Research ArticleExperimental Studies

Ephemeranthol A Suppresses Epithelial to Mesenchymal Transition and FAK-Akt Signaling in Lung Cancer Cells

NONGYAO NONPANYA, ORNJIRA PRAKHONGCHEEP, KORRAKOD PETSRI, CHOLASIT JITJAICHAM, SUCHARAT TUNGSUKRUTHAI, BOONCHOO SRITULARAK and PITHI CHANVORACHOTE
Anticancer Research September 2020, 40 (9) 4989-4999; DOI: https://doi.org/10.21873/anticanres.14502

Abstract

Background/Aim: Epithelial to mesenchymal transition (EMT) is a cellular process that facilitates cancer metastasis. Therefore, therapeutic approaches that target EMT have garnered increasing attention. The present study aimed to examine the in vitro effects of ephemeranthol A on cell death, migration, and EMT of lung cancer cells. Materials and Methods: Ephemeranthol A was isolated from Dendrobium infundibulum. Non-small cell lung cancer cells H460 were treated with ephemeranthol A and apoptosis was evaluated by Hoechst 33342 staining. Anoikis resistance was determined by soft agar assay. Wound healing assay was performed to test the migration. The regulatory proteins of apoptosis and cell motility were determined by western blot. Results: Treatment with ephemeranthol A resulted in a concentration-dependent cell apoptosis. At non-toxic concentrations, the compound could inhibit anchorage-independent growth of the cancer cells, as indicated by the decreased colony size and number. Ephemeranthol A also exhibited an inhibitory effect on migration. We further found that ephemeranthol A exerts its antimetastatic effects via inhibition of EMT, as indicated by the markedly decrease of N-cadherin, vimentin, and Slug. Furthermore, the compound suppressed the activation of focal adhesion kinase (FAK) and protein kinase B (Akt) proteins, which are key regulators of cell migration. As for the anticancer activity, ephemeranthol A induced apoptosis by decreasing Bcl-2 followed by the activation of caspase 3 and caspase 9. Conclusion: The pro-apoptotic and anti-migratory effects of ephemeranthol A on human lung cancer cells support its use for the development of novel anticancer therapies.

Lung cancer is a leading cause of cancer-related deaths (1). In 2020, an estimated 228,820 new cases of lung cancer and about 135,720 deaths from this disease are expected (2). More than half of patients diagnosed with lung cancer die within one year and the 5-year survival rate of patients is less than 18% (3). Human lung cancers are divided into small cell lung cancers (SCLC; approximately 15% of all lung cancers) and non-small cell lung cancers (NSCLC; 85% of all lung cancers) (4, 5). It has been reported that cancer metastasis is a major factor determining low 5-year survival rate and mortality in NSCLC patients (6). Therefore, inhibition of metastasis is crucial to the effectiveness of anti-cancer treatments.

It has been previously reported that during cancer cell dissemination, focal adhesion kinase (FAK), and ATP-dependent tyrosine kinase (Akt; also known as protein kinase B) are activated (7-9). Specifically, in lung cancer, FAK has been linked with increased cell motility and cell survival in detached conditions (10), while elevated levels of activated Akt have been associated with lung cancer metastasis (11, 12). Likewise, epithelial to mesenchymal transition (EMT), a cellular process during which epithelial cells acquire the characteristics of invasive mesenchymal cells, has been shown to correlate with cancer progression, cancer stem cell, invasion, and therapy resistance (13, 14).

Although chemotherapy is currently used for cancer treatment, there are several limitations such as toxic side effects, high-dose requirements, as well as the development of drug resistance after therapy (15). For example, cisplatin, an important chemotherapy for lung cancer treatment, has a dose-dependent side effects on the kidney and others organ (16-18). In addition, it was shown that chemotherapeutic drugs can induce resistance in cancer cells (19). Therefore, the new agents derived from various plants with novel anti-cancer activities as well as antimetastatic effects would be beneficial for the management of the disease.

