Biomedicine & Pharmacotherapy 68 (2014) 959–967
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Original article
Novel tetrahydroacridine derivatives inhibit human lung adenocarcinoma cell growth by inducing G1 phase cell cycle arrest and apoptosis Paulina Olszewska a, Elz˙bieta Mikiciuk-Olasik a, Katarzyna Błaszczak-S´wia˛tkiewicz a, Jacek Szyman´ski b, Paweł Szyman´ski c,* a Department of Pharmaceutical Chemistry, Drug Analysis and Radiopharmacy, Faculty of Pharmacy, Medical University, Muszyn´skiego 1, 90-151 Lodz, Poland b Central Scientific Laboratory, Medical University, Mazowiecka 6/8, 92-215 Lodz, Poland c Laboratory of Radiopharmacy, Department of Pharmaceutical Chemistry, Drug Analysis and Radiopharmacy, Faculty of Pharmacy, Medical University, Muszyn˜skiego 1, 90-151 Lodz, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 May 2014 Accepted 15 October 2014
Lung cancer is not only the most commonly diagnosed cancers worldwide but it is still the leading cause of cancer-related death. Acridine derivatives are a class of anticancer agents with the ability to intercalate DNA and inhibit topoisomerases. The aim of this study was to evaluate the effect of sixteen new tetrahydroacridine derivatives on the viability and growth of human lung adenocarcinoma cells. We compared anticancer activity of a series of eight compounds with 4-fluorobenzoic acid and eight compounds with 6-hydrazinonicotnic acid differed from each other in length of the aliphatic chain containing from 2 to 9 carbon atoms. Interestingly, tetrahydroacridine with 4-fluorobenzoic acid (compounds 9–16) showed higher anticancer activity than derivatives with 6-hydrazinonicotnic acid (compounds 1–8) and their efficacy was correlated with increasing number of carbon atoms in the aliphatic chain. The results showed that inhibition of cancer cell growth by the most effective compounds 15 and 16 was associated with induction of G1 phase cell cycle arrest followed by caspase-3 dependent apoptosis. Our findings suggest that tetrahydroacridine with 4-fluorobenzoic acid containing 8 and 9 carbon atoms may be potential candidate for treatment of lung cancer. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Tetrahydroacridine derivatives Acridine derivatives Anticancer compounds Lung cancer DNA intercalation
1. Introduction Lung cancer is the most commonly diagnosed cancers worldwide and by far is the leading cause of cancer-related death among both men and women [1,2]. There are two major types of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Approximately 85% of lung cancers are NSCLC that includes adenocarcinoma (40%). It is an aggressive tumor with a 5-year survival rate of less than 15% demonstrating that current therapy is still inefficient [3,4]. Therefore, the search for better chemotherapeutic agents with advanced activity against lung cancer is needed. * Corresponding author. 1, Muszynskiego Street, 90-151 Lodz, Poland. Tel.: +48 042 677 92 90; fax: +48 042 677 92 50. E-mail address: [email protected] (P. Szyman´ski). http://dx.doi.org/10.1016/j.biopha.2014.10.018 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
DNA targeted drugs are successful anticancer agents, which cause DNA damage and can lead to cell cycle arrest and apoptosis [5,6]. Acridine derivatives are the most extensively studied class of potential anticancer agents that interfere with DNA as intercalators [7,8]. In addition to cancer, the acridine-based compounds have a long history of treatment of other human diseases, such as bacterial, parasitic infections and Alzheimer’s disease [9]. The biological activity of acridines is mainly attributed to the planarity of aromatic structures, which can intercalate within the DNA structure [10]. The act of intercalation induces local structural changes to the DNA, including the unwinding of the double helix and lengthening of the DNA strand [11,12]. Moreover, acridines are able to decrease the activity of topoisomerase I and II [10,13,14]. Topoisomerases are involved in replication, recombination, transcription, and chromosome segregation and they are crucial for proper DNA actions [15]. For example, first synthetic
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Fig. 1. Chemical structure of tested 1,2,3,4-tetrahydro-9-aminoacridine derivatives. Sixteen tetrahydroacridine derivatives with 6-hydrazinonicotinic fragment or 4-fluorobenzoic acid moiety are shown. Series of derivatives have the aliphatic linker comprising from 2 to 9 carbon atoms (n = 2–9) between tetrahydroacridine and 6-hydrazinonicotinic (compounds 1–8) or 4-fluorobenzoic acid (compounds 9–16).
acridine-based agent to be considered as intercalator and topoisomerase II inhibitor was amsacrine [8,16]. We have previously synthetized and described a variety of tetrahydroacridine (1,2,3,4-tetrahydro-9-aminoacridine) analogues coupled with 6-hydrazinonicotinic acid (compounds 1–8) or 4-fluorobenzoic acid moiety (compounds 9–16) [17–19]. In the current study, we evaluated these new tetrahydroacridine derivatives for anticancer activity in human lung adenocarcinoma cells. The chemical structures of compounds used in this study are shown in Fig. 1. These synthesized compounds differed from each other in length of the aliphatic chain containing from 2 to 9 carbon atoms between tetrahydroacridine and 6-hydrazinonicotinic acid or 4-fluorobenzoic acid (Fig. 1). The purpose of this study was to investigate the efficacy of sixteen new tetrahydroacridine derivatives on the survival and growth of human lung adenocarcinoma cells. Moreover, we evaluated the effects of hydrazinonicotinic acid and fluorobenzoic acid moiety and different length of aliphatic chain on anticancer activity of new compounds. To characterize the mode of actions of the most effective compounds, we monitored changes in cell morphology and determined their effects on cell cycle progression and apoptosis.
2. Materials and methods 2.1. Cell culture
dimethyl sulfoxide (DMSO) to prepare the stock solution. Before use, stock solution of compounds was diluted with cultured medium to the desired concentrations and the final concentration of DMSO was 0.1%. A549 cells were treated with compounds at different concentrations from 1 to 200 mM ranges and medium containing diluent (0.1% DMSO) was added to the control cells. After 72 h treatment, WST-1 reagent was added to each well and incubated for additional 3 h at standard condition. Next, absorbance was measured using microplate reader (Synergy H1, Bio-Tek, Winooski, VT, USA) at a wavelength of 440 nm. The cell viability was expressed as a percentage of the control values. Data showed the mean SD of three independent experiments performed in triplicates. IC50 values (the concentration of tested compound that inhibited cell growth by 50%) were calculated by concentration-response curve fitting using a Microsoft Excel-based analytic method. 2.3. Cell morphology Control and tetrahydroacridine-treated A549 cells were examined for morphological changes and cell growth inhibition under light microscopy with phase-contrast (Opta-Tech). The changes in cell morphology upon treatment with tested compounds were photographed after 24 h, 48 h, and 72 h at 100 magnification. 2.4. Cell cycle analysis
Anticancer activities of the compounds were tested against A549 cells obtained from a human lung adenocarcinoma. A549 cells were purchased from the European Collection of Cell Cultures (ECACC, Salisburg, UK). The cells were grown in F12K medium (HyClone, UK) supplemented with 10% heat-inactivated fetal bovine serum, FBS (Lonza, Basel, Switzerland), 100 units/mL penicillin and 100 mg/mL streptomycin (Lonza) at 37 8C with 5% CO2. 2.2. Cell viability assay The cell viability was quantified by using WST-1 assay (Millipore, Billerica, MA, USA). The assay is based on the enzymatic cleavage of the water-soluble tetrazolium salt (WST-1) to formazan by cellular mitochondrial dehydrogenases present in viable cells. The amount of formazan dye formed directly correlates to the number of live cells. For the experiment, cells were seeded in 96-well plates at density 5000 cells per well and cultured for 24 h. The tested derivatives were dissolved in
A549 cells were seeded in 6-well plates at density 20 104 cells per well and cultured for 24 h. Next, cells were exposed to compounds 5, 15, and 16 at 30, 15, 10 mM concentrations corresponding to IC50, respectively. After 24 h treatment, control and treated cells were then collected, washed with PBS solution, fixed in cold 70% ethanol at 4 8C. Prior to analysis, the cells were washed with PBS, incubated with ribonuclease (100 mg/mL, Sigma–Aldrich, St. Louis, MO, USA) for 30 min and stained with propidium iodide (50 mg/mL, Sigma–Aldrich) in darkness at room temperature for additional 20 min. DNA content and number of cells in the individual cell cycle phases was measured by flow cytometry (FACS Canto II, Becton Dickinson, USA). Percentages of cells in each cell cycle phase were calculated from live cells that were expressed as 100%. Sub-G1 population representing cell debris was excluded from the analysis. Then, 20,000 cells were analyzed from each sample and data were presented as the mean SD of three independent experiments.
