Assessment of the anticancer function of Coronilla orientalis MILLER through comprehensive in vitro and computational studies
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Halilibrahim Ciftci
,
Ayhan Oral
,
Yalcin Coskun
,
Gülin Renda
,
Masami Otsuka
,
Mikako Fujita
and
Belgin Sever
Abstract
Objectives
Although many synthetic anticancer drugs are available, a significant proportion of human therapeutics in the anticancer armamentarium are derived from natural products. The aim of this study to examine the anticancer effects of natural compounds against non-small cell lung cancer (NSCLC) and breast cancer, which remain among the world’s greatest obstacles.
Methods
Coronilla orientalis MILLER (CO) was collected in Erzincan, Türkiye, prepared, and extracted with 70 % ethanol. CO was then tested against A549 NSCLC and MCF-7 breast cancer cells using the MTT assay. To explore its potential anticancer mechanism, the apoptotic effects of CO in A549 and MCF-7 cells and the kinase inhibitory effects of CO were investigated using the Annexin V/ethidium homodimer III staining assay and the ADP-Glo kinase assay, respectively. Molecular docking studies were also performed for several major components of CO in the ATP binding site of EGFR.
Results
The results showed that CO, with IC50 values of 2.37 ± 0.59 μg/mL and 7.60 ± 1.18 μg/mL, exhibited anticancer activity against A549 cells and MCF-7 cells, respectively. CO was also selectively cytotoxic between Jurkat cells and PBMCs (healthy). CO-treated A549 and MCF-7 cells were found to undergo significant apoptosis and CO was found to inhibit EGFR. Molecular docking studies revealed the interaction of some defined components of CO with key residues in the ATP binding site of EGFR.
Conclusions
Taken together, this research has shown that CO has a great deal of potential as an inhibitor of the anticancer function against NSCLC and breast cancer, and warrants further investigation.
Introduction
Lung neoplasia is the most common cause of cancer incidence and morbidity in the world. Depending on the cell of origin, lung cancer is roughly divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), which is further divided into adenocarcinoma, the predominant subtype, squamous cell carcinoma and large cell carcinoma [1], 2]. It is known that NSCLC harbours targeted oncogenic mutations such as epidermal growth factor (EGFR) and HER-2 whose overexpression and mutations have been shown to be reciprocal to other drivers of genetic alterations [3], 4]. Targeted drugs offer a remarkable survival advantage for NSCLC patients with genetic alterations that are amenable to drug. In patients with locally advanced or metastatic lung adenocarcinoma carrying activating EGFR mutations, EGFR inhibitors such as erlotinib have been shown to be effective. On the other hand, several drugs, such as trastuzumab and emtansine, have been shown to be effective against HER-2-driven NSCLC [5], 6].
EGFR is a cell surface protein that, together with HER-2, HER-3 and HER-4, belongs to the ErbB family of receptor tyrosine kinases. Binding of the ligand to EGFR results in dimerization, phosphorylation of intracellular tyrosine kinase domain and stimulation of downstream signalling network such as RAS-MAP kinase, PI3K-AKT-mTOR and JAK-STAT [7]. In contrast, HER-2 is unable to bind ligands and functions by forming heterodimers with other members of the family, such as EGFR, which is the dimerization partner of choice for all other ErbB members [8]. EGFR is actively involved in these signalling cascades that subsequently trigger various cancers, including NSCLC, breast cancer, glioblastoma, hepatocellular carcinoma, and pancreatic cancer [9]. EGFR inhibitors, including erlotinib and geftinib, are competitive and reversible kinase inhibitors binding to the ATP binding cleft of the intracellular tyrosine kinase domain and impede autophosphorylation, downstream signaling and EGFR-dependent cell growth [10], 11].