Natural products have been reported as an important source for the discovery and development of therapeutic agents especially for antimicrobial and anticancer agents (20, 21). Ephemeranthol A is a compound isolated from D. infundibulum (22). Plants of the genus Dendrobium (Orchidaceae) have been used in traditional Chinese medicine for the treatment of stomach, kidney, and lung disorders (23). Also, extract of D. formosum has been found to exhibit anticancer effects in T-cell lymphoma (24). However, the anticancer as well as antimetastatic potentials of ephemeranthol A are unknown. This is the first study to investigate the effects of ephemeranthol A on the human NSCLC cell line H460 cell death, migration, as well as on the EMT process.

Materials and Methods

Compound and reagents. Ephemeranthol A was isolated from D. infundibulum as previous reported (22); its purity was determined using NMR spectroscopy. In order to get various concentrations, the compound was dissolved in 0.1% dimethyl sulfoxide (DMSO) and RPMI-1640. RPMI 1640 medium, fetal bovine serum (FBS), penicillin/streptomycin, L-glutamine, phosphate buffered saline (PBS) and trypsin were purchased from GIBCO (Grand Island, NY, USA). Propidium iodide (PI) and Hoechst 33342 were obtained from Molecular Probes, Inc. (Eugene, OR, USA). 3-(4,5-dimethyl-thiazol-2-yl)-2,5-Diphenyl tetrazolium bromide (MTT) was obtained from Sigma Chemical, Inc. (St. Louis, MO, USA). Antibody for PARP, cleaved PARP, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, Mcl-1, Bcl-2, BAX, β-actin, N-cadherin, Vimentin, Slug, p473-Akt, Akt, p397-FAK, FAK and peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Cell culture. Human NSCLC H460 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cancer cells were cultured in RPMI 1640 medium, supplemented with 10% FBS, 100 units/ml penicillin/streptomycin and 2 mM L-glutamine. The cells were incubated at 37°C with 5% CO2, in humidified incubator. For subcultures, cells were washed by 1×PBS and detached using a 0.25% trypsin-0.53 mM EDTA solution.

Cytotoxicity assay. Cell viability was determined using the MTT assay. Briefly, cells were seeded in 96-well plates at a density of 1×104 cells/well. Then, cells were treated with 0-200 μM of ephemeranthol A in RPMI 1640 medium for 24 h and 48 h, and incubated with 400 μg/ml of MTT solution for 4 h at 37°C. After careful removal of the supernatants, 100 μl of DMSO were added to dissolve the formed formazan crystals. Color intensity of crystal formazan product was measured by spectrophotometry at 570 nm using a microplate reader (Anthros, Durham, NC, USA); the percentage of cell viability was calculated in relation to untreated cells.

Cell death assay. Cells were seeded in 96-well plates at a density of 1×104 cells/well and incubated at 37°C with 5% CO2 overnight. Cells were treated with various concentrations of ephemeranthol A (0-200 μM) and incubated at 37°C with 5% CO2 for 24 h. After treatments, apoptotic cell death was determined by using nuclear staining with Hoechst 33342 and PI. Cells were stained with 10 μM of Hoechst 33342 and 5 μM of PI for 30 min at 37°C. Cells were visualized and imaged using a fluorescence microscopy (Olympus DP70, Melville, NY, USA). Three random fields were captured at 20× magnification and then the percentages of apoptotic cells were calculated.

Migration assay. Migration was determined using wound healing assay. Cells were cultured as monolayer in 96-well plates. The bottom of each well was scratched using sterile 1-mm-wide pipette tips. Media was then removed and washed by PBS. The monolayer cells were incubated with ephemeranthol A at several concentrations (0-100 μM) for 24 h and 48 h and migration were observed under a phase contrast microscope (Olympus, Melville, NY, USA) and images were captured using Olympus DP70 digital camera with Olympus DP controller software (Olympus). The wound area was determined by ImageJ software. The percentage of wound closure was calculated as an equation following: Wound closure (%)=[(A0 − AΔh)/A0] ×100, where, A0 is the area of the wound measured immediately after scratching (0 h), and AΔh is the area of the wound measured at 24 or 48 h after treatment.