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Table 1 In vitro growth inhibition of A549 cells by new tetrahydroacridine derivatives. IC50 values (concentration of tested compounds causing 50% inhibition of cell growth compared to control cells) were determined after 72 h incubation with tested compounds using WST-1 assay. Data are expressed as the mean SD, n = 3. Compounds 1–8
Compounds 9–16
Compound
Number of carbon atoms (n)
IC50 (mM)
Compound
Number of carbon atoms (n)
IC50 (mM)
1 2 3 4 5 6 7 8
2 3 4 5 6 7 8 9
> 200.0 > 200.0 150.4 5.1 > 200.0 28.7 3.5 83.5 2.6 98.5 1.4 53.9 1.3
9 10 11 12 13 14 15 16
2 3 4 5 6 7 8 9
116.5 5.8 156.6 2.8 67.9 2.5 31.3 4.9 31.5 0.7 15.2 4.6 15.0 5.2 9.6 4.3
2.5. Caspase-3/7 activity assay Caspase-Glo 3/7 Assay (Promega, Madison, WI, USA) was used to assess cell apoptosis according the manufacturer instruction. Briefly, the proluminescent substrate containing the DEVD is cleaved by activated caspase-3/7 and a substrate for luciferase (Z-DEVD-aminoluciferin) is released. This results in the luciferase reaction and the production of luminescent signal. Luminescence is proportional to the amount of caspase activity. The A549 cells were seeded at a density of 5000 cells per well in 96-well plates and cultured for 24 h. Next, cells were treated with tested compounds at indicated concentrations and caspase activity was measured after 24 h and 48 h incubation. The Caspase-Glo 3/7 reagent was added to the cells at ratio 1:1. After 1 h incubation at room temperature, luminescence was recorded using a microplate reader (Synergy H1, Bio-Tek) at gain 135. 2.6. Nuclear staining with Hoechst For apoptotic nuclei evaluation, the Hoechst labelling was used. Cells were treated with compounds 5 (50 mM and 30 mM), 15 (25 mM and 15 mM) and 16 (25 mM and 10 mM). After 48 h incubation, cells were fixed and permeabilized with cold methanol. Fixed cells were washed with PBS and incubated with Hoechst 33342 [1:2500] [Invitrogen] for 20 min. The cell nuclei were photographed using fluorescence microscope (Olympus IX-51) at 200 magnification. To determine percentage of apoptotic cells, normal and apoptotic nuclei from five microscopic fields per sample were counted. 2.7. Statistical analysis Data were represented as means SD from indicated number of separate experiments. The significance difference between control group and compound-treated group was validated by a Student’s paired t-test. A P value below 0.05 (*P < 0.05) was considered statistically significant.
3. Results 3.1. Effect of new tetrahydroacridine derivatives on A549 cell growth The growth-inhibitory effects of sixteen tetrahydroacridine derivatives were analyzed in human lung adenocarcinoma A549 cells using WST-1 assay. First, we performed concentrationresponse analysis to determine concentration of tested compounds that induced a 50% decrease of cell viability (IC50) as compared to control cells. The cancer cells were treated with tested compounds at different concentrations from 1 mM to 200 mM for 72 h. As shown in Table 1, six out of sixteen tested compounds had IC50 values below 50 mM. Among tetrahydroacridine derivatives with 6-hydrazinonicotinic acid moiety, compound 5 exhibited the most
potent anticancer activity (IC50 = 28.7 mM 3.5). We found that new tetrahydroacridine derivatives containing 4-fluorobenzoic acid (compounds 9–16) were much more effective in inhibition of A549 cells growth in comparison with compounds with 6-hydrazinonicotinic acid moiety (compounds 1–8). The compound concentrations that inhibited cell growth by 50% were under 20 mM for the compounds 14, 15, and 16 containing 4-fluorobenzoic acid (Table 1). Moreover, compound 16 containing 9 carbon atoms in the aliphatic chain was the most potent to inhibit A549 cell growth (IC50 = 9.6 mM 4.3). Based on these results, we selected compound 5 (as the most effective derivative containing 6-hydrazinonicotinic acid) and compounds 15 and 16 (as the most effective derivatives coupling with 4-fluorobenzoic acid) for further biological evaluation. Fig. 2 shows the dose-response effect of selected compounds on A549 cell viability after 72 h treatment. The results were expressed as relative number of viable adherent cells in relation to control cells. For example, culture of A549 cells in the presence of 10 mM of compounds 5, 15, or 16 decreased number of viable cells to 74.6%, 67.0% and 43.2%, respectively (Fig. 2). Our results showed that the most effective compounds were tetrahydroacridine derivatives with 4-fluorobenzoic acid moiety containing lengthier aliphatic chain compromising from 7 to 9 carbon atoms (Table 1). 3.2. Microscopic study of A549 cells The effects of tetrahydroacridine derivatives on cancer cell growth and viability were also monitored and visualized using phase-contrast microscopy. Morphological examination of A549 cells treated with tested compounds showed severe compoundmediated changes. Images in Fig. 3 show the comparison of dosedependent effects of selected compounds 5, 15, and 16 on cell growth after 72 h treatment. We observed changes in number, shape, and size of the tetrahydroacridine-treated cells compared with the controls. For example, incubation of cells with compound 5 caused decrease of cell population approximately by 50% at 25 mM as compared to control cells (Fig. 3). At higher concentration, cells were round and detached from adjacent cells. The compound 15 exhibited greater anticancer activity and decreased number of cells by 50% at 10 mM (Fig. 3). Results demonstrated that compound 16 was the most effective and reduced number of viable cells by 50% at 5 mM. Fig. 4 represents the dose-dependent and time-dependent effects of the most effective compound 16 on inhibition of A549 cell growth. For example, treatments of cells with 10 mM of compound 16 inhibited cell proliferation as well as more elongated cells were presented in comparison with the control cells after 24 h. After 48 h, in addition to inhibition of cell proliferation, we observed shrinkage of some cells suggesting apoptotic changes. Then, 72 h treatment showed more apoptotic cells and cell detachment was triggered as compared to 24 h and 48 h (Fig. 4). Our data demonstrated that the effects of tested compounds on cancer cell viability, morphology and growth were dependent on dose as well as time of treatment.
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Fig. 2. Effect of tetrahydroacridine derivatives on viability of cancer cells. A549 cells were cultured in the present of indicated compounds at concentrations from 1 to 100 mM range. Cell viability was determined by WST-1 assay after 72 h treatment. Concentrations are graphed on a log scale. Data are expressed as the mean SD, n = 3.
Fig. 3. Dose-dependent effect of selected compounds on A549 cell growth after 72 h treatment. A549 cells were cultured in the presence or absence (control) of compounds 5, 15, 16 at indicated concentrations for 72 h. Representative phase-contrast cell images are shown (100 magnification).
3.3. Effect of tetrahydroacridine derivatives on cell cycle progression of A549 cells To understand the mechanisms responsible for compoundsmediated cancer cell growth inhibition, we examined their effects on cell cycle distribution. The cell cycle analysis was performed in
A549 cells exposed to the compounds 5, 15 and 16 at concentrations of IC50 for 24 h (Fig. 5A and B). Separation of cells in G0/1, S and G2/M phase was based on fluorescence intensity after cell staining with propidium iodide using flow cytometry analysis. Fig. 5A shows representative profiles of cell cycle distribution for each tested compound and control cells. When cells were treated with compounds 15 and 16, a significant increase number of cells
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Fig. 4. Time- and dose-dependent effect of compound 16 on A549 cell growth. A549 cells were cultured in the presence or absence (control) of compound 16 at indicated concentrations. Representative phase-contrast cell images are shown after 24 h, 48 h and 72 h of treatment (100 magnification).
Fig. 5. Effect of new tetrahydroacridine derivatives on cell cycle. Cells were cultured for 24 h with selected compounds at IC50 concentrations. The cell cycle progression was evaluated using flow cytometry analysis of propidium iodide stained cells. A. Representative histograms of one out of three independent experiments are shown. Percentages of cells in each cell cycle phase were calculated from live cells that were expressed as 100%. B. Data are expresses as the mean SD of three independent experiments. * P < 0.05 compared with control (CTR).
