Effects of Luteolin on Human Breast Cancer Using Gene Expression Array: Inferring Novel Genes

Taraxacum officinale (dandelion) is often used in traditional Chinese medicine for the treatment of cancer; however, the downstream regulatory genes and signaling pathways mediating its effects on breast cancer remain unclear. The present study aimed to explore the effects of luteolin, the main biologically active compound of T. officinale, on gene expression profiles in MDA-MB-231 and MCF-7 breast cancer cells. The results revealed that luteolin effectively inhibited the proliferation and motility of the MDA-MB-231 and MCF-7 cells. The mRNA expression profiles were determined using gene expression array analysis and analyzed using a bioinformatics approach. A total of 41 differentially expressed genes (DEGs) were found in the luteolin-treated MDA-MB-231 and MCF-7 cells. A Gene Ontology analysis revealed that the DEGs, including AP2B1, APP, GPNMB and DLST, mainly functioned as oncogenes. The human protein atlas database also found that AP2B1, APP, GPNMB and DLST were highly expressed in breast cancer and that AP2B1 (cut-off value, 75%) was significantly associated with survival rate (p = 0.044). In addition, a Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the DEGs were involved in T-cell leukemia virus 1 infection and differentiation. On the whole, the findings of the present study provide a scientific basis that may be used to evaluate the potential benefits of luteolin in human breast cancer. Further studies are required, however, to fully elucidate the role of the related molecular pathways.


Introduction
Breast cancer is the most common type of cancer among women worldwide;~2.1 million women are affected by breast cancer each year. In addition, breast cancer has the highest mortality rate among all cancers. In 2018, an estimated 600,000 women worldwide succumbed to this disease, accounting for~15% of all cancer-related deaths among women. According to the statistics, although the incidence of breast cancer in women in more developed regions is higher, the incidence of breast cancer is increasing in almost all regions worldwide [1,2]. Breast cancer often metastasizes to distant organs, such as the bones, liver, lungs and brain, which is the primary cause of treatment failure. Currently, mammography is the most basic screening method for detecting breast cancer; mammography can detect breast cancer at an early stage and can thus effectively reduce the mortality rate [3,4]. According to a previous statistical analysis, breast cancer is currently the leading cause of cancer-related mortality among women in Taiwan and the fourth leading cause of cancer-related mortality among women worldwide; the number of deaths is also increasing annually [5]. The early diagnosis of the disease can result in a good prognosis and a high survival rate. Thus, screening is crucial for early detection [6,7].
Luteolin (3 ,4 ,5 ,7 -tetrahydroxyflavonoid) is the main component of the leaves of Taraxacum officinale [8]. Taraxacum officinale, commonly known as dandelion, is a perennial herb belonging to the Asteraceae family [9]. It is native to Eurasia but has been found worldwide. It has been used as a traditional and modern herbal system in several countries. These ingredients are non-toxic and can be used as anti-inflammatory drugs; diuretics; digestive stimulants; insulin stimulants; and antitumor, antidiabetic and antioxidant agents as well as to prevent liver and testicular damage [10][11][12][13][14]. Luteolin is a common flavonoid [15,16] that can be found in a number of plants, fruits, vegetables and herbs [17]. It was previously found that luteolin can increase the effectiveness of paclitaxel to induce breast cancer cell apoptosis by inhibiting STAT3 [18]. In addition, Taraxacum officinale leaves include caffeic and chlorogenic acid [8]. Caffeic acid inhibits cell proliferation by mediating estrogen receptor levels in human breast cancer cells [19]. Chlorogenic acid induces apoptosis by regulating NANOG, POU5F1 and SOX2 in human lung cancer cells [20].
Although several studies have investigated the mechanisms and cellular responses of luteolin in cancer, these mechanisms are complex and have not been fully elucidated. Therefore, the present study performed a comprehensive analysis of various genetic expression modifications under treatment with luteolin using full gene array and bioinformatic analyses.

Cell Culture and Reagent
The human breast cancer cell lines MDA-MB-231 and MCF-7 were obtained from the American Type Culture Collection (ATCC). The cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin and incubated at 37 • C in 5% CO 2 . Luteolin, caffeic acid and chlorogenic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell Viability Assay
The cell viability assay was performed using a Cell Counting Kit-8 (CCK-8) according to the manufacturer's protocol. Briefly, 1 × 10 3 MCF-7 and MDA-MB-231 cells were plated in 96-well plates and incubated with luteolin at various concentrations (0, 10, 20, 40 and 80 µM) and for different periods of time (24,48 and 72 h). After specifying the conditions, the medium was removed, and the cells were incubated with CCK-8 reagent diluted in culture medium at 37 • C for 2 h. The absorbance was analyzed at 450 nm using a microplate reader.

