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Antitumor effect of chiral organotelluranes elicited in a murine melanoma model
Accepted Manuscript Antitumor effect of chiral organotelluranes elicited in a murine melanoma model Thaysa Paschoalin, Adam A. Martens, Álvaro T. Omori, Felipe V. Pereira, Luiz Juliano, Luiz R. Travassos, Glaucia M. Machado-Santelli, Rodrigo L.O.R. Cunha PII: DOI: Reference:
S0968-0896(19)30086-0 https://doi.org/10.1016/j.bmc.2019.03.032 BMC 14824
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
16 January 2019 15 March 2019 18 March 2019
Please cite this article as: Paschoalin, T., Martens, A.A., Omori, A.T., Pereira, F.V., Juliano, L., Travassos, L.R., Machado-Santelli, G.M., Cunha, R.L.O., Antitumor effect of chiral organotelluranes elicited in a murine melanoma model, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.03.032
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Antitumor effect of chiral organotelluranes elicited in a murine melanoma model.
Thaysa Paschoalin1,2*, Adam A. Martens3, Álvaro T. Omori4, Felipe V. Pereira1, Luiz Juliano2, Luiz R. Travassos1, Glaucia M. Machado-Santelli3 and Rodrigo L. O. R. Cunha4*
1Departamento
de Microbiologia, Imunologia e Parasitologia, Unidade de Oncologia Experimental (UNONEX),
Universidade Federal de São Paulo, São Paulo, Brasil 2
Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brasil
3
Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de
São Paulo, São Paulo, SP, Brasil. 4 Centro
de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, Brasil
* Corresponding author E-mail: [email protected] (RLORC), [email protected] (TP)
Abstract Protease roles in cancer progression have been demonstrated and their inhibitors display antitumor effects. Cathepsins are lysosomal cysteine proteases that have increased expression in tumor cells, and tellurium compounds were described as potent cysteine protease inhibitors and also assayed in several animal models. In this work, the two enantiomeric
forms
of
1-[Butyl(dichloro)-4-tellanyl]-2-[1S-methoxyethyl]benzene
(organotelluranes RF-13R and RF-13S) were evaluated as inhibitors of cathepsins B and L, showing significant enantiodiscrimination. We observed their cytotoxic effects on a murine melanoma model, effectively inhibiting tumor progression in vivo. The enantiomers were able to inhibit melanoma cell viability, migration and invasion in vitro. Besides, RF-13S and RF-13R were able to inhibit endothelial cell angiogenesis using a tube formation assay in vitro, in a stereodependent manner. These organotelluranes affected cell morphology, showing disassembling of the actin cytoskeleton. These results suggest organotelluranes as potential antitumor agents, acting directly on tumour cell proliferation, migration and invasion, and on endothelial cells, disrupting angiogenesis, showing low toxicity and high efficiency. Taken together our results suggest that this class of compounds should be further studied to reveal their potential as antitumoral agents.
KEY WORDS: hypervalent tellurium compounds · proteases · cancer · angiogenesis · metastasis
1. Introduction Tellurium is an element whose biological effects were put aside due to an assumed general toxicity known described only in some cases with its inorganic forms or with few organic derivatives [1]. The generalization of its toxicological properties has hampered the study of tellurium compounds in biology; nevertheless, the study of biological applications of tellurium is flourishing. Tellurium has various oxidation states and a wide range of derivatives, comprising distinct classes of compounds with particular reactivity [1-3]. Among these classes, tellurides are mainly investigated as glutathione peroxidase mimetics [1-4]. After the seminal demonstration that the inorganic hypervalent tellurium compound AS101, ammonium trichloro (dioxoethylene-O,O’) tellurate, is a potent, non-toxic immunomodulator with demonstrated synergistic effect with chemoterapeutic compounds [5], the interest in a wide systematization on the effects of hypervalent tellurium compounds (telluranes) appeared. Initially, we demonstrated the inhibition of human recombinant cathepsin B for a series of organotelluranes, which revealed that the inhibition has a clear modulation on the structural scaffold and not only on the electrophilicity of the tellurium (IV) atom [6]. Thus, a series of compounds where further studied with other cysteine cathepsins, revealing that inorganic derivatives are less potent inhibitors than organotelluranes [7]. The inhibition of cathepsins V and S by a collection of structurally related organotelluranes and organoselenuranes showed that selenium derivatives are less potent inhibitors than tellurium ones [8]. Beyond the in vitro studies in with recombinant enzymes, the organotellurane
RT04,
dichloro-[(E)
-2-chloro-1-
(2-hydroxyprop-2-yl)-vinyl]-
(4-
methoxyphenyl)- tellurium(IV) induced apoptosis and reduced the expression of antiapoptotic Bcl-2 family proteins in HL60 leukemic cells with negligible toxic effects in mice [9]. In an animal model of visceral leishmaniasis, treatment with the organotellurane RF07, 4-{2-Chloro-3-[chloromethylidene]-1-oxa-2λ4-telluraspiro[3.5]non-2-yl}phenyl methyl ether,
led to an impressive reduction of parasitemia and absence of toxic effects in hamsters [10], Similar findings were demonstrated for a mice model of cutaneous leishmaniasis [11]. The reactivity towards thiols is a common characteristic of other organometallic and organometalloid derivatives of heavy elements, whose toxicological and, in some cases, putative therapeutic effects are related to redox modulation or enzyme inhibition [12]. Due to the diverse functional roles of bioactive thiols in cellular homeostasis [13], the proposition of thiol-reactive compounds as chemotherapeutical agents is debatable due to their possible toxicity. However, covalent mechanisms of drug activity present advantageous properties related to their biochemical efficacy [14], such as avoidance of resistance mechanisms as the electrophilic moieties target residues essential for protein function [15]. Selenium and tellurium agents may act efficiently against cancer cells and pathogenic microorganisms by changing the intracellular redox state of cells [16, 17], inducing growth arrest and apoptosis [18] and by modulating cytokines profile [19, 20]. The most studied tellurium compound is the immunomodulator AS101, a nontoxic tellurium (IV) compound that presents antitumor properties in several murine models [19, 21-24]. This inorganic tellurane exerts antitumor effects on mice bearing B16 melanoma, and this effect was due, at least in part, to Fas ⁄Fas ligand-induced apoptosis [23]. AS101 exerts minimal toxicity, therefore, is safe for clinical application [25]. AS101 has a plethora of beneficial applications, which allowed for its evaluation in phase I and phase II clinical trials for agerelated macular degeneration and human papilloma virus infection, respectively [21, 26, 27]. There are other examples of tellurium compounds displaying in vivo antitumor activities. The telluride LAB027 is an organotellurium-substituted quinone that alters the activities of cellular antioxidant enzymes unbalancing the glutathione metabolism in tumor cells conducting them to a necrosis-type cell death. It was shown that LAB027 exerts prooxidative properties that impaired the development of colon cancer growth in mice model
alone or in a synergistic fashion with oxaliplatin preventing in vitro and in vivo colon cancer proliferation with a beneficial effect on decreasing the in vivo toxicity of oxyliplatin [28]. Another example consists in a β-cyclodextrin derived aryltelluride, DTCD, identified as a potent inhibitor of both mitochondrial and cytosolic thioredoxin reductase (TrxR), which augments the levels of oxidized thioredoxin-1, leading cells to a ROS sensibilization state. The effect of DTCD on ovarian cells triggers signaling pathways related to ERK1/2-MAPKSp1, which are critical to DR5 expression and sensibilization to cytotoxicity of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Altogether, these effects lead to an inhibition of tumour growth in nude mice implanted with human A2780 xenographs [29]. Finally, it was recently identified that the integrin dimer, VLA-4, is a target for telluranes as demonstrated for bis-(4-methoxyphenyl)-tellurium dichloride [30]. VLA-4 ligand binding requires divalent cations and two reduced cysteines from the α4 chain where its modification with thiol reagents hampers ligand binding [31]. This effect may be related to the antimetastatic action on liver metastases of melanoma cells [30] (Figure 1).
Figure 1. Structures of antitumoral tellurium compounds active in in vivo models. The inorganic tellurane AS101, the naphtoquinone LAB027, the β-cyclodextrin derivative DTCD, the bis-(p-methoxyphenyl) tellurium dichloride and the quiral organotelluranes used in the present study.
Organotelluranes are potent cysteine protease inhibitors [8], being effective for the inhibition of cysteine cathepsins involved in the development and progression of cancer [32, 33]. It has been extensively demonstrated that cathepsins have increased expression in tumor cells, may translocate to the cell surface and be secreted to the extracellular environment, where they can degrade components of the ECM. This activity allows tumor cells to invade surrounding tissues, blood and lymph vessels and metastasize to distant sites. In all steps of cancer development, which include cell proliferation, angiogenesis, migration and invasion, the role of cathepsins was demonstrated [32, 33]. In several cancer types, cathepsins have proven diagnostic and prognostic value [34, 35]. Cathepsin inhibitors could then be potentially effective in anticancer therapy. In the present study, we report the promising application of hypervalent organotellurium compounds as potential
anticancer agents, supported by inhibition of cysteine cathepsins, in vitro effects on tumor cell motility, adhesion, angiogenesis and invasion, leading to the inhibition of tumor development in vivo.
2. Methods 2.1. Chemicals The chiral organotelluranes used in this study, RF13-S and RF13-R (Figure 1) were prepared as previously described, starting from racemic 1-phenylethanol employing a chemoenzymatic preparative route [8]. All spectroscopic data are in accordance with the published data proving the identity and purity of the organotelluranes. The enantiomeric excess of each sample was also verified by circular dichroism of 2 mmol L-1 solutions (in dichloromethane) of RF13-S and RF13-R (see spectra in Supporting Information – S1 Figure). 1-[Butyl(dichloro)-4-tellanyl]-2-[1S-methoxyethyl]benzene (RF13-S). Colourless crystals, m.p.: (115.8-115.9)°C, []D26 = +38.2° (CHCl3, c = 1.92), enantiomeric excess: >99%. 1H NMR (500.13 MHz, CDCl3, ppm) δ 7.92 (dd, 3J 7.2 4J 1.8 Hz, 1H), 7.537,42 (m, 2H), 7.35 (dd, 3J 7.2 4J 2.1 Hz, 1H), 4.89 (q, 3J 6.6 Hz, 1H), 3.69-3.55 (m, 3H), 3.51 (s, 3H), 2.23 (qn, 3J 7.5, 2H), 1.67 (d, 3J 6.6, 3H), 1.63 (sext, 3J 7.2 Hz, 2H), 1.06 (t, 3J 7.5, 3H).
