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Flubendazole induces mitotic catastrophe and apoptosis in melanoma cells
Toxicology in Vitro 46 (2018) 313–322
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Flubendazole induces mitotic catastrophe and apoptosis in melanoma cells
MARK
Čáňová K. , Rozkydalová L. , Vokurková D. , Rudolf E. a
a b c
b
c
a,⁎
Department of Medical Biology and Genetics, Charles University, Faculty of Medicine in Hradec Králové, Czech Republic Department of Pharmacology, Charles University, Faculty of Pharmacy in Hradec Králové, Czech Republic Department of Clinical Immunology and Allergology, University Hospital in Hradec Králové, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Keywords: Melanoma Flubendazole Microtubules Mitotic catastrophe, apoptosis
Flubendazole (FLU) is a widely used anthelmintic drug belonging to benzimidazole group. Recently, several studies have been published demonstrating its potential to inhibit growth of various tumor cells including those derived from colorectal cancer, breast cancer or leukemia via several mechanisms. In the present study we have investigated cytotoxic effects of FLU on malignant melanoma using A-375, BOWES and RPMI-7951 cell lines representing diverse melanoma molecular types. In all three cell lines, FLU inhibited cell growth and proliferation and disrupted microtubule structure and function which was accompanied by dramatic changes in cellular morphology. In addition, FLU-treated cells accumulated at the G2/M phase of cell cycle and displayed the features of mitotic catastrophe characterized by formation of giant cells with multiple nuclei, abnormal spindles and subsequent apoptotic demise. Although this endpoint was observed in all treated melanoma lines, our analyses showed different activated biochemical signaling in particular cells, thus suggesting a promising treatment potential of FLU in malignant melanoma warranting its further testing.
1. Introduction Metastatic melanoma is an aggressive form of skin cancer, with a high mortality rate. Although melanoma represents < 5% of all diagnosed skin cancers, the World Health Organization has indicated that its incidence is increasing faster than any other type of malignancy, mainly due to the general population's increasing exposure to ultraviolet light (Daud et al., 2017). The extensive danger of melanoma is based not only on the increasing incidence but also on its tendency to fast metastasizing and on extensive development of drug resistance - the biggest reason of treatment failure. While the prognosis of melanoma is promising with early diagnosis, upon its systemic spread where the effective therapy is still missing survival rates are significantly lower – the main reason for ongoing searches for new treatment options. One of the promising strategies in the development of novel antioneoplastic medicines is drug repositioning (or repurposing). Drug repositioning is the process of searching for new indications of existing drugs (Shim and Liu, 2014). Repositioned drugs have the advantage of decreased development costs and decreased time to market compared to traditional discovery candidates, mainly due to the availability of previously collected pharmacokinetic, toxicology, and safety data (Padhy and Gupta, 2011). Among the potential pharmacological candidates for repurposing is also a group of benzimidazoles whose several
members have already shown antitumor activities in various preclinical models (Michaelis et al., 2015; Nygren et al., 2013). Flubendazole (FLU) is a derivative benzimidazole which has been extensively evaluated in humans and animals for the treatment of intestinal parasites as well as for the treatment of systemic worm infections (Spagnuolo et al., 2010). The mechanism of action of benzimidazole compounds in helmints can be explained by their increased affinity to tubulin, which leads to the inhibition of tubulin polymerization and results in the interference with microtubule-mediated transport in helminth tissues. Accordingly, benzimidazoles were found to interact with mammalian tubulin too, although their affinity is weaker in comparison to the helminth tubulin. Cytotoxicity of various benzimidazoles including FLU has been proved in a number of in vitro and in vivo tumor models (Canova et al., 2017). FLU first showed its anti-tumor activity in leukemia and myeloma cells where at low and pharmacologically feasible concentrations it induced mitotic catastrophe and cell death and delayed tumor growth in vivo (Spagnuolo et al., 2010). Moreover, in recently published studies, FLU inhibited proliferation of several breast cancer cell lines (Hou et al., 2015), colon cancer cell lines (Kralova et al., 2016) and neuroblastoma cells (Michaelis et al., 2015). Despite the fact that currently no information exists about FLU effects in melanoma, reports on cytotoxic activity of mebendazole, a related benzimidazole compound, in
⁎ Corresponding author at: Department of Medical Biology and Genetics, Charles University, Faculty of Medicine in Hradec Králové, Zborovská 2089, 500 03 Hradec Králové, Czech Republic. E-mail address: [email protected] (E. Rudolf).
http://dx.doi.org/10.1016/j.tiv.2017.10.025 Received 10 August 2017; Received in revised form 20 October 2017; Accepted 25 October 2017 Available online 26 October 2017 0887-2333/ © 2017 Elsevier Ltd. All rights reserved.
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concentration range 0.01 μM–10 μM. After 24-, 48- and 72-hours exposure, the medium was removed and the cells were washed twice with 150 μl PBS. Then, 100 μl of culture medium containing WST-1 (0.3 mg/ ml) was added to each well. Absorbance of the samples was measured immediately at 450 nm/650 nm wavelength (Tecan Infinite M200; Tecan, Salzburg, Austria). The samples were then placed in the incubator and the absorbance was measured again after 2 h of incubation. Each sample was assayed in 6 parallels and three independent experiments were performed. The viabilities of treated cells were expressed as percentage of untreated controls (100%).
melanoma cells as well as in xenographts appear promising (Doudican et al., 2008; Doudican et al., 2013) and suggest a wider scientific potential of other benzimidazole compounds including FLU in this type of malignancy. Based on these facts, we have investigated the effect of FLU on three melanoma cell lines A-375, BOWES and RPMI-7951 representing diverse molecular types of this malignancy. Here we report that FLU inhibited the proliferation of all three employed cell lines with a similar IC50 equalling to 0.25 μM (RPMI-7951), 0.90 μM (BOWES) and 0.96 μM (A-375) at 72 h of exposure. FLU-dependent antiproliferative activity was associated with accumulation of cells at the G2/M phase of cell cycle and their extensive morphological changes including the specific alterations of the microtubular network. Thus treated cells finally underwent mitotic catastrophe characterized by formation of giant cells with multiple nuclei, abnormal spindles and caspase-dependent apoptosis. Although this endpoint was observed in all treated melanoma lines, our analyses showed different activated biochemical signaling in particular cells, thus suggesting a promising treatment potential of FLU in malignant melanoma warranting its further testing.
2.3.2. Test of proliferation using the xCELLigence system The system measures electrical impedance across interdigitated microelectrodes integrated at the bottom of tissue culture E-plates. The impedance measurement provides quantitative information about the biological status of the cells, including cell number, viability and morphology. Ninety microliter of culture medium was added into each well and plates were inserted in the device for background measurement. Then, 100 μl of cell suspension (containing 1500 cells) was added in duplicate to the appropriate wells. The growing impedance (corresponding to cell proliferation) was measured every 30 min for 24 h. After 24 h, 10 μl of treatment medium was added into each well, so that final concentration of FLU was 1 μM. Plates were inserted back into the device and impedance was measured every hour for 72 h. Each sample was assayed in duplicate and three independent experiments were performed.
2. Materials and methods 2.1. Chemicals Flubendazole was purchased from Janssen Pharmaceutica (Prague, Czech Republic), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer, dithiotreitol (DTT), dimethylsulfoxide (DMSO), 4′,6-diamidino-2-phenylindole (DAPI), Triton-X, propidium iodide (PI), Dulbecco's Modified Eagle's Medium (DMEM), sodium dodecyl sulfate (SDS), bicinchoninic acid (BCA) kit for protein determination were purchased from Sigma-Aldrich (Prague, Czech Republic). Eagle's Minimum Essential Medium (EMEM) was supplied by LGC Standards (Lomianki, Poland), fetal bovine serum from Invitrogen (Carlsbad, CA, USA) and bovine serum albumin (BSA) from Fluka (Prague, Czech Republic). WST-1 was purchased from Roche Diagnostics (Manheim, Germany). Polyvinylidene difluoride (PVDF) membrane was obtained from BIO-RAD Laboratories (Prague, Czech Republic). All other chemicals were of highest analytical grade.
2.4. Time-lapse videomicroscopy A-375, BOWES and RPMI-7951 cell lines were seeded into plastic tissue-culture dishes with glass bottom and left for 24 h in an incubator with 5% CO2 at 37 °C. Next, the growth medium was replaced with a medium containing flubendazole. The tissue-culture dishes were transferred into a time-lapse imaging system BioStation IM (Nikon, Prague, Czech Republic) combining an incubator, a motorized microscope and a cooled CCD camera. Recording was carried out in a multipoint and multichannel manner employing various time-lapse modes and upon small as well as high magnifications to allow global as well as detailed view of changes in behavior of treated cell populations. Recorded sequences were subsequently semi automatically analyzed with the software NIS Elements AR 3.20 (Nikon, Prague, Czech Republic).