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were arrested at the G0/1 phase of the cell cycle with a corresponding decrease in the percentage of cells from the S phase. With respect to control cells, the cell population in the G0/1 phase increased from 57.4% 2.5 to 76.2% 3.2 in the presence of 15 mM compound 15 (Fig. 5B). Additionally, compound 15 induced a reduction in the percentage of cells in the G2/M phase. Similarly, in the case of derivative 16, we observed significant increase in the G0/1 fraction from 57.4% 2.7 to 68.4% 3.7 (Fig. 5B). In contrast, treatment of A549 cells with compound 5 had no significant effect on cell cycle distribution (Fig. 5B). Our data showed that tested tetrahydroacridine derivatives containing fluorobenzoic acid moiety induced arrest of cell cycle progression through G1 into S phase in A549 cells. Moreover, the effect induced by compound 15 was more pronounced as compared to compound 16 (Fig. 5). 3.4. Effect of tetrahydroacridine derivatives on apoptosis Since the inhibition of cancer cell proliferation can results in initiation of apoptosis, we next determined whether selected tetrahydroacridine derivatives repressed cancer cell growth by induction of apoptosis. The effect of tested compounds on cell apoptosis was assessed by the caspase-3/7 activity after 24 h and 48 h treatment. Twenty-four-hour treatment of cancer cells with compound 16 at 25 mM resulted in 2.7-fold increase of caspase3/7 activity as compared to control cells (Fig. 6A). In contrast, treatment of A549 cells with compounds 5 and 15 at indicated concentrations for 24 h had no significant effect on the level of activated enzyme (Fig. 6A). However, when caspase-3/7 activity was evaluated after 48 h exposure, we observed significant elevation of enzyme activity in cells treated with compounds 5 and 15 at IC50 concentration and higher (Fig. 6B). Similar to 24 h exposure, the high content of activated caspase-3/7 in cells treated with compound 16 at 25 mM was also detected after 48 h ( 2.6fold) (Fig. 6B). In addition, 48 h treatment of cells with 10 mM of compound 16 also significantly increased caspase-3/7 activity. To further confirm the induction of apoptosis in compound-treated cells, we analyzed chromatin condensation and nuclear fragmentation by fluorescence microscopy using the DNA-binding fluorescence dye, Hoechst. As shown in Fig. 6B, exposure of cells to higher tested concentrations of compounds for 48 h induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies that suggest the presence of apoptotic cells. These nuclear apoptotic changes visualized by Hoechst staining corresponded to changes in cell morphology (cell shrinkage, rounded cells) observed in phase-contrast microscopy (Fig. 6B). To determine percentage of apoptotic cells after 48 h treatment with compounds, Hoechst-stained normal and apoptotic nuclei from five microscopic fields per sample were counted. We observed appearance of some apoptotic cells in control cells (7.8%) after 48 h due to intensive cell proliferation leading to over-confluence. This may explain about 40% increase in caspase-3/7 activity in control cells measured at 48 h as compared to 24 h control cells (Fig. 6A). Exposure of A549 cells to compounds 5 and 15 at higher tested concentrations resulted in 2-fold (16.5%) and 3-fold (22.6%) increased percentage of apoptotic cells, respectively, as compared to control (7.8%). Interestingly, 48 h treatment of cells with compound 16 at 25 mM induced apoptosis in almost all cells as determined by nuclei staining (87.4%) and corresponding changes in cell morphology (Fig. 6B). This effect correlated with high content of activated caspase-3/7 detected at 24 h that was continued to be elevated after 48 h treatment (Fig. 6A). The results showed that tested compounds inhibited cancer cell growth, at least in part, by caspase-3/7-dependent apoptosis. We found that compound 16 was the strongest inducer of apoptosis in A549 cells.
4. Discussion In the present study, we investigated the efficacy of sixteen new tetrahydroacridine derivatives on inhibition human lung adenocarcinoma cell growth. We have synthetized a series of tetrahydroacridine analogues coupled with 6-hydrazinonicotinic acid (compounds 1–8) or 4-fluorobenzoic acid (compounds 9–16) to examine how different moieties and the length of the aliphatic chain influence anticancer activity of new compounds [17– 19]. Moreover, we evaluated the mechanisms of anticancer activity of the most effective compounds. Our results demonstrated that tetrahydroacridine derivatives with 4-fluorobenzoic acid moiety had a more potent growth inhibition activity in A549 cells than compounds having 6-hydrazinonicotinic acid and their efficacy correlated with increased number of carbon atoms in aliphatic chain. We found that all selected compounds (5, 15, 16) induced cancer cell death by caspase-3/7-dependent apoptosis. However, compounds containing 4-fluorobenzoic acid (15 and 16) inhibited cancer cell growth as results of cell cycle arrest in G0/1 phase. Lung cancer is one of the most common diagnosed malignancies in the world and is still the leading cause of death from cancer [1,2]. There are two major histological types of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC is the most common type of lung cancer, accounting for approximately 85% of all cases and includes adenocarcinoma (40%) [4,20]. It is an aggressive tumor type with 5-year survival rate of only about 15% [1]. The lack of effective therapy for treatment of NSCLC indicates an urgent need to search for new drugs [21]. Drugs having affinity for DNA are favoured as potential therapeutic agents for the treatment of cancer [22,23]. The acridine-based compounds are the most studied chemotherapeutic agent with DNA intercalative properties [8]. They also had ability to inhibit enzymes that are crucial for proper DNA actions, such as topoisomerase I/II and telomerase [10,24,25]. Amsacrine was the first synthetic DNA intercalating agents and topoisomerase inhibitor that was used for treatment of leukemia [8,26]. Moreover, acridine derivatives display also other interest forms of biological activity such as anti-bacterial drugs [9,16] and antiprotozoal drugs [26,27], or cholinesterase inhibitors [9,19]. To explore novel acridine-based compounds as potential chemotherapeutics for treatment of lung cancers, we have synthetized a series of sixteen new tetrahydroacridine analogues containing 6-hydrazinonicotinic acid (compounds 1–8) or 4fluorobenzoic acid moiety (compounds 9–16) [17–19]. In each group, the compounds differed from each other in the length of the aliphatic chain comprising from 2 to 9 carbon atoms. Our initial study was performed to screen the effect of these new tetrahydroacridne derivatives on growth of human lung adenocarcinoma A549 cells using WST-1 assay. We found a significant difference in the anticancer activity of the tested compounds. Results obtained in the present study demonstrated that derivatives containing 4-fluorobenzoic acid moiety were much more effective than derivatives with 6-hydrazinonicotinic acid. Moreover, we found that the length of aliphatic chain significantly influenced on the cell growth inhibition of compounds with 4-fluorobenzoic acid and the efficacy of these derivatives was increased with increase the number of carbon atoms. The most effective was compound 16, (4-fluoro-N-[9-(1,2,3,4-tetrahydroacridin-9-ylamino)nonyl]-benzamide), containing 9 carbon atoms in aliphatic linker. Recently, we have reported that cyclopentaquinoline derivatives with 4-fluorobenzoic acid also exhibit higher anticancer activity than cyclopentaquinoline derivatives with 6-hydrazinonicotinic acid and their efficacy correlate with the length of the aliphatic chain [28]. Thus the current results are consistent with our previous findings and strongly suggest that coupling of acridine-based compounds with fluorobenzoic acid by
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Fig. 6. Effects of new compounds on cell apoptosis. A549 cells were cultured in the presence or absence (CTR, control) of selected compounds (5, 15 and 16) at indicated concentrations. A. Cell apoptosis was determined by caspase-3/7 activity assay after 24 h and 48 h treatment. Caspase-3/7 activity was measured as a luminescence light unit [RLU] and expressed as the mean SD from two independent experiments performed in triplicates. *P < 0.01 compared with control. B. Visualization of apoptotic nuclei in cells treated with compounds at indicated concentrations for 48 h. The cell nuclei were stained with DNA-binding fluorescence dye, Hoechst. Representative fluorescence images are shown (200 magnifications). Arrows point apoptotic nuclei characterized by condensation of chromatin or nuclear fragmentations. Numbers represent the percentage of apoptotic cells expressed as the mean SD calculated from 5 random microscopic fields. Lower panel shows the effects of compounds on cell growth and morphology directly before Hoechst staining. Representative phase-contrast images are shown (100 magnifications).
