Cytotoxic Activity, Apoptosis Induction and Cell Cycle Arrest in Human Breast Cancer (MCF7) Cells by a Novel Fluorinated Tetrahydro-[1,2,4]Triazolo[3,4-a]Isoquinolin Chalcones

Abstract A series of fluorinated chalcones (3a-f) was synthesized and confirmed by several spectral tools. The cytotoxic effect of this series was tested against a panel of different cancer cell lines (MCF7, A549, HCT116, and PC3). MTT assay revealed that chalcone 3f has the potent cytotoxic activity against all tested cancer cell lines except A549 cells. Chalcone 3f showed the least cytotoxic activity against the normal epithelial cell line RPE-1 and the lowest IC50 at 10.96 µM relative to the IC50 of doxorubicin at 12.8 µM against the human breast cancer cell line MCF7. Molecular docking studies showed a good interaction of chalcone 3f with the active site of histone demethylase (PLU-1/JARID1B) and Carboxy-terminal binding protein1 (CtBP1) proteins. Mechanistically, chalcone 3f induced cell cycle arrest at G2/M phase and apoptosis assessed by flow cytometry, as well as DNA fragmentation in MCF7 cells. Chalcone 3f upregulated mRNA expression levels of the apoptotic genes BAX, p53, and Caspase-7, Caspase-8, and Caspase-9, whereas mRNA expression levels of the antiapoptotic gene Bcl2, metastasis-related gene matrix metalloproteinase 1 (MMP1), and the autophagic markers ATG5 and LC3B were downregulated as quantified by qPCR. This study shows a cytotoxic effect of chalcone 3f against cancer cells and emerges as a promising therapeutic drug against breast cancer. Graphical Abstract


Introduction
Breast cancer is the second leading cause of cancer-related death among females worldwide with approximately 1.67 million new cancer cases (25% of all cancer cases). 1 Despite the great current efforts to control this devastating disease, its incidence is estimated to rise in the coming years. 2 Thus, innovative therapies are urgently needed for breast cancer. Chalcones (1, 3diphenyl-2-propene-1-one) are one of the important classes of anticancer agents that have a promising effect against breast cancer. 3 They are the precursors of flavonoids and isoflavonoids and are found abundantly in edible plants. 4 They show a wide variety of activities, including anticancer, 5 anti-inflammatory, 6 and antimicrobial 7 activities. Several modifications on the chalcone nucleus have been done including methoxy, hydroxyl, and amino groups as substituents with a promising anticancer activity. 8 Halogenated chalcones showed an enhanced anti-proliferative activity including arresting of the cell cycle, induction of apoptosis, and multidrug resistance (MDR)-reversal activity. 8,9 Among these halogens, fluorine is an important element in medicinal chemistry. The uniqueness of fluorine properties as an inorganic compound is due to the ability to modulate biological activity through enhancing absorption, distribution, metabolism, and elimination (ADME) properties. 10 The fluorine can affect lipophilicity (log P), hydrogen bonding and other binding interactions, modify the pKa of neighboring groups, and in many cases block pathways leading to rapid metabolization of the drug, so that increasing its bioavailability. 10 Apoptosis is a conserved intrinsic programmed cell death that occurs in both physiological and pathological conditions. 11 The major morphological and biochemical characteristics of apoptosis are cell shrinkage, nuclear DNA fragmentation, and membrane blebbing. Activation of caspases in response to anticancer chemotherapy can be initiated through activation of either the extrinsic (receptor) pathway and/or at the mitochondria by stimulating the intrinsic pathway of apoptosis. 11 Because of the potential detrimental effects of apoptosis on cell survival, the induction of apoptosis pathways are highly important in cancer cells. The above mentioned biological role of fluorine and chalcones stimulated us to synthesize a novel series of fluorinated chalcones, evaluate their cytotoxic activity against cancer cells, and study their effect on the apoptosis process in breast carcinoma MCF7 cells using several molecular techniques.

