Comparison of Anticancer Activity of Dorycnium Pentaphyllum Extract on MCF-7 and MCF-12A Cell Line: Correlation with Invasion and Adhesion

biomolecules

Article Comparison of Anticancer Activity of Dorycnium pentaphyllum Extract on MCF-7 and MCF-12A Cell Line: Correlation with Invasion and Adhesion

Gözde Koygun 1 , Emine Arslan 2,*, Gökhan Zengin 2 , Giustino Orlando 3 and Claudio Ferrante 3

1 Department of Nanotechnology and Advanced Materials, Selcuk University, Konya 42130, Turkey; [email protected] 2 Department of Biology, Science Faculty, Selcuk University, Konya 42130, Turkey; [email protected] 3 Department of Pharmacy, Università degli Studi “Gabriele d’Annunzio”, Via dei Vestini 31, 66100 Chieti, Italy; [email protected] (G.O.); [email protected] (C.F.) * Correspondence: [email protected]

Abstract: Dorycnium pentaphyllum subsp. haussknechtii is an important medicinal plant in several countries, including Turkey. This study aimed to evaluate the cytotoxicity of a crude extract of D. pentaphyllum subsp. haussknechtii against different breast cell lines to determine invasion, adhesion, and lipid peroxidation. The cytotoxic effects on MCF-7 breast cancer and MCF-12A as the immortalized cell line were examined by the XTT assay. Invasion and adhesion studies were per-

formed according to the manufacturer’s kit procedure to IC50 values for 48 h. Lipid peroxidation was measured in the MCF-7 cell. A bioinformatics analysis was conducted to unravel the mechanism of action underlying antiproliferative effects, as well. According to XTT results, the tested extract showed a time- and a concentration-dependent cytotoxic effect. The most effective concentration was Citation: Koygun, G.; Arslan, E.; 100.5 µg/mL (48 h), which was selected for biological activities, such as apoptotic activity, invasion, Zengin, G.; Orlando, G.; Ferrante, C. adhesion, and lipid peroxidation assays. The extract caused tumoral cell death, and it did not have a Comparison of Anticancer Activity of cytotoxic effect on healthy human breast cells. Duplication times and measurement of CI analyses of Dorycnium pentaphyllum Extract on cells were performed using the real-time cell analysis system xCELLigence. Finally, the bioinformatics MCF-7 and MCF-12A Cell Line: analysis indicated the prominent role of quercetin as an extract component exerting a key role in the Correlation with Invasion and observed antiproliferative effects. This was supported by the micromolar/submicromolar afﬁnity of Adhesion. Biomolecules 2021, 11, 671. quercetin towards proto-oncogene serine/threonine–protein kinase (PIM-1) and hematopoietic cell https://doi.org/10.3390/biom11050671 kinase (HCK), both involved in breast cancer. Altogether, our ﬁndings proposed that the extraction of

Academic Editor: Vijai Kumar Gupta the plant can be an effective strategy to isolate biomolecules with promising cytotoxic effects against breast cancer cells. Received: 4 March 2021 Accepted: 27 April 2021 Keywords: Dorycnium pentaphyllum; cancer; MCF-7; MCF-12A; invasion; bioinformatics Published: 30 April 2021

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional afﬁl- It has been estimated that 3.5 million cancer-related deaths are annualized world- iations. wide [1]. Although chemotherapy aims to kill cancer cells from a patient’s body, on the other hand, it also affects normal and healthy cells causing severe side effects, which in turn, results in multiple organ dysfunction [2,3]. Breast cancer is one of the most prevalent types of cancer in women all over the world. Along with lung and cervix cancers, this Copyright: © 2021 by the authors. type of cancer is one of the most common types worldwide. According to statistics, one Licensee MDPI, Basel, Switzerland. out of every eight women has breast cancer at certain times of her life. Treatment options This article is an open access article may include one or more, depending on the stage of the disease, the characteristics of the distributed under the terms and patient, and the general health. In addition to methods, such as surgery, radiation therapy, conditions of the Creative Commons hormone therapy, chemotherapy, targeted therapy, or herbal treatments are very popular Attribution (CC BY) license (https:// and are continuing to develop rapidly [4]. The generation of invasiveness in transformed creativecommons.org/licenses/by/ cells represents a required step of carcinoma progression [5]. Tumorigenic cell invasion is 4.0/).

Biomolecules 2021, 11, 671. https://doi.org/10.3390/biom11050671 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 671 2 of 17

accompanied by various changes in cellular phenotype, which includes alterations in mor- phology, proteolytic activity and motility of the invasive cells [6]. Furthermore, a dramatic increase in the mobility of transformed epithelial cells can be observed during the adhesion test of carcinoma cells, contrasting with the low mobility of cells in normal epithelia. In these, cell migration is restricted by the different kinds of junctional organelles, which tightly bind the cells together and thus immobilize individual cells. Thus, disruption of intercellular adhesion may be a prerequisite for releasing carcinoma cells from the epithelia and a key event during progression towards a fully malignant invasive cell [7]. Oxidative stress can occur in almost any tissue, and oxidative stress is known to play a master role in carcinogenesis [8]. In the normal biological process, there is a delicate balance between the formation of reactive oxygen species (ROS) and antioxidant substances. Low ROS level has a protective role in the cell under physiological conditions. However, the high accumulation of ROS within the cell can result in oxidative stress, leading to damage, such as genomic instability, and neoplastic transformations occur in DNA, thus leading to cancer [9,10]. During oxidative stress, superoxide anions, hypochlorite radicals, singlet oxygen, hydrogen peroxide, hydroxyl radicals, nitric oxide radicals, and lipid peroxides all increased in the cell. These increased substances react with membrane lipids, nucleic acids, proteins, enzymes and other small molecules to produce cellular damage [11]. Nat- ural products have long represented a valuable source of compounds with promising applications in cancer chemotherapy due to their medicinal effectiveness, low toxicity, and many natural anticancer agents derived from different medicinal plants [12–15], that are usually rich in phenolic compounds, which could mediate, albeit partially, traditional and innovative pharmacological applications [16]. Chemoprevention by renewable phyto- chemicals could also be considered low-priced, quickly feasible, admissible, and sensible to cancer control and management. In the last years, research also deals with studying biochemical and therapeutic routes, appertaining to prevalent approaches due to natural anticancer agents in clinical practice, new candidate oncological therapy drugs from herbals, microorganisms, and promising chemopreventive agents from them [17]. Dorycnium pentaphyllum, a perennial herb of 10–80 cm height belonging to the Dorycnium genus of the Fabaceae family, is commonly found in central and southeast Europe [18]. Eth- nobotanical data indicated that D. pentaphyllum was used as an anti-hemorrhoidal [19] and antidiarrheal [20]. D. pentaphyllum was previously reported to exhibit antibacterial activity [21]. D. pentaphyllum was also found to inhibit PGE2, an inflammatory mediator, in human mono- cytes [19]. Studies have reported the presence of kaempferol, quercetin, myricetin [22], dorycnioside, kaempferol 3-O-(6”-acetyl)-β-glucopyranoside, (+)-dihydromyricetin, (−)-catechin, β-sitosterol, lotaustralin, gallic acid methyl ester, tachioside, d-pinitol, and phenylbutanone derivatives [18] in D. pentaphyllum. Many studies have investigated the antimicrobial, antibiofilm, and antioxidant potentials of D. pentaphyllum [23,24]. In the literature, two recent papers observed on cytotoxic properties of D. pentaphyllum extracts. Demir et al. [25] were reported the cytotoxic effect of this plant on human breast, liver, and lung cancer cells, and they reported IC50 values as 100.4 to 298.5 µg/mL in the tested cancer cell lines. In second study conducted by Demir et al. [26], the cytotoxic effect was investigated on the human cervix (HeLa), and colon (WiDr) cancer cells and IC50 values were found to be 46.5 and 84.5 µg/mL, respectively. However, there is still lack in scientific literature about the cytotoxic, antiproliferative effects and mechanistic approaches of this plant for inhibiting tumor growth of various cancer cell lines, including breast. In this regard, in the present study, we aimed to investigate the anticancer effect and mechanism of action of D. pentaphyllum extract against human breast carcinoma cell line (MCF-7) and compare the effects with the immortalized breast cell line (MCF-12A). Most cancer cell types are thought to have improved adhesion ability, making it easier for cells to migrate to a new site to create new tumors in the body. In addition, we preferred to use adhesion and invasion assays, which can also be used to assess the effect of the extract treatments on the MCF-7 cell line to mimic the metastatic ability of cancer cells. The results of the cell adhesion assay permitted us to examine the interactions between the cells and Biomolecules 2021, 11, 671 3 of 17

extracellular materials herein. Finally, bioinformatics analysis was conducted to unravel the mechanism of action underlying antiproliferative effects.

