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

Abstract

Background

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.

Methods

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.

Results

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.

Conclusion

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.

Electronic supplementary material

The online version of this article (doi:10.1186/s13020-016-0116-7) contains supplementary material, which is available to authorized users.

Background

In 2013, the mortality rates for male and female lung cancer patients in the United States were 28 and 26 %, respectively [1]. 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 [4]. 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 [7]. 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 [8] was shown to inhibit expression of β-catenin and the matrix metalloproteinases MMP-2 and MMP-9 in colitis-associated colon carcinoma [9], induce Bax expression in urothelial cell carcinoma [10], induce apoptosis by blocking the AMPK-mTOR-survivin pathway [11], and inhibit the anchorage-independent growth (AIG) of transformed cells [12]. 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 [22].

Trans-FA ameliorated ionizing radiation-induced inflammation and glycerol-induced nephrotoxicity [23, 24] and modulated fluoride-induced oxidative hepatotoxicity in male Wistar rats [25]. Water-soluble trans-FA sugar esters protected normal rat erythrocytes against peroxyl radical 2,2ʹ-azobis-2-amidinopropane dihydrochloride (AAPH)-induced oxidative damage [26] 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 [31], 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.

Methods

Chemicals

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.

Cell culture

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 [4].

DPPH radical-scavenging activity assay

The anti-oxidant activities of trans-FA were measured based on the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) (#D9132, Sigma-Aldrich) free radical [34, 35]. Briefly, vitamin C standards and various trans-FA concentrations were freshly prepared and diluted in methanol. Methanol (as a blank control; 10 µM) or trans-FA solution was added to 90 µL DPPH solution to yield a final trans-FA concentration of 0.15 mg/mL in a 96-well microplate. The mixture was incubated at 25 °C and protected from light. After incubation, solution absorbance was measured at 492 nm using a Multiskan Ascent 354 microplate reader (Thermo Fisher Scientific, Rockford, IL, USA). DPPH radical scavenging activity was calculated as follows:

$$\text{DPPH radical scavenging activity}\left(\%\right)=\left(1-A_0/A_1\right)\times 100$$

where A₀ and A₁ are the absorbances of the control and trans-FA solutions, respectively. Each experiment was repeated three times and found to be reproducible within experimental error margins.

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 [36]. 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 [37]. 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 [15]. 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 [38]. 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.

Wound-healing assay

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) [39].

Transwell invasion assay

Invasion assays were performed as described in our previous work, with minor modifications [40], 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.

Gelatin zymography

MMP-2 and -9 activity was assessed by gelatin zymography as previously described [41], 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).

Statistical analysis

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.

Results

Radical scavenging activity of trans-FA

The DPPH assay was used to assess the radical scavenging activity of trans-FA. Antioxidant activity was calculated by evaluating the capacity of trans-FA to scavenge DPPH. As shown in Fig. 1, trans-FA treatment produced significant radical-scavenging activity compared with the DMSO control. Vitamin C was serially diluted in methanol (500–7.8 µM) and used in triplicate as a positive control (Additional file 1).

Fig. 1.

DPPH radical-scavenging capacity of trans-FA. a Trans-FA was tested in an antioxidant assay by measuring the DPPH radical-scavenging activity. The indicated concentrations of trans-FA were incubated with DPPH respectively as described in the “Methods” section. b Quantificative analysis. The radical-scavenging capacity of trans-FA at indicated concentrations was quantified as the percentage decrease in absorbance at 492 nm against the DMSO control. *P < 0.05 and **P < 0.001, respectively

The short-term effect of trans-FA on proliferation of NSCLC cells

H1299 cells were treated with PBS (as vehicle control) or different concentrations of trans-FA for 24 or 48 h before gross morphological changes were examined by light microscopy to determine the effect of trans-FA on cell growth (Fig. 2a). Cells exhibited no significant change in morphology compared with the vehicle control. Next, measurement of cell survival was performed by trypan blue dye exclusion assay. Low doses (>0.15 mM) of trans-FA exerted no significant cytotoxic effect, but moderate cytotoxicity was observed with 0.3 and 0.6 mM treatment for 48 h (Fig. 2b).

Fig. 2.

Comparison of cell morphology and proliferation between of control and trans-FA-treated of NSCLC cells. a H1299 cells were treated with PBS as vehicle control or the indicated doses of trans-FA for 24 and 48 h, respectively. And then, morphology was examined and photographed with a light microscope. (phase contrast, X200). b 5 × 10⁴ H1299 cells were seeded onto a 12-well plate, and cells were treated with indicated doses of trans-FA for 24 and 48 h, respectively. Cell viability was determined using the trypan blue exclusion assay described in “Methods” section. *P < 0.001 against vehicle control

The long-term effect of trans-FA on colony formation and AIG assay in NSCLC cells

As shown in Fig. 3a, trans-FA inhibited colony formation in H1299 cells after 11 days of treatment. The calculated colony diameters for trans-FA concentrations of 0, 0.06, 0.15, 0.3 and 0.6 mM were 100 ± 0.00, 77.19 ± 2.70, 64.19 ± 2.75, 56.75 ± 4.23 and 4.59 ± 1.25 % (n = 3), respectively. Figure 3c shows that long-term treatment with trans-FA inhibited the AIG capacity of H1299 cells. Furthermore, the results of the colony formation showed the selectively inhibitory effect of trans-FA on cellular proliferation between lung cancer cells H1299 and lung fibroblast cells HEL-299 (Additional file 2). The calculated colony diameters for trans-FA at 0, 0.03, 0.06, 0.15, 0.3 and 0.6 mM were 100 ± 3.10, 95.34 ± 4.18, 85.64 ± 1.08, 75.51 ± 2.41, 70.41 ± 1.71 and 61.36 ± 2.70 % (n = 3), respectively.

Fig. 3.

The long-term effect of trans-FA on colony formation and anchorage-independent growth assay of NSCLC. a The assessment of colony formation, a 2D indicator of cell proliferation. Adherent H1299 cells were treated with indicated concentrations for 24 h. 5 × 10 cells treated with trans-FA were allowed to form colonies for 11 days. Afterward, colonies were fixed and stained using crystal violet. c Representative results showed the formation of tumor spheres, an indicator of 3D for anchorage-independent growth. H1299 cells were treated with indicated concentrations of trans-FA for 13 days using the soft agar assay. b, d Quantitative analysis of results from a and c showed the number and size of tumorspheres. *P < 0.05 and **P < 0.001, respectively

Trans-FA caused moderate G₀/G₁ accumulation

The effects of 48 h trans-FA treatment on cell cycle progression in H1299 cells were examined. Trans-FA treatments caused the arrest of the cell cycle at G₀/G₁ and a decrease in the percentage of the G₂/M phase (Fig. 4a, b).

Fig. 4.

Effect of trans-FA on cell-cycle progression of H1299 cells. H1299 cells were treated with the indicated doses, 0.03, 0.06, 0.15, 0.3 and 0.6 mM of trans-FA for 48 h respectively. a An accumulation of G₀/G₁ population in trans-FA-treated H1299 cells and vehicle controls at 48 h. b The quantification analysis. Data are presented as mean ± SD (n = 3). Different letter notations indicate the statistical significance between control and trans-FA treatment groups (a no significance; a vs b and a vs c, statistically significant with P < 0.05 and 0.001, respectively)

Trans-FA-induced apoptosis in lung cancer cells

We investigated whether inhibition of proliferation by trans-FA was achieved by apoptosis in H1299 cells. Only the highest concentration (0.6 mM) of trans-FA increased the proportion of Annexin V⁺/PI⁺ cells (from 1.81 to 5.4 %) (Fig. 5). Other concentrations of trans-FA used in the study did not induce a significant increase in apoptotic populations.

Fig. 5.

Effect of trans-FA on apoptosis of H1299 cells. H1299 cells were treated with indicated doses, 0.03, 0.06, 0.15, 0.3 and 0.6 mM of trans-FA for 48 h respectively. Annexin V/PI double staining was performed to detect the apoptosis

Trans-FA induced changes in intracellular ROS

The endogenous level of ROS can regulate a variety of cellular physiological processes, including survival, proliferation, angiogenesis, and signaling pathways [42]. Flow cytometry-based detection with DCFDA and DHE staining was used to detect endogenous H₂O₂ and O₂⁻, respectively. As shown in Fig. 6, H₂O₂ concentrations of 100 ± 20, 317 ± 28, 364 ± 23 and 375 ± 20 % (n = 3) were observed in H1299 cells treated with different concentrations of trans-FA (0–0.15 mM) for 24 h. Furthermore, endogenous O₂⁻ concentrations of 100 ± 9, 100 ± 2, 241 ± 19 and 392 ± 7 % (n = 3) were observed with the same trans-FA concentrations (Fig. 6b).

Fig. 6.

Trans-FA up-modulates the endogenous level of ROS in H1299 cells. 1 × 10⁵ H1299 cells were seeded onto a 6-well plate and treated with indicated doses of trans-FA (from 0.03 to 0.15 mM) 24 h respectively. Afterward, the induction of endogenous ROS was determined by a DCFDA or b DHE staining combined with a flow cytometry analysis. **P < 0.001 against vehicle control

Regulation of survival proteins by trans-FA

Western blotting was used to examine whether trans-FA treatment affected the expression of β-catenin in H1299 cells. As shown in Fig. 7, trans-FA treatment increased β-catenin phosphorylation at Thr⁴¹/Ser⁴⁵ but did not affect β-catenin protein levels. The anti-cancer effects of trans-FA might act by selective inhibition of β-catenin, a transcription factor associated with the growth and migration of H1299 cells. Anti-survival Bax protein was increased following trans-FA treatment in a dose-responsive manner, although the protein level of survivin was decreased.

Fig. 7.

Effect of trans-FA on survival protein of H1299 cells. H1299 cells were treated with the indicated doses, 0.03, 0.06, 0.15, 0.3 and 0.6 mM of trans-FA for 48 h. Western blot assay was performed

Trans-FA attenuated the motility of lung cancer cells

Wound-healing assays were performed to investigate whether trans-FA affected migration of NSCLC cells. trans-FA treatment moderately attenuated the migration of H1299 lung cancer cells (Fig. 8). The area of the denuded zone was used as an index of the migratory ability of H1299 cells. The areas measured for trans-FA concentrations of 0, 0.03, 0.06, 0.15, 0.3 and 0.6 mM were 100 % ± 5.78, 96.86 % ± 2.69, 98.99 % ± 4.39, 93.95 % ± 6.77, 85.78 % ± 5.76 and 76.87 % ± 1.76 (n = 3), respectively.

Fig. 8.

Effect of trans-FA on the migration of H1299 cells. Trans-FA inhibits cell migration of NSCLC tumor H1299 cells at the highest dose (0.6 mM). a 3 × 10⁵ cells were seeded in a 12-well plate and cells were scraped to create a clean 1-mm area within the confluent culture. Cells were treated with the indicated doses of 0.03, 0.06, 0.15, 0.3 and 0.6 mM of trans-FA for 16 h. Afterward, the wound gaps were photographed using an inverted phase-contrast microscopy. b The quantification analysis. *P < 0.05 for trans-FA treatments against vehicle control

Trans-FA exerted an anti-invasion effect

A Boyden chamber assay was used to evaluate invasion ability. After treating H1299 cells with various concentrations (from 0.03 to 0.6 mM) of trans-FA for 16 h, the percentages of invasive cells were 100 % ± 7.49, 95.79 % ± 3.42, 95.61 % ± 5.86, 88.39 % ± 7.01, 82.57 % ± 5.87 and 68.57 % ± 4.48 (n = 3) (Fig. 9).

Fig. 9.

Effect of trans-FA on the invasion of H1299 cells. a Cells were treated with the indicated doses of 0.03, 0.06, 0.15, 0.3 and 0.6 mM of trans-FA for 16 h and stained with 0.1 % w/v Giemsa stain respectively. b The results of the quantificative analysis. *P < 0.05 and **P < 0.001 for trans-FA treatments against vehicle control, respectively

Trans-FA reduced the activity of MMP-2 and MMP-9

MMP-2 and MMP-9 are gelatinases which degrade extracellular matrix and thus regulate the ability of cells to migrate. Overexpression of MMP-2 and MMP-9 promotes cancer progression and is highly correlated with poor prognosis of cancer patients [43]. Therefore, targeting of MMP-2 and -9 represents a promising strategy for cancer treatment [44]. The activity of MMP-2 and MMP-9 was determined using a gelatin zymography assay (Fig. 10a). Trans-FA treatment (0, 0.03, 0.06, 0.15, 0.3 and 0.6 mM) significantly reduced the activity of MMP-2 [100 ± 2.95, 90.59 ± 1.96, 80.43 ± 4.46, 71.99 ± 2.9, 66.04 ± 1.59 and 55.87 ± 0.26 % (n = 3)] (Fig. 10b, c). The activity of MMP-9 [100 ± 4.44, 105.13 ± 5.29, 94.04 ± 0.99, 112.08 ± 5.24, 78.04 ± 1.86 and 53.32 ± 2.38 % (n = 3)] was also reduced.

Fig. 10.

Effect of trans-FA on activities of MMP-2 and MMP-9. H1299 cells were treated with indicated concentrations of trans-FA for 24 h respectively. a The activities of MMP-2 and MMP-9 were determined by a gelatin zymography assay (n = 3). b, c The results of the quantificative analysis. b *P < 0.05 and **P < 0.001 for trans-FA treatments against vehicle respectively. c *P < 0.001 for trans-FA treatments against vehicle control

Discussion

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 [47]. 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 [47]. 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 [42]. 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⁻ [52]. Superoxide dismutase (SOD) converts O₂⁻ into H₂O₂, and is overexpressed in lung cancer compared with normal and non-malignant lung tissues [53]. 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 [54]. However, phosphorylated β-catenin is inactivated and undergoes proteasomal degradation, causing the inhibition of cell growth [55].

With respect to tumorigenesis, constitutive activation or overexpression of β-catenin is frequently observed in cancers, including rectal cancer [56], colon cancer [57], breast cancer [58], prostate cancer [59], glioma [60], and lung cancer [61]. 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 [62]. 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 [63]. Furthermore, Wnt/β-catenin signaling was shown to be a potent activator of ROS generation, resulting in DNA damage and acceleration of cellular senescence [64]. 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 [55] 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 [67]. 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 [67].

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 [68]. 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 [69]. 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 [69]. 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 [70]. Survivin expression may be an indicator of cellular proliferation but not apoptosis in HCC tissues [70]. The degradation or expression Bax may represent a threshold for inducing apoptosis [71]. 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 [72]. 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.

Based on these observations, we propose that the anti-lung cancer effects of trans-FA might act through the modulation of endogenous ROS and β-catenin stabilization. Trans-FA induced the production of endogenous ROS and may cause β-catenin phosphorylation, resulting in proteasomal degradation. In addition, trans-FA regulated the balance between pro-survival and pro-apoptosis signals and downregulated the activities of metastasis-associated MMP-2 and MMP-9 (Fig. 11).

Fig. 11.

Schematic diagram of hypothesized mechanism of trans-FA effect of lung cancer cells. Trans-FA induced ROS leading to degradation of phosphorylated β-catenin. Trans-FA induced regulation of pro-survival proteins survivin and anti-survival protein Bax caused the anti-proliferation of lung cancer H1299 cells. Trans-FA also reduced the activity of both MMP-2 and MMP-9, causing the down-regulation of migration of H1299 cells

Conclusion

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.

Abbreviations

AAPH

2,2′-azobis-2-amidinopropane dihydrochloride

AIG

anchorage-independent growth

AMPK

AMP activated protein kinase

Bax

Bcl-2-associated X protein

Bcl-2

B-cell lymphoma 2

Bcl-xL

B-cell lymphoma-extra large

DCFDA

2′,7′-dichlorofluorescin diacetate

DHE

dihydroethidium

DMSO

dimethyl sulphoxide

DPPH

2,2-diphenyl-1-picrylhydrazyl

ECL

enhanced chemiluminescence

FA

ferulic acid

NSCLC

non-small cell lung cancer

IAP

inhibitor of apoptosis

MMP

matrix metallopeptidase

mTOR

mammalian target of rapamycin

PBS

phosphate-buffered saline

PI

propidium iodide

RNase A

ribonuclease A

ROS

reactive oxygen species

SD

standard deviation

SDS

sodium dodecyl sulfate

SOD

superoxide dismutase

TCF

T cell factor

Additional files

13020_2016_116_MOESM1_ESM.pdf ^{(245.6KB, pdf)}

10.1186/s13020-016-0116-7 DPPH radical-scavenging capacity of vitamin C as a positive control. (A) Vitamin C as a positive control in DPPH assay. (B) Quantificative analysis of (A). The radical-scavenging capacity of Vitamin C at indicated concentrations was quantified as the percentage decrease in absorbance at 492 nm against the blank control. *P < 0.05 and **P < 0.001 for trans-FA treatments against vehicle respectively.

13020_2016_116_MOESM2_ESM.pdf ^{(237KB, pdf)}

10.1186/s13020-016-0116-7 Discrepant proliferative effect of trans-FA on long-term expansion of lung cancer cells and lung fibroblast. Lung fibroblast HEL-299 and NSCLC tumor cells H1299 were treated with indicated concentrations of trans-FA respectively. Afterward, cells were fixed with glutaraldehyde and stained with crystal violet.