13C
NMR (125.78 MHz, CDCl3, ppm) δ 142.2, 133.1, 131.3, 131.1, 129.4, 127.7,
78.7, 56.3, 47.6, 26.6, 24.6, 20.3, 13.7.
125Te
NMR (157.79 MHz, CDCl3, ppm) δ . IR (KBr)
cm-1 3484, 2957, 2930, 2860, 2823, 1462, 1434, 1292, 1200, 1180, 1084, 909, 757, 611. Anal cald for C13H20Cl2OTe: C, 39.95, H, 5.16; Found C, 39.68, H, 5.26. 1-[Butyl(dichloro)-4-tellanyl]-2-[1R-methoxyethyl]benzene (RF13-R). Colourless crystals, m.p.: (115.1-115.7)°C, []D26 = -34.3° (CHCl3, c = 1.81), enantiomeric excess: >99%. Anal cald for C13H20Cl2OTe: C, 39.95, H, 5.16; Found C, 39.79, H, 5.36.
2.2. Enzymes Human recombinant cathepsin B was obtained as previously described [36]. Recombinant human cathepsin L was expressed in Pichia pastoris as previously described [37]. Heterologous expression and purification of human cathepsin S was performed as previously described [38]. Active concentrations of all cathepsins were determined by active-site titration experiments using 1 mM E-64 [L-trans-epoxysuccinyl-leucylamido(4guanidino)butane] dissolved in dimethyl sulfoxide [39]. Enzyme and substrate were used with the following conditions. Cathepsin B buffer: 100 mM sodium phosphate, 1 mM EDTA, 0.025% BRIJ, substrate Z-RR-MCA; Cathepsin L buffer: 50 mM sodium acetate buffer containing 100 mM NaCl, 2.5 mM EDTA pH 5.5, substrate Z-FR-MCA; Cathepsin S buffer: 50 mM sodium phosphate, containing 100 mM NaCl, 2.5 mM EDTA, pH 6.5, substrate ZFR-MCA. Cathepsin inhibition assay was performed as previously described [7]. In brief, the inhibition kinetics was performed using cathepsins previously activated with aqueous dithiothreitol (2 mmol L-1) and the assays with the organotelluranes were performed in degassed buffer solutions in the absence of the reducing agent.
2.3. Mice and cell lineages Six- to eight-week-old male C57BL/6 mice were obtained from the Center for Development of Experimental Models (CEDEME) animal facility, Universidade Federal de São Paulo (UNIFESP), and kept in isolators, supplied with autoclaved water and food. The animal experiments were carried out in accordance with the Research Ethics Committee of UNIFESP (protocol number CEP 1306/10). The original murine melanoma cell line B16F10 was obtained from the Ludwig Institute for Cancer Research (LICR), São Paulo branch. The subline B16F10-Nex2 was isolated at the Experimental Oncology Unit, Federal University of São Paulo, and deposited in the Banco
de Células do Rio de Janeiro (BCRJ), reg. 0342. It is characterized by adherence, spindle or stellate morphology, darkly melanotic cells, fast growing in vitro and in vivo, forming black tumor masses and black nodules in the lungs when injected subcutaneously and intravenously, respectively, in syngeneic H-2b C57BL/6 mice. The cervical cancer cell line (HeLa) and human melanoma cell lines Mewo and A2058 were obtained from the LICR. The human melanoma cell lines (SK-Mel-25, SK-Mel-28) were provided by Dr. Alan N. Houghton from the Memorial Sloan Kettering Cancer Center, NY, and HT144 was obtained from ATCC and was cultivated in Dulbecco's Modified Eagle Medium/HAM-F12 (Sigma Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS – Vitrocell Embriolife). The human umbilical vein endothelial cell line (HUVEC) was provided by Dr. Julio Scharfstein from Federal University of Rio de Janeiro and human fibroblast cell line (NIH 3T3) was obtained from ATCC. All cells were maintained in complete medium consisting of RPMI 1640, pH 7.2, supplemented with 10 mM N-2-hydroxyethylpiperazineN’-2-ethanesulphonic acid (HEPES), 24 mM sodium bicarbonate, 10% heat-inactivated fetal calf serum (FCS – Gibco, Minneapolis, MN, USA) and 40 µg/mL gentamicin sulfate (Hipolabor Farmacêutica, Sabará, MG, Brazil).
2.4. In vitro angiogenesis assay on Matrigel BD MatrigelTM Matrix (B&D Biosciences, Bedford, MA, USA) thawed on ice was distributed in 96-well plates (50 L per well) and allowed to polymerize for 1 h at 37oC. HUVEC cells (1.5 x 104 cells/well) suspended in 100 µL of RPMI medium supplemented with 1% of FCS were added to each well in the presence of compounds E-64 (5 M), RF-13R (0.5 M) or RF-13S (0.062 M). The plates were incubated at 37oC for 18h and then images were captured at 8x magnification with a ProgRes C3 camera (Jenoptik, Germany) coupled to an inverted light microscope (Zeiss, Germany). The number of angiogenic structures (closed
rings) was counted from 4 different wells, and the average value was determined for each sample. As a control of the assay, HUVEC cells were plated on Matrigel without the addition of any compound.
2.5. In vitro cytotoxic activity Cell lines (5x103 cells per well) were cultivated in 96-well plates and incubated for 24 h with compound RF-13R or RF-13S, after cell attachment, in the presence or absence of 1% FCS. Organotelluranes were used in increasing and folded concentrations, ranging from 0.02 μM to 50 μM. Cell proliferation was measured using the Cell Proliferation Kit I (MTT) (Boehringer Mannheim), an MTT-based colorimetric assay for quantification of cell proliferation and viability. Readings were performed in a plate reader at 570 nm.
2.6. In vivo tumor protection assay with tellurium compounds Syngeneic B16F10-Nex2 viable cells (5x105) in 0.1 mL (per mouse) were injected into tail veins of 22 seven-to-eight week-old male C57BL/6 mice. Eight animals were used in the control group and 14 animals were equally distributed for treatment with RF-13R and RF13S compounds. For protection experiments, the animals received i.p. doses of 20 M of RF-13R or RF-13S on days 2, 4, 6, 8, 10 and 12 after tumor cell challenge, or PBS, at the same time period. After 21 days, the lungs were collected and inspected for metastatic colonization and the superficial melanotic nodules were counted at 2X magnification. Data are reported as the mean number of tumor nodules ± SE for seven or more mice per group.
2.7. Monolayer wound-healing assay HUVEC and B16F10-Nex2 melanoma cells (5x104 cells/well) were plated on 24-well plates in triplicates. After cell attachment and growth to a confluent monolayer, one scratch wound
was made with a blue P1000 tip (Fisher) in each well. RPMI with 1% FCS was added to each well in the presence of 20 M of compound RF-13R or RF-13S. The plate was placed into a culture chamber at 37 oC on an inverted microscope (AxioObserver Z1 Zeiss) and wells were photographed at high resolution with the 5X objective lens, every hour for 28 h or 36h. Measuring the width of the wound and subtracting this value from the initial halfwidth value of the wound determined the cell migration distance using AxioVision microscope software (Zeiss).
2.8. Transwell invasion assay The Matrigel invasion ability of HUVEC and B16F10-Nex2 melanoma cells pre-incubated with 20 M of RF-13R or RF-13S was tested using Biocoat Matrigel Invasion Chambers (B&D Biosciences, Bedford, MA, USA). Briefly, 80 μL of Matrigel diluted (1:6) in cold PBS was added on the upper chambers of 24-well transwells and incubated for 30 min at 37 oC for gel formation. After incubation, 500 μL of serum-free RPMI containing 5x104 cells preincubated or not with organotelluranes was added to the Matrigel-coated PET filters (8-μm pore size). The lower chamber of the transwell unit was filled with 500 μL of RPMI supplemented with 2% FCS, as chemoattractant. Plates were incubated for 24 h at 37 oC and then non-invading cells were scraped off the top of the transwell with a cotton swab. Transwell units were removed from the 24-well plates, fixed for 30 min with methanol and stained with Giemsa’s stain for 15 min. Invading cells were counted under a light microscope.
2.9. Cell-to-extracellular matrix (ECM) adhesion assay For the ECM assay, 96-well plates were coated with 40 g/ml of type I collagen and type IV collagen in 2% acetic acid and fibronectin and laminin in PBS for 16h at 4oC. Non-specific
binding was blocked by the addition of 1% bovine serum albumin PBS for 1 h at 37oC. After three washing steps with PBS, B16F10-Nex2 cells were added to the wells (5x104 cells/well) in the presence of RF-13R or RF-13S at 2 M and incubated for 3 h at 37oC. Plates were gently washed twice with PBS to remove unattached cells and the attached cells were fixed with ice-cold methanol for 5 min. Fixed cells were stained with 1% toluidine blue in 1% sodium tetraborate for 5 min and washed with PBS. Dye was solubilized in 1% SDS for 20 min at 37oC and the resulting colored solution was quantified at 540 nm using a scanning multiwell spectrophotometer. Cells incubated without dichlorides were used as control and are taken as 100% adhesion.
