The evaluation of the anti-cancer activity of ixazomib on Caco2 colon solid tumor cells, comparison with bortezomib
Selin Engür & Miriş Dikmen
To cite this article: Selin Engür & Miriş Dikmen (2017): The evaluation of the anti-cancer activity of ixazomib on Caco2 colon solid tumor cells, comparison with bortezomib, Acta Clinica Belgica, DOI: 10.1080/17843286.2017.1302623
To link to this article: http://dx.doi.org/10.1080/17843286.2017.1302623
Published online: 22 Mar 2017.
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The evaluation of the anti-cancer activity of ixazomib on Caco2 colon solid tumor cells, comparison with bortezomib
Selin Engür1, Miriş Dikmen2,3
1Graduate School of Health Sciences, Anadolu University, Eskisehir, Turkey, 2Faculty of Pharmacy, Department of Pharmacology, Anadolu University, Eskisehir, Turkey, 3Faculty of Pharmacy, Department of Clinical Pharmacy, Anadolu University, Eskisehir, Turkey
Proteasome inhibition has recently emerged as a clinically effective anticancer therapeutic approach. The first proteasome inhibitor, bortezomib (Velcade, PS-341), and new proteasome inhibitors including ixazomib have become more important in the development of targeted cancer therapies. Under physiological conditions, MLN9708 (ixazomib citrate), the stable citrate ester drug substance, hydrolyzes rapidly to MLN2238 (ixazomib), the biologically active boronic acid. It is a second-generation proteasome inhibitor, similar to the well-known proteasome inhibitor bortezomib, which is currently being investigated in phase 3 trials as a treatment for multiple Myeloma. Despite the proven efficacy of these drugs in hematologic malignancies, clinical activity is limited to solid tumors such as colon adenocarcinoma. This study is the first to investigate and compare the antiproliferative and apoptotic effects of MLN2238 and bortezomib on human colon adenocarcinoma Caco2 cells. The antiproliferative effects of MLN2238 and bortezomib were determined using WST-1; apoptotic effects of this drug were determined by caspase-3 and a mitochondrial membrane potential (JC-1) activity assay. Expression levels associated
with proteasome inhibition and apoptosis of NF-κB and c-myc mRNA were evaluated by RT-PCR. At 24 and
48 h, MLN2238 showed significant time- and concentration-dependent antiproliferative and apoptotic effects on
Caco2 cells. Depending on increasing mitochondrial depolarization and caspase-3 activation, MLN2238 induced apoptosis at level similar to that of bortezomib. In addition, MLN2238 downregulated NF-κB and c-myc mRNA expression levels. For the first time, MLN2238 was shown to induce antiproliferative and apoptotic effects on
human colon adenocarcinoma cells that are comparable with those of bortezomib; these in vitro data in Caco2 cells support the development of MLN2238 for colon cancer.
Keywords: Proteasome inhibitor, MLN9708, bortezomib, Caco2, apoptosis, ixazomib, MLN2238
Colorectal cancer is a leading cause of cancer death worldwide.1 A better understanding of colorectal cancer pathogenesis has provided an opportunity for the devel- opment of new drugs to combat this common and deadly malignancy.2 Although chemotherapy has improved the outcomes for patients with metastatic disease, new therapies with novel mechanisms of action are required. Major molecular pathways involved in the pathogenesis of colorectal carcinoma such as nuclear factor κB (NF-κB) regulated by the ubiquitin–proteasome system, provide a rationale for proteasome inhibition.3
The ubiquitin–proteasome system is a major pathway for protein degradation. Targeting this pathway using proteasome inhibitors represents a novel approach for the treatment of cancer. Proteasome inhibitors decrease cell
Correspondence to: Selin Engür, Graduate School of Health Sciences, Anadolu University, Eskisehir, Turkey. Email: [email protected]
proliferation and induce apoptosis in solid and hemato- logic malignancies through multiple mechanisms.4
