- Email: [email protected]
Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest
G Model BIOPHA 4556 No. of Pages 9
Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest Luis Felipe Buso Bortolottoa , Flávia Regina Barbosaa , Gabriel Silvaa,b , Tamires Aparecida Bitencourta , Rene Oliveira Belebonia , Seung Joon Baekb , Mozart Marinsa , Ana Lúcia Fachina,* a b
Biotechnology Unit, Ribeirão Preto University, SP, Brazil Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA
A R T I C L E I N F O
Article history: Received 29 August 2016 Received in revised form 4 November 2016 Accepted 9 November 2016 Keywords: Apoptosis CIDEA Comet assay Licochalcone A MCF-7 Trans-chalcone PCR Array
A B S T R A C T
Chalcones are precursors of flavonoids that exhibit structural heterogeneity and potential antitumor activity. The objective of this study was to characterize the cytotoxicity of trans-chalcone and licochalcone A (LicoA1 ) against a breast cancer cell line (MCF-7) and normal murine fibroblasts (3T3). Also the mechanisms of the anti-cancer activity of these two compounds were studied. The alkaline comet assay revealed dose-dependent genotoxicity, which was more responsive against the tumor cell line, compared to the 3T3 mouse fibroblast cell line. Flow cytometry showed that the two chalcones caused the cell cycle arrest in the G1 phase and induced apoptosis in MCF-7 cells. Using PCR Array, we found that trans-chalcone and LicoA trigger apoptosis mediated by the intrinsic pathway as demonstrated by the inhibition of Bcl-2 and induction of Bax. In western blot assay, the two chalcones reduced the expression of cell death-related proteins such as Bcl-2 and cyclin D1 and promoted the cleavage of PARP. However, only trans-chalcone induced the expression of the CIDEA gene and protein in these two experiments. Furthermore, transient transfections of MCF-7 using a construction of a promoter-luciferase vector showed that trans-chalcone induced the expression of the CIDEA promoter activity in 24 and 48 h. In conclusion, the results showed that trans-chalcone promoted high induction of the CIDEA promoter gene and protein, which is related to DNA fragmentation during apoptosis. ã 2016 Published by Elsevier Masson SAS.
1. Introduction Despite advances in cancer research, breast cancer is a leading cause of death among women. Surgery, radiotherapy and chemotherapy administered separately or in combination are the most common treatments for breast cancer, but are not always effective and may cause undesired side effects [1]. Doxorubicin is a chemotherapeutic agent commonly used for the treatment of breast cancer, but has limitations because of its cardiotoxicity [2] and selection of resistant tumors. Resistance to cytotoxic drugs can be caused by the reduced expression of tumor suppressor genes and by the overexpression of proto-oncogenes [3,4]. The resistance in tumor cells may prevent apoptosis by altering the modulation of essential genes involved in programmed cell death pathways, such
* Correspondence to: Unidade de Biotecnologia, Universidade de Ribeirão Preto. Av: Costábile Romano 2201, 14096-900, Ribeirão Preto, SP, Brasil. E-mail address: [email protected] (A.L. Fachin). 1 LicoA = licochalcone A.
as those of the Bcl-2 family [5]. In this regard, there is a continuous search for alternatives of cytotoxic compounds that exhibit a better therapeutic response than the classical chemotherapeutic agents. Some examples of alternative molecules under research are the chalcones, which are open-chain polyphenols that consist of two aromatic rings joined by one a, b-unsaturated propenone. These compounds occur naturally as precursors of flavonoids and possess anti-microbial, anti-oxidant and anti-inflammatory properties, among others [6]. There are numerous reports of natural and synthetic chalcones with potent anti-tumor activity against different cell lines [7], resulting from the activation of apoptotic pathways and anti-proliferative action [6–8,12,14–16]. In the present study, trans-chalcone and licochalcone A (LicoA) were found to be cytotoxic and considerably genotoxic, caused cell cycle arrest, induced apoptosis, and modulated apoptotic genes through the inhibition of Bcl-2 and induction of Bax in the breast cancer cell line MCF-7. Western blot assay confirmed the two chalcones reduced the expression of cell death-related proteins such as Bcl-2 and cyclin D1 and promoted the cleavage of PARP at
http://dx.doi.org/10.1016/j.biopha.2016.11.047 0753-3322/ã 2016 Published by Elsevier Masson SAS.
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
2
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
48 h. However, only trans-chalcone induced the expression of the CIDEA gene and protein, which is involved in the apoptotic mechanism in the MCF-7 cell.
Bio-Tek Instruments, Inc.) at 490 nm. LDH leakage was calculated according to Grosse et al. [9]. The experiments were performed three times in triplicate.
2. Materials and methods
2.5. Comet assay
2.1. Materials
The alkaline comet assay was used to determine the genotoxic potential of the chalcones against MCF-7 and 3T3 cells. Briefly, 3 105 cells/well were seeded in 6-well plates and incubated for 24 h. The cells were then treated with the lowest concentrations of the chalcones used in the MTT assay (15, 10, and 5 mg/mL) for 6 h. The treated cells were fixed on slides and submitted to lysis and electrophoresis, followed by neutralization, according to Liao et al. [10]. The slides were stained with 20 mg/mL ethidium bromide per slide. On fluorescence microscopy, 100 cells/slide were randomly selected and scored visually into five classes of DNA damage according to Cavalcanti et al. [11]. The DNA damage index (DDI) was calculated using the following formula: DDI = (0 n0) + (1 n1) + (2 n2) + (3 n3) + (4 n4). The experiments were performed three times in duplicate.
The two tested compounds, trans-chalcone and LicoA, and some of the main reagents including MTT (4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), trypsin, doxorubicin hydrochloride, dimethyl sulfoxide (DMSO), DMEM, penicillin, kanamycin, streptomycin and LDH assay TOX7 were purchased from SigmaAldrich (St. Louis, MO, USA). Fetal bovine serum was obtained from Cultilab (Campinas, SP, Brazil), and Triton1 X-100 was purchased from USB (Cleveland, OH, USA). The flow cytometry kits Cycletest Plus DNA Reagent and FITC Annexin V Apoptosis Detection I were purchased from BD Biosciences (San Jose, CA, USA). The materials used for gene expression were RNeasy1 Mini Kit, RNase-free DNase Set, RT2 SYBR1 Green ROXTM qPCR Mastermix, RT2 First Strand Kit, and RT2 ProfilerTM PCR Array PAHS-012Z (Qiagen, Germany). The BCA protein assay was purchased from Thermo Scientific (Rockford, IL, USA), jetPRIME from Polyplus-transfection (New York, NY, USA), and the 1X passive lysis buffer and DualGlo Luciferase Assay Kit from Promega (Madison, WI, USA). 2.2. Cell culture and assay conditions The cell lines 3T3 (mouse fibroblasts) and MCF-7 [human breast cancer (estrogen receptor (ER)-positive)] were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL kanamycin and 100 mg/mL streptomycin and maintained in a humidified 5% CO2 atmosphere at 37 C. Doxorubicin was used at 2.5 mg/mL (4.31 mM) as the positive control for all the tests, except for the comet assay in which a concentration of doxorubicin of 0.8 mg/mL (1.38 mM) was used. Culture medium containing 0.25% DMSO was used as the solvent control. 2.3. Growth inhibition The inhibition of cell viability by the two chalcones was evaluated by the MTT assay. Each cell line (MCF-7 and 3T3) was plated at a density of 3 104 cells/well in 96-well plates and incubated for 24 h. The cells were then treated with 5 different concentrations of the chalcones (25, 20, 15, 10, and 5 mg/mL) for 24 and 48 h. Next, 20 mL MTT solution (5 mg/mL) was added per well, and the plates were incubated for 4 h. The plates were centrifuged at 1800 g for 5 min, the culture medium was removed, and formazan dye was solubilized in 200 mL DMSO. Absorbance was read in a Thermoplate TP-Reader at 550 nm. The experiment was repeated at least three times in triplicate. The percent inhibition of cell viability was determined by the cell death formula of Szliszka et al. [8]. The estimated averages were used to calculate the IC20, IC50 and IC80 values by nonlinear regression. 2.4. Lactate dehydrogenase assay For the determination of lactate dehydrogenase (LDH) leakage, 3 104 MCF-7 cells/well were incubated in 96-well plates for 24 h and then treated with IC50 and IC80 (48-h MTT) of the chalcones for 24 and 48 h. Triton X-100 (1%, USB) was used as control. Next, 50 mL/well of the cell-free supernatant was collected and mixed with 100 mL/well reagent mixture (TOX7 Kit from Sigma-Aldrich) according to manufacturer instructions. The plates were covered with opaque material and incubated at room temperature for 30 min. Absorbance was read in a microplate reader (ELx 800 UV
2.6. Flow cytometry Cell cycle and apoptosis were analyzed in a FACSCanto II flow cytometer (BD Biosciences). Twenty-four well plates with 1 105 cells/well were used in both experiments. The tests were done using the IC50 of the chalcones determined by the 24-h MTT test in the MCF-7 cell line. 2.6.1. Apoptosis analysis using annexin V-FITC/PI Apoptosis was evaluated using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) according to manufacturer recommendations, with slight modifications. After incubation for 24 h, cells were treated with the inhibitory concentrations (IC50 and IC20) of the chalcones for 24 h. After this period, the cells were trypsinized, centrifuged (201 g for 5 min at 8 C), washed twice with cold 1X PBS, and resuspended in 500 mL of 1X binding buffer. Next, 5 mL PI and 5 mL annexin V-FITC were added and the tubes were incubated at room temperature in the dark for 15 min before analysis in the flow cytometer. The experiments were performed three times in duplicate treatments. 2.6.2. Cell cycle analysis After incubation for 24 h, the treated cells with IC50 and IC20 (24-h MTT) were collected, centrifuged, and washed as described above for apoptosis analysis. The supernatant was decanted and the cells were stained with the CycletestTM DNA Reagent Kit (BD Biosciences) according to the protocol of the manufacturer. The test was performed once in triplicate and the results were analyzed using the ModFit LT program (BD Biosciences). 2.7. RNA extraction, cDNA conversion and RT2 Profiler PCR Array The real-time PCR Array was used to screen for apoptotic genes related to chalcone-induced cell death. This quantitative real-time PCR Array permits to analyze the expression of 84 apoptosisrelated genes. First, MCF-7 cells were seeded at a density of 2.5 106 in 25-cm2 flasks and incubated for 24 h. Next, the cells were treated with the IC50 (24-h MTT) of the chalcones for 6 and 24 h. Total RNA was extracted using the RNeasy1 Mini Kit and RNase-free DNase Set (Qiagen). The integrity of the samples was assessed in a 2100 Bioanalyzer (Agilent) (RIN 8). Next, 500 ng of each RNA sample were converted into cDNA using the RT2 First Strand Kit (Qiagen). Each cDNA sample was mixed with RT2 SYBR1 Green ROXTM qPCR Mastermix (Qiagen) and a 25-mL aliquot of the mixture (cDNA and Mastermix) was added per well of the RT2
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
ProfilerTM PCR Array PAHS-012Z plates (Qiagen). The SYBR1 Green program (with dissociation curve) was executed on the Mx3005P1 thermocycler (Agilent Stratagene) (95 C for 10 min, 40 cycles of 95 C for 15 s and 60 C for 1 min, followed by 1 cycle of 95 C for 1 min, 55 C for 30 s and 95 C for 30 s). Fold regulation values were calculated from the qRT-PCR data with the RT2 Profiler PCR Array Data Analysis v. 3.5 tool (SABiosciences) using ACTB (b-actin) as the control gene.