Anchorage-independent growth assay. Soft agar colony formation assay was used to detect anchorage-independent cell growth. Cells were pre-treated with ephemeranthol A at 0-50 μM concentration for 48 h, at 37°C. Equal volumes of culture medium containing 10% FBS and 1% agarose (Bio-Rad Laboratories, Hercules, CA, USA) were mixed. The mixture was allowed to solidify as a bottom layer in a 24-well plate. Thereafter, the top layer containing 10% FBS, 0.33% agar, and cells (3×103 cells/ml) was added in each well. After the upper layer was solidified, cultured medium containing 10% FBS was added in each well and the culture plate was incubated at 37°C. Colony formation was determined after two weeks using a phase contrast microscope (Nikon ECLIPSE Ts2, Tokyo, Japan). Relative colony number and diameter were determined by dividing the values of the treated cells to those of untreated cells.

Western blot. After treatment, the cells (2×106) were washed with cold PBS twice and incubated on ice with lysis buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM sodium chloride (NaCl), 10% glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM phenylmethylsulphonyl fluoride, and a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA) for 30 min. Cell lysates were collected, and the protein content was determined using the BCA protein assay kit (Bio-Rad Laboratories). Approximately 70-100 μg of protein from each sample was denatured at 95°C for 5 min and loaded onto 7.5-15% SDS-polyacrylamide gels. After gel separation, the proteins were transferred onto 0.45 μm nitrocellulose membranes (Bio-Rad), and the transferred membranes were incubated with blocking buffer [5% non-fat dry milk in TBST (25 mM Tris-HCl (pH 7.5), 125 mM NaCl, 0.05% Tween 20)] for 1 h and subsequently incubated with the appropriate specific primary antibody at 4°C overnight. Membranes were then washed 3 times for 5 min with TBST and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG isotype-specific secondary antibodies in blocking buffer for 2 h at room temperature. Membranes were then washed again with TBST (3 times for 5 min). The SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific, Rockford, IL, USA) was used to detect immune complexes and signal was quantified by densitometry, using the ImageJ software.

Figure 1.
Figure 1.

Cytotoxicity and characteristics of ephemeranthol A. Chemical structure of ephemeranthol A (A). Dendrobium infundibulum plant (B). Cytotoxic effects of ephemeranthol A on human non small cell lung cancer H460 cells were investigated using MTT assay (*p<0.05, **p<0.01, compared to the untreated control cells) (C). IC50 of H460 cells at 24 and 48 h of treatment. Data represent the mean±SEM (n=3) (D).

Statistical analysis. Statistical differences between multiple groups were analyzed using an analysis of variance (ANOVA). The data is presented as the mean±standard error of the mean (SEM) of three independent experiments with three replicates per experiment. All p-values of less than 0.05 were considered as statistically significant.

Results

Cytotoxicity of ephemeranthol A on human lung cancer H460 cells. To test the effect of ephemeranthol A, derived from D. infundibulum (Figure 1A, B), we first determined the cytotoxic concentrations of the compound in human lung cancer H460 cells. Briefly, 80% confluent cancer cells were treated with various concentrations of ephemeranthol A (0, 5, 10, 50, 100, and 200 μM), and cell viability was determined after 24 and 48 h by MTT viability assay. Results showed that ephemeranthol A at concentrations lower than 50 μM was non-toxic to slightly toxic to the cells, while the concentrations of 100 μM caused a significant decrease in cell viability after 24 and 48 h of treatment (Figure 1C). Data analysis showed that the IC50 of ephemeranthol A was higher than 200 μM at 24 h and approximately 150 μM at 48 h (Figure 1D).

Ephemeranthol A induced apoptosis in lung cancer cells. In order to define the mechanism of cell death, we further analyzed nuclear morphology and membrane integrity by Hoechst 33342 and PI staining in ephemeranthol A-treated cells. Results suggested that ephemeranthol A at the concentrations of 50-100 μM, can induce cell death through apoptosis, as indicated by the condensed and fragmented nuclei stained with Hoechst (Figure 2A-C). Necrotic cells can be generally detected by the cell losing membrane integrity; however, only few necrotic cells were detected in response to ephemeranthol A treatment at all tested concentrations, implying that apoptosis is the main mechanism of death induction of the compound. Induction of apoptosis by ephemeranthol A was also confirmed by western blot results. Treatment of lung cancer H460 cells with ephemeranthol A significantly increased the levels of cleaved PARP (at 50 and 100 μM, 1.98- and 2.33-folds, respectively; p<0.05), cleaved Caspase 3 (at 100 μM, 4.78-folds; p<0.05), and cleaved Caspase 9 (at 50 and 100 μM, 1.52- and 1.73-folds, respectively; p<0.05) (Figure 3A, B). Although ephemeranthol A had no significant effect on Bax, a pro-apoptotic member of Bcl-2 family, and the anti-apoptotic protein Mcl-1 (Figure 3C, D), it significantly reduced the level of Bcl-2 protein (at 100 μM, 0.63-folds; p<0.05). The results suggested that ephemeranthol A induced apoptosis in lung cancer cells.

