A Comparative Study on Anticancer Effects of the Alhagi maurorum and Amygdalus haussknechtii Extracts Alone and in Combination with Docetaxel on 4T1 Breast Cancer Cells

Medicinal plants have long been studied due to their anticancer effects and use of them is commonly increased as a complementary and alternative medicine (CAM therapies) among patients with cancer. In this study, Alhagi maurorum (A.m) and Amygdalus haussknechtii (A.h) extracts were evaluated for their effects on inhibiting the growth of 4T1 breast cancer cells. Based on MTT assay results, the IC50s of A.m and A.h extracts were 57 µg/ml and 85 µg/ml, respectively. Then the cell migration, gene expression, and degree of apoptosis after 48 hours in each treated group with A.m and A.h extracts alone or in combination with docetaxel (DTX) on 4T1 cells were evaluated. A.m had a synergistic behavior with DTX (CI < 1). A.h reduced DTX IC50 but presented CI > 1. Cell migration assay showed that each extract alone or in combination with DTX prevented the migration of 4T1 cells. The Ao/EB staining and flowcytometry results confirmed that, in combination therapy, A.m + DTX and A.h + DTX induced apoptosis close to the level of DTX. Real-time PCR analysis showed that A.m + DTX (IC50 + IC25) downregulated the mRNA expression of HIF-1α and FZD7. A.m + DTX (IC50 + IC10) group decreased the expression of HIF-1α. Moreover, in A.h + DTX (IC50 + IC25) group, β-Catenin and FZD7 were downregulated and upregulated, respectively. Generally, our findings suggest that the combination of A.m and DTX possesses synergistic antitumor effects on 4T1 cells, which may be a valuable choice for CAM therapies. A.h has an acceptable antitumor activity but not in combination with DTX.


Introduction
Despite enormous medicinal developments in recent decades, a large number of people suffer from cancer. Based on the statistics adapted from the American Cancer Society and the International Union Against Cancer, 27 million diagnoses and 17 million deaths will occur by 2030 worldwide [1]. Breast cancer is the first deadly cancer among women [2]. Triple-negative breast cancer (TNBC) is one of the breast cancer subtypes that cannot be controlled with standard-ofcare therapy [3].
Chemotherapy drugs usually have a lot of side effects and limitations due to nonspecificity, and resistance to some of them is usually observed sometime after using the drug [4][5][6]. Docetaxel (Taxotere ® ) (DTX) is a semisynthetic derivative of paclitaxel which was found to be more effective because of being soluble in water. DTX is used as a treatment remedy in patients suffering from breast, ovarian, and/or metastatic cancers [7]. Today, studies have shown that many plants recommended in traditional medicine against cancer have positive effects on cell lines in laboratory or animal studies and they are great sources for discovering new drugs. e concurrent use of multiple medications can apply treatment from multiple paths to one or more targets. Combination therapy or synergistic therapy has several benefits such as increasing treatment efficacy, reducing dose and toxicity, and minimizing drug resistance. Hence, drug combinations have been widely used, especially in most dreadful diseases, such as cancer, and infectious diseases, including acquired immunodeficiency syndrome (AIDS) [8]. Also, combining medicinal plants with chemotherapeutics, including DTX, can be useful in reducing the toxicity and side effects of chemotherapy [6,9].
One of the pathways which is tightly associated with cancer and is activated in more than 50% of breast cancers is Wnt signaling [10].
is pathway consists of β-Catenin dependent and independent signaling groups [11]. In many studies, it has been shown that a number of medicinal plants or their compounds are effective on the activity of the Wnt path [12][13][14]. e use of complementary and alternative medicines (CAM) in cancer patients is growing, and pharmacokinetic interactions between CAM and chemotherapeutics that might lead to either increased or decreased plasma levels of anticancer drugs should be investigated [15].
Before starting the main study, we found the effective plants in the treatment of various types of cancer from literature to find the most effective and desired plants and then selected 20 of the available types. en we tested their inhibitory effect on the growth of 4T1 breast cancer cells, among which A. m and A. h had the most favorable effect, respectively (data are available in the supplementary file).
A. maurorum is a plant from the Fabiaceae family which has been widely used in traditional medicine in the treatment of various diseases, and scientists have recently studied its anticancer effect on some cell lines [16][17][18]. is plant has many biological properties such as antioxidant, antimicrobial, cytotoxic, anti-inflammatory, antiproliferative properties [19]. Previous studies revealed the presence of flavonoids, glycosides, saponins, tannins, alkaloids, steroids, and anthraquinone in the A. m extract [20,21].
Also Lupeol as an active fraction was detected by nuclear magnetic resonance in A. m inducing apoptosis in human breast cancer cells [22].
A. haussknechtii is one of the amygdalus species which is grown in a few provinces of Iran, including Chaharmahal and Bakhtiari. During local research, we found that indigenous people, in addition to using the fruit of this plant, use its leaves to treat some diseases. erefore, we decided to examine it in this study. In a study, the phenolic content of leaves of several types of wild almonds was examined and it was found that the highest phenolic content (gallic acid) was related to A. h [23]. Because of the benefits mentioned above for combination therapy, the aim of the current study was to evaluate the effects of A. m and A. h extracts alone and in combination with docetaxel on 4T1 breast cancer cells. DTX (20 mg) manufactured by NanoAlvand Co. (Tehran, Iran) was purchased from a drugstore.