3
2.11. Statistical analysis All experimental data were submitted to analysis of variance (ANOVA) followed by the Scott-Knott test using the Sisvar software, except for the comet assay, qPCR and luciferase assay data. The level of statistical significance was set at p < 0.05. The comet assay data are expressed as the mean standard deviation. The luciferase assay data were analyzed by the unpaired Student ttest, with statistical significance of p < 0.05, p < 0.01 and p < 0.001.
2.8. Western blot analysis 3. Results MCF-7 were grown to 80% confluence in 60-mm dishes and treated with 0, 20, 40 or 80 mM of trans-chalcone and LicoA in serum-containing media for 24 or 48 h. Briefly, cells were washed with PBS, harvested in RIPA buffer supplemented with protease (1 mM PMSF, 0.15 mM aprotinin, 2.3 mM leupeptin) and phosphatase (0.1 nM Na3VO4 and 25 mM NaF) inhibitors, and subjected to sonication. The protein concentration in the cell lysate was determined by the BCA protein assay (Thermo Scientific). Total protein (50 mg) was subjected to Western blot analysis as described previously by Silva et al. [12]. The image quantification was carried out using the ImageJ program and the b-actin bands were used for normalization. 2.9. Construction of a promoter-luciferase vector The promoter of the human CIDEA gene was cloned into the pGL3-basic luciferase vector. Briefly, DNA primers containing Hind III and Xho I linkers were designed based on regions comprising the CIDEA promoter between 1124 and +14 from the transcription start site of the CIDEA gene. Genomic DNA was isolated from human colorectal cancer cells and PCR was performed to amplify a 1138-bp DNA fragment. The PCR product was cloned into the Hind III and Xho I restriction sites of the pGL3-basic luciferase vector and the inserted promoter region was sequenced. The promoter sequence was 100% identical to the human CIDEA promoter sequence published in GenBank.
3.1. Inhibitory concentration and cytotoxicity of the chalcones We evaluated the cytotoxicity of the two chalcones (Fig. 1) against the breast cancer cell line MCF-7 and 3T3 cells treated for 24 and 48 h (Table 1). LicoA and trans-chalcone exhibited a pronounced cytotoxicity activity against MCF-7 and 3T3 cell lines in 48 h of treatment. 3.2. Chalcones cause LDH leakage in tumor cells after 48 h of treatment Cytotoxicity was also evaluated based on LDH leakage to determine plasma membrane damage in MCF-7 cells. Twenty-four hour treatment with the selected chalcones did not cause expressive enzyme leakage. However, in the case of cells treated for 48 h, differences in the percentage of LDH activity were observed for concentrations higher than the IC50, especially for LicoA. At IC80, trans-chalcone and LicoA resulted in a high percentage of LDH leakage in the MCF-7 cell line. The LDH assays Table 1 Cytotoxicities of the four chalcones toward MCF-7 and 3T3 cell lines at 48 and 24-h treatments. 48-h treatments Inhibitory concentrations (mM)
2.10. CIDEA promoter activity assay Transient transfections were performed using jetPRIME (Polyplus-transfection) according to the protocol of the manufacturer. MCF-7 cells were grown to 80% confluence in 12-well plates and plasmid mixtures containing 0.8 mg of the human CIDEA promoter-luciferase reporter construct and 0.8 mg pRL-null vector were cotransfected for 24 h in serum-containing media. The transfected cells were treated with 0, 20, 40 and 80 mM trans-chalcone in serum-containing media for 24 h and harvested in 1X passive lysis buffer (Promega). Luciferase activity in the cell lysates (50 mg protein) was measured and normalized to pRL-null luciferase activity using the DualGlo Luciferase Assay Kit (Promega) according to manufacturer instructions.
trans-chalcone Licochalcone A
IC50
IC20 MCF7
3T3
MCF7
20.79 11.11
25.83 41.53 18.59 23.32
IC80 3T3
MCF7
48.41 71.93 33.43 42.58
3T3 83.08 55.56
24-h treatments Inhibitory concentrations (mM)
trans-chalcone Licochalcone A
IC20
IC50
IC80
MCF-7
3T3
MCF-7
3T3
MCF-7
3T3
30.23 27.57
ND ND
58.25 60.46
ND ND
98.03 >73.88
ND ND
IC = Inhibitory concentrations; IC20, IC50 and IC80 are concentrations that causes, respectively, 20%, 50% and 80% growth inhibition of cell lines; results express the means of at least three experiments; ND = Non-determined. Each experiment was repeated at least three times in triplicate; the percentage of inhibition of viability was determined by a cell death formula [8]; the estimated averages were used to calculate the IC20, IC50 and IC80 values by nonlinear regression.
Fig. 1. Structures of the two chalcones tested.
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
4
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
Table 2 Percentage of LDH release of the MCF-7 cell line at treatments with the two most cytotoxic chalcones. 24-h treatments LDH release (%)
IC50
IC80
trans-chalcone Licochalcone A
15.45 4.1 a 4.61 0.8 a
28.44 4.1 b 25.83 7.8 b
48-h treatments LDH release (%)
IC50
IC80
trans-chalcone Licochalcone A
61.17 6.3 c 83.57 3.4 d
91.23 4.3 e 93.22 10.6 e
Results are shown as the means standard deviations; different letters represent statistical differences (p< 0.05); each group of treatment (24 or 48 h) were individually treated with statistical analysis. The experiments were performed three times in triplicate LDH leakage was calculated according to Grosse et al. [9].
suggest that the two chalcones damage the cell membrane of MCF7 cells in a dose- and time-dependent manner (Table 2). Thus, the results from these two assays (MTT and LDH test) indicated that trans-chalcone and LicoA induced cytotoxicity in MCF7 cells. 3.3. Both chalcones induce low to significant DNA damage The comet assay was used for the evaluation of genotoxicity. In general, the two chalcones exhibited dose-dependent genotoxicity against the MCF-7 cell line. At the three concentrations tested, the compounds induced less DNA damage in normal 3T3 cells (Table 3) compared to the MCF-7 cells. Considering that the maximum DDI
Table 3 Genotoxic potential evaluated by comet assay. Cell lines
DNA Damage Index (DDI) MCF-7
3T3
Medium Negative control (DMSO 0.5%) Doxorubicin 1.38 mM (0.8 mg/mL) trans-chalcone 72.02 mM (15 mg/mL) trans-chalcone 48.02 mM (10 mg/mL) trans-chalcone 24.01 mM (5 mg/mL) Licochalcone A 44.33 mM (15 mg/mL) Licochalcone A 29.55 mM (10 mg/mL) Licochalcone A 14.77 (5 mg/mL)
32 9 57 13.33 344.33 10.22 208.5 3.5 195 10 121 8 187.67 27.11 117 24 90.5 13.5
13.67 8,89 36 12 369 4 118 53.3 94.33 33.11 86 38 108.33 5.78 90 1.33 68.33 20.44
Comet assay was performed three times in duplicate. The DNA damage index (DDI) was calculated based on five levels of DNA damage by the formula: DI = (0 n0) + (1 n1) + (2 n2) + (3 n3) + (4 n4). Results are shown as the means standard deviations.
