In vitro Non-Small Cell Lung Cancer Inhibitory Effect by New Diphenylethane Isolated From Stems and Leaves of Dioscorea oppositifolia L. via ERβ-STAT3 Pathway

Lung cancer is the most leading cause of cancer mortality throughout the world, of which about 85% cases comprise the non-small cell lung cancer (NSCLC). Estrogen and estrogen receptors are known to be involved in the pathogenesis and development of lung cancer. Dioscorea oppositifolia L. is a traditional Chinese medicine and a nutritious food, and can be an excellent candidate as an anti-cancer agent owing to its estrogen-like effects. However, the stems and leaves of D. oppositifolia L. are piled up in the field as a waste, causing environmental pollution and waste of resources. In the present study, a new diphenylethane (D1) was isolated from the stems and leaves of D. oppositifolia L. It was observed that D1 reduced the cell viability, migration, energy metabolism, and induced apoptosis in the A549 cells. Mechanistic studies showed that D1 reduced the STAT3 nuclear localization and downregulated the expression of the STAT3 target genes like Mcl-1, Bcl-xL and MMP-2 that are involved in the cell survival and mobility. Moreover, our results indicated that D1 exhibited estrogenic activities mediated by ERβ, and antagonising ERβ decreased the cytotoxic effect of D1 in A549 cells. In addition, inhibition of the nuclear translocation of STAT3 did not interfere with the binding of D1 and ERβ. However, after antagonizing ERβ, the nuclear translocation of STAT3 increased, thereby demonstrating that STAT3 was the downstream signaling molecule of ERβ. In conclusion, the D1 mediated anti-NSCLC in vitro effects or at least in part can be attributed to the ERβ-STAT3 signaling. Our findings suggest the role of D1 in treating NSCLC on a molecular level, and can help to improve the comprehensive utilization rate of D. oppositifolia L.


INTRODUCTION
According to the Global Annual Cancer Report of 2019, lung cancer remains the most malignant tumor, in terms of both morbidity (11.6%) and mortality (18.4%). Among the different variants, the non-small cell lung cancer (NSCLC) accounts for approximately 85% of the lung cancer incidences (Cong et al., 2019;Siegel et al., 2020). At present, a significant progress has been made in the treatment of NSCLC. Targeted therapy and immunotherapy have significantly increased the 5-years survival rate (Herbst et al., 2018). However, with the emergence of new problems, such as high recurrence rate, metastasis and drug resistance, there is an urgent need of new treatment options that are highly efficient, low-cost and with weak side-effects. Some traditional Chinese medicines have certain anti-tumor effects, which imply to be detoxifying, heat-clearing and strengthening the foundation of body, according to the traditional Chinese medicine (Wang et al., 2019). These medicines typically have multiple targets, functioning at multiple stages and showing diverse effects. Furthermore, they have low toxic and side effects, improve the body's immunity, and are not prone to drug resistance. Therefore, the anti-tumour traditional Chinese medicines have become a popular area of research. With the gradual understanding of the traditional Chinese medicine, it has been realised that the active antitumour ingredients (e.g., baicalein, matrine and oridonin) extracted from the traditional Chinese medicine are suitable for multi-target therapy (Han et al., 2016).
Dioscorea oppositifolia L. is documented in the "Shennong Bencaojing" and is regarded as a model of the "chemical similarity of medicine and food" principle in ancient China, besides promoting the functions of lungs and kidneys (Ren et al., 2019a). Modern pharmacological research shows that it has the biological activities of lowering blood sugar and blood pressure, as well as the anti-inflammatory and anti-tumour effects (Hsu et al., 2014;Ma et al., 2017;Ren et al., 2019b). However, during the collecting process of D. oppositifolia L., its aerial parts (stems and leaves) are piled up in the field as waste, causing environmental pollution and a waste of resource. At present, the non-medicinal parts of many traditional Chinese medicine plants or their extracts have been used in clinical practice, such as Eucommia ulmoides, a cheap and common plant which has anti-hypertensive effects (Hu et al., 2020). Chlorogenic acid extracted from the E. ulmoides leaves is known to exhibit anti-bacterial and anti-inflammatory effects (Zhao et al., 2020). Earlier, we studied the chemical constituents in the stems and leaves of D. oppositifolia L., and isolated sesquiterpenoids, diphenylethanes and other compounds (Ren et al., 2019a;Ren et al., 2019b). We found that these metabolites exhibit estrogen-like activities, which is mediated by the estrogen receptor (ER). Several studies have shown that estrogen therapy can improve the prognosis of NSCLC patients and improve the efficacy, and has been gradually used in the treatment of NSCLC (Kawai, 2014;Samuel et al., 2019;Abdulaziz et al., 2020). STAT3 is known to regulate the proliferation, differentiation and migration of lung cancer cells by activating the genes associated with the cell cycle progression, inducing survival genes and genes playing role in the invasion and metastasis (Xiong et al., 2020). In the present study, a new diphenylethane compound was isolated from the stems and leaves of D. oppositifolia L., and its intervention on A549 cells was studied from the perspective of ER and STAT3. Our study provides important information for exploring the pharmacological actions of the stems and leaves of D. oppositifolia L., and in improving the comprehensive utilization rate of D. oppositifolia L. In addition, this study also provides a theoretical basis for the discovery of new drugs in the treatment of NSCLC.

