1. Introduction
Breast cancer is a neoplastic disease with high global mortality for the female population. It is characterized by the accumulation of mutations in the DNA structure that leads to uncontrolled development and multiplication of cells within the breast tissue[
1,
2]. This disease will continue to be the cause of many deaths due to tumour recurrence and drug resistance[
3]. Therefore, there is a need for the development and formulation of new therapeutic agents to address this type of human ailment.
Curcumin (diferuloylmethane) is one of the significant metabolites present in the rhizome of the Asian spice
Curcuma longa[
4,
5]. It has extensive therapeutic properties well documented in the scientific area because of its anti-inflammatory[
6,
7], antioxidant[
8] and cytotoxic potential[
9]. Therefore, extensive biological studies continue to be a significant task for scientists.
Recent studies have found that the curcumin molecule is considerably active against breast cancer cell lines (e.g., MCF-7 or MDA)[
10,
11] and shows antitumoral activity in the
Ovo model in nanoformulations without altering embryo development [
12]. However, disadvantages such as poor solubility[
13], low bioavailability[
14,
15], and rapid metabolism limit its use in the clinic as a successful therapeutic molecule. Consequently, research has focused on synthetic derivatives, producing a considerable increase in new molecules called curcuminoids[
8,
16,
17] or analogues.
The synthetic curcuminoid obtained from the replacement of two phenolic groups (Ph-OH) with two methoxyl groups (Ph-OCH3) is called dimethoxycurcumin (DiMeOC) and has exhibited good cytotoxic activity against cancer cells[
18]. In addition, DiMeOC has better metabolic stability [
19](compared to curcumin). Dimethoxycurcumin also presents the well-known keto-enol equilibrium at molecular half moiety, which allows it to form chelates or complexes with different metal ions. So, this chemical property has been advantageous in improving its solubility and bioavailability. Different metals of biological interest, such as copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), gallium (Ga), and indium (In), have previously been bonded to curcuminoid-type molecules forming active metal complexes with antioxidant or cytotoxic activities[
20,
21,
22,
23]. A metal ion related to breast cancer is magnesium (Mg)[
24], the second most relevant ion in the human body[
25] because it acts as a cofactor for about 300 enzymatic processes involved in maintaining energy metabolism[
26], protein synthesis, and DNA replication[
27], besides exerting essential antioxidant functions[
28].
The ailment hypomagnesemia[
29] is related to high oxidative stress and low serum concentrations of magnesium in women with breast cancer that compromises the expression and function of antioxidant enzymes, concomitantly manifesting the progression and proliferation of the breast tumour [
26,
27]. So, considering the importance and participation of magnesium in breast cancer, the synthesis of metallodrugs that contain this metal ion is essential.
On the other hand, an additional approach to solving the problem of poor solubility of curcuminoid-type molecules has been the discovery of new formulations, including liposomes[
30,
31], nanoparticles[
32,
33,
34,
35,
36], or the preparation of inclusion complexes with cyclodextrins[
37,
38]. Beta-cyclodextrin (BCD) is the first choice [
39] for the administration of drugs toward their sites of action because of its low toxicity[
40], certain hydrophilicity[
41], and adequate cavity. Beta-cyclodextrins can also help to increase the solubility[
42] and bioavailability and confer stability[
43]of different insoluble guests in aqueous media.
In this work, we designed a formulation following three concepts of molecular architecture: 1. The use of a metabolically stable curcuminoid compound (dimethoxy curcumin, DiMeOC, 3) and cytotoxic against cancer cell lines; 2. Using homoleptic metal complex with a metal (magnesium, DiMeOC-Mg, 4 ) of physiological importance related to cancer; 3. The preparation of an inclusion complex (DiMeOC-Mg-BCD, 5) with beta-cyclodextrin that has adequate cavity size for the encapsulation of a wide variety of drugs. Although the homoleptic metal complexes of curcuminoids have demonstrated high antioxidant and cytotoxic activity
in vitro [
44], there are few studies of new formulations with cyclodextrins and their biological activity
in vivo.
The overall biological screening suggested focussing on the new inclusion metal complex (5) and the free homoleptic metal complex (4) using the antitumoral model
in Ovo. Such a model is a good choice as an alternative for mammalian tumour induction to investigate the characteristics of tumour growth, metastasis, and angiogenesis[
45]. The antitumor in-Ovo assay was carried out by INOVOTION SAS (France).
4. Materials and Methods
All the chemicals were commercially available by Sigma-Aldrich and were user without purification process. Melting points were recorded using an electrothermal engineering IA9100X1 melting point apparatus, and the values are uncorrected[
68].
The spectra IR-ATR determinations were recorded in the 4000–400 cm−1 range and using an equipment FT-IR NICOLET IS-50, Thermo Fisher Scientific spectrophotometer.
