Lotus Leaf Extract Inhibits the Cell Migration and Metastasis of ER- Breast Cancer

Background: Patients with estrogen receptor negative (ER-) breast cancer have poor prognosis because of their high rates of metastasis. However, there is no effective treatment and drugs for ER − breast cancer metastasis. Our purpose of this study was to evaluate the effect and mechanism of lotus leaf alcohol extract (LAE) on the cell migration and metastasis of ER- breast cancer. Methods: The anti-migratory effect and mechanism of LAE were analysed in ER- breast cancer cells including SK-BR-3, MDA-MB-231 and HCC1806. Cell viability assay, wound-healing assay, RNA-sequence analysis and immunoblotting assay were applied in examining the cytotoxicity, anti-migratory effect and its possible pathways of LAE. To further investigate the inhibitory effect of LAE on metastasis in vivo , subcutaneous xenograft nude mice model and intravenous injection nude mice model were established. Lung and liver tissues were analysed by the Hematoxylin & eosin staining and immunoblotting assay. Results: We found that lotus leaf alcohol extract (LAE), not nuciferine, inhibited cell migration significantly in SK-BR-3, MDA-MB-231 and HCC1806 breast cancer cells, and did not change viability of breast cancer cells. The anti-migratory effect of LAE was dependent on TGF-β1 signaling, while independent of Wnt signaling and autophagy influx. Intracellular H 2 O 2 participated in the TGF-β1-related inhibition of cell migration. LAE inhibited significantly the breast cancer cells metastasis in mice models. RNA-sequence analysis showed that extracellular matrix signaling pathways are associated with LAE-suppressed cell migration. Conclusions: Our findings demonstrated that lotus leaf alcohol extract inhibits the cell migration and metastasis of ER- breast cancer via TGF-β1/Erk1/2 and TGF-β1/SMAD3 signaling, which provides a potential therapeutic strategy for ER- breast cancer.


Introduction
Breast cancer is usually classified into three subtypes according to the expression of hormone receptors: estrogen receptor positive/progesterone receptor positive (ER+/PR+), human epidermal growth factor receptor positive (HER2+) and triple negative (TNBC) [1]. Among the three subtypes of breast cancer cells, ER-breast cancer such as HER2 + and Triple negative breast cancer cells have a higher migration capacity than that of ER+/PR + breast cancer cells. In breast cancer patients, HER2 + subtypes of breast cancer accounts for 20-30%, which is an invasive tumor with shorter metastatic time and poor prognosis compared to other two subtypes of breast cancer patients [2]. In addition, Triple negative breast cancer (TNBC) is considered as the most severe subtypes of breast cancer because of high rate of recurrence and metastasis [3]. However, there is no effective treatment and drugs for ER − breast cancer metastasis.
About 90% death rates of breast cancer are resulted from metastasis. It's a complicated process associated with NF-κB, TGF-β1 and Wnt/β-catenin signaling pathways [4][5][6]. TGF-β1 signaling pathway is a pivotal signal for cell migration in breast cancer. TGF-β enhances ROS levels by enhancing production and reducing antioxidative/scavenging systems activity [7][8]. And increased ROS levels in turn may promote TGF-β expression and stimulate the release of TGF-β from the secreted complex in cell [9]. Moreover, several studies have demonstrated that ROS is associated with carcinogenesis, abnormal growth, and angiogenesis, especially metastasis [10][11][12][13][14]. Thus, inhibition of breast cancer cell metastasis by TGF-β as well as ROS pathway may become a promising strategy for the treatment of ER − breast cancer patients.
Lotus leaf (Nelumbo nucifera Gaertn) is a traditional Chinese medicine, also called He-Ye, which has a long history of usages against oxidation, diabetes, obesity and immunomodulatory effects in China [15]. Sometimes lotus leaf is also used as a seasoning in food due to its unique aroma. Considerable literatures have demonstrated that the main compounds contained in the lotus leaf are nuciferine, quercetin, quercetin-3-Oglycoside, kampherol-3-O-glycoside, and myricetin-3-O-glucoside [16]. These compounds from the extracts of lotus leaves, seed and rhizome display cytoprotective, anti-bacterial, anti-obesity especially anti-oxidant pharmacologic activities partly through the extracellular signal-regulated protein kinases like Erk1/2 and PKC [17][18][19][20][21]. However, the effect of lotus leaf extract on cancer metastasis is still remained unclear. Given that the important role of Erk1/2 and ROS in TGF-β-related cancer metastasis [22][23], we tested the effect of LAE on ER − breast cancer metastasis.
In this study, our data showed that LAE significantly inhibits the ER − breast cancer cell migration and metastasis via the SMAD3 and Erk1/2, accompanied by reduced intracellular H 2 O 2 level in ER-breast cancer cells. RNAsequence analysis also showed that extracellular matrix signaling pathways and FAK might be another possible pathways to inhibit the cell migration induced by LAE. These results suggested that LAE has the potential to screen out novel drugs for the treatment of ER − breast cancer clinically.

