WIN55,212-2 induces caspase-independent apoptosis on human glioblastoma cells by regulating HSP70, p53 and Cathepsin D

Toxicology in Vitro 57 (2019) 233–243

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WIN55,212-2 induces caspase-independent apoptosis on human glioblastoma cells by regulating HSP70, p53 and Cathepsin D

T

Ana Gabriela Silvaa, Caio Fabio Baeta Lopesa, Clóvis Gomes Carvalho Júniora, Ralph Gruppi Thoméb, Hélio Batista dos Santosb, Rui Reisc,d,e, ⁎ Rosy Iara Maciel de Azambuja Ribeiroa, a

Experimental Pathology Laboratory, Federal University of São João del Rei (UFSJ), 400, Sebastião Gonçalves Coelho, Chanadour, Divinópolis, MG, Brazil Tissue Processing Laboratory, Federal University of São João del Rei (UFSJ), 400, Sebastião Gonçalves Coelho, Chanadour, Divinópolis, MG, Brazil c Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Brazil d Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal e ICVS/3B's - PT Government Associate Laboratory, Braga, Guimarães 4806909, Portugal b

A R T I C LE I N FO

A B S T R A C T

Keywords: Acridine orange/propidium iodide Synthetic cannabinoid Colony forming assay Angiogeneses Chick chorioallantoic membrane HET-CAM

Despite the standard approaches to treat the highly aggressive and invasive glioblastoma (GBM), it remains incurable. In this sense, cannabinoids highlight as a promising tool, because this tumor overexpresses CB1 and/ or CB2 receptors and being, therefore, can be susceptible to cannabinoids treatment. Thus, this work investigated the action of the cannabinoid agonist WIN55-212-2 on GBM cell lines and non-malignant cell lines, in vitro and in vivo. WIN was selectively cytotoxic to GBM cells. These presented blebbing and nuclear alterations in addition to cell shrinkage and chromatin condensation. WIN also significantly inhibited the migration of GAMG and U251 cells. Finally, the data also showed that the antitumor effects of WIN are exerted, at least to some extent, by the expression of p53 and increased cathepsin D in addition to the decreased expression of HSP70.This data can indicate caspase-independent cell death mechanism. In addition, WIN decreased tumoral perimeter as well as caused a reduction the blood vessels in this area, without causing lysis, hemorrhage or blood clotting. So, the findings herein presented reinforce the usefulness of cannabinoids as a candidate for further evaluation in treatment in glioblastoma treatment.

1. Introduction Cannabinoids are a group of diverse substances capable of eliciting a different cellular response via cannabinoid receptors, namely CB1 and CB2, both G-protein coupled receptors (GPCRs) with tissue-specific distribution and multiple intracellular targets and effectors (Mackie, 2008). These substances can be grouped into three main classes: phytocannabinoids, derived from the plant Cannabis sativa and represented by approximately 60 different compounds, notably Δ9-

tetrahidrocanabinol (THC), known by its profound psychotropic effects mediated by CB1 agonism, and cannabidiol (CBD), a putative inverse agonist with effects that may counterbalance THC toxicity or “high”. Besides that, the endocannabinoids, endogenous ligands derived from arachidonic acid (AA), produced on demand and released for autocrine/paracrine lipid signalling, being anandamide (AEA). 2-arachidonoylglycerol (2-AG) the two best characterized. And also, the synthetic cannabinoids, a group of structurally diverse molecules with variable affinity for CB1 and/or CB2, and prototypical signalling properties

Abbreviations: 2-AG, 2-arachidonoil-glycerl; AA, arachidonic acid; AEA, anandamide; CAM, Chick Chorioallantoic Membrane; CBD, cannabidiol; DMEM, Dulbecco's modified Eagle's medium; DMSO, Dimethylsulfoxide; EDTA, Ethylenediamine Tetra Acetic acid; FBS, Fetal Bovine Serum; GBM, Glioblastoma; GPCRs, G-protein coupled receptors; HET-CAM, Hen's Egg Test - Chorioallantoic Membrane; IC50, Inhibitory Concentration of 50%; mm, Millimeter; mM, Millimolar; MMP-2, Metalloproteinases 2; MMP-9, Metalloproteinases 2; MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromite; Na3VO4, Sodium Orthovanadate; Na4P2O7, Sodium Pyrophosphate; NaCl, Sodium Chloride; NHA, Normal Human Astrocyte line; PSB, Phosphate-Saline Buffer; SDS, Sodium Dodecyl Sulfate; TBS-T, Tris Buffered Saline-Tween 20; TCA, trichloroacetic acid; THC, Δ9-tehydrocannabinol; Tris-CaCl2, Tris- Calcium chloride; TS, Tumor Specificity; WIN, WIN55-212-2; μl, Microliters ⁎ Corresponding author at: Experimental Pathology Laboratory, Federal University of São João del-Rei, 400, Sebastião Gonçalves Coelho, Chanadour, Divinópolis, MG 35501-296, Brazil. E-mail address: [email protected] (R.I.M.d.A. Ribeiro). https://doi.org/10.1016/j.tiv.2019.02.009 Received 21 November 2018; Received in revised form 2 February 2019; Accepted 8 February 2019 Available online 15 February 2019 0887-2333/ © 2019 Elsevier Ltd. All rights reserved.