Mass spectra were obtained using a JEOL SX 102 A spectrometer equipped with MALDI-Flight time technology or using the MStation JMS-700 JEOL equipment (Electron Ionization, 70 eV, 250 °C, Impact positive mode and calibration standard with perfluorokerosene) and the AccuTOF JMS-T100LC JEOL equipment (DART
+, 350 °C, positive ion mode and calibration standard with PEG 600)[
20,
69].
1H and
13C NMR liquid spectra were acquired in dimethyl sulfoxide (DMSO-
d6) or deuterium oxide (D
2O) on a Bruker Fourier 400 MHz spectrometer with TMS serving as the internal reference and NMR spectra were processed with MestreNova software 12.0.3.3.[
70].
DOSY was acquired with a JEOL 400 MHz (JNM JEOL ECZ 400S) equipment, using the pulse sequence One-shot-DOSY, with a grad_1_amp 30 [min] and grad_2_amp 280 [min], with the follow parameters: sw = 20 ppm; at = 3.0 s; d1 = 5.0 s; nt = 32; lb = 0.1 Hz, diffusion time 500 ms, for the NMR data acquisition was used a Jeol Delta 6.0 software, and for data processing was used MestreNova software 12.0.3.3.[
70]
UV–Visible. The maxima absorption measurements were recorded with an UV–Visible Shimadzu, U160 spectrophotometer. The aqueous (PBS) solubility of DiMeOC-Mg and DiMeOC-Mg-BCD were determined by the shake-flask method in triplicate (see Supporting information) and the quantification was performed by interpolation using a calibration curve previously constructed (see Supplementary material), and the determinations were analyzed by UV-vis spectroscopy at 415 nm with an enzyme-linked immunosorbent assay (ELISA) plate reader equipment (Bio-Tek Instruments, Winooski, VT, USA). Phases solubility studies were carried out according to Higuchi and Connors methods [
71](see Supplementary material). Briefly and 2 mg of DiMeOC-Mg was added to a series different mole fraction of BCD (0.0, 0.125, 0.25, 0.5, 0.75, 1.0 and 1.25) in PBS medium after being shaken for 48 hours, the solution was filtered through sintered filter of 0.45 μM and the amount dissolved in each solution was measurement at 415 nm.
HPLC chromatograms were recorded using an Agilent 1260 infinity II with diode -UV detector at 417 nm, column Spherisorb 25 mm x 4.6 mm x 5 mm, eluting with a solvent isocratic acetonitrile/water (formic acid 0.02%) 55/45 and flow 1.0 mL/min [
72].
13C CPMAS ssNMR spectra were recorded using a Jeol 600 MHz spectrometer (15.0 kHz of MAS) with adamantane as the reference (298 K).
The surface morphology images were recorded for BCD, DiMeOC, DiMeOC-Mg and DiMeOC-Mg-BCD and for analysis of Scanning Electronic Microscopy (SEM) the images were captured with a VEGA3 model microscope (TESCAN, Brno, Czech Republic).
TGA and DSC was carried out with a thermobalance (thermo analyzer Netzsch model STA 449 F3 Jupiter) using an aluminum crucible 25/40 µl, outer bottom Ø 5 mm (NETZSCH). The sample was heated from 25 ° to 550 °C at a heating rate of 10 °C min
-1 under a nitrogen atmosphere[
53].
The human breast epithelial cell lines MDA-MB-231, an estrogen receptor-negative cell line derived from a metastatic carcinoma obtained from American Type Culture Collection (ATCC; Manassas, VA, USA), were used in this study. MDA-MB-231 cells in Dulbecco’s Modified Eagle Medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10000 units/ml penicillin G sodium, 10000 μg/ml streptomycin sulfate, 25 μg Amphotericin B (Gibco) and 1% of non-essential amino acids (Gibco). The cell lines were kept at 37 °C in a humidified atmosphere of 5% CO2, and cell viability exceeded 95%.
We used a protein-binding dye sulforhodamine B (SRB) colourimetric assay[
73]. A suspension of 100 μl containing 5000-10000 cells per well were cultured into 96 micro-litre plates (Costar). Different concentrations of DiMeOC-Mg-BCD and control vehicle DMSO 1.3%, incubated at 37 °C for 48 h in a 5% CO
2 atmosphere were used. Subsequently, cells were fixed on the plastic substratum by adding 50 μl of cold aqueous 50% trichloroacetic acid for 48 h. Then, cells were removed from the tissue culture flask by treatment with trypsin and diluted with fresh media from the cell. The plates were incubated at 4 °C for one hour, washed with water, and air-dried. The addition of 4 % SRB stained trichloroacetic acid-fixed cells. The free SRB was removed by washing with 1% aqueous acetic acid, the plates were air-dried, and the dye was solubilized by adding 10 mM unbuffered Tris-base (100 μl). The plates were placed on a shaker for 10 min, and the absorption was measured at 515 nm using an ELISA plate reader (Bio Tex Instruments). The cell viability of MDA-MB-231, cells cultured in the presence of the assessed compounds was calculated as a percentage of the control cells, and the CC
50 values were obtained from dose–response curves. All experiments were performed in triplicate, and the CC
50 was calculated using GraphPad Software 6.0 (GraphPad Inc., San Diego, CA, USA). The results are expressed as the mean of CC
50 relative to vehicle and control.