Plant Material
The stems and leaves of D. oppositifolia L. were collected in September 2017 in Wenxian (112°51′39″ to 113°13′20″ east longitude, 34°52′ to 35°2′48″ north latitude), Jiaozuo city, Henan province, People's Republic of China, and was identified by Prof. Sui-qing Chen of Henan University of Chinese Medicine. A voucher specimen (No. 20171115A) was stored in the Department of Natural Medicinal Chemistry, School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, China (Ren et al., 2019b).

Cell Culture and Treatment
The human lung adenocarcinoma cells A549 and human breast cancer cells MCF-7 were obtained from the American Type Culture Collection (Manassas, VA, United States). The A549 and MCF-7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin, and 10% heatinactivated foetal bovine serum (FBS) at 37°C under a humidified atmosphere of 5% CO 2 in air. Prior to the experiments involving the treatment with estrogen, MCF-7 cells were cultured in a hormonefree medium (phenol red-free DMEM with 10% v/v charcoalstripped FBS) for 7 days (Ren et al., 2019a) Cell Viability A549 cells were seeded in 96-well plates in DMEM with 10% v/v FBS. The density was 5,000 cells/well, and cells were divided into the Control group, D1 (25 μM and 50 μM) group, and D1 (25 μM and 50 μM) + STAT3 inhibitor (AG490, 10 μM) group. After 24 or 48 h, the MTT assay was used to detect cell viability. The cell viability was proportional to absorbance. MCF-7 cells were seeded on a 96-well plates in phenol red-free DMEM with 10% v/v charcoal-stripped FBS, cells were divided into the control group, groups of D1 (25, 50 μM); in another set of experiments, the specific ERβ antagonist (THC, 1 μM) was added 30 min before D1 to evaluate, whether the observed effects elicited by D1 were mediated by ERβ (Zeng et al., 2018). After 24 h, the MTT assay was used to detect cell viability.

Cell Migration
Assay for cell migration was performed using a 96-well plate (E190236X, PerkinElmer, United States) and 16-well plate (CIM-Plate16, ACEA, Belgium). Cells (2000 cells/well) were divided into the Control group, D1 (25 μM and 50 μM) group, D1 (25 μM and 50 μM) + AG490 (10 μM) group, D1 (25 μM and 50 μM) + THC (1 μM) group, D1 (25 μM and 50 μM) + specific estrogen receptor β agonist (PPT, 1 μM) group in 96-well plates, the movement of A549 cells was monitored in real time by highcontent imaging system (Opera Phenix, PerkinElmer, United States). The displacement was normalized against the Harmony 4.8. In another experiment, 160 μl medium was added to the lower chamber of the CIM-Plate16, the upper and lower chambers were assembled, and 30 μl complete medium was added to the upper chamber. Subsequently, 100 μl the cell suspension (group as described above) was added to the chamber of the CIM-Plate16, xCELLigence system for realtime analysis of cell migration. The results were expressed by the cell migration kinetic curve (time-versus-index).

OCR and ECAR
Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse analyzer (Agilent Technologies, United States) and a Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies, United States). Cells were divided into Control group and D1 (25 μM and 50 μM) group. Before the test, the culture medium was replaced with a specific medium (Seahorse XF DMEM, pH 7.4), to which oligomycin (1 mol/L), FCCP (2 mol/L), and rotenone/antimycin A (0.5 mol/L) were added. After detection, the data were normalized through the cell count, and then the OCR value per 10,000 cells was calculated. In addition, glucose (10 mM), oligomycin (2 μM), and 2-DG (50 mM) were diluted in assay medium and injected into ports A, B, and C, respectively, of the Seahorse analyzer. The assay was performed using the glycolytic stress test assay protocol, and the ECAR value per 10,000 cells was calculated (Jung et al., 2016).