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This work aims to investigated the role of cannabinoid system activation by WIN on cell viability, proliferation, and migration of GBM cell lines (GAMG, U251), and non-malignant cell lines WI-38 and NHA, a fibroblast and astrocyte-derived lineages, respectively. 2. Methods 2.1. Preparing the standard solution Mesylate salt (R)-(+)-WIN55,212-2 or (R)-(+)-WIN55212 (W102 SIGMA) was diluted at concentrations of 1 μM, 2.5 μM, 5 μM, 10 μM and, 23 μM. The solvent used was dimethylsulfoxide (DMSO). All solutions were stored in a refrigerator (4 °C), in the dark.

Fig. 1. Chemical structure of compound (R)-(+)-WIN55,212-2.

upon these receptors, hence mimicking endogenous ligands and evoking highly specific cellular responses based on their capacity to discriminate and bind selectively to their designated targets (Shevyrin et al., 2016). The aminoalkylindole derivative WIN55,212-2 (WIN) is a synthetic cannabinoid, Fig. 1, with a strong affinity for CB1 and CB2, that produces a range of effects similar to those attributed to THC. With a Ki in the low nanomolar range, WIN represents a potent drug extensively used to investigate the endocannabinoid signalling system (Sim-Selley and Martin, 2002). WIN also binds to different cellular targets, such as peroxisome proliferator-activated receptors α-γ (PPARα-γ) (O'Sullivan, 2007) and other GPCRs and enzymes (Soderstrom et al., 2017). The ability of cannabinoids to control malignant cell fate is well documented. Studies have shown that WIN has in vitro pro-apoptotic effects on mantle cell lymphoma (Gustafsson et al., 2006), antimitogenic properties upon human Kaposi's sarcoma cells (Luca et al., 2009) and induces cell-cycle arrest on hepatocellular carcinoma cells, abrogating migration and proliferation in vitro in a CB2-dependent mechanism (Xu, 2015). Hence, WIN represents a promising tool to investigate the role of the cannabinoid system in cancer, a trend extensively reviewed and proposed to be a novel therapeutic approach (Śledziński et al., 2018), considering that many malignancies overexpress CB1 and/or CB2 and are susceptible to cannabinoids treatment (Sánchez et al., 2001; Bifulco et al., 2004; Caffarel et al., 2006). The highly aggressive and invasive glioblastoma (GBM) corresponds to a solid tumor of the central nervous system, which is the most malignant lesion among gliomas, with an overall poor prognosis and strong resistance to treatments. Standard approaches to treat GBM include maximal surgical resection, radiotherapy and adjuvant chemotherapy with temozolomide (Stupp et al., 2015). Despite these combined therapies, recurrences are common and few patients (0.05%–4.7%) survive five years after primary diagnosis (Ostrom et al., 2014). This type of cancer remains incurable, what justifies an urge to seek for new therapeutic approaches. In this sense, cannabinoids highlight as a promising tool for GBM treatment, capable of tackle this malignancy in three different axes: cell death-induction (autophagy and apoptosis), inhibition of cell proliferation and anti-angiogenic effects (Dumitru et al., 2018). Indeed, a positive correlation can be found between CB2 expression levels and GBM grading (Ellert-Miklaszewska et al., 2013), that suggests that targeting cannabinoid receptors could be a rational approach in the malignancy management. The potential applicability of cannabinoids on GBM treatment is endorsed by several lines of evidence suggesting a range of possible cytotoxic and selective effects mediated or not by CB1/CB2 receptors (Parolaro and Massi, 2008). In this regard, the pioneer work by Guzman team in 1998 (Sánchez et al., 1998) showing THC toxicity upon GBM cell lines opened new perspectives that led to a phase I clinical trial in which 9 patients with recurrent GBM were treated with intratumoral administration of THC, with significant results on tumor reduction and malignant cell viability (Guzman et al., 2006). Taken together, these aspects direct future treatments for GBM based on cannabinoid system exploration, relying on exhaustive evidence both in vitro and in vivo for the effectiveness of this approach.

