The Dietary Isothiocyanate Erucin Reduces Kidney Cell Motility by Disturbing Tubulin Polymerization

Abstract

Scope

Epidemiological evidence associates the consumption of cruciferous vegetables with reduced risk of several cancers, including renal cell carcinoma. Erucin can be generated by in vivo reduction of sulforaphane or by enzymatic hydrolysis of glucoerucin. Contrarily to sulforaphane, only limited studies have addressed the anticancer properties of erucin. This study aims at evaluating the impact of erucin on renal cell biology.

Methods and Results

The effects of erucin were assessed in 786‐O and Vero‐E6 cells, representative of human renal cancer and non‐ cancer kidney cells, respectively. Erucin induced a concentration‐dependent decrease in cell viability and cell cycle arrest at G2/Mitosis. In Vero‐E6 cells erucin modestly reduced intracellular reactive oxygen species levels while in 786‐O no effects were detected. After erucin treatment, both cell lines revealed altered morphology, with a concentration‐dependent change from an elongated shape towards a smaller round conformation. Moreover, erucin affected cell adhesion and strongly altered the tubulin network structure and specifically microtubule polymerization. These results are in line with the observed decrease in collective and single cell migration and G2/Mitosis arrest.

Conclusions

Overall, erucin may have a beneficial impact in reducing the motility of renal cancer cells. Our results contribute to explore possible dietary approaches for secondary/tertiary renal cancer chemoprevention.

Erucin is a compound found in cruciferous vegetables, such as arugula. This study demonstrates that erucin is able to decrease the motility and adhesion of kidney cells by interfering with the construction of the cytoskeleton. Reducing the motility of renal cancer cells could have beneficial effects against the progression of renal cancer.

1. Introduction

Several ancient civilizations in Europe and worldwide described the cultivation and consumption of cruciferous plants.^{[ 1 ]} Plants from the Brassicaceae family, also named Cruciferae, are commonly consumed nowadays as part of different dietary regimens. The list of these vegetables includes broccoli, Brussels sprouts, cabbage, cauliflower, collard greens, kale, kohlrabi, rutabaga, turnips, mustard, Chinese cabbage, watercress, among others.^{[ 2} , ^{3 ]}

The cruciferous vegetables are often associated with a number of beneficial health properties and are well‐established as sources of promising bioactive compounds. These include nutrients (e.g., proteins, carbohydrates, vitamins, and minerals) as well as several non‐nutritive secondary metabolites (e.g., glucosinolates, different classes of polyphenols and carotenoids). The detailed description of both nutritive and non‐nutritive compounds found in cruciferous vegetables is reviewed in the literature.^{[ 3 ]} Concerning the non‐nutritive constituents, it is important to highlight the presence of the isothiocyanates (ITCs) sulforaphane (SFN, 1‐isothiocyanato‐4‐(methylsulfinyl)butane)) and erucin (1‐isothiocyanato‐4‐(methylthio)butane), a compound metabolically and structurally related with SFN. Both ITCs have been closely associated with many of the health promoting properties of these vegetables, particularly as chemopreventors and promising anticancer molecules in both in vitro and in vivo models.^{[ 4} , ^{5 ]} In this regard, while the effects of SFN have been thoroughly reported in the literature, less information is available for erucin, especially concerning its cytotoxic, anti‐migratory, and anti‐invasive potential in cancer cell models. Nevertheless, erucin anticancer effects have been studied in vitro in human cancer cell lines of different origins, such as lung,^{[ 6 ]} breast,^{[ 7} , ^{8 ]} bladder,^{[ 1 ]} pancreas,^{[ 9 ]} liver,^{[ 10} , ^{11 ]} and prostate.^{[ 12 ]} However, to our knowledge no reports addressing the potential effects of erucin in the context of renal cell carcinoma (RCC) are available in the literature. It should be mentioned that this type of renal cancer constitutes approximately 90% of kidney malignancies, being the clear cell RCC (ccRCC) the most common subtype of RCC (75%).^{[ 13 ]} Moreover, in terms of the estimated new cases of cancer in the US, kidney & renal pelvis cancer ranks in the 6th place in men and the 9th in women, corresponding to 3–5% of all adult malignancies as reported in Siegel et al.^{[ 14 ]} Importantly, it is also well recognized that novel approaches to cope with RCC are still needed. In view of this, it is pertinent to explore the impact of erucin in RCC cells in vitro. This topic becomes even more relevant since meta‐analyses of observational studies suggested that the cruciferous vegetables intake is associated with a lower risk of renal cancer.^{[ 15} , ¹⁶ , ^{17 ]} In this context, the present study aims at exploring the effects of erucin in kidney cancer cell biology. Endpoints relevant to cancer progression were evaluated and the molecular mechanisms underlying the observed phenotypes were explored. In this work, we demonstrate that erucin reduces kidney cell motility by disturbing tubulin polymerization, and may thus contribute to the protective effects of cruciferous vegetables against kidney cancer development.