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longer aliphatic linker caused changes in the structure of compounds that increased their anticancer activity. Based on the IC50 values of tested tetrahydroacridine derivatives, we selected the most effective compounds from each group (compounds 5, 15 and 16) for further evaluation. Phase-contrast images of A549 cells culture in the presence or absence of selected compounds visualized that effects of compounds on cancer cell growth and viability were dose and time-dependent. Previous studies have shown that acridine-based compounds, such as cyclopentaquinoline derivatives or 9-aminoacridine derivatives suppress viability of lung cancer cell lines or mesothelioma, respectively [28,29]. Compounds that suppress tumor growth by induction of cell cycle arrest and apoptosis are desired strategy in cancer therapy [30–32]. Apoptosis induction is one of the main mechanisms that impede cancer growth and proliferation and is one of the major mechanisms used by various anticancer agents. Caspases play the key role in the initiation and execution of programmed cell death. Based on the functions, they have been divided into two groups: caspases-2, -8, -9, -10 are initiators and caspases-3, -6, -7 are effectors of apoptosis [33]. Caspases-3 is a major executioner of apoptosis [33,34] and is responsible for specific cleavage of many key cellular proteins, chromatin condensation and DNA fragmentation, which ultimately result in cell death [35]. To determine whether the observed decrease in cell viability induced by new compounds was associated with apoptosis, we measured levels of caspase-3/7 activity at 24 and 48 h. We found that 48 h exposure of A549 cells to all selected compounds at IC50 and higher concentrations significantly increased the caspase-3/7 activity levels. Interestingly, treatment of cells with compound 16 at 25 mM induced caspase-3/7 activation early at 24 h and the high content of activated enzyme was also detected after 48 h treatment. In addition to caspase-3/7 activity, apoptotic cells were visualized by nuclear morphological changes in Hoechststained cells. Treatment of cells with compounds 5 and 15 at higher concentrations for 48 h significantly increased percentage of apoptotic cells ( 2-fold and 3-fold, respectively). Consistent with the high caspase-3/7 activity levels, treatment of cells with compound 16 at 25 mM showed almost all apoptotic nuclei. Moreover, these effects also correlated with cellular morphological changes observed under phase-contrast microscope. Our data showed that selected compounds (5, 15, and 16) inhibited cancer cell growth, at least in part, via induction of caspase-3/7dependent apoptosis in a concentration-dependent manner. The results demonstrated that compound 16 was the strongest inducer of apoptosis in A549 cells. Previous study has reported that 9-phenylacridine derivatives led to release of cytochrome c and caspase-3/7 activation [11]. Moreover, study conducted by Goodell et al. showed that substituted 9-aminoacridine derivatives inhibit cell proliferation and induces apoptosis of pancreatic cancer cell lines [36]. Cell cycle arrest is an important signal for inhibition of cell proliferation [37]. Moreover, the induction of apoptosis can be mediated through the cell cycle arrest [36,38]. To determine whether selected compounds inhibited cancer cell growth as a result of induction of cell cycle arrest, we examined their effect on cell cycle progression. Flow cytometry analysis revealed that growth of the A549 cells was arrested at G0/1 phase in the presence of compounds 15 and 16. Interestingly, the strongest G0/1 arrest with corresponding decreased in percentages of S and G2/M cell population was observed after treatment with compound 15. It has been demonstrated that 9-aminoacridine derivatives induce a G0/1 phase arrest followed by apoptosis in pancreatic cancer cells as a consequence of topoisomerase II inhibition [24,36]. Our results also demonstrated that treatment of lung adenocarcinoma cells with compounds 15 and 16 at IC50 concentrations led initially to cell cycle arrest followed by induction of cell apoptosis. Thus, these
compounds exhibited similar effects on cell cycle and apoptosis, suggesting potential inhibition of topoisomerase II in A549 cells. In contrast, compound 5 containing 6-hydrazinonicotinic acid had no significant effect on cell cycle progression. These results suggest that compounds 15 and 16-induced G0/1 phase arrest was dependent on the presence of 4-fluorobenzoic acid. These findings are consistent with our previous study, demonstrating that cyclopentaquinoline derivatives with 4-fluorobenzoic acid induce a G0/1 phase arrest in lung cancer cells [28]. Together, these results strongly suggest that coupling of acridine-based compounds with 4-fluorobenzoic acid not only improved anticancer activity but also influenced mechanism of anticancer action. In conclusion, our present findings showed that new tetrahydroacridine derivatives containing 4-fluorobenzoic acid (compounds 9–16) were much more effective in inhibition of human lung adenocarcinoma cell growth in comparison with compounds with 6-hydrazinonicotinic acid moiety (compounds 1–8). Moreover, the anticancer activity of tetrahydroacridine derivatives with 4-fluorobenzoic acid was correlated with the number of carbon atoms in the aliphatic linker. All selected compounds (5, 15, and 16) induced cancer cell death by induction of caspase-3/7 apoptosis. However, growth inhibition of A549 cells by tested tetrahydroacridine with 4-fluorobenzoic acid was associated with a cell cycle arrest at G0/1 phase. Our data demonstrated that the most active was compound 16 with lengthier of aliphatic chain containing 9 carbon atoms between tetrahydroacridine and 4-fluorobenzoic acid. These results suggest the compound 16 may be promising molecule for treatment of lung cancer. In addition, our studies gained new knowledge that fluorobenzoic acid and length of the aliphatic linker enhance anticancer activity and have impact on mode of action of acridine-based compounds. Acknowledgements This study was supported by grant from Medical University of Lodz, Poland, Research Program No. 502-03/3-015-01/502-34-040. References [1] Jemal A, Siegel R, Xu J, Ward E. Cancer statistics 2010. CA Cancer J Clin 2010;60:277–300. [2] Ferlay J, Shin HR, Bray F, Forman D, Mathers CD, Parkin D. GLOBOCAN. Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10. Lyon, France: International Agency for Research on Cancer 2010; 2008Available from:http:// globocan.iarc.fr, . Last accessed 04 November 2013. [3] Collins LG, Haines C, Perkel R, Enck R. Lung cancer: diagnosis and management. Am Fam Phys 2007;75:56–63. [4] Dempke WCM, Suto T, Reck M. Targeted therapies for non-small cell lung cancer. Lung Cancer 2010;67:257–74. [5] Strasser A, Harris AW, Jacks T, Cory S. DNA damage can induce apoptosis in proliferating lymphoid cells via p53-independent mechanisms inhibitable by Bcl-2. Cell 1994;21:329–39. [6] Lord CJ, Ashworth A. The DNA damage response and cancer therapy. Nature 2012;481:287–94. [7] Cholewin´ski G, Dzierzbicka K, Kołodziejczyk AM. Natural and synthetic acridines/acridones as antitumor agents: their biological activities and methods of synthesis. Pharmacol Rep 2011;63:305–36. [8] Kumar R, Sharma A, Sharma S, Silakari O, Singh M, Kaur M. Synthesis, characterization and antitumor activity of 2-methyl-9-substitued acridines. Arab J Chem 2013. http://dx.doi.org/10.1016/j.arabjc.2012.12.035. [9] da Rocha Pita MG, Souza ES, Barros FWA, et al. Synthesis and in vitro anticancer activity of novel thiazacridine derivatives. Med Chem Res 2012;22:2421–9. [10] Belmont P, Bosson J, Godet T, Tiano M. Acridine and acridone derivatives, anticancer properties and synthetic methods: where are we now? Anticancer Agents Med Chem 2007;7:139–69. [11] Ghosh R, Bhowmik S, Guha D. 9-Phenyl acridine exhibits antitumour activity by inducing apoptosis in A375 cells. Moll Cell Biochem 2012;361:55–66. [12] Sondhi SM, Singh J, Rani R, Gupta PP, Agrawal SK, Saxena AK. Synthesis, antiinflammatory and anticancer activity evaluation of some novel acridine derivatives. Eur J Med Chem 2010;45:555–63. [13] Barros WA, Bezerra DP, Ferreira PMP, Cavalcanti BC, Silva TG, et al. Inhibition of DNA topoisomerase I activity and induction of apoptosis by thiazacridine derivatives. Toxicol Appl Pharm 2013;268:37–46. [14] Demeunynck M. Antitumor acridines. Expert Opin Ther Pathol 2004;14:55–70.