To evaluate the morphological alterations induced by the DiMeOC-Mg-BCD complex on MDA-MB-231. Cell lines were cultured on a tissue culture flask with DiMeOC-Mg-BCD (CC
50) and different treatments for 48 h. After incubation, cells were washed two times with PBS, fixed with methanol 100 %, and stained with Wright-Giemsa (Sigma-Aldrich, WG32-1L). The culture flasks were examined under an Olympus IX71 Inverted Fluorescence Phase Contrast Microscope. The images were recorded with a Nikon Coolpix 4300 digital camera using the 20x objectives and analyzed by ImageJ software [
74].
The esterase activity and membrane damage were determined using the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular et al., OR, USA). MDA-MB-231 (0.6 106/mL) were culture and incubated at 37 °C in 24 well plates and treated with DiMeOC-Mg-BCD (CC
50). After 48 h incubation, cells were centrifuged at 3000 rpm for 10 min to remove the supernatant medium; the pellet was next resuspended in 997 µl of cold phosphate-buffered saline (PBS), 2 µl of a solution of Calcein-AM (C-AM) 50 µM and 1 µl of ethidium homodimer (Eth-1) 2 mM. Then, samples were incubated for 45 min at room temperature and immediately analyzed in a ThermoFisher Attune Flow Cytometer with a 530/30 nm filter (FL1-H) for calcein (green fluorescence/ living cells) and a 670 nm/long-pass filter (FL3-H) for Eth-1 (red fluorescence/dead cells), 20,000 events per treatment were acquired. The data were analyzed using FlowJo 7.3.2 software[
75] and expressed as the percentage of cells for each population phenotype. The compensation was performed using live cells grown in DMSO, unstained and stained with C-AM and dead cells by heat (65 °C for 15 min) stained with Eth-1 as dead cells control. EF
Double staining for annexin V-fluorescein isothiocyanate (AV-FITC) and propidium iodide (PI) was performed with the Annexin-V apoptosis detection kit (Molecular Probes, Eugene, OR, USA). MDA-MB-231 cells were treated with DiMeOC-Mg-BCD (CC50), or control and vehicle DMSO for 48 h. Cells were washed twice in cold annexin V-buffer and centrifuged at 3000 rpm for 10 min. Pellets were resuspended in 20 μL of annexin V FITC, and after 15 min of incubation in the dark, 480 μL of annexin V-buffer containing 0.5 mg/mL of PI was added according to the manufacturer’s instructions. Annexin V-FITC labelling was recorded on a ThermoFisher Attune Flow Cytometer and analyzed using the FlowJo 7.3.2 software[
75]. (Rodríguez-Hernández et al., 2020).
To evaluate the effect of DiMeOC-Mg-BCD on cell migration, the Wound-Healing assay was carried in a mammary cancer derived cell line (MDA-MB-231), 2 x 105 cells per well were seeded in 24-well plate and cultured at 38 °C and 5% CO2 until confluence reached 90-100%. Then, an artificial space called wound was made on the cell monolayer using a 200 μL sterile micropipette tip, then washed with PBS to remove suspended cells, the supernatant was removed and replaced with new supplemented medium with DiMeOC-Mg-BCD and vehicle dimethyl sulfoxide (DMSO) and incubated for 48 h. We tested with DiMeOC-Mg-BCD (at 1μM). The images were captured by an inverted microscope (DIAPHOT 300 Nikon®, Japan) with a digital camera (AmScope MD500) at 0 h, 24 h and 48 h treatment. Wound areas were obtained using polygon selection and the measure tool of ImageJ software[
74]. The relative migration ratio (%) (RMR) = ((Wound area 0 h − Wound area 24 h or 48 h) ÷ Wound area 0 h) X 100). Results expressed triplicated experiments and significance was obtained by a two-way ANOVA with Tukey’s multiple comparisons test.
For the gene expression study, the cells were plated at a density of 150, 000 cells in six well plates with DMEM. Then, cells were incubated with DiMeOC-Mg-BCD for 48 h and RNA was extracted for gene expression studies. Gene expression was studied by extracting total RNA from treated cells using Trizol[
76]. In all cases, the amount and quality of RNA were estimated spectrophotometrically at 260/280 nm using a Synergy HT (Biotek, USA), and a constant amount of RNA (2 µg) was reverse transcribed using the Maxima First Strand cDNA Synthesis kit for RT-qPCR (Thermo Scientific™, LT) according to the manufacturer’s instructions. Primers and probes for qPCR amplifications were designed by the Universal Probe Library Assay Design Center from Roche, and respective sequences are listed in
Table 6. Identical RT-qPCR conditions were performed for all genes and in all cases, results were normalized against ribosomal protein L32 (
RPL32) used as housekeeping gene. Real time RT-qPCR amplifications were carried on a LightCycler® 480 II (Roche), as previously described[
77].