a] isoquinoline chalcones
The anticancer activity of the synthesized compounds was studied against breast, lung, colon, and prostate cancer cells, including MCF7, A549, HCT116, and PC3, respectively, and the normal human retinal epithelial cell line RPE-1. As shown in Table 1 and Figure 1, all tested compounds showed cytotoxic effects against all cancer cell lines used with an IC 50 ranging from 10.96 mM to 89.21 mM. Chalcone 3f displayed the lowest IC 50 against MCF7, HCT116, and PC3 cells at 10.96 mM, 24.25 mM, and 29.88 mM, respectively relative to the chemotherapeutic drug doxorubicin with IC 50 at 12.8 mM. Interestingly, chalcone 3f exhibited a cytotoxic effect against normal cell line RPE-1 with the highest IC 50 of 137.5 mM. The IC 50 of Chalcone 3e against the A549 cell line was 15.59 mM. The most potent activity of chalcone 3e and 3f against A549 and MCF7 cells was observed, respectively. This may be attributed to the presence of chloride and bromide substituents in 3e and 3f, respectively, and that is consistent with previous studies. 8,9 Therefore, we focus Scheme 1. Synthesis of fluorinated [1,2,4]triazolo [3,4-a]isoquinoline chalcones 3a-f. on the effect of chalcone 3f on MCF7 cells in the subsequent experiments. Our synthesized compounds showed the lowest activity against PC3 cell line. While in the literature, the (E)-1-(2-aminophenyl)-3-(4-nitrophenyl) prop-2-en-1-one had the lowest IC 50 (10.55 mmol/L), and hence the  most powerful activity, against the PC3 cell line. 24 It was reported that the chalcone containing dinitrobenzotriflouride showed comparable activity as chalcone 3f against MCF7 cell line. 25 Also Jin et al. synthesized chalcones containing pyrimidinyl group which showed cytotoxicity against various human cancer cell lines with the least IC 50 value confirmed by MTT assay. 26 Although our series is a congeneric series, the difference in one substituent among the series affects greatly the cytotoxic activity against the different cancer cell lines. It was reported that the usage of an electron releasing group enhanced cytotoxicity, as confirmed by MTT assay. 27 By applying this on our series, it was found that chalcones 3 b and 3c containing electron donating groups (methyl and methoxy substituent, respectively) showed higher cytotoxic effect than chalcone 3d which contain electron withdrawing group (nitro group). Chalcone 3f containing bromide substituent demonstrated lower IC 50 values than chalcone 3e containing chloride substituent against all the tested cancer cell lines except for A549 cell line. So, these structure activity relationship findings cannot be generalized on all cancer cell lines.

