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[email protected] Anti-Cancer Agents in Medicinal Chemistry, 2015, 15, 000-000
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Isothiouronium Salts Reduce NRAS Expression, Induce Apoptosis and Decrease Invasion of Melanoma Cells Júlia Cisilotto1, Misael Ferreira2, Fabíola Branco Filippin-Monteiro3, Adailton João Bortoluzzi2, Marcus Mandolesi Sá2 and Tânia Beatriz Creczynski-Pasa1,* 1
Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil; Departamento de Química, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil; 3Departamento de Análises Clínicas, Universidade Federal de Santa Catarina, 88040-900, Florianópolis, SC, Brazil 2
Abstract: Melanoma is a very aggressive type of skin cancer. Mutation in BRAF and NRAS are often found in patients with this disease. Therefore, in recent years the search for new molecules that inhibit these proteins has been intensified. After many years with no new treatments for melanoma, the U.S. Food and Drug Administration (FDA) recently approved vemurafenib. However, many patients have already acquired resistance and have experienced severe side effects. Therefore, this work aims to evaluate a new set of compounds including allylic isothiouronium salts (1, 2 and 3), N-phenyl-substituted analog (4) and isothiosemicarbazide salts (5 and 6) for their potential antimelanoma activity. To this end, viability assay, cell cycle analysis, expression of NRAS and BRAF, as well as migration and invasion assay were performed with different melanoma cell lines. Isothiouronium salts 1-3 presented CC50 (concentration required to reduce the cell number by 50%) in a range of 7-28 µM. Furthermore, salt 1 significantly decreased the expression of NRAS. However, cells incubated with these salts did not disturb the cell cycle phases; instead, an increase in the number of apoptotic cells was observed. Regarding potential anti-invasion effects, both 1 and 2 prevented cell migration as well as cell invasion. Finally, when salts 1 and 2 were associated with vemurafenib, a marked decrease in cell viability was observed when compared to the compounds incubated alone. Briefly, the salts exhibited interesting results, especially 1, which decreased the expression of NRAS, increased apoptotic cells and, when combined with vemurafenib, resulted in a synergistic effect. Therefore, we intend to test compound 1 in pre-clinical studies.
Keywords: anti-invasion activity, antitumoral action, cytotoxicity, expression of NRAS, isothiouronium salts, melanoma cells. 1. INTRODUCTION Metastatic melanoma is the most aggressive type of skin cancer, and overall 10-15% of patients survive 5 years after diagnosis [1]. In addition, the incidence of this cancer has increased in recent decades [2]. It arises from the malignant transformation of melanocytes cells responsible for producing melanin for skin pigmentation [3] and among several molecular pathways involved in melanoma progression, the MAPK pathway is very important. This pathway includes RAS (HRAS, KRAS and NRAS), RAF (ARAF, BRAF and CRAF), MEK1/2 and ERK1/2 serine threonine kinase cascade. These serial proteins are involved in proliferation, differentiation and cellular survival [4]. In melanoma, NRAS mutations are found in about 20% of cases and BRAF in approximately 60% [5-7]. The presence of NRAS and BRAF mutations in patients with metastatic lesion is associated with a poor prognosis and a low overall survival rate [8]. NRAS mutation in human melanoma can contribute to tumor growth, cellular proliferation and blocked apoptosis [9, 10]. Furthermore, melanoma with BRAF mutation may have defects in the checkpoints of the cell cycle [11] and may assist in the process of invasion and metastasis [12]. Unfortunately, treatment failure in malignant melanoma is a clinical problem with a profound social impact [13]. Dacarbazine (an alkylating agent) was the first drug approved in 1975 by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic melanoma [14]. This drug is still in use for many patients, and the response rates for single-agent therapy range from 5 to 28% [1, 15-18]. Since the discovery of BRAF mutations in malignant melanomas in 2002 [5], research on drugs and active compounds that alter MAPK pathway has been intensified, *Address correspondence to this author at the Departamento de Ciências Farmacêuticas, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil; Tel: +55 48 37218057; Fax: +55 48 37219542; E-mail:
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resulting in a fundamental change in the approach to melanoma therapies [19]. Thus, in 2011 the FDA approved an inhibitor of BRAFV600E, vemurafenib, for metastatic melanoma. The treatment with vemurafenib increased median survival and response rates compared with dacarbazine [1, 15]. However, many patients treated with vemurafenib developed secondary mutations lead to the development of severe adverse effects, especially cutaneous [1]. Drugs combination seems to be an alternative for decreasing the adverse effects and improving the response rate of inhibitors of MAPK pathway [20]. Therefore, new therapeutic strategies to treat metastatic melanoma are required. Isothiouronium salts, thioureas and analogs constitute a class of biologically important compounds that have been studied as inhibitors of protein kinase C [21], as well as agonists of GABAtype [22] and histamine-H3 receptors [23]. They also exhibit significant pharmacological activities, such as anesthetic [24], antimicrobial [23, 25-28], and antitumoral [29]. We recently discovered that functionalized allylic isothiouronium salts were moderately active against Mycobacterium tuberculosis [30]. The aim of the current study is to evaluate whether a class of compounds like this could also exhibit antitumoral activity. Therefore, we studied the cytotoxicity properties of six representative compounds (Fig. 1): three allylic isothiouronium salts (1, 2 and 3), an N-phenylsubstituted analog (4) and two isothiosemicarbazides (5 and 6), as well as their ability to induce cell death in different metastatic melanoma cell lines. 2. MATERIALS AND METHODS 2.1. Reagents Vemurafenib (also known as PLX4032) was obtained from Selleck Chemicals (USA). The cell culture medium was purchased from Cultilab (São Paulo, SP). Serum and antibiotics were acquired from GIBCO (Grand Island, NY). The ApopNexin™ FITC Apoptosis Detection Kit was purchased from BD (Becton Dickinson, Franklin Lakes, NJ). The DNase I, TRIzol reagent and the primers were © 2015 Bentham Science Publishers
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structural elucidation by single crystal X-ray crystallographic analysis (see crystallographic data in the Supplementary Material). Spectral Data for Novel Compounds 5 and 6 Methyl (Z)-2-[(isothiosemicarbazido)methyl]-3-(4-methoxyphenyl)2-propenoate hydrobromide (5) White solid, 91% yield, mp 160.2-162.5 °C, 1H NMR (400 MHz, DMSO-d6): mixture of isomers (88:12) coexisting in solution (data for the major isomer) δ 3.75 (s, 3H), 3.79 (s, 3H), 4.26 (s, 2H), 7.05 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 7.77 (s, 1H); 13 C NMR (100 MHz, D2O, DMSO-d6 as internal standard): δ 28.9 (CH2), 52.5 (OCH3), 55.5 (OCH3), 114.6 (2 × CH), 121.8 (C), 125.8 (C), 132.0 (2 × CH), 143.3 (=CH), 160.7 (C), 165.6 (C), 166.6 (C); IR (KBr) νmax/cm-1: 3709, 3362, 3320, 3242, 3143, 2952, 2837, 1706, 1658, 1620, 1599, 1509, 1442, 1259, 1179, 822; Elemental Analysis: calcd for C13H18BrN3O3S (%): C, 41.50; H, 4.82; N, 11.17; S, 8.52; found: C, 41.61; H, 4.80; N, 11.28; S, 8.73. Methyl (Z)-2-[(isothiosemicarbazido)methyl]-3-(4-nitrophenyl)-2propenoate hydrobromide (6)
Fig. (1). Chemical structures of isothiouronium (1-3), N-phenylisothiouronium (4) and isothiosemicarbazide (5, 6) salts evaluated as antitumoral agents.