The statistical analyses were performed using Prism 6 software (GraphPad, San Diego, CA, USA)[
78]. All the experiments represent the mean of three independent assays tested in duplicate and the data shown in the graphs are expressed as the mean ± standard deviation (SD). The data were analyzed using One-way analyses of variance (ANOVAs). Significant differences among means were identified using Dunnett's multiple comparisons tests. Values of
p < 0.001 and
p < 0.05 were considered statistically significant. For gene expression, the results are expressed as the mean ± S.D. Statistical differences were determined by one-way ANOVA followed by appropriate post hoc tests (Holm-Sidak method for pairwise comparisons), using a specialized software package [
79](SigmaPlot 11.0, Jandel Scientific). Experiments were performed from three separated cell cultures and each variable was assessed in triplicate. Differences were considered statistically significant at p < 0.05
The antitumor tests were carried out in the in-ovo model by the company INOVOTION SAS, 5 Avenue du grand sablon 38700 La Tronche, France. Results were delivered with numbers of study: STU20220110, STU20220112, and STU20221017_CU. Double-blind testing was used, labelling the chemical compounds as FOIN-REH-M3 (Phase 1, corresponding to DiMeOC-Mg) and FOINS-REH-M3 (Phase 2, corresponding to DiMeOC-Mg-BCD). The methods used are briefly described here.
In Ovo chicken embryo experiment. Fertilized white Leghorn eggs were incubated at 37.5°C with 50% relative humidity for nine days. At that moment, the CAM was dropped by drilling a small hole through the eggshell into the airndow was cut in the eggshell above the CAM. At least 15 eggs wersurgical manipulation.
The antitumor tests were carried out in the in-ovo model by the company INOVOTION SAS, 5 Avenue du grand sablon 38700 La Tronche, France. Results were delivered with numbers of study: STU20220110, STU20220112, and STU20221017_CU. Double-blind testing was used, labelling the chemical compounds as FOIN-REH-M3 (Phase 1, corresponding to DiMeOC-Mg) and FOINS-REH-M3 (Phase 2, corresponding to DiMeOC-Mg-BCD). The methods used are briefly described here.
In Ovo chicken embryo experiment. Fertilized white Leghorn eggs were incubated at 37.5°C with 50% relative humidity for nine days. At that moment, the CAM was dropped by drilling a small hole through the eggshell into the air sac, and a 1 cm2 window was cut in the eggshell above the CAM. At least 15 eggs were grafted for each group because 10-15% of death may occur by invasive surgical manipulation.
Amplification and grafting of tumor Cells. MDA-MB-231 tumor cell line was cultivated in a DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. On day 9, cells were detached with trypsin, washed with a complete medium, and suspended in a graft medium. An inoculum of 1 X 106 cells was added onto the CAM of each egg, and then eggs were randomized into groups.
Quantitative evaluation of tumour growth. On day 18, the tumor was carefully removed, washed with PBS buffer, and then directly transferred in 4% paraformaldehyde solution for fixation for 48 h. After that, the tumors were weighed. A one-way ANOVA analysis with Student-Newman-Keuls post-test was done on the data (<0.05).
Quantitative evaluation of embryonic toxicity. Embryonic viability was checked daily. The number of dead embryos was counted on day 18 in combination with the observation of eventual visible gross abnormalities to evaluate treatment-induced embryotoxicity. Any visible abnormality observed during the study was also briefly described. A Kaplan-Meyer curve was used to evaluate the final death ratio.
4.1. Synthetic Procedures
The synthon was prepared according to the previously reported methodology and the spectroscopic data correspond adequately to the target compound previusly reported[
46]. 2,2-difluoro-4,6-dimethyl-2H-1,3,2-dioxaborinin-1-ium-2-uide (Synthon, 1), Yield 95%, solid amber, melting point 40 °C.
1H NMR (400 MHz, CDCl
3, TMS): δ 5.96 (s, 1H), 2.27 (s, 6H);
13C NMR (100 MHz, CDCl
3, TMS): δ 192.63, 102.12, 24.32. This data coincided with literature
The aldolic condensation of 3,4-dimethoxybenzaldehyde (2 moles) was realized with experimental conditions reported in the literature[
80,
81].
