Antiproliferative and Proapoptotic Effects of Phenanthrene Derivatives Isolated from Bletilla striata on A549 Lung Cancer Cells

Lung cancer continues to be the world’s leading cause of cancer death and the treatment of non-small cell lung cancer (NSCLC) has attracted much attention. The tubers of Bletilla striata are regarded as “an excellent medicine for lung diseases” and as the first choice to treat several lung diseases. In this study, seventeen phenanthrene derivatives, including two new compounds (1 and 2), were isolated from the tubers of B. striata. Most compounds showed cytotoxicity against A549 cells. An EdU proliferation assay, a cell cycle assay, a wound healing assay, a transwell migration assay, a flow cytometry assay, and a western blot assay were performed to further investigate the effect of compound 1 on A549 cells. The results showed that compound 1 inhibited cell proliferation and migration and promoted cell apoptosis in A549 cells. The mechanisms might correlate with the regulation of the Akt, MEK/ERK, and Bcl-2/Bax signaling pathways. These results suggested that the phenanthrenes of B. striata might be important and effective substances in the treatment of NSCLC.


Introduction
For decades, lung cancer has been a considerable health issue owing to its high incidence and fatality rates [1,2]. Indeed, lung cancer kills more than one million people worldwide every year, with 80-90% of cases being related to NSCLC [3]. Although our understanding of targeted therapies, immunotherapy, and genetic alterations of cancers is evolving, the cure rate for NSCLC remains low [4,5]. In recent years, natural products from traditional Chinese medicines have been suggested as potential drugs to treat NSCLC [6,7]. Therefore, the discovery of anti-NSCLC active ingredients from traditional Chinese medicines has become one of the hotspots of modern lung cancer research.

Structure Elucidation
Compound 1 showed IR absorption peaks at 1455 and 1589 cm −1 for aromatic rings and 3227 cm −1 for hydroxy groups. Its molecular formula was determined as C30H26O6 with 18 degrees of unsaturation from a (−)-HR-ESI-MS ion peak at 481.1647 [M − H] − (calculated for C30H25O6, 481.1651). The 13 C-NMR data of 1 (Table 1)

Cytotoxicity of the Isolates against A549 and BEAS-2B Cells
Our prior research showed that EtOAc extract from B. striata exhibited significant cytotoxic activity against A549 cells (IC50 = 11.92 ± 0.68 μg/mL). However, water extract

Cytotoxicity of the Isolates against A549 and BEAS-2B Cells
Our prior research showed that EtOAc extract from B. striata exhibited significant cytotoxic activity against A549 cells (IC 50 = 11.92 ± 0.68 µg/mL). However, water extract and n-BuOH extract from B. striata exhibited no obvious cytotoxicity (IC 50 > 100 µg/mL) [14]. Thus, the cytotoxic effects of the phenanthrenes isolated from the EtOAc extract were investigated in this study. Table 2 presents the cytotoxicity results of the isolates against A549 lung cancer cells; compounds 11 and 15 were not detected due to limited sample amounts after structure determination. In addition, the cytotoxicity of compound 1 against normal human lung cells (BEAS-2B) was also assessed. The result showed that the cytotoxic

Discussion
Considering its high incidence and mortality, lung cancer is gradually becoming a serious health problem [26,27]. NSCLC is the most frequent type of lung cancer according to the histological type [28]. Although surgery, radiation, and immunotherapy have been used for treating NSCLC, pharmaceutical treatment is of great significance, and innovative drugs are needed. Traditional Chinese medicine (TCM) reflects the profound understanding of the Chinese people regarding life, health, and disease and has a time-honored historical tradition and unique theories and techniques. With the advantages of diverse chemical structures, a wide range of sources, and significant activities, natural products from TCM have been studied for the treatment of NSCLC [29,30]. Therefore, it is meaningful and feasible to find effective natural compounds from TCM to treat NSCLC.
The tubers of B. striata, praised as "an effective medicine to treat lung diseases", were the best choice in terms of lung disease treatment according to Shennong's Materia Medica, Essential of Materia Medica and Treasury of Words on Materia Medica. Modern studies have demonstrated that phenanthrene derivatives from B. striata exert significant cytotoxic activities against A549 cells [11,13,31,32]. Therefore, this study explored phenanthrene derivatives from EtOAc extract from B. striata in terms of cytotoxicity against A549 cells and investigated the preliminary mechanisms. As expected, 17 phenanthrene derivatives, including two new compounds (1 and 2), were isolated from the tubers of B. striata. Most of the tested compounds showed cytotoxicity, especially compounds 1, 2, 4, 6, 7, 8, and 13 (IC50 < 10 μM). Moreover, the structure-cytotoxicity relationship was also investigated. In general, the cytotoxic effects of biphenanthrenes (1-4 and 6-8) were much stronger than those of simple phenanthrenes (9, 10, 12, 14, 16, and 17). However, the IC50 value of compound 5 was over 100 μM. A comparison of the results for compound 5 and other biphenanthrenes, especially between compounds 5 and 6, indicated that the introduction of an OMe group in position eight resulted in a considerable reduction in cytotoxicity. In addition, a comparison of compounds 1 (1,1′-connection) and 4 (1,3′-