2. Results and Discussion

Observational studies suggest that cruciferous vegetables intake is associated with a lower risk of renal cancer.^{[ 15} , ¹⁶ , ^{17 ]} In this context, the impact of the poorly explored ITC erucin on renal cancer was studied. For this purpose, we selected 786‐O cells, a well‐known ccRCC model, and Vero‐E6 cells as a non‐tumor control model. As ccRCC is the most common subtype of renal cancer,^{[ 18 ]} we chose a well‐characterized human ccRCC model included in the National Cancer Institute Anticancer Drug Screen panel. The highly invasive 786‐O cells are defective in Von Hippel‐Lindau expression and produce high vascular endothelial growth factor levels, which are characteristic of ccRCC.^{[ 18 ]} In addition, the Vero‐E6 cells were used as a non‐tumor kidney cell model.

The effects of erucin on the viability of renal cells were initially explored. Erucin only reduced cell viability at a higher concentration (100 µM; Figure 1A). At this concentration, the cytotoxicity was more pronounced in 786‐O cancer cells than in the non‐tumor Vero‐E6 cells. The absence of cytotoxicity of erucin at concentrations up to 80 µM, in both cell lines, was confirmed by the lack of cell permeability to propidium iodide (PI) (Figure 1B,C). To the best of our knowledge, this is the first report on the impact of erucin on renal cancer cells viability. Nevertheless, erucin was shown to reduce the cell viability of breast,^{[ 7} , ^{8 ]} prostate,^{[ 12 ]} pancreas,^{[ 9 ]} and bladder^{[ 19 ]} cancer cell models, with IC50 values between 8 and 50 µM. In lung carcinoma cells,^{[ 6 ]} erucin was less toxic, as also observed in our study.

Figure 1.

Impact of erucin on cell viability and cell cycle. Vero‐E6 and 786‐O cell mitochondrial activity and membrane permeability following exposure to erucin for 24 h were evaluated by A) MTT and B, C) PI‐stain, respectively. D, E) Cellular DNA content was measured by flow cytometry analysis of PI‐stained cells. D) Histograms show representative Vero‐E6 and 786‐O DNA content profiles following exposure to erucin, fixation, and PI stain. E) Summary results from DNA content assays. F, G) Intracellular ROS levels were measured using the DCFH‐DA probe. Results are expressed as mean ± SD (n = 2‐3); * p < 0.05, ** p < 0.01, *** p < 0.001, when compared with control cells. PI, propidium iodide.

Although renal cells remained viable when treated with erucin up to 80 µM, we aimed to explore whether the treatment with this ITC could alter cell cycle distribution. Cell cycle progression analysis revealed an increased G2/Mitosis (G2/M) population at 50 µM in Vero‐E6 cells. In 786‐O cells, a concentration‐dependent increase in G2/M was observed, with statistical significance at 80 µM (Figure 1D,E).

ITCs may interfere with the redox status of cells and such effects could contribute to the beneficial effects observed in different cancer cell models.^{[ 20} , ^{21 ]} The data regarding the impact of erucin on intracellular reactive oxygen species (ROS) levels in cancer cells reported by other authors are somehow conflicting and appear to be cell and/or cancer type specific.^{[ 5} , ²¹ , ^{22 ]} To the best of our knowledge, the impact of erucin on the levels of intracellular ROS in kidney cells has not been reported previously. The global intracellular levels of ROS were assessed and the obtained results are depicted in Figure 1F,G. Erucin decreased the basal intracellular ROS levels in non‐tumor Vero‐E6 cells in a modest albeit significant manner. In 786‐O cancer cells, such antioxidant effect was not observed. Interestingly, the increase in ROS induced by tert‐butyl hydroperoxide (TBHP) was much more pronounced in 786‐O, suggesting a lower antioxidant defense capability of this cell line. While studies comparing the levels of ROS and antioxidant defenses of these specific cell lines are not available, our hypothesis concurs with previous reports suggesting a role for oxidative stress in the pathogenesis of different cancers, including RCC. In fact, a downregulation of the antioxidant enzymes catalase and glutathione peroxidase was previously described in human tumor kidney specimens by Pljesa‐Ercegovac et al.^{[ 23 ]} Increased oxidative/nitrosative damage was also reported in renal tissue from RCC patients, particularly in high‐grade tumors.^{[ 24 ]} Permanent oxidative stress could thus be considered a feature of RCC.^{[ 25 ]}