Dimethoxycurcumin-BF2, 2: 4,6-bis((E)-3,4-dimethoxystyryl)-2,2-difluoro-2H-1λ3,3,2λ4-dioxaborinine: yield 85%, violet powder, m.p. = 226 °C. 1H NMR (400 MHz, DMSO-d6) δ 7.97 (d, J = 15.6 Hz, 2H), 7.50 (d, J = 2.0 Hz, 2H), 7.47 (dd, J = 8.3, 2.0 Hz, 2H), 7.12 (d, J = 15.6 Hz, 2H), 7.08 (d, J = 8.3 Hz, 2H), 6.50 (s, 1H), 3.84 (s, 12H). 13C NMR (100 MHz, DMSO-d6) δ 179.07, 152.58, 149.13, 146.86, 127.13, 125.20, 118.88, 111.81, 111.23, 101.42, 55.79, 55.69. IR 3115 cm−1, 2943 cm−1, 2836 cm−1, 1615 cm−1, 1583 cm−1, 1546 cm−1, 1509 cm−1, 1267 cm−1, 1159 cm−1, 1140 cm−1, 967 cm−1, 821 cm−1, 604 cm−1. MS: m/z = 444 m/z calc = 444.23 for C23H23BF2O6
Synthesis of ligand dimetoxycurcumin: the removal of BF
2 group is obtained using metanol and alumina under similar experimental conditions previously reported[
46].
DiMeOC, 3: (1E,4Z,6E)-1,7-bis(3,4-dimethoxyphenyl)-5-hydroxyhepta-1,4,6-trien-3-one: yield 90%, orange powder, m.p. = 130°C. 1H NMR (400 MHz, DMSO-d6) δ 7.59 (d, J = 15.9 Hz, 2H), 7.35 (d, J = 2.0 Hz, 2H), 7.26 (dd, J = 8.6, 2.0 Hz, 2H), 7.03 – 6.98 (m, 2H), 6.82 (d, J = 15.9, 2H), 6.10 (s, 1H), 3.83 (s, 6H), 3.80 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 183.19, 150.96, 149.02, 140.40, 127.56, 122.90, 122.03, 111.67, 110.47, 100.99, 55.57. The IR spectrum and MS data are the same to previously analysis and coincided for the compound with molecular formula C23H24O6.
The synthesis of the magnesium metal complex, was carried our as follows[
21]: 1 mmol of DiMeOC was dissolved in 15 mL of EtAcO later 0.6 mmol of magnesium(II) acetate. 4H
2O dissolved in MeOH was added drop by drop. The mixture of reaction was stirred during 24 h. A yellow fine powder was filtered off in vacuo and washed with H
2O.
DiMeOC-Mg, 4: magnesium (1E,3Z,6E)-1,7-bis(3,4-dimethoxyphenyl)-5-oxohepta-1,3,6-trien-3-olate: yield 80%, yellow powder, m.p. = 230 °C. 1H NMR (400 MHz, DMSO-d6) δ 7.36 (d, J = 15.6 Hz, 2H), 7.25 (s, 2H), 7.13 (d, J = 8.3 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.71 (d, J = 15.6 Hz, 2H), 5.64 (s, 1H), 3.81 (s, 6H), 3.78 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ 181.24, 149.74, 148.99, 135.81, 128.77, 121.44, 111.72, 109.82, 103.06, 55.54, 55.51. IR 3349 cm−1, 2934 cm−1, 2833 cm−1, 1627 cm−1, 1598 cm−1, 1581 cm−1, 1544 cm−1, 1507 cm−1, 1439 cm−1, 1255 cm−1, 1024 cm−1, 809 cm−1, 470 cm−1. MS-MALDI-TOF: m/z = 817.118 m/z calc = 817.29 for C46H46MgO12.
The inclusion complex was prepared via the coprecipitation method[
43,
48], using the following procedure: In a round flask of 250 mL was dissolved 1 mmol of beta-cyclodextrin (BCD) in 100 mL of distilled water, later 1 mmol of magnesium metal complex (DiMeOC-Mg) dissolved in 100 mL acetone was added dropwise, the mixture was left in continuous stirring at room temperature for 12 hours. Afterwards one yellow precipitate was filtered and washed with distilled water/acetone 1:1 to remove residual BCD or DiMeOC-Mg, precipitate was dried in high vacuum.
DiMeOC-Mg-BCD, 5 (Inclusion complex), yield 75%, pale yellow powder, m.p. = 320 °C. 1H NMR (400 MHz, DMSO-d6) δ 7.34 (d, J = 15.6 Hz, 4H), 7.25 (d, J = 2.0 Hz, 4H), 7.13 (dd, J = 8.4, 1.9 Hz, 4H), 6.95 (d, J = 8.4 Hz, 4H), 6.70 (d, J = 15.6 Hz, 4H), 5.74 (s, 7H), 5.69 (s, 7H), 5.63 (s, 2H), 4.83 (d, J = 3.6 Hz, 7H), 4.46 (t, J = 5.6 Hz, 7H), 3.81 (s, 12H), 3.78 (s, 12H), 3.64 (M, 14H), 3.57(m, 7H), 3.34 (m, 21H). 13C NMR (100 MHz, DMSO-d6) δ 181.22, 149.73, 148.98, 135.79, 128.76, 121.45, 111.73, 109.81, 103.01, 101.95, 81.55, 73.06, 72.43, 72.04, 59.92, 55.54, 55.50. IR 3386 cm−1, 2930 cm−1, 2836 cm−1, 1628 cm−1, 1600 cm−1, 1582 cm−1, 1550 cm−1, 1510 cm−1, 1438 cm−1, 1420 cm−1, 1264 cm−1, 1157 cm−1, 1139 cm−1, 1026 cm−1. MS-ESI: m/z = 1949.8 m/z calc = 1950.15 for C88H116MgO47
Scheme 1.
