Inhibitory Effects of Furanocoumarins From the Roots of Angelica dahurica on Ionizing Radiation-Induced Migration of A549 Human Non-Small Cell Lung Cancer Cells

Abstract

Radiation therapy is a very effective tool for the treatment of advanced human lung cancers. However, as one of its malignancy-promoting behaviors, ionizing radiation (IR) increases cell migration and radiation resistance in several lung cancer cells, including non-small cell lung cancer (NSCLC) cells. As part of our ongoing search for potent radiotherapy enhancers from medicinal herbs, a chloroform-soluble fraction of the roots of Angelica dahurica was subjected to phytochemical investigation, leading to the isolation of 8 furanocoumarins. Of these, psoralen (1), xanthotoxin (2), and bergapten (3) inhibited IR-induced migration at a non-cytotoxic concentration (50 μM) in human NSCLC A549 cells. This study is the first to report on the inhibitory activities of these constituents of A. dahurica against IR-induced cancer metastasis.

Lung cancer is one of the most common types of cancer and is the leading cause of cancer deaths worldwide. ¹ In recent years, the annual incidence of lung cancer has been about 1.8 million people, and the annual death rate, about 1.6 million people worldwide, accounting for nearly 20% of all cancer deaths. ^2,3 Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers, and many patients are diagnosed at an advanced stage. ³ Most NSCLC patients are diagnosed with locally advanced or metastatic diseases, and the prognosis for these patients remains very inadequate. ⁴ Treatment of lung cancer includes surgical resection, chemotherapy, and radiation therapy, and among them, surgical resection is the preferred treatment but is limited to early stages (stages I and II). ⁵ Radiotherapy and chemotherapy are considered the standard therapy for inoperable stage lung cancer (stages III and IV). ⁵ However, in some cases, radiotherapy promotes malignant behaviors, such as local recurrence or distal metastasis. These consequences may cause the regrowth or spread of cancer cells that have survived radiation therapy. In vitro studies have shown that sublethal doses of ionizing radiation (IR) increase the migration and invasion of various cancer cell lines, including lung, glioma, hepatocellular carcinoma, and pancreatic cancer cells, ^{6 -9} and in vivo studies have suggested that radiotherapy of primary tumor sites may promote metastasis. ¹⁰ Therefore, it is necessary to develop radiosensitizers that can inhibit malignant behavior caused by radiotherapy and simultaneously enhance its effectiveness.

We have previously reported that sublethal doses of IR increase sulfatase 2 (SULF2) expression via the p53 transcription factor, which mediates the migration and invasion of cancer cells. This appears to occur through mechanisms that stimulate the β-catenin, interleukin-6, signal transducer and activator of transcription 3, and Bcl-X_L signaling pathway ¹¹ or the phosphoinositide 3-kinase, Akt, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. ¹² Moreover, it is reported that increased SULF2 from sublethal doses of IR regulates IR-induced cancer cell invasion via mitochondrial superoxide dismutase 2 (SOD2). ¹³ These results suggest that IR promotes cancer cell invasion by activating different signaling pathway mediators, such as Bcl-X_L, NF-κB, and SOD2.

Angelicae Dahuricae Radix, the dried root of Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. (Apiaceae) possesses medicinal properties and has been used as a traditional Chinese medicine for treating stomach ache, headache, toothache, abscesses, nose congestion, and dysmenorrhea. ¹⁴ In previous phytochemical studies on A. dahurica, furanocoumarins were identified as major constituents ¹⁵ exhibiting diverse biological activities that include antioxidant, ¹⁶ anti-inflammatory, ^17,18 anti-allergic, ¹⁹ and antitumor effects. ^20,21 As part of our ongoing search for radiotherapy enhancers from natural sources, 8 furanocoumarins were isolated from the dried root of A. dahurica. Chromatographic separation of the chloroform (CHCl₃) extract of the roots resulted in the isolation of 8 compounds which were identified as psoralen (1), ²² xanthotoxin (2), ²³ bergapten (3), ²⁴ imperatorin (4), ²⁴ phellopterin (5), ²⁴ isoimperatorin (6), ²⁴ cnidilin (7), ²⁵ and (R)-(+)-oxypeucedanin (8), ²² by analysis of their spectroscopic data as well as by comparison of their data with published values (Figure 1). In the case of compound 8, the specific rotation was found to be [α]_D ²⁶ +10.48 (c 0.1, CHCl₃), which can be referred to as R configuration at C-2″ as evidence that (R)-(+)-oxypeucedanin and (S)-(-)- oxypeucedanin showed opposite specific rotation, $[\alpha {]}_{\mathrm{D}}^{28}$ +13.2 (c 0.05, CHCl₃) ^26,27 and $[\alpha {]}_{\mathrm{D}}^{24}$ –13.5 (c 0.06, CHCl₃), ²⁷ respectively.