2.10. Fluorescent staining B16F10-Nex2 and HT-144 cells were plated onto 35mm plates at a density of 1.3x105 cells/plate and cultured for 24 h in 10% FCS DMEM/Ham-F12. Cells were then treated with 10 or 30 M RF-13R and RF-13S in 1% FCS DMEM/Ham-F12 for 24 h. Cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100 and incubated with a 20 µM phalloidin-TRITC, 20 µM tubulin-Cy2, 10µg/mL RNAse A solution for 90 min. Slides were closed with TO-PRO-3 and anti-fading agent VectaShield Mounting Media (Vector Laboratories, USA). Fluorescent images were acquired with confocal laser scanning microscope Zeiss LSM510 (Zeiss, Germany) using argon (488nm) and helium-neon (543 nm) lasers.
2.11. Statistical analysis The data are represented as mean ± SE. Statistical significance was determined by the Student’s t test. All experiments were conducted two or more times. Reproducible results were obtained and representative data are shown.
3. Results
3.1. Cathepsin activity inhibition by enantiomeric organotelluranes Organotelluranes display high reactivity towards thiols; this led to their application as inhibitors of cysteine proteases like cathepsins [2, 6, 7], caspases and more recently protein tyrosine phosphatases [40]. The chiral organotelluranes RF13-R and RF13-S were previously evaluated as inhibitors of cathepsins S and V, showing no significant enantiodiscrimination between the pair of enantiomers when in presence of small quantity of dithiothreitol [8]. On the other hand, the enantiomeric organotelluranes exhibited differences on the inhibition of cathepsins B, L, S and K in the absence of this reducing agent. The organotellurane RF-13S showed higher potency for the inhibition of cysteine cathepsins with as expressed in the values of the determined second-order inhibition rate constants values of 7400±90, 8700±120, 600000±5400 M-1s-1 for cathepsins B, L and S, respectively; in other hand its antipode, RF-13R presented values of 3700±45, 5500±90 and 39000±340 M-1s-1.
3.2. Cytotoxic effect of organotelluranes To evaluate the cytotoxic effects of the organotelluranes, murine and human tumor cell lines were incubated with dichlorides RF-13R and RF-13S for 16 h, and cell viability was measured using MTT reagent, following manufacturer’s instructions. All cell lines tested were more susceptible to dichloride RF-13S than to RF-13R, when the assay was carried out in the presence of FCS-free RPMI medium (Table 1). The IC50 values were different depending on the cell line tested, but in all cases the cytotoxicity occurred at micromolar concentrations and was dose-dependent (not shown). B16F10-Nex2 melanoma
cells and cervical cancer cells (Hela) were most sensitive to dichlorides, with IC50 values lower than 1 M to RF-13S. This organotellurane was more cytotoxic than RF-13R.
3.3. Effect of organotelluranes on tumor development Effective treatment of mice challenged intravenously with B16F10-Nex2 cells was investigated using RF-13R and RF-13S. Mice were injected with 5x104 melanoma cells/100 μl/animal and treated on days 2, 4, 6, 8 and 10 with organotelluranes at 20 M/injection. In Figure 2A, we show that animals treated with RF-13R and RF-13S had, respectively, 2-fold and 7-fold less superficial nodules than animals treated with PBS. Specimens of lungs from animals treated with dichlorides are shown in Figure 2B. These results corroborate the in vitro experiments, showing that RF-13S is more effective than RF-13R as inhibitor of tumor development in a melanoma metastatic model.
Figure 2. In vivo antitumor effects of organotelluranes RF-13R and RF-13S. C57BL/6 mice (n=10, control group; n=6, treated-group) were injected intravenously with 5x105 B16F10-Nex2 melanoma cells. Tellurium compounds were administered intraperitoneally (20 M/injection) on days 2, 4, 6, 8, 10 and 12 after tumor cell challenge. Control mice received PBS for the same period of time. Lungs were collected 21 days after tumor challenge and the superficial nodules were counted. (A) Total number of lung metastases. Data presented as mean number of lung lesions ± SE. Two independent experiments were performed. p<0.0005, RF-13R vs
control; p<0.000001, RF-13S vs control. B. Representative lungs from B16-bearing mice treated with PBS, RF-13R or RF-13S.
3.4. Organotelluranes inhibit angiogenesis in vitro The effect of dichlorides on endothelial cell angiogenesis in vitro was evaluated using HUVECs plated on Matrigel in presence of RF-13R and RF-13S or E-64, a known cysteine protease inhibitor, as a control (Fig. 3). The growth of HUVECs on Matrigel with 1% fetal calf serum led to the formation of closed intercellular compartments arising from endothelial cell sprouting (pro-angiogenic structures) independent of any other factor. Addition of RF13R, RF-13S and E-64 to the incubation mixture inhibited the formation of these structures in about 45%, 55% and 80%, respectively compared to control. Although the effective concentration was substantially different, RF-13S was able to inhibit angiogenesis with only 62 nM, while compound RF-13R and E-64 needed 0.5 M and 5 M, respectively, to exert the same effect.
Figure 3. Effect of organotelluranes RF-13R and RF-13S on pro-angiogenic closed structures formed by sprouting of endothelial cells. (A) HUVECs were plated on Matrigel in RPMI medium supplemented with 1%
FCS in the presence of dichorides RF-13R and RF-13S and cysteine protease inhibitor E-64. The number of pro-angiogenic structures was counted after 18 h. One representative picture of four different treatments is shown with the respective counts. (B) Results expressed in number of pro-angiogenic structures. Error bars, SD of quadruplicate samples. * p = 0.02 vs control; ** p = 0.004 vs control; *** p = 0.001 vs control.
3.5. Organotelluranes RF-13R and RF-13S decrease melanoma cell adhesion to extracellular matrix proteins To analyse the effect of organotelluranes on cell adhesion, B16F10-Nex2 cells were plated on 96-well plates coated with ECM proteins in the presence of 2 M RF-13R and RF13S. Incubation for 3h with organotelluranes modified adhesion of B16F10-Nex2 cells to the ECM proteins tested, fibronectin (FN), laminin, and collagen types I and IV. Adhesion of melanoma to types IV and I collagen was more affected by organotellurane treatment than to adhesion to FN and laminin (Fig. 4). In this case, dichloride RF-13S was also more effective than RF-13R.
Figure 4. Decrease in cell adhesion to ECMs after treatment with organotelluranes RF-13R and RF-13S. B16F10-Nex2 cells (5x104 cells/well) were spread on 96-well plates coated with collagen type I (Col I), collagen type IV (Col IV), laminin (Lamin) or fibronectin (Fibro) in the presence of 2 M of RF-13R or RF-13S
dichlorides. After incubation for 3h, non-adherent cells were removed by a PBS rinse and adherent cells were stained. Data are representative of three independent experiments. *p < 0.05; **p < 0.005 (values are compared with untreated control).
3.6. Modulation of cell migration and invasion by organotelluranes RF-13R and RF13S To evaluate the ability of organotelluranes to inhibit cell migration, scratch wound healing assays were performed with B16F10-Nex2 melanoma cells and endothelial cells (HUVEC). RF-13R (20 μM) inhibited by 33% the wound closure in HUVEC cells as compared to control (Fig. 5A). The inhibition of migration by organotelluranes was more effective in melanoma cells. B16F10-Nex2 cells migration was inhibited by 80% with 20 M of compounds RF-13R and RF-13S (Fig. 5B). The invasion process of melanoma and endothelial cells was significantly inhibited with 20 M of RF-13R and RF-13S as can be seen in Figure 6. RF-13S was more effective in inhibiting HUVEC invasion than RF-13R (Fig. 6A). Melanoma cells invasion decreased 80% by both organotelluranes (Fig. 6B).
Figure 5. Effect of organotelluranes RF-13R and RF-13S on cell migration. HUVEC (A) and B16F10-Nex2 melanoma cells (B) at 5x104 cells/well in 1 mL were seeded on 24-well plates and after growing to form a confluent monolayer, one scratch wound was made. Cell migration in the presence of RF-13R and RF-13S compounds at 20 M was determined by measuring the width of the wound at 0h and after 24h incubation. p values comparing to control are shown.
Figure 6. Effect of organotelluranes RF-13R and RF-13S on cell invasion. HUVEC (A) and B16F10-Nex2 melanoma cells (B) (5x103) were incubated on transwell units coated with Matrigel in the presence of RF-13R and RF-13S compounds at 20 M for 24h. Invading cells were quantified by counting after crystal violet staining. Values are expressed in percentage comparing to control. p values comparing to control are shown.
3.7. Organotelluranes RF-13R and RF-13S affect cell morphology B16F10-Nex2 and HT-144 cells were treated with 10µM RF-13R and RF-13S for 24 hours and were then stained with tubulin-Cy2 and TO-PRO-3. The fluorescently stained confocal microscope images (Fig. 7) showed that both RF-13R and RF-13S affect cell morphology. Cell lines B16F10-Nex2 and HT-144 treated with organotelluranes showed a tendency to cell rounding, with retraction of cell protrusions and depolymerisation of F-actin, as well as some membrane blebbing. HT-144 cells stained with phalloidin-TRITC, tubulin-
Cy2 and TO-PRO-3 and treated with 30µM RF-13R and RF-13S showed complete loss stress fibers (Fig 8), with cell rounding and the remaining cortical actin in close association with the microtubule network.
Figure 7. Effect of organotelluranes RF-13R and RF-13S on cell morphology. HT-144 and B16F10-Nex2 melanoma cells (1.3x105) were treated with 10 or 30 M of RF-13R and RF-13S for 24h. Cells were permeabilized and stained with tubulin-Cy2 (green) and TO-PRO-3 (blue). Fluorescent images were acquired with confocal laser scanning microscope Zeiss LSM510 (Zeiss, Germany) using argon (488nm) and heliumneon (543nm) lasers.
Figure 8. Effect of organotelluranes RF-13R and RF-13S on cell morphology. HT-144 (1.3x105) was treated with 30 M of RF-13R and RF-13S for 24h. Cells were permeabilized and stained with phalloidin-TRITC (red), tubulin-Cy2 (green) and TO-PRO-3 (blue). Fluorescent images were acquired with confocal laser scanning microscope Zeiss LSM510 (Zeiss, Germany) using argon (488nm) and helium-neon (543nm) lasers.
