Antiproliferative Activity of Novel Derivative of Thiopyran on Breast and Colon Cancer Lines and DNA Binding

Chemicals and materials

Trypsin, trypan blue, antibiotic and antimycotic agent, fetal bovine serum (FBS), sulforhodamine B (SRB), and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. Chemicals and solvents for synthesis of 4 were purchased from Sigma-Aldrich Italia. NMR spectra were obtained with a Varian Gemini 300 MHz spectrometer. Chemical shifts (d) are reported in parts per million downfield from tetramethylsilane and were referenced from solvent references. Electron impact (70 eV) mass spectra were obtained on a ThermoQuest Finnigan GCQ Plus mass spectrometer. Chromatographic separations were performed on silica gel columns by flash (Kieselgel 40, 0.04–0.06 mm; Merck) or gravity column (Kieselgel 60, 0.063–0.200 mm; Merck) chromatography. Reactions were followed by thin-layer chromatography on Merck aluminum silica gel (60 F254) sheets that were visualized under an ultraviolet (UV) lamp. Evaporation was performed in vacuo (rotating evaporator).

Chemical synthesis of 4

To a stirred solution of dimethyl acetylenedicarboxylate (DMAD [2], 2 mmol) and arylisothiocyanate (1, 2 mmol) in 10 mL of acetonitril, a mixture of 1,3-dicarbonyl (2 mmol) and sodium hydride (2 mmol) in acetonitril was added at room temprature. The reaction mixture was stirred for 8 h. The solvent was removed under reduced pressure and the residue was separated by silica gel column chromatography (Merck 230–400 mesh) using petroleum ether-EtOAc (7:1) as eluent to give 4. Oil; yield: 0.68 g (92%).

Cell culture and cell morphology

Human breast cancer MCF-7 and colon cancer HCT-15 cell lines were supplied from ATCC and grown as a monolayer in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 2 mM glutamine, 100 units/mL penicillin, and 100 mg/mL streptomycin. Cultures were maintained at 37°C with 5% CO₂ in a humidified atmosphere. Cell shape and morphology of treated and untreated cells were viewed using an inverted phase-contrast microscope model Zeiss and photographed using a Nikon camera attached to the microscope.

In vitro evaluation of cytotoxic activity

Growth activity of 4 in vitro was evaluated by the SRB assay (Mehrzad et al., 2010; Rajabi et al., 2010). The 4 stock solution (10 mM in DMSO) was stored at 4°C and diluted with DMEM to 0.1–1 μM at room temperature before treatment. The final percentage of DMSO in the reaction mixture was <1% (v/v). Cells (2×10³ cells/well) were plated in 96-well plates and incubated in medium for 24 h. Serial dilutions of 4 were added and incubated at 37°C for 96 h. The assay was terminated by the addition of 50 μL of ice-cold trichloroacetic acid (final concentration, 10% TCA) and incubated for 60 min at 4°C. The plates were washed five times with distilled water and air-dried. SRB solution (50 μL) at 0.4% (w/v) in 1% acetic acid was added to each of the wells, and plates were incubated for 30 min at room temperature. The residual dye was removed by washing five times with 1% acetic acid. The plates were air dried under a hood. Bound stain was subsequently eluted with 10 mM Trizma base, and the absorbance was read on an enzyme-linked immunosorbent assay plate reader at a wavelength of 540 nm and used as a relative measure of viable cell number. The percentage of growth inhibition was calculated using the following equation: percentage growth inhibition (1−A _t/A _c)×100, where A _t and A _c represent the absorbance in treated and control cultures, respectively. IC₅₀ was determined by interpolation from dose–response curves.

Clonogenic assay

MCF-7 and HCT-15 cells were plated at a density of 700 cells/well in 12-well plates and treated with 0, 5, 10, 25, 50, 80, and 100 μM 4 for 14 days. The plates were rinsed in PBS and colonies were fixed in methanol, stained with 10% Giemsa, and counted under a light microscope.