2. Materials and Methods 2.1. Preparation of Plant Extract The material (aerial parts of D. pentapyllum subsp. haussknechtii) for the extraction was collected in the area of Kahramanmara¸s(Andırın) (Turkey) in summer 2015. Taxonomic identiﬁcation was performed by the botanist Dr. Murad Aydin Sanda (Mus Alpaslan University, Department of Molecular Biology, Mus, Turkey). The grinding of naturally dried aerial parts of the plant was carried out by a mill in a laboratory. The extract was obtained by Soxhlet apparatus, and the procedure was given in our earlier paper [24]. The batch-to-batch consistency of the extract was being realized by mixing three different batches of extract.

2.2. Cell Culture Immortalized human breast cell line, MCF-12A (ATCC® CRL-10782™), was grown in DMEM-F12 medium (ThermoFischer, Waltham, USA) supplemented with 10% FBS (fetal bovine serum), 0.1% pen-strep, 1% NEAA (nonessential amino acids), 20 ng/mL of EGF(epidermal growth factor), 500 ng/mL of hydrocortisone, and 0.01 mg/mL human insulin. The MCF-7 breast cancer cell line (ATCC® HTB-22™) was obtained from Ameri- can Type Culture Collection (Rockville, MD, USA). They were cultured with RPMI-1640 (BioChrom, Berlin, Germany) medium containing 10% FBS (Fetal Bovine Serum Biological Industries) (Sigma Aldrich, Milan, Italy) and 0.1% gentamicin sulfate solution (Sigma Aldrich, Milan, Italy). Both cell lines were kept under sterile conditions at 37 ◦C with 5% CO2 and 95% air, also subcultured weekly using 0.02% EDTA and 0.05% trypsin. Cells proliferating in cultures gradually lose their proliferation rate after consuming a high rate of nutrient agents in the cell culture medium or using the entire surface in which they can reproduce, and cell growths and deaths occur over time. In this period, the division of culture is the most accurate process to be done, and this process is called passaging [27]. The media were changed every 2–3 days.

2.3. Detection of Cell Viability by XTT Assay The cell proliferation was determined by using a standard 2,3-bis [2-methoxy-4-nitro- 5-sulfophenyl]-2H-tetrazolium5-carboxanilide inner salt (XTT) assay kit, which measures the metabolic activity of viable cells. In brief, the tested extract was horizontally diluted, respectively, from a high dose (5000 g/mL) to a low dose (9.75 low µg/mL). The extract was dissolved in DMSO (1%), and the control group contained just DMSO (without extract). MCF-7 and MCF-12A cells (1 × 105 cells/well) were harvested from the exponential phase maintenance cultures, counted via Trypan blue exclusion assay, and seeded in 96-well plates in 50 µL volume per well by using a multichannel pipette. These procedures were repeated for 24, 48 and 72 h to see the changing of the extract how the effect on cell cultures. The assay is based on the cleavage of the tetrazolium salt XTT to form an orange soluble formazan dye by mainly mitochondrial dehydrogenase activity in living cells. Formazan formation was quantiﬁed spectrophotometrically at 490 nm using an ELISA reader (Biotek, Swindon, UK). Four replicates of each concentration were used in each assay and repeated three times. The results were compared and evaluated in terms of the response of breast cancer cells (MCF-7) and immortalized human cells (MCF-12A) for treatments.

2.4. RTCA System Measurements By trypsinization, cells were harvested from exponential phase cultures, counted and then plated in 48-well plates. Sub-concentrations were prepared in serial dilutions from the stock solution. 50 µL of the medium was added to the e-plate wells. The cells used (MCF-7/MCF-12A) were inoculated in 50 µL of medium to contain 104 cells in 50 µL, and the e-plate was incubated at room temperature for 30 min. Then the e-plate was placed in Biomolecules 2021, 11, 671 4 of 17

the plate area of the real-time cell analysis system (RTCA) device in the incubator. When the cell index (CI) measured by the device reaches 1, add different concentrations of the extract (assay concentration 100 µg/mL; 50 µg/mL, 25 µg/mL, and 12.5 µg/mL to the wells). 50 µL of medium containing 1 mL) was added. Just medium (with serum) was added to the control wells. After adding the tested extract, the experiment followed until the logarithmic phase on the CI-time graph was completed, and the CI values were measured at this time. To clarifying the cells’ response to the extract, the cells were observed every 60 min after the addition of the extract. Data analysis was performed with the RTCA software on the computer of the xCELLigence system. Each concentration was examined 3 times within the same experiment. General cell culture conditions and culture medium used for this method were similar to those applied for the xCELLigence counterpart experiments and applied cell densities (10,000 cells/cm2). Cell doubling time was calculated from the exponential phase of the growth curve.

2.5. Matrigel Invasion Assay An invasion assay was treated by using 24well (8 µm pore size) Transwell plates (EZcellTM cell invasion assay kit; Corning, Inc. Corning, NY, USA). MCF-7 cells were plated in the upper chambers in RPMI 1640 (serum-free) media at 15 × 104 cells/well, which were pre-coated with collagen I., while the extract was maintained in the bottom chamber in RPMI 1640 (serum-free). After 48 h of incubation, MCF-7 cells were detected by crystal violet staining at room temperature for 30 min, and all the results were observed using inverted light microscopy (magniﬁcation, ×10 and ×40). Carefully aspirate the medium from the inside of the insert and gently swab the interior of the inserts to remove non-invasive cells. After cleaning the chambers, we added 400 µL of cell stain solution and incubate for 10 min at room temperature. For each well invasive cells were counted with a light microscope under a high magniﬁcation objective.

2.6. Adhesion Assay MCF-7 breast cancer adhesion assay was performed by using a Vybrant™ cell adhesion assay kit (V-13181; Life Technologies, Grand Island, NY, USA) as described by the manufacturer. We confirmed this correlation by examining the MCF-7 breast cancer, which was treated with different concentrations of the tested extract and control groups. Briefly, 5 × 105 MCF-7 breast cancer cells were seeded into a 96-well plate for two days. Cells were treated with or without extract at a 50% growth inhibitory concentration (IC50); 100.5 µg/mL. Also, 25 and 50 µg/mL concentrations of the extract were chosen where the cell viability was higher than the percentage of seventy. After 48 h incubation, MCF-7 cells were labeled with calcein-AM. They were co-cultured with the breast cancer cells for 2 h at 37 ◦C. After the indicated period, non-adherent cells were removed, and then calcein-AM measured the fluorescence using a fluorescein filter set (calcein has an absorbance maximum of 494 nm and an emission maximum of 517 nm) to calculate the number of adherent cells.