2.2. Cell lines and reagents The two human glioblastoma lines, GAMG and U251, and the normal human astrocyte line (NHA) were obtained from European Collection of Cell Cultures (ECACC, Salisbury, United Kingdom). Cell lines authentication was performed by Department of Molecular Diagnostics, Barreto's Cancer Hospital. Genotyping confirmed the complete identity of all cell lines. All cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma Aldrich, ST. Louis, MO, USA), supplemented with 10% Fetal Bovine Serum (FBS Gibco-Life Technologies, Grand Island, NY, USA), 1% penicillin/streptomycin and incubated in a humidified atmosphere with 5% CO2, at 37 °C. Confluent monolayers were dissociated with 0.25% trypsin-EDTA (Life Technologies). Cells of logarithmic growth phase were used in all experiments. All experiments were performed in triplicate. 2.3. Cytotoxic activity assay The cells were plated into 96-well plates at a density of 5 × 103 cells per well and allowed to adhere overnight (24 h). Subsequently, the cells were treated with increasing concentrations of WIN in DMSO (1%) and DMEM (0.5% FBS and 1% P/S). The IC50 values were determined after incubation of the cell lines for 24 and 72 h using the MTT (3-[4,5Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromite; Thiazolyl blue). Cell viability was quantified at 570 nm. The tumor specificity (TS) was calculated by the following equation: TS = mean IC50 (normal cells)/mean IC50 tumoral cells (Ogbole et al., 2017). The IC50 value found in the 72 hs was used for all subsequent assays in vitro and in vivo. 2.4. Cellular death by propidium iodide/acridine orange The GAMG and U251 cells were seeded 7.5 × 104 cells/well in a 96well plate and incubated in a CO2 5%, for 24 h. After this time, the cells were treated using the IC50 and again, the cells were incubated. Finally, the treatment was removed, and the cells were washed in 1× PBS, trypsinized and centrifuged for 5 min at 1500 rpm. The supernatant was discarded, the cells resuspended in PBS and were added 10 μl of acridine orange and 10 μl of 0.01 mg/ml propidium iodide. These solutions were added on a histological slide and 20 images at 200× magnification were obtained. The images were analyzed, and the data analyzed using software GraphPad PRISM version 5. 2.5. Wound healing GAMG and U251 cells were cultured in a 24-well plate containing DMEM medium and 10% FBS. After confluence, two wounds were performed in each well. Wounds were washed with PBS to remove supernatant cells and then the treatment was diluted in DMEM and 2% FBS medium. Images of the wounds were obtained at times of 0, 24, 48 and 72 h (Wang et al., 2018). In the end, the images were analyzed using ZEN software and the results analyzed using GraphPad PRISM 234

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version 5 software.

Table 1 WIN and TMZ cytotoxicity in tumor and non-tumor cell lines at times of 24 and 72 h of treatment.

2.6. Cell morphology For the morphological analysis, U251 and GAMG cell lines were seeded in six-well plates at 5 × 105 cells/well. The cells were treated with IC50 for 24, 48 and 72 h. At the indicated time points, any morphological changes occurring with the cells were examined and the images were obtained (magnification 200×) in a Zeiss Vert A.1 microscope linked the Zeiss Axiocam 503 colour camera (Bin Rohin et al., 2017).

Cell type

Cytotoxicity (μM) WIN 24 h

Cytotoxicity (μM) WIN 72 h

Cytotoxicity (μM) TMZ 24 h

Cytotoxicity (μM) TMZ - 72 h

GAMG U251 WI-38 NHA

9.80 11.81 27.00 37.2

6.22 9.65 11.25 26.74

81,22 155.8 – 53.69

75,58 167.3 – 48.49

No determined.

2.7. Colony forming assay

Table 2 Selectivity of WIN and TMZ treatments by glioblastomas, GAMG and U251, tumor cell lines after 24 and 72 h of treatment.

The cell lines were examined by anchorage-independent soft agar for the clonogenic assay. Briefly, 500 μl of 1.2% agar base was loaded into each well with 500 μl of DMEM 2× (20% FBS) media and brought to the CO2 (5%) even at 37 °C, for 30 min. Then, agar (0.7%) with 5 × 103 cells was seed into wells and incubated overnight in a CO2 (5%) at 37 °C. After that, the media was discarded, and the cells were cultured in DMEM (0.5% FBS) with vegetal samples, WIN and DMSO (1%), for 21 days. After that, colonies developed were fixed with methanol and stained with 0.5% crystal violet for 5 min before being counted microscope linked the Zeiss Axiocam 503 colour camera (Geissmann, 2013).

Cell type

Cytotoxicity (μM) WIN 24 h

Cytotoxicity (μM) WIN 72 h

Cytotoxicity (μM) TMZ 24 h

Cytotoxicity (μM) TMZ 72 h

Wi-38 × GAMG Wi-38 × U251 NHA × GAMG NHA × U251

1.8 1.2 4.3 2.8

2.7 2.3 4 3.3

– – 0.66 0.34

– – 0.64 0.29

No determined.

done by ImageJ (NIH) software (Santos et al., 2018). 2.8. Invasion assay 2.10. Western blotting

Cell invasion was measured using transwell chambers (Corning, USA) containing 24-well inserts with 8 μM pores, in the presence or absence of matrigel Matrigel® (BD Matrigel Matrix, BD Biosciences, Cat. Number 354234). Fifty microlitres of Matrigel® (25%) diluted in serumfree medium and added to the upper chamber of a Transwell® nylon filter membrane inserts (Corning Inc.), incubated at 37 °C in a 5% CO2 humidified atmosphere for 30 min. Thus, 3 × 105 cells (U251 and GAMG) per well and the treatments were placed on the bottom of the membrane and allowed to invade for 24 h. Then, the cells in the upper chamber were removed, and the membranes were fixed with 4% paraformaldehyde and stained with hematoxylin and eosin. Finally, the membranes were cut from the inserts and mounted on the glass slides for microscopic analysis. Fifteen (100×) images of the membranes were obtained on a LAB. A1 microscope using the software AxioVision Rel. 4.8 and the number of cells were quantified using ImageJ software (Salo et al., 2015).