Cell invasion

Briefly, 6.5-mm-diameter polycarbonate filters of 8 μm pore size (Transwell; Costar) were coated with 20 μg of basement membrane Matrigel, dried, and reconstituted at 37°C with DMEM before use. The lower chambers were filled with a conditioned medium of fibroblasts as chemoattractant, supplemented with 2 mg/mL bovine serum albumin (BSA) and 10 ng/mL basic fibroblast growth factor. After a brief exposure to ethylenediaminetetraacetic acid, the cells were collected and then added on the upper chamber (10⁵ cells/chamber in DMEM containing 0.2 mg/mL BSA and 10% FBS). 4 was also added to the upper chamber. After 24 h of incubation at 37°C, the filters were removed and stained with a Diff-Quick kit (DADE). The number of cells that had spread on the lower surface of the filter was counted with a microscope.

Cell adhesion

Adhesion assay of tumor cells to basement membrane was performed in 96-well plates (Costar) using substrate for cell endogenous phosphatases. The wells were coated overnight at 4°C with Matrigel (20 μg/100 μL) or 1% BSA as control. Nonspecific binding sites were blocked with 1% BSA for 1 h at 37°C and washed three times with adhesion buffer (10 mM HEPES, 140 mM NaCl, 5.6 mM glucose, 5.4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, and 1 mM MnCl₂ [pH 7.4]). The cells (5×10⁴/well in adhesion buffer), pretreated or not with 4 were allowed to adhere for 20 min at 37°C. Then, nonadhered cells were removed by washing three times with adhesion buffer. The substrate for phosphatases (p-nitrophenyl phosphate; Sigma) in acetate buffer containing 0.1% Triton X-100 (pH 5.5) was added to each well and incubated for 1 h at 37°C. The reaction was stopped with 1 N NaOH, and absorbance was measured at 405 nm. Each point was determined in triplicate.

DNA titration experiments

The absorbance at 260 and 280 nm was recorded to check the protein content of DNA solution (Nafisi et al., 2008). DNA (5 mg/mL) was dissolved in distilled water (pH 7) at 4°C for 24 h with occasional stirring to ensure the formation of a homogeneous solution. The final concentration of the DNA solution was spectrophotometrically determined at 260 nm using molar extinction coefficient ɛ ₂₆₀=6600 cm⁻¹ M⁻¹ (expressed as molarity of phosphate groups). The UV absorbance at 260 nm of a diluted solution (1/187.5) of DNA used in our experiments was 0.666 and the final concentration of the DNA solution was 12.5 mM. Solutions of 4 (0.05–12.5 mM) were prepared in distilled water and added dropwise to DNA solution in order to attain the desired ligand/DNA molar ratios (r) of 1/40, 1/20, 1/10, 1/5, 1/2, and 1 with a final DNA concentration of 6.25 mM. The pH of the solutions was adjusted at 7.0±0.2 using NaOH solution.

Chemical synthesis of 4

A facile synthesis of 4 is described via reaction between 1,3-dicarbonyls, electron-deficient acetylenic compounds such as DMAD, and aryleisothiosyanate in the presence of sodium hydride as a base in acetonitril at room temperature (Fig. 1). The advantages of this protocol are that no catalyst is required for this reaction and the simplicity of the present procedure makes it an attractive alternative to the complex multistep approaches. The reaction mixture was stirred for 8 h. The solvent was removed under reduced pressure and the residue was separated by silica gel column chromatography to give 4.

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FIG. 1.

Synthesis of dimethyl-5-acetyl-4-methyl-6-(4-methylphenylimino)-6H-thiopyran-2,3-dicarboxylate (4).