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Original article
Novel tetrahydroacridine derivatives inhibit human lung adenocarcinoma cell growth by inducing G1 phase cell cycle arrest and apoptosis Paulina Olszewska a, Elz˙bieta Mikiciuk-Olasik a, Katarzyna Błaszczak-S´wia˛tkiewicz a, Jacek Szyman´ski b, Paweł Szyman´ski c,* a Department of Pharmaceutical Chemistry, Drug Analysis and Radiopharmacy, Faculty of Pharmacy, Medical University, Muszyn´skiego 1, 90-151 Lodz, Poland b Central Scientific Laboratory, Medical University, Mazowiecka 6/8, 92-215 Lodz, Poland c Laboratory of Radiopharmacy, Department of Pharmaceutical Chemistry, Drug Analysis and Radiopharmacy, Faculty of Pharmacy, Medical University, Muszyn˜skiego 1, 90-151 Lodz, Poland
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 May 2014 Accepted 15 October 2014
Lung cancer is not only the most commonly diagnosed cancers worldwide but it is still the leading cause of cancer-related death. Acridine derivatives are a class of anticancer agents with the ability to intercalate DNA and inhibit topoisomerases. The aim of this study was to evaluate the effect of sixteen new tetrahydroacridine derivatives on the viability and growth of human lung adenocarcinoma cells. We compared anticancer activity of a series of eight compounds with 4-fluorobenzoic acid and eight compounds with 6-hydrazinonicotnic acid differed from each other in length of the aliphatic chain containing from 2 to 9 carbon atoms. Interestingly, tetrahydroacridine with 4-fluorobenzoic acid (compounds 9–16) showed higher anticancer activity than derivatives with 6-hydrazinonicotnic acid (compounds 1–8) and their efficacy was correlated with increasing number of carbon atoms in the aliphatic chain. The results showed that inhibition of cancer cell growth by the most effective compounds 15 and 16 was associated with induction of G1 phase cell cycle arrest followed by caspase-3 dependent apoptosis. Our findings suggest that tetrahydroacridine with 4-fluorobenzoic acid containing 8 and 9 carbon atoms may be potential candidate for treatment of lung cancer. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Tetrahydroacridine derivatives Acridine derivatives Anticancer compounds Lung cancer DNA intercalation
1. Introduction Lung cancer is the most commonly diagnosed cancers worldwide and by far is the leading cause of cancer-related death among both men and women [1,2]. There are two major types of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Approximately 85% of lung cancers are NSCLC that includes adenocarcinoma (40%). It is an aggressive tumor with a 5-year survival rate of less than 15% demonstrating that current therapy is still inefficient [3,4]. Therefore, the search for better chemotherapeutic agents with advanced activity against lung cancer is needed. * Corresponding author. 1, Muszynskiego Street, 90-151 Lodz, Poland. Tel.: +48 042 677 92 90; fax: +48 042 677 92 50. E-mail address: [email protected] (P. Szyman´ski). http://dx.doi.org/10.1016/j.biopha.2014.10.018 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
DNA targeted drugs are successful anticancer agents, which cause DNA damage and can lead to cell cycle arrest and apoptosis [5,6]. Acridine derivatives are the most extensively studied class of potential anticancer agents that interfere with DNA as intercalators [7,8]. In addition to cancer, the acridine-based compounds have a long history of treatment of other human diseases, such as bacterial, parasitic infections and Alzheimer’s disease [9]. The biological activity of acridines is mainly attributed to the planarity of aromatic structures, which can intercalate within the DNA structure [10]. The act of intercalation induces local structural changes to the DNA, including the unwinding of the double helix and lengthening of the DNA strand [11,12]. Moreover, acridines are able to decrease the activity of topoisomerase I and II [10,13,14]. Topoisomerases are involved in replication, recombination, transcription, and chromosome segregation and they are crucial for proper DNA actions [15]. For example, first synthetic
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Fig. 1. Chemical structure of tested 1,2,3,4-tetrahydro-9-aminoacridine derivatives. Sixteen tetrahydroacridine derivatives with 6-hydrazinonicotinic fragment or 4-fluorobenzoic acid moiety are shown. Series of derivatives have the aliphatic linker comprising from 2 to 9 carbon atoms (n = 2–9) between tetrahydroacridine and 6-hydrazinonicotinic (compounds 1–8) or 4-fluorobenzoic acid (compounds 9–16).
acridine-based agent to be considered as intercalator and topoisomerase II inhibitor was amsacrine [8,16]. We have previously synthetized and described a variety of tetrahydroacridine (1,2,3,4-tetrahydro-9-aminoacridine) analogues coupled with 6-hydrazinonicotinic acid (compounds 1–8) or 4-fluorobenzoic acid moiety (compounds 9–16) [17–19]. In the current study, we evaluated these new tetrahydroacridine derivatives for anticancer activity in human lung adenocarcinoma cells. The chemical structures of compounds used in this study are shown in Fig. 1. These synthesized compounds differed from each other in length of the aliphatic chain containing from 2 to 9 carbon atoms between tetrahydroacridine and 6-hydrazinonicotinic acid or 4-fluorobenzoic acid (Fig. 1). The purpose of this study was to investigate the efficacy of sixteen new tetrahydroacridine derivatives on the survival and growth of human lung adenocarcinoma cells. Moreover, we evaluated the effects of hydrazinonicotinic acid and fluorobenzoic acid moiety and different length of aliphatic chain on anticancer activity of new compounds. To characterize the mode of actions of the most effective compounds, we monitored changes in cell morphology and determined their effects on cell cycle progression and apoptosis.
2. Materials and methods 2.1. Cell culture
dimethyl sulfoxide (DMSO) to prepare the stock solution. Before use, stock solution of compounds was diluted with cultured medium to the desired concentrations and the final concentration of DMSO was 0.1%. A549 cells were treated with compounds at different concentrations from 1 to 200 mM ranges and medium containing diluent (0.1% DMSO) was added to the control cells. After 72 h treatment, WST-1 reagent was added to each well and incubated for additional 3 h at standard condition. Next, absorbance was measured using microplate reader (Synergy H1, Bio-Tek, Winooski, VT, USA) at a wavelength of 440 nm. The cell viability was expressed as a percentage of the control values. Data showed the mean SD of three independent experiments performed in triplicates. IC50 values (the concentration of tested compound that inhibited cell growth by 50%) were calculated by concentration-response curve fitting using a Microsoft Excel-based analytic method. 2.3. Cell morphology Control and tetrahydroacridine-treated A549 cells were examined for morphological changes and cell growth inhibition under light microscopy with phase-contrast (Opta-Tech). The changes in cell morphology upon treatment with tested compounds were photographed after 24 h, 48 h, and 72 h at 100 magnification. 2.4. Cell cycle analysis
Anticancer activities of the compounds were tested against A549 cells obtained from a human lung adenocarcinoma. A549 cells were purchased from the European Collection of Cell Cultures (ECACC, Salisburg, UK). The cells were grown in F12K medium (HyClone, UK) supplemented with 10% heat-inactivated fetal bovine serum, FBS (Lonza, Basel, Switzerland), 100 units/mL penicillin and 100 mg/mL streptomycin (Lonza) at 37 8C with 5% CO2. 2.2. Cell viability assay The cell viability was quantified by using WST-1 assay (Millipore, Billerica, MA, USA). The assay is based on the enzymatic cleavage of the water-soluble tetrazolium salt (WST-1) to formazan by cellular mitochondrial dehydrogenases present in viable cells. The amount of formazan dye formed directly correlates to the number of live cells. For the experiment, cells were seeded in 96-well plates at density 5000 cells per well and cultured for 24 h. The tested derivatives were dissolved in
A549 cells were seeded in 6-well plates at density 20 104 cells per well and cultured for 24 h. Next, cells were exposed to compounds 5, 15, and 16 at 30, 15, 10 mM concentrations corresponding to IC50, respectively. After 24 h treatment, control and treated cells were then collected, washed with PBS solution, fixed in cold 70% ethanol at 4 8C. Prior to analysis, the cells were washed with PBS, incubated with ribonuclease (100 mg/mL, Sigma–Aldrich, St. Louis, MO, USA) for 30 min and stained with propidium iodide (50 mg/mL, Sigma–Aldrich) in darkness at room temperature for additional 20 min. DNA content and number of cells in the individual cell cycle phases was measured by flow cytometry (FACS Canto II, Becton Dickinson, USA). Percentages of cells in each cell cycle phase were calculated from live cells that were expressed as 100%. Sub-G1 population representing cell debris was excluded from the analysis. Then, 20,000 cells were analyzed from each sample and data were presented as the mean SD of three independent experiments.
