Inhibitory effect of trans-ferulic acid on proliferation and migration of human lung cancer cells accompanied with increased endogenous reactive oxygen species and β-catenin instability
- Yao Fong†1,
- Chia-Chun Tang†2,
- Huei-Ting Hu3,
- Hsin-Yu Fang4,
- Bing-Hung Chen3, 10,
- Chang-Yi Wu3, 5,
- Shyng-Shiou Yuan6,
- Hui-Min David Wang7,
- Yen-Chun Chen3,
- Yen-Ni Teng8 and
- Chien-Chih Chiu3, 5, 6, 9Email author
© The Author(s) 2016
Received: 18 March 2015
Accepted: 19 September 2016
Published: 1 October 2016
Trans-ferulic (FA) acid exhibits antioxidant effects in vitro. However, the underlying mechanism of trans-FA activity in cellular physiology, especially cancer physiology, remains largely unknown. This study investigated the cellular physiological effects of trans-FA on the H1299 human lung cancer cell line.
The 2,2-diphenyl-1-picrylhydrazyl assay was used to determine free radical scavenging capability. Assessment of intracellular reactive oxygen species (ROS) was evaluated using oxidized 2ʹ,7ʹ-dichlorofluorescin diacetate and dihydroethidium staining. Trypan blue exclusion, colony formation, and anchorage-independent growth assays were used to determine cellular proliferation. Annexin V staining assay was used to assess cellular apoptosis by flow cytometry. Wound healing and Boyden’s well assays were used to detect the migration and invasion of cells. Gelatin zymography was used to detect matrix metalloproteinase (MMP-2 and MMP-9) activity. Western blotting was used to detect expression levels of various signaling pathway proteins.
DPPH assay results indicated that trans-FA exerted potent antioxidant effects. However, trans-FA increased intracellular ROS levels, including hydrogen peroxide and superoxide anion, in H1299 cells. Trans-FA treatment inhibited cellular proliferation and induced moderate apoptotic cell death at the highest concentration used (0.6 mM). Furthermore, trans-FA moderately inhibited the migration of H1299 cells at the concentrations of 0.3 and 0.6 mM and attenuated MMP-2 and MMP-9 activity. Trans-FA caused the phosphorylation of β-catenin, resulting in proteasomal degradation of β-catenin. Conversely, trans-FA treatment increased the expression of pro-apoptotic factor Bax and decreased the expression of pro-survival factor survivin.
Various concentrations (0.06–0.6 mM) of trans-FA exert both anti-proliferation and anti-migration effects in the human lung cancer cell line H1299.
In 2013, the mortality rates for male and female lung cancer patients in the United States were 28 and 26 %, respectively . At present, chemotherapy is the primary treatment for lung cancer [2–4], with both carboplatin and cisplatin commonly used as chemotherapy drugs [5, 6]. However, the combination of cisplatin or vinblastine with irradiation increases the level of unexpected toxicity in the body . Pemetrexed (Alimta®), a next-generation antifolate drug, is used for treating malignant pleural mesothelioma and non-small cell lung cancer (NSCLC) but can induce scleroderma-like induration of the lower extremities . It is therefore necessary to develop alternative treatment strategies for selective inhibition of lung cancer cells growth.
The carcinogenic process may be driven by mutations, leading to alterations in phenotypes, genetics, and epigenetics. The induction of oxidative stress in various cancer cells such as human pancreatic and colon adenocarcinoma cancer cell lines  was shown to inhibit expression of β-catenin and the matrix metalloproteinases MMP-2 and MMP-9 in colitis-associated colon carcinoma , induce Bax expression in urothelial cell carcinoma , induce apoptosis by blocking the AMPK-mTOR-survivin pathway , and inhibit the anchorage-independent growth (AIG) of transformed cells . Chemopreventive treatment is moderate or non-cytotoxic to normal cells, but significantly inhibits cancer cell growth or metastasis. Accumulating evidence shows that anti-oxidative compounds isolated from plants exert potentially chemopreventive effects. Among these compounds are carotenoids, curcumin, and hesperidin [13, 14]. Recently, anticancer compounds derived from plants, including goniothalamin and feruloyl-l-arabinose, were shown to inhibit the growth and metastasis of human lung cancer cells [15, 16]. Additionally, moscatilin, isolated from the orchid Dendrobrium loddigesii, inhibited metastasis of both human breast and lung cancer cells [17, 18].