Discussion
Considering its high incidence and mortality, lung cancer is gradually becoming a serious health problem [26,27]. NSCLC is the most frequent type of lung cancer according to the histological type [28]. Although surgery, radiation, and immunotherapy have been used for treating NSCLC, pharmaceutical treatment is of great significance, and innovative drugs are needed. Traditional Chinese medicine (TCM) reflects the profound understanding of the Chinese people regarding life, health, and disease and has a time-honored historical tradition and unique theories and techniques. With the advantages of diverse chemical structures, a wide range of sources, and significant activities, natural products from TCM have been studied for the treatment of NSCLC [29,30]. Therefore, it is meaningful and feasible to find effective natural compounds from TCM to treat NSCLC.
The tubers of B. striata, praised as "an effective medicine to treat lung diseases", were the best choice in terms of lung disease treatment according to Shennong's Materia Medica, Essential of Materia Medica and Treasury of Words on Materia Medica. Modern studies have demonstrated that phenanthrene derivatives from B. striata exert significant cytotoxic activities against A549 cells [11,13,31,32]. Therefore, this study explored phenanthrene derivatives from EtOAc extract from B. striata in terms of cytotoxicity against A549 cells and investigated the preliminary mechanisms. As expected, 17 phenanthrene derivatives, including two new compounds (1 and 2), were isolated from the tubers of B. striata. Most of the tested compounds showed cytotoxicity, especially compounds 1, 2, 4, 6, 7, 8, and 13 (IC 50 < 10 µM). Moreover, the structure-cytotoxicity relationship was also investigated. In general, the cytotoxic effects of biphenanthrenes (1-4 and 6-8) were much stronger than those of simple phenanthrenes (9, 10, 12, 14, 16, and 17). However, the IC 50 value of compound 5 was over 100 µM. A comparison of the results for compound 5 and other biphenanthrenes, especially between compounds 5 and 6, indicated that the introduction of an OMe group in position eight resulted in a considerable reduction in cytotoxicity. In addition, a comparison of compounds 1 (1,1 -connection) and 4 (1,3 -connection) showed that the manner of the connection between the two dihydrophenanthrene monomers did not significantly affect the cytotoxicity. In the simple phenanthrenes, when compared with compound 9, the introduction of an additional OH (10) or an additional p-hydroxybenzyl (12) at C-1 significantly improved the cytotoxicity. The introduction of another p-hydroxybenzyl at C-6 (14) further notably increased the cytotoxicity. These structure-activity relationships may serve as a reference in the investigation of anti-NSCLC phenanthrenes from B. striata.
The novel compound 1, possessing good cytotoxicity, was used to study the preliminary mechanisms. EdU immunofluorescence staining and cell cycle analysis were carried out to detect the antiproliferation effect of compound 1 on A549 cells. The data showed that, at concentrations of 3.13, 6.25, and 12.5 µM, compound 1 significantly inhibited the proliferation of A549 cells and induced cell cycle arrest at the G2/M phase. The checkpoint at the G2/M transition is a crucial regulatory gate during cell-cycle progression, but the cell will die if the cell-cycle checkpoint is lost before the completion of DNA repair [33,34]. In addition, cell migration is an essential procedure when the metastatic dissemination of cancer cells is mentioned [35]. Compound 1 exhibited an inhibitory effect with respect to A549 cell migration, as determined by the migration-related assays. Since Akt dominates the growth, cycle, metabolism, and death of cells by regulating various downstream substrates [36], the effect of compound 1 on AKT phosphorylation in A549 cells was investigated. Meanwhile, MAPKs acts as one of the significant serine/threonine protein kinases, which exhibit a crucial role in receiving signals and transmitting them into cytomembrane [37]. ERK1/2 is identified as an important mitogen-activated factor, which participates in numerous biological processes including both cell proliferation and survival. MEK1/2 is a crucial upstream protein of ERK1/2 [38]. Both of them are important members of the MAPK family, and the MEK/ERK signaling pathway has been proven to be crucial for NSCLC research [39,40]. Thus, the expression ratios of p-MEK/MEK and p-ERK/ERK were evaluated. These results disclosed that the Akt and MEK/ERK signaling pathways were involved in the antiproliferation effect of compound 1 on A549 cells.
Unlike necrosis, apoptosis is a type of programmed cell death. Apoptosis disorder can cause pathological events. Among them, tumors and autoimmune diseases are representative examples [41]. The flow cytometry results indicated that compound 1 dramatically promoted the apoptosis of A549 cells. Thus, the proapoptotic effect of compound 1 was further studied. Bcl-2 and Bax are regarded as important apoptotic regulatory proteins. Bcl-2 promotes cell survival and suppresses cell death, while the effects of Bax are the opposite [42,43]. Our results pointed out that the proapoptotic effect of compound 1 on A549 cells was associated with a decrease in the Bcl-2/Bax ratio.