Synthesis of Dimethoxycurcumin (DiMeOC, 3).
Scheme 1.
Synthesis of Dimethoxycurcumin (DiMeOC, 3).
Scheme 2.
Synthesis of magnesium metal complex (DiMeOC-Mg, 4) and inclusion complex with beta-cyclodextrin (DiMeOC-Mg-BCD, 5).
Scheme 2.
Synthesis of magnesium metal complex (DiMeOC-Mg, 4) and inclusion complex with beta-cyclodextrin (DiMeOC-Mg-BCD, 5).
Figure 1.
1H NMR of 1) BCD inclusion complex, 2) BCD free (400 MHz, D2O δ = 4.8 ppm).
Figure 1.
1H NMR of 1) BCD inclusion complex, 2) BCD free (400 MHz, D2O δ = 4.8 ppm).
Figure 2.
1H NMR DOSY spectra a) DiMeOC-Mg, b) DiMeOC-Mg-BCD 1:1 (400MHz, DMSO-d6).
Figure 2.
1H NMR DOSY spectra a) DiMeOC-Mg, b) DiMeOC-Mg-BCD 1:1 (400MHz, DMSO-d6).
Figure 3.
150 MHz 13C ssNMR spectra of 1) DiMeOC, 2) DiMeOC-Mg, 3) BCD, 4) DiMeOC-Mg-BCD.
Figure 3.
150 MHz 13C ssNMR spectra of 1) DiMeOC, 2) DiMeOC-Mg, 3) BCD, 4) DiMeOC-Mg-BCD.
Figure 4.
SEM of a) BCD, b) DiMeOC, c) DiMeOC-Mg and d) DiMeOC-Mg-BCD.
Figure 4.
SEM of a) BCD, b) DiMeOC, c) DiMeOC-Mg and d) DiMeOC-Mg-BCD.
Figure 5.
TGA A) beta-cyclodextrin (BCD), B) DiMeOC-Mg and C) DiMeOC-Mg-BCD.
Figure 5.
TGA A) beta-cyclodextrin (BCD), B) DiMeOC-Mg and C) DiMeOC-Mg-BCD.
Figure 6.
DSC curves of BCD and complexes.
Figure 6.
DSC curves of BCD and complexes.
Figure 7.
DiMeOC-Mg-BCD induced morphological changes in the MDA-231-MB breast cancer cell line. Giemsa stain of cells incubated with CC50 concentrations for 48 h. Cell size reduction, rounding and pyknotic nuclei (*), cytoplasm vacuolization (+), chromatin condensation, apoptotic bodies (black arrow) Bar: 20 µm (20x).
Figure 7.
DiMeOC-Mg-BCD induced morphological changes in the MDA-231-MB breast cancer cell line. Giemsa stain of cells incubated with CC50 concentrations for 48 h. Cell size reduction, rounding and pyknotic nuclei (*), cytoplasm vacuolization (+), chromatin condensation, apoptotic bodies (black arrow) Bar: 20 µm (20x).
Figure 8.
Effect of DiMeOC-Mg-BCD on MDA-231-MB breast cancer cell line, esterase activity and cell membrane damage. Cells were incubated with different treatments for 48 h and stained with calcein fluorescence stain (C-AM+) and ethidium homodimer (Eth-1+). (a) Flow cytometry dot plots: 1) Unstained cells (DMEM, 2) Vehicle and live cells/C-AM+, 3) cell membrane damage shown or dead cells/Eth-1+ after heating (65ºC), 4) vehicle used and 5) DiMeOC-Mg-BCD (CC50). (b) Percentage of esterase activity, c) Percentage of dead cells. The results correspond to mean±SD in three independent duplicated experiments. One-way analysis of variance and Tukey multiple comparison test were applied. Asterisks represent significant differences compared with the Vehicle DMSO, * p< 0.001.
Figure 8.
Effect of DiMeOC-Mg-BCD on MDA-231-MB breast cancer cell line, esterase activity and cell membrane damage. Cells were incubated with different treatments for 48 h and stained with calcein fluorescence stain (C-AM+) and ethidium homodimer (Eth-1+). (a) Flow cytometry dot plots: 1) Unstained cells (DMEM, 2) Vehicle and live cells/C-AM+, 3) cell membrane damage shown or dead cells/Eth-1+ after heating (65ºC), 4) vehicle used and 5) DiMeOC-Mg-BCD (CC50). (b) Percentage of esterase activity, c) Percentage of dead cells. The results correspond to mean±SD in three independent duplicated experiments. One-way analysis of variance and Tukey multiple comparison test were applied. Asterisks represent significant differences compared with the Vehicle DMSO, * p< 0.001.