Overexpression of the EGFR and HER-2 genes has also been reported as a hallmark of breast cancer, the leading cause of cancer death in women all over the world and the second most common cancer overall [12], 13]. The survival rate of people with breast cancer is reduced if the cancer metastasizes to other parts of the body, and metastatic breast carcinoma is currently deemed incurable. A disproportionately high number of breast cancer deaths are due to forms of the disease that are particularly prone to metastasize to the lungs and brain. Approximately 25–30 % of breast cancer patients have HER-2 gene amplification or overexpression, making HER-2 one of the most imperative pathways in breast cancer [14], [15], [16], [17]. Despite the high levels of EGFR in breast cancer, the anti-breast cancer effects of EGFR inhibitors are generally lower than expected. Both in vitro and in vivo studies have shown that lapatinib, an inhibitor of EGFR and HER-2, is effective against HER-2-driven tumour cells. Lapatinib can be given as an adjuvant treatment to patients with breast cancer that is either positive or negative for lymph nodes. Apart from NSCLC cells, erlotinib also exhibited anticancer activity against breast cancer cells along with head-and-neck and ovarian cancer cells [18], 19].
Natural products from medicinal plants have been in use since ancient times for the treatment of various ailments including cancer, as they generally have protective and selective therapeutic effects, and are beneficial in the production of nutrient replenishment in people at risk [20]. In the current development pipeline, a number of plant-based compounds, such as vincristine, paclitaxel (taxol) and irinotecan, are currently being used successfully in the treatment of cancer [21]. Natural phytochemicals with anticancer activity have been reported to exert their effects through the modulation of downstream signal transduction and host gene expression [22], [23], [24], [25]. Some natural ingredients from plants were found effective against EGFR-driven lung and breast cancer including luteolin, curcumin, silibinin, plumbagin, berberine, and quercetin [26], 27]. On the other hand, some natural compounds such as 2-O-caffeoyl tartaric acid, 2-O-feruloyl tartaric acid, and salvianolic acid have been confirmed through ligand- and structure-based studies to have a marked ligand potency and consistent binding affinities to HER-2 [28]. Plumbagin was also identified to exhibit apoptosis in HER-2 overexpressing breast cancer types [29]. In addition, a number of natural substances have been discovered in other studies as potential HER-2 inhibitors on the basis of computational methods [30], 31].
Fabaceae species are important sources of phytochemicals, found in every genus of the Fabaceae family, with anticancer activity against several cancers, including lung and breast cancer [32], 33]. Coronilla species (Fabaceae) have long been sought for their ability to treat cancer, as well as colds, diabetes and pain [34], [35], [36], [37], [38], [39], [40]. The anticancer effects of Coronilla varia L. (C. varia L.) have been mechanistically identified against the breast cancer cells MDA-MB-231 and MCF-7. The methanol, ethanol, and water extracts of C. varia L. were determined as 597.7 μg/mL, 386.4 μg/mL and 824.1 μg/mL, respectively [40]. On the other hand, some cardiac glycosides of C. varia L. were found to exert highest selective anticancer activity against lung and colorectal carcinoma cells [39].
In the current work, initially Coronilla orientalis MILLER (CO) was collected and extracted properly. Subsequently, the extract of CO was tested for its antiproliferative activity against the human lung adenocarcinoma cell line A549 and the human breast cancer cell line MCF-7. The selectivity of the cytotoxic effects of CO was affirmed between the human leukaemic T-cell line Jurkat and peripheral blood mononuclear cells (PBMCs) (healthy). Moreover, CO has been evaluated in its mechanism of action against NSCLC and breast cancer, including apoptosis activity in A549 and MCF-7 cells, and inhibition profile against a broad panel of kinases including EGFR and HER-2. Molecular docking studies were also employed to understand the EGFR and HER-2 binding potential of key CO moieties in EGFR and HER-2 ATP-binding clefts.
