Background
Lung cancer is a leading malignancy in the world, especially in the Taiwan area [
1]. Human non-small cell lung cancer (NSCLC) accounts for around 80% of total lung cancer cases [
2]. The primary treatments for NSCLC patient are chemotherapeutics; however, the chemoresistance of NSCLC cells is frequently reported, resulting in poor prognosis and low survival rate of NSCLC patients [
3]. Therefore, novel and improved chemotherapies for NSCLC cells are still being developed [
3‐
7].
Compounds with a quinoline backbone have been shown to exert many bioactivities such as anti-autoimmune [
8], anti-inflammatory [
9] and anti-carcinogenic modalities [
9‐
13]. For example, camptothecin (CPT), isolated from
Camptotheca acuminata, exerts potent inhibitory activities against cancer cells, and two CPT derivatives topotecan and irinotecan are used for treating cancers clinically [
14‐
16]. Accordingly, CPT-based derivatives are being developed for improving the anti-tumor activities [
17,
18]. Our previous study demonstrated that
2,9-Bis[2-(pyrrolidin-1-yl)ethoxy]-6-{4-[2-(pyrrolidin-1-yl)ethoxy] phenyl}-11
H-indeno[1,2-
c]quinoline-11-one (BPIQ), a synthetic quinoline, exerts anti-growth and apoptosis-inducing potential against cancer cell lines including hepatocellular carcinoma cells [
10,
12], non-small cell lung cancer (NSCLC) [
19] and retinoblastoma cells [
20]. Recently, our work further showed the BPIQ-induced apoptosis of cancer cells was mitochondrial-dependent [
19].
Mitogen-activated protein kinase (MAPK) signaling pathways are involved in mediating processes of cell growth, survival, and death. There are three members of MAPK, JNK, p38 and ERK. Among MAPK members, JNK and p38 are activated in response to various intrinsic and extrinsic stresses [
21,
22]. Additionally, activated p38 MAPK may induce apoptosis by phosphorylating or indirectly down-regulating pro-survival Bcl-2 family proteins under conditions such as cellular stress including ROS [
23], DNA adducts [
24] and starvation [
25]. Previous studies indicate the mechanisms of many anti-cancer drugs are closely correlated with the stimulation of MAPK JNK and p38 [
23,
26,
27].
On the contrary, the third member of MAPK, ERK is crucial for cell proliferation and survival and is activated by mitogenic stimuli, such as growth factors and cytokines [
28]. Constitutive activation and overexpression of ERK are frequently observed in many cancer cells [
29]. For example, more than 50% of acute myeloid leukemias and acute lymphocytic leukemias exert activated ERK pathways [
30]. Additionally, the activated ERK pathway in lung cancer cells has also been reported [
31].
Therefore, ERK targeting strategies against cancer have been used for treating cancer cells in vivo [
32] and clinically [
29].
On the contrary, ERK activation is not always correlated with pro-cellular survival. A recent study showed the interplays of ERK signaling and cell death, including apoptosis, autophagy, and senescence [
33]. In a comparison of ERK targeting strategies, accumulating evidence demonstrated that activating ERK could take effect in cancer treatments [
34‐
36]. Additionally, ERK signaling has also been involved in cell death induced by anti-cancer compounds including quercetin [
37], betulinic acid [
38] and miltefosine [
39]. Besides, apoptosis induced by SU11274, a small molecule inhibitor of c-Met in NSCLC A549, has been associated with ERK-dependent p53 activation and Bcl-2 inactivation [
36].
Contrarily, ERK has been shown to play an important role in cancer metastasis [
40‐
42]. Likewise, our previous work also demonstrated that cardiotoxin III (CTX III), a basic polypeptide isolated from the venom of the Taiwan cobra (
Naja naja atra) inhibits ERK-dependent migration and invasion of breast cancer cells MDA-MB-231 through down-regulating the signaling pathways of Src [
43] and EGF/EGFR pathway [
44].
In this study, we first examined whether the members of MAPK JNK, p38, and ERK involve in BPIQ-induced anti-NSCLC cells, and the dual roles of ERK in BPQI-induced anti-proliferation and anti-migration in NSCLC H1299 cells are also demonstrated. Furthermore, the possible mechanisms underlying ERK-mediated apoptosis of NSCLC cells induced by BPIQ are also discussed.
Methods
Preparation of BPIQ
BPIQ was synthesized described as previously published [
10,
12]. BPIQ was freshly dissolved in DMSO (<0.01% final concentration) before assays.