Wound Healing Assay
Breast cancer cells (5 × 10 5 ) were seeded in six-well plates. Following 24 h of incubation, scratches were created in each well using a sterile yellow pipette tip, and images of the edge of the scratch to the wounded area in the wells were acquired using an inverted microscope at 0 and 48 h.

Array Hybridization and Data Analysis
Gene expression profiles were analyzed using an Agilent Technologies array SurePrint G3 Human Gene Expression 8 × 60 K microarray according to the manufacturer's instructions. For transcription, Cy3-labeled RNA was amplified with a Low Input Quick-Amp Labeling kit (Agilent Technologies, Inc., Santa Clara, CA, USA). Fragmented, labeled cRNA was hybridized to an Agilent SurePrint Microarray, and scanned images were analyzed using Feature Extraction 10.7.3.1 software (Agilent Technologies, Inc.). Differential gene expression was normalized by quantile normalization of the raw signal data. The cluster-Profiler package was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses by Welgen, Inc. (Worcester, MA, USA) A flowchart illustrating the gene expression profiling process is shown that total RNA was extracted from the cells and sent for whole-genome microarray analysis followed by bioinformatics analyses.

Statistical Analysis
The data are the mean ± SD from three independent experiments. * p < 0.05 vs. untreated control (the data were analyzed using a two-tailed Student's t-test). In addition, data from the human protein atlas database were analyzed by descriptive statistics programs in GraphPad.

Luteolin Inhibits the Viability and Wound Healing Ability of Human Breast Cancer Cells
To examine the effects of luteolin on breast cancer, three main ingredients of Taraxacum officinale leaves including luteolin, caffeic and chlorogenic acid were used and cell viability was examined using CCK-8 assays. The results revealed that only luteolin significantly inhibited the viability of the MDA-MB-231 estrogen receptor (ER) -/progesterone receptor (PR) -/human epidermal growth factor receptor (HER -) and MCF-7 (ER + /PR + ) cells ( Figure 1A,B). The concentration-and time-dependent effects of luteolin on the viability of MCF-7 and MDA-MB-231 cells were further analyzed. Similarly, it was found that luteolin more effectively inhibited cell viability in a time-and concentration-dependent manner ( Figure 1C,D). In addition, the effects of luteolin on cell motility were analyzed using a wound healing assay. The results revealed that luteolin inhibited cell motility and migration ( Figure 1E-H). These results suggested that luteolin can effectively inhibit breast cancer cell proliferation and motility.

Identification of Potential Gene Expression Alterations in Luteolin-Treated Breast Cancer Cells
In the past, the majority of studies on luteolin in breast cancer focused on triplenegative breast cancer. Therefore, in the present study, a whole-genome microarray was used to analyze the genes regulated by luteolin in the MCF-7 and MDA-MB-231 cells ( Figure 2).

Validation of Differentially Expressed Genes (DEGs) in Response to Luteolin
Subsequently, network analyses of the 41 genes with a differential expression mediated by luteolin were performed using ingenuity pathway analysis (IPA) software. The biological processes and molecular function terms, i.e., 'molecular function (MF)', 'biological process (BP)' and 'cellular component (CC)', associated with the DEGs in the luteolin-treated breast cancer cells are listed in Figure 4 and Table 2. The biological processes and molecular functions associated with DEGs in the luteolin-treated breast cancer cells are listed in Table 2   Further assessment of the microarray analysis results revealed that the AP2B1, APP, GPNMB and DLST genes were consistently identified in the MF, BP and CC categories ( Figure 4A). To confirm the reliability of the fold changes identified with microarray analysis, the AP2B1, APP, GPNMB and DLST genes were analyzed using RT-qPCR. The results revealed that all the examined genes were downregulated by luteolin treatment in the MDA-MB-231 and MCF-7 human breast cancer cell lines ( Figure 4B).

External Validation of AP2B1, APP, GPNMB and DLST in Cell Viability, Human Protein Atlas (HPA) Databases and KEGG Pathways
To further verify the effects of four genes AP2B1, APP, GPNMB and DLST on breast cancer, plasmids of the four genes were transfected into breast cancer cell lines and gene overexpression was analyzed using RT-qPCR ( Figure 5A). Cell viability assay revealed that AP2B1, APP, GPNMB and DLST increased the growth of the MDA-MB-231 ( Figure 5B) and MCF-7 cells ( Figure 5C). In addition, the HPA database demonstrated [21] that AP2B1, APP, GPNMB and DLST were highly expressed in breast cancer ( Figure 5D); the increased expression of AP2B1 (cut-off value, 75%) was significantly associated with the survival rate (p = 0.044) of 1075 patients with breast cancer ( Figure 5E). The data suggested that the four genes, AP2B1, APP, GPNMB and DLST, were highly expressed in breast cancer and that AP2B1 was a suitable prognostic marker for breast cancer. The pathway network of these candidate genes mediating the response to luteolin is illustrated schematically in Figure 6.  Table 3).