Materials and methods
Sample collection and extraction
C. orientalis MILLER was collected from Erzincan (Coordinates: 39.6077936, 39.830847), Türkiye, on June, 2019. Three plants were selected from the fresh rooted plants collected, photographed and herbarium was created for identification. The remaining plants were wrapped in paper towels and delivered to Çanakkale in cardboard boxes the next day. Tulay Tutenocaklı described the plant material and confirmed it with Çanakkale Onsekiz Mart University Çanakkale Botanical Garden Herbarium (CBB 00003137). The plant material, including roots, branches, leaves, and flowers, was washed once with tap water and three times with distilled water to remove foreign matter including soil, blotted the wash water with paper towels, and then allowed to dry. The 800 g fresh material was dried at 50 °C for 72 h using the dryer machine (Simsek Laborteknik ST-055, Ankara, Türkiye). At the end of drying, 137 g of dry material was obtained. Dried plant material was ground using a herbal grinder. The ethanol extract was prepared by immersion of 20 g of the dried material in 100 mL of ethanol (70 % purity) for 24 h at room temperature (rt). The mixture was then filtered. The process was repeated with the remaining residue using 60 mL ethanol. The two filtrates were combined and the mixture was concentrated under reduced pressure by means of a rotary evaporator. The ethanol was evaporated and the samples were redissolved in dimethyl sulfoxide (DMSO) (10 mg/mL). After nitrogen gas was pressed onto the extract placed in a colored bottle, it was closed and kept in the refrigerator (+4 °C) until the experiments [41], [42], [43].
Biochemistry
Cytotoxicity
DMEM (Wako Pure Chemical Industries, Osaka, Japan) and RPMI 1640 (Wako Pure Chemical Industries, Osaka, Japan), which were supplemented with 10 % fetal bovine serum (Biosera, Kansas City, MO, USA) were used for A549 cell culture, and MCF-7, Jurkat and PBMCs cultures, respectively. Then, drugs were treated as previously explained [42], [43], [44], [45]. In experiments, PBMCs were cultured in 96-well plates at 1 × 106 cells/mL concentrations for 72 h. A549 and MCF-7 cells (2 × 104 cells/mL - 24-well plate) and Jurkat cells (4 × 104 cells/mL - 24-well plate) were plated and incubated for 72 h (our previous studies have identified the optimal cell number) [42], [43], [44], [45]. The cytotoxic effects of CO on A549, MCF-7, Jurkat cells and PBMCs were assessed by using MTT (Dojindo Molecular Technologies, Kumamoto, Japan) in accordance with previously described procedures. After approximately 100 µL of MTT solution was added to each well and further incubation for 4 h, formazan crystals formed. Then, the medium was removed and the formazan crystals were solubilized by adding 100 µL of DMSO to each well. Absorbance was read on an Infinite M1000 plate reader (Tecan, Mannedorf, Switzerland) at a wavelength of 595 nm for formazan absorbance and 650 nm for background absorbance. All experiments were performed in triplicate and IC50 values were defined as the drug concentrations that decreased the absorbance to 50 % of the control values [44], [45], [46], [47].
Apoptotic activity
A549 and MCF-7 cell lines (2 × 104 cells/well) were incubated in each well of 24-well plate with CO at IC50 concentrations (24 h). Apoptotic/necrotic/healthy/detection kit (PromoKine, Heidelberg, Germany) was applied related to manufacturer`s directions with some amendments. A549 and MCF-7 cell lines were exposed twice to 1 × binding buffer, a staining solution containing 50 μL of 1 × binding buffer, 4 μL solutions of FITC-Annexin V, ethidium homodimer III and Hoechst 33342 and incubated for 30 min at rt in the dark. The apoptotic, late apoptotic/necrotic and necrotic cells were then analysed using a Biorevo Fluorescence BZ-9000 fluorescence microscope (Keyence, Osaka, Japan) [48], [49], [50], [51].
Kinase inhibitory activity
The protocol of the EGFR enzyme system (Promega V3831, Madison, WI, USA) and kinase selectivity profiling systems (Promega TK-1 and TK-2, Madison, WI, USA) were performed according to the manufacturer’s protocol. The kinase reaction was carried out using 4 µL of the kinase solution, 4 µL ATP/substrate solution and 2 µL of plant extract solution (at 10 and 1 µM concentrations) or 5 % DMSO solution in the 384-well plate. After 1.5 h of incubation at rt, the kinase activity was quantified using ADP-Glo Kinase Assay (Promega Corporation, Madison, WI, USA). Briefly, 5 μL of ADP-Glo reagent was added to each well of the 384-well plate, and the mixture was incubated at rt for 40 min to stop the kinase reaction and consume the remaining ATP. Then, 10 μL of Kinase Detection Reagent was added, and the mixture was incubated for another 30 min to convert the ADP to ATP and then to light. The inhibitory kinase activity of the compounds was measured with a luminescence plate reader Infinite M1000 (Tecan, Grödig, Austria) in dose-response mode, and the % EGFR kinase inhibition effects of plant extract at 1 μg/mL and 10 μg/mL concentrations were calculated [48], [49], [50], [51].