4. Discussion Cathepsins have a well-characterized role in tumor development as described in clinical reports and experimental murine cancer models [32, 41, 42]. Up-regulation of cathepsin expression and/or activity and extracellular localization are associated with cancer progression and positively correlated with a poor prognosis for cancer patients [4345], including those with melanoma [46, 47]. Cathepsins B, L and some others are able to promote cancer cell migration and invasion [48, 49]. Also, cathepsin B expression correlates with increased metastatic potential of tumors [50]. Therefore, selective inhibitors of cathepsins could offer new possibilities for the diagnosis and treatment of human cancers [34]. In the present study, we report on the inhibition by chiral tellurium compounds
of cysteine cathepsins and the possible application of these compounds as anticancer agents. The inhibition of cathepsins B and L by organotelluranes RF-13R and RF-13S was time- and concentration-dependent. The distinguished feature of cysteine proteases inhibition by organic telluranes consists in its reactivation by reducing thiols or by the decrease of inhibition efficacy in a reducing medium (not shown). This aspect is particularly noteworthy because it can be expected that hypervalent tellurium compounds exert a low influence in molecular targets in the reducing cytoplasmic environment. Nevertheless, it was found that some organotelluranes can inhibit intracellular proteolysis of Plasmodium falciparum parasites suggesting that these thiol-reactive compounds can be directed to more reactive thiols within the cell [51]. Tumor
cell
lines,
endothelial
cells
and
fibroblasts
were
susceptible
to
organotelluranes in micromolar concentrations and the cytotoxic effect was dosedependent. B16F10-Nex2 murine melanoma cells and the cervical cancer cell line (HeLa) were the cell lines more susceptible to organotelluranes, while human umbilical vein endothelial cell (HUVEC) and human melanoma HT-144 were more resistant. Corroborating the in vitro cytotoxic assay, RF-13S was more effective than RF-13R in the inhibition of tumor development in vivo; this could be related to a differential reactivity of the organotelluranes with tissular, cysteine-targets. Intraperitoneal administration of organotelluranes at 20 M for five non-consecutive days after melanoma challenge, reduced lung metastases in 85% and 58% for RF-13S and RF-13R treatment, respectively. According with this, RF-13S was more effective than its enantiomer, in the inhibition of endothelial cell angiogenesis in vitro. It is unclear by what mechanism the organotelluranes inhibit the angiogenic process. The inorganic tellurane AS101 was reported to inhibit angiogenesis in an indirect manner, regulating the expression and secretion of VEGF,
MMP-2, and MMP-9 [24]. The organotelluranes tested here were efficient inhibiting the activity of cathepsins V and S [8]. The role of cathepsins in angiogenesis modulation has been studied in a wide variety of tumor models. Extracellularly active cathepsins B and L were identified in highly invasive melanoma cells [52]. It was shown that cathepsin L facilitates high metastatic melanoma cell invasion and migration [49], and cathepsin B inhibitor (CA-074) significantly reduced human melanoma growth and number of lung metastases [53]. Cathepsin B was also described as a regulator of the concentration of angiomodulators at the level of the endothelium, being able to modulate the angiogenic process [54]. Here, we observed that HUVEC and B16F10-Nex2 cells express cathepsins B and L, which could be another possible target for angiogenesis inhibition. The death of most cancer patients is due to metastasis events. The process by which cancer cells escape from the primary tumor lesion, invade the blood or lymphatic vessels and colonize distant organs is very complex and is controlled by numerous mediators. Fundamental steps in this process are the tumor cell capability to migrate and invade surrounding tissues. Loss of cell-cell and cell-matrix adhesion and degradation of ECM components predispose cells to migration and invasion [55]. We show that 2 M RF13R and RF-13S decreased melanoma cell adhesion to ECM proteins and significantly inhibited migration and invasion of tumour and endothelial cells, being able to significantly decrease angiogenesis and metastasis. This could be due to the effect of the organotelluranes on the cell cytoskeleton, disassembling actin and reducing cell protrusions. In human melanoma cells (HT-144) organotellurane treatment led to round cells with remaining cortical actin.
5. Conclusion
The angiogenic switch is a fundamental step that allows tumors to expand in size and acquire metastatic potential. Anti-angiogenic therapies are very promising, since they inhibit primary tumor growth and prevent metastatic spread. Adhesion inhibition is also an effective strategy for preventing proliferation and metastasis of melanomas. The results shown in the present work depict organotelluranes as potential antitumor agents, acting directly on tumour cell proliferation, migration and invasion, and on endothelial cells, disrupting angiogenesis. Organotelluranes display low toxicity and high antitumor efficiency, thus being encouraging candidates to be developed as anti-cancer drugs.
Acknowledgements This work was supported by grants from FAPESP (2004/14426-0, 2006/51880-7, 2009/53840-0) and CNPq (487012/2012-7, 477525/2011-3, 474440/2013-3), Brazil. LRT and LJ are CNPq career fellowship.
Authors’ contributions
Conceived and designed the experiments: TP RLORC GMMS. Performed the experiments: TP AAM ATO FVP RLORC. Analyzed the data: TP AAM LJ LRT GMMS RLORC. Contributed reagents/materials/analysis tools: TP ATO GMMS LRT LJ RLORC. Wrote the paper: TP AAM GMMS LRT LJ RLORC.
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Figure legends
Figure 1: Structures of antitumoral tellurium compounds active in in vivo models. The inorganic tellurane AS101, the naphtoquinone LAB027, the β-cyclodextrin derivative DTCD, the bis-(p-methoxyphenyl) tellurium dichloride and the quiral organotelluranes used in the present study.
Figure 2: In vivo antitumor effects of organotelluranes RF-13R and RF-13S. C57BL/6 mice (n=10, control group; n=6, treated-group) were injected intravenously with 5x105 B16F10Nex2 melanoma cells. Tellurium compounds were administered intraperitoneally (20 M/injection) on days 2, 4, 6, 8, 10 and 12 after tumor cell challenge. Control mice received PBS for the same period of time. Lungs were collected 21 days after tumor challenge and the superficial nodules were counted. (A) Total number of lung metastases. Data presented as mean number of lung lesions ± SE. Two independent experiments were performed. p<0.0005, RF-13R vs control; p<0.000001, RF-13S vs control. B. Representative lungs from B16-bearing mice treated with PBS, RF-13R or RF-13S.
Figure 3: Effect of organotelluranes RF-13R and RF-13S on pro-angiogenic closed structures formed by sprouting of endothelial cells. (A) HUVECs were plated on Matrigel in RPMI medium supplemented with 1% FCS in the presence of dichorides RF-13R and RF13S and cysteine protease inhibitor E-64. The number of pro-angiogenic structures was counted after 18 h. One representative picture of four different treatments is shown with the respective counts. (B) Results expressed in number of pro-angiogenic structures. Error bars, SD of quadruplicate samples. * p = 0.02 vs control; ** p = 0.004 vs control; *** p = 0.001 vs control.
Figure 4. Decrease in cell adhesion to ECMs after treatment with organotelluranes RF-13R and RF-13S. B16F10-Nex2 cells (5x104 cells/well) were spread on 96-well plates coated with collagen type I (Col I), collagen type IV (Col IV), laminin (Lamin) or fibronectin (Fibro) in the presence of 2 M of RF-13R or RF-13S dichlorides. After incubation for 3h, nonadherent cells were removed by a PBS rinse and adherent cells were stained. Data are representative of three independent experiments. *p < 0.05; **p < 0.005 (values are compared with untreated control).
Figure 5: Effect of organotelluranes RF-13R and RF-13S on cell migration. HUVEC (A) and B16F10-Nex2 melanoma cells (B) at 5x104 cells/well in 1 mL were seeded on 24-well plates and after growing to form a confluent monolayer, one scratch wound was made. Cell migration in the presence of RF-13R and RF-13S compounds at 20 M was determined by measuring the width of the wound at 0h and after 24h incubation. p values comparing to control are shown.
Figure 6: Effect of organotelluranes RF-13R and RF-13S on cell invasion. HUVEC (A) and B16F10-Nex2 melanoma cells (B) (5x103) were incubated on transwell units coated with Matrigel in the presence of RF-13R and RF-13S compounds at 20 M for 24h. Invading cells were quantified by counting after crystal violet staining. Values are expressed in percentage comparing to control. p values comparing to control are shown.
Figure 7: Effect of organotelluranes RF-13R and RF-13S on cell morphology. HT-144 and B16F10-Nex2 melanoma cells (1.3x105) were treated with 10 or 30 M of RF-13R and RF13S for 24h. Cells were permeabilized and stained with tubulin-Cy2 (green) and TO-PRO-3
(blue). Fluorescent images were acquired with confocal laser scanning microscope Zeiss LSM510 (Zeiss, Germany) using argon (488nm) and helium-neon (543nm) lasers.
Figure 8: Effect of organotelluranes RF-13R and RF-13S on cell morphology. HT-144 (1.3x105) was treated with 30 M of RF-13R and RF-13S for 24h. Cells were permeabilized and stained with phalloidin-TRITC (red), tubulin-Cy2 (green) and TO-PRO-3 (blue). Fluorescent images were acquired with confocal laser scanning microscope Zeiss LSM510 (Zeiss, Germany) using argon (488nm) and helium-neon (543nm) lasers.
S1 Figure: CD spectra of dichlorides RF-13R and RF-13S in chloroform.