acquired from Invitrogen (Carlsbad, CA). The High-Capacity cDNA Reverse Transcription Kit and the Power SYBR-Green PCR Master Mix were purchased from Thermo Scientific (Waltham, Massachusetts, USA). The others reagents were purchased from Sigma-Aldrich (St. Louis, MO). 2.2. Chemistry Isothiouronium salts 1-4 were prepared as previously described by Silveira et al. and Sá et al. [30, 31] (Scheme 1). For the synthesis of isothiosemicarbazide salts 5 and 6, a typical procedure was employed: to a stirred solution of allylic bromide 7d or 7e (1.0 mmol) in 3.0 mL of acetonitrile at 25 °C was added thiosemicarbazide (8c; 0.95 mmol). After stirring for 1 h, the insoluble solid formed was separated by filtration and washed with ethyl ether/CH2Cl2 (4:1) to obtain the expected products 5 or 6 in pure form. The methoxy-substituted isothiosemicarbazide salt 5 slowly crystallized in ethyl acetate, which allowed for its unequivocal
Yellowish solid, 84% yield, mp 175.0-177.0 °C, 1H NMR (400 MHz, DMSO-d6): mixture of isomers (90:10) coexisting in solution (data for the major isomer) δ 3.79 (s, 3H), 4.15 (s, 2 H), 7.73 (d, J = 8.4 Hz, 2H), 7.89 (s, 1H), 8.30 (d, J = 8.4 Hz, 2H); 13C NMR (100 MHz, DMSO-d6, CDCl3 as internal standard): δ 28.2 (CH2), 52.6 (OCH3), 123.8 (2 × CH), 129.0 (C), 130.4 (2 × CH), 140.2 (=CH), 140.4 (C), 147.4 (C), 164.0 (C), 165.8 (C); IR (KBr) νmax/cm-1: 3476, 3346, 3274, 3129, 3082, 2956, 2851, 1710, 1651, 1609, 1516, 1343, 1262, 1074, 665; Elemental Analysis: calcd for C12H15BrN4O4S (%): C, 36.84; H, 3.86; N, 14.32; S 8.20; found: C, 36.82; H, 3.76; N, 14.33; S, 7.99. 2.3. Cell Culture The murine melanoma (B16-F10), human melanoma (SKMEL-28) and human melanocyte cells (NGM) were obtained from Rio de Janeiro Cell Bank. The human melanoma cell lines SKMEL-147 and SK-MEL-19 were kindly provided by Professor Ana Campa of Universidade de São Paulo (USP). The melanoma cells (B16-F10, SK-MEL-28, SK-MEL-147 and SK-MEL-19) were cultured in DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 10 mM HEPES. The non-tumoral cell line (NGM) was cultured in HAM-F12/DMEM (1:1) supplemented with 20% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 1.4 µM hydrocortisone, 1 nM triiodothyronine, 10 µg/mL insulin, 10 µg/mL transferring and 10 ng/mL epidermal growth factor (EGF). Cells were maintained at 37 °C in a 5% CO2 humidified atmosphere and pH 7.4. Salts 1 and 3 (Fig. 1) were solubilized in water and salts 2, 4, 5 and 6 (Fig. 1) were solubilized in DMSO, and posterior dilutions were performed in cell culture media. The final DMSO concentration was maintained below 1% in cell cultures. 2.4. Cytotoxicity
Scheme 1. Synthetic routes for isothiouronium (1-3), N-phenylisothiouronium (4) and isothiosemicarbazide (5, 6) salts.
The cytotoxicity of salts 1-6 was evaluated using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described by Mosmann [32], with some modifications. Briefly, cells (1×104/well) were seeded in 96-well plates and incubated for 24 h with increasing concentrations of the tested compound, ranging from 5 µM to 350 µM. For the control group, cells were incubated without treatment. DMSO was used to dissolve the formazan crystals. After that, the plates were read at 540nm on a microplate reader (BIOTEK EL800). The concentration required to reduce the cell number by 50% (CC50) was determined by nonlinear regression analysis of the logarithm of concentration as a function of the
Isothiouronium Salts Reduce NRAS Expression, Induce Apoptosis and Decrease
normalized response (percentage of cell viability, at incubation time of 24 h), using the software Prism 5.0 (GraphPad Software).
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into the wound area was monitored at 0 and 24 h microscopically (Nikon, Eclipse, TS100), and the measurement was performed using Image J software.