2.2. Cell culture and treatment conditions Human melanoma cell lines were purchased from ATCC. A-375 (ATCC® CRL1619™) are adherent, epithelial cells hypotriploid with a modal number of 62 chromosomes whose BRAF and CDKN2A genes are mutated. BOWES (ATCC® CRL9607™) are adherent, epithelial, heteroploid cells with wild-type BRAF. RPMI-7951 (ATCC® HTB66™) are adherent, epithelial-like cells derived from metastasis to the lymph node, hyperdiploid (47–66 chromosomes) with mutant BRAF, TP53, CDKN2A and PTEN. A-378 and BOWES were maintained in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. RPMI-7951 was cultivated in EMEM medium with 10% FBS and 1% penicillin/streptomycin. Cells were maintained upon standard conditions (37 °C, 5% CO2 in an incubator) and passaged twice a week upon 90% cells confluence using 0.05% EDTA/trypsin. Only mycoplasma-free cells were used for experiments. Stock solutions of FLU were prepared in DMSO and stored in aliquots at 6 °C. Cells were treated with medium containing 1 μM FLU, the final concentration of DMSO in the medium was 0.1%. Control samples were treated with medium containing 0.1% DMSO.
2.5. Cell cycle distribution analysis 2.5.1. Flow cytometry Cells were cultured in 75 cm2 culture flasks in 20 ml of culture medium for 12 and 24 h. Then the culture medium was replaced by fresh medium containing 1 μM of FLU or 0.1% DMSO as a control. After 12 or 24 h of treatment, the cells were trypsinized, collected by centrifugation, washed with PBS and fixed with ice-cold 70% ethanol while gently vortexing. Fixed samples were stored overnight in 4 °C. Then they were washed with PBS and incubated with 0.5 ml of the Vindelov solution (1.2 g/l TRIS, 0.6 g/l NyCl, 0.01 g/l RNase and 0.05 g/l propidium iodide) for 50 min at 37 °C. The samples were analyzed at the FC500 Cytomics Flow cytometer (Beckman Coulter,Hialeah, FL, USA) with PI fluorescence detected in FL3 channel. Cell cycle distributions in control and treated samples were analyzed with MultiCycle AV for Windows (Phoenix Flow Systems, San Diego, USA) and three independent experiments were performed. 2.5.2. Percentage of cells in S-phase by EdU labeling EdU (5-ethynyl-2′-deoxyuridine) is incorporated into DNA during active DNA synthesis. The cells were cultured in 96-well plates and treated with FLU in concentration 1 μM. After 24 hours exposure, the cells were labeled with EdU (Click-iT® EdU Flow Cytometry Assay Kit,
2.3. Cytotoxicity assay 2.3.1. The WST assay The cells were cultured in 96-well plates and treated with FLU in 314
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Fig. 1. Concentration-dependent effect of flubendazole (FLU) on cell proliferation in melanoma cell lines A-375, BOWES and RPMI-7951. Cells were exposed to FLU and proliferation (up to 72 h) was measured by WST-1 assay (A) and xCELLigence system (B) as described in Materials and methods section. Values represent means ± SD of at least three experiments *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001 compared to untreated control cells at the same treatment interval with one way-Anova test and Bonferroni's post test for multiple comparisons.
of cells in the medium containing 1 μM FLU, cells were lysed using buffer containing 50 mM HEPES, 5 mM CHAPS, 5 mM DTT. Then the lysates were collected into microtubes. Caspases 2 and 3/7 reagents were prepared as described in manufacturer's instruction. The lysates (25 μl in each well) were transferred into white-walled 384-well-plate for luminometer. Then, 25 μl of Caspase 2 or Caspase 3/7 reagents were added to each well. Plate was gently mixed for 30 s and then incubated for 30 min at room temperature. The luminescence was measured in three independent experiments using luminometer (Tecan Infinite M200; Tecan, Salzburg, Austria).
Invitrogen-Molecular Probes, Inc., Carlsbad, CA, USA) in concentration 10 μM and incubated for 2 h. After EdU labeling the medium was removed and the cells were washed in PBS and fixed with paraformaldehyde and incubated for 15 min. Then were washed again with PBS + 3% BSA and permeabilized with 0.5% Triton-X for 20 min in room temperature. After removing permeabilization solution, cells were washed twice in PBS + 3% BSA and to the plates were added EdU buffer additive and incubated for 30 min in the dark, 5 min before incubation was over was added DAPI (1 μg/ml). Cells were washed again and determined by using Image Xpress Micro XLS Widefield HighContent Analysis System. Specific fluorescence was visualized, recorded and analyzed by Cell scoring module of MetaXpress® Image Acquisition and Analysis Software (Molecular Devices, Sunnyvale, CA, USA). Three independent experiments were performed.
2.7. Immunofluorescence and image analysis Cells in cytospine chambers were exposed to 1 μM FLU for 12 and 24 h, then were washed by 1 ml pre-warm PBS and fixed with 4% paraformaldehyde (10 min, 25 °C), rinsed with phosphate saline buffer with 1% Triton X (PBS-T) and blocked in 5% BSA for 1 h at 25 °C. Immunofluorescence was performed at 4 °C for 1 h using α-tubulin (Cell Signaling Technology, 1: 100), β-actin (1:1000, Sigma Aldrich) and p21 (1:100, Santa Cruz biotechnology) antibodies. After washing with cold
2.6. Caspase assay The activities of caspases 2 and 3/7 were assayed using Promega Caspase-Glo Assay (Madison, USA). The A-375, BOWES and RPMI-7951 cells were cultivated in 96-well plates. After 12, 24 and 48 h incubation 315
Fig. 2. The effect of 1 μM flubendazole (FLU) on cell cycle of melanoma cell lines during 24 h. Melanoma cells were exposed to FLU and their cell cycle analysis was carried out using FC500 Cytomics Flow cytometer. Cell cycle distributions in control and treated samples were analyzed with MultiCycle AV for Windows. (A) Flow charts of analyzed control and 1 μM FLU-exposed melanoma cells A-375, BOWES and RPMI-7951. (B) Calculated distribution of the treated melanoma cells in respective cell cycle phases. (C) Percentage of S-phase cells in 1 μM FLU exposed melanoma cells during 24 h as measured by EdU assay (Materials and methods). Values represent means ± SD of at least three experiments *P < 0.05, ***P < 0.001, **** P < 0.0001 compared to untreated control cells at the same treatment interval with one way-Anova test and Bonferroni's post test for multiple comparisons.
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Fig. 3. The effect of 1 μM flubendazole (FLU) on cell cycle inhihibitor p21 expression and cellular localization in melanoma cell lines A-375, BOWES and RPMI-7951 during 24 h. Cells were exposed to FLU during up to 24 h and the expression and subcellular localization of p21 was determined as described in Materials and methods section. Fluorescence microscopy 600 ×. Bar 25 μm.
PBS (5 min, 25 °C), secondary antibody (anti-rabbit/anti-mouse, 1:250) was added for additional 1 h (25 °C). The specimens were then rinsed three times in PBS, post-labeled with DAPI (10 μg/ml, 15 min, 25 °C), mounted into ProLong® Gold medium (Invitrogen-Molecular Probes, Inc., Carlsbad, CA, USA) and examined under fluorescence microscope Nikon Eclipse E 400 (Nikon, Prague, Czech Republic) (excitation filter 330–380 nm and emission filter 420 nm) equipped with a digital color matrix camera COOL 1300 (VDS, Vosskűhler, Germany). Photographs were taken using the software NIS Elements AR 3.20. Samples were prepared in duplicates, three independent experiments were performed.
DAKO), the incubation with primary antibody lasted for 1 h in room temperature. Subsequently, the membranes were washed six times in TBST for 9 min, followed by 1 hour incubation with secondary antibody in room temperature (anti-rabbit secondary antibody, 1:2000 for BAX, p53 and phospho-p53 (ser15), and anti-mouse secondary antibody 1:800 for BCL2 and 1:10,000 for β-actin). After washing the membrane six times (9 min) with TBST, the chemiluminescence proces and quantification of immunoreactive bands on the exposed films were carried out. For each antibody used, three independent experiments were performed.