LAE treament
The dry lotus leaf (Nelumbo nucifera Gaertn, http://www.theplantlist.org) powder was purchased from Tong Ren Tang Group in Wuxi, China, in in November 2017 (Latitude: 31°33′17.44″N;Longitude:120°19′15.24″E, organization code 101461180). The company is approved by the competent department, ensuring the good production and quality according to the approved content. All production lines had passed the national GMP recognition. Then, the lotus leaf were identified in National Functional Food Engineering Technology Research Center in Jiangnan University (China).
Lotus leaf alcohol extract: The lotus leaf powder and the 75% aqueous ethanol solution were uniformly mixed at a ratio of 1:20, and shaken at 37 o C for 12 hours. Then separated by filtration, and the obtained residue was repeatedly extracted twice. All the collected filtrate was concentrated to dryness by evaporation on a rotary evaporator, and then dissolved in DMSO and filtered by a membrane filter with 0.22 µm pore size (Millipore, USA). Finally LAE was diluted into several concentrations (10,25,50, 100, 250 µg/ml) for cell treatments and stored at 4 o C.

Human breast cancer xenograft model
This study was approved by the ethics committee of Jiangnan University with protocol number SYXK2016-0045. Female BALB/c nude mice, 4 weeks of age, were purchased from Lingchang Biotechnology Co., Ltd. (Shanghai, China). All the experiments were in accordance with national institution guidelines. MDA-MB-231 cells (1 × 10 6 ) suspending in medium were subcutaneously injected in the right flank of mice. After one week of injection, the mice bearing tumor were randomly subdivided into 2 groups and treated with either vehicle (model group) or LAE (0.5% w/w). Tumor dimensions were measured with vernier caliper and tumor volumes were estimated by the formula: length × width^2 × 0.5. Body weights and tumor growth were recorded twice a week. The mice were sacrificed after 56 days, and visible metastatic tumors were counted and subjected to further analysis. suspending in PBS were injected through the tail vein of mice. Then, all the mice were randomly subdivided into 2 groups (10 mice/group) and treated with either vehicle (model group) or LAE (0.5% w/w). Body weights were scaled once a week. The mice were sacrificed after 41 days, and the metastatic tumors and the organs with visible metastatic tumors were analyzed.

Cell viability assay
Cell Counting Kit 8 (CCK8) was purchased from Dojindo Molecular Technology (Tokyo, Japan). Cells were cultured in 96-well plates at a density of 5000 cells per well in 100 µl medium. LAE were added into the wells and incubated for 48 h. Then, cells were added 10 µl CCK8 substrate and incubated for another 2 h at 37 °C. The optical density was measured at 450 nm on a microplate reader Multiskan GO (Thermo Scientific, USA).

Wound-healing assay
Cells were seeded in dish to form a confluent monolayer. Then 200 µl pipette tip was used to make a scratch in the middle of plate, and wash twice with PBS. The monolayer was maintained with the FBS-free media with LAE supplement. Wound closure was monitored after 0 and 36 h. The percent of wound closed are calculated by image J. TGF-β1 was purchased from Peprotech (USA, # 100-21-10) and dissolved in 10 mM citric acid, pH3.0 at 0.1-1.0 mg/ml. Chloroquine diphosphate (CQ) was purchased from Medchem Express (Shanghai, China, cat# HY-17589 ) and dissolved in H 2 O to 10 mg/ml. LiCl was purchased from Sigma-Aldrich (Sigma, St. Louis, MO, USA, # 213233) and dissolved in cell culture medium to 20 mM. Nuciferine was purchased from Medchem Express (Shanghai, China, cat# HY-N0049) and dissolved in DMSO to 1.6925 mM.

H 2 O 2 content
The content of H 2 O 2 was measured by the Hydrogen Peroxide assay kit (Beyotime, # S0038) according to the manufacturer protocols. In brief, test tubes containing 50 µl test solutions were placed at room temperature for 30 min and measured immediately with a spectrophotometer at a wavelength of 560 nm. Absorbance values were calibrated with a standard curve generated with known concentration of H 2 O 2 .