Molecular docking
Molecular docking is a computational tool which is used as an alternative to high-throughput random screening. Molecular docking facilitates the drug discovery through scanning a database of compounds for ligands which exploit some aspect of complementarity to the protein of interest. 28 So, this computational technique helped us to save our time and effort in selecting the most active compounds for further studies.
In this study, to understand the possible molecular interactions of chalcone 3f, the molecular simulations were done on two different proteins, histone demethylase enzyme (PLU-1/ JARID1B), which is a transcriptional repressor implicated in breast cancer, 29 and the transcriptional co-repressor Carboxy-terminal binding protein1 (CtBP1), which is antiapoptotic protein. 30 The PDB codes were obtained from the protein data bank which are 4LCE and 5FUP for CtBP1 and (PLU-1/JARID1B), respectively. Figures 2 and 3 showed the binding mode of interaction between the standard ligand and chalcone 3f, respectively with C-terminal binding protein 1. Chalcone 3f interacted with the active site of CtBP1 with a binding score S ¼ À20.28 Kcal/mol, which is more negative than the standard (S ¼ À5.1 Kcal/ mol), where there are two types of interactions, one hydrogen bond between oxygen of carbonyl group and Thr128 with bond distance 3.42 A o , arene-arene interaction between fluoro-benzene ring and Trp318. Figures 4 and 5 demonstrated the type of interactions of standard and chalcone 3f respectively with histone demethylase enzyme. Chalcone 3f interacted with the active site of histone demethylase enzyme with a binding score S ¼ À10.99 Kcal/mol comparing to the standard (S ¼ À9.5 Kcal/mol). It was obvious that chalcone 3f fits the active sites of the protein through two interactions; the first one was aren-cation interaction between benzene ring and Arg98, the second was arene-cation interaction between bromo-benzene ring and Arg98. Based on these findings, we can conclude that chalcone 3f may inhibit both tested proteins with a good energy score and hence may suggest a promising cytotoxic activity of chalcone 3f against cancer cells via inhibition of anti-apoptosis protein. One possible explanation for the observed higher activity of chalcone 3f is the halogen content evidenced by its interaction modes via the bromo and fluoro benzenes.    Chalcone 3f induces expression of apoptosis-related genes and downregulated antiapoptotic-, autophagic-and metastasis-related genes in MCF7 cells Since our molecular docking suggested a potential role for chalcone 3f in apoptosis, we quantified the expression of the mRNA level of key genes involved in apoptosis in MCF7 treated with chalcone 3f using qPCR. Cisplatin (IC 50 ¼ 393.3 mM) was used as a positive control and untreated MCF7 cells as a negative control. As shown in Figure 6, the expression mRNA levels of the proapoptotic gene BAX, caspase-7, caspase-8, caspase-9, and the tumor suppressor p53 were up-regulated in both chalcone 3fand cisplatin-treated MCF7 cells relative to the negative controls. On the other hand, the anti-apoptotic gene Bcl2 was down-regulated in chalcone 3fand cisplatintreated MCF7 cells relative to controls. The expression of matrix metalloproteinase 1 (MMP1), which is responsible for the metastasis and proliferation, was downregulated upon treatment with chalcone 3f and cisplatin compared to controls. The mRNA expression levels of ATG5 and LC3B, markers of autophagy (type II programmed cell death) were downregulated in chalcone 3fand cisplatin-treated MCF7 cells in comparison to controls. Together, this suggests that chalcone 3f evoked apoptosis-related genes, and suppressed autophagy-and metastsiss-related factors in MCF7 cells. This is consistent with different reports: licochalcone A and trans-chalcone treatment  induced the overexpression of BAX and downregulated the expression of Bcl2 gene in MCF7 cells. 31 The synthetic chalcone (E)-3-(3, 5-dimethoxyphenyl) 1-(2-hydroxy-5-methoxyphenyl)prop-2-en-1-one (2-hydroxy-3 0 ,5,5 0 -trimethoxychalcone stimulated the mitochondrial death pathway through upregulation of caspase-7 and caspase-9 in A549 lung carcinoma cells. 32 Two dihydrotriazine-chalcone derivatives exerted an inhibitory effect on the migration of invasive MDA-MB-231 cells via down-regulation of MMP9 expression. 33 In contrast, 3 0 ,5 0 -Diprenylated chalcone ((E)-1-(2-Hydroxy-4-methoxy-3,5-diprenyl)(phenyl-3-(3-pyridinyl)-propene-1-one)) induced autophagy in the human leukemia HEL and K562 cell lines through increasing the expression of LC3A/B, 34 this suggests that chalcone derivative have a cellcontext-dependent effect with different mode of actions.
Chalcone 3f increases cell cycle arrest at the G2/M phase in MCF-7 cells Next, we examined whether chalcone 3f had an impact on the cell cycle. We used two concentrations of chalcone 3f and cisplatin, IC 50 and IC 25 . As shown in Table 2 and Figure 7, after the 48 h of treatment with IC 50 , the percentage of cells in the G2/M phase was increased from 11.72% in the untreated control MCF7 cells to 34.117% in the chalcone 3f-treated cells. A lower percentage of chalcone 3f-treated cells were observed in the G0-G1 and S phases with 35.18% and 30.7% compared with the untreated control with 50.2% and 38.08%, respectively. Cisplatin as a positive control caused the cell cycle arrest at G2/M with 40.31%. Also, in MCF7 cells treated with IC 25 (5.48 mM), both compound 3f and cisplatin caused cell cycle arrest at G2/M phase relative to the negative control. This is in agreement with the action of other chalcone derivatives on the cell cycle. 35 It was found that quinazolinone-chalcone derivative ( Chalcone 3f induces apoptosis in MCF-7 cells Next, we examined if chalcone 3f induces apoptosis. Our flow cytometric analysis indicated that the IC 50 treatment of chalcone 3f increased the percentage of the Annexin V-positive early-phase apoptotic cells (8.85%) when compared to the untreated control cells (0.53%) ( Figure 8). The percentage of late-stage apoptotic cells was 23.4% relative to the control (0.25%). Cisplatin (IC 50 ¼ 393.3 mM) was included as a positive control and showed comparable results to those of chalcone 3f. It was also noticed that there was an increase in the percentage of necrotic cells in chalcone 3f-treated cells (11.39%) and cisplatin-treated cells (9.373%) relative to the untreated control MCF7 cells (0.9%). A comparable result was shown after the treatment with the IC 25 concentration (Table 3)  Chalcone 3f potentiates damage of DNA integrity in MCF7 cells DNA fragmentation is considered a late event that takes place in the apoptosis process. 38 Since we proved that chalcone 3f is an apoptosis inducer, we tested its impact on DNA fragmentation. Indeed, as shown in Table 4 and Figure 9, When MCF7 cells were treated with 10.96 mM of chalcone 3f for 48 h, the percentage of fragmented DNA was increased approximately by 6-fold (26.83%) in comparison to negative control MCF7 cells (4.56%). The effect of chalcone 3f was comparable to that of cisplatin as a positive control (31.61%). This is consistent with the apoptosis results. A previous report indicated that the novel derivatives of chalcone, imidazolone     (E)-1-(8,9-Dimethoxy-1-phenyl-1,5,6,10b-tetrahydro- [1,2,4] (E)-1-(1-(4-Bromophenyl)-8,9-dimethoxy-1,5,6,10b-tetrahydro- [1,2,4]