in the test is 400, the positive control (doxorubicin) was markedly genotoxic in the two cell lines even at a low concentration (1.38 mM), while the DNA damage induced by the chalcones was lower. 3.4. Licochalcone A and trans-chalcone induce cell cycle arrest and apoptosis in MCF-7 cells The flow cytometry experiments showed that treatment with the two chalcones for 24 h caused cell cycle arrest in G1 when compared to the control, particularly the treatment with IC50 of trans-chalcone and LicoA (55.91 0.2% and 54.9 1.5%, respectively) (Figs. 2 and 3). In the treatments, cell cycle arrest in G1 is explained by the accumulation of cells in this phase, with a consequent reduction in the proportion of cells in the S and G2/M phases. As shown in Figs. 4 and 5, both chalcones induced apoptosis of the breast cancer cell line. The proportion of apoptotic cells for the different treatments was 27 6.1% for IC20 of LicoA, 67.3 10.9% for IC20 of trans-chalcone, 72.3 8.5% of IC50 of LicoA and 85.6 2.3% of IC50 of trans-chalcone, which characterizes a dose-dependent phenomenon with better efficacy of trans-chalcone. The greatest difference between treatments was observed in terms of the stage of apoptosis (early vs. late), i.e., the IC20 concentrations resulted in a larger number of cells in early apoptosis (38.2 3.9% for transchalcone and 14.3 4.7% for LicoA) and viable cells, while the IC50 concentrations resulted in a larger proportion of cells in late apoptosis (69.9 5.2% for trans-chalcone and 46.4 11.9% for LicoA) (Fig. 5). Thus, treatment of cells with chalcones resulted in induction of apoptosis in a dose-dependent manner (Fig. 4). Taken together, our data indicate that the two chalcones affects cell cycle arrest as well as apoptosis, thereby facilitating anti-tumorigenic activity in MCF-7 cells. 3.5. trans-chalcone and licochalcone A modulate apoptosis-related genes in MCF-7 cells In view of the data obtained by flow cytometry, the expression of apoptosis-related genes was investigated using a PCR Array. Fourteen of the 84 genes were modulated (Table 4), including the anti-apoptotic genes AKT1, BAG1, Bcl-2, BFAR, BIRC2, BIRC3, BRAF, IGF1R and XIAP and the pro-apoptotic genes AIFM1, APAF1, BAX, BAK1 and CIDEA, all of them related to the intrinsic pathway. In general, trans-chalcone yielded the best results after 24 h, with greater inhibition of Bcl-2, induction of APAF1 and BAX, and strong induction of CIDEA, which was observed exclusively for this drug.
Fig. 2. Flow cytometry evaluation of cell cycle arrest of MCF-7 treated with both chalcones using CycleTESTTM DNA reagent kit (BD Biosciences). Asterisks represent statistical differences (p < 0,05 vs. control). Equal number of asterisks represent statistical equivalence in the same cell cycle phase. Each phase was individually treated by statistical analysis (ANOVA).
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
5
Fig. 3. Percentage of MCF-7 cells treated with Licochalcone A and trans-chalcone on each cell cycle phase (G1, S, G2/M) evaluated by flow cytometry. Results are shown as the means standard deviations.
In the two treatments of 24 h, the induction of the pro-apoptotic gene BAX together with the inhibition of the pro-survival gene Bcl2 indicates the induction of apoptosis mediated by the intrinsic pathway [5]. Both substances induced strong inhibition of BAK1, an essential apoptotic effector gene, after 6 h. However, the repression of this gene was no longer observed at the later time point (24 h). Furthermore, the caspase inhibitors BIRC2 (LicoA) and XIAP (transchalcone) that had been induced at 6 h were not highly expressed at 24 h, different from BIRC3 (Table 4, 24 h).
3.6. Effect of the chalcones on the expression of apoptosis-related proteins In the MCF-7 cell line, the higher concentrations of the two chalcones reduced the expression of the apoptosis-related protein Bcl-2 at the two time points tested (Fig. 6A and B), confirming PCR Array results (Table 4). Furthermore, there was marked degradation of cyclin D1 at 48 h (Fig. 6B) and the two chalcones induced cleavage of PARP in this time (Fig. 6A and B).
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
6
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
3.7. Effect of trans-chalcone on the expression of the CIDEA promoter and protein In order to confirm the induction of the CIDEA gene by transchalcone in the PCR Array experiment, a luciferase assay was performed to determine the promoter activity of the CIDEA gene in the MCF-7 cancer cell line. The results showed that trans-chalcone induced the expression of the CIDEA gene by affecting the activity of the promoter of this gene. This was observed in the MCF-7 cell line from a concentration of 20 mM trans-chalcone and was more marked at 80 mM (Fig. 7C). Moreover, the induction of the CIDEA protein by trans-chalcone was observed in all treatments (Fig. 7A and B). 4. Discussion
Fig. 4. Apoptosis induction of all of the treatments on MCF-7 using Apoptosis Detection Kit. Different letters represent statistical differences (p < 0.05).
Breast cancer is the second most common type of cancer in the world, with approximately 92,000 deaths on the American continent in 2012, and 57,120 new cases in 2014 in Brazil alone [13]. The two chalcones with different structures were investigated
Fig. 5. Flow cytometry (PI + Annexin V-FITC) of all of the treatments on MCF-7 cell line.
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx Table 4 Apoptotic genes modulated on MCF-7 treated with IC50 (24-h) of chalcones evaluated by PCR Array. Genes
AIFM1 AKT1 APAF1 BAG1 BAK1 BAX Bcl-2 BFAR BIRC2 BIRC3 BRAF CIDE-A IGF1R XIAP
6h
24 h
Licochalcone A
trans-chalcone
1.96 1.93 1.06 2.53 18.13 1.39 1.85 1.23 3.71 1.54 1.07 0 1.41 0
1.32 1.80 1.69 3.60 25.81 1.51 1.03 1.03 0 1.92 1.47 0 1.10 1.31
Licochalcone A 2.08 0 1 0 0 2.75 31.12 1.36 0 8.81 1.74 0 1.26 0
trans-chalcone 1.76 1.26 2.55 0 0 5.62 44.63 3.18 0 2.89 1.94 71.01 3.97 0
Results show fold regulation values. PCR Array was performed once per treatment. Fold regulation values were calculated from the qRT-PCR data with the RT2 Profiler PCR Array Data Analysis v. 3.5 tool (SABiosciences), considering the expression of ACTB (b-Actin) used as the housekeeping gene.
in the present study, although their backbone structure is the same (Fig. 1). The MTT cytotoxicity test (Table 1) showed a dosedependent response of MCF-7 cells to treatment with the compounds trans-chalcone and LicoA. trans-chalcone has not been studied in detail in tumorigenesis, on the other hand there are reports showing the anticancer activity of LicoA toward different human tumor cell lines such as oral cancer cells, hepatocellular carcinoma and gastric cancer [14–16]. LicoA exhibits antiproliferation activity in a time-dependent manner in most assays described here. The alkaline comet assay using trans-chalcone or LicoA demonstrated that DNA damage induced by the chalcones was lower than the doxorubicin-induced damage. The dose-dependent genotoxicity was more noticeable in the MCF-7 cells compared to the 3T3 cells (Table 3). DNA damage can activate of the immune system to eliminating damaging cells. However, activation of immune system to reduce DNA damage cells, can also contribute to chronic inflammation [17]. On the other hand, the cell response to DNA damage can be related to induction of the intrinsic apoptotic pathway [18]. The G1 phase is the state preceding DNA replication in which factors such as cellular conditions (metabolism, signaling and cell size) influence cell cycle progression, leading to DNA repair in the cell or inducing apoptosis [19]. The IC50 of LicoA (60.46 mM) and the two treatments with trans-chalcone induced arrest at the G1 checkpoint (Fig. 4) and increased apoptotic cells as assessed by annexin V (Fig. 5). We propose that the apoptotic pathway triggered by the treatments with these chalcones is the mitochondrial pathway, considering the induction of crucial genes of this
7
pathway after 24 h (Table 4), such as the adapter protein apoptotic protease-activating factor-1 (APAF-1) and Bcl-2-associated X protein (BAX). Syam et al. [20] reported the induction of apoptosis by synthetic chalcones in the same cell line (MCF-7) through the generation of reactive oxygen species and loss of mitochondrial membrane potential, resulting in the release of cytochrome C. These results are similar to the findings of the present study since the induction of the APAF1 gene is an indicator of cytochrome C release from mitochondria, thus forming the apoptosome. Furthermore, the main evidence of apoptosis through the intrinsic pathway over the same period of treatment (24 h) is the elevated repression of the Bcl-2 gene. Although the literature has shown that the MCF-7 cell line is deficient in caspase-3 [21,22], in the present work the treatment with chalcones induced the cleavage of PARP protein (Fig. 6A and B), a polymerase of which the cleavage into two fragments is an apoptosis marker [23]. Repression of the Bcl-2 gene by the two chalcones in the PCR Array experiment was also observed at the protein level in a dose-dependent manner in the MCF-7 cell line, supporting the data that apoptosis is mediated by the intrinsic pathway. Cyclin D1 was another protein that was repressed in the MCF-7 cell line in the presence of the two chalcones. This protein is crucial for progression from the G1 to the S phase and is an important biomarker of some types of cancer, including breast cancer [24]. Thus, the degradation of cyclin D1 is attractive for the development of new anti-tumor agents [25], and may be related to cell cycle arrest. A major difference between the two chalcones was the marked positive modulation of the CIDEA gene by only trans-chalone (24 h) in the PCR Array experiment (Table 4). The results confirmed that trans-chalone induced the expression of the CIDEA promoter activity (Fig. 7C) and protein (24 and 48 h) (Fig. 7A and B). Proteins of the CIDE family (cell death-inducing DNA fragmentation factor alpha-like effector) possess homology to DNA fragmentation factors (DFF). The latter consists of two subunits, a DNA fragmentation subunit (DFF40) and an inhibitor subunit (DFF45) [26,27]. Such functions were therefore initially attributed to CIDEA. A relationship between the expression of CIDEA and apoptosis induction in different types of tumors, such as hepatocellular carcinoma [28] and breast carcinoma [29], has been previously reported. For example, Omae et al. [30] demonstrated a directly proportional relationship between apoptotic DNA fragmentation and the expression of CIDEA during the treatment of murine pancreatic cells (MIN6) with palmitic acid. Moreover, Yong et al. [31] using the same qRT-PCR array demonstrated that the combination of treatment with Gemcitabine and a-radiation in LS-174T tumor xenograft model of disseminated intraperitoneal disease, resulted in the differential expression of apoptotic genes, including, CIDEA. We therefore suggest that trans-chalcone could induce the expression of CIDEA, facilitating the induction of cell death by apoptosis of MCF-7 cells.