2.8. Western blot analysis
2.9. Statistics
Treated and control cells were washed with PBS and harvested in ice-cold lysis buffer (50 mM Tris/HCl, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, b-glycerolphosphate, 50 mM NaF, 10 mM sodium pyrophosphate, 200 lM sodium orthovanadate, 2 mM DTT). The lysates were resuspended and supernatants were obtained after a 13,000 rpm centrifugation at 4 °C for 10 min. Amount of protein in supernatant was determined by BCA assay. The SDS sample buffer (Tris–HCl pH 6.8, 40% glycerol, 6% SDS, 0.2 M DTT, 0.1 g bromphenol blue) and lysis buffer were added accroding to BCA assay results to obtain the same protein concentration of all the samples. Fifteen micrograms of protein were separated on 10%, alternatively on 15% (based on protein size) polyacrylamide gels. After electrophoresis (15 mA per gel, 200 V, 75 min), proteins were transfered to a PVDF membrane (170 mA, 25 V,20 min for 1 gel) and blocked for 1 h with 5% nonfat dry milk in TBST (Tris-buffered saline containing 0.05% Tween 20). Then, the membranes were incubated with primary antibody overnight at 4 °C with gentle shaking, these conditions were used for BAX (polyclonal rabbit antibody, 1:1000; Cell Signaling Technology), p53 (polyclonal rabbit antibody, 1:1000; Cell Signaling Technology), phospho-p53 (ser15) (polyclonal rabbit antibody, 1:1000; Cell Signaling Technology). For β-actin (polyclonal mouse antibody, 1:8000; Sigma-Aldrich) and BCL2 (polyclonal mouse antibody, 1:400;
Statistical analysis was carried out with a statistical program GraphPad Prism (GraphPad Software version 6.0, Inc. San Diego, U.S.A.). We used one-way Anova test with Bonferroni's test for multiple comparisons. 3. Results 3.1. Antiproliferative effect of FLU in different melanoma cell lines The effect of FLU at a concentration range of 0.01–10 μM on viability and proliferation of three human melanoma cell lines A-375, BOWES and RPMI-7951 during up to 72 h of exposure was evaluated with formazan assay WST-1. In all three cell lines FLU showed a concentration and time-dependent cytotoxicity, with the earliest statistically significant observable inhibition of viability and proliferation seen after treatment with 1 μM (BOWES and RPMI-7951) and 5 μM (A-375), respectively at 24 h of exposure. Subsequent experiments using xCELLigence system confirmed these observations and helped to determine FLU IC50 values for each model cell line (A375 − IC50 = 0.96 μM, BOWES − IC50 = 0.90 μM and RPMI7951 − IC50 = 0.25 μM) (Fig. 1). In view of these results, the FLU concentration of 1 μM was used for subsequent experiments. 317
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Fig. 4. Multinucleation induced by 1 μM flubendazole (FLU) in melanoma cell lines A-375, BOWES and RPMI-7951 during 12 h. Cells were exposed to FLU and their morphological changes were recorded using (A) time-lapse imaging system BioStation IM and (B) fluorescent microscopy as described in Materials and methods section. Phase contrast 600 ×. Bar 25 μm. Fluorescence microscopy 600 ×. Bar 20 μm.
(Fig. 3).
3.2. Effect of FLU on melanoma lines cell cyle To gain further insight into effects of 1 μM FLU on melanoma cells, cell cycle analysis using flow cytometry was carried out. As early as at 12 h after the beginning of treatment, significant changes in the distribution of cells in particular cell cycle phases were recorded when compared to controls. While individually varying, these changes were still present in all three tested cell lines. Universally, both G1 and S phase cells declined in their numbers, with a marked increase in G2/M fraction cells. This trend continued into 24 hours treatment interval and resulted in the majority of cells accumulated in G2/M phase (A-375 79%, Bowes 75% and RPMI-7951 71%) while G1 (A-375 2%, Bowes 9% and RPMI-7951 15%) and S phase cells (A-375 8%, Bowes 11% and RPMI-7951 25%) were severely reduced (with exception of RPMI-7951 cells in the S phase) (Fig. 2). These observations corresponded with the increased expression and nuclear localization of cell-cycle inhibitor p21
3.3. Effect of FLU on cellular and nuclear morphology Time-lapse morphological studies of treated melanoma cells revealed that during first 12–24 h of exposure cells grew and divided although less intensively than in controls. Approximately at 12 h of exposure, gradually larger cells with multiple nuclei started to appear, in particular in RPMI-7951 cultures. These multinuclear cells were not of the uniform dimensions and their nuclei differed in the size, shape and numbers too. Besides multinucleation, in A-375 and RPMI-7951 cells but very little in Bowes cells, the perinuclear vacuolization developed (Fig. 4A and B). Such vacuolated cells persisted for another 12–24 h and then lost their attachment, became rounded and their membrane exhibited a series of rapidly bulging protrusions resembling blebs. Cell rounding and membrane blebbing led to the final 318
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Fig. 5. Morphological changes in by 1 μM flubendazole (FLU) in melanoma cell lines A-375, BOWES and RPMI-7951 during 48 h. Cells were exposed to FLU and their morphological changes were recorded using time-lapse imaging system BioStation IM. Phase contrast 400 ×. Bar 10 μm.
significant variability, which included a marked increase in the p53 protein abundance following the treatment with 1 μM FLU (A-375 cells) as well as stable p53 protein levels (Bowes). Moreover, in RPMI-7951 cells, we could not detect any p53 protein expression in both controls and treated cells, which seems to confirm its already reported p53 nullstatus (Warenius et al., 2000). Protein p53 phosphorylations at Ser-15 and Ser-46 which are regarded as instrumental posttranslational modifications following DNA damage and activation of apoptosis were not significantly elevated either. Similar to these findings, 1 μM FLU activation of cell death did not involve BAX and BCL-2 proteins either (Fig. 8).
fragmentation of cell bodies into small elements. Alternatively, some rounded cells did not show membrane blebbing and quickly underwent fast cell demise without any marked morphological features (Fig. 5). 3.4. Effect of FLU on cytoskeleton With respect to the main FLU cellular target – microtubules, our next efforts were aimed at investigations of the structure and topography of this type of cytoskeletal fibers in FLU-treated melanoma cultures. In controls, microtubules of the interphase cells were typically organized from the single centrosome placed near the nucleus from which individual microtubule fibers extended to the periphery in a sheet-like pattern. In 1 μM FLU treated malignant melanocytes, microtubular cytoskeleton was affected in several ways. Firstly, the number and the distribution of centrosomes in exposed cells changed which resulted in the presence of aberrant mitotic spindles and subsequent multipolar mitoses (Fig. 6A). Secondly, microtubular network in interphase cells became disorganized; individual filaments were twisted and disrupted and their density decreased which accompanied the often present overall change of the cells´ shape (Fig. 6B). In addition to altered microtubular topography, some changes in actin filaments, namely their concentration in the subcortical regions of the cells, were noted (data not shown).
4. Discussion Classical microtubule addressing agents such as Vinca alcaloids or taxanes still remain the mainstay of cytotoxic chemotherapy of several solid tumors. Their clinical efficiciency is, however, often limited by toxic side-effects and, in particular by developed chemoresistance in tumor cells. Such a chemoresistence and the aggressive tumor expansion are significant characteristics of advanced malignant melanoma which generally warrants the need for further researches into possible new treatment options. In our present work we demonstrated that FLU potently inhibits the growth and proliferation of three melanoma cell lines with diverse molecular phenotypes. Still, our discovered FLU IC50 values in 72 h treatment interval ranged in individual exposed cells from 0.25 μM in RPMI-7951 to 0.96 μM in A-375 cells. These achieved different IC50 values are attributable to the varying sensitivity of the tested melanoma cells as was already demonstrated with other benzimidazole derivatives and melanoma cell lines (Doudican et al., 2008). While showing antiproliferative effects towards tested melanoma cells, FLU also significantly changed their cell cycle distribution. It increased G2/M fraction cells upon corresponding decrease of the G1 phase cells which was further underscored by the elevated expression and nuclear localization of the cell cycle inhibitor p21. Cell cycle arrest at G2/M phase is often associated with so called mitotic catastrophe where damaged DNA or spindle apparatuses activate the spindle checkpoint whose prolonged activity leads to aberrant or disruptive mitosis (Roninson et al., 2001). FLU as well as other benzimidazoles bind to the colchicine site on β-tubulin, thus preventing polymerization of microtubules and resulting in the disruption of mitotic spindle (Russell and Lacey, 1995). Accordingly, in the employed
3.5. Effect of FLU on cell death To evaluate the final phenotype of 1 μM FLU treated melanoma cells, first morphological analyses were carried out. As mentioned in Section 3.3. of Results, exposed cells shriveled and their membrane displayed protrusions – blebs. In the studied time frame of 48 h of exposure, the numbers of thus affected cells steadily grew in all tested cell lines albeit their proportions showed individual variability (Fig. 7A). This trend was further confirmed by the corresponding presence of cells with fragmented chromatin as well as by elevated activities of caspase-2 and caspase-3/7 (Fig. B, C). 3.6. Effect of FLU on expression of selected stress-related proteins To determine whether 1 μM FLU-dependent cell death proceeds via p53-mediated signaling as suggested before (Michaelis et al., 2015), we analyzed potential changes in the expression of p53 in exposed melanoma cells with differing p53 status. Our data suggest rather a 319
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Fig. 6. The effect of 1 μM flubendazole (FLU) on microtubules in melanoma cell lines A-375, BOWES and RPMI-7951 during 24 h. Cells were incubated and treated as described in Materials and methods section. (A) Control and FLU-treated interphase microtubular cytoskeleton (B) Control and FLU-treated spindle microtubules in mitotic cells. Fluorescence microscopy 600 ×. Bar 10 μm.