Figure 1

Chemical structures of furanocoumarins 1-8 isolated from Angelica dahurica.

In our previous report, ¹² we found that the natural product-derived compound, linarin inhibited IR-induced cancer cell migration and invasion by suppression of NF-κB activation in A549 cells. In these contexts, to search for other potent radiotherapy enhancers from medical herbs, we used a CHCl₃-soluble fraction of the roots of A. dahurica. First, to investigate the potential inhibitory effect of each compound on the migration of γ-irradiated A549 cells, wound healing assays were performed. As shown in Figure 2 (left panels), wounded A549 cells were irradiated with 10 Gy (γ-rays) and treated with or without 50 µM of each compound for 0, 24, and 48 hours, after which cell motility was monitored. To quantify cell migration, wound width was measured and calculated at the indicated time point, as shown in Figure 2 (right panels). All tested compounds significantly suppressed wound healing of γ-irradiated A549 cells in comparison with the untreated γ-irradiated A549 cells. These findings indicate that isolated compounds 1-8 inhibited IR-induced A549 cell migration.

Figure 2

Effects of furanocoumarins 1-8 on ionizing radiation (IR)-induced A549 cell migration (a–h). (left panels) Wound healing assay was performed to examine the effects of furanocoumarins (50 µM) on the IR-induced migration of A549 cells. (right panels). Relative wound width was calculated as the ratio of the remaining wound width at the given time point to the original width at 0 hours. Data represent the mean ± standard deviation (n = 3); ^# P < 0.05; ^## P < 0.005; *P > 0.05 vs the control.

Next, to investigate whether the inhibitory effects on IR-induced A549 cell migration were actually due to cytotoxicity, cell viability assays were performed. The results demonstrated that compounds 4-8 exhibited cytotoxic effects at 50 µM, but compounds 1-3 did not (Figure 3). Consequently, it was assumed that the antimigratory activity of compounds 4-8 on γ-irradiated A549 cells was due to their cytotoxicities, while 1-3 appear to have inhibitory effects that are independent of cytotoxicity.

Figure 3

Effects of furanocoumarins 1-8 on the viability of A549 cells (a–h). Cell viability of A549 (2 × 10² cells/well) was estimated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after treatment with the indicated compounds (0.78-200 μM) for 24 hours. Values are expressed as mean ± standard deviation of 3 independent experiments.

In several published studies, compounds 1-3 have shown potent anticancer activity in numerous cancer cell lines but these have not included lung cancer. ^{28 -33} For example, psoralen (1) inhibited proliferation by modulating the Wnt/β-catenin pathway in breast cancer cells. ²⁸ Xanthotoxin (2) has been reported to possess antitumor activity in breast cancer cells ²⁹ and inhibits the activation of AP-1 and NF-κB subunits. ³⁰ NF-κB has been implicated in numerous age-related diseases, including cancer. ³¹ Finally, bergapten (3) decreased cell proliferation of breast cancer cells by inducing apoptosis via p53 signaling. ^32,33 Although the cancer-inhibiting properties of these compounds were documented, to the best of our knowledge, this is the first report on their biological activities relating specifically to cancer metastasis. Therefore, we suggest that compounds 1-3 may be effective in improving the therapeutic effects of radiotherapy. However, further study on the anti-invasive effects and the mechanisms of these compounds is required in order for them to become useful as radiosensitizers and enhancers during radiotherapy.