Considering the importance of cell motility for cancer progression and taking into account the previous reports on the impact of ITCs at this level,^{[ 26 ]} the effects of non‐cytotoxic concentrations (up to 80 µM) of erucin in cell migration were studied. Collective cell migration was assessed by the wound healing assay. The basal wound closure rate of 786‐O cells was higher than that of Vero‐E6 cells, which is consistent with the malignant nature of the former. Erucin significantly reduced collective cell migration in both cell lines in a concentration‐dependent manner (Figure 2A–D). Erucin (50 µM) also decreased single cell chemotaxis and chemoinvasion through extracellular matrix to ≈75% of untreated cells, as measured by transwell‐based assays (Figure 2E‐H).

Figure 2.

Erucin reduces collective cell migration, chemotaxis, and chemoinvasion. Collective cell migration of A, C) Vero‐E6 and B, D) 786‐O erucin‐treated cells was evaluated by the wound healing assay. A, B) Images show representative fields at different time points post scratching. Scale bar 800 µm. E, G) Chemotaxis and F, H) chemoinvasion were measured on 786‐O erucin‐treated cells (50 µM) using transwell‐based assays. E, F) Representative images of migrating/invading cells. Scale bar 400 µm. Results are expressed as mean ± SD (n = 3); * p < 0.05, ** p < 0.01, when compared with control cells.

As erucin reduced intracellular ROS of Vero‐E6 cells and decreased cell migration and invasion, we investigated whether those alterations could be ascribed to the inhibition of lysyl oxidase like‐2 (LOXL2) enzymatic activity. This enzyme catalyzes the cross‐linking reactions of elastin and collagen in the extracellular matrix of tumors, facilitating the processes of cell migration and invasion. During the catalytic reaction, hydrogen peroxide is generated as a by‐product, further contributing to the pro‐invasive effects of LOXL2. This enzyme was previously shown to interfere with cell growth, migration, and invasion of ccRCC cell lines and its expression is correlated with the pathologic stages of ccRCC patients.^{[ 27 ]} We have thus evaluated the effect of erucin in LOXL2 activity. A slight reduction of 15% in LOXL2 activity was observed, but only for a very high erucin concentration (300 µM; data not shown). This result excludes LOXL2 inhibition as a possible mechanism implicated in the effects observed in cell motility.

Changes in cell motility are often associated with alterations in cytoskeleton dynamics and cell adhesion properties. To explore the impact of erucin on these important aspects, cell morphology and adhesion were evaluated (Figure 3 ).

Figure 3.

Erucin affects cell morphology. A–F) The morphology of Vero‐E6 and 786‐O cells was analyzed using contrast phase microscopy. A, B) Microscopy images show representative Vero‐E6 and 786‐O cells after 8 h of incubation with erucin. Summary results show cell C, E) area and D, F) circularity relative to control. G–J) The percentage of cells attached to matrigel after 3 and 12 h was assessed using CV after a 24 h incubation with erucin. Results are expressed as mean ± SD (n = 2–3); * p < 0.05, ** p < 0.01, *** p < 0.001 when compared with control cells. Scale bar 400 µm.

Treatment with erucin, particularly at higher concentrations, dramatically altered cell morphology (Figure 3A–F). A strong reduction of cell area was observed in both cell lines (Figure 3C,E), accompanied by an increase in cell circularity. It should be noticed that under the same conditions, cell viability was maintained (Figure 1B,C), suggesting that the observed alterations are not a consequence of cell death‐associated mechanisms. The ability of cells to adhere was also reduced by erucin (Figure 3G–J), as expected considering the increase in cell circularity observed in Figure 3. This effect was observed at 3 h post‐seeding and persisted after 12 h. The impairment of cell adhesion was more pronounced in 786‐O than in Vero‐E6 cells. The marked changes in cell morphology measured as increased circularity and decreased cell area (Figure 3A‐F) and loss of cell adhesion (Figure 3G‐J) may explain the reduction in cell motility induced by erucin. To further explore the impact of erucin on cell adhesion dynamics, cell spread was assessed in cells after exposure to erucin for 24 h. Incubation with erucin strongly impaired cell spread (Figure 4 ), as demonstrated by a slower decrease in circularity and slower increase in cell area, compared with non‐treated cells.

