Antiproliferative, Antiangiogenic, and Antimigrastatic Effects of Oroxylum indicum (L.) Kurz Extract on Breast Cancer Cell

Breast cancer recurrence continues to pose a major clinical problem, despite significant advancements in early diagnosis and an aggressive mode of treatment. This study aimed at investigating the anticancer activity of Oroxylum indicum extract (OIE) by assessing cell proliferation, cell migration, and angiogenesis in metastatic breast cancer MDA-MB-231 cell lines. This study also estimated the phytochemical profiles of OIE by LC-QTOF-MS. The extract was found to contain six identified flavonoid substances, and baicalein was the most abundant substance in the extract. Cell proliferation capacity was performed by cell counting kit-8 (CCK-8) and colony formation assays. The effect of OIE on cell migration was determined using wound healing and transwell assays. Meanwhile, MDA-MB-231-induced angiogenesis on chick chorioallantoic membrane (CAM) was applied to investigate the ex vivo antiangiogenesis activity of the extracts. OIE at concentrations lower than 600 μg/mL had no cytotoxic effects against MDA-MB-231 cells. OIE was found to inhibit the long-term colony formation ability of MDA-MB-231 cells in a concentration-dependent manner. Antimigration and antiangiogenesis activities were further investigated using noncytotoxic concentrations of OIE ranging from 25 to 150 μg/mL. OIE greatly reduced the migration of MDA-MB-231 breast cancer cells in a dose-dependent manner. OIE significantly suppressed the MDA-MB-231-induced angiogenesis, and there was no substantial toxic effect on natural angiogenesis. Interestingly, the concentration of OIE at 150 μg/mL was as practically potent as pazopanib, the positive anticancer drug, at 4.37 μg/mL in inhibiting MDA-MB-231 cell migration and angiogenesis induced by these cells. Therefore, the inhibitory effects of OIE in cell proliferation and cell migration, together with antiangiogenesis in MDA-MB-231 breast cancer cells, suggesting that OIE has the potential to be a novel adjunct candidate for breast cancer chemotherapeutic agents.


Introduction
Breast cancer is the leading cause of cancer-associated mortality among women [1]. Breast cancer treatment can become complicated if the tumor cells metastasize to distant body organs [1,2]. According to Hanahan and Weinberg, inducing angiogenesis and the ability to invade surrounding tissues and metastasize are hallmarks of tumor malignancy [3]. Typically, cancer cells secrete various proangiogenic factors such as vascular endothelial growth factors (VEGFs), fbroblast growth factor-2 (FGF-2), and transforming growth factor-alpha (TGF-α), which can activate proangiogenic signalling pathways to promote tumor growth, invasion, metastasis, and angiogenesis [4]. An increased level of angiogenesis is associated with decreased survival in breast cancer patients [5]. If angiogenesis is a critical and rate-limiting step in tumor progression, then blocking angiogenesis should inhibit cancer progression. It is, therefore, essential to develop new agents with high efcacy to block cancer angiogenesis and metastasis processes explicitly.
Oroxylum indicum (L.) Kurz (O. indicum) has been used in the Indian Ayurvedic medicine system to cure stomach problems, diarrhea, dysentery, and rheumatism [6,7]. Numerous studies conducted on phytochemical investigations of O. indicum clearly demonstrated the abundance of natural favonoids such as baicalein, chrysin, and oroxylin A [6,8,9]. O. indicum and its favonoid constituents have been reported to possess a wide range of biological and pharmacological activities [8,[10][11][12][13][14][15]. Over recent years, O. indicum has attracted a growing interest in treating various cancer cells, focusing on inhibiting cell division and proliferation [6,[16][17][18][19][20]. Talari et al. have demonstrated the antiangiogenic efect of O. indicum on VEGF-induced neovascularization in the CAM model [21]. However, aggressive breast cancer cells may also express many proangiogenic factors which induce cancer cells to form new blood vessels as a route for metastasis [4]. Terefore, we frst investigated the antiangiogenesis efect of OIE on aggressive MDA-MB-231-induced neovascularization in the CAM model and assessed the efect of OIE on cell proliferation, colony formation, and migration of these cells.

Cell Lines.
Te human breast cancer MDA-MB-231 cell line was purchased from the American Type Culture Collection (ATCC). Te cells were grown in L-15 supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 IU/ mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine. Cultures were maintained in a humidifed atmosphere with 5% CO 2 at 37°C.

Preparation of O. indicum Extract (OIE). O. indicum
(fruit pods) fresh samples were purchased from the local market in Wang Nam Khiao District, Nakhon Ratchasima Province, Tailand. Te voucher specimens were kept in the fora of the Suranaree University of Technology Herbarium (SOI0808U). Te plant extraction was conducted according to a previous study [22]. Te O. indicum extract (OIE) was stored at −20°C until use in subsequent experiments.

Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF-MS) Analysis.
Te phytochemical profling of OIE was analyzed using LC-QTOF-MS/MS in negative mode. Te analyses were performed on the Dionex Ultimate 3000 UHPLC system (Dionex, USA) coupled with an electrospray ionization (ESI) tandem mass spectrometer (micrOTOF-Q II) (Bruker, Germany). A sample injection volume of 5 μL was used for chromatographic separation of analytes using a Zorbax SB-C18 (250 mm × 4.6 mm × 3.5 μm (Agilent Technologies, USA)) and a gradient program including deionized water containing 0.1% formic acid (FA) as solvent A and acetonitrile containing 0.1% formic acid (FA) as solvent B. Te fow rate of the mobile phase was fxed at 0.8 mL/min and the temperature of the column was fxed at 35°C. Te gradient program was optimized by passing through the reservoir 30% B, reaching 80% B at 30 min, and holding until 38 min, reducing at 30% B in 2 min, and holding until the run ended at 45 min. Te LC-QTOF data were collected and processed by Compass 1.3 software (Bruker, Germany). Apiin, luteolin, quercetin, apigenin, kaempferol, baicalein, and oroxylin A were used as standard reference compounds. Te calibration curves were constructed from peak areas of diferent concentrations of the reference standard (from 1 μg/mL to 100 μg/mL), and the concentrations of targeted compounds were calculated based on the equation for linear regression obtained from the calibration curves. . Te cell counting kit-8 (CCK-8, Cat. No. E-CK-A362, Elabscience Biotechnology Inc.) was performed to determine the cell viability of MDA-MB-231 cells after being treated with OIE, as previously described by Paquette et al. [23]. Briefy, MDA-MB-231 cells (1.5 × 10 4 cells/well) were seeded in a 96-well plate for 24 hrs and treated with various concentrations of OIE. After 24 hrs, 10 μL of CCK-8 solution was added to the cells and further incubated for 2 hrs at 37°C. Te absorbance was measured at 450 nm using a microplate reader (Bio-Rad, USA).

Colony Formation Assay.
Te efect of OIE on the ability of MDA-MB-231 cells to form colonies was investigated using a colony formation assay as previously described by Brix et al. [24]. MDA-MB-231 cells (500 cells/well) were seeded for 24 hrs. Ten, the cells were treated with the indicated concentration of OIE (50, 150, 300, and 600 μg/mL), baicalein 3.75 μg/mL, or pazopanib 4.37 μg/mL for 24 hrs. After incubation, the culture medium was removed and washed twice with PBS. Te culture medium was replaced every 2 days for two weeks. Ten, the cells were washed twice with PBS, fxed with methanol: acetone (3 : 1), and stained with a 0.5% crystal violet solution for 15 min at room temperature. Te number of colonies was photographed and counted.

Cell Migration Assay
2.6.1. Wound Healing Assay. Te efect of OIE on directional cell migration in vitro was evaluated by the wound healing assay [25]. Briefy, MDA-MB-231 cells were seeded at a density of 1 × 10 5 cells/well in a 6-well plate and grown for 24 hrs. Next, a linear scratch wound was created across the middle of the well's surface using a 200 μL pipette tip. After that, the culture medium was replaced with a fresh culture medium containing OIE (50, 150, 300, and 600 μg/ mL), baicalein 3.75 μg/mL, or pazopanib 4.37 μg/mL, and further incubated for 24 hrs. At predetermined time points (18 and 24 hrs), the images of cells that migrated into the denuded areas were taken using an inverted microscope with 40× magnifcation equipped with a DinoEye digital eyepiece. Te widths of the wounds were measured using ImageJ software.

Transwell Migration Assay.
Te efect of OIE on the migration ability of MDA-MB-231 cells and the in vitro migration assay were performed using the Transwell chamber system (6.5 mm diameter, 0.4 μm pore size). Briefy, MDA-MB-231 cells (1 × 10 4 cells/well) were seeded on the upper well of chambers placed in a 24-well plate in 450 μL of serum-free medium containing indicated concentrations of OIE (25, 50, 100, and 150 μg/mL), baicalein 3.75 μg/mL or pazopanib 4.37 μg/mL. Te lower chamber was flled with 750 μL of culture medium supplemented with 10% FBS. Cells were incubated undisturbed in the CO 2 incubator for 18 hrs. Te inserts' cells on the upper side were removed with a cotton swab. Te migrated cells on the lower surface were fxed and stained with crystal violet. Te images were captured, and the numbers of migrated cells were counted in four diferent microscopic felds. Results are expressed as the percentage of migrated cells compared to the control.