Ferulic acid (FA) is an aromatic compound, abundant in plant cell walls [19, 20]. Both isomers of FA, cis-FA and trans-FA, show a potent ability to remove reactive oxygen species (ROS) and inhibit lipid peroxidation [20, 21]. Unlike cis-FA, trans-FA is abundant in plant cells and easily isolated from various plants, thus significantly reducing the cost of its preparation .
Trans-FA ameliorated ionizing radiation-induced inflammation and glycerol-induced nephrotoxicity [23, 24] and modulated fluoride-induced oxidative hepatotoxicity in male Wistar rats . Water-soluble trans-FA sugar esters protected normal rat erythrocytes against peroxyl radical 2,2ʹ-azobis-2-amidinopropane dihydrochloride (AAPH)-induced oxidative damage  and exhibited protective capacity against oxidative damage caused by diabetes [27, 28]. Furthermore, trans-FA exhibited antiproliferative effects in colon cancer cells [29, 30], increased the radiosensitivity of cervical cancer cells , and exerted protective effects against chemical-induced DNA strand breaks [32, 33]. However, the effects of trans-FA in lung cancer have not been reported to date, and its biological mechanism remains unknown.
The study investigated the cellular and physiological effects of trans-FA in human lung cancer cells.
Trans-FA (4-hydroxy-3-methoxycinnamic acid) was purchased from Sigma-Aldrich Chemicals (#128708, St. Louis, MO, USA). Trans-FA was freshly dissolved in 0.01 % dimethyl sulfoxide (DMSO) and aliquoted before use.
The human non-small cell lung cancer (NSCLC) cell line H1299 and lung fibroblast cells HEL-299 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in DMEM:F-12 medium (1:1 ratio) supplemented with 8 % fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin (Gibco BRL, Gaithersburg, MD, USA) at 37 °C in a humidified atmosphere of 5 % CO2 .
DPPH radical-scavenging activity assay
Cell viability and proliferation assay
Briefly, 5 × 104 cells were seeded into wells of a 12-well plate and treated with phosphate-buffered saline (PBS) (Sigma-Aldrich) as control or concentrations of trans-FA (0.03–0.6 mM) for 24 or 48 h. After incubation, cell viability and proliferation were analyzed by trypan blue assay and an automated cell counter (Countess™) according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA).
Colony formation assay
Fifty cells were seeded into wells of a 6-well plate and, after 24 h of incubation, were treated with different concentrations of trans-FA (0.03–0.6 mM). After incubation for 11 days, cell colonies were glutaraldehyde-fixed and stained with crystal violet (0.1 % w/v) for 10 min. Colony diameter was determined using Image-Pro v3.0 software (Media Cybernetics, Silver Spring, MD, USA).
Anchorage-independent growth (AIG) assay
The assay procedure was performed as described in our previous work, with minor modifications . Briefly, 1 × 103 cells were mixed with 0.75 % low melting agarose (MDBio Inc, Taipei, Taiwan). The mixtures were placed on a solidified layer of 1.5 % agarose with medium in a 12-well plate. After incubation for 13 days, cells in the upper layer were fixed with 1 % glutaraldehyde and stained with 0.1 % w/v Giemsa (Merck, Darmstadt, Germany). Colony diameter was determined using Image-Pro v3.0 software.
Cell cycle analysis
Cell cycle distribution was assessed using propidium iodide (PI; Sigma-Aldrich) staining described previously . Briefly, 1 × 105 cells were treated with PBS (as vehicle control) or various trans-FA concentrations (0.03–0.6 mM) for 48 h. Next, cells were detached using 0.05 % trypsin (Biological Industries, Kibbutz Beit Haemek, Israel) for 5 min, harvested, and washed with PBS. Cells were then fixed with 75 % ethanol overnight. After centrifugation (Microfuge® 16, Beckman Coulter Life Sciences, Taipei, Taiwan) at 664×g for 15 min at 4 °C, the resulting supernatants were decanted. Cell pellets were stained with 10 µg/mL PI and 10 µg/mL RNase A (Sigma-Aldrich) in PBS buffer for 30 min at 37 °C in the dark. The samples were assayed using a FACScan flow cytometer (Becton–Dickinson, Mansfield, MA, USA) and the results were analyzed using FlowJo v7.5.5 software (Tree Star Inc., San Carlos, CA).