Figure 2.
Figure 2.

Ephemeranthol A induced apoptosis and necrosis response of the cells. H460 cells were treated with ephemeranthol A (0-100 μM) for 48 h. Hoechst 33342 and propidium iodide (PI) were added. Cells were visualized under an inverted fluorescence microscope (scale bar=50 μm) (A). Merge image of H460 cells treated with ephemeranthol A at 100 μM (scale bar=50 μm) (B). Data are presented as percentage of apoptotic nuclei in ephemeranthol A-treated cells [mean±SEM (n=3)] (C). **p<0.01, compared to the untreated control.

Figure 3.
Figure 3.

The effect of ephemeranthol A on the expression of apoptosis-associated proteins. H460 cells were treated with ephemeranthol A for 48 h. The expression levels of poly (ADP-ribose) polymerase (PARP), cleaved PARP, caspase-9, cleaved caspase-9, caspase-3, and cleaved caspase-3 proteins in H460 cells were defined by western blot (A). Relative protein levels and protein levels were calculated by densitometry (B). The expression levels of the anti-apoptotic proteins Mcl-1 and Bcl-2, and the pro-apoptotic protein, Bax, were determined by western blot assay (C). Relative protein levels and protein levels were quantified by densitometry (D). Blots were reprobed for β-actin to confirm equal protein loading of each sample. Data represent the mean±SEM (n=3). *p<0.05 compared to the untreated control.

Figure 4.
Figure 4.
Figure 4.

Ephemeranthol A-treatment inhibited anchorage-independent growth and motility in lung cancer cells. Cells treated with ephemeranthol A (0-50 μM) for 48 h were subjected to colony-formation assay for two weeks. Then, colony formation was assessed by microscopy (4×) (A). Colony number (B) and colony size (C) in the wells with treated cells were evaluated and calculated as relative to untreated control wells. Data represent the mean±SEM (n=3). *p<0.05, **p<0.01, compared to the untreated control. Wound-healing assay was performed to evaluate the migration ability the H460 lung cancer cells, treated with 0-100 μM of ephemeranthol A for 24 and 48 h (D). The migration rate was expressed as the percentage of wound closure (E). Data represent the mean±SEM (n=3). **p<0.01 compared to the untreated control.

Ephemeranthol A suppressed anchorage-independent growth and inhibited lung cancer cell migration. The ability of cancer cells to grow in semisolid media (anchorage-independent cell growth) is important for the oncogenic phenotype, and has been associated with the metastatic potential (25). In order to evaluate the antimetastatic activity of ephemeranthol A, the cells were treated with various concentrations of the compound (0-50 μM) for 48 h and then were subjected to anchorage-independent growth assay. After 14 days, the colony number and colony size were determined and calculated relative to untreated control cells. Ephemeranthol A at the concentrations of 5-50 μM significantly decreased the ability of the cancer cells to grow and form spheroids, as indicated by the dramatic decline in the percentage of colony formation (at 5, 10, and 50 μM: 52.03%; p<0.05, 30.95%, p<0.05, and 14.55%, p<0.01, respectively; compared to the control 100%) and the percentage of colony size (at 5, 10, and 50 μM: 73.12%; p<0.05, 59.79%; p<0.05, and 29.59%; p<0.01, respectively; compared to the control 100%) (Figure 4A-C). Furthermore, a wound-healing assay was performed to test the effect of ephemeranthol A on migration of the cells. Results showed that ephemeranthol A significantly inhibited H460 cell migration at the concentrations of 50 and 100 μM at 48 h, by reducing the motility of H460 cells to the wound area (at 50 and 100 μM: 33.77% and 25.06%, respectively, vs. control 38.40%; p<0.05), whereas the same concentration of ephemeranthol A at 24 h had no significant impact on cell migration, compared to the untreated control at 24 and 48 h (Figure 4D, E).