2.7. Lipid Peroxidation Two different procedures were used for lipid peroxidation analysis. First, MDA (malondialdehyde) determination: The determination of MDA, an in vitro marker of lipid peroxidation, was determined according to the method proposed by [28]. According to this method, MCF-7 cells were treated with extract for 48 and 72 h, and 1 M HCl was added to cell lysates, followed by the addition of thiobarbituric acid (TBA) reagent and the resulting mixture was incubated at 95 ◦C. At the end of incubation, the absorbance of MDA was measured at 535 nm and 600 nm. The results obtained were corrected by subtracting nonspeciﬁc turbidity at 600 nm. Data are expressed as nmol/mg protein [28]. The second method, reduced glutathione (GSH) determination: GSH analysis, was performed using the method proposed by [29]. In this method, all proteins containing sulfhydryl in the cell lysates were precipitated with precipitation solution. The resulting sulfhydryl groups were treated with Ellman’s reagent [DTNB (5,50-2-dithiobis nitrobenzoic Biomolecules 2021, 11, x 5 of 18

added to cell lysates, followed by the addition of thiobarbituric acid (TBA) reagent and the resulting mixture was incubated at 95 °C. At the end of incubation, the absorbance of MDA was measured at 535 nm and 600 nm. The results obtained were corrected by subtracting nonspecific turbidity at 600 nm. Data are expressed as nmol/mg protein [28]. The second method, reduced glutathione (GSH) determination: GSH analysis, was performed using the method proposed by [29]. In this method, all proteins containing sulfhydryl in the cell lysates were precipitated with precipitation solution. The resulting sulfhydryl groups were treated with Ellman’s reagent [DTNB (5,5′-2-dithiobis nitroben-

Biomolecules 2021, 11, 671 zoic acid)], and the absorbance of the resulting yellow color at 412 nm was measured.5 of The 17 values obtained will be expressed in mg/100 mL [29].

2.8. Bioinformatics acid)],Putative and the targets absorbance were identified of the resulting according yellow to the color bioinformatic at 412 nm was method measured. recently The de- valuesscribed obtained [30,31]. willBriefly, be expressed human proteins in mg/100 targeted mL [29 by]. the extract phytocompounds were predicted using bioinformatics platform STITCH. Docking calculations were conducted 2.8. Bioinformatics through the AutoDock Vina of PyRx 0.8 software. Crystal structures of target proteins werePutative derived targets from the were Protein identified Data accordingBank (PDB) to with the bioinformatic PDB IDs as follows: method 3BGZ recently (proto de-- scribedoncogene [30 , serine/threonine31]. Briefly, human–protein proteins kinase targeted (PIM- by1)) the, 1QFC extract (hematopoietic phytocompounds cell kinase were predicted(HCK)). Discovery using bioinformatics studio 2020 platformvisualizer STITCH. was employed Docking to investigate calculations the were protein conducted–ligand throughnon-bonding the AutoDock interactions. Vina of PyRx 0.8 software. Crystal structures of target proteins were derived from the Protein Data Bank (PDB) with PDB IDs as follows: 3BGZ (proto- oncogene2.9. Statistical serine/threonine–protein Analysis kinase (PIM-1)), 1QFC (hematopoietic cell kinase (HCK)). Discovery studio 2020 visualizer was employed to investigate the protein–ligand non-bondingStatistical interactions. significance between the control and treated MCF-7 cells and MCF12 A human immortalized cells were evaluated by using Student’s t-test to determine differ- 2.9.ences Statistical between Analysis mean values for normal distribution. All analyzed results were calculated withStatistical GraphPad significance Prism 6 Software between (San the Diego, control CA and, USA); treated a probability MCF-7 cells level and <0.05 MCF12 was con- A humansidered immortalized significant throughout cells were the evaluated analysis. byExper usingiments Student’s were repeatedt-test to at determine least four differ- times. encesContinuous between variables mean values are given for normal as mean distribution. ±1 standard All deviation analyzed (SD). results were calculated with GraphPad Prism 6 Software (San Diego, CA, USA); a probability level <0.05 was considered3. Results significant throughout the analysis. Experiments were repeated at least four times.3.1. Cytotoxicity Continuous Assay variables on MCF are-7 givenand MCF as mean-12A Cells±1 standard deviation (SD). Figure 1a,b the MCF-7 breast cancer and MCF-12A breast cell lines, which were ex- 3. Results posed to the extracts to evaluate antiproliferative effects. Specifically, the XTT test was 3.1.used Cytotoxicity to determine Assay the on cytotoxicity MCF-7 and effects MCF-12A of the Cells extract in the range 9.765–5000 µg/mL. As showFiguren in Figure1a,b the 2, after MCF-7 24 breasth of exposition cancer and to extracts, MCF-12A the breast extract cell anti lines,-proliferative which were effect exposedwas evident to the in extractsMCF-7 cells to evaluate with an antiproliferativeIC50 value of 78.69 effects. µg/mL. Specifically, In MCF-12A the cells, XTT the test extract was useddid not to determinedetermine thea viability cytotoxicity fall below effects 80%, of the thus extract resulting in the biocompatible range 9.765–5000 (p <µ 0g/mL..0001). AsAfter shown 48 and in Figure72 h of2, treatment, after 24 h ofthe exposition XTT results to showed extracts, that the extractthe extract anti-proliferative IC50 value was effect100.5 wasµg/mL evident and 20.08 in MCF-7 µg/mL, cells respectively. with an IC50 Onvalue the ofother 78.69 hand,µg/mL. no toxic In MCF-12A effect was cells, ob- theserved extract in MCF did- not12A determine cells after 48 a viabilityand 72 h fallexposition below 80%,to the thus extract. resulting biocompatible (p < 0.0001)The results. After of 48 the and XTT 72 hassay of treatment, showed that the XTTthe extract results induced showed thattime- the and extract concentra- IC50 valuetion-dependent was 100.5 µ cytotoxicg/mL and effects 20.08 onµg/mL, the MCF respectively.-7 cell line, On whereas the other MCF hand,-12A no cells toxic were effect un- wasaffected observed by any in concentration MCF-12A cells of after the 48extract. and 72 h exposition to the extract.

Figure 1. (a) Microscopic view of MCF-7 breast cancer cell cultures. (b) MCF-12A immortalized human breast cell cultures (Leica, 10× objective). The results of the XTT assay showed that the extract induced time- and concentration- dependent cytotoxic effects on the MCF-7 cell line, whereas MCF-12A cells were unaffected by any concentration of the extract.

3.2. RTCA System Results After adhering to the cells-plate surface, different concentrations of the extract prepared in the medium were applied to the cells and followed for 3 more days. In Figure3a, the graph showing the duplication time of the extract is given on the MCF12 A with immortalized cell lines and on the MCF-7 breast cancer cell line in Figure3b. Biomolecules 2021, 11, x 6 of 18

Biomolecules 2021, 11, 671 6 of 17 Figure 1. (a) Microscopic view of MCF-7 breast cancer cell cultures. (b) MCF-12A immortalized human breast cell cultures (Leica, 10× objective).

a. b.

FigureFigure 2. Effect 2. Effect of the extract of the on extract the viability on the of MCF-7viability and of MCF-12A MCF-7 cellsandassessed MCF-12A with cells the XTT assessed assay after with (a )the 24, (XTTb) 48 and (c) 72 h of treatment graph. assay after (a) 24,(b) 48 and (c) 72 h of treatment graph. In cell culture studies with MCF-7 and MCF-12A cells models, Dorycnium extract 3.2. RTCA System concentrationsResults less than 50 µg/mL (50, 25, and 12.5 µg/mL) were found to be safe in terms of cytotoxicity and duplication time. All data recorded using the xCELLigence RTCA After adheringsystem to the were cells processed-plateusing surface, MS Excel different to obtain concentrations data series with of the the same extract resolution. pre- This pared in the mediumwas performedwere applied by selecting to the onlycells the and xCELLigence-generated followed for 3 more values days. corresponding In Figure 3 toa, the the graph showingtime the points duplication used in the time reference of the methods, extract thus is given leading on to the a reconstruction MCF12 A ofwith the studiedim- mortalized cell linesprocess and dynamics on the MCF at a lower-7 breast time resolution.cancer cell line in Figure 3b. In cell culture studiesBased with on the MCF obtained-7 and graphs, MCF the-12A tested cells extract models, exhibited Doryc anium cytotoxic extract effect con- with a concentration of 50 µg/mL and above. The concentrations of 12.5 and 25 µg/mL did not centrations less thanexhibit 50 µg/mL cytotoxic (50, effects, 25, andand these 12.5 concentrations µg/mL) were were found the sameto be as safe control. in terms In addition, of cytotoxicity and duplicationduplication times time. were All calculateddata recorded for each using cell line, the andxCELLigence the results areRTCA given system in Table 1. were processed usingAccording MS Excel to the duplicationto obtain times,data series <25 µg/mL with concentrations the same resolution. were found This as safe was on the performed by selectingMCF-12A only cell the line. xCELLigence-generated values corresponding to the time points used in theTable reference 1. MCF-7 methods, and MCF-12A thus duplication leading time to chart.a reconstruction of the studied process dynamics at a lower time resolution. Well Doubling Time (h) ± Standard Deviation (SD) Based on the obtainedMCF-12A graphs, cell the control tested extract exhibited a cytotoxic35.66 ± 3.5 effect with a concentration of 50 µg/mLMCF-12A—50 and above.µg/mL The concentrations of 12.5 and23.42 ±250.37 µg/mL did not exhibit cytotoxic effects, andMCF-12A—25 these concentrationsµg/mL were the same as28.45 control.± 2.72 In addition, duplication times were calculatedMCF-12A—12.5 forµ g/mLeach cell line, and the results31.57 are± given3.80 in Table 1. ± According to the duplicationMCF-7 times, cell control<25 µg/mL concentrations were46.53 found2.97 as safe on the MCF-7—50 µg/mL 29.43 ± 0.74 MCF-12A cell line. MCF-7—25 µg/mL 34.74 ± 1.12 MCF-7—12.5 µg/mL 39.94 ± 1.54 Biomolecules 2021, 11, 671 7 of 17