The cells were collected and scrapped in buffer lyses (50 mM Tris (pH 7.6–8), 150 mM NaCl, 5 mM EDTA, 1 mM Na3VO4, 10 mM Na4P2O7, 1% NP-40 and protease inhibitors). The supernatants were collected after centrifugation (13,000 rpm) at 4 °C for 15 min. Protein concentration in the cell lysate was determined by Bradford method. The protein samples were filtered and separated by SDS-PAGE (10% and 15% acrylamide gel), at 140 V for 4 h and transferred on to nitrocellulose membrane (GE Healthcare Life Sciences) using semi-dry transfer (Amersham). The membranes were blocked (5% non-fat milk in TBS-T) for 1 h at room temperature. Then, the membranes were incubated overnight with a primary antibody [(RIP (Cell Signalling), FADD (Cell Signalling), hILP/XIAP (BD Biosciences), Caspase 3 (Cell Signalling), APAF-1 (BD Biosciences), Caspase 8 (Cell Signalling), Beclin (Cell Signalling), Caspase 7 (Cell Signalling), p53 (BD Biosciences), PARP (Cell Signalling), FAS (Cell Signalling), HSP70 (Cell Signalling) and, Cathepsin D (Cell Signalling). After to wash (TBS-T) the membranes, they were incubated with antibody secondary anti-mouse or anti-rabbit (Cell Signalling))] for 1 h. Then, the membrane was again washed (TBS-T) and developed by the chemiluminescence method using an equimolar mixture of two solutions (first solution = 100 mM Tris [pH = 8.5], 2.5 mM Luminol and 0.396 mM coumaric acid and second solution = 100 mM Tris [pH = 8.5] and 0.06% H2O2). The membrane was developed on a radiographic film (Medical Xray FilmKODAK). Densitometric quantification was done by ImageJ (NIH) software (Pereira et al., 2018). The expression of the target protein was expressed as the ratio of the absorbance of the target protein band to the absorbance of the loading control band (Gürtler et al., 2013).

2.9. Zymography The proteolytic enzyme activity of pro-MMP-2 was measured by gelatin zymography. Matrix metalloproteinase 2 and 9 (MMP-2 and MMP-9) (Sigma M9445) was used as standard and gelatinases were obtained from human saliva. Saliva collected for the first 2 min was discarded and the remainder was placed in mini-cooled tubes, centrifuged at 14000 g, before discarding the pellet and retaining the supernatant. The total protein was measured by the Bradford method using bovine serum albumin (Sigma) as standard (Kruger, 2002). Equivalent amounts (20 μg) of proteins from the saliva were mixed with an equal volume of non-denaturing buffer (2% SDS, 125 mM Tris-HCl, pH 6.8, 10% glycerol and 0.001% bromophenol blue) and the protein samples were subjected to electrophoresis through a 7% Zymogram Ready Gel (Bio-Rad Laboratories). After electrophoresis, the gel was incubated twice in 2.5% TritonX-100 for 60 min at room temperature and then, incubated at 37 °C for 18 h in activation buffer (10 mM TrisHCl buffer (pH 8.0), containing 5 mM (Tris-CaCl2). Gels were stained (0.25% Comassie blue G-250, 30% ethanol, 10% acetic acid) for 1 h and destained (30% ethanol, 10% acetic acid), for 2 h. Gelatinolytic activity was detected as unstained bands and densitometric quantification was

2.11. Chick chorioallantoic membrane (CAM) assay To assess the in vivo tumor proliferation and angiogenesis, we used the CAM assay as previously described (Martinho et al., 2013). Fertilized chicken eggs (Gallus gallus) were incubated at 37 °C and 70% humidity. After 3 days, a small hole (1 mm) was drilled into the acute in each pole and then, another hole (3 mm) was made in eggshell, exposing the chorioallantoic membrane. On the 9th day, 2 × 106 tumor 235