Te Toxicity of OIE on Natural Angiogenesis in the
Chick Chorioallantoic Membrane (CAM) Model. Te CAM assay was performed to evaluate the efect of OIE on natural angiogenesis at 24 and 48 hrs postexposure. Briefy, the fertilized chicken eggs were used after incubating them for 4 days. A 2 cm 2 window was made in the shell to create a pocket to expose the CAM. A flter disc in the presence or absence of OIE with indicated concentrations (25, 50, 100, and 150 μg/mL), baicalein 3.75 μg/mL or pazopanib 4.37 μg/ mL was placed upon the CAM, and 20 μL of 100 U/mL penicillin was immediately added. Te exposed hole in each egg was closed with tape and further incubated for 24 and 48 hrs. After incubation, the CAM images were taken using a stereomicroscope with 40× magnifcation equipped with a DinoEye digital eyepiece and photographed. Angiogenesis was quantitated by counting the number of blood vessels in direct contact with the flter disk.

Te Antiangiogenic Efect of OIE on MDA-MB-231-Induced Angiogenesis in the CAM Model.
Te inhibitory efect of OIE on MDA-MB-231-induced angiogenesis was evaluated in the CAM model. Shortly, MDA-MB-231 cells at a density of 1 × 10 6 cells were placed directly onto the CAM of the fertilized chicken eggs. Ten, a flter disc in the presence or absence of OIE (25, 50, 100, and 150 μg/mL), baicalein 3.75 μg/mL, or pazopanib 4.37 μg/mL, was placed upon the CAM before adding 20 μL of 100 U/mL penicillin. Te window was sealed with tape and incubated for 24 and 48 hrs. Angiogenesis at each time point was quantifed.

Statistical
Analysis. All statistical signifcance (Statistical Package for the Social Sciences, version 19) was determined by performing a one-way analysis of variance (ANOVA) with a Tukey's post hoc analysis to determine diferences among each treated and control group. Values were considered statistically signifcant when P < 0.05, and data was representative of at least three independent experiments.

LC-QTOF-MS Analysis of OIE.
Te peak chromatograms of OIE and reference standards are presented in Figure 1, while the quantitative results are presented in Table 1. Among the seven reference standards used, it was clear that the six major identifed favonoid compounds were luteolin, quercetin, apigenin, kaempferol, baicalein, and oroxylin A. Te highest of these compounds was baicalein, and it was found that an OIE concentration of 94.22 μg/mL consisted of baicalein at 3.75 μg/mL.

OIE Inhibited the Colony-Forming Ability of MDA-MB-231 Cells.
Te results showed that OIE treatment signifcantly inhibited the colony-forming ability of MDA-MB-231 cells starting from 150 to 600 μg/mL compared to the control (P < 0.05; Figures 3(a) and 3(b)). Te result showed that baicalein decreased the MDA-MB-231 colony formation by approximately 10% (Figure 3(b)). Interestingly, OIE at a concentration of 600 μg/mL was virtually potent as the anticancer drug, pazopanib, in inhibiting MDA-MB-231 colony formation.

Discussion
Breast cancer is the second-most common life-threatening disease in women worldwide [26]. Te two main reasons for the high mortality rates associated with breast cancer and the leading causes of poor clinical outcomes are invasion and metastasis [27]. Tis study was particularly interested in using highly metastatic, aggressive breast cancer, MDA-MB-231 cell lines, as a model for investigating the anticancer activity of OIE. Before investigating anticancer activity, we tested the cytotoxicity of OIE against MDA-MB-231 cell lines. In a subsequent experiment, the noncytotoxic concentration of OIE up to 600 μg/mL was chosen to treat MDA-MB-231. Cancer is any one of many diseases characterized by the development of abnormal cells that divide uncontrollably [3].