Table 1: Estimated IC50 of RF-13R and RF-13S cytotoxic effect in murine and human cell lines IC50 (mM) Cell
line[a]
IC50 ratio (R) : (S)
RF-13R
RF-13S
B16F10-Nex2
2.3 + 1.1
0.9 + 0.3
2.6
HUVEC
20.3 + 3.3
2.8 + 0.9
7.25
3T3
11.5 + 2.1
3.1 + 0.8
3.7
SKMel-25
18.0 + 2.5
2.5 + 1.0
7.2
SKMel-28
6.2 + 0.7
3.1 + 0.5
2.0
A2058
5.5 + 1.8
1.5 + 0.8
3.7
Mewo
9.5 + 1.1
3.2 + 0.5
3.0
Hela
2.3 + 0.2
0.8 + 0.3
2.9
HT-144
18.1+ 5.3
15.3 + 3.0
1.2
S0968-0896(19)30086-0 https://doi.org/10.1016/j.bmc.2019.03.032 BMC 14824
To appear in:
Bioorganic & Medicinal Chemistry
Received Date: Revised Date: Accepted Date:
16 January 2019 15 March 2019 18 March 2019
Please cite this article as: Paschoalin, T., Martens, A.A., Omori, A.T., Pereira, F.V., Juliano, L., Travassos, L.R., Machado-Santelli, G.M., Cunha, R.L.O., Antitumor effect of chiral organotelluranes elicited in a murine melanoma model, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.03.032
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Antitumor effect of chiral organotelluranes elicited in a murine melanoma model.
Thaysa Paschoalin1,2*, Adam A. Martens3, Álvaro T. Omori4, Felipe V. Pereira1, Luiz Juliano2, Luiz R. Travassos1, Glaucia M. Machado-Santelli3 and Rodrigo L. O. R. Cunha4*
1Departamento
de Microbiologia, Imunologia e Parasitologia, Unidade de Oncologia Experimental (UNONEX),
Universidade Federal de São Paulo, São Paulo, Brasil 2
Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, Brasil
3
Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de
São Paulo, São Paulo, SP, Brasil. 4 Centro
de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André, Brasil
* Corresponding author E-mail: [email protected] (RLORC), [email protected] (TP)
Abstract Protease roles in cancer progression have been demonstrated and their inhibitors display antitumor effects. Cathepsins are lysosomal cysteine proteases that have increased expression in tumor cells, and tellurium compounds were described as potent cysteine protease inhibitors and also assayed in several animal models. In this work, the two enantiomeric
forms
of
1-[Butyl(dichloro)-4-tellanyl]-2-[1S-methoxyethyl]benzene
(organotelluranes RF-13R and RF-13S) were evaluated as inhibitors of cathepsins B and L, showing significant enantiodiscrimination. We observed their cytotoxic effects on a murine melanoma model, effectively inhibiting tumor progression in vivo. The enantiomers were able to inhibit melanoma cell viability, migration and invasion in vitro. Besides, RF-13S and RF-13R were able to inhibit endothelial cell angiogenesis using a tube formation assay in vitro, in a stereodependent manner. These organotelluranes affected cell morphology, showing disassembling of the actin cytoskeleton. These results suggest organotelluranes as potential antitumor agents, acting directly on tumour cell proliferation, migration and invasion, and on endothelial cells, disrupting angiogenesis, showing low toxicity and high efficiency. Taken together our results suggest that this class of compounds should be further studied to reveal their potential as antitumoral agents.
KEY WORDS: hypervalent tellurium compounds · proteases · cancer · angiogenesis · metastasis
1. Introduction Tellurium is an element whose biological effects were put aside due to an assumed general toxicity known described only in some cases with its inorganic forms or with few organic derivatives [1]. The generalization of its toxicological properties has hampered the study of tellurium compounds in biology; nevertheless, the study of biological applications of tellurium is flourishing. Tellurium has various oxidation states and a wide range of derivatives, comprising distinct classes of compounds with particular reactivity [1-3]. Among these classes, tellurides are mainly investigated as glutathione peroxidase mimetics [1-4]. After the seminal demonstration that the inorganic hypervalent tellurium compound AS101, ammonium trichloro (dioxoethylene-O,O’) tellurate, is a potent, non-toxic immunomodulator with demonstrated synergistic effect with chemoterapeutic compounds [5], the interest in a wide systematization on the effects of hypervalent tellurium compounds (telluranes) appeared. Initially, we demonstrated the inhibition of human recombinant cathepsin B for a series of organotelluranes, which revealed that the inhibition has a clear modulation on the structural scaffold and not only on the electrophilicity of the tellurium (IV) atom [6]. Thus, a series of compounds where further studied with other cysteine cathepsins, revealing that inorganic derivatives are less potent inhibitors than organotelluranes [7]. The inhibition of cathepsins V and S by a collection of structurally related organotelluranes and organoselenuranes showed that selenium derivatives are less potent inhibitors than tellurium ones [8]. Beyond the in vitro studies in with recombinant enzymes, the organotellurane
RT04,
dichloro-[(E)
-2-chloro-1-
(2-hydroxyprop-2-yl)-vinyl]-
(4-
methoxyphenyl)- tellurium(IV) induced apoptosis and reduced the expression of antiapoptotic Bcl-2 family proteins in HL60 leukemic cells with negligible toxic effects in mice [9]. In an animal model of visceral leishmaniasis, treatment with the organotellurane RF07, 4-{2-Chloro-3-[chloromethylidene]-1-oxa-2λ4-telluraspiro[3.5]non-2-yl}phenyl methyl ether,
led to an impressive reduction of parasitemia and absence of toxic effects in hamsters [10], Similar findings were demonstrated for a mice model of cutaneous leishmaniasis [11]. The reactivity towards thiols is a common characteristic of other organometallic and organometalloid derivatives of heavy elements, whose toxicological and, in some cases, putative therapeutic effects are related to redox modulation or enzyme inhibition [12]. Due to the diverse functional roles of bioactive thiols in cellular homeostasis [13], the proposition of thiol-reactive compounds as chemotherapeutical agents is debatable due to their possible toxicity. However, covalent mechanisms of drug activity present advantageous properties related to their biochemical efficacy [14], such as avoidance of resistance mechanisms as the electrophilic moieties target residues essential for protein function [15]. Selenium and tellurium agents may act efficiently against cancer cells and pathogenic microorganisms by changing the intracellular redox state of cells [16, 17], inducing growth arrest and apoptosis [18] and by modulating cytokines profile [19, 20]. The most studied tellurium compound is the immunomodulator AS101, a nontoxic tellurium (IV) compound that presents antitumor properties in several murine models [19, 21-24]. This inorganic tellurane exerts antitumor effects on mice bearing B16 melanoma, and this effect was due, at least in part, to Fas ⁄Fas ligand-induced apoptosis [23]. AS101 exerts minimal toxicity, therefore, is safe for clinical application [25]. AS101 has a plethora of beneficial applications, which allowed for its evaluation in phase I and phase II clinical trials for agerelated macular degeneration and human papilloma virus infection, respectively [21, 26, 27]. There are other examples of tellurium compounds displaying in vivo antitumor activities. The telluride LAB027 is an organotellurium-substituted quinone that alters the activities of cellular antioxidant enzymes unbalancing the glutathione metabolism in tumor cells conducting them to a necrosis-type cell death. It was shown that LAB027 exerts prooxidative properties that impaired the development of colon cancer growth in mice model
alone or in a synergistic fashion with oxaliplatin preventing in vitro and in vivo colon cancer proliferation with a beneficial effect on decreasing the in vivo toxicity of oxyliplatin [28]. Another example consists in a β-cyclodextrin derived aryltelluride, DTCD, identified as a potent inhibitor of both mitochondrial and cytosolic thioredoxin reductase (TrxR), which augments the levels of oxidized thioredoxin-1, leading cells to a ROS sensibilization state. The effect of DTCD on ovarian cells triggers signaling pathways related to ERK1/2-MAPKSp1, which are critical to DR5 expression and sensibilization to cytotoxicity of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Altogether, these effects lead to an inhibition of tumour growth in nude mice implanted with human A2780 xenographs [29]. Finally, it was recently identified that the integrin dimer, VLA-4, is a target for telluranes as demonstrated for bis-(4-methoxyphenyl)-tellurium dichloride [30]. VLA-4 ligand binding requires divalent cations and two reduced cysteines from the α4 chain where its modification with thiol reagents hampers ligand binding [31]. This effect may be related to the antimetastatic action on liver metastases of melanoma cells [30] (Figure 1).
Figure 1. Structures of antitumoral tellurium compounds active in in vivo models. The inorganic tellurane AS101, the naphtoquinone LAB027, the β-cyclodextrin derivative DTCD, the bis-(p-methoxyphenyl) tellurium dichloride and the quiral organotelluranes used in the present study.
Organotelluranes are potent cysteine protease inhibitors [8], being effective for the inhibition of cysteine cathepsins involved in the development and progression of cancer [32, 33]. It has been extensively demonstrated that cathepsins have increased expression in tumor cells, may translocate to the cell surface and be secreted to the extracellular environment, where they can degrade components of the ECM. This activity allows tumor cells to invade surrounding tissues, blood and lymph vessels and metastasize to distant sites. In all steps of cancer development, which include cell proliferation, angiogenesis, migration and invasion, the role of cathepsins was demonstrated [32, 33]. In several cancer types, cathepsins have proven diagnostic and prognostic value [34, 35]. Cathepsin inhibitors could then be potentially effective in anticancer therapy. In the present study, we report the promising application of hypervalent organotellurium compounds as potential
anticancer agents, supported by inhibition of cysteine cathepsins, in vitro effects on tumor cell motility, adhesion, angiogenesis and invasion, leading to the inhibition of tumor development in vivo.
2. Methods 2.1. Chemicals The chiral organotelluranes used in this study, RF13-S and RF13-R (Figure 1) were prepared as previously described, starting from racemic 1-phenylethanol employing a chemoenzymatic preparative route [8]. All spectroscopic data are in accordance with the published data proving the identity and purity of the organotelluranes. The enantiomeric excess of each sample was also verified by circular dichroism of 2 mmol L-1 solutions (in dichloromethane) of RF13-S and RF13-R (see spectra in Supporting Information – S1 Figure). 1-[Butyl(dichloro)-4-tellanyl]-2-[1S-methoxyethyl]benzene (RF13-S). Colourless crystals, m.p.: (115.8-115.9)°C, []D26 = +38.2° (CHCl3, c = 1.92), enantiomeric excess: >99%. 1H NMR (500.13 MHz, CDCl3, ppm) δ 7.92 (dd, 3J 7.2 4J 1.8 Hz, 1H), 7.537,42 (m, 2H), 7.35 (dd, 3J 7.2 4J 2.1 Hz, 1H), 4.89 (q, 3J 6.6 Hz, 1H), 3.69-3.55 (m, 3H), 3.51 (s, 3H), 2.23 (qn, 3J 7.5, 2H), 1.67 (d, 3J 6.6, 3H), 1.63 (sext, 3J 7.2 Hz, 2H), 1.06 (t, 3J 7.5, 3H).