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Fig. 2. The morphological alterations after the treatment with WIN demonstrated more cell shrinkage, chromatin condensation, and fragmentation of the nucleus. These data together indicate that GAMG and U251 cells are in initial death. A and B - Images representative of the initial death of the GAMG and U251 glioblastoma cells, respectively. Blank arrows point to chromatin condensation and fragmentation (200× magnification). C and D - WIN treatment cause death of GAMG and U251 cells, respectively, after 24 h. Statistical analysis by t-test, ***p < .0001. E and F - Photographic representation of morphological alterations after WIN treatment on GAMG and U251 lines at times of 0, 24, 48 and 72 h. The higher detail shows the cells with normal morphology (in blue) and cell shrinkage and chromatin condensation (in red) (200× magnification). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. WIN inhibited cell motility in U251 and GAMG cells. Control and WIN-treated glioma cells were allowed to form monolayers before wounding with a micropipette. A and B - The same frame was photographed directly in 0, 24, 48 and 72 h. The border of the wound is highlighted in red. C and D - Graph representing the degree of wound closure/cell migration for each treatment group following 24, 48 and 72 h incubation post wounding (magnification 100×). The data were analyzed by GraphPad PRISM version 5 software. Data are represented as the mean ± SD ***values with different superscripts are significantly different (p < .001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

inappropriately result in an irritant response. The treatments (300 μl) were applied to the membrane for observation of the effects caused such as hemorrhage, coagulation, and lysis of vessels. The endpoints at fixed time intervals of 0.5, 2, and 5 min were used for obtaining the images (Solano et al., 2018).

cells resuspended in 20 μl were placed in the CAM. The tumors were treated with WIN (20 μl topically in the tumor mass) in the 14th day. The images of the formed tumor in ovo were obtained using a stereomicroscope (MOTIC) in the 17th day. The perimeter of the tumors was measured with ZEN software and the size of the growth was calculated by the measurement of the tumor in the 17th - measurement of the tumor in the 14th. The CAMs were removed and histologically analyzed by staining of hematoxylin and eosin. Images were obtained at 200× magnification and the area of tumor perimeter and quantified blood vessels with ZEN software. The results analyzed using GraphPad PRISM version 5 software. Ethical approval was obtained by the Ethics Committee of the Federal University of São João Del Rei under protocol number 039/ 2017.

3. Results 3.1. Cell viability is differentially affected by different concentrations of win in glioblastoma and normal human cell line The WIN was evaluated in two lines of glioblastoma (GAMG and U251) and two no malignant cell (WI-38 and NHA) at 24 h. The IC50 obtained were 9.8 μM, 11.81 μM, for GAMG and U251 respectively. For the no malignant cells, the IC50 were 27 μM and 37,2 μM, for WI-38 and NHA, respectively (Table 1), while temozolomide (TMZ) presented IC50 of 81.22 μM, 155.8 μM and 53.69 μM for GAMG, U251, and NHA, respectively. The compound was selective for tumor cells at 1.84 and 1.53 for GAMG and U251, respectively, when compared to the WI-38 lung fibroblast line. In addition, the selective index was 4.3 and 2.8 for GAMG and U251, respectively, when compared to the NHA astrocyte cell line respectively (Table 2). At 72 h, the IC50 obtained were 6.22 μM, 9.65 μM, 11.25 μM and 26.74 μM for GAMG, U251, WI-38 and NHA, respectively (Table 1),

2.12. Hen's egg test - chorioallantoic membrane (HET-CAM) test method Fertilized chicken eggs (Gallus gallus) were incubated at 37 °C and at 70% humidity, for 10 days. Then, the eggshell was opened at the side of the air chamber and the inner egg membrane was carefully removed to avoid any damage to the fine blood vessels of the chorioallantoic membrane. The 0.9% NaCl (negative control) and 1% SDS (positive control) were included in each experiment to provide a baseline for the assay endpoints and to ensure that the assay conditions do not 237

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Fig. 4. WIN treatment decreases the invasion potential in the adjacent tissue by Glioblastoma cells. The inhibition of the activity of MMP-2 and MMP-9, important enzymes present in the process of tumor invasion, was observed through the zymogram. A, B - The detection of the migration of U251 following a 24 h incubation period was evaluated by Transwell assay. The U251 cells treated with WIN were less effective at invading when compared to the control group (magnification 200×). Statistical analysis by t-test, *p < .0027. C, D- MMP-2, and MMP-9 treated with WIN have their gelatinolytic activity decreased. Data are presented as mean ± SD, *p < .01; **p < .001.