Plant Extraction.
Ethanol (Merck, Hohenbrunn, Germany) was used for plants extraction. Hydroalcoholic extract (ethanol 70%) was prepared and maceration method was used for plants extraction. To this purpose, the dried powdered plants were soaked in a hydroalcoholic solution for 48 hours, and, after passing through the filter paper, they were concentrated with a rotary machine and the solvent was removed.
e concentrated extracts were dried in freeze dryer and the resulting powders were kept at 2-8°C for future assays. e extract powders were dissolved in DMSO to make the stock solutions of each sample (5 mg mL −1 ). Serial dilutions were made from the stock solutions to reach final concentrations (DMSO 1%) [24].

Assessment of Cell Growth after Treatment.
For quantification of cell viability using MTT assay, after trypsinization of 4T1 cells, they were seeded onto 96-well plates at 6 × 10 5 and incubated at 37°C. After 24 h of incubation and when the cells had about 35% confluences, they were treated with fresh medium containing serial dilutions of each plant extract. e control cells medium also contained 1% DMSO vehicle. After 48 h of treatment, the supernatants were removed and MTT solution (0.5 mg mL −1 ) was added and incubated for 4 h. en, the supernatants were removed, and DMSO was added. e plates were then placed on a shaker for 20 minutes. e absorbance of each well was measured in Stat Fax2100 microtiter plate reader at the wavelength of 570 nm. In this way, cell survival was measured in the form of [A] samples/[A] control * 100, where [A] was the absorbance of test samples and control, respectively. e doseviability curves were fitted by Excel software and IC50 values were calculated. In such studies, the extracts with IC50 greater than 100 μg/mL were considered as ineffective extracts [24]. All experiments were carried out in 3 individual plates with at least three parallel wells.

Assessment of Combined Treatment Using Median-Effect
Plot Analysis. CompuSyn software (set up by Dr. Dorothy Chou, ComboSyn Inc., 2005) was used for analysis of results of the MTT assay. Briefly, dose-response curves were generated for individual drugs, and then combination indexes (CI) were calculated for each combination. CI < 1 represents synergy, CI � 1 shows additive effect, and CI > 1 indicates antagonism.

