT) process in AGS cells. a – Analysis of CDH2 gene expression in AGS cells under the influence of OLE, 5-FU, Cis and their combinations. b and c – Study of the ability of OLE, 5-FU, Cis and their combinations to inhibit the EMT process in AGS cells using Western blotting. Analysis of the expression of N-cadherin, E-cadherin, vimentin and claudin-1 involved in the EMT process is shown. β-Actin reflects the protein level in the samples. The graphs show the expression of the studied EMT proteins, expressed in pixels. g – Analysis of miR-23b-3p expression in AGS cells cultured for 72 h in the presence of OLE, 5-FU, Cis and their combinations; d – Analysis of ZEB1 gene expression in AGS cells cultured for 72 h in the presence of OLE, 5-FU, Cis and their combinations; e – Analysis of miR-200c expression in AGS cells cultured for 72 h in the presence of OLE, 5-FU, Cis and their combinations; g – Analysis of SNAI1 gene expression in AGS cells cultured for 72 h in the presence of OLE, 5-FU, Cis and their combinations. OLE – Olea europaea leaf extract; 5-fluorouracil – 5-FU; cisplatin – Cis. The level of statistical significance (p) was calculated using Student's t-test for independent samples; * p < 0.05; n = 3

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7. Fig. 6. Study of the effect of OLE (1.5 mg/ml), 5-FU (25 μM), Cis (20 μM) and their combinations on SOX2 protein expression in AGS cells by immunofluorescence microscopy. a and b – AGS cells were incubated with OLE for 24 h, with chemotherapy drugs – for 48 h, with a combination of OLE + chemotherapy drugs – 24 h. SOX2 protein expression – green fluorescence (determined at 488 nm wavelength). The cell nucleus was visualized using DAPI dye (blue staining). c – SOX2 fluorescence intensity in AGS cells. The fluorescence intensity index (H-score) for each sample was calculated using the formula: number of stained cells (%) × cell staining intensity. OLE – Olea europaea leaf extract; 5-fluorouracil – 5-FU; cisplatin – Cis. The level of statistical significance (p) was calculated using Student's t-test for independent samples; * p < 0.05; n = 3

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8. Fig. 7. Study of the effect of OLE (1.5 mg/ml), 5-FU (25 μM), Cis (20 μM) on the migration rate of AGS cells. a – Light microscopy of the damaged area of ​​the AGS cell monolayer after different time intervals (upon damage – 0 h, after 24 h, after 48 h). 1 – Control; 2 – OLE; 3 – 5-FU; 4 – OLE + 5-FU; 5 – Cis; 6 – OLE + Cis; 7 – 5-FU + Cis; 8 – OLE + 5-FU + Cis. b – Change in the “wound area” (damaged area of ​​the AGS cell monolayer) under the influence of OLE, chemotherapy drugs and their combinations. On the right of the graph is the change in the “wound area” under the influence of OLE, chemotherapy drugs and their combinations in the first 24 hours. The level of statistical significance (p) was calculated using one-way analysis of variance (ANOVA); * p < 0.05; n = 3

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