Biomolecules 2021, 11, x 7 of 18

(a)

(b) 50 45 40 35 30 25 20 15 Doubling Time (hr) Doubling 10 5 0 cell control 50 μg / mL 25 μg / mL 12.5 μg / mL

MCF-7 MCF-12A

(c)

Figure 3. Normalized cell index (a,b) and duplication graph (c) of the effect of Dorycnium on MCF-7 breast cancer cells and MCF12 A human immortalized cells.

3.3. Role of the Extract on MCF-7 Breast Cancer Cells for Cell Differentiation and Invasion MCF-7 control group cells are highly invasive compared with MCF-7 cells with different concentrations of the extract. An EZ TM cell invasion (Matrigel) assay was used to determine the invasion potentials of the breast cancer cell line with the extract. Cells were seeded into either a porous 8 µm membrane insert (control) or Matrigel-layered membrane insert and incubated over its 50% growth inhibitory concentration (IC50) and two more different doses of extract concentrations for 48 h. Cells were ﬁxed on the membrane, stained, photographed, and counted. Representative images of cells that invaded through the Matrigel layer (Figure4) three concentrations of the extract were counted and graphed for cell invasion assay. Biomolecules 2021, 11, 671 8 of 17 Biomolecules 2021, 11, x 9 of 18

Figure 4. Cont. Biomolecules 2021, 11, 671 9 of 17 Biomolecules 2021, 11, x 10 of 18

Figure 4. Cells were then incubated for 48 h. Photographs were taken at 0 h (A,C,E,G), and 48 h Figure 4. Cells were then incubated for 48 h. Photographs were taken at 0 h (A,C,E,G), and 48 h (B,D,F,H) using inverted microscope with 10× magnification and the fluorescent microplate reader × absorbance(B,D,F,H) using invasion inverted results microscope of MCF-7 withcells treated 10 magnification with different and concentration the fluorescent of microplateDorycnium readerex- tractabsorbance (I) **** p invasion < 0.0001, results *** p < of 0.001 MCF-7 vs. cellscontrol treated group with (CC different). concentration of Dorycnium extract (I) **** p < 0.0001, *** p < 0.001 vs. control group (CC). 3.4. Effect of Cell Adhesion Secretion on MCF-7 CELLS We characterized the invasive phenotype of MCF-7 cells by comparing them to MCF-7 cellsThe with ability three differentof malignancy extract to concentrations invade and metastasize to the control largely group. depends The highest on the percentage degree ofof epithelial cell death differentiation for the extract within was the observed tumors, after i.e., defectively 48 h incubation, segregate and being its 50% more growth invasive than well-segregated malignant tumor [32]. The conditions for MCF-7 cell adhesion inhibitory concentration (IC50) was 100.5 µg/mL. The extracts were pretty effective on the assayMCF-7 were breast examined cancer cell in 96 line.-well 100 cellµg/mL culture extract-treated treated with MCF-7 different breast concentration cancer cell lines of wasthe extractobserved in microplates to be highly for invasive an effort compared to determine to the rapid, MCF-7 cost control-effective group. methods The effect to identify of the potential therapeutic compounds. After harvesting, dilutions of the extract (200 µL, 100 extract (100 µg/mL) (IC50value) concentration results was found to be 5-times less invasive µLfor and collagen 50 µL gels) were than the seeded control in group. MCF-7 cell cultures per 96 wells, respectively. The Vybrant™ Cell Adhesion Assay was used to assess the effect of the quantity of how the extract3.4. Effect effected of Cell on Adhesion MCF-7 Secretioncells seeded on MCF-7 on the CELLSnumber of adherent cells at 48 h incubation. This suggestsThe ability that of decreasing malignancy the to concentration invade and metastasize of the extract largely MCF depends-7 cells onseeded the degree leads to of anepithelial increased differentiation number of adherent within the cells tumors, (Figure i.e., 5). defectively segregate being more invasive thanTwo well-segregated fields of view malignant were counted tumor in [32 each]. The well conditions (20× magnification) for MCF-7 cell, shown adhesion in Figure assay 6.were Experiments examined were in 96-well conducted cell culture a coupl treatede of times, with both different in triplicate. concentrations Our data of showed the extract that in itmicroplates expressed as for the an absolute effort to determinenumber of rapid,cells adhered cost-effective to the methodsendothelial to identifylayer and potential as the percentagetherapeutic of compounds. cells adhered After relative harvesting,ly to control. dilutions of the extract (200 µL, 100 µL and 50 µInL) addition were seeded to evaluating in MCF-7 adhesion cell cultures for control per 96 wells,and the respectively. extract applied The to Vybrant MCF-7™ cells,Cell assaysAdhesion were Assay also performed was used toat assessapplying the only effect tumor of the cells quantity with the of how extract the (200 extract µL, effected100 µL andon MCF-750 µL) cellsfor 48 seeded h (Figure on the6). number of adherent cells at 48 h incubation. This suggests that decreasing the concentration of the extract MCF-7 cells seeded leads to an increased number of adherent cells (Figure5). Two fields of view were counted in each well (20× magnification), shown in Figure6. Experiments were conducted a couple of times, both in triplicate. Our data showed that it expressed as the absolute number of cells adhered to the endothelial layer and as the percentage of cells adhered relatively to control. In addition to evaluating adhesion for control and the extract applied to MCF-7 cells, assays were also performed at applying only tumor cells with the extract (200 µL, 100 µL and 50 µL) for 48 h (Figure6). Biomolecules 2021, 11, x 11 of 18

Biomolecules 2021, 11, 671 10 of 17 Biomolecules 2021, 11, x 11 of 18

Figure 5. Representative photomicrographs of tumor cell adhesion.(A) MCF-7 cell control group. The Vybrant™ cell adhesion assay was used to assess the effect of the quantity of how dilutions of the tested extracts (B) 200 µL, (C) 100 µL and (D) 50 µL) effected on MCF-7 cells seeded on the number of adherent cells at 48 h incubation.