Apoptosis Analysis Using Annexin V Staining.
Double-staining was done using the Annexin V and Propidium Iodide kit (BD Pharmingen ™ ) according to the manufacturer's protocol in order to determine percentages of viable, apoptotic, and necrotic cells following 48-hour exposure to DTX, A. m and A. h extracts, and combination of each extract with DTX. For this purpose, 4T1 cells were seeded onto 6 well plates, and, after treatment with each of the above items, they were trypsinized with repetitive pipetting and washed with PBS (centrifuged at 1200 rpm for 5 min). Untreated cells were used as negative controls. After counting them, they were resuspended in 100 μl of the binding buffer solution per 100000 cells. After adding annexin V and PI, they were incubated for 15 min in the dark at 25°C. Annexin V-FITC binding was detected by flow cytometer (space Partec, Germany) (Ex � 488 nm, Em � FITC 495-519 nm, PI 535-617 nm, FL1 filter for annexin V/FITC and FL2 filter for PI). e data were analyzed using FloMax software version 2.70 (Partec GmbH, Münster, Germany).

Acridine Orange/Ethidium Bromide (AO/EB) Staining.
Acridine Orange/Ethidium Bromide (AO/EB) was purchased from Sigma Chemical Company (Becton Dickinson, San, CA, USA). is test is done to visualize nuclear changes and apoptotic body formation and observation of characteristics of apoptosis. Cells were viewed under Zeiss fluorescence microscope connected to a CCD camera. Briefly, 4T1 cells were seeded onto 6-well plates and treated for 48 hours with DTX, A. m and A. h extracts, and combination of each extract with DTX. en, they were harvested and 25 µl of cell suspension (0.5 × 10 6 to 2.0 × 10 6 cells/ml) was incubated with 1 µl of AO/ EB solution. Each sample was mixed just prior to microscopy. en, 10 µl of cell suspension was placed onto a microscopic slide, covered with a glass coverslip, and examined using a fluorescein filter and a 40X objective.

Cell Migration Assay.
e 4T1 cells were seeded onto 12well culture plates to form a monolayer, and then identical straight strip scratches were created using sterile yellow tips. After that, the cells were washed with PBS and incubated with RPMI containing 2% FBS (this FBS concentration allows cell survival with no cell proliferation) and different concentrations of hydroalcoholic extract of A. m (IC10), A. h (IC10), and DTX (IC10) and the combination groups of each of these two extracts with DTX (IC10 of DTX with IC10 extracts). After 48 h of incubation, the 4T1 cells were washed with PBS and photographed using a camera connected to an inverted microscope (Nikon, Eclipse TS100) at 10× magnifications. Relative width of scratch was obtained using ImageJ software.

Quantitative Real-Time PCR.
Total RNA was extracted from 4T1 cells treated with each of A. m, A. h, and DTX as well as A. m and A. h combined with DTX (IC50 of each of extracts with IC10 and IC25 of DTX) for 48 h using the total RNA extraction kit (Pars Tous, Iran). e quantity of the isolated RNA was characterized by NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE). e complementary DNA was synthesized by cDNA synthesis kit (Pars Tous, Iran). Primers for genes, frizzled7 (FZD7), hypoxia inducible factor 1α (HIF-1α), vascular endothelial growth factor-a (VEGF-a), beta-Catenin (β-Catenin), and the housekeeping gene glyceraldehydes 3phosphate dehydrogenase (GAPDH) were designed using Oligo7 software (Table 1). SYBR Premix Ex Taq TM II (Tli RNase H Plus) (Takara BIO, Inc., Otsu City, Shiga, Japan) was used. Real-time PCR amplifications were run using a Rotor-Gene 3000 (Corbett Research, Sydney, Australia). Normalization of the expression levels of the target genes to the housekeeping gene GAPDH was done using REST software (QIAGEN GmbH, 2009, v2.0.13). Finally, the fold change in expression level of each mRNA was quantified based on Pfaffl method.

Statistical
Analysis. Experimental data were expressed by mean ± standard deviation of three independent assays. e results of gene expression were shown as mean-± standard error of the mean.

Combined Effect of A.m and A.h Extracts with DTX on 4T1
Cells. A. m and A. h extracts both showed IC50 < 100 µg ml −1 in 4T1 cells. is represents very good potential of these plants to inhibit the growth of this cancer cell line. Since some medicinal plants have been shown to have synergistic effects in combination with chemical drugs or they have been able to reduce their side effects or drug resistance [9,25,26], we investigated the combination of these two plants with DTX, which is used to treat breast cancer. First, using MTTassay, the IC50 of DTX was calculated to be about 11 µM (Figure 1(c)). en, by selecting some concentrations lower and higher than IC50 for each extract or DTX, MTTassay was done again in combination mode. e combination index (CI) was calculated by entering doses and their effects in CompuSyn software.