Morphological features of melanoma cells treated with FLU suggested apoptotic cell death mode. We have sought to confirm these indices by investigating the presence of chromatin condensation and caspase-3/7 activity in the treated cells. Our results show that while there were differences in the expression of the followed apoptotic markers between individual melanoma cell lines, these markers were significantly increased already at 24 h of exposure in all cells and at 72 h almost all treated cells were dead. Caspase-dependent apoptosis in melanoma cells treated with benzimidazoles which has been reported before (Doudican et al., 2008) could be activated via several mechanisms. One of them is the involvement of BCL-2 family of proteins whose activity is also a key determinant of cell fate following the disrupted mitosis. BCL-2 protein is reported to be amply expressed in melanoma where it is thought to promote chemoresistance via its interaction with proapoptic BAX (Selzer et al., 1998). To this effect, it has been discovered that mebendazole, a benzimidazole derivative related to FLU, promoted rapid phosphorylation of BCL-2 which rendered this protein
model melanoma lines FLU altered mirotubular cytoskeleton in dividing cells with an increased presence of aberrant multipolar spindles and abnormal mitoses (Fig. 6B). However, in interphase cells FLU produced visible alterations in microtubular topography too (Fig. 6A). These data together with our demonstrated early emergence of giant multinucleated cells as well as activated caspase-2 prove that FLU activated mitotic catastrophe in our model and underscored the efficiency of FLU in both actively proliferating as well as resting cells. Mitotic catastrophe as a sensor of mitotic damage may direct the defective cell to several endpoint fates which include cell death or senescence (Denisenko et al., 2016; Mc Gee, 2015). Since the morphological changes occurring in FLU-exposed melanoma cells were indicative of cell demise, i.e. the appearance of intracellular vacuoles, cell volume shrinkage, cell rounding, loss of adherence and membrane blebbing, we next focused on the identification of the nature of the stimulated death process. In addition, we attempted to elucidate the possible mechanism linking mitotic catastrophe and final cell fate. 320
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Fig. 7. Apoptosis in melanoma cell lines A-375, BOWES and RPMI-7951 exposed to 1 μM flubendazole (FLU) as measured by (A) cellular morphology (B) caspase-2 and-3/7 activity and (C) chromatin condensation during 48 h. Cells were exposed to FLU and individual parameters were determined as described in Materials and methods section. Values represent means ± SD of at least three experiments *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the beginning of treatment with one way-Anova test and Bonferroni's post test for multiple comparisons. Fluorescence microscopy 600 ×. Bar 10 μm.
anticipated pleiotropic effect in malignant cells. The promising potential of FLU in the treatment of melanoma is further potentiated by the fact that it interacts with tubulin through a mechanism similar to colchicine but distinct from vinca alkaloids. In addition, FLU is not a substrate of PgP proteins whose everexpression and activity are responsible for resistance against paclitaxel (Dupuy et al., 2010; Spagnuolo et al., 2010). These facts along with an already published clinical experiences on albendazole – a related benzmidazole compound (Morris et al., 2001; Pourgholami et al., 2010) strongly argue for further testing of the antineoplastic potential of FLU in melanoma.
inactive and promoted apoptosis in melanoma cells. Furthermore, mebendazole also reduced expression of antiapoptotic XIAP via mitochondrial SMAC/DIABLO to induce apoptosis in melanoma xenographts (Doudican et al., 2013). Our own findings could not entirely confirm these reports as we found no marked changes in BCL-2 as well as in BAX expressions. On the other hand, it is very likely that mitochondria were involved in the killing process in our model since we detected changes in mitochondrial organization due to the collapsing microtubules along with a loss of mitochondrial membrane potential and release of cytochrome c (data not shown). The exact sequence of steps linking the affected microtubules and mitochondria is not clear but it could also involve TP53 via a number of its targets. It appears to be the case at least in A-375 cells where TP53 was dramatically upregulated at 24 and 48 h of exposure with concomitant phosphorylation at Ser-15 but not Ser-48, suggesting the non-direct involvement of this protein in induced apoptosis. Ultimately, FLU like other benzimidazole compounds are structurally similar to nucleotides which explains their possible interaction with many biomolecules and intracellular targets as reported before (Cumino et al., 2009; Lacey, 1990). Also, the fact that FLU affects microtubules which are involved in intracellular transport of several cargos, cell stabilization as well as movement should reflect its
5. Conclusion FLU demonstrated its cytotoxicity towards employed melanoma cell lines rergardless of their molecular background. Its effects occurred via induced mitotic catastrophe characterized by G2/M phase arrest, emergence of giant multinuclear cells with abnormal spindles and elevated caspase-2 activity. In the end, mitotic catastrophe led to mitochondria and caspase-3/7 dependent apoptosis. The exact sequence of steps linking mitotic catastrophe and resulting apoptosis was not completely elucidated and thus should be addressed in the future 321
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Fig. 8. The expression of stress-related proteins BAX, BCL-2 and TP53 in melanoma cell lines A-375, BOWES and RPMI-7951 exposed to 1 μM flubendazole (FLU) as measured by immunoblotting during 48 h. Obtained cell lysates were subjected to SDS-PAGE, immunoblotted and probed with antibodies against the above-mentioned proteins. The loading was normalized to β-actin and quantitative analysis of bands was carried out by GelQuant Ver 2.7 software (DNR Bio-Imaging Systems, Jerusalem, Israel). Numbers in individual blot bands represent differences in band densities compared to non-treated cells. Shown is one typical result of at least three independent experiments. Long, Z.J., Wang, X.T., Li, G.H., Wan, X.Y., Yang, Y.L., Liu, Q., 2015. Flubendazole, FDA-approved anthelmintic, targets breast cancer stem-like cells. Oncotarget 6, 6326–6340. Kralova, V., Hanusova, V., Rudolf, E., Canova, K., Skalova, L., 2016. Flubendazole induces mitotic catastrophe and senescence in colon cancer cells in vitro. J. Pharm. Pharmacol. 68, 208–218. Lacey, E., 1990. Mode of action of benzimidazoles. Parasitol. Today 6, 112–115. Mc Gee, M.M., 2015. Targeting the mitotic catastrophe signaling pathway in cancer. Mediat. Inflamm. 2015, 146282. Michaelis, M., Agha, B., Rothweiler, F., Loschmann, N., Voges, Y., Mittelbronn, M., Starzetz, T., Harter, P.N., Abhari, B.A., Fulda, S., Westermann, F., Riecken, K., Spek, S., Langer, K., Wiese, M., Dirks, W.G., Zehner, R., Cinatl, J., Wass, M.N., Cinatl Jr., J., 2015. Identification of flubendazole as potential anti-neuroblastoma compound in a large cell line screen. Sci Rep 5, 8202. Morris, D.L., Jourdan, J.L., Pourgholami, M.H., 2001. Pilot study of albendazole in patients with advanced malignancy. Effect on serum tumor markers/high incidence of neutropenia. Oncology 61, 42–46. Nygren, P., Fryknas, M., Agerup, B., Larsson, R., 2013. Repositioning of the anthelmintic drug mebendazole for the treatment for colon cancer. J. Cancer Res. Clin. Oncol. 139, 2133–2140. Padhy, B.M., Gupta, Y.K., 2011. Drug repositioning: re-investigating existing drugs for new therapeutic indications. J. Postgrad. Med. 57, 153–160. Pourgholami, M.H., Szwajcer, M., Chin, M., Liauw, W., Seef, J., Galettis, P., Morris, D.L., Links, M., 2010. Phase I clinical trial to determine maximum tolerated dose of oral albendazole in patients with advanced cancer. Cancer Chemother. Pharmacol. 65, 597–605. Roninson, I.B., Broude, E.V., Chang, B.D., 2001. If not apoptosis, then what? Treatmentinduced senescence and mitotic catastrophe in tumor cells. Drug Resist. Updat. 4, 303–313. Russell, G.J., Lacey, E., 1995. Inhibition of [3H]mebendazole binding to tubulin by structurally diverse microtubule inhibitors which interact at the colchicine binding site. Biochem. Mol. Biol. Int. 35, 1153–1159. Selzer, E., Schlagbauer-Wadl, H., Okamoto, I., Pehamberger, H., Potter, R., Jansen, B., 1998. Expression of Bcl-2 family members in human melanocytes, in melanoma metastases and in melanoma cell lines. Melanoma Res. 8, 197–203. Shim, J.S., Liu, J.O., 2014. Recent advances in drug repositioning for the discovery of new anticancer drugs. Int. J. Biol. Sci. 10, 654–663. Spagnuolo, P.A., Hu, J., Hurren, R., Wang, X., Gronda, M., Sukhai, M.A., Di Meo, A., Boss, J., Ashali, I., Beheshti Zavareh, R., Fine, N., Simpson, C.D., Sharmeen, S., Rottapel, R., Schimmer, A.D., 2010. The antihelmintic flubendazole inhibits microtubule function through a mechanism distinct from Vinca alkaloids and displays preclinical activity in leukemia and myeloma. Blood 115, 4824–4833. Warenius, H.M., Jones, M., Gorman, T., McLeish, R., Seabra, L., Barraclough, R., Rudland, P., 2000. Combined RAF1 protein expression and p53 mutational status provides a strong predictor of cellular radiosensitivity. Br. J. Cancer 83, 1084–1095.