Figure 9.
Flow cytometry dot plots of the effect of DiMeOC-Mg-BCD on MDA-MB-231 breast cancer cell lines on apoptosis. Cells were incubated with different treatments for 48 h and stained with fluorescent stain Annexin V-FITC/ Propidium iodide (AV/PI). The heat renders necrotic positive cells (AV-/PI+). DMEM live cells were negative for both stains (AV-/PI-), and DMSO 5% is a positive control for early apoptosis (AV+/PI-). Vehicle DMSO 1.3% corresponds to the negative controls of the treatment. The dot plots are representatives of three independent experiments performed in duplicate.
Figure 9.
Flow cytometry dot plots of the effect of DiMeOC-Mg-BCD on MDA-MB-231 breast cancer cell lines on apoptosis. Cells were incubated with different treatments for 48 h and stained with fluorescent stain Annexin V-FITC/ Propidium iodide (AV/PI). The heat renders necrotic positive cells (AV-/PI+). DMEM live cells were negative for both stains (AV-/PI-), and DMSO 5% is a positive control for early apoptosis (AV+/PI-). Vehicle DMSO 1.3% corresponds to the negative controls of the treatment. The dot plots are representatives of three independent experiments performed in duplicate.
Figure 10.
Effect of DiMeOC-Mg-BCD on MDA-MB-231 breast cancer cell lines on apoptosis. Cells were incubated with different treatments for 48 h and stained with fluorescent stain Annexin V-FITC/ Propidium iodide (AV/PI). Heat was used as necrotic positive cells (AV-/PI+), DMEM live cells were negative for both stains (AV-/PI-), and DMSO 5% was the positive control of early apoptosis (AV+/PI-). Vehicle/ DMSO 1.3% corresponds to the negative control of the treatment. Data are represented by mean and SD of three independent experiments performed in duplicate. One-way analysis of variance and Tukey multiple comparison test were applied. Asterisks represent significant differences from vehicle control. *** p< 0.001.
Figure 10.
Effect of DiMeOC-Mg-BCD on MDA-MB-231 breast cancer cell lines on apoptosis. Cells were incubated with different treatments for 48 h and stained with fluorescent stain Annexin V-FITC/ Propidium iodide (AV/PI). Heat was used as necrotic positive cells (AV-/PI+), DMEM live cells were negative for both stains (AV-/PI-), and DMSO 5% was the positive control of early apoptosis (AV+/PI-). Vehicle/ DMSO 1.3% corresponds to the negative control of the treatment. Data are represented by mean and SD of three independent experiments performed in duplicate. One-way analysis of variance and Tukey multiple comparison test were applied. Asterisks represent significant differences from vehicle control. *** p< 0.001.
Figure 11.
Effect of DiMeOC-Mg-BCD on MDA-MB-231 cell migration. The wound-healing assay in the migration of MDA-MB-231 cancer cell lines. Vehicle/ DMSO 1.3% corresponds to the negative control of the treatment. Data are represented by mean and SD of three independent experiments performed in duplicate. One-way analysis of variance and Tukey multiple comparison test were applied. Asterisks represent significant differences from vehicle control. * p< 0.05 (24h), *** p< 0.001 (48h).
Figure 11.
Effect of DiMeOC-Mg-BCD on MDA-MB-231 cell migration. The wound-healing assay in the migration of MDA-MB-231 cancer cell lines. Vehicle/ DMSO 1.3% corresponds to the negative control of the treatment. Data are represented by mean and SD of three independent experiments performed in duplicate. One-way analysis of variance and Tukey multiple comparison test were applied. Asterisks represent significant differences from vehicle control. * p< 0.05 (24h), *** p< 0.001 (48h).
Figure 12.
Effect DiMeOC-Mg-BCD on MMP-2, MMP-9, IL-6 and STAT3 gene expression of MDA-MB-231 breast cancer cell lines. The effect on gene expression is measured after 48 h of treatment. Each bar represents the mean ± S.D. of n = 3. Values for the Vehicle control DMSO were set at 1.3% and set to 1. One-way analysis of variance and Tukey multiple comparison test were applied. Asterisks represent significant differences compared with the Vehicle, DMSO 1.3%, * p< 0.001.
Figure 12.
Effect DiMeOC-Mg-BCD on MMP-2, MMP-9, IL-6 and STAT3 gene expression of MDA-MB-231 breast cancer cell lines. The effect on gene expression is measured after 48 h of treatment. Each bar represents the mean ± S.D. of n = 3. Values for the Vehicle control DMSO were set at 1.3% and set to 1. One-way analysis of variance and Tukey multiple comparison test were applied. Asterisks represent significant differences compared with the Vehicle, DMSO 1.3%, * p< 0.001.
Figure 13.