As a result of adhesion analysis using the Vybrant cell adhesion analysis kit, application of the extract at concentrations of 50 µM, 100 µM 200 µM in MCF-7 cell line was decreased 82.4%, 50% and 27.2%, respectively compared to the control group. A significant result was determined at an IC50 concentration of 100 µM. These findings indicate that using the extract can generate dedifferentiation and invasiveness of MCF-7 cells, and they suggest further that cytotoxic concentration of the extract acts as an invasion suppressor. In addition, quantification of tumor cell adhesion Figure 5. Representative photomicrographs of tumor cell adhesion.(A) MCF-7 cell control group. strengthFigure 5. couldRepresentative operate as photomicrographs a predictor of the of metastatic tumor cell potential adhesion.( ofA) a MCF-7 solid tumor. cell control However, group. The Vybrant™ cell adhesion assay was used to assess the effect of the quantity of how dilutions of The Vybrant™ cell adhesion assay was used to assess the effect of the quantity of how dilutions of the thethe obtainedtested extracts results (B) 200must µL , confirm(C) 100 µL by and gene (D) 50 and µL ) protein effected expressionon MCF-7 cells analysis seeded, onand the we µ µ µ stronglytestednumber extracts of suggested adherent (B) 200 cells thatL, (atC further )48 100 h incubation L analysis and (D). 50 needL) aeffected clear onexplanation MCF-7 cells of seeded this case on the, and number our findingsof adherent could cells provide at 48 h incubation. a light in the road. As a result of adhesion analysis using the Vybrant cell adhesion analysis kit, application of the extract at concentrations of 50 µM, 100 µM 200 µM in MCF-7 cell line was decreased 82.4%, 50% and 27.2%, respectively compared to the control group. A significant result was determined at an IC50 concentration of 100 µM. These findings indicate that using the extract can generate dedifferentiation and invasiveness of MCF-7 cells, and they suggest further that cytotoxic concentration of the extract acts as an invasion suppressor. In addition, quantification of tumor cell adhesion strength could operate as a predictor of the metastatic potential of a solid tumor. However, the obtained results must confirm by gene and protein expression analysis, and we strongly suggested that further analysis need a clear explanation of this case, and our findings could provide a light in the road.

Figure 6. Cell adhesion results of the extract at concentrations of 50 µg/mL, 100 µg/mL, 200 µg/mL in MCF-7 cell line. Statistical analysis was performed using GraphPad Prism 9, using a two-way ANOVA with Tukey’s multiple comparison posttest. Only signiﬁcant differences are displayed: ** p < 0.01, **** p < 0.0001.

Biomolecules 2021, 11, 671 11 of 17

Biomolecules 2021, 11, x 12 of 18

These findings indicate that using the extract can generate dedifferentiation and Biomolecules 2021, 11, x 12 of 18 invasiveness of MCF-7 cells, and they suggest further that cytotoxic concentration of the Figureextract 6. Cell acts adhesion as an invasionresults of suppressor.the extract at Inconcentrations addition, quantification of 50 µg/mL, 100 of tumorµg/mL, cell 200adhesion µg/mL in MCFstrength-7 cell couldline. Statistical operate asanalysis a predictor was performed of the metastatic using GraphPad potential Prism of a 9, solid using tumor. a two However,-way ANOVA with Tukey’s multiple comparison posttest. Only significant differences are displayed: ** Figurethe 6. Cell obtained adhesion results results must of the confirm extract by at gene concentrations and protein of 50 expression µg/mL, 100 analysis, µg/mL, and200 µg/mL we strongly p < 0.01, **** p < 0.0001. in MCFsuggested-7 cell line. that Statistical further analysis analysis was need performed a clear explanationusing GraphPad of this Prism case, 9, using and our a two findings-way could ANOVAprovide with Tukey’s a light inmultiple the road. comparison posttest. Only significant differences are displayed: ** p 3.5.< 0.01, Effect **** ofp < the 0.0001 Extract. on MDA and GSH Levels 3.5.Regarding Effect of thethe Extract activity on of MDA extract and on GSH MDA Levels and GSH levels in MCF-7 cells, it was ob- 3.5.served Effect a ofRegarding significant the Extract the increaseon activityMDA ofand ofMDA GSH extract levelsLevels on 48 MDA and and72 h GSH following levels treatment in MCF-7 (Figure cells, it 7 was). However,Regardingobserved the aextract the significant activity significantly increaseof extract reduced of on MDA MDA GSH levels and 48 48hGSH following and levels 72 h in followingtreatment, MCF-7 cells,treatmentwhereas it was the(Figure ob- levels7 ) . servedof GSHHowever, a weresignificant unchanged the extractincrease compared significantly of MDA to levelsthe reduced control 48 and GSHgroup 72 48h 72 following h h following after the treatment stimulation treatment, (Figure (Figure whereas 7). 8). the However,levels the of extract GSH were significantly unchanged reduced compared GSH 48 to h the following control treatment, group 72 hwhereas after the the stimulation levels of GSH(Figure were8 unchanged). compared to the control group 72 h after the stimulation (Figure 8).

Figure 7. Effects of the extract (37.5–150 µM) on MDA levels in MCF-7 cells 48 and 72 h following

treatment. **** p < 0.0001, *** p < 0.001, ** p < 0.05 vs. control group (CC). FigureFigure 7. Effects 7. Effects of the ofextract the extract (37.5–150 (37.5–150 µM) onµ M)MDA on levels MDA in levels MCF in-7 MCF-7 cells 48 cells and 4872 andh following 72 h following treatment.treatment. **** p ****< 0.0001p < 0.0001,, *** p <*** 0.001,p < 0.001,** p < 0.05** p

Figure 8. Effects of the extract (37.5–150 µM) on GSH levels in MCF-7 cells 48 and 72 h following

treatment. **** p < 0.0001 vs. control group (CC). Figure 8. Effects of the extract (37.5–150 µM) on GSH levels in MCF-7 cells 48 and 72 h following 3.6. Bioinformatics treatment. **** p < 0.0001 vs. control group (CC). Figure 8. EffectsThe resultsof the extract of the (37.5 target–150 prediction µM) on GSH yielded levels byin MCF the STITCH-7 cells 48 platformand 72 h following are reported in treatment. **** p < 0.0001 vs. control group (CC). 3.6. FigureBioinformatics8 and highlight the prominent position of quercetin in the network pharmacology analysis. It is interesting that quercetin was predicted to interact with PIM-1 and HCK 3.6. BioinformaticsThe results of the target prediction yielded by the STITCH platform are reported in proteins involved in breast cancer. The capability of quercetin to interact with these proteins Figure 8 and highlight the prominent position of quercetin in the network pharmacology Thewas results also demonstrated, of the target albeitprediction partially, yielded by docking by the STITCH runs yielding platform afﬁnity are reported constants in (Ki) in analysis. It is interesting that quercetin was predicted to interact with PIM-1 and HCK Figurethe 8 and range highlight of 0.8–3.8 theµ Mprominent (Figures9 position–11). of quercetin in the network pharmacology analysis.proteins It involved is interesting in breast that cancer.quercetin The was capabilit predictedy of quercetinto interact to with interact PIM with-1 and these HCK pro- proteinsteins was involved also demonstrated, in breast cancer. albeit The partially, capabilit byy of docking quercetin runs to yieldinginteract with affinity these constants proteins(Ki) wasin the also range demonstrated, of 0.8–3.8 µM albeit (Figures partially, 9–1 1by). docking runs yielding affinity constants (Ki) in the range of 0.8–3.8 µM (Figures 9–11). Biomolecules 2021, 11, x 13 of 18

BiomoleculesBiomolecules2021, 11, 671 2021, 11, x 12 of 17 13 of 18

Figure 9. Components–targetsFigure analysis 9. Components–targets underlying the prominent analysis position underlying of quercetin, the prominent among position all phytocompounds of quercetin, among iden- all tified andFigure quantified 9. Components in thephytocompounds extracts.–targets Noteworthy analysis identiﬁed underlying are the and putative quantiﬁed the prominent interactions in the position extracts. towards of Noteworthy quercetin, proto-oncogene among are the serine/threonineall putative phytocompounds interactions– iden- protein kinasetified (PIMand quantified-1) and hematopoietic in the extracts. cell Noteworthy kinase (HCK), are both the putativeinvolved interactions in breast cancer. towards proto-oncogene serine/threonine– towards proto-oncogene serine/threonine–protein kinase (PIM-1) and hematopoietic cell kinase protein kinase (PIM-1) and hematopoietic cell kinase (HCK), both involved in breast cancer. (HCK), both involved in breast cancer.