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Table 1 In vitro growth inhibition of A549 cells by new tetrahydroacridine derivatives. IC50 values (concentration of tested compounds causing 50% inhibition of cell growth compared to control cells) were determined after 72 h incubation with tested compounds using WST-1 assay. Data are expressed as the mean SD, n = 3. Compounds 1–8
Compounds 9–16
Compound
Number of carbon atoms (n)
IC50 (mM)
Compound
Number of carbon atoms (n)
IC50 (mM)
1 2 3 4 5 6 7 8
2 3 4 5 6 7 8 9
> 200.0 > 200.0 150.4 5.1 > 200.0 28.7 3.5 83.5 2.6 98.5 1.4 53.9 1.3
9 10 11 12 13 14 15 16
2 3 4 5 6 7 8 9
116.5 5.8 156.6 2.8 67.9 2.5 31.3 4.9 31.5 0.7 15.2 4.6 15.0 5.2 9.6 4.3
2.5. Caspase-3/7 activity assay Caspase-Glo 3/7 Assay (Promega, Madison, WI, USA) was used to assess cell apoptosis according the manufacturer instruction. Briefly, the proluminescent substrate containing the DEVD is cleaved by activated caspase-3/7 and a substrate for luciferase (Z-DEVD-aminoluciferin) is released. This results in the luciferase reaction and the production of luminescent signal. Luminescence is proportional to the amount of caspase activity. The A549 cells were seeded at a density of 5000 cells per well in 96-well plates and cultured for 24 h. Next, cells were treated with tested compounds at indicated concentrations and caspase activity was measured after 24 h and 48 h incubation. The Caspase-Glo 3/7 reagent was added to the cells at ratio 1:1. After 1 h incubation at room temperature, luminescence was recorded using a microplate reader (Synergy H1, Bio-Tek) at gain 135. 2.6. Nuclear staining with Hoechst For apoptotic nuclei evaluation, the Hoechst labelling was used. Cells were treated with compounds 5 (50 mM and 30 mM), 15 (25 mM and 15 mM) and 16 (25 mM and 10 mM). After 48 h incubation, cells were fixed and permeabilized with cold methanol. Fixed cells were washed with PBS and incubated with Hoechst 33342 [1:2500] [Invitrogen] for 20 min. The cell nuclei were photographed using fluorescence microscope (Olympus IX-51) at 200 magnification. To determine percentage of apoptotic cells, normal and apoptotic nuclei from five microscopic fields per sample were counted. 2.7. Statistical analysis Data were represented as means SD from indicated number of separate experiments. The significance difference between control group and compound-treated group was validated by a Student’s paired t-test. A P value below 0.05 (*P < 0.05) was considered statistically significant.
3. Results 3.1. Effect of new tetrahydroacridine derivatives on A549 cell growth The growth-inhibitory effects of sixteen tetrahydroacridine derivatives were analyzed in human lung adenocarcinoma A549 cells using WST-1 assay. First, we performed concentrationresponse analysis to determine concentration of tested compounds that induced a 50% decrease of cell viability (IC50) as compared to control cells. The cancer cells were treated with tested compounds at different concentrations from 1 mM to 200 mM for 72 h. As shown in Table 1, six out of sixteen tested compounds had IC50 values below 50 mM. Among tetrahydroacridine derivatives with 6-hydrazinonicotinic acid moiety, compound 5 exhibited the most
potent anticancer activity (IC50 = 28.7 mM 3.5). We found that new tetrahydroacridine derivatives containing 4-fluorobenzoic acid (compounds 9–16) were much more effective in inhibition of A549 cells growth in comparison with compounds with 6-hydrazinonicotinic acid moiety (compounds 1–8). The compound concentrations that inhibited cell growth by 50% were under 20 mM for the compounds 14, 15, and 16 containing 4-fluorobenzoic acid (Table 1). Moreover, compound 16 containing 9 carbon atoms in the aliphatic chain was the most potent to inhibit A549 cell growth (IC50 = 9.6 mM 4.3). Based on these results, we selected compound 5 (as the most effective derivative containing 6-hydrazinonicotinic acid) and compounds 15 and 16 (as the most effective derivatives coupling with 4-fluorobenzoic acid) for further biological evaluation. Fig. 2 shows the dose-response effect of selected compounds on A549 cell viability after 72 h treatment. The results were expressed as relative number of viable adherent cells in relation to control cells. For example, culture of A549 cells in the presence of 10 mM of compounds 5, 15, or 16 decreased number of viable cells to 74.6%, 67.0% and 43.2%, respectively (Fig. 2). Our results showed that the most effective compounds were tetrahydroacridine derivatives with 4-fluorobenzoic acid moiety containing lengthier aliphatic chain compromising from 7 to 9 carbon atoms (Table 1). 3.2. Microscopic study of A549 cells The effects of tetrahydroacridine derivatives on cancer cell growth and viability were also monitored and visualized using phase-contrast microscopy. Morphological examination of A549 cells treated with tested compounds showed severe compoundmediated changes. Images in Fig. 3 show the comparison of dosedependent effects of selected compounds 5, 15, and 16 on cell growth after 72 h treatment. We observed changes in number, shape, and size of the tetrahydroacridine-treated cells compared with the controls. For example, incubation of cells with compound 5 caused decrease of cell population approximately by 50% at 25 mM as compared to control cells (Fig. 3). At higher concentration, cells were round and detached from adjacent cells. The compound 15 exhibited greater anticancer activity and decreased number of cells by 50% at 10 mM (Fig. 3). Results demonstrated that compound 16 was the most effective and reduced number of viable cells by 50% at 5 mM. Fig. 4 represents the dose-dependent and time-dependent effects of the most effective compound 16 on inhibition of A549 cell growth. For example, treatments of cells with 10 mM of compound 16 inhibited cell proliferation as well as more elongated cells were presented in comparison with the control cells after 24 h. After 48 h, in addition to inhibition of cell proliferation, we observed shrinkage of some cells suggesting apoptotic changes. Then, 72 h treatment showed more apoptotic cells and cell detachment was triggered as compared to 24 h and 48 h (Fig. 4). Our data demonstrated that the effects of tested compounds on cancer cell viability, morphology and growth were dependent on dose as well as time of treatment.
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Fig. 2. Effect of tetrahydroacridine derivatives on viability of cancer cells. A549 cells were cultured in the present of indicated compounds at concentrations from 1 to 100 mM range. Cell viability was determined by WST-1 assay after 72 h treatment. Concentrations are graphed on a log scale. Data are expressed as the mean SD, n = 3.
Fig. 3. Dose-dependent effect of selected compounds on A549 cell growth after 72 h treatment. A549 cells were cultured in the presence or absence (control) of compounds 5, 15, 16 at indicated concentrations for 72 h. Representative phase-contrast cell images are shown (100 magnification).
3.3. Effect of tetrahydroacridine derivatives on cell cycle progression of A549 cells To understand the mechanisms responsible for compoundsmediated cancer cell growth inhibition, we examined their effects on cell cycle distribution. The cell cycle analysis was performed in
A549 cells exposed to the compounds 5, 15 and 16 at concentrations of IC50 for 24 h (Fig. 5A and B). Separation of cells in G0/1, S and G2/M phase was based on fluorescence intensity after cell staining with propidium iodide using flow cytometry analysis. Fig. 5A shows representative profiles of cell cycle distribution for each tested compound and control cells. When cells were treated with compounds 15 and 16, a significant increase number of cells
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Fig. 4. Time- and dose-dependent effect of compound 16 on A549 cell growth. A549 cells were cultured in the presence or absence (control) of compound 16 at indicated concentrations. Representative phase-contrast cell images are shown after 24 h, 48 h and 72 h of treatment (100 magnification).
Fig. 5. Effect of new tetrahydroacridine derivatives on cell cycle. Cells were cultured for 24 h with selected compounds at IC50 concentrations. The cell cycle progression was evaluated using flow cytometry analysis of propidium iodide stained cells. A. Representative histograms of one out of three independent experiments are shown. Percentages of cells in each cell cycle phase were calculated from live cells that were expressed as 100%. B. Data are expresses as the mean SD of three independent experiments. * P < 0.05 compared with control (CTR).