Statistical analysis
All results were presented as means±SD One-way analysis of variance was used for the analysis of data. Differences were defined as significant at *p<0.05, **p<0.005, ***p<0.001. GraphPad Prism7 (GraphPad Software, San Diego, CA, USA) was used for the determination of the IC50 values.
Docking assessment
The RSCB database was used to retrieve the crystal structures of EGFR (PDB ID: 1M17) [52] and HER-2 (PDB ID: 3RCD) [53]. The raw file was prepared for docking using Maestro’s PrepWizard. Prime automatically added missing chains and PropKa determined protonated states at physiological pH. The LigPrep module was used to prepare the main components of CO using energy minimization at physiological pH after sketching and cleaning them in the Maestro workspace. Maestro’s grid generation feature was used to generate the docking grid. The grid was then applied to the docking experiments using the Glide/XP docking protocols [48], [49], [50, 54].
Results
The MTT assay was used to investigate the anticancer effects of CO against A549 and MCF-7 cells compared to erlotinib, a potential anticancer agent acting as an EGFR inhibitor. Figure 1A explained that the cytotoxic effects of CO against A549 and MCF-7 cells were dose-dependent at different concentrations. The IC50 values of CO against A549 and MCF-7 cells were detected as 2.37 ± 0.59 μg/mL and 7.60 ± 1.18 μg/mL, respectively. The Selectivity Index (SI) was calculated between the IC50 values of CO against A549 and MCF-7 cells, indicating a more than 3-fold selective profile of CO against NSCLC cells (Table 1). The selectivity of cytotoxicity of CO was determined between Jurkat cells and PBMCs (healthy). Results showed that CO displayed superior selectivity with a SI value of 32.92 (Table 1) and Figure 1B also outlined the high selectivity of CO against cancer cells.
Cell viability of A549 and MCF-7 cells (A) and Jurkat cells and PBMCs (B) exposed to different concentrations of CO.
The IC50 values of CO against A549, MCF-7, Jurkat cells and PBMCs compared to erlotinib.
Compound |
IC50 values, μg/mL | SI1a | SI2b | |||
|---|---|---|---|---|---|---|
| A549 cells | MCF-7 cells | Jurkat cells | PBMCs | |||
| CO | 2.37 ± 0.59 | 7.60 ± 1.18 | 4.67 ± 1.06 | 153.73 ± 16.88 | 32.92 | 3.21 |
| Erlotinib | 7.91 ± 1.65 | 5.85 ± 1.40 | 3.50 ± 1.22 | 23.89 ± 2.95 | 6.83 | 0.74 |
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aSI1=IC50 for PBMC/IC50 for Jurkat cell line. bSI2=IC50 for MCF-7 cell line/IC50 for A549 cell line.
The cells stained with Annexin V but not stained with Ethidium homodimer III are judged to be early apoptotic cells. After exposing A549 and MCF-7 cells with CO, cells were stained green for fluorescent microscopic apoptosis monitoring (Figure 2A and B). Annexin V staining data showed apoptosis levels of 22.5 and 9.8 % in A549 and MCF-7 cells, respectively, after 24 h CO treatment (Figure 2C).
Apoptotic (green), necrotic or late apoptotic (yellow) and necrotic (red) cells were shown in A549 (A) and MCF-7 (B) cells after exposure to IC50 concentration of CO for 24 h. In each experiment, 100 randomly selected stained cells were analysed to quantify the percentage of apoptotic cell ratio (C). (***p<0.001).