Figure 10. Putative interactions between quercetin and human and proto-oncogene serine/threo- Figure 10. Putative interactions between quercetin and human and proto-oncogene serine/threonine– nine–proteinFigure kinase 10. Putative (PIM-1; PDB:interactions 3BGZ). between Free energy quercetin of binding and human (ΔG) and and affinity proto- (Ki)oncogene are −7.4 serine/threo- protein kinase (PIM-1; PDB: 3BGZ). Free energy of binding (∆G) and affinity (Ki) are −7.4 kcal/mol kcal/mol nineand –3.8protein µM, respectively. kinase (PIM -1; PDB: 3BGZ). Free energy of binding (ΔG) and affinity (Ki) are −7.4 and 3.8 µM,kcal/mol respectively. and 3.8 µM, respectively. Biomolecules 2021, 11, x 14 of 18

Biomolecules 2021, 11, 671 13 of 17

FigureFigure 11. 1Putative1. Putative interactions interactions between between quercetin quercetin and human and and hematopoietichuman and cellhematopoietic kinase (HCK; cell kinase PDB:(HCK; 1QFC). PDB: Free 1QFC). energy ofFree binding energy (∆G) of and binding affinity (Ki) (ΔG) are and−8.3 kcal/molaffinity (Ki) and 0.8 areµM, −8.3 respectively. kcal/mol and 0.8 µM, respectively. 4. Discussion Herbal remedies play an important role in cancer prevention and treatment [33]. 4. Discussion Anticancer study on MCF 7 cells showed that the death rate of cancer cell lines increases with a riseHerbal in the remedies concentration play of anDorycnium importantextract. role in cancer prevention and treatment [33]. An- ticancerThe Dorycnium study ongenus MCF is 7 an cells important showed group that of the medicinal death plantsrate of that cancer have beencell lines em- increases with ployed in traditional medicine for a long time [19,26,34,35]. It is known that the plants are a a rise in the concentration of Dorycnium extract. significant source of flavonoids, and previous phytochemical investigations identified five flavonoidsThe in Dorycnium the phytocomplex: genus myricitrin, is an important quercitrin, group kaempferol of medicinal 3-O-β-glucopyranoside, plants that have been em- kaempferolployed in 3-O-(6”-acetyl)- traditional medicineβ-glucopyranoside, for a long (+)-dihydromyricetin time [19,26,34,35] [18. It]. is known that the plants are a significantMultiple studies source conducted of flavonoids, on the genus and also previous showed antimicrobial, phytochemical antibiofilm, investigations and identified antioxidantfive flavonoids activity, in which the phytocomplex: were related, albeit myricitrin, partially, to quercitrin, the content ofkaempferol phenolic com- 3-O-β-glucopyra- pounds [19,23,24]. However, the members of this genus have not yet been fully explored as cytotoxic.noside, Inkaempferol this context, 3 the-O- present(6″-acetyl) results-β contribute-glucopyranoside to a better, understanding (+)-dihydromyricetin of their [18]. propertiesMultiple and potential studies application conducted as antioxidant on the genus agents. also showed antimicrobial, antibiofilm, and antioxidantIn our earlier activity, paper [24 which], the chemical were related, composition, albeit antioxidant partially, and to enzyme the content inhibitory of phenolic com- propertiespounds of[19,23,24] the extract. However, tested in the the present members study were of this determined. genus have Catechin, not quercetinyet been fully explored and rutin were found to be the main phenolics in the chemical composition. From this as cytotoxic. In this context, the present results contribute to a better understanding of point, the cytotoxic effects of the extract could be attributed to the presence of these compounds.their properties Following and ourpotential approaches, application catechin as exhibited antioxidant significant agents. anticancer and anti-lipidIn peroxidationour earlier abilitiespaper [24] [36–,38 the]. In chemical addition, somecomposition, authors reported antiox thatidant catechin and enzyme inhibi- inducedtory properties apoptosis of severalthe extract cell lines tested [39 ,in40 ].the Similar present findings study were were also determined. observed for Catechin, quer- rutincetin [41 and,42] andrutin quercetin were found [43]. Taken to be together, the mainD. pentaphyllum phenolicssubsp. in thepentaphyllum chemicalcould composition. From be considered as a valuable source of anticancer compounds in the management of several cancerthis point, types. the cytotoxic effects of the extract could be attributed to the presence of these compounds.Therefore, the Following cytotoxic effect our approaches, of the tested extract catechin was examined exhibited on MCF-significant 7 breast anticancer can- and anti- cerlipid cell linesperoxidation and MCF12A abilities immortalized [36–38 cell]. In lines addition, using the XTTsome assay. authors In an earlierreported study, that catechin in- theduced effective apoptosis antitumor of effect several has alsocell been lines proven [39,40 against]. Similar HeLa findings and WiDr were cells compared also observed for rutin to normal fibroblast and colon cells from the D. pentaphyllum extracts. D. pentaphyllum extracts[41,42] inhibited and quercetin the cell viability [43]. Take of HeLan together, and WiDr D. cells pentaphyllum in a concentration-dependent subsp. pentaphyllum could be mannerconsidered with lower as a IC valuable50 values comparedsource of to normalanticancer colon andcompounds fibroblast cells. in the Furthermore, management of several cancer types.

Biomolecules 2021, 11, 671 14 of 17

the IC50 values of the extract in HeLa cells were higher than cisplatin, but the selectivity index of the extract on HeLa cells (9.7-fold) was found higher than cisplatin (6.9-fold) [26]. Based on the results of the present study, it is confirmed that D. pentaphyllum extracts can be used as an alternative for the extraction of biomolecules for anticancer therapy. Over 90% of human tumors are malignant tumors; in these, transformed epithelial cells grow in an uncontrolled fashion, break through the basement membrane, and invade the underlying mesenchyme [5]. According to this knowledge, we tried to figure out the invasiveness of MCF-7 breast cancer cells treated with the extract in vitro. Carcinomas can be subdivided by morphological and functional criteria: the first is that well-differentiated carcinomas retain epithelial tissue structures, which show well-developed intercellular junctions, and are generally weakly mobile and invasive. On the other hand, the second one is that poorly differentiated carcinomas are characterized by a more amorphous tissue structure, with fewer cell-to-cell junctions, thus resulting in more highly invasive [44]. In our study, 48 h application of the extract decreased (%) cell adhesion of MCF-7 breast cancer cells, thus further strengthening the importance of herbal extracts as sources of antiproliferative biomolecules against breast cancer [44–46]. Specifically, we used cell adhesion assay to see the extract affects the metastatic ability of MCF-7 breast cancer cells. So here, we wanted to show whether the cell adhesion was weakened on MCF-7 cells seeded on the number of adherent cells at 48 h incubation. As shown in Figure4B, the number of adherent cells gradually decreased within a concentration-dependent manner, but the number of adherent cells treated with the tested extract for 48 h was significantly less than that of the control cells with significant statistical differences. This study provides the first evidence that the extract plays an important role in the development and progression of human cancer, MCF-7. In the present study, the extract was effective, in the concentration range 37.5–150 µg/mL, to significantly increase the MDA levels in MCF-7 cells. Conversely, the GSH levels tend to decrease following extract treatment. Collectively, these findings showed pro-oxidant effects in MCF-7 cells, thus suggesting that the antiproliferative effects induced by the extract in this cell line could be the result of increased oxidative stress [45]. However, the pro-oxidant effect induced by the extract is contrasting with our previous observations of intrinsic/scavenging effects [24]. Nevertheless, we cannot exclude paradoxical pro-oxidant effects induced by the phenolic compounds present in the extract, particularly in vitro [46]. Based on the phytochemical composition of the extract, a bioinformatics investigation was conducted on the platform STITCH. The components-targets analysis considered all phytocompounds identified and quantified in the extracts and fully listed in Table1. The results of the target prediction yielded by the STITCH platform highlight the prominent position of quercetin in the network pharmacology analysis. It is of noteworthy interest that quercetin was predicted to interact with PIM-1 and HCK proteins. In breast cancer, these target proteins could play pivotal roles in regulating cytokine signaling and cell proliferation, respectively [47,48]. The capability of quercetin to interact with PIM-1 and HCK was also demonstrated, albeit partially, by docking runs yielding affinity constants (Ki) in the micromolar range that agrees with the quercetin level in the extract. These results add to the observed inhibitory effects on MCF7 cell proliferation and lipid peroxidation induced by the extract, as well. Additionally, literature data showed the capability of quercetin to contrast breast cancer proliferation via multiple mechanisms, including the inhibition of lipid peroxidation and the modulation of apoptosis and inflammatory pathways [49]. To the best of our knowledge, this is the first in silico study suggesting the quercetin affinity towards PIM-1 and HCK proteins. In this regard, future studies are required to confirm these putative interactions via independent biochemical analyses.