Fig. 6. Western blot assays for the expression of the proteins Bcl-2, PARP and Cyclin D1 on MCF-7 breast cancer cell line through the treatments of different concentrations of trans-Ch and LicoA. MCF-7 cells were exposed to the chalcones for 24-h (A) and 48-h treatments (B).
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
8
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
Fig. 7. The link between the exposure of trans-Ch and the expression of CIDEA protein was determined by western blot analysis after cells (MCF-7) were exposed to different concentrations of the chalcone at 24-h (A) and 48-h treatments (B). The CIDEA-promoter activity was evaluated on MCF-7 breast cancer cell lines by the luciferase-promoter assay at different concentrations of trans-Ch (Student unpaired t-test with statistical significance of *p < 0.05, **p < 0.01 and ***p < 0.001) (C).
In conclusion, LicoA and particularly trans-chalcone are cytotoxic for MCF-7 and 3T3 cell lines but exhibit higher genotoxicity to the breast cancer cell. The two chalcones caused cell cycle arrest at G1 phase with cyclin D1 suppression and induced apoptosis mediated by the intrinsic pathway. transchalcone induced the expression of the CIDEA gene and protein, which is related to DNA fragmentation during apoptosis. Competing interests The authors declare that they have no competing interests. Acknowledgements This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2012/06889-7), CNPq (PhD fellowship granted to F.R.B.), CAPES (PhD fellowship granted to G.S) and FAPESP Master’s and PhD fellowships granted to L.F.B.B. (FAPESP 2012/15862-5) and T.A.B. (FAPESP 2012/02920-7), respectively. References [1] M. Niyazi, C. Maihoefer, M. Krause, et al., Radiotherapy and new drugs-new side effects? Radiat. Oncol. 6 (2011) 1–19, doi:http://dx.doi.org/10.1186/1748717X-6-177. [2] A. Schlitt, K. Jordan, D. Vordermark, et al., Cardiotoxicity and oncological treatments, Dtsch. Arztebl. Int. 111 (2014) 161–168, doi:http://dx.doi.org/ 10.3238/arztebl.2014.0161. [3] H. Zahreddine, K.L.B. Borden, Mechanisms and insights into drug resistance in cancer, Front. Pharmacol. 4 (2013) 1–8, doi:http://dx.doi.org/10.3389/ fphar.2013.00028. [4] C.M. Croce, Oncogenes and cancer, N. Engl. J. Med. 358 (2008) 502–511, doi: http://dx.doi.org/10.1056/nejmra072367. [5] P.E. Czabotar, G. Lessene, A. Strasser, J.M. Adams, Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy, Nat. Rev. Mol. Cell Biol. 15 (2014) 49–63, doi:http://dx.doi.org/10.1038/nrm3722.
[6] N.K. Sahu, S.S. Balbhadra, J. Choudhary, Exploring pharmacological significance of chalcone scaffold: a review, Curr. Med. Chem. 19 (2012) 209–225, doi:http://dx.doi.org/10.2174/092986712803414132. [7] C. Karthikeyan, Moorthy NSHN, S. Ramasamy, et al., Advances in chalcones with anticancer activities, Recent Pat. Anticancer Drug Discov. 10 (2015) 97– 115, doi:http://dx.doi.org/10.2174/1574892809666140819153902. [8] E. Szliszka, Z.P. Czuba, B. Mazur, et al., Chalcones and dihydrochalcones augment TRAIL-mediated apoptosis in prostate cancer cells, Molecules 15 (2010) 5336–5353, doi:http://dx.doi.org/10.3390/molecules15085336. [9] S. Grosse, L. Evje, T. Syversen, Silver nanoparticle-induced cytotoxicity in rat brain endothelial cell culture, Toxicol. In Vitro 27 (2013) 305–313, doi:http:// dx.doi.org/10.1016/j.tiv.2012.08.024. [10] W. Liao, M.A. McNutt, W.-G. Zhu, The comet assay: a sensitive method for detecting DNA damage in individual cells, Methods 48 (2009) 46–53, doi: http://dx.doi.org/10.1016/j.ymeth.2009.02.016. [11] B.C. Cavalcanti, C.M.L. Sombra, J.H.H.L. de Oliveira, et al., Cytotoxicity and genotoxicity of ingenamine G isolated from the Brazilian marine sponge Pachychalina alcaloidifera, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 147 (2008) 409–415, doi:http://dx.doi.org/10.1016/j.cbpc.2008.01.005. [12] G. Silva, M. Marins, A.L. Fachin, et al., Anti-cancer activity of trans-chalcone in osteosarcoma: involvement of Sp1 and p53, Mol. Carcinog. (2015), doi:http:// dx.doi.org/10.1002/mc.22386. [13] Instituto Nacional de Câncer José Alencar Gomes da Silva (INCA) – Tipos de câncer – Mama. http://www2.inca.gov.br/wps/wcm/connect/tiposdecancer/ site/home/mama. Accessed 18 August 2015. [14] J.-S. Kim, M.-R. Park, S.-Y. Lee, et al., Licochalcone A induces apoptosis in KB human oral cancer cells via a caspase-dependent FasL signaling pathway, Oncol. Rep. 31 (2014) 755–762, doi:http://dx.doi.org/10.3892/or.2013.2929. [15] J.-P. Tsai, P.-C. Hsiao, S.-F. Yang, et al., Licochalcone A suppresses migration and invasion of human hepatocellular carcinoma cells through downregulation of MKK4/JNK via NF-kB mediated urokinase plasminogen activator expression, PLoS One 9 (2014) 1–12, doi:http://dx.doi.org/10.1371/journal.pone.0086537. [16] X. Xiao, M. Hao, X. Yang, et al., Licochalcone A inhibits growth of gastric cancer cells by arresting cell cycle progression and inducing apoptosis, Cancer Lett. 302 (2011) 69–75, doi:http://dx.doi.org/10.1016/j.canlet.2010.12.016. [17] M.A. Ermolaeva, B. Schumacher, Systemic DNA damage responses: organismal adaptations to genome instability, Trends Genet. 30 (3) (2014) 95–102. [18] K.H. Khan, M. Blanco-Codesido, L.R. Molife, Cancer therapeutics: targeting the apoptotic pathway, Crit. Rev. Oncol. Hematol. 90 (2014) 200–219, doi:http:// dx.doi.org/10.1016/j.critrevonc.2013.12.012. [19] E. Xiaofei, T.F. Kowalik, The DNA damage response induced by infection with human cytomegalovirus and other viruses, Viruses 6 (2014) 2155–2185, doi: http://dx.doi.org/10.3390/v6052155.
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx [20] S. Syam, S.I. Abdelwahab, M.A. Al-Mamary, S. Mohan, Synthesis of chalcones with anticancer activities, Molecules 17 (2012) 6179–6195, doi:http://dx.doi. org/10.3390/molecules17066179. [21] Y. Liang, C. Yan, N.F. Schor, Apoptosis in the absence of caspase 3, Oncogene 20 (2001) 6570–6578, doi:http://dx.doi.org/10.1038/sj.onc.1204815. [22] R.U. Jänicke, MCF-7 breast carcinoma cells do not express caspase-3, Breast Cancer Res. Treat. 117 (2009) 219–221, doi:http://dx.doi.org/10.1007/s10549008-0217-9. [23] A.R. Green, D. Caracappa, A.A. Benhasouna, et al., Biological and clinical significance of PARP1 protein expression in breast cancer, Breast Cancer Res. Treat. 149 (2015) 353–362, doi:http://dx.doi.org/10.1007/s10549-014-3230-1. [24] M. Sana, H.J. Malik, Current and emerging breast cancer biomarkers, J. Cancer Res. Ther. 11 (2015) 508–513, doi:http://dx.doi.org/10.4103/09731482.163698. [25] J.P. Alao, The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention, Mol. Cancer 6 (2007) 1–16, doi: http://dx.doi.org/10.1186/1476-4598-6-24. [26] N. Inohara, T. Koseki, S. Chen, et al., CIDE, a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor, EMBO J. 17 (1998) 2526–2533, doi:http://dx.doi.org/10.1093/emboj/17.9.2526.
9
[27] T. Yonezawa, R. Kurata, M. Kimura, H. Inoko, Which CIDE are you on? Apoptosis and energy metabolism, Mol. Biosyst. 7 (2011) 91–100, doi:http://dx.doi.org/ 10.1039/c0mb00099j. [28] L. Zhang, G. Jiang, F. Yao, et al., Growth inhibition and apoptosis induced by osthole, a natural coumarin, in hepatocellular carcinoma, PLoS One 7 (2012) 1– 9, doi:http://dx.doi.org/10.1371/journal.pone.0037865. [29] J.C. Silva, J. Ferreira-Strixino, L.C. Fontana, et al., Apoptosis-associated genes related to photodynamic therapy in breast carcinomas, Lasers Med. Sci. 29 (2014) 1429–1436, doi:http://dx.doi.org/10.1007/s10103-014-1547-y. [30] N. Omae, M. Ito, S. Hase, et al., Suppression of FoxO1/cell death-inducing DNA fragmentation factor a-like effector A (Cidea) axis protects mouse b-cells against palmitic acid-induced apoptosis, Mol. Cell Endocrinol. 348 (2012) 297– 304, doi:http://dx.doi.org/10.1016/j.mce.2011.09.013. [31] K.J. Yong, D.E. Milenic, K.E. Baidoo, M.W. Brechbiel, Impact of a-targeted radiation therapy on gene expression in a pre-clinical model for disseminated peritoneal disease when combined with paclitaxel, PLoS One 30 (9) (2014) e108511, doi:http://dx.doi.org/10.1371/journal.pone.0108511.
