Antiproliferative activity and p53 upregulation effects of chalcones on human breast cancer cells

Abstract Chalcones are valuable structures for drug discovery due to their broad bioactivity spectrum. In this study, we evaluated 20 synthetic chalcones against estrogen-receptor-positive breast cancer cells (MCF-7 line) and triple-negative breast cancer (TNBC) cells (MDA-MB-231 line). Antiproliferative screening by MTT assay resulted in two most active compounds: 2-fluoro-4’-aminochalcone (11) and 3-pyridyl-4’-aminochalcone (17). Their IC50 values ranged from 13.2 to 34.7 µM against both cell lines. Selected chalcones are weak basic compounds and maintained their antiproliferative activity under acidosis conditions (pH 6.7), indicating their resistance to ion-trapping effect. The mode of breast cancer cells death was investigated and chalcones 11 and 17 were able to induce apoptosis rather than necrosis in both lines. Antiproliferative target investigations with MCF-7 cells suggested 11 and 17 upregulated p53 protein expression and did not affect Sp1 protein expression. Future studies on chalcones 11 and 17 can define their in vivo therapeutic potential.


Introduction
Breast cancer is the most common type of cancer that affects women around the world, corresponding to 25% of cases. It is also the main cause of cancer death among women [1][2][3] . Its classification is based on the presence of cellular receptors: (i) Hormone-Receptor (HR), with Estrogen (ER) and/or Progesterone-Receptors (PR); (ii) Human Epidermal Growth Factor-2 receptor (HER2); and (iii) Triple-Negative Breast Cancer (TNBC), which does not express ER, PR, or HER2 receptors [4][5][6] . Chemotherapy choices for breast cancer are made according to its classification aiming to reach specific targets. ER-positive cancer treatments include ER modulators, such as tamoxifen or aromatase inhibitors. HER2-positive cancer is treated with monoclonal antibodies, such as trastuzumab, which is administered alongside tyrosine-kinase inhibitors [7][8][9] . TNBC cancer treatment is focused on cytotoxic agents, such as taxanes or doxorubicin. This cancer has poor response to chemotherapy, particularly at metastatic sites, with survival rates below 2 years. Altogether, TNBC cancer has been considered the most severe and difficult to treat due to a lack of targeted therapy 10-12 . Chalcones are valuable structures for drug discovery due to their broad bioactivity spectrum, as well as their versatile and simple synthesis. Their structures, bearing two benzene rings (A and B), are linked by an enone bridge. They have demonstrated antiproliferative activity against cancer cells, including breast cancer [13][14][15] . Mai et al. described chalcone was effective against ERpositive breast cancer cells (MCF-7 line) targeting 20 apoptotic markers 16 . Iftikhar et al. reported that chalcone bearing chlorine at position 2 was effective against breast cancer cells (CAL-51 line) and induced accumulation of p53 protein 17 . Silva and coauthors described unsubstituted chalcone increased p53 protein activity in osteosarcoma cells (U2OS line) through the induction of heat shock protein DNAJB1 18 .
In our ongoing search for anticancer compounds with structures based on chalcone framework, we evaluated the antiproliferative activity of 20 chalcones against ER-positive cells (MCF-7 line) and TNBC (MDA-MB-231 line). The two most active chalcones (11 and 17) were selected to antiproliferative evaluation under acidosis conditions (pH 6.7) and pro-apoptotic activity in MCF-7 and MDA-MB-231 lines. In addition, we investigated tumour molecular targets of 11 and 17 in MCF-7 line, which were able to upregulate p53 protein expression.

Materials and methods
Chemical procedure for synthesis of chalcones 1-20 Reagents and solvents were purchased from Merck V R (Kenilworth, NJ). Series of 20 chalcones was synthesised by Claisen-Schmidt aldol condensation reaction, according to protocol described by Santos and coauthors, with minor modifications 19,20 . Reactions were carried out at room temperature using 3.0 mmol of 4'-aminoacetophenone and 3.0 mmol of aldehydes, which were dissolved in ethanol (30 mL). Sodium hydroxide in ethanol (1.0 mol/L) was added as catalyst solution. Reagents conversion was monitored using thin layer chromatography. Crude product was poured onto ice (from distilled and deionised water) and filtered. All compounds were purified over silica gel chromatography column eluted with mixture of hexane and ethyl acetate (3:2). Melting points were determined in TecnoponPFM-II V R apparatus (MS Tecnopon Instrumentação, Piracicaba, Brazil) and were uncorrected. Structure of compounds was confirmed by 1 H and 13 C nuclear magnetic resonance (NMR) spectra analyses. Spectral data were obtained in Bruker Avance III V R (14 Tesla, 600 MHz) equipment (Bruker Corporation, Billerica, MA) using deuterated dimethyl sulfoxide (DMSO-d 6 ) as solvent. Chalcones had their UV-vis spectra and chromatograms obtained in High Performance Liquid Chromatography with Diode Array Detector (HPLC-DAD) Agilent Technologies V R 1220 Infinity equipment (Agilent Technologies, Palo Alto, CA) coupled with a photodiode array system (1260-Infinity V R ) and Agilent Zorbax Eclipse Plus C-18 V R column (250 mm Â 4.6 mm, 5 lm), using methanol:water (3:1) as mobile phase (1.0 mL/min).