5. Conclusions Medicinal plants have a great place in cancer therapy and are a valuable resource for discovering small-molecule inhibitors that target signal transduction proteins, e., kinases, which regulate the proliferation and invasion of cancer cells. We are convinced that the Biomolecules 2021, 11, 671 15 of 17

inclusion of these assays in relevant quantitative methods will be a powerful tool for the preclinical screening of new therapeutic drugs designed to characterize cancer metastatic prognosis and disrupt metastatic progression. In summary, our data demonstrate the efficacy of D. pentaphyllum extract in inhibiting cell proliferation, invasion and adhesion, which could be related, albeit partially, to the quercetin content. This further adds to the importance of validating using traditional medicinal plants and herbs in breast cancer therapy.

Author Contributions: Conceptualization, G.K., E.A. and G.Z.; methodology, G.K.; software, G.O.; C.F.; validation, G.K., E.A.; formal analysis, E.A.; investigation, G.K.; resources, G.Z.; data curation, G.Z.; writing—original draft preparation, G.K.; writing—review and editing, E.A.; visualization, G.O., C.F.; supervision, E.A.; project administration, E.A.; funding acquisition, C.F. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References 1. Ferlay, J.; Ervik, M.; Dikshit, R.; Eser, S.; Mathers, C.; Rabelo, M. Cancer Incidence and Mortality Worlwide: IARC Cancer Base No 11; International Agency for Research on Cancer: Lyon, France, 2012. 2. Bose, S.; Panda, A.K.; Mukherjee, S.; Sa, G. Curcumin and tumor immune-editing: Resurrecting the immune system. Cell Div. 2015, 10, 1–13. [CrossRef] 3. Das, T.; Sa, G.; Saha, B.; Das, K. Multifocal signal modulation therapy of cancer: Ancient weapon, modern targets. Mol. Cell. Biochem. 2010, 336, 85–95. [CrossRef][PubMed] 4. Torre, L.A.; Sauer, A.M.G.; Chen Jr, M.S.; Kagawa-Singer, M.; Jemal, A.; Siegel, R.L. Cancer statistics for Asian Americans, Native Hawaiians, and Pacific Islanders, 2016: Converging incidence in males and females. CA A Cancer J. Clin. 2016, 66, 182–202. [CrossRef] 5. Behrens, J.; Weidner, K.M.; Frixen, U.H.; Schipper, J.H.; Sachs, M.; Arakaki, N.; Daikuhara, Y.; Birchmeier, W. The role of E-cadherin and scatter factor in tumor invasion and cell motility. In Cell Motility Factors; Springer: Berlin/Heidelberg, Germany, 1991; pp. 109–126. 6. Frixen, U.H.; Behrens, J.; Sachs, M.; Eberle, G.; Voss, B.; Warda, A.; Löchner, D.; Birchmeier, W. E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells. J. Cell Biol. 1991, 113, 173–185. [CrossRef] 7. Weinstein, R.S.; Merk, F.B.; Alroy, J. The structure and function of intercellular junctions in cancer. Adv. Cancer Res. 1976, 23, 23–89. [PubMed] 8. Mollica, A.; Stefanucci, A.; Zengin, G.; Locatelli, M.; Macedonio, G.; Orlando, G.; Ferrante, C.; Menghini, L.; Recinella, L.; Leone, S.; et al. Polyphenolic composition, enzyme inhibitory effects ex-vivo and in-vivo studies on two Brassicaceae of north-central Italy. Biomed. Pharmacother. 2018, 107, 129–138. [CrossRef][PubMed] 9. Deferme, L.; Briede, J.; Claessen, S.; Cavill, R.; Kleinjans, J. Cell line-specific oxidative stress in cellular toxicity: A toxicogenomics- based comparison between liver and colon cell models. Toxicol. In Vitro 2015, 29, 845–855. [CrossRef][PubMed] 10. Saw, C.L.-L.; Wu, Q.; Kong, A.-N. Anti-cancer and potential chemopreventive actions of ginseng by activating Nrf2 (NFE2L2) anti-oxidative stress/anti-inflammatory pathways. Chin. Med. 2010, 5, 37. [CrossRef] 11. Lobo, V.; Patil, A.; Phatak, A.; Chandra, N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacogn. Rev. 2010, 4, 118. [CrossRef] 12. Canter, P.H.; Ernst, E. Herbal supplement use by persons aged over 50 years in Britain. Drugs Aging 2004, 21, 597–605. [CrossRef] 13. Cohen, P.A.; Ernst, E. Safety of herbal supplements: A guide for cardiologists. Cardiovasc. Ther. 2010, 28, 246–253. [CrossRef] 14. Loya, A.M.; Gonzalez-Stuart, A.; Rivera, J.O. Prevalence of polypharmacy, polyherbacy, nutritional supplement use and potential product interactions among older adults living on the United States-Mexico border. Drugs Aging 2009, 26, 423–436. [CrossRef] 15. Qato, D.M.; Alexander, G.C.; Conti, R.M.; Johnson, M.; Schumm, P.; Lindau, S.T. Use of prescription and over-the-counter medications and dietary supplements among older adults in the United States. JAMA 2008, 300, 2867–2878. 16. Lin, D.; Xiao, M.; Zhao, J.; Li, Z.; Xing, B.; Li, X.; Kong, M.; Li, L.; Zhang, Q.; Liu, Y. An overview of plant phenolic compounds and their importance in human nutrition and management of type 2 diabetes. Molecules 2016, 21, 1374. [CrossRef] 17. Greenwell, M.; Rahman, P. Medicinal plants: Their use in anticancer treatment. Int. J. Pharm. Sci. Res. 2015, 6, 4103. [PubMed] 18. Kazantzoglou, G.; Magiatis, P.; Panoutsopoulos, G.; Skaltsounis, A.-L. Dorycnioside, a New Phenylbutanone Glucoside from Dorycnium pentaphyllum subsp. herbaceum. Z. Für Nat. C 2004, 59, 23–26. [CrossRef] Biomolecules 2021, 11, 671 16 of 17