Fig. 5. Clonogenic assays: WIN treatment effects on the in vitro growth of GAMG and U251 cell lines. The colonies formed were fixed with 10% formaldehyde and stained with crystal violet. Representative pictures (A, C) and graph (B, D) show GAMG and U251 colonies, respectively, after 21 days of WIN treatment. The images were obtained in MOTIC stereomicroscope (50× magnification). The data are presented as mean ± SD. Statistical analysis by the test T *p < .001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

while temozolomide (TMZ) presented IC50 values were 75.58 μM, 167.3 μM and 48.4 ± 9 μM for GAMG, U251, and NHA, respectively. The compound was selective by tumor cells in 1.8 and 1.16 for GAMG and U251, respectively, when compared to the WI-38 pulmonary fibroblast line, and 4.29 and 2.77 for GAMG and U251, respectively,

when compared to the lineage normal human astrocyte NHA (Table 2). 3.2. WIN triggers apoptosis Morphological changes associated with apoptosis were evaluated by 238

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Fig. 6. Altered expression levels of the apoptosis proteins by WIN in two glioblastoma cells. The effects of the WIN on GAMG or U251 cells were determined at the translational levels for apoptosis protein in two glioblastoma cell lines at 24 h by Western blot assay. A- Representative images of western blotting. B, D, E, F, G and H: Quantitative analysis of the WIN treatment showed reduced expression of PARP, hILP/XIAP, APAF-1, cleaved-caspase 8, cleaved -caspase 3 and cleaved -caspase 7 proteins. There was no alteration in FADD protein expression. Densitometric quantification was done by ImageJ (NIH) software. The value of the vehicle control (1% DMSO) was set as 100%. The data represented in the graph are the means ± SEM of experiments that were conducted at least three times. *Data are represented with the mean ± SD. *p < .05, *p < .01 and **p < .001.

using the Acridine Orange (AO) and Propidium Iodide (PI) staining (Fig. 2). We observed that after AO/PI staining the viable cells were uniformly stained by AO, a cell-permeable fluorescent dye which stains the nuclear DNA into cells that have the intact plasmatic membrane. The cells in initial death were also staining by AO, but differently, they showed bubbles, chromatin condensation, and fragmentation (Fig. 2 A and B). Few cells were stained with PI (Fig. 2C and D), this dye only stains nuclear DNA when plasma membrane integrity is impaired, indicating late cell death. Due to the cytotoxicity of the WIN on glioma cell lines, we concerned in know the kind of cell death. Thus, we observed the cell morphological changes microscopically after the exposure of the cells to IC50 for 24, 48 and 72 h. As shown in Fig. 2 (E and F), WIN induced alterations morphological as cells shrinkage and condensation of the chromatin occurred. These results indicate cell death by apoptosis.

3.6. WIN induces caspase-independent apoptosis of the glioblastoma cells

3.3. WIN inhibits migration of glioblastoma cells

The chicken chorioallantoic membrane assay is used to evaluate the tumor perimeter and angiogenesis. Regarding the tumor perimeter, there was a significant reduction after treatment (p < .001). Regarding the histological analysis, H.E. staining and the tumor perimeter were performed, the number and area of the vessels were quantified using the Zen Lite software. Reduction of the tumor mass formed by U251 cell line was observed, as well as macroscopic analysis. There was a reduction around the blood vessels, but it was not statistically significant (Fig. 9).

To investigate cell signalling involved in apoptosis, we examined whether WIN treatment led to the activation of proteins involved with this pathway of death in glioblastoma cells. Treatment with WIN did not result in increased expression of PARP, FADD, hILP/XIAP, APAF-1, cleaved Caspase 8, cleaved Caspase 3, cleaved Caspase 7 (Fig. 6) proteins related to canonic apoptosis pathway. Thus, we also verified the non-modification of the expression of the Beclin and RIP proteins, related to autophagy (Fig. 7). Finally, we checked the increase the expression of p53. From this finding, we analyzed proteins that are related to oxidative stress, HSP70, and cathepsin D, and found the decrease and increase of these proteins, respectively (Fig. 8). 3.7. Assay of chicken coralalantid membrane with compound (R) - (+) WIN55,21-2, in vivo

WIN inhibits the invasion and migration of glioblastoma cells (GAMG and U251). These processes are essential for the tumor cells to invade the other tissues. The treatment inhibited cell migration significantly after the treatment from time 24 h for the GAMG and U251 cell lines (Fig. 3).

3.4. WIN reduces cell invasion, and inhibits the activity of matrix metalloproteinases 2 and 9

3.8. Irritation test of the chalioalantic membrane of chicken egg after the treatment with the compound WIN

The cell invasion assay indicated that WIN significantly inhibited cell locomotion dependent on extracellular matrix degradation. Thus, the action of WIN on the activity of MMP-2 and MMP-9 was evaluated by zymography assay. Our results showed that WIN inhibited the activities of these two enzymes, reinforcing the effect found in the invasion assay. These findings demonstrate that WIN works by reducing processes that are important in cell metastasis (Fig. 4).

