Anti-Cancer Effect of Tacrine-Coumarin Derivatives on Diverse Human and Mouse Cancer Cell Lines

Acridine derivatives were first used as antibacterial and antiparasitic agents, later as antimalarial and anti-HIV drugs, and now as potentially anticancer agents due to their high cytotoxic activity. Due to their serious adverse effects, new synthetic derivatives were introduced and tested based on modified naturally occurring substances, such as acridone derivatives, which also exhibit potential anti-tumor activity. Most of them are DNA-damaging substances, causing relatively strong and selective destruction of tumor cells. We have tested in vitro anti-proliferative effects of newly-synthesized tetrahydroacrid derivatives, namely tacrine-coumarin hybrid molecules, on various human and mouse cancer cell lines. Our results showed that tacrine-coumarin hybrids with seven, eight, and nine methylene groups in spacer reduce proliferation of cancer cells. A hybrid with nine methylene groups had the most significant anti-cancer effect.


Introduction
Coumarin and coumarin derivatives are well known anticancer agents.The anticancer activity of coumarin derivatives, 3-, 4-, 7-, 8-substituted biscoumarins and fused coumarins, has been described according to the type and position of the side chain of the coumarin core structure. 1he number, location, and length of the side chain have important effects on anti-tumor activities. 2 Fig. 1 shows the chemical structure of 4-substituted coumarins that reveal interesting anti-cancer activity.
Since the anti-cancer activity of hybrids in which the coumarin ring is connected with tacrine has not yet been reported, based on the knowledge that coumarin functional groups as well as tetrahydroacridine derivatives have anti-tumor effects, we decided to test such effects using newly synthesized tacrine-coumarin hybrids 7a-g (Fig. 2).Both the synthesis and the biological activity of relevant tacrine-coumarin hybrids, except for anti-cancer ones, have already been published. 8Little work has been done on the anti-tumor effects of tacrine.However, published results clearly demonstrated that only high concentration of tacrine can induce apoptosis via lysosome-and mitochondrial-dependent pathway in HepG2 cells. 9,10On the other hand, despite high concentration, tacrine did not show any cytotoxic effect on the promyelocytic leukemia HL-60 cell line. 11In this study, we decided to test the anti-cancer effects of tacrine-coumarin hybrids on highly invasive (4T1), less invasive (A549, HCT) and non-invasive (MCF-7) cell lines.To reinforce the hypothesis of different effects of tacrine-coumarin hybrids on cancer cells compared to normal ones, we compared human cancer cell lines (MCF-7, A549, HCT) with normal HUVEC cells and mouse tumor cell line 4T1 with the normal mouse cells NMuMG.

1. Cell lines and Cell Culture
Human colorectal carcinoma HCT116 (HCT), human breast adenocarcinoma MCF-7, human A549 lung carcinoma, and mouse mammary carcinoma 4T1, as well as non-cancer mouse mammary gland cells NMuMG and human endothelial cells isolated from umbilical vein HUVEC, were used in our experiments.The HCT, MCF-7, and 4T1 cell lines were cultured in RPMI medium (Sigma-Aldrich, St. Louis, MO, USA), A549 and NMuMG in DMEM medium with high glucose (4.5 g/l), and L-glutamine (GE Healthcare, Little Chalfont, UK) and HUVEC cells in HMEC medium (Sigma-Aldrich) with added vascular endothelial growth factor and bovine fibroblast growth factor.All media contained 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and antibiotics (Sigma-Aldrich), except 4T1 and NMuMG cells, which did not contain antibiotics.The cells were incubated at 37 °C in a humidified 5% CO2 (v/v) atmosphere.Dr. Hamuľaková of the Institute of Chemistry, Faculty of Science, Pavol Jozef Šafárik University in Košice, synthesized Tacrine-coumarin derivatives.