Figure 4.

Erucin impacts cell spreading dynamics. The ability of cells to spread after a 24 h incubation with erucin was evaluated by contrast phase microscopy. Images show representative fields of A) Vero‐E6 and B) 786‐O cells at 2 and 6 h post seeding. Cell area and circularity were measured every 2 h for 12 h. Results are expressed as mean ± SEM (750 cells per condition from three independent experiments); *p < 0.05, **p < 0.01, ***p < 0.001 when compared with control cells at each time‐point. Scale bar 25 µm.

The phenotypic alterations observed in kidney cell morphology, motility, adhesion, and spread are consistently suggestive of changes in the cytoskeleton. Additionally, the observed G2/M arrest observed (Figure 1E) may also be the result of alterations in microtubule dynamics. To explore this hypothesis, the analysis of microtubule network morphology was carried out using fluorescence microscopy. Erucin induced profound concentration‐dependent alterations in the microtubule network, in both cell lines (Figure 5A‐F). At 50 µM, and as early as 4 h of incubation with erucin, a strong reduction in the microtubule density and an increase in the number of cells with aberrant microtubules were observed. At 80 µM, microtubules were not visible in most cells. Instead, small tubulin aggregates were detected on the cytoplasm. These observations were accompanied by the loss of identifiable microtubule structure characteristics of the microtubule‐organizing center (MTOC). Importantly, no relevant changes were observed in the actin cytoskeleton other than those induced by the loss of microtubules. Additionally, the number of cells undergoing mitosis increased in cultures treated with erucin 50 and 80 µM (Figure S1, Supporting Information). This is in agreement with the cell cycle distribution of cells treated with erucin. Moreover, this may also explain why cells become larger for several hours after treatment with 50 µM erucin (Figure 4E,F) despite remaining more circular (Figure 4C,D). Thus, suggesting that cells are arrested at G2/M phase due to the loss of microtubule function. In general, these observations in kidney cells are in accordance with a previous study carried out in MCF7 breast cancer cells^{[ 7 ]} where erucin also suppressed microtubule dynamics.

Figure 5.

Erucin disrupts the cellular microtubule network and directly impairs tubulin polymerization. A, D) The microtubule network of A–C) Vero‐E6 and D–F) 786‐O cells treated with erucin was analyzed by fluorescence microscopy. Images show representative cells treated for 4 and 8 h with erucin and stained with an anti‐tubulin antibody, phalloidin and DAPI. Scale bar 20 µm. The presence of B, E) microtubule‐organizing centers (MTOC) and C, F) microtubule (MTs) presence and morphology were assessed. B, C, E, F) Summary results show mean percentage of cells ± SD (150 cells from three independent assays). G) In vitro tubulin polymerization was measured in the presence of erucin or paclitaxel as a positive control. *, &, @ p < 0.05; **, ##, &&, @@ p < 0.01; ***, ###, @@@ p < 0.01 when comparing the percentages of present/normal (*),dubious (#), aberrant (&), and absent(@) MTs or MTOC in treated cells versus controls.

To explore the mechanisms underlying erucin interference on microtubules, an in vitro tubulin polymerization assay was performed in the presence of different concentrations of erucin. Erucin strongly reduced tubulin polymerization in a concentration‐dependent manner (Figure 5G), thus suggesting a direct effect of erucin on tubulin. Although additional effects on cellular regulatory cytoskeleton controlling mechanisms cannot be excluded, an interference in the tubulin polymerization process seems to play a major role. These results are in accordance with those obtained by Azarenko et al.^{[ 7 ]} using an equivalent methodology. Although we have observed a considerable reduction in the in vitro microtubule polymerization rate at concentrations as low as 10 µM.