Discussion
Natural herbs and plants continue to play an important role in drug research and development, particularly in cancer research. Over the past 20 years, 50% of all anticancer drugs approved worldwide are natural products or components of natural products [22]. The present study found that luteolin inhibited breast cancer cell proliferation and invasion and comprehensively surveyed the differential expression of mRNAs in mediating the effects of luteolin on MDA-MB-231 and MCF-7 cells using microarray and bioinformatics analyses. Previous studies have found that luteolin inhibits inflammation, allergies and cancer through its antioxidant and pro-oxidative abilities. It has been used in traditional Chinese medicine to prevent and treat various diseases including hypertension, inflammatory diseases, Alzheimer's disease [23] and many types of cancer [17]. Recent studies have also found that luteolin significantly inhibits cancer cell proliferation and metastasis through the AKT/mTOR/MMP9 signaling pathway in androgen receptor-positive triplenegative breast cancer. It has also been demonstrated that luteolin inhibits the in vitro and in vivo proliferation of lung cancer cells by targeting LIMK1 [24]. The present study identified four genes (AP2B1, APP, GPNMB and DLST) that were significantly downregulated by luteolin in breast cancer cell lines. These genes were associated with drug resistance, cell proliferation, macrophages, apoptosis and HDAC inhibition.
AP2B1 is a subunit of adaptor-related protein complex 2, a clathrin assembly protein complex, and is involved in clathrin-mediated endocytosis. The resistance phenotype to cancer drugs is associated with an increase in the protein level of AP2B1 in non-small cell lung cancer [25]. A previous study found that luteolin induced apoptosis by blocking the PI3K/AKT/mTOR pathway in tamoxifen-resistant breast cancer cells [26]. This result led to the hypothesis that luteolin can influence drug resistance by regulating AP2B1. APP is an evolutionarily conserved protein. APP is overexpressed in several types of cancer, including breast, colon, lung and pancreatic cancer, and promotes cell proliferation, invasion and migration. In breast cancer, APP has been shown to mediate cell proliferation and motility through the AKT signaling pathway [27,28]. In addition, APP has been shown to regulate proliferation through the ERK signaling pathway in colon cancer [29,30]. APP also increases proliferation and leads to cell size abnormalities in both lung and pancreatic cancer [31][32][33]. The same results were also found in the present study; in addition, it was found that luteolin inhibited the proliferation and invasion of breast cancer cells.
GPNMB triggers the self-renewal of spheroids to increase cell survival and the tumorforming ability of macrophages interacting with tumor cells, and GPNMB activates the expression of the IL-33cytokine and its receptor, IL-1R1L, through CD47 and is sufficient to induce tumor spheroid formation [34]. In addition, GPNMB is a prognostic indicator and is assessed to identify recurrence in triple-negative breast cancers [35]. A previous study also found that the HDAC inhibitor, trichostatin A, induced apoptosis by inhibiting GPNMB expression in gastric cancer [36]. Dihydrolipoamide S-succinyltransferase (DLST) is the E2 transferase of the α-ketoglutarate (α-KG) dehydrogenase complex. Silencing DLST induces apoptosis and decreases cell growth in T-cell acute lymphoblastic leukemia [37]. It has been demonstrated that luteolin induces apoptosis and autophagy by regulating the p38, JNK and Akt pathways in macrophage-related diseases [38]. In addition, luteolin mediates apoptosis through the modulation of histone H3 acetylation activity in leukemia cells [39].
In KEGG pathway analysis, the present study found that luteolin-mediated DEGs were involved in T-cell leukemia virus 1 infection and differentiation. A previous study demonstrated that luteolin mediated the Th1/Th2 imbalance through the TLR4/NF-κB signaling pathway. Furthermore, luteolin inhibited the expression of Th2 transcription factor GATA-3 to inhibit the differentiation of T cells into a Th2 subset. Therefore, it is suggested that luteolin plays a crucial role in the differentiation of T cells [40]. Luteolin has also been shown to significantly activate the expressions of cyclooxygenase 2, phosphorylated STAT1 and phosphorylated STAT3 and to promote the differentiation of Th cells into Th17 cells [41]. Therefore, it was considered that luteolin plays a main role in the differentiation of T cells.

Conclusions
In conclusion, the present study reported the upregulated/downregulated mRNAs regulated by luteolin in MDA-MB-231 and MCF-7 breast cancer cells. These results provide novel insight into the effects of luteolin on breast cancer and may aid in the development of novel therapeutic strategies to prevent and treat breast cancer.