studies involving more melanoma cells, patients´ samples and in vivo models. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgment This work was supported by the program PROGRESS Q40 01 and SVV 2017 of Charles University, Faculty of Medicine in Hradec Králové. Conflict of interest statement None declared. References Canova, K., Rozkydalova, L., Rudolf, E., 2017. Anthelmintic flubendazole and its potential use in anticancer therapy. Acta Med. (Hradec Kralove) 60, 5–11. Cumino, A.C., Elissondo, M.C., Denegri, G.M., 2009. Flubendazole interferes with a wide spectrum of cell homeostatic mechanisms in Echinococcus granulosus protoscoleces. Parasitol. Int. 58, 270–277. Daud, A., Gill, J., Kamra, S., Chen, L., Ahuja, A., 2017. Indirect treatment comparison of dabrafenib plus trametinib versus vemurafenib plus cobimetinib in previously untreated metastatic melanoma patients. J. Hematol. Oncol. 10, 3. Denisenko, T.V., Sorokina, I.V., Gogvadze, V., Zhivotovsky, B., 2016. Mitotic catastrophe and cancer drug resistance: a link that must to be broken. Drug Resist. Updat. 24, 1–12. Doudican, N., Rodriguez, A., Osman, I., Orlow, S.J., 2008. Mebendazole induces apoptosis via Bcl-2 inactivation in chemoresistant melanoma cells. Mol. Cancer Res. 6, 1308–1315. Doudican, N.A., Byron, S.A., Pollock, P.M., Orlow, S.J., 2013. XIAP downregulation accompanies mebendazole growth inhibition in melanoma xenografts. Anti-Cancer Drugs 24, 181–188. Dupuy, J., Alvinerie, M., Menez, C., Lespine, A., 2010. Interaction of anthelmintic drugs with P-glycoprotein in recombinant LLC-PK1-mdr1a cells. Chem. Biol. Interact. 186, 280–286. Hou, Z.J., Luo, X., Zhang, W., Peng, F., Cui, B., Wu, S.J., Zheng, F.M., Xu, J., Xu, L.Z.,
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Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
Flubendazole induces mitotic catastrophe and apoptosis in melanoma cells
MARK
Čáňová K. , Rozkydalová L. , Vokurková D. , Rudolf E. a
a b c
b
c
a,⁎
Department of Medical Biology and Genetics, Charles University, Faculty of Medicine in Hradec Králové, Czech Republic Department of Pharmacology, Charles University, Faculty of Pharmacy in Hradec Králové, Czech Republic Department of Clinical Immunology and Allergology, University Hospital in Hradec Králové, Czech Republic
A R T I C L E I N F O
A B S T R A C T
Keywords: Melanoma Flubendazole Microtubules Mitotic catastrophe, apoptosis
Flubendazole (FLU) is a widely used anthelmintic drug belonging to benzimidazole group. Recently, several studies have been published demonstrating its potential to inhibit growth of various tumor cells including those derived from colorectal cancer, breast cancer or leukemia via several mechanisms. In the present study we have investigated cytotoxic effects of FLU on malignant melanoma using A-375, BOWES and RPMI-7951 cell lines representing diverse melanoma molecular types. In all three cell lines, FLU inhibited cell growth and proliferation and disrupted microtubule structure and function which was accompanied by dramatic changes in cellular morphology. In addition, FLU-treated cells accumulated at the G2/M phase of cell cycle and displayed the features of mitotic catastrophe characterized by formation of giant cells with multiple nuclei, abnormal spindles and subsequent apoptotic demise. Although this endpoint was observed in all treated melanoma lines, our analyses showed different activated biochemical signaling in particular cells, thus suggesting a promising treatment potential of FLU in malignant melanoma warranting its further testing.
1. Introduction Metastatic melanoma is an aggressive form of skin cancer, with a high mortality rate. Although melanoma represents < 5% of all diagnosed skin cancers, the World Health Organization has indicated that its incidence is increasing faster than any other type of malignancy, mainly due to the general population's increasing exposure to ultraviolet light (Daud et al., 2017). The extensive danger of melanoma is based not only on the increasing incidence but also on its tendency to fast metastasizing and on extensive development of drug resistance - the biggest reason of treatment failure. While the prognosis of melanoma is promising with early diagnosis, upon its systemic spread where the effective therapy is still missing survival rates are significantly lower – the main reason for ongoing searches for new treatment options. One of the promising strategies in the development of novel antioneoplastic medicines is drug repositioning (or repurposing). Drug repositioning is the process of searching for new indications of existing drugs (Shim and Liu, 2014). Repositioned drugs have the advantage of decreased development costs and decreased time to market compared to traditional discovery candidates, mainly due to the availability of previously collected pharmacokinetic, toxicology, and safety data (Padhy and Gupta, 2011). Among the potential pharmacological candidates for repurposing is also a group of benzimidazoles whose several
members have already shown antitumor activities in various preclinical models (Michaelis et al., 2015; Nygren et al., 2013). Flubendazole (FLU) is a derivative benzimidazole which has been extensively evaluated in humans and animals for the treatment of intestinal parasites as well as for the treatment of systemic worm infections (Spagnuolo et al., 2010). The mechanism of action of benzimidazole compounds in helmints can be explained by their increased affinity to tubulin, which leads to the inhibition of tubulin polymerization and results in the interference with microtubule-mediated transport in helminth tissues. Accordingly, benzimidazoles were found to interact with mammalian tubulin too, although their affinity is weaker in comparison to the helminth tubulin. Cytotoxicity of various benzimidazoles including FLU has been proved in a number of in vitro and in vivo tumor models (Canova et al., 2017). FLU first showed its anti-tumor activity in leukemia and myeloma cells where at low and pharmacologically feasible concentrations it induced mitotic catastrophe and cell death and delayed tumor growth in vivo (Spagnuolo et al., 2010). Moreover, in recently published studies, FLU inhibited proliferation of several breast cancer cell lines (Hou et al., 2015), colon cancer cell lines (Kralova et al., 2016) and neuroblastoma cells (Michaelis et al., 2015). Despite the fact that currently no information exists about FLU effects in melanoma, reports on cytotoxic activity of mebendazole, a related benzimidazole compound, in
⁎ Corresponding author at: Department of Medical Biology and Genetics, Charles University, Faculty of Medicine in Hradec Králové, Zborovská 2089, 500 03 Hradec Králové, Czech Republic. E-mail address: [email protected] (E. Rudolf).
http://dx.doi.org/10.1016/j.tiv.2017.10.025 Received 10 August 2017; Received in revised form 20 October 2017; Accepted 25 October 2017 Available online 26 October 2017 0887-2333/ © 2017 Elsevier Ltd. All rights reserved.