Assessment of apoptosis
Apoptotic cell death was assessed by annexin V and PI double staining (Pharmingen, San Diego, CA, USA) according to our previous paper . Briefly, 1 × 106 cells were seeded into a 100-mm petri dish and treated with vehicle or trans-FA at doses of 0–0.6 mM for 48 h. Next, cells were inoculated with 10 µg/mL of annexin V-fluorescein isothiocyanate (FITC) and 20 µg/mL of PI and analyzed using a FACScan flow cytometer FlowJo v7.5.5 software.
Detection of endogenous ROS
Changes in endogenous ROS levels were assessed using the fluorescent indicators 2′,7′-dichlorofluorescin diacetate (DCFDA, Sigma-Aldrich) for hydrogen peroxide (H2O2) and dihydroethidium (DHE) (Invitrogen) for superoxide anion (O2 −). A total of 1 × 105 H1299 cells were seeded into wells of a 6-well culture plate and treated with various concentrations (from 0.03 to 0.15 mM) of trans-FA for 24 h. Next, cells were harvested and stained with 100 nM DCFDA or 1 µM DHE for 30 min at 37 °C in PBS and then washed twice with PBS. Fluorescence was measured by flow cytometry.
Western blot assay
Western blot assays were performed as described previously . Briefly, cells were harvested and lysed, lysates were centrifuged, and protein concentrations of cell pellets were determined. Next, 40 mg quantities of protein lysate were resolved by 10 % SDS–polyacrylamide gel electrophoresis and electro-transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5 % nonfat milk and incubated with the following primary antibodies: Thr41/Ser45 phosphorylated β-catenin (#2377-S, Epitomics, CA, USA), β-catenin (#sc-7963, Santa Cruz Biotech., CA, USA), Bax (#ab32503, Abcam Inc., Cambridge, MA, USA), survivin (#614701, Biolegend, CA, USA), and β-actin (#AM1021b, Abgent, San Diego, CA, USA), followed by appropriate secondary antibodies (anti-Mouse, #074-1806; anti-Rabbit, #074-1506, KPL, Gaithersburg, MD, USA). The WesternBright™ ECL kit (Advansta, CA, USA) was used for signal detection.
Quantities of 3 × 105 H1299 cells were seeded in wells of a 12-well plate, treated with PBS (as vehicle control) or trans-FA (0.03–0.6 mM), and grown to 100 % confluence. Culture monolayers were scratched using a pipette tip to create a clean 1-mm-wide wound area. After further incubation for 16 h, the wound gaps were photographed and analyzed using TScratch software (CSE Lab, Zurich, Switzerland) .
Transwell invasion assay
Invasion assays were performed as described in our previous work, with minor modifications , using 8 µm-pore Transwell® chambers (Greiner Bio-One, Frickenhausen, Germany). Control and various concentrations (from 0.03 to 0.6 mM) of trans-FA treated cells were cultured in triplicate at 5 × 104 cells/well in the upper inserts of 24-well Transwell® culture plates. Next, cells were fixed for 5 min and stained with 0.1 % w/v Giemsa. The cells which had invaded the lower inserts were counted by arbitrarily selecting five fields from each well. The experiments were repeated three times.
MMP-2 and -9 activity was assessed by gelatin zymography as previously described , with minor modification. Briefly, 3 × 105 H1299 cells were seeded into wells of 12-well plates and cultured with various concentrations of trans-FA for 24 h, after which aliquots of culture medium were harvested for gelatin zymography analysis. Samples were prepared in standard SDS-PAGE loading buffer containing 0.01 % SDS without β-mercaptoethanol and were not boiled before use. Next, the samples were subjected to electrophoresis (150 V for 3 h) on 10 % SDS–polyacrylamide gels containing 1 % gelatin. After electrophoresis, the gels were thoroughly washed with distilled water containing 2.5 % Triton X-100 on a gyratory shaker for 30 min at room temperature. Gels were incubated in 100 mL reaction buffer (40 mM Tris–HCl, pH 8.0; 10 mM CaCl2, and 0.02 % NaN3) at 37 °C overnight, followed by staining with Coomassie brilliant blue R-250 (Bio basic Inc., Markham, Ontario, Canada) and de-staining with methanol-acetic acid–water (50/75/875, v/v/v). The gelatinase activities of MMP-2 and -9 were determined by analyzing signal intensity using Gel Pro v.4.0 software (Media Cybernetics, Silver Spring, MD, USA).
All data are mean ± SD from at least three experiments, with three replicates per experiment. The significance of the differences was analyzed using one-way analysis of variance (ANOVA) and SigmaPlot v12.0 software (Systat Software Inc., San Jose, CA, USA). P values less than 0.05 were considered statistically significant.