Antiproliferative activity of selected chalcones 11 and 17 under acidosis
Cell viability was evaluated under acidosis conditions by MTT assay, using 4-morpholine-ethanesulfonic acid to produce a pH value of 6.7 24,25 . Cells were treated with selected chalcones 11 and 17 at their respective IC 50 values. All procedures were performed in triplicate and three independent experiments.

Pro-apoptotic activity of selected chalcones 11 and 17
Pro-apoptotic activity of selected chalcones 11 and 17 was evaluated using FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen V R , BD Biosciences, Franklin Lakes, NJ) by flow cytometry 26 . MCF-7 and MDA-MB-231 cells were seeded and cultivated in 6-well plates (6.5 Â 10 5 cells/well), for 24 h, and treated with 40 lM selected chalcones in DMEM containing 10% FBS for 48 h. As negative control 0.1% DMSO was used. Adherent and floating cells were collected, submitted to centrifugation, and washed with PBS (Sigma Aldrich V R , St. Louis, MO). Apoptotic cells were determined by flow cytometry in BD FACSCalibur V R Flow Cytometer (BD Biosciences V R Franklin Lakes, NJ), after double staining (Annexin V/ propidium iodide) using FITC Annexin V Apoptosis Detection kit I (BD Pharmigen V R , BD Biosciences, Franklin Lakes, NJ) according to instructions of the manufacturer. All procedures were performed in triplicate and in three independent experiments.

Effect of selected chalcones 11 and 17 on Sp1 and p53 proteins expressions
Protein expression was assessed using western blot assay. Breast cancer cells (MCF-7) were seeded in 6-well plates at 7.5 Â 10 5 cells/well, treated with selected chalcones 11 and 17 at 10 and 20 lM or with negative control (0.1% DMSO) for 24 h 26 . Cells were suspended in PBS solution, collected in Radioimmunoprecipitation Assay buffer -(RIPA buffer at 4 C), supplemented with proteinase inhibitors, sonicated for lysis, and submitted to centrifugation (14,000 rpm, 4 C, 20 min, Eppendorf V R , Hamburg, Germany). Protein total concentration was obtained using Pierce TM BCA Protein Assay reagent (Thermo Scientific V R , Waltham, MA). Total proteins (30 lg) in sodium dodecyl sulphate (SDS) buffer were warmed at 95 C, cooled at 4 C and submitted to sodium dodecyl sulphate-polyacrylamide gel electrophoresis SDS-PAGE (12%, 100 V, 360 mA) for 90 min at room temperature, and were transferred to nitrocellulose membrane (PALL Corporation V R , Port Washington, NY). Membranes were blocked using TBST buffer (25 mM Tris, 3 mM KCl, 0.14 M NaCl, 0.05% Tween 20) containing 5% nonfat milk at room temperature for 1 h, followed by overnight incubation at 4 C, with primary antibodies to Sp1 (MilliPore V R , Burlington, MA), p53, and b-actin (Santa Cruz Biotechnology V R , Dallas, TX). Subsequently, membranes were washed with TBST (3Â) and incubated with peroxidase-conjugated secondary antibodies diluted in TBST buffer-5% nonfat milk (1:5000) at room temperature and washed several times with TBST buffer. Proteins were detected through chemiluminescence using Enhanced Chemiluminescent (ECL V R ) western blotting detection reagent (Amersham Biosciences V R , Little Chalfont, UK) in LAS 500 luminescence analyser (GE Healthcare V R , Chicago, IL) 26 . Bands intensity in image was quantified using ImageJ V R software (NIH Image J system, Bethesda, MD).

Statistical analysis
Statistical analyses were carried out using one-way ANOVA multiple comparison tests followed by Tukey's HSD test 19,27 . Statistical significance difference was considered if p< 0.05.