¹H-NMR (500 MHz, CDCl₃): δ=2.07 (3 H, s, Me), 2.15 (3 H, s, Me), 2.54 (3 H, s, Me), 3.75 (3 H, s, MeO), 3.84 (3 H, s, MeO), 7.14 (2 H, d, ³ J=7.4 Hz, 2 CH), 7.95 (2 H, d, ³ J=7.4 Hz, 2 CH). ¹³C-NMR (125.7 MHz, CDCl₃): δ=16.5 (Me), 17.8 (Me), 31.2 (Me), 52.6 (MeO), 53.4 (MeO), 121.4 (2 CH), 125.8 (2 CH), 127.6 (C), 133.4 (C), 135.2 (C), 138.3 (C), 142.5 (C), 153.8 (C-N), 155.2 (C=N), 161.2 (C=O), 167.3 (C=O), 208.9 (C=O). IR (KBr) (ν _max/cm⁻¹): 1742 (C=O), 1727 (C=O), 1664 (C=O), 1575, 1454, 1335, 1247, 1120 cm⁻¹. Anal. Calcd. for C₁₉H₁₉NO₅S (373.42): C, 61.11; H, 5.13; N, 3.75; found: C, 60.97; H, 5.00; N, 3.67. Oil; yield: 0.59 g (80%).

Cytotoxic evaluation in vitro and clonogenic assay

One of the factors of importance in determining the killing of mammalian cells following exposure to a variety of anticancer agents is the proliferative state of cell population (Valeriote and van Putten, 1975). The compound 4 was evaluated for its growth inhibitory effect on HCT-15 colon and MCF-7 breast cancer cell lines. The cells were treated with 4 at different concentrations, ranging from 1 to 100 μM, for 4 days using the SRB assay (Fig. 2A, B). In the presence of different doses of 4, the cells were inhibited ranging from 10% to 90% with a loss of viable cells (Fig. 2C, D). The compound 4 inhibited the proliferation of MCF-7 and HCT-15 cells in a concentration- and time-dependent manner. The IC₅₀ was determined by interpolation from dose–response curves. The percentage of growth inhibition was calculated by comparison of the absorbance of treated cells versus control cells. The viability of the HCT-15 cells was unaffected by less than 0.2 μM of 4, but changed to 80% in response to 0.5 μM 4. However, the viability of HCT-15 decreased to 50% (IC₅₀) in response to 3.5 μM of 4 and continued to decrease as the concentration of 4 was increased; viability was 20% and 5% with exposure to 10 μM and 50 μM of 4, respectively. Subsequently, the viability of the MCF-7 cells was also unaffected by less than 0.5 μM of 4 treatment but changed to 60% in response to 1 μM 4, decreased to 50% (IC₅₀) in response to 1.5 μM 4, and continued to decrease as the concentration of 4 was increased; viability was 20% with 10 μM. Therefore, the IC₅₀ calculated for HCT-15 was 3.5 and 1.5 μM for MCF-7 (Fig. 2C, D). Control groups showed regular polygonal shape, and cell antennas were short. Cell morphology was affected by 4 treatment and included loss of adhesion, rounding, cell shrinkage, and detachment from the substratum. Analysis of clonogenic activity in HCT-15 and MCF-7 cells treated with 4 at concentrations ranging from 0.5 to 100 μM of 4 for 14 days revealed a complete inhibition of colony formation at 4 μM for HCT-15 and 2 μM for MCF-7 cells, whereas concentrations lower than 5 μM were ineffective (Fig. 2E, F).

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FIG. 2.

Effects of 4 on the proliferation of HCT-15 and MCF-7 cells. (A) Effect of 4 on the proliferation of HCT-15; (B) effects of 4 on the proliferation of MCF-7; (C) inhibition of 4 on HCT-15 growth; (D) inhibition of 4 on MCF-7 growth. The percentage of growth inhibition was calculated using the following equation: (1−A _t/A _c)×100, where A _t and A _c represent the absorbance in treated and control cultures, respectively. IC₅₀ was determined by interpolation from dose–response curves. Dose–response curve of 4-mediated inhibition of HCT-15 (E) and MCF-7 (F) cell colony formation.