Hematoxylin and eosin staining
For histopathology analysis, lung and liver tissues were fixed by paraformaldehyde and then were dehydrated, embedded in paraffin and cut into serial sections at 5 µm. Sections were stained with hematoxylin and eosin (H&E) solution and observed them under an optical microscope (DP73, OLUMPUS, Japan).

Statistical Analysis
All experiments were performed at least three times and data were presented as mean ± SEM. One-way ANOVA with Dunnett's post-test was performed (*P < 0.05; **P < 0.01; ***P < 0.001).

LAE inhibits ER − breast cancer cells migration independent of nuciferine
To investigate LAE effects on cell migration, metastatic SK-BR-3, MDA-MB-231 and HCC1806 ER − breast cancer cell lines were used in the study. Previous studies have shown that cell proliferation interferes with cell migration. To address this issue, the proliferation of breast cancer cells was examined by CCK8 assay. The viability of SK-BR-3, MDA-MB-231 and HCC1806 cells was similarly unchanged between with low dose (50 and 100 µg/ml) of LAE supplement and without LAE (Fig. 1a). All of SK-BR-3, MDA-MB-231 and HCC1806 cells exhibited no cytotoxicity in morphological observation with 100 µg/ml or less LAE supplement (Fig. 1b). However, cell number was significantly reduced by high doss (250 µg/ml) of LAE supplement for 48 hours (Fig. 1a). These results indicated that low dose LAE did not alter breast cancer cell proliferation.
Next, we examined the LAE role in cell migration by wound healing assays with 50 and 250 µg/ml LAE supplement for 36 hours. Previous studies found that FBS promote cell proliferation to alter cell migration. To rule out the effects of FBS, all experiments were performed in an FBS-free medium. Cell wound healing distance analysis showed that LAE supplement significantly reduced the wound healing distance in three subtypes cells, suggesting cell migration was suppressed (Fig. 1c). Interestingly, 50 or 250 µg/ml LAE supplement showed similarly inhibitory effect on SK-BR-3 cells migration, while LAE supplement inhibited cell migration of MDA-MB-231 and HCC1806 cells in a dose dependent manner.
Nuciferine, a major bioactive component of lotus, inhibits the growth of cancer cells and breast cancerassociated bone loss [24][25]. To verify if nuciferine is involved in the inhibition of cell migration, SK-BR-3, MDA-MB-231 and HCC1806 cells were treated with nuciferine supplement. Cell viability (Supplementary Fig. 1a) and migration ( Supplementary Fig. 1b) were unchanged in the presence of high dose of nuciferine, indicating that nuciferine was not associated with LAE-inhibited migration. Together, these results indicate that LAE supplement suppresses ER − breast cancer cells migration independent of nuciferine.

LAE-inhibited cell migration is independent of autophagy and Wnt signaling
Autophagic process is recognized to suppress cancer metastasis through decreasing EMT [26]. To determine if LAE-suppressed cell migration was regulated by autophagy and EMT-related Wnt/β-catenin signaling pathways directly, we examined β-catenin, snail, p62 and LC3 proteins levels by immunoblotting. Elevated LC3 protein level was detected in cells with high dose (100, 250 µg/ml) of LAE supplement. However, p62 protein level was similarly in cells with low and high dose of LAE supplement ( Supplementary Fig. 1a). To address the controversial results, we used autophagy/lysosome inhibitor chloroquine (CQ) to inhibit autophagy flux. Inhibition of autophagy flux did not alter cell migration, revealed by wound healing distance ( Supplementary Fig. 1b). Unchanged β-catenin and snail protein levels were detected in three subtypes of breast cancer cells with low or high dose of LAE supplement ( Supplementary Fig. 1c). To further determine the role of Wnt signaling on the effect of LAE-inhibited cell migration, LiCl was used to activate the Wnt signaling. Wound healing assays revealed that the activation of Wnt signaling did not reverse the cell migration inhibited by LAE ( Supplementary  Fig. 1d), suggesting LAE-suppressed cell migration was independent of Wnt signaling. Thus, LAE-suppressed cell migration is independent of autophagy and Wnt signaling.