Bortezomib is a well characterized first-generation pro- teasome inhibitor antitumor drug that was approved by the Food and Drug Administration (FDA) for clinical use in patients with refractory multiple myeloma and mantle cell lymphoma.5 It is a specific and reversible inhibitor of the chymotryptic activity of the 26S proteasome and has a wide range of molecular effects, including inhibition of nuclear factor-kB (NF-κB), abrogation of tumor growth and survival, and induction of apoptosis.6 Despite the clin- ical success of bortezomib, it has also been associated with many unfavorable outcomes, including cytotoxic side effects and the development of drug resistance.7
Recently, several new agents have been introduced into the field, including ixazomib (MLN9708-MLN2238), and trials investigating these second-generation protea- some inhibitors have demonstrated promising results.8
© Acta Clinica Belgica 2017
DOI 10.1080/17843286.2017.1302623 Acta Clinica Belgica 2017 1
Ixazomib is an investigational small-molecule proteasome inhibitor currently being developed for a broad range of human malignancies. In several studies, ixazomib showed extremely high potency against several cancer cell lines and it has selectivity and potency similar to that of borte- zomib.4,9 Ixazomib is an improved new proteasome inhib- itor currently being evaluated in multiple clinical studies for both solid tumors and hematologic malignancies. Based on preclinical studies, upon exposure to aqueous solutions or plasma, MLN9708 immediately hydrolyzes to its biologically active form, MLN2238.4,9,10 MLN2238 potently, reversibly, and selectively inhibits the proteas- ome and has been developed as an orally bioavailable drug with a lower toxicity profile.11
In this study, the antiproliferative and apoptotic effects of MLN2238, a proteasome inhibitor, on the human colon adenocarcinoma cell line Caco2 were investigated for the first time in a direct comparison with those of bortezomib.
Materials and methods
Cell culture and treatment
Caco2 cells were obtained from the American Type Culture Collection (ATCC number HTB-37™). The cells were grown in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum and 1% pen- icillin/streptomycin at 37 °C in a humidified incubator with a 5% CO2 atmosphere. MLN2238 and bortezomib were dissolved in dimethyl sulfoxide (DMSO) and a stock solution diluted to the required concentrations. In total, 70–80% confluent cells (after 24 h) were trypsinized and treated with MLN2238 and bortezomib (0.01, 0.1, 1, 5,
10, 20, 30 μM) for 24 and 48 h in the growth medium.
WST-1 cytotoxicity test
The viability of cells was measured using a 4-[3-(4-Iodo- phenyl)-2-(4-nitrophenyl)-2H-5 tetrazolio]-1,3-benzene disulphonate (WST-1) assay (Roche). The test is based on the cleavage of the tetrazolium salt WST-1 in for- mazan by mitochondrial dehydrogenases in viable cells. The formazan dye was quantified in a scanning multiwell spectrophotometer by measuring the absorbance of the dye at 420 nm. Caco2 cells were inoculated into 96-well culture plates at densities of 5 × 103 cells per well. After 24 h, cells were treated with the specified concentrations of MLN2238 and bortezomib for 24 and 48 h. After the incubation periods, the cell proliferation reagent WST-1 (10 μl per well) was added to wells; and absorbances were measured after 3 h. With an ELISA reader (wavelength 420 nm). The measured absorbances directly correlated to the number of viable cells. Cell viability rates were expressed as the percentage of the controls.12
Graphics were drawn with Graphpad Prism 5.0 soft- ware and statistically analyzed using one way ANOVA and Tukey’s post hoc test. Results are expressed as mean ± standard deviation and the means of three inde- pendent experiments (n = 8), n.s; p > 0.05, *p < 0.05,
**p < 0.01, and ***p < 0.001 were considered significant
compared to the control group.