MTT cytotoxicity assay
Cell viability was assessed using MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] (Bio Basic Canada Inc., Canada). Briefly, cells were seeded into a 96-well plate at the concentration of 10 4 cells/well and allowed to adhere for 24 h. Different concentrations of the chemical compounds tested at 100, 50, 25 and 12.5 mM were added to the cell monolayer in triplicate and incubated for 48 h. Subsequently, the medium was aspirated and fresh media with 40 ll MTT (2.5 lg/ml) was added to each well and incubated for a further 4 h. Then, 200 ll of 10% sodium dodecyl sulfate (SDS) was added to each well and incubated overnight at 37 C to stop the reaction and dissolve the formed formazan crystals. The amount of formazan product was measured at a wavelength of 595 nm with a reference wavelength of 620 nm using a microplate reader (Bio-Rad Laboratories, model 3350, USA). AdrinamycinV R (doxorubicin) was used as a positive control. Dimethyl sulfoxide (DMSO) was the vehicle used for dissolving all chalcone compounds whereby, its final concentration was less than 0.2%. IC 50 was evaluated using the Prism software program (Graph Pad software incorporated, version 3).

Molecular docking
The molecular docking studies were performed using the ''Molecular Operating Environment (MOE) version 2009.10 release of Chemical Computing Group's''. The target compound was drawn using the MOE builder interface and subjected to energy minimization using the included MOPAC. The resulted model was subjected to Systematic Conformational Search where all items were set as default with RMS gradient of 0.01 kcal/mole and RMS distance of 0.1 A o . The X-ray crystallographic structure of the C-terminal binding protein (CtBP) 1 and histone demethylase (PLU-1/JARID1B) complexed with their ligands (PDB ID: 4LCE and 5FUP, respectively) were obtained from the protein data bank. The proteins were prepared for molecular simulations where: Firstly, the hydrogen atoms were added to the target protein with their standard geometry. Then, the water chains and unwanted co-ligands were removed from the protein active site. The active site was determined by MOE alpha site finder and dummy atoms were made from the resulted alpha spheres. Finally, after the self-docking of the prepared protein with the co-crystallized ligand, it was then docked with the target compound to predict the ligand-protein interactions at the active site. The final results obtained in 2 D form, which were then visualized through BIOVIA Discovery Studio V6.1.0.15350 in the 3 D form.
Cell cycle assay DNA content was quantitatively assessed using propidium iodide (ab139418, Abcam, Cambridge, UK) and Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL). Firstly, cells were treated with chalcone 3f at IC 50, IC 25 and incubated for 48 h. Subsequently, cells were collected in a single cell suspension and fixed in 70% ethanol on ice. Then, the cells were centrifuged at 500 xg for 5 minutes, the pellet was washed by 1 ml 1x phosphate-buffered saline (PBS) and the supernatant was discarded. Afterward, the cells were centrifuged, the cell pellet was re-suspended gently in 200 ml 1X propidium iodide and incubated in dark at 37 C for 20-30 minutes. Finally, the cells were analyzed using a flow cytometer. The cell cycle profile was analyzed using MultiCycle software (Phoenix Flow Systems, San Diego, CA).