Evidence-Based Complementary and Alternative Medicine
According to the calculations performed by software, it was found that A. m at doses below 100 µg/ml in combination with DTX had a CI < 1, indicating synergy. e results of isobologram analysis (Figure 2(a)) were in agreement with fraction affected (FA) versus CI; in this way A. m at doses below 100 µg/ ml in combination with DTX was below the line of additive effects, demonstrating the synergistic antiproliferative effect of DTX + A. m combination (Figure 2(c)). A. m concentrations above 100 µg/ml had antagonistic effects. e results of combining A. h extract with DTX showed that most of the points were above the additive effect line and had antagonistic effects.
ese results of isobologram analyses and FA versus CI are shown in Figures 2(b) and 2(d), respectively. Table 2 shows the doses used for extracts and DTX, as well as the values of CI obtained from the software analysis for each combination.
In other experiments, we investigated the effects of the increased concentrations of A. m extract on a range of DTX concentrations (

Effects of A.m and A.h Extracts on Migration of 4T1 Cells.
As shown in Figure 3, in treated groups, the scratch closure was delayed after 24 hours. At time 0, the relative width of injury line (scratch width) in the untreated group and in the other treated groups is considered 100%. In the untreated group, after 12 and 24 hours, significant scratch closure was observed due to the migration of 4T1 cells. In the group treated with A. m (IC10), the relative width of the scratch did not change much after 24 hours. A. h (IC10) yielded a relative width of ∼92.68% and DTX (IC10) had a relative width of about 90.13% after 24 h. In the group treated with A. h + DTX (IC10 of both of them) and A. m + DTX (IC10 of both of them), the scratch remained approximately the same after 24 hours with relative width of  Results are reported as a percentage of viability compared with control and are presented as mean ± SD from three independent experiments. Significance was set at * P < 0.05, * * P < 0.01, * * * P < 0.001 and * * * * P < 0.0001. 99.48% and 96.14%, respectively, but the cells were a little deformed and some of the cell debris was seen in the gap. ese results indicate that the used treatments can significantly impede the migration of 4T1 cells. In the untreated group, the gap was significantly closed, but, in other groups, except for DTX and A. h, which slightly reduced the scratch width, there was no significant difference across the scratch after 24 hours.

Apoptosis Analysis after Treatment with DTX, A.m, and A.h Extracts Alone or in Combination with DTX in 4T1 Cell
Line. In the case of A. m, IC50 of this extract significantly induced apoptosis (45.12%) compared to control group (13.53%). But A. m + DTX (IC50+IC25) and A. m + DTX (IC50 + IC10) groups increased apoptosis (68.92% and 65.5%, respectively) to a level close to DTX (74.85%) and this effect was significantly higher than that of A. m alone. A. m + DTX (IC10 + IC10) group (46.2% apoptosis) had the least necrosis. A. h + DTX (IC10 + IC10) (40.8%) increased the extent of apoptosis close to the amount of DTX (41.6%). A. h + DTX (IC50 + IC10) group (63.3%) increased apoptosis even more than DTX alone. In all groups, apoptosis was increased significantly compared to the control. e obtained results are shown in Figure 4.