13C
NMR (125.78 MHz, CDCl3, ppm) δ 142.2, 133.1, 131.3, 131.1, 129.4, 127.7,
78.7, 56.3, 47.6, 26.6, 24.6, 20.3, 13.7.
125Te
NMR (157.79 MHz, CDCl3, ppm) δ . IR (KBr)
cm-1 3484, 2957, 2930, 2860, 2823, 1462, 1434, 1292, 1200, 1180, 1084, 909, 757, 611. Anal cald for C13H20Cl2OTe: C, 39.95, H, 5.16; Found C, 39.68, H, 5.26. 1-[Butyl(dichloro)-4-tellanyl]-2-[1R-methoxyethyl]benzene (RF13-R). Colourless crystals, m.p.: (115.1-115.7)°C, []D26 = -34.3° (CHCl3, c = 1.81), enantiomeric excess: >99%. Anal cald for C13H20Cl2OTe: C, 39.95, H, 5.16; Found C, 39.79, H, 5.36.
2.2. Enzymes Human recombinant cathepsin B was obtained as previously described [36]. Recombinant human cathepsin L was expressed in Pichia pastoris as previously described [37]. Heterologous expression and purification of human cathepsin S was performed as previously described [38]. Active concentrations of all cathepsins were determined by active-site titration experiments using 1 mM E-64 [L-trans-epoxysuccinyl-leucylamido(4guanidino)butane] dissolved in dimethyl sulfoxide [39]. Enzyme and substrate were used with the following conditions. Cathepsin B buffer: 100 mM sodium phosphate, 1 mM EDTA, 0.025% BRIJ, substrate Z-RR-MCA; Cathepsin L buffer: 50 mM sodium acetate buffer containing 100 mM NaCl, 2.5 mM EDTA pH 5.5, substrate Z-FR-MCA; Cathepsin S buffer: 50 mM sodium phosphate, containing 100 mM NaCl, 2.5 mM EDTA, pH 6.5, substrate ZFR-MCA. Cathepsin inhibition assay was performed as previously described [7]. In brief, the inhibition kinetics was performed using cathepsins previously activated with aqueous dithiothreitol (2 mmol L-1) and the assays with the organotelluranes were performed in degassed buffer solutions in the absence of the reducing agent.
2.3. Mice and cell lineages Six- to eight-week-old male C57BL/6 mice were obtained from the Center for Development of Experimental Models (CEDEME) animal facility, Universidade Federal de São Paulo (UNIFESP), and kept in isolators, supplied with autoclaved water and food. The animal experiments were carried out in accordance with the Research Ethics Committee of UNIFESP (protocol number CEP 1306/10). The original murine melanoma cell line B16F10 was obtained from the Ludwig Institute for Cancer Research (LICR), São Paulo branch. The subline B16F10-Nex2 was isolated at the Experimental Oncology Unit, Federal University of São Paulo, and deposited in the Banco
de Células do Rio de Janeiro (BCRJ), reg. 0342. It is characterized by adherence, spindle or stellate morphology, darkly melanotic cells, fast growing in vitro and in vivo, forming black tumor masses and black nodules in the lungs when injected subcutaneously and intravenously, respectively, in syngeneic H-2b C57BL/6 mice. The cervical cancer cell line (HeLa) and human melanoma cell lines Mewo and A2058 were obtained from the LICR. The human melanoma cell lines (SK-Mel-25, SK-Mel-28) were provided by Dr. Alan N. Houghton from the Memorial Sloan Kettering Cancer Center, NY, and HT144 was obtained from ATCC and was cultivated in Dulbecco's Modified Eagle Medium/HAM-F12 (Sigma Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS – Vitrocell Embriolife). The human umbilical vein endothelial cell line (HUVEC) was provided by Dr. Julio Scharfstein from Federal University of Rio de Janeiro and human fibroblast cell line (NIH 3T3) was obtained from ATCC. All cells were maintained in complete medium consisting of RPMI 1640, pH 7.2, supplemented with 10 mM N-2-hydroxyethylpiperazineN’-2-ethanesulphonic acid (HEPES), 24 mM sodium bicarbonate, 10% heat-inactivated fetal calf serum (FCS – Gibco, Minneapolis, MN, USA) and 40 µg/mL gentamicin sulfate (Hipolabor Farmacêutica, Sabará, MG, Brazil).
2.4. In vitro angiogenesis assay on Matrigel BD MatrigelTM Matrix (B&D Biosciences, Bedford, MA, USA) thawed on ice was distributed in 96-well plates (50 L per well) and allowed to polymerize for 1 h at 37oC. HUVEC cells (1.5 x 104 cells/well) suspended in 100 µL of RPMI medium supplemented with 1% of FCS were added to each well in the presence of compounds E-64 (5 M), RF-13R (0.5 M) or RF-13S (0.062 M). The plates were incubated at 37oC for 18h and then images were captured at 8x magnification with a ProgRes C3 camera (Jenoptik, Germany) coupled to an inverted light microscope (Zeiss, Germany). The number of angiogenic structures (closed
rings) was counted from 4 different wells, and the average value was determined for each sample. As a control of the assay, HUVEC cells were plated on Matrigel without the addition of any compound.
2.5. In vitro cytotoxic activity Cell lines (5x103 cells per well) were cultivated in 96-well plates and incubated for 24 h with compound RF-13R or RF-13S, after cell attachment, in the presence or absence of 1% FCS. Organotelluranes were used in increasing and folded concentrations, ranging from 0.02 μM to 50 μM. Cell proliferation was measured using the Cell Proliferation Kit I (MTT) (Boehringer Mannheim), an MTT-based colorimetric assay for quantification of cell proliferation and viability. Readings were performed in a plate reader at 570 nm.
2.6. In vivo tumor protection assay with tellurium compounds Syngeneic B16F10-Nex2 viable cells (5x105) in 0.1 mL (per mouse) were injected into tail veins of 22 seven-to-eight week-old male C57BL/6 mice. Eight animals were used in the control group and 14 animals were equally distributed for treatment with RF-13R and RF13S compounds. For protection experiments, the animals received i.p. doses of 20 M of RF-13R or RF-13S on days 2, 4, 6, 8, 10 and 12 after tumor cell challenge, or PBS, at the same time period. After 21 days, the lungs were collected and inspected for metastatic colonization and the superficial melanotic nodules were counted at 2X magnification. Data are reported as the mean number of tumor nodules ± SE for seven or more mice per group.
2.7. Monolayer wound-healing assay HUVEC and B16F10-Nex2 melanoma cells (5x104 cells/well) were plated on 24-well plates in triplicates. After cell attachment and growth to a confluent monolayer, one scratch wound
was made with a blue P1000 tip (Fisher) in each well. RPMI with 1% FCS was added to each well in the presence of 20 M of compound RF-13R or RF-13S. The plate was placed into a culture chamber at 37 oC on an inverted microscope (AxioObserver Z1 Zeiss) and wells were photographed at high resolution with the 5X objective lens, every hour for 28 h or 36h. Measuring the width of the wound and subtracting this value from the initial halfwidth value of the wound determined the cell migration distance using AxioVision microscope software (Zeiss).
2.8. Transwell invasion assay The Matrigel invasion ability of HUVEC and B16F10-Nex2 melanoma cells pre-incubated with 20 M of RF-13R or RF-13S was tested using Biocoat Matrigel Invasion Chambers (B&D Biosciences, Bedford, MA, USA). Briefly, 80 μL of Matrigel diluted (1:6) in cold PBS was added on the upper chambers of 24-well transwells and incubated for 30 min at 37 oC for gel formation. After incubation, 500 μL of serum-free RPMI containing 5x104 cells preincubated or not with organotelluranes was added to the Matrigel-coated PET filters (8-μm pore size). The lower chamber of the transwell unit was filled with 500 μL of RPMI supplemented with 2% FCS, as chemoattractant. Plates were incubated for 24 h at 37 oC and then non-invading cells were scraped off the top of the transwell with a cotton swab. Transwell units were removed from the 24-well plates, fixed for 30 min with methanol and stained with Giemsa’s stain for 15 min. Invading cells were counted under a light microscope.
2.9. Cell-to-extracellular matrix (ECM) adhesion assay For the ECM assay, 96-well plates were coated with 40 g/ml of type I collagen and type IV collagen in 2% acetic acid and fibronectin and laminin in PBS for 16h at 4oC. Non-specific
binding was blocked by the addition of 1% bovine serum albumin PBS for 1 h at 37oC. After three washing steps with PBS, B16F10-Nex2 cells were added to the wells (5x104 cells/well) in the presence of RF-13R or RF-13S at 2 M and incubated for 3 h at 37oC. Plates were gently washed twice with PBS to remove unattached cells and the attached cells were fixed with ice-cold methanol for 5 min. Fixed cells were stained with 1% toluidine blue in 1% sodium tetraborate for 5 min and washed with PBS. Dye was solubilized in 1% SDS for 20 min at 37oC and the resulting colored solution was quantified at 540 nm using a scanning multiwell spectrophotometer. Cells incubated without dichlorides were used as control and are taken as 100% adhesion.
2.10. Fluorescent staining B16F10-Nex2 and HT-144 cells were plated onto 35mm plates at a density of 1.3x105 cells/plate and cultured for 24 h in 10% FCS DMEM/Ham-F12. Cells were then treated with 10 or 30 M RF-13R and RF-13S in 1% FCS DMEM/Ham-F12 for 24 h. Cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100 and incubated with a 20 µM phalloidin-TRITC, 20 µM tubulin-Cy2, 10µg/mL RNAse A solution for 90 min. Slides were closed with TO-PRO-3 and anti-fading agent VectaShield Mounting Media (Vector Laboratories, USA). Fluorescent images were acquired with confocal laser scanning microscope Zeiss LSM510 (Zeiss, Germany) using argon (488nm) and helium-neon (543 nm) lasers.