TGF-β1 signaling is associated with LAE-suppressed cell migration
Considerable literatures showed that TGF-β1 is recognized as a major regulator of EMT to regulate tumor metastasis [27][28][29]. Then we tested the protein level of TGF-β1 after treating with LAE. The results showed that TGF-β1 was obviously reduced in SK-BR-3 and HCC1806 cells at both low (even 10 µg/ml) and high concentrations (Fig. 2a). To further determine whether TGF-β1 signal was required for LAE-inhibited cell migration, 10 ng/ml TGF-β1 growth factor with 100 µg/ml LAE was used to treat SK-BR-3, MDA-MB-231 and HCC1806 cells. LAE-inhibited cell migration was significantly restored by the addition of TGF-β1 growth factor, as revealed by wound healing assays (Fig. 2b). Consistent with literature, TGF-β1 growth factor changed cells morphology from epithelial to mesenchymal transition (EMT), which was suppressed by LAE supplement (Fig. 2c). Thus, these observations demonstrate that TGF-β1 signal is involved in LAE-inhibited cell migration via inhibition of EMT.

SMAD3 and Erk1/2 phosphorylation are inhibited
To explore the signaling pathway involved in LAE-inhibited cell migration, we examined the canonical TGF-β1/SMAD3 signaling pathway and the non-canonical TGF-β1/PTEN/AKT and MAPKs signaling pathways [30][31]. Immunoblotting analysis showed that AKT phosphorylation and PTEN levels were unchanged in three subtypes of breast cancer cells with LAE supplement (Fig. 3a). Similar p38 and JNK phosphorylation levels were detected in cells with and without LAE supplement (Fig. 3a). However, the phosphorylated Erk1/2 level was significantly decreased in SK-BR-3 and HCC1806 cells even at a low LAE supplement (10 µg/ml), and mildly reduced in MDA-MB-231 cells (Fig. 3b). Interestingly, with LAE supplement, the phosphorylated SMAD3 level was significantly reduced in SK-BR-3 and MDA-MB-231 cells, but not in HCC1806 cells (Fig. 3c). Moreover, TGF-β1 supplement significantly restored Erk1/2 and SMAD3 phosphorylation levels reduced by LAE supplement in three subtypes of breast cancer cells (Fig. 3d).
Since mutant p53 mediates the TGF-β1 signaling pathway via Erk1/2 as well as SMAD3 signals, and the p53 in all of the three subtypes of breast cancer cells are mutant [32][33][34][35]. To address this issue, we examined the mutant p53 protein level in three subtypes of breast cancer cells with LAE supplement. Although p53 level was unchanged in SK-BR-3 and HCC1806 cells with LAE supplement compared to cells without LAE supplement, immunoblotting analysis showed that p53 level was significantly decreased in MDA-MB-231 cells with LAE supplement in a dose dependent manner (Fig. 3e). It provides another explanation for the effect of LAE on the inhibition of the metastasis in MDA-MB-231 cells via Erk1/2 as well as SMAD3 signals. Above all, these results demonstrate that LAE inhibited the cell migration of ER-breast cancer cells via suppressing the phosphorylation of TGF-β1/Erk1/2 as well as TGF-β1/SMAD3 signals.

LAE inhibits TGF-β1-related cell migration via downregulating hydrogen peroxide
Reactive oxygen species (ROS), especially hydrogen peroxide (H 2 O 2 ) are recognized as associated with tumor metastasis [36][37][38]. TGF-β-related signaling promotes hydrogen peroxide production in several types of cells [39][40][41]. Simultaneously, H 2 O 2 enhances TGF-β-mediated EMT via SMAD and MEK/ERK signaling [42]. To determine if intracellular H 2 O 2 participate in the TGF-β1-related inhibition of cell migration, we first measured H 2 O 2 level in cells with LAE supplement for 48 hours. With LAE supplement, intracellular H 2 O 2 level was significantly downregulated in MDA-MB-231 and HCC1806 cells, but not in SK-BR-3 (Fig. 4a). Consistently, H 2 O 2 scavenger catalase protein level was significantly increased even at a low concentration LAE supplement (10 µg/ml) (Fig. 4b). However, the proteins levels of SOD1, NOX2 and NOX4, responsible for the production of large amounts of ROS, were similarly in cells with or without LAE supplement. Thus, LAE supplement reduced and HCC1806 cells (Fig. 4c). However, SK-BR-3 cells displayed similarly cell migration with or without H 2 O 2 supplement, suggesting other signals involved in LAE-inhibited cell migration. Interestingly, elevated intracellular H 2 O 2 level was significantly detected in MDA-MB-231 and HCC1806 cells with 10 ng/ml TGF-β1 and 100 µg/ml LAE compared to cells with single LAE supplement, but not in SK-BR-3 cells (Fig. 4d). These findings indicate that LAE inhibits TGF-β1-related cell migration via downregulating hydrogen peroxide in MDA-MB-231 and HCC1806.