The EGFR and HER-2 inhibitory effects of CO were also screened. CO was detected to inhibit EGFR (Promega V3831) significantly with 56.8 % at 10 μg/mL concentration (Figure 3A). The results of CO against kinase selectivity profiling systems (Promega TK-1 and TK-2) showed that CO inhibited EGFR with the highest inhibitory level as 54.5 % at 10 μg/mL concentration compared to a large panel of kinases including EGFR, HER-2, HER-4, IGF1R, InsR, KDR, PDGFR, PDGFR, Abl-1, BRK, BTK, CSK, FYNA, LCK, LYN B, SRC. CO inhibited HER-2 with 34.5 % (Figure 3B).
EGFR inhibitory effects of CO at 10 μg/mL and 1 μg/mL concentrations (A). The inhibition of a panel of tyrosine kinases by CO at 10 μg/mL concentration (B).
Previously, Renda et al. 2019 [37] identified several major components of CO obtained by hydrodistillation and solid-phase microextraction techniques by means of gas chromatography-mass spectrometry (GC/MS) analysis. They showed that CO was rich with limonene, γ-terpinene, hexanal, linalool, naphthalene, (E)-ethyl cinnamate, α-muurolol, α-(E)-bergamotene, and octen-3-ol. Based on this study, we conducted molecular docking of these components in the ATP binding clefts of EGFR and HER-2. Results indicated that generally all components occupied the binding sites of EGFR (Figure 4A and B) and HER-2 (Figure 5A and B) presenting high affinity. Among these compounds, the most promising results were achieved with (E)-ethyl cinnamate, octen-3-ol and hexanal in the binding site of EGFR.
Docking poses of limonene, γ-terpinene, hexanal, linalool, naphthalene, (E)-ethyl cinnamate, α-muurolol, α-(E)-bergamotene, and octen-3-ol and erlotinib in the ATP binding cleft of EGFR. Ribbon presentation (A) surface presentation (B).
Docking poses of limonene, γ-terpinene, hexanal, linalool, naphthalene, (E)-ethyl cinnamate, α-muurolol, α-(E)-bergamotene, and octen-3-ol and lapatinib in the ATP binding cleft of HER-2. Ribbon presentation (A) surface presentation (B).
The docking scores of (E)-ethyl cinnamate, octen-3-ol and hexanal were found as −7.328, −6.795, and −6.524 kcal/mol, respectively compared to erlotinib (−7.996 kcal/mol) (Table 2).
Docking score (kcal/mol) values of compounds in the binding sites of EGFR and HER-2.
| Compound | Kinase | |
|---|---|---|
| EGFR | HER-2 | |
| Docking score | Docking score | |
| (E)-ethyl cinnamate | −7.328 | −6.192 |
| Octen-3-ol | −6.795 | −6.392 |
| Linalool | −6.524 | −6.694 |
| Hexanal | −6.725 | −5.998 |
| α-muurolol | −5.919 | −4.624 |
| Naphthalene | −5.332 | −4.777 |
| Limonene | −4.852 | −3.393 |
| α-(E)-bergamotene | −4.207 | −2.270 |
| γ-terpinene | −3.587 | −2.757 |
| Erlotinib | −7.996 | – |
| Lapatinib | – | −8.033 |
All these three components formed a key hydrogen bonding with Met769 that erlotinib also formed similarly (Supplemental Figure 1). Linalool and α-muurolol interacted with Thr830 through hydrogen bonding, whereas naphthalene established a π-cation bond with Lys721. Limonene, α-(E)-bergamotene and γ-terpinene could not contribute to the binding of CO to EGFR, although these compounds were detected with the highest amount in CO.
On the other hand, the binding potential of these compounds to HER-2 was found weaker compared to that of EGFR. Linalool and octen-3-ol presented the most significant binding to HER-2 forming a hydrogen bonding with Asp863. The docking scores of linalool and octen-3-ol were found as −6.694 and −6.392 kcal/mol, respectively compared to lapatinib (−8.033 kcal/mol) (Table 2). (E)-ethyl cinnamate and hexanal served a distinct binding mode via interactions with Thr862 and Thr798, respectively. Limonene, α-(E)-bergamotene, γ-terpinene were also not be able to form any inteactions in the HER-2 binding site along with naphthalene and α-muurolol (Supplemental Figure 2).