2.11. Statistical analysis The data are represented as mean ± SE. Statistical significance was determined by the Student’s t test. All experiments were conducted two or more times. Reproducible results were obtained and representative data are shown.
3. Results
3.1. Cathepsin activity inhibition by enantiomeric organotelluranes Organotelluranes display high reactivity towards thiols; this led to their application as inhibitors of cysteine proteases like cathepsins [2, 6, 7], caspases and more recently protein tyrosine phosphatases [40]. The chiral organotelluranes RF13-R and RF13-S were previously evaluated as inhibitors of cathepsins S and V, showing no significant enantiodiscrimination between the pair of enantiomers when in presence of small quantity of dithiothreitol [8]. On the other hand, the enantiomeric organotelluranes exhibited differences on the inhibition of cathepsins B, L, S and K in the absence of this reducing agent. The organotellurane RF-13S showed higher potency for the inhibition of cysteine cathepsins with as expressed in the values of the determined second-order inhibition rate constants values of 7400±90, 8700±120, 600000±5400 M-1s-1 for cathepsins B, L and S, respectively; in other hand its antipode, RF-13R presented values of 3700±45, 5500±90 and 39000±340 M-1s-1.
3.2. Cytotoxic effect of organotelluranes To evaluate the cytotoxic effects of the organotelluranes, murine and human tumor cell lines were incubated with dichlorides RF-13R and RF-13S for 16 h, and cell viability was measured using MTT reagent, following manufacturer’s instructions. All cell lines tested were more susceptible to dichloride RF-13S than to RF-13R, when the assay was carried out in the presence of FCS-free RPMI medium (Table 1). The IC50 values were different depending on the cell line tested, but in all cases the cytotoxicity occurred at micromolar concentrations and was dose-dependent (not shown). B16F10-Nex2 melanoma
cells and cervical cancer cells (Hela) were most sensitive to dichlorides, with IC50 values lower than 1 M to RF-13S. This organotellurane was more cytotoxic than RF-13R.
3.3. Effect of organotelluranes on tumor development Effective treatment of mice challenged intravenously with B16F10-Nex2 cells was investigated using RF-13R and RF-13S. Mice were injected with 5x104 melanoma cells/100 μl/animal and treated on days 2, 4, 6, 8 and 10 with organotelluranes at 20 M/injection. In Figure 2A, we show that animals treated with RF-13R and RF-13S had, respectively, 2-fold and 7-fold less superficial nodules than animals treated with PBS. Specimens of lungs from animals treated with dichlorides are shown in Figure 2B. These results corroborate the in vitro experiments, showing that RF-13S is more effective than RF-13R as inhibitor of tumor development in a melanoma metastatic model.
Figure 2. In vivo antitumor effects of organotelluranes RF-13R and RF-13S. C57BL/6 mice (n=10, control group; n=6, treated-group) were injected intravenously with 5x105 B16F10-Nex2 melanoma cells. Tellurium compounds were administered intraperitoneally (20 M/injection) on days 2, 4, 6, 8, 10 and 12 after tumor cell challenge. Control mice received PBS for the same period of time. Lungs were collected 21 days after tumor challenge and the superficial nodules were counted. (A) Total number of lung metastases. Data presented as mean number of lung lesions ± SE. Two independent experiments were performed. p<0.0005, RF-13R vs
control; p<0.000001, RF-13S vs control. B. Representative lungs from B16-bearing mice treated with PBS, RF-13R or RF-13S.
3.4. Organotelluranes inhibit angiogenesis in vitro The effect of dichlorides on endothelial cell angiogenesis in vitro was evaluated using HUVECs plated on Matrigel in presence of RF-13R and RF-13S or E-64, a known cysteine protease inhibitor, as a control (Fig. 3). The growth of HUVECs on Matrigel with 1% fetal calf serum led to the formation of closed intercellular compartments arising from endothelial cell sprouting (pro-angiogenic structures) independent of any other factor. Addition of RF13R, RF-13S and E-64 to the incubation mixture inhibited the formation of these structures in about 45%, 55% and 80%, respectively compared to control. Although the effective concentration was substantially different, RF-13S was able to inhibit angiogenesis with only 62 nM, while compound RF-13R and E-64 needed 0.5 M and 5 M, respectively, to exert the same effect.
Figure 3. Effect of organotelluranes RF-13R and RF-13S on pro-angiogenic closed structures formed by sprouting of endothelial cells. (A) HUVECs were plated on Matrigel in RPMI medium supplemented with 1%
FCS in the presence of dichorides RF-13R and RF-13S and cysteine protease inhibitor E-64. The number of pro-angiogenic structures was counted after 18 h. One representative picture of four different treatments is shown with the respective counts. (B) Results expressed in number of pro-angiogenic structures. Error bars, SD of quadruplicate samples. * p = 0.02 vs control; ** p = 0.004 vs control; *** p = 0.001 vs control.
3.5. Organotelluranes RF-13R and RF-13S decrease melanoma cell adhesion to extracellular matrix proteins To analyse the effect of organotelluranes on cell adhesion, B16F10-Nex2 cells were plated on 96-well plates coated with ECM proteins in the presence of 2 M RF-13R and RF13S. Incubation for 3h with organotelluranes modified adhesion of B16F10-Nex2 cells to the ECM proteins tested, fibronectin (FN), laminin, and collagen types I and IV. Adhesion of melanoma to types IV and I collagen was more affected by organotellurane treatment than to adhesion to FN and laminin (Fig. 4). In this case, dichloride RF-13S was also more effective than RF-13R.
Figure 4. Decrease in cell adhesion to ECMs after treatment with organotelluranes RF-13R and RF-13S. B16F10-Nex2 cells (5x104 cells/well) were spread on 96-well plates coated with collagen type I (Col I), collagen type IV (Col IV), laminin (Lamin) or fibronectin (Fibro) in the presence of 2 M of RF-13R or RF-13S
dichlorides. After incubation for 3h, non-adherent cells were removed by a PBS rinse and adherent cells were stained. Data are representative of three independent experiments. *p < 0.05; **p < 0.005 (values are compared with untreated control).
3.6. Modulation of cell migration and invasion by organotelluranes RF-13R and RF13S To evaluate the ability of organotelluranes to inhibit cell migration, scratch wound healing assays were performed with B16F10-Nex2 melanoma cells and endothelial cells (HUVEC). RF-13R (20 μM) inhibited by 33% the wound closure in HUVEC cells as compared to control (Fig. 5A). The inhibition of migration by organotelluranes was more effective in melanoma cells. B16F10-Nex2 cells migration was inhibited by 80% with 20 M of compounds RF-13R and RF-13S (Fig. 5B). The invasion process of melanoma and endothelial cells was significantly inhibited with 20 M of RF-13R and RF-13S as can be seen in Figure 6. RF-13S was more effective in inhibiting HUVEC invasion than RF-13R (Fig. 6A). Melanoma cells invasion decreased 80% by both organotelluranes (Fig. 6B).
Figure 5. Effect of organotelluranes RF-13R and RF-13S on cell migration. HUVEC (A) and B16F10-Nex2 melanoma cells (B) at 5x104 cells/well in 1 mL were seeded on 24-well plates and after growing to form a confluent monolayer, one scratch wound was made. Cell migration in the presence of RF-13R and RF-13S compounds at 20 M was determined by measuring the width of the wound at 0h and after 24h incubation. p values comparing to control are shown.
Figure 6. Effect of organotelluranes RF-13R and RF-13S on cell invasion. HUVEC (A) and B16F10-Nex2 melanoma cells (B) (5x103) were incubated on transwell units coated with Matrigel in the presence of RF-13R and RF-13S compounds at 20 M for 24h. Invading cells were quantified by counting after crystal violet staining. Values are expressed in percentage comparing to control. p values comparing to control are shown.
3.7. Organotelluranes RF-13R and RF-13S affect cell morphology B16F10-Nex2 and HT-144 cells were treated with 10µM RF-13R and RF-13S for 24 hours and were then stained with tubulin-Cy2 and TO-PRO-3. The fluorescently stained confocal microscope images (Fig. 7) showed that both RF-13R and RF-13S affect cell morphology. Cell lines B16F10-Nex2 and HT-144 treated with organotelluranes showed a tendency to cell rounding, with retraction of cell protrusions and depolymerisation of F-actin, as well as some membrane blebbing. HT-144 cells stained with phalloidin-TRITC, tubulin-
Cy2 and TO-PRO-3 and treated with 30µM RF-13R and RF-13S showed complete loss stress fibers (Fig 8), with cell rounding and the remaining cortical actin in close association with the microtubule network.
Figure 7. Effect of organotelluranes RF-13R and RF-13S on cell morphology. HT-144 and B16F10-Nex2 melanoma cells (1.3x105) were treated with 10 or 30 M of RF-13R and RF-13S for 24h. Cells were permeabilized and stained with tubulin-Cy2 (green) and TO-PRO-3 (blue). Fluorescent images were acquired with confocal laser scanning microscope Zeiss LSM510 (Zeiss, Germany) using argon (488nm) and heliumneon (543nm) lasers.
Figure 8. Effect of organotelluranes RF-13R and RF-13S on cell morphology. HT-144 (1.3x105) was treated with 30 M of RF-13R and RF-13S for 24h. Cells were permeabilized and stained with phalloidin-TRITC (red), tubulin-Cy2 (green) and TO-PRO-3 (blue). Fluorescent images were acquired with confocal laser scanning microscope Zeiss LSM510 (Zeiss, Germany) using argon (488nm) and helium-neon (543nm) lasers.