The proposed mechanism of action of DiMeOC-Mg-BCD on the IL-6/ STAT3 pathway is that this compound downregulates IL-6 and STAT3 expression, which could inhibit STAT translocation into the nucleus, resulting in the suppression of cell proliferation, invasion, and metastasis in hormone receptor-negative breast cancer. DiMeOC-Mg-BCD, JAK: Janus kinase, STAT: signal transducer and activator of transcription, IL-6: interleukin-6.
Figure 13.
The proposed mechanism of action of DiMeOC-Mg-BCD on the IL-6/ STAT3 pathway is that this compound downregulates IL-6 and STAT3 expression, which could inhibit STAT translocation into the nucleus, resulting in the suppression of cell proliferation, invasion, and metastasis in hormone receptor-negative breast cancer. DiMeOC-Mg-BCD, JAK: Janus kinase, STAT: signal transducer and activator of transcription, IL-6: interleukin-6.
Figure 14.
The mean tumor weights (%) obtained for DiMeOC-Mg and DiMeOC-Mg-BCD treated groups at several dosages. .
Figure 14.
The mean tumor weights (%) obtained for DiMeOC-Mg and DiMeOC-Mg-BCD treated groups at several dosages. .
Figure 15.
Kaplan-Meier curve of embryo survival by exposition to DiMeOC-Mg at 0.375, 0.75, and 1.5 mg/Kg and DiMeOC-Mg-BCD at 0.065 and 0.65 mg/Kg, P=0.6540.
Figure 15.
Kaplan-Meier curve of embryo survival by exposition to DiMeOC-Mg at 0.375, 0.75, and 1.5 mg/Kg and DiMeOC-Mg-BCD at 0.065 and 0.65 mg/Kg, P=0.6540.
Table 1.
1H NMR chemical shifts of the BCD (D2O, 400 MHz).
Table 1.
1H NMR chemical shifts of the BCD (D2O, 400 MHz).
Proton |
BCD free (ppm) |
BCD complex (ppm) |
Δδppm |
H1 |
5.11 |
5.06 |
-0.05 |
H2 |
3.68 |
3.63 |
-0.05 |
H3 |
3.99 |
3.93 |
-0.06 |
H4 |
3.62 |
3.58 |
-0.04 |
H5 H6 |
3.88 3.91 |
3.80 3.89 |
-0.08 -0.02 |
Table 2.
Diffusion coefficients (D) of DiMeOC-Mg and BCD.
Table 2.
Diffusion coefficients (D) of DiMeOC-Mg and BCD.
Compound |
Dfree |
Dinclusion complex |
HOD |
DiMeOC-Mg |
1.20 x10-10 cm2/s |
9.82 x10-11 cm2/s |
6.61 x10-10 cm2/s |
BCD |
9.6 x10-11 cm2/s |
8.34 x10-11 cm2/s |
7.5 x10-10 cm2/s |
Table 3.
Solubility in aqueous media (PBS) of complexes.
Table 3.
Solubility in aqueous media (PBS) of complexes.
Compounds |
Solubility μg /mL |
DiMeOC-Mg |
15.6 ± 0.048 |
DiMeOC-Mg-BCD |
98.2 ± 0.038 |
Table 4.
CC50 (μM) for cancer cell lines with DiMeOC and their complexes.
Table 4.
CC50 (μM) for cancer cell lines with DiMeOC and their complexes.
Compounds |
MDA-MB-231 |
DiMeOC |
>100 |
DiMeOC-Mg |
22.04 ± 0.06 |
DiMeOC-Mg-BCD |
10.73 ± 0.1 |
Table 5.
Treatments and final dosages for study in ovo.
Table 5.
Treatments and final dosages for study in ovo.
Group |
Injected concentration (μM) |
In ovo final dose (mg/Kg) |
Control (DMSO 1%) |
- |
- |
Paclitaxel |
10.0 |
0.014 |
DiMeOC-Mg-1 |
276.1 |
0.375 |
DiMeOC-Mg-2 |
552.3 |
0.750 |
DiMeOC-Mg-3 |
1104.5 |
1.5 |
DiMeOC-Mg-BCD-1 |
10 |
0.065 |
DiMeOC-Mg-BCD-2 |
100 |
0.650 |
Table 6.
Oligonucleotides used.
Table 6.
Oligonucleotides used.
Gene |
Forward |
Reverse |
*Probe |
Accession Number |
RPL32 |
GAAGTTCCTGGTCCACAACG |
GAGCGATCTCGGCACAGTA |
17 |
NM_000994.3 |
MMP-2 |
ATAACCTGGATG CCGTCGT |
AGGCACCCTTGAA GAAGTAGC |
70 |
NM_001302510.1 |
MMP-9 |
GCCACCCGAGTGTAACCATA |
GAACCAATCTCAC CGACAGG |
6 |
NM_004994.2 |
IL-6 |
GATGAGTACAAAAGTCCTGATCCA |
CTGCAGCCACTGGTTCTGT |
40 |
NM 000600.1 |
STAT3 |
CCTCTGCCGGAGAAACAGT |
CATTGGGAAGCTGTCACTGTAG |
1 |
NM_139276.2 |