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concentration range 0.01 μM–10 μM. After 24-, 48- and 72-hours exposure, the medium was removed and the cells were washed twice with 150 μl PBS. Then, 100 μl of culture medium containing WST-1 (0.3 mg/ ml) was added to each well. Absorbance of the samples was measured immediately at 450 nm/650 nm wavelength (Tecan Infinite M200; Tecan, Salzburg, Austria). The samples were then placed in the incubator and the absorbance was measured again after 2 h of incubation. Each sample was assayed in 6 parallels and three independent experiments were performed. The viabilities of treated cells were expressed as percentage of untreated controls (100%).
melanoma cells as well as in xenographts appear promising (Doudican et al., 2008; Doudican et al., 2013) and suggest a wider scientific potential of other benzimidazole compounds including FLU in this type of malignancy. Based on these facts, we have investigated the effect of FLU on three melanoma cell lines A-375, BOWES and RPMI-7951 representing diverse molecular types of this malignancy. Here we report that FLU inhibited the proliferation of all three employed cell lines with a similar IC50 equalling to 0.25 μM (RPMI-7951), 0.90 μM (BOWES) and 0.96 μM (A-375) at 72 h of exposure. FLU-dependent antiproliferative activity was associated with accumulation of cells at the G2/M phase of cell cycle and their extensive morphological changes including the specific alterations of the microtubular network. Thus treated cells finally underwent mitotic catastrophe characterized by formation of giant cells with multiple nuclei, abnormal spindles and caspase-dependent apoptosis. Although this endpoint was observed in all treated melanoma lines, our analyses showed different activated biochemical signaling in particular cells, thus suggesting a promising treatment potential of FLU in malignant melanoma warranting its further testing.
2.3.2. Test of proliferation using the xCELLigence system The system measures electrical impedance across interdigitated microelectrodes integrated at the bottom of tissue culture E-plates. The impedance measurement provides quantitative information about the biological status of the cells, including cell number, viability and morphology. Ninety microliter of culture medium was added into each well and plates were inserted in the device for background measurement. Then, 100 μl of cell suspension (containing 1500 cells) was added in duplicate to the appropriate wells. The growing impedance (corresponding to cell proliferation) was measured every 30 min for 24 h. After 24 h, 10 μl of treatment medium was added into each well, so that final concentration of FLU was 1 μM. Plates were inserted back into the device and impedance was measured every hour for 72 h. Each sample was assayed in duplicate and three independent experiments were performed.
2. Materials and methods 2.1. Chemicals Flubendazole was purchased from Janssen Pharmaceutica (Prague, Czech Republic), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffer, dithiotreitol (DTT), dimethylsulfoxide (DMSO), 4′,6-diamidino-2-phenylindole (DAPI), Triton-X, propidium iodide (PI), Dulbecco's Modified Eagle's Medium (DMEM), sodium dodecyl sulfate (SDS), bicinchoninic acid (BCA) kit for protein determination were purchased from Sigma-Aldrich (Prague, Czech Republic). Eagle's Minimum Essential Medium (EMEM) was supplied by LGC Standards (Lomianki, Poland), fetal bovine serum from Invitrogen (Carlsbad, CA, USA) and bovine serum albumin (BSA) from Fluka (Prague, Czech Republic). WST-1 was purchased from Roche Diagnostics (Manheim, Germany). Polyvinylidene difluoride (PVDF) membrane was obtained from BIO-RAD Laboratories (Prague, Czech Republic). All other chemicals were of highest analytical grade.
2.4. Time-lapse videomicroscopy A-375, BOWES and RPMI-7951 cell lines were seeded into plastic tissue-culture dishes with glass bottom and left for 24 h in an incubator with 5% CO2 at 37 °C. Next, the growth medium was replaced with a medium containing flubendazole. The tissue-culture dishes were transferred into a time-lapse imaging system BioStation IM (Nikon, Prague, Czech Republic) combining an incubator, a motorized microscope and a cooled CCD camera. Recording was carried out in a multipoint and multichannel manner employing various time-lapse modes and upon small as well as high magnifications to allow global as well as detailed view of changes in behavior of treated cell populations. Recorded sequences were subsequently semi automatically analyzed with the software NIS Elements AR 3.20 (Nikon, Prague, Czech Republic).
2.2. Cell culture and treatment conditions Human melanoma cell lines were purchased from ATCC. A-375 (ATCC® CRL1619™) are adherent, epithelial cells hypotriploid with a modal number of 62 chromosomes whose BRAF and CDKN2A genes are mutated. BOWES (ATCC® CRL9607™) are adherent, epithelial, heteroploid cells with wild-type BRAF. RPMI-7951 (ATCC® HTB66™) are adherent, epithelial-like cells derived from metastasis to the lymph node, hyperdiploid (47–66 chromosomes) with mutant BRAF, TP53, CDKN2A and PTEN. A-378 and BOWES were maintained in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. RPMI-7951 was cultivated in EMEM medium with 10% FBS and 1% penicillin/streptomycin. Cells were maintained upon standard conditions (37 °C, 5% CO2 in an incubator) and passaged twice a week upon 90% cells confluence using 0.05% EDTA/trypsin. Only mycoplasma-free cells were used for experiments. Stock solutions of FLU were prepared in DMSO and stored in aliquots at 6 °C. Cells were treated with medium containing 1 μM FLU, the final concentration of DMSO in the medium was 0.1%. Control samples were treated with medium containing 0.1% DMSO.
2.5. Cell cycle distribution analysis 2.5.1. Flow cytometry Cells were cultured in 75 cm2 culture flasks in 20 ml of culture medium for 12 and 24 h. Then the culture medium was replaced by fresh medium containing 1 μM of FLU or 0.1% DMSO as a control. After 12 or 24 h of treatment, the cells were trypsinized, collected by centrifugation, washed with PBS and fixed with ice-cold 70% ethanol while gently vortexing. Fixed samples were stored overnight in 4 °C. Then they were washed with PBS and incubated with 0.5 ml of the Vindelov solution (1.2 g/l TRIS, 0.6 g/l NyCl, 0.01 g/l RNase and 0.05 g/l propidium iodide) for 50 min at 37 °C. The samples were analyzed at the FC500 Cytomics Flow cytometer (Beckman Coulter,Hialeah, FL, USA) with PI fluorescence detected in FL3 channel. Cell cycle distributions in control and treated samples were analyzed with MultiCycle AV for Windows (Phoenix Flow Systems, San Diego, USA) and three independent experiments were performed. 2.5.2. Percentage of cells in S-phase by EdU labeling EdU (5-ethynyl-2′-deoxyuridine) is incorporated into DNA during active DNA synthesis. The cells were cultured in 96-well plates and treated with FLU in concentration 1 μM. After 24 hours exposure, the cells were labeled with EdU (Click-iT® EdU Flow Cytometry Assay Kit,
2.3. Cytotoxicity assay 2.3.1. The WST assay The cells were cultured in 96-well plates and treated with FLU in 314
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Fig. 1. Concentration-dependent effect of flubendazole (FLU) on cell proliferation in melanoma cell lines A-375, BOWES and RPMI-7951. Cells were exposed to FLU and proliferation (up to 72 h) was measured by WST-1 assay (A) and xCELLigence system (B) as described in Materials and methods section. Values represent means ± SD of at least three experiments *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001 compared to untreated control cells at the same treatment interval with one way-Anova test and Bonferroni's post test for multiple comparisons.
of cells in the medium containing 1 μM FLU, cells were lysed using buffer containing 50 mM HEPES, 5 mM CHAPS, 5 mM DTT. Then the lysates were collected into microtubes. Caspases 2 and 3/7 reagents were prepared as described in manufacturer's instruction. The lysates (25 μl in each well) were transferred into white-walled 384-well-plate for luminometer. Then, 25 μl of Caspase 2 or Caspase 3/7 reagents were added to each well. Plate was gently mixed for 30 s and then incubated for 30 min at room temperature. The luminescence was measured in three independent experiments using luminometer (Tecan Infinite M200; Tecan, Salzburg, Austria).
Invitrogen-Molecular Probes, Inc., Carlsbad, CA, USA) in concentration 10 μM and incubated for 2 h. After EdU labeling the medium was removed and the cells were washed in PBS and fixed with paraformaldehyde and incubated for 15 min. Then were washed again with PBS + 3% BSA and permeabilized with 0.5% Triton-X for 20 min in room temperature. After removing permeabilization solution, cells were washed twice in PBS + 3% BSA and to the plates were added EdU buffer additive and incubated for 30 min in the dark, 5 min before incubation was over was added DAPI (1 μg/ml). Cells were washed again and determined by using Image Xpress Micro XLS Widefield HighContent Analysis System. Specific fluorescence was visualized, recorded and analyzed by Cell scoring module of MetaXpress® Image Acquisition and Analysis Software (Molecular Devices, Sunnyvale, CA, USA). Three independent experiments were performed.