Ephemeranthol A suppressed EMT. The effect of ephemeranthol A on EMT was first determined by the general cell morphology observation. The cells were treated with various non-toxic concentrations of ephemeranthol A for 24 h. We found that the control untreated cells exhibited mesenchymal-like spindle morphology as compared to the hexagonal epithelial-like morphology in the ephemeranthol A-treated cells (Figure 5A).

The EMT suppression was further confirmed by analyzing EMT related proteins by western blot analysis. The EMT regulatory proteins as well as EMT markers including vimentin, Slug, and N-cadherin were determined in H460 treated cells treated with ephemeranthol A for 48 h. Results showed that ephemeranthol A essentially decreased those EMT markers compared with untreated control cells. In the cells treated with 50 and 100 μM of the compound as compared to the control cells, N-cadherin was decreased 1.43- and 1.67-folds (p<0.05), respectively; vimentin was reduced 1.69- and 4.35-folds (p<0.05), respectively; and Slug was decreased 1.82- and 12.5-folds (p<0.05), respectively (Figure 5B, C).

Moreover, the regulatory proteins for EMT and cell migration such as p-FAK and p-Akt were analyzed. The results demonstrated that ephemeranthol A decreased the levels of p-FAK (phosphorylated at Tyr397), while had minimal effect on total FAK (decrease of p-FAK/total FAK at 50 and 100 μM: 1.22- and 1.70-folds, respectively; p<0.05). Likewise, the active p-Akt (phosphorylated at Ser473) was found to be reduced by treatment with 100 μM ephemeranthol A (decrease of p-Akt/total Akt: 1.93 folds: p<0.05) (Figure 5D, E). These results indicated that ephemeranthol A suppressed EMT, as well as the FAK/Akt signaling in lung cancer cells.

Discussion

Lung cancer is the most prevalent cancer worldwide, accounting for high mortality (26, 27). The augmented ability of lung cancer to metastasis has been proposed as a critical factor contributing to high mortality rates. Therefore, novel anticancer strategies focusing on inhibition of cancer cell metastasis are pivotal for improving the clinical outcome. Based on previous evidence supporting that EMT facilitates metastasis in many cancers (28-30), this study explored the anti-EMT and anti-metastatic properties of ephemeranthol A in human lung cancer cells. We found that ephemeranthol A exerts its anti-migratory activity through its ability to inhibit EMT, specified by the reduction of N-cadherin, vimentin, and Slug (Figure 5B, C). In addition, the migratory pathway of FAK and Akt was suppressed, resulting in the inhibition of cell movement (Figure 5D, E).

Figure 5.
Figure 5.

The effect of ephemeranthol A on the epithelial to mesenchymal transition (EMT) of lung cancer cells. H460 cells were treated with ephemeranthol A for 48 h. Morphology of the cells was observed under light microscope (A). The expression levels of EMT markers were investigated by western blot (B). Results were quantified by densitometry (C). The expression levels of FAK and Akt proteins, and their phosphorylated forms, were investigated (D). Protein levels and relative protein levels were calculated by densitometry (E). β-Actin was used as loading control. Data represent the mean±SEM (n=3). *p<0.05, **p<0.01, compared to the untreated control.

Figure 6.
Figure 6.

Schematic overview of the proposed mechanism of action of ephemeranthol A, based on evidence from the present study, as well as previous literature. Ephemeranthol A can suppress FAK and Akt signaling that in turn affect the EMT status of the cells. Akt also controls cell survival via Bcl-2 regulation. The imbalance of Bcl-2 family proteins toward reduction of anti-apoptotic members renders the activation of mitochondrial apoptosis through activation of caspases. Ephemeranthol A decreased the protein expression levels of EMT markers such as Slug, vimentin and N-cadherin.