Molecular Docking
The ERβ protein was used as the receptor (PDB: 1U3Q), and D1 was employed as a ligand. Sphere ball (active pocket) was defined according to the ligand. Molecular docking was performed using the Discovery Studio Client V3.0 (Accelrys software Inc.).

Statistical Analysis
Data were analyzed using the SPSS software 20.0 (IBM, United States). Statistical significance was assessed in comparison with the respective control for each experiment using one-way analysis of variance. p values less than 0.05 were accepted as significant.  . These spectroscopic features suggested that the spectra of D1 was very similar to those of 3,5-dihydroxy-4-methoxybibenzyl (Yang et al., 2014) with an extra hydroxyl group at C-2′. Therefore, the structure of D1 was identified to be 2′,3,5-trihydroxy-4methoxybibenzyl ( Figure 1A). 1 H and 13 C NMR spectroscopic data of compound D1 is shown in Table 1. The content of D1 was 0.38% ( Figures 1B,C).

D1 Reduces Viability, Migration, Energy Metabolism, and Induced Apoptosis in A549 Cells
The cytotoxicity of D1 was observed in the A549 cell line from the MTT assay. As shown in Figure 2A, D1 decreased A549 cell viabilities in both time and dose-dependent manner, with IC50 values of 38.9 µM and 29.2 µM after 24 and 48 h treatment, respectively. In addition, the high-content imaging system and xCELLigence system was performed to determine the effects of D1 on A549 cell migration. As shown in Figure 2D, after 48 h treatment, D1 significantly inhibited the migratory ability of A549 cells at the concentration of 25 or 50 µM. Likewise, a significant reduction of the cell displacement was observed in the D1 treatment groups ( Figure 2C). Figure 2B shows that D1 induced apoptosis in a dose-dependent manner in the A549 cells.
In addition, the results of Seahorse XF24 Analyzer showed that treatment with D1 transformed the extracellular acidification rate and oxygen consumption rate ( Figures 2E,F).

D1 Downregulates the STAT3 Target Gene Expression in A549 Cells
STAT3 dimerizes upon tyrosine phosphorylation at site 705, which leads to its nuclear translocation, and directly regulates a panel of tumor-promoting genes. As expected, the protein levels of STAT3 in nuclear fractions of A549 cells were significantly reduced by D1 in a dose-dependent manner ( Figure 3A), moreover, we investigated, whether D1 affected the expression of STAT3 target genes including Mcl-1, Bcl-xL (involved in cell survival), and MMP-2 (involved in cell migration) (Su et al., 2017). As shown in Figure 3B, D1 dose-dependently decreased the levels of MMP-2, Mcl-1 and Bcl-xL. In addition, after nuclear translocation of STAT3 was inhibited by treatment with AG490 (a specific inhibitor of STAT3), D1 further reduced the viability, migration and induced apoptosis in A549 cells ( Figures 3D-F). Figure 4B shows that D1 improved the proliferation rate of the MCF-7 cells, and THC (specific ERβ antagonist, 1 µM) blocked the effect of D1 (25 or 50 µM) on the MCF-7 cell proliferation. Moreover, D1 (25 µM) upgraded the expression of ERβ in MCF-7 cells ( Figure 4A). To further investigate the molecular interaction between D1 and ERβ, the structure of ERβ (PDB: 1U3Q) and D1 was studied using the molecular simulations. As shown in

D1 Inhibits Migration and Induced Apoptosis via ERβ-STAT3 Pathway in A549 Cells
Specific estrogen receptor β blocker (THC), specific estrogen receptor β agonist (PPT), and STAT3 inhibitor (AG490) were administered respectively 30 min before the treatment of D1 to evaluate whether the observed effects elicited by D1 were mediated via the ERβ-STAT3 pathway. Results of the immunofluorescence experiments revealed that THC increased the nuclear translocation of STAT3, but AG490 was unable to block the expression of ERβ ( Figure 5A). Moreover, PPT decreased the nuclear translocation of STAT3. In addition, the downregulation of migration and the upregulation of apoptosis by D1 in A549 cells was found to be efficiently blocked by THC. Meanwhile, D1 promoted the migration and induced apoptosis in A549 cells, which was further activated by PPT ( Figures 5B-D).