Radical scavenging activity of trans-FA
The short-term effect of trans-FA on proliferation of NSCLC cells
The long-term effect of trans-FA on colony formation and AIG assay in NSCLC cells
Trans-FA caused moderate G0/G1 accumulation
Trans-FA-induced apoptosis in lung cancer cells
Trans-FA induced changes in intracellular ROS
Regulation of survival proteins by trans-FA
Trans-FA attenuated the motility of lung cancer cells
Trans-FA exerted an anti-invasion effect
Trans-FA reduced the activity of MMP-2 and MMP-9
The radical scavenger assay findings indicated the potent anti-oxidant activity of trans-FA (Fig. 1). Treatment with trans-FA for 24 or 48 h did not affect cellular morphology and proliferation of lung cancer H1299 cells (Fig. 2). However, long-term treatment with trans-FA attenuated colony formation and AIG, characteristics of advanced cancer, in H1299 cells (Fig. 3). Trans-FA induced moderate cell proliferation at the lowest concentration tested (0.03 mM). These results were consistent with previous reports that trans-FA promoted proliferation of MCF7 and BT20 breast cancer cells and neural progenitor cells [45, 46]. The colony formation assay also showed the discrepant inhibitory effect of trans-FA on cellular proliferation of lung cancer cells H1299 and lung fibroblast cells HEL-299 (Additional file 2), suggesting the potential of trans-FA for selectively inhibiting lung cancer. Furthermore, trans-FA significantly inhibited AIG capability in a dose-responsive manner.
Trans-FA caused an accumulation of the G0/G1 population and induced a moderate increase in the apoptotic population at the highest concentration (0.6 mM) (Fig. 4). G0/G1 cell cycle arrest is usually associated with the upregulation of cell cycle regulatory protein p15 INK4b and p21 WAF1/Cip1 . A recent study showed that perillyl alcohol, a natural compound purified from citrus fruits and herbs, causes G1 arrest and inhibits proliferation of human immortalized keratinocyte HaCaT cells through inducing p15 INK4b and p21 WAF1/Cip1 . Annexin V staining confirmed that the anti-cancer effects of Trans-FA were not mediated through apoptosis (Fig. 5).
Excess endogenous ROS may inhibit cellular growth or cause cell death [48–51]. The anti-cancer effects of trans-FA might correlate with increased levels of ROS in H1299 cells (Fig. 6). ROS content is higher in cancer cells than in normal cells, and ROS are reported to be involved in cancer cell migration . In this study, trans-FA treatment caused the accumulation of both H2O2 and O2−. Trans-FA (0.03 mM) induced an increase in H2O2, but not O2 −. Changes in endogenous ROS levels were assessed using the fluorescent indicators DCFDA for H2O2 and DHE for O2 − . Superoxide dismutase (SOD) converts O2 − into H2O2, and is overexpressed in lung cancer compared with normal and non-malignant lung tissues . Therefore, a moderate increase in O2 − might be rapidly converted into H2O2 in lung cancer cells. However, the significant increase in endogenous O2 − induced by trans-FA (>0.03 mM) may cause saturation of SOD capacity, preventing further conversion of O2 − to H2O2. Accordingly, increased levels of H2O2 might be the product of O2 − conversion by SOD in H1299 cells following low dose (0.03 mM) trans-FA treatment.
β-catenin is a transcription factor involved in cell growth and cell migration pathways. Wnt/β-catenin signaling is thus essential for the maintenance of neuronal progenitor proliferation . However, phosphorylated β-catenin is inactivated and undergoes proteasomal degradation, causing the inhibition of cell growth .
With respect to tumorigenesis, constitutive activation or overexpression of β-catenin is frequently observed in cancers, including rectal cancer , colon cancer , breast cancer , prostate cancer , glioma , and lung cancer . Furthermore, overexpression of β-catenin enhances the expression of cyclin D1, a critical factor for G1/S transition during cell cycle progression in colon carcinoma cells . S-adenosylmethionine and its metabolite, methylthioadenosine, inhibited β-catenin signaling by multiple mechanisms in colon cancer, and thus might have the potential to prevent tumorigenesis . Furthermore, Wnt/β-catenin signaling was shown to be a potent activator of ROS generation, resulting in DNA damage and acceleration of cellular senescence . Furthermore, Wnt/β-catenin signaling potently activated ROS generation in mesenchymal stem cells [64–66].