Discussion
A variety of natural phytochemicals involved in the treatment of numerous diseases have become increasingly important in drug discovery and research, mainly due to their high-accessibility, cost-effectiveness and reduced side effects. One disease for which plants have long been used is cancer, exemplified by Taxol, the well-known herbal anticancer drug approved for ovarian and breast cancer.
The potential anticancer effects of Coronilla species inspired us to examine antiproliferative activity of CO against NSCLC and breast cancer cells. CO showed a higher toxicity profile to both NSCLC and breast cancer cells displaying lower cytotoxicity to PBMCs (healthy).
The mechanism of the antiproliferative effect of CO against NSCLC and breast cancer cells was further investigated. Apoptosis is a morphologically defined mode of cell death to eliminate unwanted cells, including cancer cells, from the cell community of multicellular organisms. CO was determined to induce apoptosis in both A549 and MCF-7 cells.
Molecularly targeted therapy has been acknowledged to enhance survival in patients with NSCLC and breast cancer and led to substantial improvements at a breakneck pace. Notably, EGFR and HER-2 mutations appear to be the driver molecular aberrations in the oncogenesis of both NSCLC and breast cancer. To date, numerous synthetic EGFR and HER-2 targeted inhibitors have been designed, synthesized, clinically evaluated and approved for marketing. On the other hand, several natural compounds have been reported to inhibit EGFR and HER-2, key regulators of cell proliferation, survival, migration, and differentiation. CO inhibited EGFR and HER-2, significantly.
In the challenging process of natural product drug discovery, which begins with the collection of plants and ends with the isolation of the basic components and the identification of their targeted biological activity, in silico approaches could be helpful in exploring the binding affinity of compounds to a particular target [25]. (E)-Ethyl cinnamate, octen-3-ol and hexanal displayed the highest affinity in the ATP binding site of EGFR. On the other hand, linalool and octen-3-ol demonstrated the most significant affinity to the ATP binding site of HER-2.
Conclusions
NSCLC and breast cancer are the leading causes of cancer deaths globally. Molecular targets, such as aberrations in the EGFR and HER-2 oncogenes, are being evaluated in the treatment of NSCLC and breast cancer to afford the unmet clinical needs. Natural products are useful sources for developing new molecules with enhanced selective pharmacology and improved pharmacokinetics, as they often co-evolve with the target sites in biological systems. In the current work, we have collected, prepared and extracted CO and then evaluated the mechanistic antiproliferative effects against NSCLC and breast cancer. CO achieved a broad and durable antiproliferative response against NSCLC and breast cancer cells with its promising cytotoxic and further apoptotic effects in A549 and MCF-7 cells. CO was also a significant inhibitor of EGFR and a moderate inhibitor of HER-2 from a large panel of kinases. The potential ability of CO to bind to the active sites of HER-2 and in particular EGFR has also been highlighted in molecular docking studies. All in vitro and in silico assays showed that CO is of great interest for future research with potential cytotoxic effects against NSCLC and breast cancer cells.
Funding source: HORIZON EUROPE Marie Sklodowska-Curie Actions
Award Identifier / Grant number: 101061939
Award Identifier / Grant number: 122Z775
Acknowledgments
This publication has been produced benefiting from the 1001 Scientific and Technological Research Projects Funding Program of TÜBİTAK (Project No: 122Z775). The authors thank to TÜBIİTAK for their supports. 
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Research ethics: The local Institutional Review Board deemed the study exempt from review.
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Informed consent: Informed consent was obtained from all individuals included in this study.
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Use of Large Language Models, AI and Machine Learning Tools: None declared.
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Conflict of interest: Authors state no conflict of interest.
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Research funding: 1001 Scientific and Technological Research Projects Funding Program of TÜBİTAK (Project No: 122Z775).
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Data availability: Not applicable.
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