Ephemeranthol A is a pure compound isolated from D. infundibulum (22). Interestingly, plants in the genus Dendrobium have been found to exhibit anticancer effects in T-cell lympoma (24). Moreover, phenolic compounds isolated from D. ellipsophyllum Tang and Wang (Orchidaceae) have displayed cytotoxic activity against human lung cancer, in vitro. In addition, these compounds have shown apoptosis induction and anoikis-sensitizing effects at non-toxic concentrations (31). Denbinobin, a major phenanthrene compound isolated from stems of D. nobile, has been shown to decrease Bcl-2, induce apoptosis, and inhibit cell migration (32, 33).

During metastasis, several signaling proteins, such as FAK and Akt are activated (7-9). The induction of FAK as well as Akt has been shown to increase metastatic potentials of cancer cells by activating the survival of the cell in anchorage-independent condition and enhancing cell motility (10). In lung cancer, increased Akt phosphorylation is associated with metastatic behavior (11, 12). Several reports have indicated that Akt is a down-stream target of FAK (34, 35). Epithelial to mesenchymal transition (EMT) has been shown to be involved in the progression and metastasis of lung cancer (36). EMT is an essential process for development as well as for tissue repair (37). During EMT, cells exhibit a switch from E-cadherin to N-cadherin expression and upregulation of Snail, Slug, and vimentin (38). EMT has been shown to play critical roles in cancer progression, metastasis, and resistance to chemotherapeutic drugs (6, 39, 40). EMT is a down-stream target of Akt (41); importantly, the active status of Akt has been associated with increased level of EMT inducers and markers, including Slug, vimentin, and N-cadherin (42). Moreover, Slug and Snail are potential transcription factors for EMT regulation and have also been involved in cell migration induction in NSCLC, via the secreted protein acidic and rich in cysteine/lysine-deficient protein kinase 1 (SPARC/WNK1) signaling pathway (43). Consistently, we found that treatment of the lung cancer cells with ephemeranthol A resulted in the reduction of active Akt and decreased the expression levels of EMT markers such as Slug, vimentin, and N-cadherin. In addition, a recent study has reported that the inhibition of Akt and EMT pathways resulted in suppression of NSCLC metastasis (44). Herein, we have demonstrated that ephemeranthol A could inhibit migration and EMT in lung cancer cells. In agreement with these findings, previous studies have shown that many compounds from orchids can induce cell death and inhibit migration in various cancer cells (32, 33, 45). In this study, ephemeranthol A extracted from D. infundibulum, also induced apoptosis, confirmed by the activation of caspase-9 and caspase-3 after treatment with ephemeranthol A.

Conclusion

This study provides supporting information regarding the antimetastatic potential of ephemeranthol A. More specifically, in vitro treatment of human NSCLC cells with ephemeranthol A inhibited cell migration and growth in anchorage-independent condition, as well as EMT, and induced apoptosis. These effects were mediated through inhibition of FAK and its co-regulator Akt activation. The proposed mechanism of action is illustrated in Figure 6. Considering these properties, ephemeranthol A appears to be a promising natural anti-cancer agent that warrants further research for potential clinical application.

Acknowledgements

This work was supported by Ratchadaphiseksomphot Fund, Chulalongkorn University for the Postdoctoral Fellowship and Chulalongkorn University. The authors express their gratitude to the Faculty of Pharmaceutical Sciences, Chulalongkorn University for providing research fund (Grant number Phar2562-RG002) to Prof. Dr. Pithi Chanvorachote.

Footnotes

  • ↵* These Authors contributed equally to this study.

  • Authors' Contributions

    OR, and NN performed experiments and drafted the article. KP, CJ and ST performed the experiments. BS isolated the tested compound. PC designed and supervised the research and wrote the article.

  • Conflicts of Interest

    The Authors declare that there are no conflicts of interest.

  • Received February 11, 2020.
  • Revision received April 27, 2020.
  • Accepted April 29, 2020.

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Ephemeranthol A Suppresses Epithelial to Mesenchymal Transition and FAK-Akt Signaling in Lung Cancer Cells
NONGYAO NONPANYA, ORNJIRA PRAKHONGCHEEP, KORRAKOD PETSRI, CHOLASIT JITJAICHAM, SUCHARAT TUNGSUKRUTHAI, BOONCHOO SRITULARAK, PITHI CHANVORACHOTE
Anticancer Research Sep 2020, 40 (9) 4989-4999; DOI: 10.21873/anticanres.14502
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