3. Cell cycle Analysis
Flow cytometric DNA analysis was used to estimate the distribution of monitored cancer and normal cells at different stages of the cell cycle.All the cell lines were harvested after 72 h incubation at 10 µM concentration of 1b/sh_7, 1c/sh_8 or 1d/sh_9, washed with phosphate-buffered saline (PBS), fixed in 70% ice cold ethanol, and stored at 4 °C for 24 h.Fixed cells were centrifuged, washed with PBS, stained with staining solution [20 µg/mL propidium iodide, 137 µg/mL RNAse A, and 0.1% Triton X-100 (Sigma-Aldrich Co.) in PBS] in the dark for 30 min, and measured with a flow cytometer (FACSCalibur, Becton Dickinson, San Diego, CA, USA).For each sample, a minimum of 15 × 10 3 cells was evaluated and analysed using FlowJo software (FLOWJO, LLC; Ashland, OR, USA).Cells characterized by DNA content lower than diploid (subG0/G1 population) were considered as apoptotic cells.

4. IncuCyte ZOOM System
Experiments were performed using an IncuCyte ZOOM system (Essen BioScience, Ann Arbor, MI, USA), which consists of a microscope gantry that resides in a humidified incubator at 37 °C and 5% CO 2 , and a networked external controller hard drive that gathers and processes image data.The cells were seeded in 96-well plates in sextuplicates at 5000 cells/well (as 100 µl cell suspension/well) and placed in the IncuCyte ZOOM system.After attachment (24 h), the cells were treated with 1b/sh_7, 1c/sh_8 a 1d/sh_9 test substances (dissolved in media).The cells growing in media without tested substances were used as controls.The IncuCyte zooM system automatically monitored the cell confluence in each well through a 4X objective (Nikon) every 2 h up to 130 h of the substance treatment.The experiment was performed two times.

5. Statistical Analysis
Data were analyzed using one-way ANOVA followed by Tukey's multiple comparison tests in ORIGIN analysis software (OriginLab Co., Northampton, MA, USA).