LAE inhibits tumor metastasis
To investigate the inhibitory effects of LAE on metastasis in vivo, we used subcutaneous xenograft nude mice model to examine tumor metastasis. The schedule of experiments was showed in Figure. 5a. To exclude the toxicity of LAE in vivo, xenograft nude female mice were fed with 0.5% LAE for 56 days and body weight of mice was monitored. Body weight in mice with and without LAE supplement was similar (Fig. 5b), indicating that LAE supplement did not cause significant systemic toxicity. Consistent with the above results, LAE supplement did not affect the growth of in-situ tumor compared to controls (Fig. 5c). However, the number and weight of new metastatic tumors derived from the primary tumor were significantly decreased in mice with LAE supplement compared to controls (Fig. 5d). Immunoblotting assays showed that LAE did not inhibit the phosphorylation of Erk1/2 and SMAD3 in liver. However, LAE supplement significantly downregulated the phosphorylation of Erk1/2 but not the phosphorylation of SMAD3 in lung (Fig. 5e). These data indicate that the effect of LAE on SMAD3 and Erk1/2 is dependent on tissue environment or extracellular signal stimulation.
Next, we used intravenous injection nude mice model to investigate the anti-metastatic role of LAE. The experiments schedule was showed in Fig. 6a. Strikingly, LAE supplement significantly suppressed mice body weight loss induced by tumor compared to control mice (Fig. 6b), which indicated that LAE may have the effect of improving the weight loss in patients with advanced breast cancer. And the number and weight of new formed metastatic tumors were significantly decreased in mice with LAE supplement compared to control animals (Fig. 6c). Interestingly, we found less lung congestion in mice with LAE supplement than that of control animals (Fig. 6d). The number and size of metastatic tumor nodules in lung and liver was decreased significantly. H&E staining revealed inflammatory infiltration and vascular invasion in lung of mice, while LAE supplement improved inflammatory infiltration and vascular invasion in lung of mice (Fig. 6e). In liver, LAE supplement significantly decreased the number of large metastatic tumors. Mice with LAE supplement showed significantly downregulated the phosphorylation of SMAD3 in lung and liver, but not the phosphorylation of Erk1/2 (Fig. 6f). Taken together, these data suggest that LAE inhibited tumor metastasis via suppressing the phosphorylation of SMAD3 and Erk1/2, which is dependent on different tissue environment and extracellular signal.

Extracellular matrix signaling are associated with LAE-suppressed cell migration
To further explore the role of LAE in inhibiting cell migration, we performed RNA-sequence to analysis genes change in SK-BR-3, HCC1806 and MDA-MB-231. After LAE treatment, all the differentially expressed genes were analyzed by (Gene Ontology) GO method in DAVID with the standard of P < 0.001 and FDR < 1. Results showed that extracellular matrix (ECM) organization, oxidation-reduction process, negative regulation of cell migration and cell adhesion were significantly enriched by LAE supplement in MDA-MB-231 ( Supplementary Fig. 2a), and oxidation-reduction process, extracellular matrix organization, cell-cell adhesion and cell migration were significantly enhanced by LAE supplement in SK-BR-3 ( Supplementary Fig. 2b), while extracellular region part and extracellular matrix part were significantly changed by LAE supplement in HCC1806 ( Supplementary  Fig. 2c). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis found that pathways involved in ECM-receptor interaction and focal adhesion as well as their related genes were significantly enriched ( Supplementary Fig. 2d-f) in SK-BR-3, HCC1806 and MDA-MB-231. Notably, the ECM components and cell adhesion could be induced by the TGF-β1 and FAK signaling pathway [43][44][45][46]. Taken together, our results demonstrate that LAE-suppressed cell migration are associated with multiple different pathways in different cell lines, likely via extracellular matrix signaling.