Reagents
DMEM and F12 medium, fetal bovine serum (FBS), trypan blue, penicillin G, and streptomycin were obtained from Invitrogen (Gaithersburg, MD, USA). Dimethyl sulphoxide (DMSO), ribonuclease A (RNase A), and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against JNK, p38 (sc-7149), p-p38 (Tyr182, sc-7973), ERK, p-ERK (Tyr204, sc-7976), COX-2, and β-actin (sc-7963) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against SP-1 (5407-S) was purchased from Epitomics. Antibody against p-JNK (Thr183/Tyr185, #07-175) was purchased from Millipore. Anti-rabbit, anti-goat and anti-mouse IgG peroxidase-conjugated secondary antibodies were purchased from Pierce (Rockford, IL, USA). Annexin V-FITC staining kit was purchased from Strong Biotech Co. Ltd. (Taipei, Taiwan).
Cell culture
Human non-small cell lung cancer (NSCLC) cell line H1299 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were maintained in 1:1 ratio of DMEM: F-12 supplemented with 8% FBS, 2 mM glutamine, and the antibiotics (100 μg/ml streptomycin and 100 units/ml penicillin) at 37 °C in a humidified atmosphere of 5% CO
2. All cells were tested to ensure the mycoplasma contamination-free using a PCR-based assay [
45].
Assessment of cell viability and morphological changes
Briefly, 1 × 105 cells were seeded and treated with vehicle or various concentrations for 24 h. After incubation, the morphological changes of cells were observed by an inverted phase-contrast microscopy. For cell viability assessment, cells were trypsinized and stained with 0.2% trypan blue to count by Countess™ the automated cell counter (Invitrogen, Carlsbad, CA, USA).
Western Blot analysis
Western Blot assay was conducted according to a previously published article [
46]. In brief, cells were harvested and lysed. A total of 20 μg protein lysate was resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred. The nitrocellulose membrane was blocked with 5% non-fat milk and incubated with primary and secondary antibodies sequentially. The signals for specific proteins were detected using a chemiluminescence-based ECL™ detection kit (Amersham Piscataway, NJ, USA).
Apoptosis assessment
The Annexin V/PI double staining assay recognizes the externalization of phosphatidylserine (PS) on the cell membrane, a hallmark of apoptotic cells. In brief, 5 × 105 cells were seeded on a 100-mm petri dish and treated with BPIQ alone or 2 h pre-treatment of an ERK inhibitor PD98059 for 24 h respectively. Cells were suspended with trypsin, harvested and stained with Annexin V/PI. Afterward, the cells were analyzed by a flow cytometer (FACS Calibur; Becton–Dickinson, Mountain View, CA, USA).
Assessment of cell migration
3 × 10
5 H1299 cells were seeded into a 12-well plate, then treated with indicated concentrations of BPIQ and a 1-mm wide wound area was created using a 200 µl plastic tip. After 16 h incubation, the wound areas were photographed and automatically calculated using the free software tool “TScratch” [
47].
Boyden’s chamber assay
The invasion of cancer cells was performed by a 24-well transwell unit with Matrigel™ (Greiner Bio-One, Frickenhausen, Germany) coated on the upper side of polycarbonate filters into 8 μm filter pore size transwell inserts. The lower well was injected with 800 µl medium containing 10% FBS, without or with indicated concentrations of BPIQ. 1 × 105 H1299 cells was resuspended in 200 µl of serum-free medium were seeded onto a transwell insert and allowed to invade for 16 h. Non-invaded cells on the upper part of the membrane were removed. Cells on the bottom surface of the filters were fixed with 4% paraformaldehyde, stained with Giemsa (Merck), and counted under a microscope. Each experiment was done in triplicate, and the results from three independent experiments were expressed as mean ± SD.
Assessment of MAPK inhibitors
To determine the effects of MAPK ERK, p38, and JNK on BPIQ-induced apoptosis, three specific inhibitors (50 μM), PD98059 (Sigma) for ERK, SB203580 (Sigma) for p38 and SP600125 (Sigma) for JNK, were dissolved in DMSO respectively. The assessment has been described previously [
46]. In brief, seeded cells were pre-treated with MAPK inhibitors for 2 h respectively. Afterwards, cells were administrated with 24 h treatment of BPIQ for cell proliferation assay and annexin V staining, and 16 h for Boyden’s chamber assay.