The chicken egg chorioallantoic membrane (HET-CAM) irritation test was used to assess the potential vessels irritancy by WIN as measured by its ability to cause toxicity in the chorioallantoic membrane of a chicken (Fig. 10). 4. Discussion

3.5. WIN reduces clonogenic capacity of glioblastoma tumor cells

Tumor selectivity is related to greater cytotoxicity to tumor cells than to normal cells. The aminoalkylindole derivative WIN55,212-2 (WIN) was selectively cytotoxic for both tumor lines at both time 24 and 72 h. Indeed, this feature is desirable, if not a prerequisite, for cancer chemotherapeutics. Despite this premise, the standard

The clonogenic assay was performed to evaluate the ability of a tumoral cell to form a tumor mass. The WIN treatment reduced the colonies number of the GAMG and U251 cell lines (Fig. 5). 239

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Fig. 7. Altered expression levels of the of autophagyrelated proteins by WIN in two glioblastoma cells. The effects of the WIN on GAMG or U251 cells were determined at the translational levels for autophagyrelated proteins in two glioblastoma cell lines at 24 h by Western blot assay. A- Representative images of western blotting. B and C: WIN treatment showed reduced expression of RIP and Beclin proteins. Densitometric quantification was done by ImageJ (NIH) software. The value of the vehicle control (1% DMSO) was set as 100%. The data represented in the graph are the means ± SEM of experiments that were conducted at least three times. *Data are represented with the mean ± SD. *p < .05, **p < .01 and ***p < .001.

capability of these drugs to function as anti-metastatic agents (Roberto et al., 2018). Cell invasiveness is dependent on functional secreted enzymes, especially MMP-2 and MMP-9, capable of catalyzing the proteolytic cleavage of the extracellular matrix and basal lamina components (Lin et al., 2018). In this study, we found that WIN inhibits in vitro gelatinolytic activity of both MMP-2 and 9, which a process that could also impact in the metastatic potential of GBM cells. In addition, the clonogenic capacity also was inhibited, which directly affects the ability of cellular self-renewal (Franken et al., 2006). Thus, the activity demonstrated by the win provides the need for compounds that impact on the replicative phenotype of invasion and metastasis that becomes so imperative. Morphological characterization of WIN-treated cells, investigated with PI/AO staining, displayed significant augmentation in the number of cells on early death stages, revealing apoptotic features such as chromatin condensation, cell, and nuclear shrinkage and apoptotic bodies formation. Therefore, altogether, from these several observations, we can suggest that probable an apoptosis event happens after the

pharmacological therapeutic approach to treat Glioblastoma, the alkylating agent Temozolomide does not display significant selectivity for malignant cells (Silva et al., 2018; Suffness and Pezzuto, 1990). Unquestionably, the TS index calculated for these cells indicate specific toxicity against malignant cells, an effect trend desirable for cancer drug candidates but not always achieved, as observed for TMZ. GBM lesions present aggressive cell phenotypes, responsible for enhanced cell proliferation rates and great capability to migrate and to invade surrounding tissues, processes that account for the high incident of metastasis in GBM patients. The obtained results revealed that WIN significantly reduced, both GAMG and U251 cell migration rates in the wound healing assay. Moreover, WIN significantly decreased cell invasiveness degrading the Matrigel that has extracellular matrix components and also, the clonogenicity of treated cells, corroborating the relevance of our previous finding. This finding is relevant considering that this could impact in the metastatic process of GBM. Indeed, these findings were also observed in a recent study with prostate cancer cell lines treated with cannabinoids, reinforcing the

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Fig. 8. The effects of the WIN on GAMG or U251 cells were determined at the translational levels for HSP70, cathepsin D and p53 at 24 h by Western blot assay. Treatment with WIN reduced the expression of HSP70 that is related to cell protection to stress. There was also an increased expression of cathepsin D that is released into the cytosol when loss of stress protection occurs. In addition, there was an increase in p53 that is related to DNA damage. A- Representative images of western blotting. B- Quantitative analysis of WIN treatment showed reduced expression of HSP70 proteins. C and D: Quantitative analysis of WIN treatment showed increased expression of Cathepsin D and p53 proteins. Densitometric quantification was done by ImageJ (NIH) software. The value of the vehicle control (1% DMSO) was set as 100%. The data represented in the graph are the means ± SEM of experiments that were conducted at least three times. *Data are represented with the mean ± SD. *p < .05, **p < .01 and ***p < .001.

Fig. 9. The CAM assay was used to evaluate the growth of the tumor mass formed by the U251 glioblastoma line, and WIN treatment was performed on the 14th day and evaluated in 72 h. It was observed in vivo and the histological analysis by H.E. the reduction of the tumor perimeter. There was no change in the statistical difference concerning the area of the blood vessels in the histological examination. A - Representative figures of the tumor mass, U251, on the 14th and 17th day in vivo (50× magnification). B- Representative graph of reduction of tumor perimeter after treatment with WIN. The data are represented by the mean ± SD. Values with statistical analysis by the test T, **p < .001. C- Representative figures of the tumor mass, U251, and area of vessels of CAM on the 17th day, by histology H.E. D- Representative graph of reduction of the tumor perimeter evaluated microscopically by histology H.E (increase 200×). The data are represented by the mean ± SD. Values with statistical analysis by the t-test, **p < .05. E- Area of blood vessels evaluated microscopically by histology H.E. Data are represented by mean ± SD. Values with statistical analysis by the t-test, n.s. not significant.