Effects of 4 on cell invasion and cell adhesion

We next examined the effect of 4 on HCT-15 and MCF-7 cell invasion through Matrigel, a reconstituted extracellular matrix (ECM). Cell invasion involves a series of proteolytic events involving proteolytic enzymes such as urokinase-plasminogen activator (u-PA) and matrix metalloproteinases (MMPs). The concept that these proteases are involved in invasion and metastasis, as well as tumor angiogenesis, has been well accepted (Mignatti and Rifkin, 1996). As shown in Figure 3A, 3.5 μM and 1.5 μM of 4 greatly reduced the invasiveness of HCT-15 cells and MCF-7, respectively (Fig. 3B): 60% inhibition for HCT-15 and 75% inhibition for MCF-7 compared with control. Therefore, compound 4 reduced tumor cell invasiveness through Matrigel, and we propose that this may relate to a decrease in protease production (Shao et al., 1998; Lindenmeyer et al., 2001). However, the reduced invasiveness and migration of cells induced by 4 was not simply due to insufficient protease-dependent matrix degradation. Although cell surface saturation by exogenous scu-PA did increase the invasion capacity of the 4-treated cells, it was still reduced compared with that of scu-PA–saturated control cells.

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FIG. 3.

(A, B) Invasion through Matrigel. Cells were added in upper chamber with 4 and assay time was 24 h. Inhibitory effect of 4 on HCT-15 cell (C) and MCF-7 cell (D) adhesion to Matrigel. Pretreated cells were allowed to adhere for 20 min, and absorbance was measured at 405 nm. (E) The plot of 1/(A−A ₀) versus 1/[L] for DNA and iPA complexes at different drug concentrations.

Indeed, 4 also altered both cells' adhesive behavior in ECM proteins and our results showed that the ability of 4-treated cells to adhere to Matrigel was reduced in a dose-dependent manner, demonstrating 50% inhibition at 1.25 μM 4 for both cell lines (Fig. 3C, D). Pretreated cells were allowed to adhere for 20 min, and absorbance was measured at 405 nm. Values are means±standard deviation of three separate experiments each carried out in triplicate. As 4 altered the cells' adhesive behavior in ECM proteins, this inhibitory effect on cell adhesion seems not to be related to the u-PA system, inasmuch as it was not influenced by saturating the cells with scu-PA. The reason for this reduced adhesion is unclear, and further study is needed to identify the cell surface adhesive molecules and the matrix proteins involved. Our results reveal that 4, which inhibits the expression of proteases involved in degradation of the ECM, may also regulate other cellular activities associated with the invasive phenotype, including cell migration and cell-matrix adhesion. The decrease in invasiveness may be also due to modification of the ultrastructure of these cells, which was not restored by u-PA.

Calculation of binding constant with DNA

Small molecules that bind reversibly to DNA are among the antitumor drugs currently used in chemotherapy. In the pursuit of a more rational approach to cancer chemotherapy based upon these molecules, it is necessary to exploit the interdependency between DNA-binding affinity, sequence selectivity, and cytotoxicity. Although there are previous studies considering that large and more complex molecules are more potent antitumor agents (Huang et al., 2006), there is no clear association between biological potency and the strength of binding to DNA, but there are some examples illustrating that changes in DNA binding among structurally related molecules can be accompanied by abrupt changes in biological activity (Denny et al., 1983). The strategy followed here is the calculation of the overall binding constant, which was carried out on the basis of UV absorption, and the equilibrium for 4 and DNA complex can be described as follows: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}\begin{align*} [ { \rm DNA} + {\bf 4} ] \leftrightarrow [ { \rm DNA} - {\bf 4} ] K_{\bf 4} \\ K_{\bf 4} = [ { \rm DNA} - {\bf 4} ] / [ { \rm DNA} ] [ {\bf 4} ] \end{align*}\end{document}

The double reciprocal plot of 1/[A−A ₀] versus 1/[ligand] is linear and the association binding constant (K) is calculated from the ratio of the intercept on the vertical coordinate axis to the slope (Tajmir-Riahi et al., 1995; Mandeville et al., 2010; Froehlich et al., 2011). Concentrations of the complexed ligand were determined by subtracting absorbance of free DNA at 260 nm from those of the complexed DNA. Concentration of the free ligand was determined by subtraction of complexed ligand from total ligand used for the experiment. Our data show that 1/[complexed ligand] almost proportionally increases as a function of 1/[free ligand] (Fig. 3E). Therefore, an overall binding constant K _4-DNA=9.8×10⁴ M⁻¹ was estimated for 4-DNA.