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were arrested at the G0/1 phase of the cell cycle with a corresponding decrease in the percentage of cells from the S phase. With respect to control cells, the cell population in the G0/1 phase increased from 57.4% 2.5 to 76.2% 3.2 in the presence of 15 mM compound 15 (Fig. 5B). Additionally, compound 15 induced a reduction in the percentage of cells in the G2/M phase. Similarly, in the case of derivative 16, we observed significant increase in the G0/1 fraction from 57.4% 2.7 to 68.4% 3.7 (Fig. 5B). In contrast, treatment of A549 cells with compound 5 had no significant effect on cell cycle distribution (Fig. 5B). Our data showed that tested tetrahydroacridine derivatives containing fluorobenzoic acid moiety induced arrest of cell cycle progression through G1 into S phase in A549 cells. Moreover, the effect induced by compound 15 was more pronounced as compared to compound 16 (Fig. 5). 3.4. Effect of tetrahydroacridine derivatives on apoptosis Since the inhibition of cancer cell proliferation can results in initiation of apoptosis, we next determined whether selected tetrahydroacridine derivatives repressed cancer cell growth by induction of apoptosis. The effect of tested compounds on cell apoptosis was assessed by the caspase-3/7 activity after 24 h and 48 h treatment. Twenty-four-hour treatment of cancer cells with compound 16 at 25 mM resulted in 2.7-fold increase of caspase3/7 activity as compared to control cells (Fig. 6A). In contrast, treatment of A549 cells with compounds 5 and 15 at indicated concentrations for 24 h had no significant effect on the level of activated enzyme (Fig. 6A). However, when caspase-3/7 activity was evaluated after 48 h exposure, we observed significant elevation of enzyme activity in cells treated with compounds 5 and 15 at IC50 concentration and higher (Fig. 6B). Similar to 24 h exposure, the high content of activated caspase-3/7 in cells treated with compound 16 at 25 mM was also detected after 48 h ( 2.6fold) (Fig. 6B). In addition, 48 h treatment of cells with 10 mM of compound 16 also significantly increased caspase-3/7 activity. To further confirm the induction of apoptosis in compound-treated cells, we analyzed chromatin condensation and nuclear fragmentation by fluorescence microscopy using the DNA-binding fluorescence dye, Hoechst. As shown in Fig. 6B, exposure of cells to higher tested concentrations of compounds for 48 h induced chromatin condensation, nuclear fragmentation and formation of apoptotic bodies that suggest the presence of apoptotic cells. These nuclear apoptotic changes visualized by Hoechst staining corresponded to changes in cell morphology (cell shrinkage, rounded cells) observed in phase-contrast microscopy (Fig. 6B). To determine percentage of apoptotic cells after 48 h treatment with compounds, Hoechst-stained normal and apoptotic nuclei from five microscopic fields per sample were counted. We observed appearance of some apoptotic cells in control cells (7.8%) after 48 h due to intensive cell proliferation leading to over-confluence. This may explain about 40% increase in caspase-3/7 activity in control cells measured at 48 h as compared to 24 h control cells (Fig. 6A). Exposure of A549 cells to compounds 5 and 15 at higher tested concentrations resulted in 2-fold (16.5%) and 3-fold (22.6%) increased percentage of apoptotic cells, respectively, as compared to control (7.8%). Interestingly, 48 h treatment of cells with compound 16 at 25 mM induced apoptosis in almost all cells as determined by nuclei staining (87.4%) and corresponding changes in cell morphology (Fig. 6B). This effect correlated with high content of activated caspase-3/7 detected at 24 h that was continued to be elevated after 48 h treatment (Fig. 6A). The results showed that tested compounds inhibited cancer cell growth, at least in part, by caspase-3/7-dependent apoptosis. We found that compound 16 was the strongest inducer of apoptosis in A549 cells.
4. Discussion In the present study, we investigated the efficacy of sixteen new tetrahydroacridine derivatives on inhibition human lung adenocarcinoma cell growth. We have synthetized a series of tetrahydroacridine analogues coupled with 6-hydrazinonicotinic acid (compounds 1–8) or 4-fluorobenzoic acid (compounds 9–16) to examine how different moieties and the length of the aliphatic chain influence anticancer activity of new compounds [17– 19]. Moreover, we evaluated the mechanisms of anticancer activity of the most effective compounds. Our results demonstrated that tetrahydroacridine derivatives with 4-fluorobenzoic acid moiety had a more potent growth inhibition activity in A549 cells than compounds having 6-hydrazinonicotinic acid and their efficacy correlated with increased number of carbon atoms in aliphatic chain. We found that all selected compounds (5, 15, 16) induced cancer cell death by caspase-3/7-dependent apoptosis. However, compounds containing 4-fluorobenzoic acid (15 and 16) inhibited cancer cell growth as results of cell cycle arrest in G0/1 phase. Lung cancer is one of the most common diagnosed malignancies in the world and is still the leading cause of death from cancer [1,2]. There are two major histological types of lung cancer: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC is the most common type of lung cancer, accounting for approximately 85% of all cases and includes adenocarcinoma (40%) [4,20]. It is an aggressive tumor type with 5-year survival rate of only about 15% [1]. The lack of effective therapy for treatment of NSCLC indicates an urgent need to search for new drugs [21]. Drugs having affinity for DNA are favoured as potential therapeutic agents for the treatment of cancer [22,23]. The acridine-based compounds are the most studied chemotherapeutic agent with DNA intercalative properties [8]. They also had ability to inhibit enzymes that are crucial for proper DNA actions, such as topoisomerase I/II and telomerase [10,24,25]. Amsacrine was the first synthetic DNA intercalating agents and topoisomerase inhibitor that was used for treatment of leukemia [8,26]. Moreover, acridine derivatives display also other interest forms of biological activity such as anti-bacterial drugs [9,16] and antiprotozoal drugs [26,27], or cholinesterase inhibitors [9,19]. To explore novel acridine-based compounds as potential chemotherapeutics for treatment of lung cancers, we have synthetized a series of sixteen new tetrahydroacridine analogues containing 6-hydrazinonicotinic acid (compounds 1–8) or 4fluorobenzoic acid moiety (compounds 9–16) [17–19]. In each group, the compounds differed from each other in the length of the aliphatic chain comprising from 2 to 9 carbon atoms. Our initial study was performed to screen the effect of these new tetrahydroacridne derivatives on growth of human lung adenocarcinoma A549 cells using WST-1 assay. We found a significant difference in the anticancer activity of the tested compounds. Results obtained in the present study demonstrated that derivatives containing 4-fluorobenzoic acid moiety were much more effective than derivatives with 6-hydrazinonicotinic acid. Moreover, we found that the length of aliphatic chain significantly influenced on the cell growth inhibition of compounds with 4-fluorobenzoic acid and the efficacy of these derivatives was increased with increase the number of carbon atoms. The most effective was compound 16, (4-fluoro-N-[9-(1,2,3,4-tetrahydroacridin-9-ylamino)nonyl]-benzamide), containing 9 carbon atoms in aliphatic linker. Recently, we have reported that cyclopentaquinoline derivatives with 4-fluorobenzoic acid also exhibit higher anticancer activity than cyclopentaquinoline derivatives with 6-hydrazinonicotinic acid and their efficacy correlate with the length of the aliphatic chain [28]. Thus the current results are consistent with our previous findings and strongly suggest that coupling of acridine-based compounds with fluorobenzoic acid by
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Fig. 6. Effects of new compounds on cell apoptosis. A549 cells were cultured in the presence or absence (CTR, control) of selected compounds (5, 15 and 16) at indicated concentrations. A. Cell apoptosis was determined by caspase-3/7 activity assay after 24 h and 48 h treatment. Caspase-3/7 activity was measured as a luminescence light unit [RLU] and expressed as the mean SD from two independent experiments performed in triplicates. *P < 0.01 compared with control. B. Visualization of apoptotic nuclei in cells treated with compounds at indicated concentrations for 48 h. The cell nuclei were stained with DNA-binding fluorescence dye, Hoechst. Representative fluorescence images are shown (200 magnifications). Arrows point apoptotic nuclei characterized by condensation of chromatin or nuclear fragmentations. Numbers represent the percentage of apoptotic cells expressed as the mean SD calculated from 5 random microscopic fields. Lower panel shows the effects of compounds on cell growth and morphology directly before Hoechst staining. Representative phase-contrast images are shown (100 magnifications).