DISCUSSION
Lung cancer has become the type of cancer with the highest incidence among all the other types cancers in the world, and it appears to affect more among the young people (Xiao et al., 2020). The existing treatment methods have disadvantages such as poor selectivity, high toxic side-effects and a rapid development of drug resistance . Therefore, the exploration of new drugs and new methods for the treatment of lung cancer requires an urgent attention. A large number of basic research and clinical practice have shown that natural products can play an important role in the tumour prevention and rehabilitation of cancer patients (Kim et al., 2020;Liu et al., 2020;Yeh et al., 2020). Their reliable source and high safety are the features, which make Frontiers in Pharmacology | www.frontiersin.org February 2021 | Volume 12 | Article 622681 6 FIGURE 3 | D1 downregulates the STAT3 target gene expression in A549 cells. A549 cells were treated with D1 (25 or 50 µM) for 24 h, cellular immunofluorescence by using antibodies specific to STAT3 (A), Mcl-1, Bcl-xL and MMP-2 (B). (C): Cell viability was measured by the MTT assay. A549 cells were treated with D1 or AG490 for 24 h. Data were shown as mean ± SD from three independent experiments, **p < 0.01 vs. Control (-AG490); ## p < 0.01 vs. Control (+ AG490). (D): A549 cells were treated with D1 and AG490 for 24 h. Apoptosis was analyzed by flow cytometry after 7-AAD/PE double staining. The percentage of apoptotic cells was presented as the mean ± SD of three independent experiments, **p < 0.01 vs. Control. (E): A549 cells were plated in 96-well plates, the movement of A549 cells was monitored in real time by high-content imaging system followed by D1 and AG490 treatment. The displacement was normalized against the Harmony 4.8. **p < 0.01 vs. Control (24 h). (F): A549 cells were plated in CIM-Plate16, xCELLigence system for real-time analysis of cell migration followed by D1 and AG490 treatment.
Frontiers in Pharmacology | www.frontiersin.org February 2021 | Volume 12 | Article 622681 7 them the main source of new drug development. Currently, the anti-cancer preparations derived from the plant source account for approximately 32% of the total anti-cancer drugs, such as colchicine, vinblastine, paclitaxel are all isolated from natural products (Fatima et al., 2019;Zhou et al., 2019;Modi et al., 2020). In the present study, a new diphenylethane compound (D1) was obtained for the first time, its effects on A549 cells were studied, and its possible mechanism of action was explored. We aimed to unearth compounds with effective anti-NSCLC efficacy from stems and leaves of Dioscorea oppositifolia L., just like the discovery of artemisinin from a traditional anti-malarial Chinese medicinal herb Artemisia annua. We hope to screen out a bioactive components-enriched fraction from the studied stems and leaves of Dioscorea oppositifolia L. for developing an anti-NSCLC agent, because it has been well accepted that natural products with multi-components and multi-targets natures have advantages in managing the complex disease such as cancer (Su et al., 2017).
STAT3 is an important member of the signal transduction and activation protein (STAT) family (Kadye et al., 2020). It is known to participate in the regulation of the transcription of multiple target genes related to the tumour occurrence and development, and cause abnormal expression of proteins related to proliferation, differentiation, cycle and apoptosis, thereby causing tumour cell anti-apoptosis, promoting cell proliferation and migration (Sen et al., 2020). In the present study, it was found that D1 reduced the viability, migration, energy metabolism and induced the apoptosis in A549 cells. It is known that, upon phosphorylation, STAT3 dimerizes and translocates into the nucleus to regulate the transcription of its target genes. Therefore, we speculated that the inhibition of the STAT3 phosphorylation could result in the reduced STAT3 nuclear localization and decreased expression of genes that are transcriptionally upregulated by STAT3 (Su et al., 2017). Indeed, D1 treatment reduced the nuclear localization of STAT3 and lowered the expression of STAT3 targeted Bcl-xL, MMP-2 and Mcl-1 that are involved in A549 cell proliferation, survival and mobility.
Phytoestrogens are a class of natural plant compounds with weak estrogen effects. Its main mechanism is to exert estrogen-like or antiestrogen effects by binding to estrogen receptors (ERα, ERβ). They have two-way regulatory functions and are effective on the prevention and treatment of hormone-related diseases (Francesco and Petropoulos, 2019;Najafi, 2019). Using the estrogen-deprived MCF-7 cell model is a common method to test whether drugs have the estrogenic activity in vitro. When drugs with estrogen- Frontiers in Pharmacology | www.frontiersin.org February 2021 | Volume 12 | Article 622681 8 like activity are co-cultured with the de-estrogen MCF-7 cells, the deestrogen MCF-7 cells proliferate significantly, and the interference of exogenous estrogen is excluded (Kewen et al., 2016;Zeng et al., 2018). In this study, we found that D1 exhibited an estrogenic activity and promoted the proliferation of estrogen-deprived MCF-7 cells, and that the pro-proliferation effect could be blockedby THC (a ERβ specific antagonist) (Watanabe et al., 2004). Cellular immunofluorescence results showed that D1 increased the expression of ERβ, while the molecular docking results showed that D1 binds to the ERβ significantly, indicating that the estrogen-like activity of D1 is mediated by ERβ. SPR assay will be used to explore D1 targeting on ERβ in subsequent research.
Studies have shown that the survival time of premenopausal female NSCLC patients was significantly shorter than that of postmenopausal female patients, and that the tumour was more aggressive (Moore et al., 2003;Hsu et al., 2015). The local estrogen content of cancer tissues in postmenopausal female patients was significantly lower than that of men (Niikawa et al., 2008). Therefore, it is speculated that endogenous hormones of the female patients can promote the occurrence and development of NSCLC. D1 has estrogenlike activity, and we speculate that it can compete with the endogenous estrogen to bind to the estrogen receptor, thereby preventing the further development of NSCLC. We found that D1 can significantly reduce the viability and migration of A549 cells, and that this effect was blocked by THC (a ERβ specific antagonist) and be further activated by PPT (a ERβ specific agonist) (Brenna et al., 2016). This indicated that D1 exerts its anti-NSCLC effect through the ERβ pathway. Moreover, our results showed that inhibiting the nuclear translocation of STAT3 did not interfere with the binding of D1 to the ERβ receptor. On the contrary, after antagonizing ERβ, the nuclear translocation of STAT3 increased, indicating that STAT3 could be a downstream signalling molecule of ERβ, and that D1 could exhibit its anti-NSCLC function through the ERβ-STAT3 pathway.