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest Luis Felipe Buso Bortolottoa , Flávia Regina Barbosaa , Gabriel Silvaa,b , Tamires Aparecida Bitencourta , Rene Oliveira Belebonia , Seung Joon Baekb , Mozart Marinsa , Ana Lúcia Fachina,* a b
Biotechnology Unit, Ribeirão Preto University, SP, Brazil Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA
A R T I C L E I N F O
Article history: Received 29 August 2016 Received in revised form 4 November 2016 Accepted 9 November 2016 Keywords: Apoptosis CIDEA Comet assay Licochalcone A MCF-7 Trans-chalcone PCR Array
A B S T R A C T
Chalcones are precursors of flavonoids that exhibit structural heterogeneity and potential antitumor activity. The objective of this study was to characterize the cytotoxicity of trans-chalcone and licochalcone A (LicoA1 ) against a breast cancer cell line (MCF-7) and normal murine fibroblasts (3T3). Also the mechanisms of the anti-cancer activity of these two compounds were studied. The alkaline comet assay revealed dose-dependent genotoxicity, which was more responsive against the tumor cell line, compared to the 3T3 mouse fibroblast cell line. Flow cytometry showed that the two chalcones caused the cell cycle arrest in the G1 phase and induced apoptosis in MCF-7 cells. Using PCR Array, we found that trans-chalcone and LicoA trigger apoptosis mediated by the intrinsic pathway as demonstrated by the inhibition of Bcl-2 and induction of Bax. In western blot assay, the two chalcones reduced the expression of cell death-related proteins such as Bcl-2 and cyclin D1 and promoted the cleavage of PARP. However, only trans-chalcone induced the expression of the CIDEA gene and protein in these two experiments. Furthermore, transient transfections of MCF-7 using a construction of a promoter-luciferase vector showed that trans-chalcone induced the expression of the CIDEA promoter activity in 24 and 48 h. In conclusion, the results showed that trans-chalcone promoted high induction of the CIDEA promoter gene and protein, which is related to DNA fragmentation during apoptosis. ã 2016 Published by Elsevier Masson SAS.
1. Introduction Despite advances in cancer research, breast cancer is a leading cause of death among women. Surgery, radiotherapy and chemotherapy administered separately or in combination are the most common treatments for breast cancer, but are not always effective and may cause undesired side effects [1]. Doxorubicin is a chemotherapeutic agent commonly used for the treatment of breast cancer, but has limitations because of its cardiotoxicity [2] and selection of resistant tumors. Resistance to cytotoxic drugs can be caused by the reduced expression of tumor suppressor genes and by the overexpression of proto-oncogenes [3,4]. The resistance in tumor cells may prevent apoptosis by altering the modulation of essential genes involved in programmed cell death pathways, such
* Correspondence to: Unidade de Biotecnologia, Universidade de Ribeirão Preto. Av: Costábile Romano 2201, 14096-900, Ribeirão Preto, SP, Brasil. E-mail address: [email protected] (A.L. Fachin). 1 LicoA = licochalcone A.
as those of the Bcl-2 family [5]. In this regard, there is a continuous search for alternatives of cytotoxic compounds that exhibit a better therapeutic response than the classical chemotherapeutic agents. Some examples of alternative molecules under research are the chalcones, which are open-chain polyphenols that consist of two aromatic rings joined by one a, b-unsaturated propenone. These compounds occur naturally as precursors of flavonoids and possess anti-microbial, anti-oxidant and anti-inflammatory properties, among others [6]. There are numerous reports of natural and synthetic chalcones with potent anti-tumor activity against different cell lines [7], resulting from the activation of apoptotic pathways and anti-proliferative action [6–8,12,14–16]. In the present study, trans-chalcone and licochalcone A (LicoA) were found to be cytotoxic and considerably genotoxic, caused cell cycle arrest, induced apoptosis, and modulated apoptotic genes through the inhibition of Bcl-2 and induction of Bax in the breast cancer cell line MCF-7. Western blot assay confirmed the two chalcones reduced the expression of cell death-related proteins such as Bcl-2 and cyclin D1 and promoted the cleavage of PARP at
http://dx.doi.org/10.1016/j.biopha.2016.11.047 0753-3322/ã 2016 Published by Elsevier Masson SAS.
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
2
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
48 h. However, only trans-chalcone induced the expression of the CIDEA gene and protein, which is involved in the apoptotic mechanism in the MCF-7 cell.
Bio-Tek Instruments, Inc.) at 490 nm. LDH leakage was calculated according to Grosse et al. [9]. The experiments were performed three times in triplicate.
2. Materials and methods
2.5. Comet assay
2.1. Materials
The alkaline comet assay was used to determine the genotoxic potential of the chalcones against MCF-7 and 3T3 cells. Briefly, 3 105 cells/well were seeded in 6-well plates and incubated for 24 h. The cells were then treated with the lowest concentrations of the chalcones used in the MTT assay (15, 10, and 5 mg/mL) for 6 h. The treated cells were fixed on slides and submitted to lysis and electrophoresis, followed by neutralization, according to Liao et al. [10]. The slides were stained with 20 mg/mL ethidium bromide per slide. On fluorescence microscopy, 100 cells/slide were randomly selected and scored visually into five classes of DNA damage according to Cavalcanti et al. [11]. The DNA damage index (DDI) was calculated using the following formula: DDI = (0 n0) + (1 n1) + (2 n2) + (3 n3) + (4 n4). The experiments were performed three times in duplicate.
The two tested compounds, trans-chalcone and LicoA, and some of the main reagents including MTT (4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), trypsin, doxorubicin hydrochloride, dimethyl sulfoxide (DMSO), DMEM, penicillin, kanamycin, streptomycin and LDH assay TOX7 were purchased from SigmaAldrich (St. Louis, MO, USA). Fetal bovine serum was obtained from Cultilab (Campinas, SP, Brazil), and Triton1 X-100 was purchased from USB (Cleveland, OH, USA). The flow cytometry kits Cycletest Plus DNA Reagent and FITC Annexin V Apoptosis Detection I were purchased from BD Biosciences (San Jose, CA, USA). The materials used for gene expression were RNeasy1 Mini Kit, RNase-free DNase Set, RT2 SYBR1 Green ROXTM qPCR Mastermix, RT2 First Strand Kit, and RT2 ProfilerTM PCR Array PAHS-012Z (Qiagen, Germany). The BCA protein assay was purchased from Thermo Scientific (Rockford, IL, USA), jetPRIME from Polyplus-transfection (New York, NY, USA), and the 1X passive lysis buffer and DualGlo Luciferase Assay Kit from Promega (Madison, WI, USA). 2.2. Cell culture and assay conditions The cell lines 3T3 (mouse fibroblasts) and MCF-7 [human breast cancer (estrogen receptor (ER)-positive)] were grown in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL kanamycin and 100 mg/mL streptomycin and maintained in a humidified 5% CO2 atmosphere at 37 C. Doxorubicin was used at 2.5 mg/mL (4.31 mM) as the positive control for all the tests, except for the comet assay in which a concentration of doxorubicin of 0.8 mg/mL (1.38 mM) was used. Culture medium containing 0.25% DMSO was used as the solvent control. 2.3. Growth inhibition The inhibition of cell viability by the two chalcones was evaluated by the MTT assay. Each cell line (MCF-7 and 3T3) was plated at a density of 3 104 cells/well in 96-well plates and incubated for 24 h. The cells were then treated with 5 different concentrations of the chalcones (25, 20, 15, 10, and 5 mg/mL) for 24 and 48 h. Next, 20 mL MTT solution (5 mg/mL) was added per well, and the plates were incubated for 4 h. The plates were centrifuged at 1800 g for 5 min, the culture medium was removed, and formazan dye was solubilized in 200 mL DMSO. Absorbance was read in a Thermoplate TP-Reader at 550 nm. The experiment was repeated at least three times in triplicate. The percent inhibition of cell viability was determined by the cell death formula of Szliszka et al. [8]. The estimated averages were used to calculate the IC20, IC50 and IC80 values by nonlinear regression. 2.4. Lactate dehydrogenase assay For the determination of lactate dehydrogenase (LDH) leakage, 3 104 MCF-7 cells/well were incubated in 96-well plates for 24 h and then treated with IC50 and IC80 (48-h MTT) of the chalcones for 24 and 48 h. Triton X-100 (1%, USB) was used as control. Next, 50 mL/well of the cell-free supernatant was collected and mixed with 100 mL/well reagent mixture (TOX7 Kit from Sigma-Aldrich) according to manufacturer instructions. The plates were covered with opaque material and incubated at room temperature for 30 min. Absorbance was read in a microplate reader (ELx 800 UV
2.6. Flow cytometry Cell cycle and apoptosis were analyzed in a FACSCanto II flow cytometer (BD Biosciences). Twenty-four well plates with 1 105 cells/well were used in both experiments. The tests were done using the IC50 of the chalcones determined by the 24-h MTT test in the MCF-7 cell line. 2.6.1. Apoptosis analysis using annexin V-FITC/PI Apoptosis was evaluated using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) according to manufacturer recommendations, with slight modifications. After incubation for 24 h, cells were treated with the inhibitory concentrations (IC50 and IC20) of the chalcones for 24 h. After this period, the cells were trypsinized, centrifuged (201 g for 5 min at 8 C), washed twice with cold 1X PBS, and resuspended in 500 mL of 1X binding buffer. Next, 5 mL PI and 5 mL annexin V-FITC were added and the tubes were incubated at room temperature in the dark for 15 min before analysis in the flow cytometer. The experiments were performed three times in duplicate treatments. 2.6.2. Cell cycle analysis After incubation for 24 h, the treated cells with IC50 and IC20 (24-h MTT) were collected, centrifuged, and washed as described above for apoptosis analysis. The supernatant was decanted and the cells were stained with the CycletestTM DNA Reagent Kit (BD Biosciences) according to the protocol of the manufacturer. The test was performed once in triplicate and the results were analyzed using the ModFit LT program (BD Biosciences). 2.7. RNA extraction, cDNA conversion and RT2 Profiler PCR Array The real-time PCR Array was used to screen for apoptotic genes related to chalcone-induced cell death. This quantitative real-time PCR Array permits to analyze the expression of 84 apoptosisrelated genes. First, MCF-7 cells were seeded at a density of 2.5 106 in 25-cm2 flasks and incubated for 24 h. Next, the cells were treated with the IC50 (24-h MTT) of the chalcones for 6 and 24 h. Total RNA was extracted using the RNeasy1 Mini Kit and RNase-free DNase Set (Qiagen). The integrity of the samples was assessed in a 2100 Bioanalyzer (Agilent) (RIN 8). Next, 500 ng of each RNA sample were converted into cDNA using the RT2 First Strand Kit (Qiagen). Each cDNA sample was mixed with RT2 SYBR1 Green ROXTM qPCR Mastermix (Qiagen) and a 25-mL aliquot of the mixture (cDNA and Mastermix) was added per well of the RT2
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
ProfilerTM PCR Array PAHS-012Z plates (Qiagen). The SYBR1 Green program (with dissociation curve) was executed on the Mx3005P1 thermocycler (Agilent Stratagene) (95 C for 10 min, 40 cycles of 95 C for 15 s and 60 C for 1 min, followed by 1 cycle of 95 C for 1 min, 55 C for 30 s and 95 C for 30 s). Fold regulation values were calculated from the qRT-PCR data with the RT2 Profiler PCR Array Data Analysis v. 3.5 tool (SABiosciences) using ACTB (b-actin) as the control gene.