Results and discussion
Chemistry Chalcones 1-20 were synthesised by Claisen-Schmidt reaction with yields of 30-93% after chromatography purification. Series of compounds has chalcones with amino at position 4' (ring A) and benzene ring B substituted by electron-withdrawing (EWG) and electron-donating groups (EDG). Also, aryl analogues with benzene ring B replaced for furan, thiophene, pyridine, and naphthyl rings were synthesised (Scheme 1).
All NMR parameters, including hydrogen and carbon chemical shifts (d H and d C in ppm), integrations, multiplicities, and coupling constants (J in Hz), corresponded to proposed structures of chalcones 1-20. Two main signals in 1 H NMR spectra were diagnosed: (i) a pair of doublets with J ranging from 15 to 16 Hz (7.43-8.10 ppm), attributed to methyne hydrogens of trans carbon-carbon double bonds and (ii) broad singlets between 6.18 and 6.27 ppm related to geminal hydrogens of amino group. In 13 C NMR spectra, signals in 185.9-186.1 confirmed ketone carbonyls, which are shifted to downfield due to conjugation with carbon-carbon double bonds. UV-Vis had k max at 298-378 nm, which were related to a pivotal chromophore of chalcone framework (conjugation between ring B and enone bridge). HPLC-DAD analyses of peak areas integrations indicated chalcones purity of 94.0-99.9%. All chromatograms and spectra are presented in supplemental data (Figures S1-S62).  Table 1. Series of compounds demonstrated active (e.g. 20) and inactive (e.g. 3) chalcones, suggesting molecular variations were relevant for antiproliferative screening against both cell lines. Experiments at 20 lM allowed us to derive preliminary structural features related to antiproliferative activity [28][29][30] .
The five most antiproliferative compounds against MCF-7 were 9, 11, 17, 19, and 20 (%MVC values lower than 60). Against MDA-MB-231, the five most potent ones were 1, 6, 8, 11, and 17 (%MVC values lower than 85). These seven compounds were selected to determine IC 50 values for both cell lines (Table 1) 24,32 . This extracellular acidosis has been related to Warburg effect, in which tumour cells are in intense anaerobic metabolism, producing and exporting acid compounds through transporters. The biological central interest in acidosis is due to clear relationship with invasiveness and metastasis potential 24,33 . Tumour cells in acidic extracellular microenvironment can block membrane permeation of weak basic compounds, leading to iontrapping effect. This phenomenon is caused by protonation of basic functionalities, converting neutral into cationic compounds. Cationic form exhibits reduced passive permeation through membrane phospholipids when compared to neutral form. Doxorubicin is antineoplastic and weak basic drug and has demonstrated low efficacy in acidosis microenvironment due to iontrapping effect 34 .
Chalcones 11 and 17 were submitted to antiproliferative assays under acid microenvironment (at pH 6.7) against both breast cancer cell lines (Figure 1(a,b). In experiments with MCF-7 line, both chalcones maintained their antiproliferative effect, showing iontrapping resistance. In MDA-MB-231 line, chalcone 17 maintained similar %MVC rate at both pH values. Interestingly, chalcone 11 was five times more active at pH 6.7 (acidosis) than at 7.4, with %MVC of 14.4 and 73.7, respectively.

Pro-apoptotic activity of selected chalcones 11 and 17
Antiproliferative compounds can induce tumour cell death by several mechanisms 35 . Among these, apoptosis and necrosis are the most common and studied ones. Apoptosis events have been induced by chalcones towards different cancer cell lines 36,37 . In order to investigate antiproliferative effect mechanism of selected chalcones 11 and 17 towards MCF-7 and MDA-MB-231 lines, Annexin V/PI staining and flow cytometry were used to detect whether cells were dying due to apoptosis or necrosis events ( Figure 2).
Chalcones are known to induce apoptosis in several cancer cells, being able to up regulate more than 15 pro-apoptotic markers expression, such as Bad, Bax, Bid, Bim, CD40, Fas, IGFBP-5, IGFBP-6, p21, and sTNF-R116. Hsu  Sp1 protein is transcription factor involved in cell proliferation and differentiation. In breast cancers, it acts on invasion and metastasis processes 41,42 , and has been classified as marker for poor prognosis 43 . Chalcones 11 and 17 were not able to modulate Sp1 protein expression, at either concentration, presenting similar effect to DMSO 0.1% (negative control).
p53 protein is tumour suppressor and its mutated status has been related to several types of cancers. Expression of p53 in breast cancer cells varies according to its classification 44,45 . ER-positive and ER-negative cancer types have wild-type p53 and mutated protein forms, respectively 46 . Activation and stabilisation of wild-type p53 induce cell-cycle arrest and cell death through apoptosis, reducing cancer progression. This cell pathway has been recognised as attractive target to simple and low-weight-molecular compounds with promising antineoplastic potential. Chalcones 11 and 17 induced 5-fold upregulation of p53 expression in MCF-7 cells (Figure 3), indicating these compounds are able to activate and stabilise p53 protein expression. This result is the first evaluation of low-molecular-weight compounds against breast cancer cells. In this context, methoxychalcones and naphthylchalcones have been described as agents of p53 activation and stabilisation in prostate cancer and osteosarcoma cell lines, respectively 26,47 .

Conclusions
In summary, we reported activity of series of 20 chalcones against two types of breast cancer cells, ER-positive (MCF-7 line) and TNBC (MDA-MB-231 line). Preliminary investigations suggested halogens on ring B and additional benzene rings play central role in antiproliferative activity. Basic chalcones 11 and 17 were antiproliferative agents under acidosis (at pH 6.7), displaying resistance to iontrapping effect. These compounds induced apoptosis rather than necrosis in both cells, upregulating p53 expression in ER-positive cells (MCF-7 line).

Disclosure statement
No potential conflict of interest was reported by authors.