APAF-1 PARP, and Beclin were also found in cells treated with WIN. The reduction of the expression levels of the apoptosis suppressor, RIP, is related with sensitization to apoptosis (Humphries et al., 2015). Cell signalling to apoptosis is also dependent on several other proteins such

WIN treatment. (Cascioferro et al., 2019). To further characterize, WB investigated the cell death mechanism induced by WIN, such as apoptotic, autophagic and cell stress protein markers. Significant reduction in the expression levels of RIP, hILP/XIAP, cleaved-caspase-3 and 8, 241

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Fig. 10. The chorionic membrane did not show bleeding or lysis of the vessels after treatment with the WIN compound at the time of 0.5, 2 and 5 min. A Representative images after treatment with the positive control (SDS 1%), being observed the results of 2 and 5 min. B - Representative images of the negative control (0.9% NaCl). C- Representative images of treatment with compound WIN at the times of 0, 0.5; 2 and 5 min.

due to its ability to reduce tumor and reduce angiogenesis in vivo. HET-CAM technique was initially employed to assess the risk of irritation of the skin and mucosa (ocular, for example) exposed to new compounds. Additionally, this assay also allows the evaluation of the risk of irritation at the infusion site, reducing the use of in vivo test (Eichenbaum et al., 2013). One of the routes of administration of chemotherapy is the intravenous route. During the administration of the chemotherapeutic, its extravasation of the venous network may occur. This extravasation is defined as an inverted installation in the perivascular space during infusion and can be classified according to the subcutaneous toxicity in vesicant agents (produce tissue damage, ulcerations, and necrosis). WIN55-212-2 treatment showed once again an excellent performance since it did not cause irritability, hemorrhage and blood clotting (Pluschnig et al., 2016), can be administered parenterally safely. All data together allow us to conclude that WIN is an attractive candidate as a new chemotherapeutic agent. Thus, we think that this work has good futures perspectives such as the combination of WIN with temozolomide, seeking a potentiation of the effect and reduction of the doses administered, besides performing the 3D cell culture tests with this combination, because that allows mimicking the tumor, in vivo.

as hILP/XIAP, a pan-caspase endogenous inhibitor. Therefore, the lower expression levels of hILP/XIAP on WIN-treated cells are indicative signals of apoptosis (Kaufmann et al., 2012). Despite this finding, no difference in clevaded-caspase-3, 7 and 8 expression levels were found when WIN-treated cells were compared to controls. The same result was observed for APAF-1, another key pro-apoptotic protein (Grivicich et al., 2007). On the other hand, it was found that the cell cycle controller, p53 (Li et al., 2015), and the caspase-independent apoptosis marker, Cathepsin-D (Bröker et al., 2005), displayed significantly enhanced expression levels in GBM cell lines treated with WIN, in comparison to non-treated controls. p53 levels are strictly related to DNA damage, what induces G1 cell cycle arrest and consequently blockade of cell fate on G2/M checkpoint (Li et al., 2015). In addition, p53 affects lysosomal permeabilization (Yuan et al., 2002). An additional finding that corroborates the hypothesis of caspase-independent apoptosis is the lowered expression of HSP70 on WIN treated cells, a heat-shock chaperone which depletion is related to the release of lysosomal enzymes on cytosol, such as Cathepsin-D, and consequent apoptosis not related to procaspase cleavage (Nylandsted et al., 2000), also called caspase-independent apoptosis (Bröker et al., 2005; Serrano-Puebla and Boya, 2018), a mechanism that could account for the observed morphology observed in the WIN-treated GBM cells. The in vitro experiments exhibited here clearly revealed that WIN treatment acts on relevant cancer processes such as migration and clonogenic ability, which justifies the subsequent in vivo tests. Through the CAM, we observed that compound decreased the tumor size, a finding that, in addition to reinforcing the in vitro results demonstrates the ability of this compound to inhibit the unlimited replication potential of tumor cells, in vivo (Hanahan and Weinberg, 2011). Concerning angiogenesis, there was a significant reduction in the area of the blood vessels. It is known that tumor growth is dependent on tumor angiogenesis, with inhibition of angiogenesis being a new therapeutic possibility in the control of tumors (Kruger et al., 2001). Thus, WIN possesses potential as a novel drug in the treatment of glioblastomas,

5. Conclusion The results obtained in the present study indicate strong and selective cytotoxicity of the synthetic cannabinoid WIN against glioblastoma cell lines. Considering the aggressiveness, resistance to treatment, migration capacity and invasiveness potential of this malignancy, along with the findings in vitro that show the inhibition of migration, invasion, and clonogenicity of GBM cell lines by WIN, we can reinforce the potential usefulness of cannabinoids on Glioblastoma management. The apoptotic cell death caspase-independent is intriguing and a matter of further investigation. Furthermore, tumor growth was inhibited in vivo and it can be safely administered parenterally. All data together make WIN an interesting candidate as a new 242

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chemotherapeutic agent.

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