2.7. Immunofluorescence and image analysis Cells in cytospine chambers were exposed to 1 μM FLU for 12 and 24 h, then were washed by 1 ml pre-warm PBS and fixed with 4% paraformaldehyde (10 min, 25 °C), rinsed with phosphate saline buffer with 1% Triton X (PBS-T) and blocked in 5% BSA for 1 h at 25 °C. Immunofluorescence was performed at 4 °C for 1 h using α-tubulin (Cell Signaling Technology, 1: 100), β-actin (1:1000, Sigma Aldrich) and p21 (1:100, Santa Cruz biotechnology) antibodies. After washing with cold
2.6. Caspase assay The activities of caspases 2 and 3/7 were assayed using Promega Caspase-Glo Assay (Madison, USA). The A-375, BOWES and RPMI-7951 cells were cultivated in 96-well plates. After 12, 24 and 48 h incubation 315
Fig. 2. The effect of 1 μM flubendazole (FLU) on cell cycle of melanoma cell lines during 24 h. Melanoma cells were exposed to FLU and their cell cycle analysis was carried out using FC500 Cytomics Flow cytometer. Cell cycle distributions in control and treated samples were analyzed with MultiCycle AV for Windows. (A) Flow charts of analyzed control and 1 μM FLU-exposed melanoma cells A-375, BOWES and RPMI-7951. (B) Calculated distribution of the treated melanoma cells in respective cell cycle phases. (C) Percentage of S-phase cells in 1 μM FLU exposed melanoma cells during 24 h as measured by EdU assay (Materials and methods). Values represent means ± SD of at least three experiments *P < 0.05, ***P < 0.001, **** P < 0.0001 compared to untreated control cells at the same treatment interval with one way-Anova test and Bonferroni's post test for multiple comparisons.
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Fig. 3. The effect of 1 μM flubendazole (FLU) on cell cycle inhihibitor p21 expression and cellular localization in melanoma cell lines A-375, BOWES and RPMI-7951 during 24 h. Cells were exposed to FLU during up to 24 h and the expression and subcellular localization of p21 was determined as described in Materials and methods section. Fluorescence microscopy 600 ×. Bar 25 μm.
PBS (5 min, 25 °C), secondary antibody (anti-rabbit/anti-mouse, 1:250) was added for additional 1 h (25 °C). The specimens were then rinsed three times in PBS, post-labeled with DAPI (10 μg/ml, 15 min, 25 °C), mounted into ProLong® Gold medium (Invitrogen-Molecular Probes, Inc., Carlsbad, CA, USA) and examined under fluorescence microscope Nikon Eclipse E 400 (Nikon, Prague, Czech Republic) (excitation filter 330–380 nm and emission filter 420 nm) equipped with a digital color matrix camera COOL 1300 (VDS, Vosskűhler, Germany). Photographs were taken using the software NIS Elements AR 3.20. Samples were prepared in duplicates, three independent experiments were performed.
DAKO), the incubation with primary antibody lasted for 1 h in room temperature. Subsequently, the membranes were washed six times in TBST for 9 min, followed by 1 hour incubation with secondary antibody in room temperature (anti-rabbit secondary antibody, 1:2000 for BAX, p53 and phospho-p53 (ser15), and anti-mouse secondary antibody 1:800 for BCL2 and 1:10,000 for β-actin). After washing the membrane six times (9 min) with TBST, the chemiluminescence proces and quantification of immunoreactive bands on the exposed films were carried out. For each antibody used, three independent experiments were performed.
2.8. Western blot analysis
2.9. Statistics
Treated and control cells were washed with PBS and harvested in ice-cold lysis buffer (50 mM Tris/HCl, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 2 mM EGTA, b-glycerolphosphate, 50 mM NaF, 10 mM sodium pyrophosphate, 200 lM sodium orthovanadate, 2 mM DTT). The lysates were resuspended and supernatants were obtained after a 13,000 rpm centrifugation at 4 °C for 10 min. Amount of protein in supernatant was determined by BCA assay. The SDS sample buffer (Tris–HCl pH 6.8, 40% glycerol, 6% SDS, 0.2 M DTT, 0.1 g bromphenol blue) and lysis buffer were added accroding to BCA assay results to obtain the same protein concentration of all the samples. Fifteen micrograms of protein were separated on 10%, alternatively on 15% (based on protein size) polyacrylamide gels. After electrophoresis (15 mA per gel, 200 V, 75 min), proteins were transfered to a PVDF membrane (170 mA, 25 V,20 min for 1 gel) and blocked for 1 h with 5% nonfat dry milk in TBST (Tris-buffered saline containing 0.05% Tween 20). Then, the membranes were incubated with primary antibody overnight at 4 °C with gentle shaking, these conditions were used for BAX (polyclonal rabbit antibody, 1:1000; Cell Signaling Technology), p53 (polyclonal rabbit antibody, 1:1000; Cell Signaling Technology), phospho-p53 (ser15) (polyclonal rabbit antibody, 1:1000; Cell Signaling Technology). For β-actin (polyclonal mouse antibody, 1:8000; Sigma-Aldrich) and BCL2 (polyclonal mouse antibody, 1:400;
Statistical analysis was carried out with a statistical program GraphPad Prism (GraphPad Software version 6.0, Inc. San Diego, U.S.A.). We used one-way Anova test with Bonferroni's test for multiple comparisons. 3. Results 3.1. Antiproliferative effect of FLU in different melanoma cell lines The effect of FLU at a concentration range of 0.01–10 μM on viability and proliferation of three human melanoma cell lines A-375, BOWES and RPMI-7951 during up to 72 h of exposure was evaluated with formazan assay WST-1. In all three cell lines FLU showed a concentration and time-dependent cytotoxicity, with the earliest statistically significant observable inhibition of viability and proliferation seen after treatment with 1 μM (BOWES and RPMI-7951) and 5 μM (A-375), respectively at 24 h of exposure. Subsequent experiments using xCELLigence system confirmed these observations and helped to determine FLU IC50 values for each model cell line (A375 − IC50 = 0.96 μM, BOWES − IC50 = 0.90 μM and RPMI7951 − IC50 = 0.25 μM) (Fig. 1). In view of these results, the FLU concentration of 1 μM was used for subsequent experiments. 317
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Fig. 4. Multinucleation induced by 1 μM flubendazole (FLU) in melanoma cell lines A-375, BOWES and RPMI-7951 during 12 h. Cells were exposed to FLU and their morphological changes were recorded using (A) time-lapse imaging system BioStation IM and (B) fluorescent microscopy as described in Materials and methods section. Phase contrast 600 ×. Bar 25 μm. Fluorescence microscopy 600 ×. Bar 20 μm.
(Fig. 3).
3.2. Effect of FLU on melanoma lines cell cyle To gain further insight into effects of 1 μM FLU on melanoma cells, cell cycle analysis using flow cytometry was carried out. As early as at 12 h after the beginning of treatment, significant changes in the distribution of cells in particular cell cycle phases were recorded when compared to controls. While individually varying, these changes were still present in all three tested cell lines. Universally, both G1 and S phase cells declined in their numbers, with a marked increase in G2/M fraction cells. This trend continued into 24 hours treatment interval and resulted in the majority of cells accumulated in G2/M phase (A-375 79%, Bowes 75% and RPMI-7951 71%) while G1 (A-375 2%, Bowes 9% and RPMI-7951 15%) and S phase cells (A-375 8%, Bowes 11% and RPMI-7951 25%) were severely reduced (with exception of RPMI-7951 cells in the S phase) (Fig. 2). These observations corresponded with the increased expression and nuclear localization of cell-cycle inhibitor p21
3.3. Effect of FLU on cellular and nuclear morphology Time-lapse morphological studies of treated melanoma cells revealed that during first 12–24 h of exposure cells grew and divided although less intensively than in controls. Approximately at 12 h of exposure, gradually larger cells with multiple nuclei started to appear, in particular in RPMI-7951 cultures. These multinuclear cells were not of the uniform dimensions and their nuclei differed in the size, shape and numbers too. Besides multinucleation, in A-375 and RPMI-7951 cells but very little in Bowes cells, the perinuclear vacuolization developed (Fig. 4A and B). Such vacuolated cells persisted for another 12–24 h and then lost their attachment, became rounded and their membrane exhibited a series of rapidly bulging protrusions resembling blebs. Cell rounding and membrane blebbing led to the final 318
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Fig. 5. Morphological changes in by 1 μM flubendazole (FLU) in melanoma cell lines A-375, BOWES and RPMI-7951 during 48 h. Cells were exposed to FLU and their morphological changes were recorded using time-lapse imaging system BioStation IM. Phase contrast 400 ×. Bar 10 μm.
significant variability, which included a marked increase in the p53 protein abundance following the treatment with 1 μM FLU (A-375 cells) as well as stable p53 protein levels (Bowes). Moreover, in RPMI-7951 cells, we could not detect any p53 protein expression in both controls and treated cells, which seems to confirm its already reported p53 nullstatus (Warenius et al., 2000). Protein p53 phosphorylations at Ser-15 and Ser-46 which are regarded as instrumental posttranslational modifications following DNA damage and activation of apoptosis were not significantly elevated either. Similar to these findings, 1 μM FLU activation of cell death did not involve BAX and BCL-2 proteins either (Fig. 8).
fragmentation of cell bodies into small elements. Alternatively, some rounded cells did not show membrane blebbing and quickly underwent fast cell demise without any marked morphological features (Fig. 5). 3.4. Effect of FLU on cytoskeleton With respect to the main FLU cellular target – microtubules, our next efforts were aimed at investigations of the structure and topography of this type of cytoskeletal fibers in FLU-treated melanoma cultures. In controls, microtubules of the interphase cells were typically organized from the single centrosome placed near the nucleus from which individual microtubule fibers extended to the periphery in a sheet-like pattern. In 1 μM FLU treated malignant melanocytes, microtubular cytoskeleton was affected in several ways. Firstly, the number and the distribution of centrosomes in exposed cells changed which resulted in the presence of aberrant mitotic spindles and subsequent multipolar mitoses (Fig. 6A). Secondly, microtubular network in interphase cells became disorganized; individual filaments were twisted and disrupted and their density decreased which accompanied the often present overall change of the cells´ shape (Fig. 6B). In addition to altered microtubular topography, some changes in actin filaments, namely their concentration in the subcortical regions of the cells, were noted (data not shown).