Gelatin zymography
The gelatin zymography [
48] was performed for detecting the gelatinases MMP-2 and -9 using 10% polyacrylamide gels contained 0.1% gelatin. After electrophoresis, SDS was replaced using 2.5% Triton X-100, followed by incubation in a Tris-based buffer containing NaCl, CaCl
2, and ZnCl
2 at 37 °C overnight. The gel was then stained with Coomassie Brilliant Blue R-250, and gelatinase activity was detected as unstained gelatin-degradation zones within the gel. The signals were analyzed using Gel-Pro 3.0 software (Media Cybernetics, Silver Spring, MD, USA).
Statistical analysis
Differences between cells treated with vehicle were analyzed in at least triplicate. The statistical differences were analyzed by one-way analysis of variance (ANOVA) using SigmaPlot v12 (Systat Software Inc.) and *p < 0.05 vehicle vs. BPIQ treatment was considered statistically significant.
Discussion
Mitogen-activated protein kinase (MAPK) signaling pathways are involved in mediating processes of cell growth, survival and death [
21,
22]. MAPK members p38 and JNK pathways have been reported to induce apoptosis under various cellular stresses [
59,
60]. Therefore, many anti-cancer drugs are designed for stimulating JNK and p38-mediated apoptosis of cancer cells such as breast cancer [
26], colon cancer [
23] and lung cancer cells [
27]. However, the role of MAPK members in anti-cancer drugs-induced apoptosis may depend on cell types and the stimuli, and studies suggesting the pro-survival role of p38 MAPK in cancer cells toward anti-cancer drugs were also reported [
61,
62]. For example, Bruzzese’s work reported that the activation of p38 MAPK was associated with the resistance of prostate cancer (PCa) and multiple myeloma (MM) cells towards zoledronic acid (ZOL), a nitrogen-containing bisphosphonate. In addition, panobinostat, a histone deacetylase inhibitor, was shown to render both PCa and MM sensitive to ZOL by inhibiting the activity of p38 MAPK [
61]. Furthermore, DU145R80, a ZOL-resistant prostate cancer cell line, expresses p38 MAPK-dependent survival pathway accompanied with an enhanced potential for epithelial-mesenchymal transition (EMT) and the increased expression of metalloproteases MMP-2/-9 compared to its parental cell line, suggesting the essential role of p38 MAPK in acquiring chemoresistance of prostate cancer cells [
62].
Despite the potential of BPIQ on anti-proliferation of cancer cells, the role of MAPK in BPIQ-induced growth inhibition is not clear. To further clarify the mechanism underlying MAPK-induced apoptosis and anti-proliferation induced by BPIQ. The cellular and molecular parameters about BPIQ-induced apoptosis were studied using three NSCLC tumor cells H1299. The results of Western Blot showed the activation of two MAPK members JNK and ERK was detected after BPIQ treatment (Fig.
2).
Therefore, we determined whether JNK or ERK plays a role in BPIQ-induced apoptosis and anti-proliferation and performed the MAPK inhibitor assays. The inhibitor assay showed that blockade of ERK activity significantly rescued BPIQ-induced anti-proliferation (Fig.
3) and apoptotic cell death (Fig.
4) of NSCLC tumor cells. Regarding the correlation between ERK signaling and the process of cell death, ERK is thought to be critical for cell survival and mediating a survival response that counteracts with cell death and its activation being frequently observed in cancer cells [
29]. For example, Caraglia’s work demonstrated that the combination of ZOL and R115777, a non-peptidomimetic farnesyl transferase inhibitor, exerted a synergistic effect on apoptosis induction in cell lines of prostate adenocarcinoma through dramatically attenuating Ras signaling and its downstream targets, namely the ERK and Akt survival pathways [
63]. Likewise, the combination of Simvastatin, an HMG–coenzyme A reductase inhibitor, and R115777 (Tipifarnib) exerted a cooperative effect on anti-proliferation and apoptosis induction of two NSCLC cell lines GLC-82 (adenocarcinoma) and CALU-1 (squamous-carcinoma) by inhibiting Ras/Raf/MEK/ERK signaling [
64].
On the contrary, many studies also showed the correlations of ERK signaling and stimulating the process of cell deaths [
33,
65,
66]. ERK pathways may induce apoptosis through promoting caspase-8 signaling and the activation, or potentiating the activation of death receptors by increasing the level of death ligands such as TNFα or FasL, or death receptors such as Fas, DR4 or DR5. For example, llimaquinone, an anti-cancer agent, was found to upregulate the expression of death receptor DR-4/-5 through ERK activation in colon cancer cell lines HCT116 and HT-29 [
67].