To clarify the underlying mechanism of trans-FA-induced anti-lung cancer activities, we examined whether trans-FA could affect the expression of cell proliferation-related transcription factor β-catenin using western blotting (Fig. 7). Our results demonstrated that trans-FA treatment promoted the phosphorylation of β-catenin at residues Thr41 and Ser45  and led to the proteasomal degradation of cytoplasmic β-catenin, causing the downregulation of β-catenin protein levels. The Wnt pathway regulated MMP-2/-9 expression by directly targeting the MMP promoter through T-cell factor (TCF), a β-catenin interacting partner, therefore promoting cellular migration . In effector T cells, endothelial cell-derived Wnt induced the expression of MMP-2/-9 through activating the Frizzled receptors to regulate the transmigration of T cells. In contrast, Wnt signaling blockade reduced the migration of effector T cells in vitro .
In addition to β-catenin, we also examined the role of pro-survival protein Bax, a key anti-survival factor, can promote apoptosis by binding to and antagonizing pro-survival Bcl-2 proteins such as Bcl-2 or Bcl-xL . Conversely, survivin is a member of the inhibitor of apoptosis (IAP) family and acts as an inhibitor of caspase activation, thereby negatively regulating apoptosis or programmed cell death . Both the Bcl-2 family and IAP proteins are critical regulators of cell proliferation and survival. In our study, the significant changes in Bax and survivin expression occurred alongside the anti-proliferation effects observed following trans-FA treatment (Fig. 7). As shown using colony formation and AIG assays, trans-FA treatment might impair cell proliferation of H1299 cells. Apart from in cells treated with higher concentrations (0.3 and 0.6 mM) of trans-FA, no significant increase in the population of apoptotic cells was detected. Survivin is considered an apoptosis inhibitor which promotes cellular proliferation, although decreased expression of survivin may not always cause apoptosis . Ito et al. showed that both human hepatocellular carcinoma (HCC) cell lines and patient tissues expressed high levels of survivin mRNA, with detectable levels not found in normal and non-tumor areas of liver . Survivin expression may be an indicator of cellular proliferation but not apoptosis in HCC tissues . The degradation or expression Bax may represent a threshold for inducing apoptosis . These results might explain how trans-FA treatment caused an increase in Bax protein expression but did not significantly induce apoptosis in H1299 cells at most concentrations tested (0.015–0.15 mM). These observations suggest that trans-FA treatment attenuates cellular proliferation rather than cellular survival. Therefore, the results of the proliferation assay imply that the anti-migratory effect of trans-FA may also be mediated by regulating the balance of pro-survival and anti-survival proteins in lung cancer cells.
Extracellular matrix-degrading MMPs, especially MMP-2 and MMP-9, are involved in the metastasis of cancer cells . Trans-FA treatment inhibited the migration and invasion of lung cancer cells and concurrently attenuated the activities of both MMP-2 and MMP-9 (Figs. 8, 9, 10). These observations suggest a positive correlation between MMP activity and trans-FA-induced anti-migration in lung cancer cells.
Various concentrations (0.06–0.6 mM) of trans-FA exert both anti-proliferation and anti-migration effects in the human lung cancer cell line H1299.
AMP activated protein kinase
Bcl-2-associated X protein
B-cell lymphoma 2
B-cell lymphoma-extra large
non-small cell lung cancer
inhibitor of apoptosis
mammalian target of rapamycin
- RNase A:
reactive oxygen species
sodium dodecyl sulfate
T cell factor
YF, CCT, BHC and CCC designed the study. CCT, HTH, HYF, CYW and SSY performed the experiments. CYW, SSY, HMW and YNT analyzed and organized the data. YF, YCC and CCC wrote the manuscript. All authors read and approved the final manuscript.
The study was financially supported by Grants NSC101-2320-B-037-046-MY3, NSC102-2632-B-037-001-MY3 and MOST105-2311-B-037-001 from the Ministry of Science and Technology (MOST), Taiwan; Grants 101-CM-KMU-11, 102-CM-KMU-09 and 104-CM-KMU-006 from the ChiMei-KMU Joint Research Project, Grants NSYSU-KMU104-P031 from the NSYSU-KMU Joint Research Project, and Grant MOHW103-TD-B-111-05 from the Ministry of Health and Welfare, Taiwan; by the grants Aim for the Top Universities Grant. KMU-TP103A17, KMU-TP104A03, KMU-TP105A03 and KMU-M104008 from Kaohsiung Medical University, Taiwan; Grant Ten Chan General Hospital, Chung-Li and KMU Joint Research Project (ST102004), Taiwan.
The authors declare that they have no competing interests.
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