4. Discussion Classical microtubule addressing agents such as Vinca alcaloids or taxanes still remain the mainstay of cytotoxic chemotherapy of several solid tumors. Their clinical efficiciency is, however, often limited by toxic side-effects and, in particular by developed chemoresistance in tumor cells. Such a chemoresistence and the aggressive tumor expansion are significant characteristics of advanced malignant melanoma which generally warrants the need for further researches into possible new treatment options. In our present work we demonstrated that FLU potently inhibits the growth and proliferation of three melanoma cell lines with diverse molecular phenotypes. Still, our discovered FLU IC50 values in 72 h treatment interval ranged in individual exposed cells from 0.25 μM in RPMI-7951 to 0.96 μM in A-375 cells. These achieved different IC50 values are attributable to the varying sensitivity of the tested melanoma cells as was already demonstrated with other benzimidazole derivatives and melanoma cell lines (Doudican et al., 2008). While showing antiproliferative effects towards tested melanoma cells, FLU also significantly changed their cell cycle distribution. It increased G2/M fraction cells upon corresponding decrease of the G1 phase cells which was further underscored by the elevated expression and nuclear localization of the cell cycle inhibitor p21. Cell cycle arrest at G2/M phase is often associated with so called mitotic catastrophe where damaged DNA or spindle apparatuses activate the spindle checkpoint whose prolonged activity leads to aberrant or disruptive mitosis (Roninson et al., 2001). FLU as well as other benzimidazoles bind to the colchicine site on β-tubulin, thus preventing polymerization of microtubules and resulting in the disruption of mitotic spindle (Russell and Lacey, 1995). Accordingly, in the employed
3.5. Effect of FLU on cell death To evaluate the final phenotype of 1 μM FLU treated melanoma cells, first morphological analyses were carried out. As mentioned in Section 3.3. of Results, exposed cells shriveled and their membrane displayed protrusions – blebs. In the studied time frame of 48 h of exposure, the numbers of thus affected cells steadily grew in all tested cell lines albeit their proportions showed individual variability (Fig. 7A). This trend was further confirmed by the corresponding presence of cells with fragmented chromatin as well as by elevated activities of caspase-2 and caspase-3/7 (Fig. B, C). 3.6. Effect of FLU on expression of selected stress-related proteins To determine whether 1 μM FLU-dependent cell death proceeds via p53-mediated signaling as suggested before (Michaelis et al., 2015), we analyzed potential changes in the expression of p53 in exposed melanoma cells with differing p53 status. Our data suggest rather a 319
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Fig. 6. The effect of 1 μM flubendazole (FLU) on microtubules in melanoma cell lines A-375, BOWES and RPMI-7951 during 24 h. Cells were incubated and treated as described in Materials and methods section. (A) Control and FLU-treated interphase microtubular cytoskeleton (B) Control and FLU-treated spindle microtubules in mitotic cells. Fluorescence microscopy 600 ×. Bar 10 μm.
Morphological features of melanoma cells treated with FLU suggested apoptotic cell death mode. We have sought to confirm these indices by investigating the presence of chromatin condensation and caspase-3/7 activity in the treated cells. Our results show that while there were differences in the expression of the followed apoptotic markers between individual melanoma cell lines, these markers were significantly increased already at 24 h of exposure in all cells and at 72 h almost all treated cells were dead. Caspase-dependent apoptosis in melanoma cells treated with benzimidazoles which has been reported before (Doudican et al., 2008) could be activated via several mechanisms. One of them is the involvement of BCL-2 family of proteins whose activity is also a key determinant of cell fate following the disrupted mitosis. BCL-2 protein is reported to be amply expressed in melanoma where it is thought to promote chemoresistance via its interaction with proapoptic BAX (Selzer et al., 1998). To this effect, it has been discovered that mebendazole, a benzimidazole derivative related to FLU, promoted rapid phosphorylation of BCL-2 which rendered this protein
model melanoma lines FLU altered mirotubular cytoskeleton in dividing cells with an increased presence of aberrant multipolar spindles and abnormal mitoses (Fig. 6B). However, in interphase cells FLU produced visible alterations in microtubular topography too (Fig. 6A). These data together with our demonstrated early emergence of giant multinucleated cells as well as activated caspase-2 prove that FLU activated mitotic catastrophe in our model and underscored the efficiency of FLU in both actively proliferating as well as resting cells. Mitotic catastrophe as a sensor of mitotic damage may direct the defective cell to several endpoint fates which include cell death or senescence (Denisenko et al., 2016; Mc Gee, 2015). Since the morphological changes occurring in FLU-exposed melanoma cells were indicative of cell demise, i.e. the appearance of intracellular vacuoles, cell volume shrinkage, cell rounding, loss of adherence and membrane blebbing, we next focused on the identification of the nature of the stimulated death process. In addition, we attempted to elucidate the possible mechanism linking mitotic catastrophe and final cell fate. 320
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Fig. 7. Apoptosis in melanoma cell lines A-375, BOWES and RPMI-7951 exposed to 1 μM flubendazole (FLU) as measured by (A) cellular morphology (B) caspase-2 and-3/7 activity and (C) chromatin condensation during 48 h. Cells were exposed to FLU and individual parameters were determined as described in Materials and methods section. Values represent means ± SD of at least three experiments *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to the beginning of treatment with one way-Anova test and Bonferroni's post test for multiple comparisons. Fluorescence microscopy 600 ×. Bar 10 μm.
anticipated pleiotropic effect in malignant cells. The promising potential of FLU in the treatment of melanoma is further potentiated by the fact that it interacts with tubulin through a mechanism similar to colchicine but distinct from vinca alkaloids. In addition, FLU is not a substrate of PgP proteins whose everexpression and activity are responsible for resistance against paclitaxel (Dupuy et al., 2010; Spagnuolo et al., 2010). These facts along with an already published clinical experiences on albendazole – a related benzmidazole compound (Morris et al., 2001; Pourgholami et al., 2010) strongly argue for further testing of the antineoplastic potential of FLU in melanoma.
inactive and promoted apoptosis in melanoma cells. Furthermore, mebendazole also reduced expression of antiapoptotic XIAP via mitochondrial SMAC/DIABLO to induce apoptosis in melanoma xenographts (Doudican et al., 2013). Our own findings could not entirely confirm these reports as we found no marked changes in BCL-2 as well as in BAX expressions. On the other hand, it is very likely that mitochondria were involved in the killing process in our model since we detected changes in mitochondrial organization due to the collapsing microtubules along with a loss of mitochondrial membrane potential and release of cytochrome c (data not shown). The exact sequence of steps linking the affected microtubules and mitochondria is not clear but it could also involve TP53 via a number of its targets. It appears to be the case at least in A-375 cells where TP53 was dramatically upregulated at 24 and 48 h of exposure with concomitant phosphorylation at Ser-15 but not Ser-48, suggesting the non-direct involvement of this protein in induced apoptosis. Ultimately, FLU like other benzimidazole compounds are structurally similar to nucleotides which explains their possible interaction with many biomolecules and intracellular targets as reported before (Cumino et al., 2009; Lacey, 1990). Also, the fact that FLU affects microtubules which are involved in intracellular transport of several cargos, cell stabilization as well as movement should reflect its
5. Conclusion FLU demonstrated its cytotoxicity towards employed melanoma cell lines rergardless of their molecular background. Its effects occurred via induced mitotic catastrophe characterized by G2/M phase arrest, emergence of giant multinuclear cells with abnormal spindles and elevated caspase-2 activity. In the end, mitotic catastrophe led to mitochondria and caspase-3/7 dependent apoptosis. The exact sequence of steps linking mitotic catastrophe and resulting apoptosis was not completely elucidated and thus should be addressed in the future 321
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