In liver cells, it was suggested that the impact of erucin on cell cycle arrest may be due to increases in intracellular ROS and microtubule dynamics disturbance.^{[ 22 ]} Here, we demonstrate that two kidney cell lines exposed to erucin showed a strong G2/M arrest without (786‐O) or very limited changes (Vero‐E6) in intracellular ROS. These data strongly suggest that the observed effects of erucin on cell cycle arrest in kidney cells are mostly a direct consequence of the strong impairment of the microtubule network dynamics.

3. Concluding Remarks

The inverse association between the dietary intake of cruciferous vegetables and cancer risk is generally attributed to ITCs. These phytochemical compounds may prevent cancer initiation and progression by suppressing tumor progression‐related processes, including cell proliferation and migration.^{[ 21 ]} As far as erucin is concerned, only few studies are available and no previous reports have addressed the effects of this bioactive compound in kidney cells. Our data demonstrate that erucin directly interferes with the microtubule network of these cells, leading to alterations in cell cycle progression, morphology, motility, and adhesion. These results contribute to explore possible dietary approaches for secondary/tertiary renal cancer chemoprevention.

4. Experimental Section

Cell Culture

The Vero‐E6 green monkey kidney cells and 786‐O human renal cancer cells were obtained from ATCC (Manassas, VA, USA). Both cell lines were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Biowest, USA), 100 U mL⁻¹ penicillin, and 0.1 mg mL^‐1 streptomycin (Sigma‐Aldrich, St Louis, MO, USA). Cells were kept at 37 °C, under a humidified atmosphere containing 5% CO₂.

Erucin Treatment

Erucin ethanolic solution was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). Working solutions of erucin at 2 mM in PBS with 2% ethanol were freshly prepared before each experiment. A solution of PBS with 2% ethanol was used as vehicle. During the treatment of cell cultures, the final concentration of ethanol was 0.1% v/v in every case.

Cell Viability

The effect of erucin on cell viability was assessed by the thiazolyl blue tetrazolium bromide (MTT) assay. Briefly, 6 × 10³ 786‐O and Vero‐E6 cells were cultured in 190 µL of complete medium in 96‐well plates. Cells were grown for 24 h, and then exposed to different concentrations of erucin (0–100 µM) for 24 h. MTT reduction assay was performed as previously described by Flórido et al.^{[ 28 ]} Each condition was tested in three independent experiments, each one comprising three replicates.

The percentage of dead cells was assessed by cell permeability to PI. Cells were seeded in 12‐well plates, and 24 h later incubated with different concentrations of erucin (10, 50, and 80 µM) for 24 h. Cells were then exposed to PI (10 µg mL^‐1) for 15 min at 37 °C, 5% CO₂ without removing the initial media to avoid the loss of detached or poorly attached cells. Imaging was performed on a Zeiss Axio Observer microscope with a 20× objective. The quantification of PI‐positive cells was carried out using Fiji ImageJ software.^{[ 29 ]}

Cell Cycle

The effect of erucin on cell cycle distribution was analyzed by PI staining of fixed cells as previously described by Saraiva et al.^{[ 30 ]} Briefly, Vero‐E6 and 786‐O cells were seeded in complete medium in six‐well plates and left to sit for 24 h. Cells were then exposed to vehicle or erucin (10, 50, and 80 µM) in complete medium, for a 24 h period. Growth medium containing detached cells was collected and the remaining adherent cells were detached and collected using 8 µM EDTA in PBS. Cells were washed in PBS, fixed in cold 80% ethanol at 4 °C, for 2 h, rewashed in cold PBS, and resuspended in PBS with 1% FBS. Following an RNase‐A treatment (50 µg mL⁻¹; Sigma‐Aldrich, St Louis, MO, USA) and PI (25 µg mL⁻¹) staining, for 20 min, cell DNA content was analyzed using a FACSCalibur flow cytometer (BD). Data acquisition and analysis were performed using CellQuest software (BD) and FlowJo (Tree Star, San Carlos, CA, USA), respectively.

Intracellular ROS

The intracellular ROS levels were assessed by the dichloro‐dihydro‐fluorescein diacetate (DCFH‐DA) method, based on the ROS‐dependent oxidation of DCFH‐DA to dichlorofluorescein according to Saraiva et al.^{[ 31 ]} Vero‐E6 and 786‐O cells were seeded in a black 96‐well plate with clear flat bottom, left to sit for 24 h at 37 °C, 5% CO₂, and then exposed to vehicle or erucin (10, 50, and 80 µM). After a 22 h period, TBHP (1 mM) was added as the positive control, and the plate was left to incubate for another 2 h at 37 °C. DCFH‐DA (10 µM in PBS) was added to each well and incubated for 30 min at 37 °C. Fluorescence emission was measured (λ _exc = 485 nm and λ _em = 530 nm) using a Synergy HTX Multi‐Mode Microplate Reader (BioTek Instruments, VT, USA). Three independent experiments were performed, containing four culture replicates for each condition.