Additionally, ERK activity was reported to promote the induction of FADD, an adaptor of caspase-8 for death receptors. Furthermore, an antibiotic fluoroquinolone was reported to induce the apoptosis of pancreatic cancer through ERK-dependent mitochondrial pathways, including the proteolytic activation of caspase-9, the loss of mitochondrial membrane potential, and the up-regulated expression of pro-apoptotic Bax and Bak [
68].
Wang’s work suggested that ERK activation may contribute to activin A-induced apoptosis of NSCLC cells A549 [
69]. Similarly, piperlongumine, a bioactive compound isolated from large peppers, was reported to induce the cell death of colon cancer HT-29 cells through MEK-ERK signaling [
70]. Furthermore, recent studies also showed that antitumor compounds, such as quercetin [
37], betulinic acid [
38], miltefosine [
39] induce ERK-dependent apoptosis. Besides, a cytotoxin VacA secreted by
Helicobacter pylori, a Gram-negative bacterium, was reported to induce apoptosis of gastric cancer cells. [
34]. Likewise, SU11274, a small molecule inhibitor of c-Met was reported to induce apoptosis of lung cancer cells A549 through ERK-p53 and ERK-mediated Bcl-2 phosphorylation [
36], indicating the pro-apoptotic role of ERK and its applications in cancer treatment. Accordingly, the activation of ERK-dependent apoptotic signaling may be a promising treatment for chemoresistant cancer cells especially those that overexpress ERK.
Many anti-cancer drugs have been shown to exert multi-effects against cancer cells. For example, curcumin, a diferuloylmethane, induces both apoptosis and anti-migration of human medulloblastoma cells [
71]. We therefore examined whether BPIQ exerts anti-cancer activities beyond anti-growth and the induction of apoptosis. Both the wound healing and Boyden’s chamber assays demonstrate that sub-IC
50 of BPIQ (below 2 μM) significantly inhibits the cellular mobility of H1299 cells. We next tried to depict the mechanism underlying BPIQ-induced anti-migration in lung cancer. The upregulation of pro-inflammatory COX-2 expression, MMP-2 and -9 have been reported to be associated with the progression of malignant tumors [
72,
73]. Moreover, the expressions of MMP-2 and MMP-9 are regulated by SP-1 [
54,
74].
As shown in Fig.
7, the inactivation of migration-associated factor SP-1 following BPIQ treatment was also observed. Furthermore, the protein level of COX-2 and the activity of MMP-9 and -2 were also decreased. In cell signal pathways, the phosphorylation of many signaling proteins is thought to be dynamic and transient [
75]. Our previous work showed that magnolol, a compound isolated from the herbal plant Magnolia induced apoptosis of NSCLC A549 cells through upregulating the activity of MAPK p38 and JNK. Both the phosphorylations of MAPK p38 and JNK were increased following magnolol treatment in a dose-responsive manner, whereas the dramatic decrease of phosphorylation was observed at the highest dose [
76]. Likewise, metformin which exert anticancer activities, was reported to increase a dose-responsive phosphorylation of ERK but decrease at the highest dosage in neuroendocrine tumor cells BON1 and NCI-H727 [
77]. Similarly, the results of Western Blot assay showed that the phosphorylation of ERK following BPIQ treatments (from 1 to 5 μM) for 24 h was dose-responsive. We therefore suggested that the highest concentration (10 μM) of BPIQ may cause the phosphorylation of ERK earlier than 24 h of treatment and return to the un-phosphorylated or hypo-phosphorylated status after 24 h.
These above observations suggest that the orchestrate signaling modulated by BPIQ eventually led to the inhibition of cellular migration.
Accumulating evidence showed that the cell migration was promoted by MPAK ERK, and the inhibition of cell migration was often accompanied by attenuated activities of ERK in cancer cells [
57,
58]. Consistently, the results of our study indicate the pro-migration role of ERK and ERK blockade enhances the inhibitory effect of BPIQ on migration and invasion of H1299 cells (Fig.
8a, b). Accordingly, it will be an advantage to inhibit ERK activation combining BPIQ treatment against cancer cells in further study. A low- or non-cytotoxic dose of BPIQ combining the inhibitors of ERK such as PD98059 may be anti-metastatic or chemopreventive strategies for NSCLC treatment in future.
Authors’ contributions
Experimental designs: YF, YLC and CCC; Reagents and Materials: CYW, BHC and HMDW; Synthesis of BPIQ: YLC and CHT; Bioassays: KFC, YCC and WJC; Manuscript preparation and writing: CCC. All authors read and approved the final manuscript.