Detection of 3caspase-3 activity
Caspase-3 level alterations of the cells were examined by the PE Active Caspase-3 Apoptosis Kit (BD Pharmingen, cat. no:550914). Caspase-3 is a key protease that is acti- vated during the early stages of apoptosis and is synthe- sized as an inactive pro-enzyme that is processed in cells undergoing apoptosis by self-proteolysis and/or cleavage by another protease. In short, the cells (1 x 105 per well) were treated with 1.0, 5.0, 10.0 and 20.0 μM of MLN2238 and bortezomib, determined based on WST-1 results, for 24 and 48 h. After the incubation periods, analysis was performed as specified by the kit procedure and analyzed on a Becton-Dickinson FACS Aria flow cytometer using FACSDIVA Version 6.1.1. Software. At least 10,000 cells were analyzed per sample.12,13
Determination of mitochondrial membrane potential by JC-1 dye on flow cytometer
The loss of mitochondrial membrane potential in response to MLN2238 and bortezomib in cells were examined by Flow Cytometry Mitochondrial Membrane Potential Detection Kit (BD Pharmingen, cat. no: 551302). Caco2 cells (1 x 105 per well) were seeded on six-well plates and treated with 1.0, 5.0, 10.0 and 20.0 μM concentra- tions of MLN2238 and bortezomib for 24 and 48 h. At the end of the treatment period, 1 ml of each cell suspension was transfered into a centrifuge tube and centrifuged at 1500 rpm for 5 min. Each pellet was resuspended with JC-1 Working Solution and incubated for 15 min at 37 °C in a CO2 incubator. After incubation, Caco2 cell pellets were washed with 1000 μl assay buffer and centrifuged again. Finally, the samples were resuspended with 250 μl assay buffer and processed for data acquisition, and ana- lyzed on a Becton-Dickinson FACS Aria flow cytometer using FACSDIVA Version 6.1.1. Software. At least 10,000 cells were analyzed per sample.12,14
RNA isolation, cDNA synthesis and real time PCR
To see the effects of MLN2238 and bortezomib on the expression levels of NF-κB, c-myc genes, the real time PCR was carried out. Approximately, 106 Caco2 cells were harvested and treated with concentrations of MLN2238 and bortezomib for RNA isolation. After 24 h, cells were prepared and transferred into MagNA Lyser Green Beads tubes and the cell homogenization process was started using a MagNA Lyser Instrument. Then MagNA Pure Compact RNA Isolation Kit (Roche, Reference: 04 802 993 001) procedure was performed by using the MagNA Pure LC 2.0 system. The high quality of the RNA sam- ples was confirmed by using the NanoDrop Instrument. For each RNA population, a total of 500 ng RNA was used for cDNA synthesis according to the Transcriptor
24h 48h 24h 48h
Figure 1 Cytotoxic effects of MLN228 and Bortezomib on Caco2 cells at 24 and 48 h (The error bars shows the standard deviations).
Statistical analysis: WST-1 experiment was repeated three times and all data were recorded as mean ± standard deviation of mean (S.D.). All the data were statistically analyzed using the one-way ANOVA, tukey test. Differences were considered significantly, if
*p < 0.05, **p < 0.01, ***p < 0.001.
High Fidelity cDNA Synthesis Kit procedure (Roche,
Reference: 05 081 963 001).12,15,16
Total cDNA was used to measure the mRNA lev- els of NF-κB1 (Roche, Lot: 90007129, Accession ID: ENST00000394820, Amplicon Length: 71 bps), c-myc (Roche, Lot: 90007128, Accession ID:ENST00000377970,
Amplicon Length: 92 bps) and GAPDH (Roche, Lot:90006719, Accession ID ENST00000229239,
Amplicon Length: 112 bps) genes. The mRNA levels of GAPDH were used as the internal positive control.
The primer sequences were NF-κB1 forward:
5′-TCCAGGTCATAGAGAGGCTCA-3′; c-myc for-
ward: 5′-GCTGCTTAGACGCTGGATT T-3′, reverse: 5′-TAACGTTGAGGGGCATCG-3′and GAPDH for-
ward: 5′-CTCTGCTCCTC CTGTTCGAC-3′, reverse: 5′- ACGACCAAATCCGTTGACTC-3. qPCR was per-
formed using the Universal ProbeLibrary detection for- mat on the LightCycler® 480 Instrument to quantify gene expressions. The realtime PCR mixture, containing 10 μl 2x LightCycler® 480 Probes Master, 1 μL of each primer (Real Time Ready Assay, Roche), 4 μl PCR grade water, and 5 μl of cDNA were prepared. The cycling conditions included an initial incubation step at 95 °C for 10 min, followed by a 45 cycles of amplification with 10 s at 95 °C, 30 s at 60 °C and 1 s at 72 °C. The final cooling step was holding at 40 °C for 30 s.
Cytotoxic effects of MLN2238 and bortezomib in Caco2 cells
Cytotoxic effects of MLN2238 and bortezomib in Caco2 cells were determined by a WST-1 cell proliferation assay. MLN2238 and bortezomib inhibited cell growth depended on both concentration and time. Both MLN2238 and bortezomib decreased cell viability. The effect of both
drugs was in the same range. The Caco2 cell viability was
significantly decreased 54.89, 49.55, 45.11, and 40.27%
with 5.0, 10.0, 20.0, and 30.0 μM MLN2238 concentra- tions, respectively, for 48 h (*p < 0.001). Bortezomib also decreased the cell viability of 62.99, 55.26, 53.83, 53.45,
and 48.83% at 1.0, 5.0, 10.0, 20.0, and 30.0 μM concen- trations, respectively, for 48 h (*p < 0.001) similar to the effect of MLN2238 (Figure 1).