Results and Discussion
In the view of biological significance of tacrine and coumarin derivatives, potential cytotoxic and/or anti-cancer activities of tacrine-coumarin heterodimers 7a-g were tested using tumor as well as normal cell lines.In this re- gard, the responses of 4T1 (mouse mammary carcinoma), MCF-7 (human breast adenocarcinoma), HCT116 (human colorectal carcinoma), A549 (human lung carcinoma), NMuMG (normal mouse mammary gland cells), and HUVEC (human endothelial cells isolated from umbilical vein) were compared.Application of compounds 7a and 7e-g did not show toxic effects on either cancer or normal cells (not shown).Thus, only 7b-d compounds were included in further analysis.The concentrations of 7b-d hybrids used in both cell cycle as well as cell proliferation assays were derived based on the extensive screening of the cell metabolism and/or cell growth response (MTT).The concentrations of 7b-d derivatives inhibiting 50% (IC 50 ) of the metabolic activity and/or the cell growth are shown in supplementary data (Table S01).Based on IC 50 values, the synthesized compounds showed moderate to significant activity in the µM range from 5.7 to > 100 µM.The effect of chain lengths linking tacrine and coumarin skeleton was examined.Compounds 7d and 7c showed significant anti-metabolic activity against 4T1 cell lines, with IC 50 values of 5.7 µM and 7.0 µM, respectively (Fig. 3).The compound 7d also showed promising activity against MCF-7 cell line, with IC 50 value of 6.0 µM (Fig. 3).Furthermore, derivatives 7b and 7c showed stronger effect compared to 7d in the reduction of HUVEC cells metabolism (Fig. 3).Interestingly, based on MTT results, HCT, A549, and NMuMG cells did not respond to the effect of 7b-d compounds (Fig. 3).These results demonstrate that the length of the alkyl spacer influences, at least to some degree, the metabolic activity and/or the cell growth of breast cancer cell lines 4T1 and MCF-7.
Flow cytometric analysis was performed to evaluate the cell cycle progression in all monitored cell lines after their incubation with 7b-d derivatives at the concentration of 10 µM.Data showed that 7d hybrid had the most significant effect on the cell cycle progression (Fig. 4).This compound increased apoptotic sub G0/G1 population in 4T1, MCF-7, HCT, and HUVEC cells and reduced G1 population in 4T1 and MCF-7.On the other hand, all derivatives 7b-d increased G1 and decreased S population of HUVEC cells (Fig. 4).Without the change in the cell cycle progression stayed cancer A549 and normal NMuMG cells (Fig. 4).
The synthesized compounds 7b-d were also evaluated for their in vitro anti-proliferative activity monitored by IncuCyte system.The most sensitive tumor cell line was the MCF-7 (Supplementary data, Fig. S01), for which the decrease of proliferative activity depended on the increase of carbon number in the tested compounds 7b-d.HCT (Supplementary data, Fig. S02) and 4T1 (Supplementary data, Fig. S03) tumor cell lines showed lower sensitivity to the tested hybrids 7b-d with similar responses.On the other hand, A549 tumor cells (Supplementary data, Fig. S04) proved to be the most resistant, with proliferation not significantly different from other cell lines after the administration of the above-mentioned three tacrine-coumarin derivatives.In addition, HUVEC cells (Supplementary data, Fig. S05) responded only to the 7d derivative while the normal mouse mammary gland cells of NMuMG (Supplementary data, Fig. S06) did not demonstrate a decrease in proliferation after the application of the tested substances.
Among the synthesized compounds, the tacrine-coumarin heterodimer 7d with nine methylene groups between the two amino groups in the side chain exhibited the greatest efficiency.According to the results, in the case of tacrine-coumarin heterodimers 7e-g with longer side chains, replacing some methylene groups with amine moiety dramatically decreased the anti-cancer activity.
Emami et al. 1 have also confirmed that the anti-proliferative activity depends on the presence of the quinone skeleton.The structure-activity relationship of compound 4-(4-((4-fluorophenoxy)methyl)-1,2,3-triazol-1-yl)-7-methoxycoumarin 4 (Fig. 1) suggested that -C-4 position of 1,2,3-triazole core is the optimal position for CH 2 -Obridge because of its bioactivity.A hydrogen bond acceptor at C-4 position of phenyl is indispensable for the improvement of the potency while a hydrogen bond acceptor at C-7 position of coumarin can positively contribute to the activity. 6or bis-coumarin derivatives possessing one amino side chain 6 (Fig. 1), the length of the linker (dicarboxylic acid) had profound effects on their anti-proliferative activities.Compounds linked by the malonic amide (three carbons) showed the best anti-proliferative activities against MCF-7, A549, K562, and Hela cell lines; however, as the linker increased to five, six, or ten carbons, their anti-proliferative activities clearly decreased.In this regard, the incorporation of the aromatic or heterocyclic ring into the chain also decreased anti-proliferative activity. 2

Conclusions
In conclusion, structural modification of natural substances can yield new analogues, the biological efficacy of which can be higher compared to that of the parent compounds.In our experimental study, we found that tacrine-coumarin hybrids with seven, eight, and nine methylene groups in spacer have significant anti-cancer activity in all cancer cell lines tested, with the exception of A549 cells.Indeed, hybrid with nine methylene groups had the most significant anti-cancer effect.Interestingly, the sensitivity of tumor cells was greater compared to the sensitivity of normal cells, which points to the importance of further research on the anti-tumor effects of tacrine-coumarin hybrid molecules.

Conflict of Interest
The authors declare no conflict of interest.

Figure 3 .
Figure 3.The effect of 7b-d tacrine-coumarine derivatives on the metabolic activity of 4T1, MCF-7, HCT, A549, NMuMG, and HUVEC cells.The metabolic activity of 4T1, MCF-7, HCT, A549, NmuMG, and HUVEC cells was analyzed by MTT assay after their treatment with 7b-d derivatives for 72 h.Data are expressed as mean ± SEM (% of control) across three independent experiments.The statistical significance is designated as follows: * p < 0.05 and ** p < 0.01 for 7b derivative vs. control (C); x p < 0.05 and xx p < 0.01 for 7c derivative vs. C; and °°° p < 0.001 for 7d derivative vs. C.