19. Bremner, P.; Rivera, D.; Calzado, M.; Obón, C.; Inocencio, C.; Beckwith, C.; Fiebich, B.; Muñoz, E.; Heinrich, M. Assessing medicinal plants from South-Eastern Spain for potential anti-inflammatory effects targeting nuclear factor-Kappa B and other pro-inflammatory mediators. J. Ethnopharmacol. 2009, 124, 295–305. [CrossRef] 20. Rigat, M.; Bonet, M.À.; Garcia, S.; Garnatje, T.; Valles, J. Studies on pharmaceutical ethnobotany in the high river Ter valley (Pyrenees, Catalonia, Iberian Peninsula). J. Ethnopharmacol. 2007, 113, 267–277. [CrossRef] 21. Usta, C.; Yildirim, A.B.; Turker, A.U. Antibacterial and antitumour activities of some plants grown in Turkey. Biotechnol. Biotechnol. Equip. 2014, 28, 306–315. [CrossRef] 22. Pistelli, L.; Noccioli, C.; Martera, M.; Giamperi, L.; Bucchini, A.; Fraternale, D.; Ricci, D. Antioxidant flavonol glycosides from Dorycnium hirsutum. Chem. Nat. Compd. 2006, 42, 281–284. [CrossRef] 23. Stefanović,O.D.; Tešić,J.D.; Comić,L.R.ˇ Melilotus albus and Dorycnium herbaceum extracts as source of phenolic compounds and their antimicrobial, antibiofilm, and antioxidant potentials. J. Food Drug Anal. 2015, 23, 417–424. [CrossRef] 24. Uysal, S.; Aktumsek, A.; Picot, C.M.N.; Sahan, A.; Mollica, A.; Zengin, G.; Fawzi Mahomoodally, M. A comparative in vitro and in silico study of the biological potential and chemical fingerprints of Dorcycinum pentapyllum subsp. haussknechtii using three extraction procedures. New J. Chem. 2017, 41, 13952–13960. [CrossRef] 25. DemIr,˙ S.; Turan, I.;˙ Misir, S.; AlIyazicio˙ Glu,˘ Y. Selective cytotoxic effect of Dorycnium pentaphyllum extract on human breast, liver, and lung cancer cells. Kahramanmara¸sSütçü Imam˙ Üniversitesi Tarım Do˘gaDergisi 2019, 22, 473–479. 26. Demir, S.; Yaman, S.O.; Sener, S.O.; Ayazoglu Demir, E.; Aliyazicioglu, R.; Ozgen, U.; Mentese, A.; Deger, O.; Aliyazicioglu, Y. Dorycnium pentaphyllum extract has antiproliferative effect on human cervix and colon cancer cells. Nutr. Cancer 2020, 72, 504–512. [CrossRef] 27. Çiçek, C.; Bilgiç, A. Klinik Viroloji laboratuvarinda uzmanlik ö˘grencisine verilen hücre kültürü e˘gitimprogrami: Bir model. Infeksiyon˙ Derg. 2006, 20, 231–241. 28. Del Rio, D.; Pellegrini, N.; Colombi, B.; Bianchi, M.; Serafini, M.; Torta, F.; Tegoni, M.; Musci, M.; Brighenti, F. Rapid fluorimetric method to detect total plasma malondialdehyde with mild derivatization conditions. Clin. Chem. 2003, 49, 690–692. [CrossRef] 29. Beutler, E. Improved method for the determination of blood glutathione. J. Lab. Clin. Med. 1963, 61, 882–888. 30. Angelini, P.; Venanzoni, R.; Angeles Flores, G.; Tirillini, B.; Orlando, G.; Recinella, L.; Chiavaroli, A.; Brunetti, L.; Leone, S.; Di Simone, S.C. Evaluation of Antioxidant, Antimicrobial and Tyrosinase Inhibitory Activities of Extracts from Tricholosporum goniospermum, an Edible Wild Mushroom. Antibiotics 2020, 9, 513. [CrossRef][PubMed] 31. Ferrante, C.; Angelini, P.; Venanzoni, R.; Angeles Flores, G.; Tirillini, B.; Recinella, L.; Chiavaroli, A.; Brunetti, L.; Leone, S.; Di Simone, S.C. Antimicrobial, Antioxidant, and Antiproliferative Effects of Coronilla minima: An Unexplored Botanical Species. Antibiotics 2020, 9, 611. [CrossRef][PubMed] 32. Arvelo, F.; Sojo, F.; Cotte, C. Tumour progression and metastasis. Ecancermedicalscience 2016, 10. [CrossRef] 33. Xiong, Y.; Wu, X.; Rao, L. Tetrastigma hemsleyanum (Sanyeqing) root tuber extracts induces apoptosis in human cervical carcinoma HeLa cells. J. Ethnopharmacol. 2015, 165, 46–53. [CrossRef][PubMed] 34. Kumar, R.S.; Murugananthan, G.; Rajkapoor, B. Anticarcinogenic potential of ethanol extract of Indigofera cordifolia Roth.(Fabales: Fabaceae) on diethylnitrosamine induced hepatocarcinogenesis in rats. Braz. J. Biol. Sci. 2017, 4, 53–66. [CrossRef] 35. Lacerda, L.P.; Malaquias, G.; Peron, A.P. Antiproliferative action of aqueous extracts of Hymenaea stigonocarpa Mart.(Fabaceae) on the cell cycle of Allium cepa L. Anais Acad. Bras. Ciências 2014, 86, 1147–1150. [CrossRef] 36. Al-Hazzani, A.A.; Alshatwi, A.A. Catechin hydrate inhibits proliferation and mediates apoptosis of SiHa human cervical cancer cells. Food Chem. Toxicol. 2011, 49, 3281–3286. [CrossRef][PubMed] 37. Yang, C.S.; Wang, H.; Chen, J.X.; Zhang, J. Effects of Tea Catechins on Cancer Signaling Pathways. Enzymes 2014, 36, 195–221. 38. Yu, Y.; Deng, Y.; Lu, B.M.; Liu, Y.X.; Li, J.; Bao, J.K. Green tea catechins: A fresh flavor to anticancer therapy. Apoptosis 2014, 19, 1–18. [CrossRef][PubMed] 39. Sari, L.M. Catechin: Molecular mechanism of Anti-Cancer Effect: Katekin: Mekanisme Molekular Efek Antikanker. Dentika Dent. J. 2019, 22, 20–25. [CrossRef] 40. Silva, C.; Correia-Branco, A.; Andrade, N.; Ferreira, A.C.; Soares, M.L.; Sonveaux, P.; Stephenne, J.; Martel, F. Selective pro- apoptotic and antimigratory effects of polyphenol complex catechin:lysine 1:2 in breast, pancreatic and colorectal cancer cell lines. Eur. J. Pharmacol. 2019, 859, 172533. [CrossRef] 41. ben Sghaier, M.; Pagano, A.; Mousslim, M.; Ammari, Y.; Kovacic, H.; Luis, J. Rutin inhibits proliferation, attenuates superoxide production and decreases adhesion and migration of human cancerous cells. Biomed. Pharmacother. 2016, 84, 1972–1978. [CrossRef] 42. Caparica, R.; Júlio, A.; Araújo, M.E.M.; Baby, A.R.; Fonte, P.; Costa, J.G.; Santos de Almeida, T. Anticancer Activity of Rutin and Its Combination with Ionic Liquids on Renal Cells. Biomolecules 2020, 10, 233. [CrossRef] 43. Rauf, A.; Imran, M.; Khan, I.A.; ur-Rehman, M.; Gilani, S.A.; Mehmood, Z.; Mubarak, M.S. Anticancer potential of quercetin: A comprehensive review. Phytother. Res. 2018, 32, 2109–2130. [CrossRef] 44. Gabbert, H.; Wagner, R.; Moll, R.; Gerharz, C.-D. Tumor dedifferentiation: An important step in tumor invasion. Clin. Exp. Metastasis 1985, 3, 257–279. [CrossRef][PubMed] 45. Alamer, A.; Ali, D.; Alarifi, S.; Alkahtane, A.; Al-Zharani, M.; Abdel-Daim, M.M.; Albasher, G.; Almeer, R.; Al-Sultan, N.K.; Almalik, A.; et al. Bismuth oxide nanoparticles induce oxidative stress and apoptosis in human breast cancer cells. Environ. Sci. Pollut. Res. Int. 2020.[CrossRef][PubMed] Biomolecules 2021, 11, 671 17 of 17

46. Halliwell, B.; Clement, M.V.; Ramalingam, J.; Long, L.H. Hydrogen peroxide. Ubiquitous in cell culture and in vivo? IUBMB Life 2000, 50, 251–257. [CrossRef][PubMed] 47. Poh, A.R.; O’Donoghue, R.J.; Ernst, M. Hematopoietic cell kinase (HCK) as a therapeutic target in immune and cancer cells. Oncotarget 2015, 6, 15752. [CrossRef][PubMed] 48. Zhang, X.; Song, M.; Kundu, J.K.; Lee, M.H.; Liu, Z.Z. PIM Kinase as an Executional Target in Cancer. J. Cancer Prev. 2018, 23, 109–116. [CrossRef] 49. Ezzati, M.; Youseﬁ, B.; Velaei, K.; Safa, A. A review on anti-cancer properties of Quercetin in breast cancer. Life Sci. 2020, 117463. [CrossRef]