MLN2238 and bortezomib increased caspase-3 activity in Caco2 cells
In the mitochondria-mediated apoptosis pathway, caspase-3 is essential for apoptosis formation. In order to determine the apoptotic effects of MLN2238 and bortezomib, Caco2 cells were incubated with increasing concentrations of these compounds for 24 and 48 h and caspase-3 enzyme activities were analyzed by using flow cytometry. Results of caspase-3 activity analysis related to percent values are shown in Figures 2 and 3.
After incubation with 1, 5, 10, and 20 μM MLN2238 con- centrations for 48 h, the percent of caspase-3 positive cells were 42.1, 52.1, 50.0, and 53.7%, respectively, whereas it
was 7.4% in the control group, leading to a 5.6, 7.0, 6.7, and 7.2-fold an increase in activity. With the same concentra- tions of bortezomib, 48.4, 52.1, 51.6, and 55.0% caspase-3 positive cells were found, respectively, and thus caspase-3 activity was increased to 6.54, 7.0, 6.9, and 7.4-fold.
MLN2238 and bortezomib decreased mitochondrial membrane potential on Caco2 cells
In order to determine the effects of MLN2238 and borte- zomib on the loss of mitochondrial membrane potential, Caco2 cells were incubated with 1, 5, 10, and 20 μM MLN2238 and bortezomib concentrations for 24 and 48 h. JC-1 Mitochondrial Membrane Potential analysis was performed by using flow cytometry. Results are shown in Figures 4 and 5.
Figure 2 Typical guadrant analysis of caspase-3 flow cytometry of Caco2 cells treated with MLN2238 and Bortezomib. Caco2 cells were cultured for 24 and 48 h in medium with MLN2238 and Bortezomib at concentration of 1, 5, 10, and 20 μM. At least 10,000 cells were analyzed per sample and quadrant analysis was performed. The proportion of cell number is shown in each quadrant, Q3, viable cells and Q4, caspase-3 positive cells.
MLN2238 Concentration (µM)
Bortezomib Concentration (µM)
Figure 3 Effects of MLN2238 and bortezomib on caspase-3 enzyme activity. The data are indicated as the mean of at least two independent experiments and the error bars shows the standard error.
Figure 4 Typical guadrant analysis of JC-1 flow cytometry of Caco2 cells treated with MLN2238 and Bortezomib. Caco2 cells were cultured for 24 and 48 h in medium with MLN2238 and Bortezomib at concentration of 1, 5, 10, and 20 μM. At least 10,000 cells were analyzed per sample and quadrant analysis was performed. The proportion of cell number is shown in each quadrant, Q3, viable cells and Q4, JC-1 positive cells.
A 1.44, 1.5, 1.6, and 1.7-fold increase in loss of mito- chondrial membrane potential were determined with 1.0, 5.0, 10.0, and 20.0 μM MLN2238 concentrations, respec- tively. Loss of mitochondrial membrane potentials were decreased 1.9-, 1.6-, 1.8-, and 1.9-fold with same borte- zomib concentrations after a 48 h incubation.
MLN2238 and bortezomib downregulated expression levels of NF-κB1 and c-myc
Caco2 cells were treated with 1.0, 5.0, 10.0, and 20.0 μM concentrations of both MLN2238 and bortezomib, and consequently, expression levels of NF-κB1 and c-myc genes were determined by the qRT-PCR method. At 20 μM
MLN2238 Concentration (µM) Bortezomib Concentration (µM)
Figure 5 Effects of MLN2238 and Bortezomib on loss of mitochondrial membrane potential. The data are indicated as the mean of three independent experiments and the error bars shows the standard error, ***p < 0.001 was considered to be significant.
MLN2238 Concentration (24h.)
Bortezomib Concentration (24 h.)
Figure 6 Changes in mRNA levels of NF-kB1 and c-myc genes for 24 h.