Apoptosis assay
Apoptosis was analyzed using the Annexin V-FITC kit (catalog number # 4830-01 K, R&D Systems, Minneapolis, MN, USA). MCF7 cell line was treated with chalcone 3f at IC 50 value (10.65 mM) and IC 25 (5.325 mM) for 48 h. The cells were centrifuged at approximately 300 xg at room temperature for 10 min. Cells were washed in 500 ll cold 1X PBS buffer and then were collected by centrifugation as mentioned before. Then, the cells were gently resuspended in the annexin V incubation reagent comprising of 10 lL binding buffer (10X) þ 10 lL propidium iodide þ 1 lL annexin V-FITC þ 79 lL deionized water. A total of 10 5 -10 6 cells in 100 lL annexin V incubation reagent was incubated in dark at room temperature for 15 min. Finally, 400 ll of 1X binding buffer was added and samples were acquired on flow cytometry.

DNA fragmentation assay
The percentage of fragmented DNA was determined using Diphenylamine (DPA) assay. The monolayer cell cultures were harvested directly into centrifuge tubes and centrifuged at 300 xg at 4 C for 10 min. Then, the cell pellet was resuspended in 0.8 mL of 10 mM PBS, pH 7.4, and 0.7 mL of ice-cold lysis buffer ((5 mM Tris-HCl, 20 mM EDTA, pH 8.0, 0.5% (v/v) Triton X-100. Afterward, the cell lysate was transferred to microfuge tubes and incubated on ice for 15 min. The lysate was centrifuged at 13,000 xg at 4 C for 15 min to separate fragmented DNA from high-molecular-weight DNA. The entire supernatant (about 1.5 ml containing fragmented DNA) was transferred to a 5 ml glass tube. The pellet containing intact DNA was resuspended in 1.5 ml TE buffer (1 mM EDTA, 10 mM Tris, pH 8.0), and again transferred to another 5 ml glass tube. 1.5 mL of 10% Trichloroacetic acid (TCA) was added to each tube and incubated for 10 min at room temperature. The tubes were centrifuged at 500 xg at 4 C for 15 min and the supernatant was discarded. The 10% TCA precipitates were resuspended in 0.7 ml of 5% TCA and boiled at 100 C for 15 min, then cooled to room temperature, and centrifuged at 300 xg at 4 C for 15 min. 0.5 mL of the supernatant was transferred without disturbing the precipitate to a new glass tube. 1 mL of DPA was added and incubated overnight at 30 C. The absorbance was measured at 600 nm. The percentage of DNA fragmentation was calculated using the following equation: % fragmented DNA ¼ OD 600 of the supernatant/[OD 600 of the supernatant þ OD 600 of the pellet] Â 100.

Statistical analysis
The data are demonstrated as the mean ± SD of replicates from three independent experiments. The statistical significance was determined by one-way analysis of variance (ANOVA) using Graphpad prism, 7.0 software. The values were considered statistically significant when p < 0.05.

Conclusion
In conclusion, in this study, a novel series of chalcone derivatives (3)(4)(5)(6)(7)(8) was synthesized and evaluated as potent anticancer agents against a set of cancer cell lines (MCF7, A549, HCT116, and PC3). The most active compound chalcone 3f inhibited proliferation of MCF7 cell line with better potency compared to doxorubicin and exhibited the least cytotoxicity against the normal epithelial cell line RPE-1. Molecular docking showed the inhibitory activity of chalcone 3f against histone demethylase (PLU-1/JARID1B) and Carboxy-terminal binding pro-tein1 (CtBP1) proteins. Mechanistically, we proved experimentally that chalcone 3f arrested the cell cycle at the G2/M phase and induced apoptosis and DNA fragmentation in MCF7 cells. Furthermore, chalcone 3f upregulated the mRNA expression levels of BAX, p53, and caspase-7, caspase-8, and caspase-9, whereas Bcl2, the metastasis-related factor MMP1, and the autophagic markers ATG5 and LC3B mRNA levels were downregulated. Overall, this study suggests that chalcone 3f may have the potential to serve as a promising therapeutic drug for breast cancer.