AO/EB Staining Assay for Detection of Apoptotic Cells.
Apoptotic and necrotic cells are detectable under fluorescence microscope based on the cell morphology and membrane integrity. By penetrating into viable cells, AO causes the nucleus of the cells to be seen in green with organized structure nuclei. However, nonliving cells take up EtBr in the absence of membrane integrity and are seen in a range of yellow-orange-red [27]. Accordingly, cells with early apoptosis have green nuclei with condensed or fragmented chromatin and have a crescent-shaped or granular yellow-green staining. But cells with late apoptosis have orange-to-red nuclei with condensed or fragmented chromatin. Necrotic cells will show increased volume and orange-to-red organized structure nuclei [28]. As shown in Figure 5, the result of fluorescence microscopy of nuclear dye stained 4T1 cells showed that, in the untreated group (negative control), the nuclei were homogeneous and round, and the cells were seen in green. Green and red nuclei with condensed chromatin were observed in the groups treated with DTX, A. m, and A. h, indicating early and late apoptosis.
In combination groups, there were more apoptosis in groups e (IC50 A. m + IC50 DTX) and specially f (IC50 A. m + IC10 DTX) and apoptotic bodies were clearly evident. In group g (IC25 A. m + IC25 DTX), the red color of the nuclei and the increase of cell volume indicated necrosis. In group h (IC10 A. m + IC10 DTX), the nuclei with orange fluorescence showed late apoptosis. In the case of A. h extract + DTX combination, groups j (IC50 A. h + IC10 DTX) and l (IC10 A. h + IC10 DTX) exhibited more apoptosis than group i (IC50 A. h + IC50 DTX). Group k (IC25 A. h + IC25 DTX) showed necrosis. ese data are consistent with the results of flow cytometry.

Effect of DTX, A.m, and A.h Extracts and eir Combinations on Wnt/β-Catenin Pathway Gene Expression.
β-Catenin expression decreased in A. m, A. h, DTX, and A. h + DTX groups compared to control, but this decrease was significant in A. h + DTX (IC50 + IC25) group. FZD7 expression in A. m + DTX (IC50 + IC25) group showed a significant decrease, but, in A. h + DTX (IC50 + IC25) group, there was a significant increase. e expression of VEGF-a did not change significantly in any of the groups. e expression of HIF-1α increased in A. h + DTX (IC50 + IC10) group, but this increase was not significant. e combination treatment in A. m + DTX (IC50 + IC10) and (IC50 + IC25) groups showed a significant downregulation in mRNA expression levels of HIF-1α compared to the control. Data are shown in Figure 6

Discussion
In this in vitro study, we investigated the combined effect of A. m and A. h extracts with DTX-based chemotherapy on the growth suppression of 4T1 cancer cell line. e results of this study with A. m and A. h extracts showed acceptable IC50 values in inhibiting the growth of 4T1 cancer cells including 57 µg/ml and 85 µg/ml, respectively. 8