MLN2238 and bortezomib concentrations, the expression levels of the NF-κB1 gene were downregulated approxi- mately 3.5- and 4- fold, respectively, after 24 h. Similarly, the expression levels of c-myc gene were downregulated approximately 2.5- and 2.7- fold, respectively, with the same concentrations after 24 h. (Figure 6).
The ubiquitin–proteasome system is one of the major pro- cesses that occur during protein homeostasis in which specific proteins are targeted for destruction via the attachment of ubiquitin. Highly regulated members of the critical signaling cascades, including proteins involved in cell cycle regula- tion, growth control and apoptosis, and misfolded proteins are substrates of these proteasomes. The stabilization and accumulation of these substrates ends up with the activation of antiproliferative signals and apoptotic pathways, and ulti- mately, cell death by proteasome inhibition.4,10,11
Proteasome inhibitors have a 20 year history in cancer therapy. Bortezomib, the first proteasome inhibitor, is a reversible boronic acid that inhibits the chymotrypsin-like activity of the proteasome. Bortezomib (Velcade, PS-341)
was approved by the U.S. Food and Drug Administration for the treatment of refractory multiple myeloma in 2003.10,17 Subsequently, bortezomib received regular approval from FDA for the treatment of multiple myeloma in 2008 and relapsed or refractory mantle cell lymphoma treatment in 2014. 17
Bortezomib is by far the most extensively evaluated pro- teasome inhibitor in clinical trials for hematologic malig- nancies.10 Preclinical studies have shown that bortezomib exhibits anti-tumor activity in solid tumor malignancies.18 Among solid tumors, preclinical activity has been observed in models of non-small cell lung cancer,19 head and neck squamous cell carcinoma,20 hepatocellular carcinoma,21 melanoma,22 prostate cancer,23 colon cancer,24 renal cell carcinoma,25 and pancreatic cancer.26 Several in vitro and in vivo studies are investigating the effects of bortezomib on solid tumors. Antiproliferative action of bortezomib in a wide variety of cancer cell lines derived from solid tum- ors, including prostate, lung, breast, pancreas, gastric, and colon has been shown previously.27,28 In vitro data from a panel of 60 cell lines derived from human tumors indicated that bortezomib has significant growth-inhibitory activity
in a wide variety of malignancies including cell lines of solid tumors.29,30 In addition, bortezomib induces apoptosis in human hepatic, mantle-cell, prostate, colorectal, ovar- ian, and breast cancer cells.29,31–35 Bortezomib is cytotoxic for the human PC-3 prostate cancer cell line, leading to cell cycle arrest and apoptosis and in nude mice it inhibits PC-3 tumor growth.30 Bortezomib induces apoptosis and inhibits the proliferation in head and neck squamous cell carcinoma Ca9-22, SAS, and SCC-25 cells.36 Bortezomib inhibits proliferation and induces apoptosis in Hep-2 cells, a cell line of laryngeal squamous cell carcinoma.29 In an animal model of lung carcinoma, bortezomib inhibits both tumor growth and the development of metastases.37 The antiproliferative effects of bortezomib on HCT116, HT-29, and CaCo2 cells were evaluated bortezomib displayed a 60% decreased cell proliferation of Caco2 cells by MTT assay.2 Bortezomib also promotes cell death in the colon cancer cell line DLD-1 both through apoptosis and necro- sis, as evaluated by flow cytometry.38 Bortezomib has also been shown to induce cell growth inhibition in human Colo320HSR, HT29, and DLD1 colon cancer cells.35
By contrast, clinical trials of bortezomib treatment in solid tumors have generally resulted in less promising result. This may derive from its inability to penetrate into tissues and achieve therapeutically relevant concentra- tions at those target sites. Furthermore, the therapeutic efficacy of bortezomib is hindered by various qualities, including nonspecificity and associated adverse toxicities, inherent and acquired resistance, and short-term reversible inhibition. Therefore, there is a great need for identifying proteasome inhibitors that have different physicochemical or pharmacokinetic properties.10 Each of these limitations of bortezomib is being considered in second generation proteasome inhibitors including ixazomib (MLN2238), derived from MLN9708.18 MLN9708 was selected from a large pool of boron containing proteasome inhibitors based on a physico-chemical profile that was distinct from bortezomib. MLN9708 has a shorter 20S proteas- ome dissociation half-life than bortezomib, which plays an important role in its improved tissue distribution. Direct comparison with bortezomib revealed that MLN9708 has an improved pharmacokinetic and pharmacodynamic pro- file and shows superior antitumor activity in solid tumors, as shown previously.10
In this study, the antiproliferative and apoptotic effects of MLN2238 were demonstrated on Caco2 human colon cancer cells and compared directly with bortezomib. The cytotoxic effects were determined by WST-1 cell prolif- eration assay, and apoptotic effects were determined by flow cytometry. MLN2238 and bortezomib showed sig- nificant antiproliferative effects at similar concentrations. The highest cytotoxic effects were measured at 48 h with 20 μM MLN2238 and bortezomib.