Collective Migration

The in vitro wound‐healing assay, used to evaluate collective cell migration, was performed as previously described by Costa et al.^{[ 32 ]} Briefly, Vero‐E6 and 786‐O cells were seeded in 12 well‐plates in complete medium and left to grow for 24 h. After this period, each well was scratched using a 200 µL pipette tip, leaving a vertical gap of approximately 0.8 mm width. Growth medium was removed, and cells were washed twice with PBS to remove detached cells and cell debris. Cells were kept in medium with 2% FBS containing vehicle or erucin (10, 50, and 80 µM). The distance between the two limits of the scratch was monitored using an Olympus CKX41SF inverted microscope at 0, 2, 4, 6, 8, and 12 h after treatment. At each time point, two images of each scratch were captured, using IC capture 2.5 software (Germany), and three measurements were performed per image, using Fiji ImageJ software.^{[ 29 ]}

Chemotaxis and Chemoinvasion

The chemotactic migration of 786‐O cells was evaluated in 24‐well plates with transwell inserts with transparent PET membranes containing 8 µm pores (BD Falcon, USA) according to Almeida et al.^{[ 33 ]} Briefly, cells (1 × 10⁵ cells in 200 µL of FBS‐free medium) were seeded on the insert without FBS, and complete medium with 10% FBS was placed in the lower chamber of the culture well. Erucin was added to both chambers and cultures were incubated for 24 h. Non‐migrating cells were removed from the upper side of the inserts with a cotton swab. Cells that migrated to the underside of the inserts were fixed with cold 96% ethanol and stained with 0.1% crystal violet (CV) in 10% ethanol. The cells were photographed using the 10× objective at an Olympus CKX41SF inverted microscope and the number of cells per field was counted. The chemoinvasion assay was performed as described for chemotaxis measurements, but in this case the membrane filter was overlaid with Matrigel (Corning, USA) diluted in serum‐free medium (1:30), which blocked non‐invasive cells from migrating through. Three independent experiments were performed.

Cell Adhesion

Cell adhesion was assessed as previously described by Saraiva et al.^{[ 34 ]} Vero‐E6 and 786‐O cells were seeded in six‐well plates in complete medium and left to grow for 24 h. Vehicle or erucin (10, 50, and 80 µM) were then added to each well. After a 24 h period, cells were detached using trypsin (0.05%), resuspended in complete growth medium, and re‐seeded in Matrigel‐coated (1:30) 24 well‐plates. After 3 and 12 h, cells were gently washed with PBS and the remaining adherent cells were fixed in 80% ethanol for 10 min, washed with PBS, and stained with 0.1% CV in ddH₂O for 10 min. The wells were washed with water and the dye was eluted using 1% acetic acid. Absorbance was measured at 595 nm in a Synergy HTX Multi‐Mode Microplate Reader (BioTek Instruments, VT, USA). Three replicates were tested for each condition in three independent experiments.

Morphometric Analysis

Cells were seeded at a density of 2 × 10⁵ cells per well in 500 mL culture medium in 24‐well plates and incubated for 24 h. Cells were then incubated for 8 h with erucin (0–80 µM) and images were acquired using an inverted phase contrast microscope (Olympus CKX41) coupled to a digital camera (Olympus SC20). The morphometric analysis (cell area and circularity) was performed using Fiji ImageJ software.^{[ 29 ]}

Cell Spreading

Cell spread was assessed as previously described by Saraiva et al.^{[ 34 ]} Vero‐E6 and 786‐O cells were seeded in six‐well plates in complete medium and left to sit for 24 h at 37 °C, 5% CO₂. Vehicle or erucin (10, 50, and 80 µM) were then added to each well. After a 24 h period, cells were detached using trypsin (0.05%), resuspended in complete growth medium, and re‐seeded in 24 well plates containing round glass coverslips (VWR). Cells were then fixed with 4% PFA 1, 2, 4, 6, 8, and 12 h after re‐seeding and mounted using Mowiol 4–88 (EMD Millipore) containing DAPI. A total of 750 cells were measured for each condition in three independent experiments. Images were acquired using an Olympus CKX41SF inverted microscope with a 40× objective. The morphometric analysis (cell area and circularity) was performed using Fiji ImageJ software.^{[ 29 ]}