Discussion
Breast cancer, especially the ER-breast cancer, does harm to the health of women because of its metastasis. It's recognized that more than 90% of breast cancer deaths are the results of tumor metastasis [47], primarily to the bone, lung, liver, brain and lymph nodes [48]. Tumor metastasis is the dissemination of cancer cells from the initial site to distant organs, including epithelial-mesenchymal transition (EMT), cell migration, local tissue invasion, intravasation, extravasation, and metastatic niche formation. EMT endues cells not only migration ability but invasive properties and stem-like cells functions [49][50][51]. In the present study, we found that LAE inhibited EMT and cell migration via Erk1/2 and SMAD3 and reducing H 2 O 2 production in ER-breast cancer even at a low concentration of 10 µg/ml. Our findings provided a new possible therapeutic approach for ER-breast cancer.
Although the anti-bacterial, anti-oxidant, and anti-obesity effects of lotus and its extracts have been known for many years [24,52], little is known about the activity of LAE on cancer cell migration. We demonstrated that LAE had the potential inhibitory activity for cell migration and EMT in vitro and in vivo. However, few components of lotus leaf with the effects of anticancer have revealed so far. Previous studies showed that nuciferine, a major bioactive component of lotus, inhibits the growth of cancer cells and breast cancerassociated bone loss [25,53]. However, our results showed that nuciferine did not participate in LAE-inhibited migration. Thus, further experiments are required for identification of bioactive compound in LAE of inhibiting cell migration and metastasis.
Many signaling pathways are associated with the migration of tumor cells. TGF-β1 signaling pathway has been recognized to be the main inducer. TGF-β1 modulates the EMT process by the canonical TGF-β1/SMAD3 signaling pathway and the non-canonical TGF-β1/PTEN/AKT and MAPKs signaling pathways. Our results showed that LAE inhibited TGF-β1 signaling pathway via Erk1/2 and SMAD3 in SK-BR-3, MDA-MB-231 and HCC1806 breast cancer cells. Furthermore, several studies reported that SMAD3 increases the adhesive ability of hepatocellular carcinoma (HCC) cells by the form of exosome, which promotes the lung metastasis [54]. These might explain the reason why LAE treatment significantly changed the ECM and cell-cell adhesive signaling pathway, which were mediated by the TGF-β1 and FAK signaling pathway according to the results of GSEA. Therefore, the different effect of LAE on SMAD3 and Erk1/2 in mice model could be possibly explained by the different extracellular matrix compounds and environment in liver and lung (Fig. 5e, Fig. 6e-f). Thus, ECM and FAK might be another targets to inhibit the cell migration and metastasis induced by LAE.
Literatures have demonstrated that activated Erk1/2 stimulates the metastasis in cancers by targeting Snail, Slug and matrix metalloproteinases (MMPs) [55]. However, in the present work, we found that LAE suppressed the activation of Erk1/2 and SMAD3 as well as reduced the intracellular H 2 O 2 levels, which were regulated by TGF-β1 signaling pathway in MDA-MB-231 and HCC1806 cells, but not in SK-BR-3 cells. Exogenous H 2 O 2 significantly restored LAE-inhibited cell migration in MDA-MB-231 and HCC1806 cells, but not SK-BR-3, revealed that H 2 O 2 was not involved in LAE-inhibited cell migration in SK-BR-3 cells. Interestingly, although LAE treatment significantly reduced the phosphorylation levels of Erk1/2 and SMAD3, the expression of TGF-β1 protein which regulated them both did not change significantly in MDA-MB-231 cells. This might be explained by the significantly decreased expression of mutant p53 in MDA-MB-231 cells because the essential effects of mutant p53 in mediating the TGF-β1 signaling pathway [32]. Taken together, our findings strongly suggested that LAE inhibit EMT by primarily targeting the TGF-β1 pathway, and the underlying mechanism of LAE-suppressed cell migration is differently dependent on cell lines.
Lotus leaf has been used as "Chinese medicine" for a long history [56]. In the present study, we demonstrated that LAE inhibited the cell migration and metastasis of ER-breast cancer without significant cytotoxicity. Further identification and clinical studies are required to make LAE as a novel anticancer agent in clinical therapy, and our findings strongly contribute to achieve this goal.

Conclusion
In this study, our data showed that LAE significantly inhibits the ER − breast cancer cell migration and metastasis via the TGF-β1/Erk1/2 and TGF-β1/SMAD3 signaling, accompanied by reduced intracellular H 2 O 2 level in ERbreast cancer cells. RNA-sequence analysis also showed that extracellular matrix signaling pathways and FAK might be another possible pathways to inhibit the cell migration induced by LAE. These results suggested that LAE has the potential to screen out novel drugs for the treatment of ER − breast cancer clinically.

Supplementary Files
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