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longer aliphatic linker caused changes in the structure of compounds that increased their anticancer activity. Based on the IC50 values of tested tetrahydroacridine derivatives, we selected the most effective compounds from each group (compounds 5, 15 and 16) for further evaluation. Phase-contrast images of A549 cells culture in the presence or absence of selected compounds visualized that effects of compounds on cancer cell growth and viability were dose and time-dependent. Previous studies have shown that acridine-based compounds, such as cyclopentaquinoline derivatives or 9-aminoacridine derivatives suppress viability of lung cancer cell lines or mesothelioma, respectively [28,29]. Compounds that suppress tumor growth by induction of cell cycle arrest and apoptosis are desired strategy in cancer therapy [30–32]. Apoptosis induction is one of the main mechanisms that impede cancer growth and proliferation and is one of the major mechanisms used by various anticancer agents. Caspases play the key role in the initiation and execution of programmed cell death. Based on the functions, they have been divided into two groups: caspases-2, -8, -9, -10 are initiators and caspases-3, -6, -7 are effectors of apoptosis [33]. Caspases-3 is a major executioner of apoptosis [33,34] and is responsible for specific cleavage of many key cellular proteins, chromatin condensation and DNA fragmentation, which ultimately result in cell death [35]. To determine whether the observed decrease in cell viability induced by new compounds was associated with apoptosis, we measured levels of caspase-3/7 activity at 24 and 48 h. We found that 48 h exposure of A549 cells to all selected compounds at IC50 and higher concentrations significantly increased the caspase-3/7 activity levels. Interestingly, treatment of cells with compound 16 at 25 mM induced caspase-3/7 activation early at 24 h and the high content of activated enzyme was also detected after 48 h treatment. In addition to caspase-3/7 activity, apoptotic cells were visualized by nuclear morphological changes in Hoechststained cells. Treatment of cells with compounds 5 and 15 at higher concentrations for 48 h significantly increased percentage of apoptotic cells ( 2-fold and 3-fold, respectively). Consistent with the high caspase-3/7 activity levels, treatment of cells with compound 16 at 25 mM showed almost all apoptotic nuclei. Moreover, these effects also correlated with cellular morphological changes observed under phase-contrast microscope. Our data showed that selected compounds (5, 15, and 16) inhibited cancer cell growth, at least in part, via induction of caspase-3/7dependent apoptosis in a concentration-dependent manner. The results demonstrated that compound 16 was the strongest inducer of apoptosis in A549 cells. Previous study has reported that 9-phenylacridine derivatives led to release of cytochrome c and caspase-3/7 activation [11]. Moreover, study conducted by Goodell et al. showed that substituted 9-aminoacridine derivatives inhibit cell proliferation and induces apoptosis of pancreatic cancer cell lines [36]. Cell cycle arrest is an important signal for inhibition of cell proliferation [37]. Moreover, the induction of apoptosis can be mediated through the cell cycle arrest [36,38]. To determine whether selected compounds inhibited cancer cell growth as a result of induction of cell cycle arrest, we examined their effect on cell cycle progression. Flow cytometry analysis revealed that growth of the A549 cells was arrested at G0/1 phase in the presence of compounds 15 and 16. Interestingly, the strongest G0/1 arrest with corresponding decreased in percentages of S and G2/M cell population was observed after treatment with compound 15. It has been demonstrated that 9-aminoacridine derivatives induce a G0/1 phase arrest followed by apoptosis in pancreatic cancer cells as a consequence of topoisomerase II inhibition [24,36]. Our results also demonstrated that treatment of lung adenocarcinoma cells with compounds 15 and 16 at IC50 concentrations led initially to cell cycle arrest followed by induction of cell apoptosis. Thus, these
compounds exhibited similar effects on cell cycle and apoptosis, suggesting potential inhibition of topoisomerase II in A549 cells. In contrast, compound 5 containing 6-hydrazinonicotinic acid had no significant effect on cell cycle progression. These results suggest that compounds 15 and 16-induced G0/1 phase arrest was dependent on the presence of 4-fluorobenzoic acid. These findings are consistent with our previous study, demonstrating that cyclopentaquinoline derivatives with 4-fluorobenzoic acid induce a G0/1 phase arrest in lung cancer cells [28]. Together, these results strongly suggest that coupling of acridine-based compounds with 4-fluorobenzoic acid not only improved anticancer activity but also influenced mechanism of anticancer action. In conclusion, our present findings showed that new tetrahydroacridine derivatives containing 4-fluorobenzoic acid (compounds 9–16) were much more effective in inhibition of human lung adenocarcinoma cell growth in comparison with compounds with 6-hydrazinonicotinic acid moiety (compounds 1–8). Moreover, the anticancer activity of tetrahydroacridine derivatives with 4-fluorobenzoic acid was correlated with the number of carbon atoms in the aliphatic linker. All selected compounds (5, 15, and 16) induced cancer cell death by induction of caspase-3/7 apoptosis. However, growth inhibition of A549 cells by tested tetrahydroacridine with 4-fluorobenzoic acid was associated with a cell cycle arrest at G0/1 phase. Our data demonstrated that the most active was compound 16 with lengthier of aliphatic chain containing 9 carbon atoms between tetrahydroacridine and 4-fluorobenzoic acid. These results suggest the compound 16 may be promising molecule for treatment of lung cancer. In addition, our studies gained new knowledge that fluorobenzoic acid and length of the aliphatic linker enhance anticancer activity and have impact on mode of action of acridine-based compounds. Acknowledgements This study was supported by grant from Medical University of Lodz, Poland, Research Program No. 502-03/3-015-01/502-34-040. References [1] Jemal A, Siegel R, Xu J, Ward E. Cancer statistics 2010. CA Cancer J Clin 2010;60:277–300. [2] Ferlay J, Shin HR, Bray F, Forman D, Mathers CD, Parkin D. GLOBOCAN. Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10. Lyon, France: International Agency for Research on Cancer 2010; 2008Available from:http:// globocan.iarc.fr, . Last accessed 04 November 2013. [3] Collins LG, Haines C, Perkel R, Enck R. Lung cancer: diagnosis and management. Am Fam Phys 2007;75:56–63. [4] Dempke WCM, Suto T, Reck M. Targeted therapies for non-small cell lung cancer. Lung Cancer 2010;67:257–74. [5] Strasser A, Harris AW, Jacks T, Cory S. DNA damage can induce apoptosis in proliferating lymphoid cells via p53-independent mechanisms inhibitable by Bcl-2. Cell 1994;21:329–39. [6] Lord CJ, Ashworth A. The DNA damage response and cancer therapy. Nature 2012;481:287–94. [7] Cholewin´ski G, Dzierzbicka K, Kołodziejczyk AM. Natural and synthetic acridines/acridones as antitumor agents: their biological activities and methods of synthesis. Pharmacol Rep 2011;63:305–36. [8] Kumar R, Sharma A, Sharma S, Silakari O, Singh M, Kaur M. Synthesis, characterization and antitumor activity of 2-methyl-9-substitued acridines. Arab J Chem 2013. http://dx.doi.org/10.1016/j.arabjc.2012.12.035. [9] da Rocha Pita MG, Souza ES, Barros FWA, et al. Synthesis and in vitro anticancer activity of novel thiazacridine derivatives. Med Chem Res 2012;22:2421–9. [10] Belmont P, Bosson J, Godet T, Tiano M. Acridine and acridone derivatives, anticancer properties and synthetic methods: where are we now? Anticancer Agents Med Chem 2007;7:139–69. [11] Ghosh R, Bhowmik S, Guha D. 9-Phenyl acridine exhibits antitumour activity by inducing apoptosis in A375 cells. Moll Cell Biochem 2012;361:55–66. [12] Sondhi SM, Singh J, Rani R, Gupta PP, Agrawal SK, Saxena AK. Synthesis, antiinflammatory and anticancer activity evaluation of some novel acridine derivatives. Eur J Med Chem 2010;45:555–63. [13] Barros WA, Bezerra DP, Ferreira PMP, Cavalcanti BC, Silva TG, et al. Inhibition of DNA topoisomerase I activity and induction of apoptosis by thiazacridine derivatives. Toxicol Appl Pharm 2013;268:37–46. [14] Demeunynck M. Antitumor acridines. Expert Opin Ther Pathol 2004;14:55–70.
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