2.5. Selectivity Index The selectivity index (SI) was calculated as the ratio of the CC50 of non-tumoral cell line to the CC50 of tumoral cell line, as presented in the following formula: SI =
CC50 non - tumoral cell line CC50 tumoral cell line
A selectivity index higher than 1 indicates that cytotoxicity on tumoral cells surpassed healthy non-tumoral ones [33]. 2.6. Flow Cytometry Analysis of Cell Cycle with Propidium Iodide (PI) To analyze the cell cycle after treatment with the tested compounds 1-6, flow cytometry analysis was carried out as described by Pozarowski and Darzynkiewicz [34]. For this, SK-MEL-147 cells (3×105/well) were incubated with CC50 for 24 h in 12-well plates. The fluorescence emitted was measured by flow cytometry (FACSCanto II - BD Biosciences). The cell population in each phase of the cell cycle was determined using Flowing (2.5) software. 2.7. NRAS and BRAF Gene Expression To determinate the expression of NRAS and BRAF genes, SKMEL-147 cells were incubated with isothiouronium salt 1 (18 µM) or 2 (14 µM) for 4 h. Total RNA was extracted from each sample using TRIzol® (Invitrogen™), and reversely transcripted into cDNA using the High-Capacity cDNA Reverse Transcription Kit (Invitrogen™) according to the manufacturer's instructions. The primers for NRAS gene were: sense 5'-ACAAACTGGTGGTGG TTGGA-3' and antisense 5'-ATTGTCAGTGCGCTTTTC-3'. The primers for BRAF gene were: sense 5'-CTCCTTGAATCGGGCT GGTT-3' and antisense 5'-GCCTGGATGGGTGTTTG-3'. The GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as the constitutive control with the specific primers: sense 5'-CAATG ACCCCTTCATTGACC-3' and antisense 5'-GACAAGCTTCCC GTTCG-3'. Quantitative PCR was performed using Power SYBR Green PCR Master Mix (Thermo Scientific®) in the Real Time PCR System (Applied Biosystems, USA). The PCR cycling profile was as follows: one cycle at 50 °C for 2 min and 95 °C for 10 min, 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The gene expression data were normalized to the constitutive control GAPDH. The value of threshold cycle (CT) for each reaction was recorded. The relative expression levels of genes were measured according to the formula 2-∆∆Ct, where ∆∆Ct is the difference between the ΔCt of the targets and the ΔCt of GAPDH [35]. 2.8. Flow Cytometry Analysis of Cell Death with Double Labeling The occurrence of apoptosis and necrosis was evaluated using double staining (Annexin V-FITC/PI) as described by Vermes et al. [36]. For this analysis, SK-MEL-147 cells (3×105/well) were incubated with the CC50 for 24 h in 12-well plates, and the cell death was analyzed using FACSCanto II (BD Biosciences) flow cytometry equipment and Flowing (2.5) software. 2.9. Cell Migration Assay Wound-healing assay was used to evaluate the ability of cell migration under exposure to the tested compounds 1-6. Cell monolayers (80 – 90% confluence) were scratched with a sterile 10 µL pipette across the center of the well to generate a clean straight wound area. After that, cells were incubated with the CC50 concentrations of each compound in DMEM. Migration of cells
2.10. Cell Invasion Assay Cell invasion was determined using inserts Millicell® with a polycarbonate filter (8 µm). The upper side of the polycarbonate filter was coated with Matrigel® (Corning) diluted 1:10 in cold DMEM. The chambers were incubated at 37 °C for 30 min to allow the matrix to form a continuous thin layer. Cells (6×104cells/inserts) were incubated for 48 h with isothiouronium salts 1 and 2. After that, cells on the upper surface of the filter were removed with a cotton swab. Cells that invaded the Matrigel® and reached the lower surface of the filter were fixed with 5% glutaraldehyde, and stained in 0.5% crystal violet solution. Cells were counted in ten random fields per filter under a light microscope at 100× magnification (Nikon, Eclipse, TS100). 2.11. Combination Index To evaluate the effects of the combination of isothiouronium salt 1 with vemurafenib in SK-MEL-28 and SK-MEL-147 cells, we used the Combination Index (CI) method of Chou and Talalay [37, 38]. For this, the cells (SK-MEL-147 and SK-MEL-28) were incubated with different concentrations of the compounds (1, 2 and vemurafenib), alone or combined, for 72 h. Following that, cell viability was assessed by MTT assay. Drug interactions were analyzed using the software CompuSyn (version 1.0.CI values from 0.9 to 1.10 indicate an additive effect; a CI lower than 0.9 indicates synergy and a CI higher than 0.9 indicates antagonistic effect. 2.12. Statistical Analysis Data are shown as means ± standard deviation, and analyzed statistically using the Graphpad Prism® software, by an analysis of variance (ANOVA) one-way, followed by Dunnett's post-test. Values of p