Cytotoxic Activity Assay
The purity of the tested compounds was more than 98%. The cytotoxic effects of isolated phenanthrenes on A549 and BEAS-2B cells were examined by MTT experiments as described in our previous report [14].

EdU Proliferation Experiment
The cells were digested by trypsin and cultivated in 96-well plates (3.5 × 10 3 cells per well) for 24 h. They were incubated with compound 1 (3.13, 6.25, and 12.5 µM) for 48 h. Next, prepared EdU-labeling solution (10 µM) was used to stain the cells in an incubator at 37 • C for 2 h. The cells were fixed for 15 min with 4% cold paraformaldehyde. After that, Triton X-100 (0.3%) and Click-iT reaction cocktail were successively used to handle cells. Finally, DAPI staining was applied to the cells to counterstain them. A Leica DMI3000B inverted fluorescence microscope (Leica, Wetzlar, Germany) was used to capture the fluorescent images. In addition, to count the cells, ImageJ software (version 1.8.0) (National Institutes of Health, Bethesda, MD, USA) was used. EdU positive cells (%) = (green EdU-stained cells/blue DAPI-stained cells) × 100.

Cell Cycle Analysis
According to the manufacture's instructions, the cells were treated with compound 1 (3.13, 6.25, and 12.5 µM) for 48 h. Then, they were cultured with 500 µL staining solution (RNase A:PI = 1:9) for 1 h at room temperature without light after fixation with 70% prechilled ethanol for 2 h. The DNA content was instantly detected with a BD FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Wound Healing Assay
Cells were placed in 6-well plates for the wound healing test. The scratches were formed using a sterile 200-µL pipette tip when the cells treated with compound 1 (3.13, 6.25, and 12.5 µM) had grown to 90% confluence. After a 24-h incubation, the images were obtained with the Leica microscope and the scratch area was calculated with the ImageJ. Migration rate (%) = [(A0 − A1)/A0] × 100, where A0 represents scratch area at 0 h, and A1 suggests scratch area at 24 h.

Transwell Migration Assay
The migratory effect of compound 1 (3.13, 6.25, and 12.5 µM) on A549 cells was further detected by using transwell chambers. Briefly, the cells treated with compound 1 were placed into the upper wells, while the medium with 10% FBS was added to the bottom wells. After a 24-h incubation, non-migrated cells were cleaned up, and the migrated cells were fixed for 30 min with 4% paraformaldehyde and dyed by 0.1% crystal violet. The images and the cell numbers were obtained by the Leica microscope and ImageJ, respectively.

Apoptosis by Flow Cytometry Assay
The A549 cells were treated with compound 1 (3.13, 6.25, and 12.5 µM), and the cell apoptosis was detected by flow cytometry assay as described in our previous report [14].

Statistical Analysis
Data are shown as mean ± standard deviation (SD) with three biological replicates. One-way ANOVA and Tukey's post-hoc test were used to evaluate the significant differences (p < 0.05). Figures were obtained by GraphPad Prism software Version 5.0 (GraphPad Software, Inc., San Diego, CA, USA).
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27113519/s1, Figures S1-S16 are the 1D and 2D NMR, HR-ESI-MS, IR, and UV spectra for two new compounds, Table S1: the original western blots in three repetitions for Figure 6 in the paper, Table S2: the original western blots in three repetitions for Figure 7 in the paper.