3
2.11. Statistical analysis All experimental data were submitted to analysis of variance (ANOVA) followed by the Scott-Knott test using the Sisvar software, except for the comet assay, qPCR and luciferase assay data. The level of statistical significance was set at p < 0.05. The comet assay data are expressed as the mean standard deviation. The luciferase assay data were analyzed by the unpaired Student ttest, with statistical significance of p < 0.05, p < 0.01 and p < 0.001.
2.8. Western blot analysis 3. Results MCF-7 were grown to 80% confluence in 60-mm dishes and treated with 0, 20, 40 or 80 mM of trans-chalcone and LicoA in serum-containing media for 24 or 48 h. Briefly, cells were washed with PBS, harvested in RIPA buffer supplemented with protease (1 mM PMSF, 0.15 mM aprotinin, 2.3 mM leupeptin) and phosphatase (0.1 nM Na3VO4 and 25 mM NaF) inhibitors, and subjected to sonication. The protein concentration in the cell lysate was determined by the BCA protein assay (Thermo Scientific). Total protein (50 mg) was subjected to Western blot analysis as described previously by Silva et al. [12]. The image quantification was carried out using the ImageJ program and the b-actin bands were used for normalization. 2.9. Construction of a promoter-luciferase vector The promoter of the human CIDEA gene was cloned into the pGL3-basic luciferase vector. Briefly, DNA primers containing Hind III and Xho I linkers were designed based on regions comprising the CIDEA promoter between 1124 and +14 from the transcription start site of the CIDEA gene. Genomic DNA was isolated from human colorectal cancer cells and PCR was performed to amplify a 1138-bp DNA fragment. The PCR product was cloned into the Hind III and Xho I restriction sites of the pGL3-basic luciferase vector and the inserted promoter region was sequenced. The promoter sequence was 100% identical to the human CIDEA promoter sequence published in GenBank.
3.1. Inhibitory concentration and cytotoxicity of the chalcones We evaluated the cytotoxicity of the two chalcones (Fig. 1) against the breast cancer cell line MCF-7 and 3T3 cells treated for 24 and 48 h (Table 1). LicoA and trans-chalcone exhibited a pronounced cytotoxicity activity against MCF-7 and 3T3 cell lines in 48 h of treatment. 3.2. Chalcones cause LDH leakage in tumor cells after 48 h of treatment Cytotoxicity was also evaluated based on LDH leakage to determine plasma membrane damage in MCF-7 cells. Twenty-four hour treatment with the selected chalcones did not cause expressive enzyme leakage. However, in the case of cells treated for 48 h, differences in the percentage of LDH activity were observed for concentrations higher than the IC50, especially for LicoA. At IC80, trans-chalcone and LicoA resulted in a high percentage of LDH leakage in the MCF-7 cell line. The LDH assays Table 1 Cytotoxicities of the four chalcones toward MCF-7 and 3T3 cell lines at 48 and 24-h treatments. 48-h treatments Inhibitory concentrations (mM)
2.10. CIDEA promoter activity assay Transient transfections were performed using jetPRIME (Polyplus-transfection) according to the protocol of the manufacturer. MCF-7 cells were grown to 80% confluence in 12-well plates and plasmid mixtures containing 0.8 mg of the human CIDEA promoter-luciferase reporter construct and 0.8 mg pRL-null vector were cotransfected for 24 h in serum-containing media. The transfected cells were treated with 0, 20, 40 and 80 mM trans-chalcone in serum-containing media for 24 h and harvested in 1X passive lysis buffer (Promega). Luciferase activity in the cell lysates (50 mg protein) was measured and normalized to pRL-null luciferase activity using the DualGlo Luciferase Assay Kit (Promega) according to manufacturer instructions.
trans-chalcone Licochalcone A
IC50
IC20 MCF7
3T3
MCF7
20.79 11.11
25.83 41.53 18.59 23.32
IC80 3T3
MCF7
48.41 71.93 33.43 42.58
3T3 83.08 55.56
24-h treatments Inhibitory concentrations (mM)
trans-chalcone Licochalcone A
IC20
IC50
IC80
MCF-7
3T3
MCF-7
3T3
MCF-7
3T3
30.23 27.57
ND ND
58.25 60.46
ND ND
98.03 >73.88
ND ND
IC = Inhibitory concentrations; IC20, IC50 and IC80 are concentrations that causes, respectively, 20%, 50% and 80% growth inhibition of cell lines; results express the means of at least three experiments; ND = Non-determined. Each experiment was repeated at least three times in triplicate; the percentage of inhibition of viability was determined by a cell death formula [8]; the estimated averages were used to calculate the IC20, IC50 and IC80 values by nonlinear regression.
Fig. 1. Structures of the two chalcones tested.
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
4
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
Table 2 Percentage of LDH release of the MCF-7 cell line at treatments with the two most cytotoxic chalcones. 24-h treatments LDH release (%)
IC50
IC80
trans-chalcone Licochalcone A
15.45 4.1 a 4.61 0.8 a
28.44 4.1 b 25.83 7.8 b
48-h treatments LDH release (%)
IC50
IC80
trans-chalcone Licochalcone A
61.17 6.3 c 83.57 3.4 d
91.23 4.3 e 93.22 10.6 e
Results are shown as the means standard deviations; different letters represent statistical differences (p< 0.05); each group of treatment (24 or 48 h) were individually treated with statistical analysis. The experiments were performed three times in triplicate LDH leakage was calculated according to Grosse et al. [9].
suggest that the two chalcones damage the cell membrane of MCF7 cells in a dose- and time-dependent manner (Table 2). Thus, the results from these two assays (MTT and LDH test) indicated that trans-chalcone and LicoA induced cytotoxicity in MCF7 cells. 3.3. Both chalcones induce low to significant DNA damage The comet assay was used for the evaluation of genotoxicity. In general, the two chalcones exhibited dose-dependent genotoxicity against the MCF-7 cell line. At the three concentrations tested, the compounds induced less DNA damage in normal 3T3 cells (Table 3) compared to the MCF-7 cells. Considering that the maximum DDI
Table 3 Genotoxic potential evaluated by comet assay. Cell lines
DNA Damage Index (DDI) MCF-7
3T3
Medium Negative control (DMSO 0.5%) Doxorubicin 1.38 mM (0.8 mg/mL) trans-chalcone 72.02 mM (15 mg/mL) trans-chalcone 48.02 mM (10 mg/mL) trans-chalcone 24.01 mM (5 mg/mL) Licochalcone A 44.33 mM (15 mg/mL) Licochalcone A 29.55 mM (10 mg/mL) Licochalcone A 14.77 (5 mg/mL)
32 9 57 13.33 344.33 10.22 208.5 3.5 195 10 121 8 187.67 27.11 117 24 90.5 13.5
13.67 8,89 36 12 369 4 118 53.3 94.33 33.11 86 38 108.33 5.78 90 1.33 68.33 20.44
Comet assay was performed three times in duplicate. The DNA damage index (DDI) was calculated based on five levels of DNA damage by the formula: DI = (0 n0) + (1 n1) + (2 n2) + (3 n3) + (4 n4). Results are shown as the means standard deviations.
in the test is 400, the positive control (doxorubicin) was markedly genotoxic in the two cell lines even at a low concentration (1.38 mM), while the DNA damage induced by the chalcones was lower. 3.4. Licochalcone A and trans-chalcone induce cell cycle arrest and apoptosis in MCF-7 cells The flow cytometry experiments showed that treatment with the two chalcones for 24 h caused cell cycle arrest in G1 when compared to the control, particularly the treatment with IC50 of trans-chalcone and LicoA (55.91 0.2% and 54.9 1.5%, respectively) (Figs. 2 and 3). In the treatments, cell cycle arrest in G1 is explained by the accumulation of cells in this phase, with a consequent reduction in the proportion of cells in the S and G2/M phases. As shown in Figs. 4 and 5, both chalcones induced apoptosis of the breast cancer cell line. The proportion of apoptotic cells for the different treatments was 27 6.1% for IC20 of LicoA, 67.3 10.9% for IC20 of trans-chalcone, 72.3 8.5% of IC50 of LicoA and 85.6 2.3% of IC50 of trans-chalcone, which characterizes a dose-dependent phenomenon with better efficacy of trans-chalcone. The greatest difference between treatments was observed in terms of the stage of apoptosis (early vs. late), i.e., the IC20 concentrations resulted in a larger number of cells in early apoptosis (38.2 3.9% for transchalcone and 14.3 4.7% for LicoA) and viable cells, while the IC50 concentrations resulted in a larger proportion of cells in late apoptosis (69.9 5.2% for trans-chalcone and 46.4 11.9% for LicoA) (Fig. 5). Thus, treatment of cells with chalcones resulted in induction of apoptosis in a dose-dependent manner (Fig. 4). Taken together, our data indicate that the two chalcones affects cell cycle arrest as well as apoptosis, thereby facilitating anti-tumorigenic activity in MCF-7 cells. 3.5. trans-chalcone and licochalcone A modulate apoptosis-related genes in MCF-7 cells In view of the data obtained by flow cytometry, the expression of apoptosis-related genes was investigated using a PCR Array. Fourteen of the 84 genes were modulated (Table 4), including the anti-apoptotic genes AKT1, BAG1, Bcl-2, BFAR, BIRC2, BIRC3, BRAF, IGF1R and XIAP and the pro-apoptotic genes AIFM1, APAF1, BAX, BAK1 and CIDEA, all of them related to the intrinsic pathway. In general, trans-chalcone yielded the best results after 24 h, with greater inhibition of Bcl-2, induction of APAF1 and BAX, and strong induction of CIDEA, which was observed exclusively for this drug.