Experimental

General

Optical rotations were measured on a JASCO P-2000 polarimeter. One-dimensional (1D) and 2D NMR spectra were recorded on a UNITY INOVA 400 MHz FT-NMR instrument with tetramethylsilane as an internal standard. Mass spectrometry (MS) was performed on an Agilent 6550 ifunnel liquid chromatography/MS-quadrupole time-of-flight system. Silica gel (230-400 mesh, Merck, Germany), RP-C₁₈ (YMC gel ODS-A, 12 nm, S-150 μm, YMC Co., Japan), and Sephadex LH-20 (GE Healthcare Bio-Science AB, Uppsala, Sweden) were used for column chromatography (CC). Thin-layer chromatographic analysis was performed on Kieselgel 60 F₂₅₄ (silica gel, 0.25 mm layer thickness, Merck, Germany) and RP-18 F_254s (Merck, Germany) plates, with visualization under ultraviolet light (254 and 365 nm) and 10% (v/v) sulfuric acid spray followed by heating (120°C, 5 minutes).

Plant Materials

Angelicae Dahuricae Radix (the roots of A. dahurica (Fisch. ex Hoffm.) Benth. et Hook. were purchased from the Nonglim Oriental Herbal market in Seoul, South Korea, in July 2014 and identified by Professor Je-Hyun Lee (College of Oriental Medicine, Dongguk University). A voucher specimen (no. EA347) has been deposited at the College of Pharmacy, Ewha Woman’s University.

Extraction and Isolation

The roots of A. dahurica (4.8 kg) were extracted with CHCl₃ (3 × 6 L) overnight at room temperature. The CHCl₃ extract (115 g) was separated by silica gel column chromatography (CC) using CHCl₃–acetone (1:0 to 0:1, v/v), affording 7 fractions (F1–F7). Fraction F2 (43 g) was subjected to silica gel CC using hexanes–EtOAc (9:1 to 1:1, v/v) to obtain subfractions F0201–F0219. Compound 6 (3.3 g, 0.69% w/w) was precipitated from F0208 with EtOAc. Fraction F0214 (6 g) was subjected to RP-C₁₈ CC with acetonitrile (MeCN)–water (H₂O) (1:1, v/v) as a solvent system to yield 1 (325.8 mg, 0.0068% w/w), 3 (9.5 mg, 0.0002% w/w), 4 (659.3 mg, 0.014% w/w), and 5 (761.5 mg, 0.016% w/w). Fraction F0215 (0.1 g) was subjected to RP-C₁₈ CC with MeCN–H₂O (1:1, v/v), to furnish 2 (0.9 mg, 0.000019% w/w). Fraction F0212 (0.3 g) was chromatographed over ODS-A using gradient mixtures of MeCN–H₂O (1:1 to 2:1, v/v) to yield 7 (9.1 mg, 0.00019% w/w). Compound 8 (497.5 mg, 0.01% w/w) was precipitated from F4 in acetone.

Cell Culture

NSCLC A549 cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) at 37°C in a humidified atmosphere with 5% carbon dioxide.

Wound Healing Assay

Wound healing assays were performed as described previously. ¹² Briefly, A549 cells were seeded in a 24-well plate (3.5 × 10⁵ cells/well) containing plastic inserts (Cell Biolabs Inc., San Diego, CA, USA) for the generation of a wound field and incubated for 24 hours. After removing the inserts from the wells, cells were exposed to 10 Gy of γ-irradiation using a ¹³⁷Cs γ-ray source (Atomic Energy of Canada, Mississauga, Canada) at a dose rate of 3 Gy/min. The irradiated cells were incubated with 50 µM of each compound. Images were taken at time points of 0, 24, and 48 hours and analyzed for cell migration using an AE31 microscope (Motic, Hong Kong).

Cell Viability Assay

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used for determining the cytotoxicity of compounds in A549 cells. The cells (2 × 10³ cells/well) were seeded in 96-well plates and incubated for 24 hours and treated with various concentrations of each compound (0, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 µM) for 24 hours. Subsequently, 50 µL of MTT solution (2 mg/mL) was added to each well and incubated for 3 hours. Formazan crystals generated in living cells were dissolved in 200 µL/well of dimethyl sulfoxide, and the absorbance of individual wells was measured at 570 nm using the Gemini XPS Microplate Reader (Molecular Devices, Sunnyvale, CA, USA).

Statistical Analysis

All experiments were replicated at least 3 times. Statistical significance was determined using Student’s t-test or one-way analysis of variance using GraphPad software (LaJolla, CA, USA). The 50% inhibitory concentration was calculated from a concentration–response analysis performed using GraphPad software (LaJolla, CA, USA).