Cancer cells evade apoptosis because of their disregu- lated apoptotic signaling pathways. The key factors con- trolling apoptosis, are regulated by the 26S proteasome
complex.39 Inhibition of the proteolytic functions of the 26S proteasome activates caspases and inhibits Nf-κB, leading to a broad spectrum of anti-proliferative and pro-apoptotic activities.40 In the present study, the apop- totic effects of MLN2238 and bortezomib in various con- centrations were compared by measuring the activation of caspase-3 and loss of mitochondrial membrane potential (JC-1) using flow cytometry. At the highest (20 μM) con- centration, MLN2238 and bortezomib increased caspase-3 levels 7.2- and 7.4- fold and showed a 1.7- and 1.9- fold increased loss of mitochondrial membrane potential, respectively.
NF-κB is a transcription factor involved in the acti- vation of the genes encoding for cytokines, growth fac- tors, chemokines, cell-adhesion molecules, and surface receptors.40,41 Inhibitor of kappa B (IκB) binds to NF-κB and inhibits the translocation of NF-κB to the nucleus for gene activation in the cytoplasm. External stimuli, including pathogens, stress, free radicals, and cytokines, initialize phosphorylation of IκB, and this phosphorylation induces polyubiquitylation of IκB for degradation by the 26S proteasome complex. The translocation of NF-κB to the nucleus that switches on the transcription of its tar- get genes is promoted by the proteasomal degradation of IκB. Uncontrolled upregulation of the NF-κB function would lead to various types of cancers, including colon cancer.42 Therefore, NF-κB is likely to have a prominent role in colorectal and colitis associated tumorigenesis. Abnormal NF-κB activation has been detected in 50% of colorectal- and colitis- associated tumors. Cancer cells with activated NF-κB are resistant to chemotherapeutic agents; inhibition of NF-κB activity greatly increases cell sensitivity to chemotherapy agents.43 Next to NF-κB, c-myc mRNA expression increases during cancer forma- tion. The proto-oncogene c-myc encodes a transcription factor c-myc. The c-myc oncogene is a ‘master regulator’ that controls many aspects of cellular growth regulation and vitality.44 Drug treatments that cause apoptotic death of several cell types have been shown to greatly lower c-myc expression.45 In this study, the effects of MLN2238 and bortezomib on NF-κB and c-myc mRNA expression levels were compared. The expression of both genes was down- regulated with bortezomib and MLN2238 at the highest concentration. The downregulation of NF-κB and c-myc expression indicates a decrease in cell viability and acti- vation of apoptosis. Two in vitro studies with bortezomib showing inhibition of NF-κB on different cell lines,46,47 but to our knowledge, our study is the first study to prove a decrease in NF-κB and c-myc mRNA expression levels in the Caco2 cell line with MLN2238 and bortezomib.
In summary, the data of the present study show that MLN2238 has antiproliferative and apoptotic effects similar to bortezomib. In conclusion, we believe that our results may open the path to treatment of colon cancer with MLN2238 and other novel second generation pro- teasome inhibitors. Our results show that proteasome
inhibitors have the potential to inhibit uncontrolled growth of colorectal cancer cells. After positive in vivo experi- ments with MLN2238 treatment, clinical trials with colon cancer patients would be suggested.
The flow cytometry analysis of this research was studied in the Anadolu University Medicinal Plants, Drugs, and Scientific Research Center.
Disclosure statement Contributors
SE conceived and designed the study, collected and ana- lysed the data and revised the article. MD conceived and designed the study, obtained funded, and revised the article.
This study was carried out as a part and support of Anadolu University Scientific Research [Project number 1207S120].
Conflict of interest
No potential conflict of interest was reported by the
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