Fig. 2. Flow cytometry evaluation of cell cycle arrest of MCF-7 treated with both chalcones using CycleTESTTM DNA reagent kit (BD Biosciences). Asterisks represent statistical differences (p < 0,05 vs. control). Equal number of asterisks represent statistical equivalence in the same cell cycle phase. Each phase was individually treated by statistical analysis (ANOVA).
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
5
Fig. 3. Percentage of MCF-7 cells treated with Licochalcone A and trans-chalcone on each cell cycle phase (G1, S, G2/M) evaluated by flow cytometry. Results are shown as the means standard deviations.
In the two treatments of 24 h, the induction of the pro-apoptotic gene BAX together with the inhibition of the pro-survival gene Bcl2 indicates the induction of apoptosis mediated by the intrinsic pathway [5]. Both substances induced strong inhibition of BAK1, an essential apoptotic effector gene, after 6 h. However, the repression of this gene was no longer observed at the later time point (24 h). Furthermore, the caspase inhibitors BIRC2 (LicoA) and XIAP (transchalcone) that had been induced at 6 h were not highly expressed at 24 h, different from BIRC3 (Table 4, 24 h).
3.6. Effect of the chalcones on the expression of apoptosis-related proteins In the MCF-7 cell line, the higher concentrations of the two chalcones reduced the expression of the apoptosis-related protein Bcl-2 at the two time points tested (Fig. 6A and B), confirming PCR Array results (Table 4). Furthermore, there was marked degradation of cyclin D1 at 48 h (Fig. 6B) and the two chalcones induced cleavage of PARP in this time (Fig. 6A and B).
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
6
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
3.7. Effect of trans-chalcone on the expression of the CIDEA promoter and protein In order to confirm the induction of the CIDEA gene by transchalcone in the PCR Array experiment, a luciferase assay was performed to determine the promoter activity of the CIDEA gene in the MCF-7 cancer cell line. The results showed that trans-chalcone induced the expression of the CIDEA gene by affecting the activity of the promoter of this gene. This was observed in the MCF-7 cell line from a concentration of 20 mM trans-chalcone and was more marked at 80 mM (Fig. 7C). Moreover, the induction of the CIDEA protein by trans-chalcone was observed in all treatments (Fig. 7A and B). 4. Discussion
Fig. 4. Apoptosis induction of all of the treatments on MCF-7 using Apoptosis Detection Kit. Different letters represent statistical differences (p < 0.05).
Breast cancer is the second most common type of cancer in the world, with approximately 92,000 deaths on the American continent in 2012, and 57,120 new cases in 2014 in Brazil alone [13]. The two chalcones with different structures were investigated
Fig. 5. Flow cytometry (PI + Annexin V-FITC) of all of the treatments on MCF-7 cell line.
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx Table 4 Apoptotic genes modulated on MCF-7 treated with IC50 (24-h) of chalcones evaluated by PCR Array. Genes
AIFM1 AKT1 APAF1 BAG1 BAK1 BAX Bcl-2 BFAR BIRC2 BIRC3 BRAF CIDE-A IGF1R XIAP
6h
24 h
Licochalcone A
trans-chalcone
1.96 1.93 1.06 2.53 18.13 1.39 1.85 1.23 3.71 1.54 1.07 0 1.41 0
1.32 1.80 1.69 3.60 25.81 1.51 1.03 1.03 0 1.92 1.47 0 1.10 1.31
Licochalcone A 2.08 0 1 0 0 2.75 31.12 1.36 0 8.81 1.74 0 1.26 0
trans-chalcone 1.76 1.26 2.55 0 0 5.62 44.63 3.18 0 2.89 1.94 71.01 3.97 0
Results show fold regulation values. PCR Array was performed once per treatment. Fold regulation values were calculated from the qRT-PCR data with the RT2 Profiler PCR Array Data Analysis v. 3.5 tool (SABiosciences), considering the expression of ACTB (b-Actin) used as the housekeeping gene.
in the present study, although their backbone structure is the same (Fig. 1). The MTT cytotoxicity test (Table 1) showed a dosedependent response of MCF-7 cells to treatment with the compounds trans-chalcone and LicoA. trans-chalcone has not been studied in detail in tumorigenesis, on the other hand there are reports showing the anticancer activity of LicoA toward different human tumor cell lines such as oral cancer cells, hepatocellular carcinoma and gastric cancer [14–16]. LicoA exhibits antiproliferation activity in a time-dependent manner in most assays described here. The alkaline comet assay using trans-chalcone or LicoA demonstrated that DNA damage induced by the chalcones was lower than the doxorubicin-induced damage. The dose-dependent genotoxicity was more noticeable in the MCF-7 cells compared to the 3T3 cells (Table 3). DNA damage can activate of the immune system to eliminating damaging cells. However, activation of immune system to reduce DNA damage cells, can also contribute to chronic inflammation [17]. On the other hand, the cell response to DNA damage can be related to induction of the intrinsic apoptotic pathway [18]. The G1 phase is the state preceding DNA replication in which factors such as cellular conditions (metabolism, signaling and cell size) influence cell cycle progression, leading to DNA repair in the cell or inducing apoptosis [19]. The IC50 of LicoA (60.46 mM) and the two treatments with trans-chalcone induced arrest at the G1 checkpoint (Fig. 4) and increased apoptotic cells as assessed by annexin V (Fig. 5). We propose that the apoptotic pathway triggered by the treatments with these chalcones is the mitochondrial pathway, considering the induction of crucial genes of this
7
pathway after 24 h (Table 4), such as the adapter protein apoptotic protease-activating factor-1 (APAF-1) and Bcl-2-associated X protein (BAX). Syam et al. [20] reported the induction of apoptosis by synthetic chalcones in the same cell line (MCF-7) through the generation of reactive oxygen species and loss of mitochondrial membrane potential, resulting in the release of cytochrome C. These results are similar to the findings of the present study since the induction of the APAF1 gene is an indicator of cytochrome C release from mitochondria, thus forming the apoptosome. Furthermore, the main evidence of apoptosis through the intrinsic pathway over the same period of treatment (24 h) is the elevated repression of the Bcl-2 gene. Although the literature has shown that the MCF-7 cell line is deficient in caspase-3 [21,22], in the present work the treatment with chalcones induced the cleavage of PARP protein (Fig. 6A and B), a polymerase of which the cleavage into two fragments is an apoptosis marker [23]. Repression of the Bcl-2 gene by the two chalcones in the PCR Array experiment was also observed at the protein level in a dose-dependent manner in the MCF-7 cell line, supporting the data that apoptosis is mediated by the intrinsic pathway. Cyclin D1 was another protein that was repressed in the MCF-7 cell line in the presence of the two chalcones. This protein is crucial for progression from the G1 to the S phase and is an important biomarker of some types of cancer, including breast cancer [24]. Thus, the degradation of cyclin D1 is attractive for the development of new anti-tumor agents [25], and may be related to cell cycle arrest. A major difference between the two chalcones was the marked positive modulation of the CIDEA gene by only trans-chalone (24 h) in the PCR Array experiment (Table 4). The results confirmed that trans-chalone induced the expression of the CIDEA promoter activity (Fig. 7C) and protein (24 and 48 h) (Fig. 7A and B). Proteins of the CIDE family (cell death-inducing DNA fragmentation factor alpha-like effector) possess homology to DNA fragmentation factors (DFF). The latter consists of two subunits, a DNA fragmentation subunit (DFF40) and an inhibitor subunit (DFF45) [26,27]. Such functions were therefore initially attributed to CIDEA. A relationship between the expression of CIDEA and apoptosis induction in different types of tumors, such as hepatocellular carcinoma [28] and breast carcinoma [29], has been previously reported. For example, Omae et al. [30] demonstrated a directly proportional relationship between apoptotic DNA fragmentation and the expression of CIDEA during the treatment of murine pancreatic cells (MIN6) with palmitic acid. Moreover, Yong et al. [31] using the same qRT-PCR array demonstrated that the combination of treatment with Gemcitabine and a-radiation in LS-174T tumor xenograft model of disseminated intraperitoneal disease, resulted in the differential expression of apoptotic genes, including, CIDEA. We therefore suggest that trans-chalcone could induce the expression of CIDEA, facilitating the induction of cell death by apoptosis of MCF-7 cells.
Fig. 6. Western blot assays for the expression of the proteins Bcl-2, PARP and Cyclin D1 on MCF-7 breast cancer cell line through the treatments of different concentrations of trans-Ch and LicoA. MCF-7 cells were exposed to the chalcones for 24-h (A) and 48-h treatments (B).