CONCLUSION
In the present study, we identified a new diphenylethane (D1) from the stems and leaves of D. oppositifolia L., which reduced the cell viability, migration, energy metabolism and induced cell apoptosis in A549 cells. Mechanistic studies showed that D1 reduced the STAT3 nuclear localization and downregulated the expression STAT3 target genes. It was FIGURE 5 | D1 inhibits migration and induced apoptosis via ERβ-STAT3 pathway in A549 cells. (A): A549 cells were plated in 96-well plates, and the movement of the cells was monitored in real time by high-content imaging system after D1 and THC or PPT treatment. The displacement was normalized against the Harmony 4.8. **p < 0.01 vs. Control (total 24 h). (B): A549 cells were treated with D1 and THC for 24 h. Apoptosis was analyzed by flow cytometry after 7-AAD/PE double staining. Data was presented as the mean ± SD of three independent experiments. (C): A549 cells were plated in CIM-Plate16, xCELLigence system for real-time analysis of cell migration followed by D1 and THC or PPT treatment. (D): A549 cells were treated with D1 and AG490, THC or PPT for 24 h, cellular immunofluorescence by using antibodies specific to ERβ and STAT3.
Frontiers in Pharmacology | www.frontiersin.org February 2021 | Volume 12 | Article 622681 observed that D1 exhibited estrogenic activities mediated by ERβ, and the antagonism of ERβ decreased the cytotoxic effect of D1 in A549 cells. Additionally, inhibiting the nuclear translocation of STAT3 did not interfere with the binding of D1 and ERβ, but after antagonizing ERβ, the nuclear translocation of STAT3 increased, thereby demonstrating that STAT3 was the downstream signaling molecule of ERβ.
Our results indicate that D1 exhibited an in vitro anti-NSCLC effect, which could be partially due to the modulation of the ERβ-STAT3 signaling pathway. We speculate the potential of D1 to be developed as a modern alternative and/or complimentary agent in the management of NSCLC, and also improve the comprehensive utilization rate of D. oppositifolia L.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.