Evidence-Based Complementary and Alternative Medicine
Te ability to self-renew and diferentiate gives rise to the heterogeneous phenotype of the tumor cells. Tese cells are believed to be involved in cancer metastasis, recurrence, and therapy resistance [28]. Te colony formation assay was applied to detect the ability of single cells to survive and reproduce to form colonies after OIE treatment. Tis result revealed that OIE demonstrated a long-term suppressive efect on cell proliferation of MDA-MB-231 cells in a concentration-dependent manner. Cell migration and invasion play an essential role in the development of cancer. Regardless of advancements in local cancer treatments, there is a higher chance of clinical challenges in combating systemic metastatic disease [29].
Te wound healing assay is straightforward and gives valuable initial information about cell front migration but does not provide dynamic information. Te transwell migration was further used to analyze the ability of a single cell to respond to chemoattractant directionally. Te wound healing and transwell migration experiments were performed on MDA-MB-231 breast cancer cells to investigate the antimigration activity of OIE. To compromise on excluding the cytotoxic MDA-MB-231, a concentration of up to 150 μg/mL of OIE was selected for investigation in the experiment. Te wound healing assays showed that OIE signifcantly suppressed cell migration of MDA-MB-231 breast cancer cells in a dose-dependent manner (Figures 4(a [19,20]. Expressions of MMP-9 and ICAMP1 are closely linked to the growth, invasion, metastasis, and angiogenesis of cancer cells [30][31][32]. Tis literature triggers us to believe that O. indicum may also be able to reduce angiogenesis. Terefore, the antiangiogenic efects of OIE were further investigated using the CAM model. Angiogenesis is a term that describes the formation of new blood and lymphatic vessels from pre-existing vasculature. Angiogenesis plays a crucial role in tumor growth,   Evidence-Based Complementary and Alternative Medicine invasion, and metastasis of cancer diseases; therefore, inhibiting angiogenesis could be a strategy to arrest tumor growth and metastasis [33,34]. Te present study elucidated for the frst time the antiangiogenesis properties of O. indicum toward breast cancer cells-induced angiogenesis. Te antiangiogenesis study revealed that OIE showed a full antiangiogenesis efect without any substantial toxic efect on the cells. Tese results substantially agree with the results of Talari et al. [21], showing that O. indicum exhibits the antiangiogenic efect on VEGF-induced neovascularization in the CAM model. In addition, this study also elucidates for the frst time the antiangiogenesis efect of O. indicum on breast cancer cell-induced angiogenesis in the CAM model. In this study, the analysis of phytochemical constituents using the LC-QTOF-MS instrument attested to the presence of baicalein (39.75 μg/mg of OIE), with a high amount in OIE. It was found that an OIE concentration of 94.22 μg/mL consisted of baicalein at 3.75 μg/mL. Tese results correspond with the previous study of Dunkhunthod et al. [8,11] that the amount of baicalein contains in OIE with a concentration range of 25.50-32.85 μg/mg of the extract. To clarify whether baicalein contributed to the anticancer activity of OIE or not. So baicalein was used as a positive control. Many studies have shown that baicalein exhibits anticancer properties, which are attributed at least partially to the inhibition of cell proliferation, cell migration, and invasion in many cell types, including AGS human gastric cancer cells, DLD1 colorectal cancer cells, B16F10 melanoma cells, vein endothelial cells (HUVECs), and MDA-MB-231 breast cancer cells [28,[35][36][37]. Likewise, many researchers have also reported the antiangiogenic activity of baicalein [38][39][40]. Similar to other investigators, this study further confrmed the anticancer activity of baicalein since baicalein showed the inhibition of colony formation, cell migration, and angiogenesis in breast cancer MDA-MB-231 cells. Compared to OIE, baicalein (3.75 μg/ mL) alone showed a lower anticancer potency than OIE at 100 and 150 μg/mL concentrations throughout each investigation. Te higher strength of OIE than baicalein may be due to OIE's other bioactive compounds, such as luteolin, apigenin, quercetin, and other compounds. Tese compounds have been demonstrated to possess anticancer properties and suppress metastasis and angiogenesis progression in several cancers [41][42][43][44]. Tese reports lend support to the assumption that synergistic activity may occur between baicalein and other favonoid compounds in enhancing the anticancer activity of OIE.
In the present study, the anticancer mechanism of OIE was compared by using pazopanib as a positive anticancer drug. Pazopanib is a pan-vascular endothelial growth factor receptor inhibitor, and preclinical work indicates that pazopanib exerts an anticancer efect by inhibiting both angiogenic and oncogenic signalling pathways [45,46]. As expected, pazopanib exhibits a strong anticancer potency by suppressing colony formation, cell migration, and angiogenesis in breast cancer MDA-MB-231 cells. Interestingly, 24

Conclusions
Te fndings in the current study lead us to believe that OIE possesses anticancer activity by suppressing colony formation, cell migration, and MDA-MB-231-induced angiogenesis. Te present comprehensive study provides evidence that baicalein is also responsible for OIE's anticancer activity. Te underlying mechanism behind the antimigratory and antiangiogenic efects of OIE in MDA-MB-231 breast cancer cells should be further investigated. Nevertheless, this inhibition of cell proliferation, cell migration, and tumor-induced angiogenic activities in OIE could explain, at least in part, the claimed anticancer activity of O. indicum.

Data Availability
Te datasets used and analyzed during this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that there are no conficts of interest.