Evidence-Based Complementary and Alternative Medicine
A. m as a highly valuable plant has numerous secondary metabolites and flavonoids and has shown many medicinal properties [29]. Over the past years, numerous experimental studies have demonstrated that diethyl ether and petroleum ether extracts of A. m had IC50 values of 114.7 µg/ml and 105.7 µg/ml against MCF7 human breast cancer cell line [16]. A. m extract showed IC50s > 100 µg/ml against A549, MCF7, HepG2, HT-29, and MDBK cell lines [30].
Synergy is a process in which the combined effect of the participating elements is greater than the sum of their individual effects. Examining these interactions is important because it may have advantages for treatment, even in complex diseases such as cancer [31]; for example, ginseng increases the cytotoxicity of docetaxel, cisplatin, and doxorubicin [32].
In our study, A. m at concentrations below 100 µg/ml, in combination with the DTX, showed a synergistic behavior and reduced the IC50 value of DTX with a significant and steep gradient. A. m at concentrations above 100 µg/ml alone showed a higher suppression of cancer cell growth, so the CompuSyn software did not offer synergy in comparison to individual and combined modes. A. m extract also inhibited the migration of 4T1 cells both alone and in combination with DTX. Additionally, we found the increased percentages of apoptotic cells in A. m + DTX (IC50 + IC25 and IC50 + IC10) combinations compared to A. m extract alone, which were close to the percentage of apoptosis caused by DTX. In A. m + DTX (IC50 + IC10) group, apoptotic bodies were also clearly seen. It appears that when the amount of A. m extract in combination therapy is greater than DTX, the amount of apoptotic cells increases. However, A. m + DTX 0.0 * * Figure 6: Effect of DTX, A.m, and A.h extracts and A.m or A.h + DTX combination groups on expression levels of β-Catenin, FZD7, VEGFa, and HIF-1α genes accomplished by real-time PCR. Data are represented as the relative expression level compared to GAPDH on the basis of Pfaffl method, presented as mean ± SD. Significance was set at * P < 0.05. IC values were as follows: for A.m : IC10 � 32 µg/ml, IC25 � 40 µg/ml, and IC50 � 57 µg/ml; for A.h : IC10 � 10 µg/ml, IC25 � 33 µg/ml, and IC50 � 85 µg/ml; and for DTX : IC10 � 0.4 µM, IC25 � 3.5 µM, and IC50 � 11 µM.
(IC10 + IC10) combination group showed the least necrosis, and when the amount of DTX increased, the necrosis also was increased. e A. m + DTX (IC50 + IC10 and IC50 + IC25) combination groups also significantly reduced the expression level of the HIF-1α gene. It seems that IC10 of DTX in combination with IC50 of A. m was more effective than IC25 of DTX. Also, A. m + DTX (IC50 + IC25) group downregulated the expression of FZD7 gene. In other studies, Lupeol from A. m extracts reduced mRNA expression level of p53, caspase-3, and bax genes in MCF-7 and MDA-MB-231 cell lines; however, bcl-2 gene expression was decreased [22].
Docetaxel is generally accepted as the chemotherapy agent for patients with advanced breast cancer [7], but its clinical efficacy can be compromised by serious related side effects. So, controlling DTX therapy-related side effects may improve clinical outcomes in patients undergoing chemotherapy. Considering that A. m extract in combination with DTX showed synergistic behavior in concentrations below 100 µg/ml, this combination may be inhibiting cancer cell growth in breast cancer and reduce the side effects of chemotherapy. According to previous reviewed information, A. m has got great scope for phytochemical and pharmacological studies to prove its therapeutic potential [19]. In combination with DTX, A. m in concentrations below 100 µg/ml can be a potential complementary therapeutic option in breast cancer.
A. h fruits, oil, and even leaves in traditional medicine have been used for numerous treatments, but the efficacy of the extract in breast cancer has not been studied. A. h, though alone, had an acceptable IC50 against 4T1 cells but showed a contradictory behavior in combination therapy. In the combination of A. h with DTX, CI > 1 was obtained, indicating antagonism. Although it was able to reduce the IC50 value of DTX, it was, of course, less steep than that of A. m. A. h + DTX (IC50 + IC10 and IC10 + IC10) combination groups induced apoptosis even more than DTX alone, and A. h + DTX (IC50 + IC10) group presented 63.3% apoptosis.
e gene expression analysis showed that A. h + DTX (IC50 + IC25) significantly downregulated β-Catenin. However, A. h + DTX (IC50 + IC25) increased expression of FZD gene; this group showed the least apoptosis compared to other groups. e good potential of A. h in inhibiting the migration of 4T1 cells and inducing apoptosis was quantitatively and qualitatively evident. Since A. h in combination mode slowly reduced the IC50 value of DTX, the synergistic behavior could be observed probably by increasing the treatment duration or changing the concentrations. In fact, the concentrations or treatment time we used for this study may not have been adequate or sufficient to create a synergistic state for A. h.

Conclusion
Briefly, we investigated the combination effect of A. m and A. h extracts with DTX for the first time. A. m and A. h extracts inhibited the growth of 4T1 cancer cells. Having definitive synergistic effect in concentrations below 100 µg/ ml, A. m extract can be a good alternative in combination with DTX. Natural products are a huge source of diverse elements in drug research. ese types of studies help to identify susceptible plants in this context and develop these research studies. A. m and A. h extracts may have a good potential for becoming drugs given their acceptable IC50s in inhibiting the growth of 4T1 cancer cells. Nevertheless, it is necessary to examine the effects of these medicinal plants individually and in combination with DTX in animal model and clinical trial.
Data Availability e data supporting the findings of this study are available within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest.