Background
Lung cancer is the leading cause of cancer-related mortality worldwide [
1], because most patients have already progressed to an advanced stage or have developed distant metastasis when diagnosed. More than 85% of lung cancers are histologically of non-small cell lung cancer (NSCLC) [
2], and the prognosis of NSCLC patients with metastatic tumors or at stage IV is very poor, with only a median survival time of 8~ 10 months [
3]. Currently, chemotherapy, radiotherapy, and targeted therapy are treatment options for advanced NSCLC. The response rate of standard first-line chemotherapy (platinum-based combined with third-generation cytotoxic agents) of advanced lung cancers has significantly improved survival times but with low short-term efficacy, high toxicity, and ultimately the development of drug resistance [
4]. When chemotherapy is no longer effective, some targeted medicines such as gefitinib (an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI)) can be used for advanced NSCLC patients with EGFR mutations (deletions in exon 19 and L858R in exon 21) to further prolong patient survival [
5]. However, most patients eventually have no effective response to gefitinib, because cancer cells develop a second mutation, such as T790 M in the
EGFR gene [
6]. Therefore, searching for new drugs with high efficacy and low toxicity is urgently needed.
Tumor metastasis is a continuous multi-step process, and the epithelial-to-mesenchymal transition (EMT) is one of the most important mechanisms in the initiation and promotion of tumor metastasis [
7]. In NSCLC, the EMT of cells was reported to promote metastasis and also determine chemoresistance [
8] and insensitivity to EGFR inhibitors [
9]. The serine-threonine protein kinase, Akt, was reported to play a crucial role in NSCLC invasion [
10], but the underlying molecular mechanisms of NSCLC invasion mediated by the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is not completely understood. At present, the EMT is known to be a cellular process subject to Akt kinase regulation. Activated Akt was shown to regulate several steps of the EMT, such as loss of cell-cell adhesion and polarization, morphological changes, induction of cell motility, and changes in the production of various proteins [
11‐
13]. For example, Snail and Slug (Snail2), the most thoroughly investigated EMT regulators in lung cancer, are reportedly regulated by activated Akt [
14]. PI3K/Akt can inhibit the degradation of Snail and Slug by targeting glycogen synthase kinase (GSK)-3β or by directly upregulating Snail expression in different cancer types [
15‐
17]. Actually, the PI3K/Akt signaling pathway which mediates the EMT process has garnered widespread attention as a potential target for preventing and treating metastatic tumors. Therefore, investigating compounds with medicinal effects on Akt activation and the Snail family-mediated EMT should be a good strategy for NSCLC.
CD26, a 110-kDa type II transmembrane glycoprotein with dipeptidyl peptidase IV (DPPIV) activity in its extracellular domain, can cleave N-terminal dipeptides from polypeptides with an alanine or proline at the penultimate position [
18]. Previously, CD26 was shown to participate in T-cell biology as a marker of T-cell activation or as a costimulatory molecule able to regulate signaling transduction pathways [
19,
20]. Recently, CD26 was shown to play a critical role in cancer biology. For example, CD26 overexpression was associated with tumor aggressiveness in many cancer types such as astrocytomas [
21], lymphomas [
22], urothelial carcinoma [
23], colorectal cancer [
24], and gastrointestinal stromal tumors [
25]. For example, CD26-positive colorectal cancer stem cells, which are mediators of the EMT, contribute to the invasive phenotype and metastatic capacity [
24]. An in vivo study further showed that vildagliptin, a CD26 inhibitor, significantly suppressed metastasis of colorectal cancer [
26]. These data emphasize the involvement of CD26 in cancer metastasis. So far, little information is known about the role of CD26 and its underlying mechanisms in regulating metastasis and invasion of NSCLC in vitro and in vivo.
Flavonoids are plentiful in fruits and vegetables and are a class of plant secondary metabolites with a ubiquitous phenolic structure. Recent cancer research studies have shown that flavonoids are highly promising compounds alone or in combination with other therapeutic agents against the growth and/or metastasis of different cancer cells in vitro and in vivo [
27]
. Apigenin (API), 4′,5,7-trihydroxyflavone, is one of the most common flavonoids and is abundantly present in various fruits, vegetables, and Chinese medicinal herbs [
28]. API recently received much attention in cancer treatment, due to its potent anticancer activities in different cancer types in vitro and in vivo, including inducing apoptosis or autophagy, regulating the cell cycle, inhibiting migration/invasion, attenuating drug resistance, and stimulating immune responses [
29]. The PI3K/Akt signaling pathway was reported to play a critical role during most anticancer processes induced by API [
29].
Although API was recently shown to inhibit proliferation by targeting Akt in NSCLC cells harboring the wild-type EGFR [
30], whether API has a broad impact on NSCLC with different EGFR mutation statuses, how API impacts the metastatic ability of NSCLC cells in vivo, and what the underlying mechanisms of API are in regulating Akt activity and modulating cell motility are still undefined. In the present study, we further investigated the antimetastatic effect of API on four NSCLC cell lines which harbor the wild-type (WT) or mutant EGFR and defined its underlying mechanisms in vitro and in an orthotopic xenograft model.
Methods
Materials
API (A3145) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS), antibiotics, molecular weight standards, trypsin-EDTA, trypan blue stain, and all medium additives were obtained from Life Technologies (Gaithersburg, MD). An enhanced chemiluminescence kit was purchased from Amersham (Arlington Heights, IL). An anti-matrix metalloproteinase (MMP)-3 antibody was purchased from Epitomic (Burlington, CA). Antibodies specific for fibronectin and MMP-9 were obtained from Abcam (Cambridge, MA). Antibodies specific for CD26, MMP-2, Snail, Slug, Twist, phosphatase and tensin homolog (PTEN), and unphosphorylated and phosphorylated (p-) forms of the corresponding Akt and epidermal growth factor receptor (EGFR) were obtained from Cell Signaling Technology (Danvers, MA). Antibodies specific for vimentin and N-cadherin were purchased from BD Biosciences (San Jose, CA). Antibodies specific for presenilin-1 and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyvinylidene fluoride (PVDF) membranes for Western blotting were purchased from Bio-Rad (Hercules, CA). Unless otherwise specified, other chemicals used in this study were purchased from Sigma Chemical (St. Louis, MO).
Cell lines and cell culture
The A549, H1975, and HCC827 NSCLC cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA), and a series of NSCLC cell lines, CL1–0, CL1–3, and CL1–5, in ascending order of invasiveness, were established in the National Health Research Institute laboratory [
31]. PC9 cells were developed by Lee and colleagues at National Cancer Center Hospital (Tokyo, Japan) [
32]. All cells were maintained in RPMI 1640 supplemented with 10% FBS and 1% penicillin-streptomycin-glutamine. All cells were incubated at 37 °C in a humidified 5% CO
2 atmosphere.
Cell viability assay (MTS assay)
A549, CL1–5, HCC827, and H1975 NSCLC cells (5 × 103) were seeded in 96-well plates, treated with various concentrations of API (5~ 80 μM) for 24 and 48 h, and then subjected to a cell-viability assay (MTS assay; Promega, Madison WI) according to the manufacturer’s instructions. Data were collected from three replicates.
Plate clonogenic assay
NSCLC cells were diluted and seeded at 1000 cells/well in six-well plates and incubated for 24 h. Subsequently, various concentrations (5~ 40 μM) of API were added for 24 h, and then continuously incubated in new fresh medium at 37 °C. After incubation for 7~ 10 days, cells were stained with crystal violet, and a colony was defined as consisting of more than 50 cells.
Transwell migration and invasion assays
Migration and invasion assays were performed according to our previous study [
33]. Briefly, 3 × 10
4 cells were plated in a uncoated top chamber (24-well insert; pore size, 8 μm; Corning Costar, Corning, NY) for the transwell migration assay. The invasion assay used 4 × 10
4 cells (A549 and HCC827) or 3 × 10
4 cells (CL1–5 and H1975) plated in a Matrigel (BD Biosciences, Bedford, MA)-coated top chamber. In both assays, cells which had been pretreated for 24 h with API (5~ 40 μM) were plated in medium without serum or growth factors, and medium supplemented with 10% serum was used as a chemoattractant in the lower chamber. Cells that were allowed to migrate or invade for 24 h were fixed with methanol and stained with crystal violet. The number of cells migrating through or invading through the membrane was counted under a light microscope (× 100 or × 200, three random fields per well).
Immunofluorescence (IF) microscopy
IF techniques were used to observe actin rearrangement after treatment of NSCLC cells with API or the vehicle. Cells were grown on coverslips and fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with Alexa Fluor 594 Phalloidin (Thermo Fisher Scientific, Rockford, IL) in the dark. Slides were examined and photographed using a Zeiss Axiophot fluorescence microscope (Carl Zeiss Microimaging, Gottingen, Germany). Nuclei were counterstained with 4′,6-diamino-2-phenylindole (DAPI).
Preparation of total cell extracts and western blot analysis
Protein lysates were prepared as described previously [
34]. A Western blot analysis was performed with indicated primary antibodies and horseradish peroxidase-conjugated secondary antibodies. After washing, blots were incubated with the Western blotting reagent ECL (TOOLS, New Taipei City, Taiwan), and chemiluminescence was detected by the chemiluminescence imaging system, MultiGel-21 (TOP BIO, New Taipei City, Taiwan). Furthermore, the same blots were stripped by stripping buffer (TOOLS, New Taipei City, Taiwan) and reprobed with the β-actin antibody as an internal control.
Transient transfection of DNA and small interfering (si)RNA
siRNA for human CD26 (s4245) was obtained from Thermo Fisher Scientific. The pENTER-CD26 plasmid was obtained from Vigene Biosciences (Rockville, MD), and the myr-Akt, pLEX-Snail, and pCIneo-Slug plasmids were obtained from Dr. C.C. Chen and Dr. T.C. Kuo (National Taiwan University, Taipei, Taiwan). To knock down CD26 or overexpress CD26, Akt, Snail, or Slug, semiconfluent cultures of NSCLC cells in a 6-mm2 Petri dish were transfected with 50 nM of siRNA using GenMute™ siRNA Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD) or 3 μg of an empty or expression vector using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA) for 6 h according to the manufacturer’s instructions. At 24 h after transfection, cells were analyzed for invasion/migration and expressions of CD26, Snail, Slug, and p-Akt.
Lentiviral production and infection
Short hairpin (sh) RNAs were purchased from the National RNAi Core Facility at Academic Sinica (Taipei, Taiwan). The target sequence of CD26 shRNA was 5’-ACACTCTAACTGATTACTTAA-3. The shRNA lentivirus was produced as previously described [
35].
RNA preparation and reverse-transcriptase polymerase chain reaction (RT-PCR)
Messenger (m) RNA was isolated and amplified as described previously [
35]. Primer sequences of CD26 were F: 5’-GAATGCCAGGAGGAAGGAATCT-3′ and R: 5’-TATTCCACACTTGAACACGCCA-3′.
In vivo lung cancer orthotopic model
All animal experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee of Taipei Medical University. For the CD26 overexpression and knockdown experiments in an orthotopic xenograft model, 5-week-old nonobese diabetic (NOD)-SCID male mice were anesthetized with isoflurane and placed in the right lateral decubitus position; then the A549-mock-luciferase, A549-CD26-luciferase, or A549-sh-CD26-luciferase (sh-775 and sh-777) stable cell lines (106 cells) were resuspended in a 1:1 mixture of phosphate-buffered saline (PBS) and GFR-Matrigel and injected into the left lung parenchyma of a NOD-SCID mouse using a 30-gauge needle. After 7 days, the mice were randomized into six groups according to bioluminescence images taken using the Xenogen IVIS-Spectrum system (Caliper; Xenogen, CA), and treatment was initiated according to similar mean tumor sizes in each group. Subsequently, mice were intraperitoneally (IP) administered 3 mg/kg API or the vehicle (10% DMSO in PBS) 5 days/week. The day after API treatment, the mice were injected with D-luciferin and imaged for 1~5 min using this live imaging device to monitor the tumor size and location in real time. After 28 days, A549-injected mice were sacrificed, and luciferase activities in the excised lungs were further determined using the In Vivo Imaging System (IVIS)-Spectrum system. Mouse lungs were also fixed, sectioned, and stained with hematoxylin and eosin (H&E).
Statistical analysis
Values are presented as the mean ± standard deviation (SD). The statistical analysis was performed using Statistical Package for Social Science software, vers. 16 (SPSS, Chicago, IL). Data were analyzed using Student’s t-test when two groups were compared. A one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used to analyze three or more groups. Statistical analyses of the correlation between CD26 and the invasiveness of NSCLC cells were performed using Spearman rank correlations. Differences were considered significant at a 95% confidence interval (p < 0.05).
Discussion
Dissemination of cancer cells and resistance to drug treatment are two main causes for the poor prognosis of lung cancer patients. Cancer cells undergoing the EMT were reported to have increased resistance to targeted therapy and chemotherapy, higher invasive abilities, and acquisition of stem cell features [
42]. Recently, API was used as a traditional medicine for its anticancer activities and low toxicity to normal cells. In both in vitro and in vivo models, API was shown to suppress tumor growth and metastasis in various cancer types. The antiproliferative activities involve inducing apoptosis and autophagy, modulating the cell cycle and stemness, stimulating an immune response, and enhancing chemosensitivity [
29]. Moreover, API was also shown to inhibit migration, invasion, and metastasis of various cancer cells through suppressing the EMT or modulating ECM-degrading enzymes [
29]. However, compared to other tumor types, little information on the effects of API on the migration and invasiveness of NSCLC cells is available. Until now, only one report indicated that API can inhibit the in vitro cell motility of NSCLC A549 cells by targeting Akt-mediated MMP-9 expression [
30]. Our present results showed that API inhibited the migratory and invasive abilities of a series of NSCLC cell lines harboring WT (A549 and CL1–5) or different mutant EGFR statuses (HCC827 and H1975). Moreover, our results showed that API-mediated downregulation of MMP-2 and MMP-9 was only observed in A549 cells, but not in other NSCLC cell lines (CL1–5 and H1975), suggesting that inhibition of MMP-2 and MMP-9 by API might be cell-type specific. In contrast to MMPs, our study identified that CD26/DPPIV plays a critical role in regulating the invasive abilities of several NSCLC cell lines (A549, CL1–5, CL1–0, and H1975) and can be downregulated by API treatment in these cell lines. Moreover, the antimetastatic effect of API and the metastasis-promoting effect of CD26 were also observed in a human A549 xenograft model. These results suggested that suppression of CD26 might be a general phenomenon in API-regulated cell motility of NSCLC cells.
CD26 is a multifunctional cell-surface glycoprotein with intrinsic DPPIV activity, and it is widely expressed in most cell types. Cell-surface proteases participate in cancer progression and malignant transformation by facilitating tumor cell invasion and metastasis [
43]. Clinical studies of urothelial carcinoma, thyroid cancer, colorectal cancer, and gastrointestinal stromal tumors indicated that CD26 expression is associated with distant metastasis or recurrence after resection [
23,
25,
44]. Similar to previous findings, we also observed that lung cancer patients with a high CD26 expression level had significantly worse recurrence-free survival than those with a lower level. Pang et al. recently identified that CD26-positive colon cancer cells were associated with enhanced invasiveness and chemoresistance, which might be due to EMT induction [
24]. Our results showed abundant CD26 expression in highly invasive NSCLC cell lines (A549, H1975, and CL1–5), but low CD26 expression in the poorly invasive CL1–0 cell line and found a significant correlation between CD26 protein levels and the invasive abilities of a set of NSCLC cell lines (A549, CL1–0, CL1–3, CL1–5, HCC827, PC9, and H1975). A similar correlation between CD26 expression and invasive abilities was also reported in T-cell malignancies [
45]. Moreover, CD26 knockdown in NSCLC cells caused decreases in the invasive abilities and EMT-related markers (Snail, Slug, and fibronectin). Based on these results, we suggest that the CD26 level is a potential marker for predicting the invasive ability of NSCLC cells and the prognosis of lung cancer patients, and CD26 may regulate invasion of cells through EMT induction.
According to the oncogenic role of CD26 in cancers, CD26 itself appears to be a novel therapeutic target. For example, an anti-CD26 monoclonal antibody or CD26 inhibitor treatment was shown to inhibit growth and invasiveness against several tumor types such as renal cell carcinoma and colon cancer [
26,
46]. Recently, several flavonoids, particularly luteolin, API, and resveratrol, were shown to inhibit the in vitro enzyme activity of CD26 [
47]. Our present study further demonstrated that API also suppressed the protein and mRNA expressions of CD26 and the EMT-mediated cell invasion in several NSCLC cell lines (A549, CL1–5, and H1975). These results suggested that inhibition of CD26 might be a general phenomenon in API-regulated cell motility of NSCLC cells. Moreover, our studies also showed that API treatment attenuated growth and metastasis via targeting CD26 in an A549 orthotopic graft model. Taken together, our data suggest that the ability of API to inhibit cell invasiveness might be attributable to its capacity to suppress CD26 expression followed by inhibiting the EMT, and API has potential value in clinical applications for advanced NSCLC. Furthermore, as CD26 was reported to be a possible stem cell marker that induced the EMT in colon cancer [
24], the role of CD26 and the effect of API on the stemness of NSCLC will be further investigated in the future.
Transcription factors of the Snail family (Snail and Slug) were associated with EMT progression during lung cancer metastasis [
42]. Our results showed that protein levels of Snail family members exhibited dramatic decreases after API treatment of NSCLC cells. PI3K/Akt was reported to play a crucial role in regulating expressions of Snail family members through multiple mechanisms. For example, activation of PI3K/Akt can phosphorylate GSK-3β to promote GSK-3β degradation and further maintain the stability of Snail and Slug [
14]. Moreover, activation of Akt can induce an increase of nuclear factor (NF)-κB subunit p65, further leading to increased Snail transcription [
48]. Our present study demonstrated that API treatment could suppress Akt activation in several NSCLC cell lines (A549, CL1–5, and H1975). Moreover, overexpressing activated Akt in A549 cells induced upregulation of Snail and Slug and reversed API-mediated inhibition of the invasive ability, suggesting that Akt inhibition by API is a general phenomenon in NSCLC cells and may be the main cause for the API-mediated suppression of Snail family-induced cell motility. However, the effects of API on GSK-3β phosphorylation and p65 expression in NSCLC cells need to be further investigated in the future. Moreover, Akt activation was recently defined as a convergent feature of acquired EGFR TKI resistance, and increased p-Akt levels were observed in clinical specimens obtained from EGFR-mutant NSCLC patients who acquired EGFR TKI resistance [
49,
50]. Combined treatment of Akt inhibitor with EGFR TKI had shown to exhibit the synergistic growth inhibitory effect in several EGFR TKI-resistant NSCLC models [
49]. From the inhibitory effect of API on invasion and colony formation of NSCLC cells, our data also showed that EGFR mutant cells, HCC827 and H1975, were more sensitive to API treatment compared to the EGFR WT cells, A549 and CL1–5. Among these NSCLC cells, TKI-resistant H1975 cell was the most sensitive cells to API treatment. In addition to Akt, our study further demonstrated that API also suppressed the expression of p-EGFR in EGFR mutant cells (Additional file
1: Figure S4), suggesting the combination of API and EGFR TKI for the treatment of TKI-resistant NSCLC cells is worthy of further investigation.
As to upstream factors of PI3K/Akt signaling in cancer cells with EMT progression, transforming growth factor (TGF)-β was reported to act as an important inducer to stimulate the Akt-mediated EMT. TGF-β was reported to activate Akt directly or through upregulating hyaluronan synthase (HAS) expression, promoting CD44/EGFR expression and co-localization, and subsequently activating Akt in NSCLC cells [
14,
51]. The effect of API on TGF-β expression and its downstream signaling molecules will be further investigated in NSCLC cells. In addition to TGF-β, the present study showed that knockdown of CD26 also suppressed Akt activation in NSCLC cells, suggesting that CD26 suppression by API might contribute to API-mediated inhibition of the Akt-induced EMT in NSCLC cells. Recent studies indicated that expression of CD26 in T cell lines leads to increased stromal-cell-derived factor (SDF)-1-α-mediated invasion through inducing PI-3 K/Akt pathways. Moreover, CD45 was reported to be associated with CD26 to regulate CD26’s enhancement of invasion [
45]. Hence, the roles of SDF-1-α and CD45 in CD26-mediated invasive abilities of NSCLC and the effect of API on SDF-1-α and CD45 warrant further study in our future work.
In contrast to Akt-regulated expressions of Snail family members, our results also showed that overexpressing Snail or Slug in NSCLC cells could induce activation of Akt and reverse the API-mediated inhibition of Akt, suggesting that Snail family members showed positive feedback regulation of Akt activation. Previous studies reported that Snail or Slug can regulate Akt activation to induce prostate cancer cell motility and drug resistance through transcriptional inhibition of several tumor suppressors which target Akt, including maspin [
52] and PTEN [
53,
54]. In greater detail, Snail was reported to cooperate with lysine-specific demethylase 1 (LSD1) to repress PTEN through removing histone H3 lysine 4 [
55]. Maspin and PTEN were shown to suppress survival of lung cancer cells through modulating the Akt pathway [
56,
57]. Actually, our present and previous studies showed that Snail or Slug overexpression in A549 cells decreased maspin or PTEN expression [
58], but the functional roles of maspin, PTEN, and LSD1 in Snail/Slug-regulated Akt activity and the effects of API on these regulators in NSCLC cells with different EGFR statuses should be further evaluated in the future.