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
8
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx
Fig. 7. The link between the exposure of trans-Ch and the expression of CIDEA protein was determined by western blot analysis after cells (MCF-7) were exposed to different concentrations of the chalcone at 24-h (A) and 48-h treatments (B). The CIDEA-promoter activity was evaluated on MCF-7 breast cancer cell lines by the luciferase-promoter assay at different concentrations of trans-Ch (Student unpaired t-test with statistical significance of *p < 0.05, **p < 0.01 and ***p < 0.001) (C).
In conclusion, LicoA and particularly trans-chalcone are cytotoxic for MCF-7 and 3T3 cell lines but exhibit higher genotoxicity to the breast cancer cell. The two chalcones caused cell cycle arrest at G1 phase with cyclin D1 suppression and induced apoptosis mediated by the intrinsic pathway. transchalcone induced the expression of the CIDEA gene and protein, which is related to DNA fragmentation during apoptosis. Competing interests The authors declare that they have no competing interests. Acknowledgements This study was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2012/06889-7), CNPq (PhD fellowship granted to F.R.B.), CAPES (PhD fellowship granted to G.S) and FAPESP Master’s and PhD fellowships granted to L.F.B.B. (FAPESP 2012/15862-5) and T.A.B. (FAPESP 2012/02920-7), respectively. References [1] M. Niyazi, C. Maihoefer, M. Krause, et al., Radiotherapy and new drugs-new side effects? Radiat. Oncol. 6 (2011) 1–19, doi:http://dx.doi.org/10.1186/1748717X-6-177. [2] A. Schlitt, K. Jordan, D. Vordermark, et al., Cardiotoxicity and oncological treatments, Dtsch. Arztebl. Int. 111 (2014) 161–168, doi:http://dx.doi.org/ 10.3238/arztebl.2014.0161. [3] H. Zahreddine, K.L.B. Borden, Mechanisms and insights into drug resistance in cancer, Front. Pharmacol. 4 (2013) 1–8, doi:http://dx.doi.org/10.3389/ fphar.2013.00028. [4] C.M. Croce, Oncogenes and cancer, N. Engl. J. Med. 358 (2008) 502–511, doi: http://dx.doi.org/10.1056/nejmra072367. [5] P.E. Czabotar, G. Lessene, A. Strasser, J.M. Adams, Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy, Nat. Rev. Mol. Cell Biol. 15 (2014) 49–63, doi:http://dx.doi.org/10.1038/nrm3722.
[6] N.K. Sahu, S.S. Balbhadra, J. Choudhary, Exploring pharmacological significance of chalcone scaffold: a review, Curr. Med. Chem. 19 (2012) 209–225, doi:http://dx.doi.org/10.2174/092986712803414132. [7] C. Karthikeyan, Moorthy NSHN, S. Ramasamy, et al., Advances in chalcones with anticancer activities, Recent Pat. Anticancer Drug Discov. 10 (2015) 97– 115, doi:http://dx.doi.org/10.2174/1574892809666140819153902. [8] E. Szliszka, Z.P. Czuba, B. Mazur, et al., Chalcones and dihydrochalcones augment TRAIL-mediated apoptosis in prostate cancer cells, Molecules 15 (2010) 5336–5353, doi:http://dx.doi.org/10.3390/molecules15085336. [9] S. Grosse, L. Evje, T. Syversen, Silver nanoparticle-induced cytotoxicity in rat brain endothelial cell culture, Toxicol. In Vitro 27 (2013) 305–313, doi:http:// dx.doi.org/10.1016/j.tiv.2012.08.024. [10] W. Liao, M.A. McNutt, W.-G. Zhu, The comet assay: a sensitive method for detecting DNA damage in individual cells, Methods 48 (2009) 46–53, doi: http://dx.doi.org/10.1016/j.ymeth.2009.02.016. [11] B.C. Cavalcanti, C.M.L. Sombra, J.H.H.L. de Oliveira, et al., Cytotoxicity and genotoxicity of ingenamine G isolated from the Brazilian marine sponge Pachychalina alcaloidifera, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 147 (2008) 409–415, doi:http://dx.doi.org/10.1016/j.cbpc.2008.01.005. [12] G. Silva, M. Marins, A.L. Fachin, et al., Anti-cancer activity of trans-chalcone in osteosarcoma: involvement of Sp1 and p53, Mol. Carcinog. (2015), doi:http:// dx.doi.org/10.1002/mc.22386. [13] Instituto Nacional de Câncer José Alencar Gomes da Silva (INCA) – Tipos de câncer – Mama. http://www2.inca.gov.br/wps/wcm/connect/tiposdecancer/ site/home/mama. Accessed 18 August 2015. [14] J.-S. Kim, M.-R. Park, S.-Y. Lee, et al., Licochalcone A induces apoptosis in KB human oral cancer cells via a caspase-dependent FasL signaling pathway, Oncol. Rep. 31 (2014) 755–762, doi:http://dx.doi.org/10.3892/or.2013.2929. [15] J.-P. Tsai, P.-C. Hsiao, S.-F. Yang, et al., Licochalcone A suppresses migration and invasion of human hepatocellular carcinoma cells through downregulation of MKK4/JNK via NF-kB mediated urokinase plasminogen activator expression, PLoS One 9 (2014) 1–12, doi:http://dx.doi.org/10.1371/journal.pone.0086537. [16] X. Xiao, M. Hao, X. Yang, et al., Licochalcone A inhibits growth of gastric cancer cells by arresting cell cycle progression and inducing apoptosis, Cancer Lett. 302 (2011) 69–75, doi:http://dx.doi.org/10.1016/j.canlet.2010.12.016. [17] M.A. Ermolaeva, B. Schumacher, Systemic DNA damage responses: organismal adaptations to genome instability, Trends Genet. 30 (3) (2014) 95–102. [18] K.H. Khan, M. Blanco-Codesido, L.R. Molife, Cancer therapeutics: targeting the apoptotic pathway, Crit. Rev. Oncol. Hematol. 90 (2014) 200–219, doi:http:// dx.doi.org/10.1016/j.critrevonc.2013.12.012. [19] E. Xiaofei, T.F. Kowalik, The DNA damage response induced by infection with human cytomegalovirus and other viruses, Viruses 6 (2014) 2155–2185, doi: http://dx.doi.org/10.3390/v6052155.
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047
G Model BIOPHA 4556 No. of Pages 9
L.F.B. Bortolotto et al. / Biomedicine & Pharmacotherapy xxx (2016) xxx–xxx [20] S. Syam, S.I. Abdelwahab, M.A. Al-Mamary, S. Mohan, Synthesis of chalcones with anticancer activities, Molecules 17 (2012) 6179–6195, doi:http://dx.doi. org/10.3390/molecules17066179. [21] Y. Liang, C. Yan, N.F. Schor, Apoptosis in the absence of caspase 3, Oncogene 20 (2001) 6570–6578, doi:http://dx.doi.org/10.1038/sj.onc.1204815. [22] R.U. Jänicke, MCF-7 breast carcinoma cells do not express caspase-3, Breast Cancer Res. Treat. 117 (2009) 219–221, doi:http://dx.doi.org/10.1007/s10549008-0217-9. [23] A.R. Green, D. Caracappa, A.A. Benhasouna, et al., Biological and clinical significance of PARP1 protein expression in breast cancer, Breast Cancer Res. Treat. 149 (2015) 353–362, doi:http://dx.doi.org/10.1007/s10549-014-3230-1. [24] M. Sana, H.J. Malik, Current and emerging breast cancer biomarkers, J. Cancer Res. Ther. 11 (2015) 508–513, doi:http://dx.doi.org/10.4103/09731482.163698. [25] J.P. Alao, The regulation of cyclin D1 degradation: roles in cancer development and the potential for therapeutic invention, Mol. Cancer 6 (2007) 1–16, doi: http://dx.doi.org/10.1186/1476-4598-6-24. [26] N. Inohara, T. Koseki, S. Chen, et al., CIDE, a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor, EMBO J. 17 (1998) 2526–2533, doi:http://dx.doi.org/10.1093/emboj/17.9.2526.
9
[27] T. Yonezawa, R. Kurata, M. Kimura, H. Inoko, Which CIDE are you on? Apoptosis and energy metabolism, Mol. Biosyst. 7 (2011) 91–100, doi:http://dx.doi.org/ 10.1039/c0mb00099j. [28] L. Zhang, G. Jiang, F. Yao, et al., Growth inhibition and apoptosis induced by osthole, a natural coumarin, in hepatocellular carcinoma, PLoS One 7 (2012) 1– 9, doi:http://dx.doi.org/10.1371/journal.pone.0037865. [29] J.C. Silva, J. Ferreira-Strixino, L.C. Fontana, et al., Apoptosis-associated genes related to photodynamic therapy in breast carcinomas, Lasers Med. Sci. 29 (2014) 1429–1436, doi:http://dx.doi.org/10.1007/s10103-014-1547-y. [30] N. Omae, M. Ito, S. Hase, et al., Suppression of FoxO1/cell death-inducing DNA fragmentation factor a-like effector A (Cidea) axis protects mouse b-cells against palmitic acid-induced apoptosis, Mol. Cell Endocrinol. 348 (2012) 297– 304, doi:http://dx.doi.org/10.1016/j.mce.2011.09.013. [31] K.J. Yong, D.E. Milenic, K.E. Baidoo, M.W. Brechbiel, Impact of a-targeted radiation therapy on gene expression in a pre-clinical model for disseminated peritoneal disease when combined with paclitaxel, PLoS One 30 (9) (2014) e108511, doi:http://dx.doi.org/10.1371/journal.pone.0108511.
Please cite this article in press as: L.F.B. Bortolotto, et al., Cytotoxicity of trans-chalcone and licochalcone A against breast cancer cells is due to apoptosis induction and cell cycle arrest, Biomed Pharmacother (2016), http://dx.doi.org/10.1016/j.biopha.2016.11.047