4. Discussion Cathepsins have a well-characterized role in tumor development as described in clinical reports and experimental murine cancer models [32, 41, 42]. Up-regulation of cathepsin expression and/or activity and extracellular localization are associated with cancer progression and positively correlated with a poor prognosis for cancer patients [4345], including those with melanoma [46, 47]. Cathepsins B, L and some others are able to promote cancer cell migration and invasion [48, 49]. Also, cathepsin B expression correlates with increased metastatic potential of tumors [50]. Therefore, selective inhibitors of cathepsins could offer new possibilities for the diagnosis and treatment of human cancers [34]. In the present study, we report on the inhibition by chiral tellurium compounds
of cysteine cathepsins and the possible application of these compounds as anticancer agents. The inhibition of cathepsins B and L by organotelluranes RF-13R and RF-13S was time- and concentration-dependent. The distinguished feature of cysteine proteases inhibition by organic telluranes consists in its reactivation by reducing thiols or by the decrease of inhibition efficacy in a reducing medium (not shown). This aspect is particularly noteworthy because it can be expected that hypervalent tellurium compounds exert a low influence in molecular targets in the reducing cytoplasmic environment. Nevertheless, it was found that some organotelluranes can inhibit intracellular proteolysis of Plasmodium falciparum parasites suggesting that these thiol-reactive compounds can be directed to more reactive thiols within the cell [51]. Tumor
cell
lines,
endothelial
cells
and
fibroblasts
were
susceptible
to
organotelluranes in micromolar concentrations and the cytotoxic effect was dosedependent. B16F10-Nex2 murine melanoma cells and the cervical cancer cell line (HeLa) were the cell lines more susceptible to organotelluranes, while human umbilical vein endothelial cell (HUVEC) and human melanoma HT-144 were more resistant. Corroborating the in vitro cytotoxic assay, RF-13S was more effective than RF-13R in the inhibition of tumor development in vivo; this could be related to a differential reactivity of the organotelluranes with tissular, cysteine-targets. Intraperitoneal administration of organotelluranes at 20 M for five non-consecutive days after melanoma challenge, reduced lung metastases in 85% and 58% for RF-13S and RF-13R treatment, respectively. According with this, RF-13S was more effective than its enantiomer, in the inhibition of endothelial cell angiogenesis in vitro. It is unclear by what mechanism the organotelluranes inhibit the angiogenic process. The inorganic tellurane AS101 was reported to inhibit angiogenesis in an indirect manner, regulating the expression and secretion of VEGF,
MMP-2, and MMP-9 [24]. The organotelluranes tested here were efficient inhibiting the activity of cathepsins V and S [8]. The role of cathepsins in angiogenesis modulation has been studied in a wide variety of tumor models. Extracellularly active cathepsins B and L were identified in highly invasive melanoma cells [52]. It was shown that cathepsin L facilitates high metastatic melanoma cell invasion and migration [49], and cathepsin B inhibitor (CA-074) significantly reduced human melanoma growth and number of lung metastases [53]. Cathepsin B was also described as a regulator of the concentration of angiomodulators at the level of the endothelium, being able to modulate the angiogenic process [54]. Here, we observed that HUVEC and B16F10-Nex2 cells express cathepsins B and L, which could be another possible target for angiogenesis inhibition. The death of most cancer patients is due to metastasis events. The process by which cancer cells escape from the primary tumor lesion, invade the blood or lymphatic vessels and colonize distant organs is very complex and is controlled by numerous mediators. Fundamental steps in this process are the tumor cell capability to migrate and invade surrounding tissues. Loss of cell-cell and cell-matrix adhesion and degradation of ECM components predispose cells to migration and invasion [55]. We show that 2 M RF13R and RF-13S decreased melanoma cell adhesion to ECM proteins and significantly inhibited migration and invasion of tumour and endothelial cells, being able to significantly decrease angiogenesis and metastasis. This could be due to the effect of the organotelluranes on the cell cytoskeleton, disassembling actin and reducing cell protrusions. In human melanoma cells (HT-144) organotellurane treatment led to round cells with remaining cortical actin.
5. Conclusion
The angiogenic switch is a fundamental step that allows tumors to expand in size and acquire metastatic potential. Anti-angiogenic therapies are very promising, since they inhibit primary tumor growth and prevent metastatic spread. Adhesion inhibition is also an effective strategy for preventing proliferation and metastasis of melanomas. The results shown in the present work depict organotelluranes as potential antitumor agents, acting directly on tumour cell proliferation, migration and invasion, and on endothelial cells, disrupting angiogenesis. Organotelluranes display low toxicity and high antitumor efficiency, thus being encouraging candidates to be developed as anti-cancer drugs.
Acknowledgements This work was supported by grants from FAPESP (2004/14426-0, 2006/51880-7, 2009/53840-0) and CNPq (487012/2012-7, 477525/2011-3, 474440/2013-3), Brazil. LRT and LJ are CNPq career fellowship.
Authors’ contributions
Conceived and designed the experiments: TP RLORC GMMS. Performed the experiments: TP AAM ATO FVP RLORC. Analyzed the data: TP AAM LJ LRT GMMS RLORC. Contributed reagents/materials/analysis tools: TP ATO GMMS LRT LJ RLORC. Wrote the paper: TP AAM GMMS LRT LJ RLORC.
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Figure legends
Figure 1: Structures of antitumoral tellurium compounds active in in vivo models. The inorganic tellurane AS101, the naphtoquinone LAB027, the β-cyclodextrin derivative DTCD, the bis-(p-methoxyphenyl) tellurium dichloride and the quiral organotelluranes used in the present study.
Figure 2: In vivo antitumor effects of organotelluranes RF-13R and RF-13S. C57BL/6 mice (n=10, control group; n=6, treated-group) were injected intravenously with 5x105 B16F10Nex2 melanoma cells. Tellurium compounds were administered intraperitoneally (20 M/injection) on days 2, 4, 6, 8, 10 and 12 after tumor cell challenge. Control mice received PBS for the same period of time. Lungs were collected 21 days after tumor challenge and the superficial nodules were counted. (A) Total number of lung metastases. Data presented as mean number of lung lesions ± SE. Two independent experiments were performed. p<0.0005, RF-13R vs control; p<0.000001, RF-13S vs control. B. Representative lungs from B16-bearing mice treated with PBS, RF-13R or RF-13S.
Figure 3: Effect of organotelluranes RF-13R and RF-13S on pro-angiogenic closed structures formed by sprouting of endothelial cells. (A) HUVECs were plated on Matrigel in RPMI medium supplemented with 1% FCS in the presence of dichorides RF-13R and RF13S and cysteine protease inhibitor E-64. The number of pro-angiogenic structures was counted after 18 h. One representative picture of four different treatments is shown with the respective counts. (B) Results expressed in number of pro-angiogenic structures. Error bars, SD of quadruplicate samples. * p = 0.02 vs control; ** p = 0.004 vs control; *** p = 0.001 vs control.
Figure 4. Decrease in cell adhesion to ECMs after treatment with organotelluranes RF-13R and RF-13S. B16F10-Nex2 cells (5x104 cells/well) were spread on 96-well plates coated with collagen type I (Col I), collagen type IV (Col IV), laminin (Lamin) or fibronectin (Fibro) in the presence of 2 M of RF-13R or RF-13S dichlorides. After incubation for 3h, nonadherent cells were removed by a PBS rinse and adherent cells were stained. Data are representative of three independent experiments. *p < 0.05; **p < 0.005 (values are compared with untreated control).
Figure 5: Effect of organotelluranes RF-13R and RF-13S on cell migration. HUVEC (A) and B16F10-Nex2 melanoma cells (B) at 5x104 cells/well in 1 mL were seeded on 24-well plates and after growing to form a confluent monolayer, one scratch wound was made. Cell migration in the presence of RF-13R and RF-13S compounds at 20 M was determined by measuring the width of the wound at 0h and after 24h incubation. p values comparing to control are shown.
Figure 6: Effect of organotelluranes RF-13R and RF-13S on cell invasion. HUVEC (A) and B16F10-Nex2 melanoma cells (B) (5x103) were incubated on transwell units coated with Matrigel in the presence of RF-13R and RF-13S compounds at 20 M for 24h. Invading cells were quantified by counting after crystal violet staining. Values are expressed in percentage comparing to control. p values comparing to control are shown.
Figure 7: Effect of organotelluranes RF-13R and RF-13S on cell morphology. HT-144 and B16F10-Nex2 melanoma cells (1.3x105) were treated with 10 or 30 M of RF-13R and RF13S for 24h. Cells were permeabilized and stained with tubulin-Cy2 (green) and TO-PRO-3
(blue). Fluorescent images were acquired with confocal laser scanning microscope Zeiss LSM510 (Zeiss, Germany) using argon (488nm) and helium-neon (543nm) lasers.
Figure 8: Effect of organotelluranes RF-13R and RF-13S on cell morphology. HT-144 (1.3x105) was treated with 30 M of RF-13R and RF-13S for 24h. Cells were permeabilized and stained with phalloidin-TRITC (red), tubulin-Cy2 (green) and TO-PRO-3 (blue). Fluorescent images were acquired with confocal laser scanning microscope Zeiss LSM510 (Zeiss, Germany) using argon (488nm) and helium-neon (543nm) lasers.
S1 Figure: CD spectra of dichlorides RF-13R and RF-13S in chloroform.
Table 1: Estimated IC50 of RF-13R and RF-13S cytotoxic effect in murine and human cell lines IC50 (mM) Cell
line[a]
IC50 ratio (R) : (S)
RF-13R
RF-13S
B16F10-Nex2
2.3 + 1.1
0.9 + 0.3
2.6
HUVEC
20.3 + 3.3
2.8 + 0.9
7.25
3T3
11.5 + 2.1
3.1 + 0.8
3.7
SKMel-25
18.0 + 2.5
2.5 + 1.0
7.2
SKMel-28
6.2 + 0.7
3.1 + 0.5
2.0
A2058
5.5 + 1.8
1.5 + 0.8
3.7
Mewo
9.5 + 1.1
3.2 + 0.5
3.0
Hela
2.3 + 0.2
0.8 + 0.3
2.9
HT-144
18.1+ 5.3
15.3 + 3.0
1.2