Immunofluorescence

Immunofluorescence was performed based on the method previously described by Saraiva et al.^{[ 34 ]} Briefly, cells were fixed with 4% paraformaldehyde and stained with phalloidin conjugated to Alexa Fluor 594 (1:1000, Abcam) and with an anti‐tubulin antibody (1:100, Termofisher), followed by a secondary antibody conjugated to Alexa Fluor 488 (1:300, Invitrogen). Coverslips were mounted in Mowiol 4–88 (EMD Millipore) containing DAPI. Cells were imaged using a 40× objective on a Zeiss Axio Observer microscope. The evaluation of the presence and status of the MTOC and microtubules was performed using Fiji ImageJ software.^{[ 29 ]} The microtubules were considered: i) normal, when organized in a network branching from the MTOC and presented a size (thickness) and shape typical of the microtubule network; ii) aberrant, when the size of shape of the microtubules was altered, typically increased thickness and more tortuous shape; iii) dubious, when only shape or size were altered and/or the alteration observed was less obvious; and iv) absent when no microtubules were visible. The MTOC was considered: i) present when an agglomeration of microtubules from which the microtubule network branched off was visible; ii) absent, when this structure was not detectable; iii) and dubious when this structure was present but not clearly defined or was considerably smaller and/or disorganized.

Tubulin Polymerization

In vitro tubulin polymerization was evaluated using the commercial HTS tubulin polymerization assay biochem kit (Cytoskeleton, Inc.), according to the manufacturer's instructions. Briefly, the assay was prepared in the provided buffer, on a pre‐warmed 96 well‐plate at 37 °C. Tubulin polymerization was evaluated in the presence of different concentrations of erucin. Absorbance measurements were carried out on a Synergy HTX Multi‐Mode Microplate Reader (BioTek Instruments, VT, USA). Readings were performed at 340 nm for 60 min, with one measurement per min. Paclitaxel (10 µM) was used as control. Two independent assays were performed.

LOXL2 Inhibitory Activity

The ability of erucin to inhibit the activity of human LOXL2 (hLOXL2; SinoBiological) was evaluated by assessing the H₂O₂ release during the enzymatic reaction, using a protocol adapted from Rowbottom et al.^{[ 35 ]} Erucin and hLOXL2 (100 ng) were incubated in assay buffer (50 mM sodium borate, urea 1.2 M, CaCl₂ 10 mM, pH 8.0) for 15 min at 37 °C, in black‐wall 96‐well microplates. Cadaverine (500 µM), Amplex Ultra Red (AUR; 50 µM), and horseradish peroxidase (0.5 U mL⁻¹) were added. Fluorescence was measured using a BioTeck Synergy HTX (λ _exc = 560/15 nm and λ _em = 590/20 nm), every 2 min over a 50 min kinetics. β‐aminopropionitrile was used as positive control. The enzymatic activity was calculated using the slope of the linear portion of the curve. Two independent assays were performed.

Statistical Analysis

The normality and the homogeneity of the variances of continuous variables were assessed. The differences in mean values of the results observed in different conditions were evaluated by Student's t‐test. The Mann–Whitney U test was applied for non‐normal variables. Statistical significance was evaluated by comparing each group to the respective control group. All analyses were performed using SPSS statistical package (version 25, SPSS Inc. Chicago, IL, USA).

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

N.S. and A.S.F. both shared senior authorship. A.S.F., N.S., and N.G.O conceived the study. A.S.F. and N.S. designed the experiments. I.G., B.V., M.M., and S.F. performed the experiments. J.G.C., I.G., A.S.F. and N.S. analyzed the data. I.G., N.G.O, J.G.C., A.S.F. and N.S. wrote the paper. All authors discussed the results, interpreted the data, and reviewed the manuscript.

Supporting information

Supporting Information

Guerreiro Í., Vidovic B., Costa J. G., Martins M., Ferreira S., Oliveira N. G., Saraiva N., Fernandes A. S., The Dietary Isothiocyanate Erucin Reduces Kidney Cell Motility by Disturbing Tubulin Polymerization. Mol. Nutr. Food Res. 2023, 67, 2200581. 10.1002/mnfr.202200581

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.