Preclinical report 923
HA-1077 inhibits cell migration/invasion of oral squamous cell carcinoma Simone de Sales Costa Moreira Carbonia, Nathália Alves Rodrigues Limaa, Nanci Mendes Pinheiroa, Beatriz Martins Tavares-Murtab and Virgínia Oliveira Cremaa Oral squamous cell carcinoma (OSCC) is the most malignant lesion occurring in the head and neck. The Rho-kinases (ROCKs), effectors of Rho proteins, are involved in actin cytoskeletal organization, cell migration, and maintenance cortex. The HA-1077 inhibits the ROCKs. This study aimed to evaluate the effect of treatment with HA-1077 on cell motility in SCC-4 cells, a cell line originating from human OSCC. F-actin of SCC-4 cells treated or not with HA-1077 (1, 50 and 100 μmol/l), and also HA-1077 50 μmol/l and/or inhibitors Y-27632 30 μmol/l was stained with rhodamine-conjugated phalloidin and analyzed by confocal microscopy. Approximately 1 × 105 cells/well, control and treated with HA-1077 (25, 50, and 100 μmol/l) were added to the migration plate assay. In addition, 1 × 105 cells/well, control and treated with HA-1077 50 μmol/l, were tested by invasion assays (plate coated with Matrigel). The inhibition of ROCKs with HA-1077 and/or Y-27632 leads to morphological changes, affecting the organization of the actin. The inhibitory effect of HA-1077 (P < 0.0001) was dose dependent as the number of cells
Introduction Oral squamous cell carcinoma (OSCC) is an epithelial neoplasm responsible for most malignant lesions occurring in the head and neck [1,2]. It represents about 3% of all malignancies of the body [3]. It is a tumor that leads to invasion and metastasis. The degrees of invasion and metastasis adversely affect the prognosis and treatment of patients. The complications associated with metastasis are the main causes of mortality. Therefore, there is a need to identify molecular pathways that are essential for the invasion and metastasis of cancer cells and to thus develop pharmacologic compounds that can block these pathways effectively [4]. The Rho-kinases (ROCKs), serine threonine protein kinase family belonging to the AGC (kinase linked to G protein), play an important role in many cellular functions through the phosphorylation of a specific substrate [5]. Some in-vitro studies and animal experiments suggest that the interruption pathway Rho/ROCKs affects invasion and metastasis [6]. The ROCKs are the effectors of RhoA, RhoB, and RhoC proteins, which are best characterized; their activation occurs through an activated form of Rho and these 0959-4973 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
migrated at 100 μmol/l was statistically different: 25 μmol/l (P < 0.0001) and 50 μmol/l (P < 0.01). The number of cells treated with HA-1077 50 μmol/l decreased compared with control cells that invaded through Matrigel (P < 0.0001). This study shows an inhibitory effect of HA-1077 on cell migration and invasion, suggesting that the use of HA-1077 can be a potential therapy for OSCC. Anti-Cancer Drugs 26:923–930 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Anti-Cancer Drugs 2015, 26:923–930 Keywords: cell invasion, cell migration, HA-1077, oral squamous cell carcinoma, Rho GTPases, ROCKs, SCC-4 Departments of aStructural Biology and bPharmacology, Institute of Natural and Biological Sciences, Federal University of Triângulo Mineiro, Uberaba, MG, Brazil Correspondence to Virgínia Oliveira Crema, Department of Structural Biology, Institute of Natural and Biological Sciences, Federal University of Triângulo Mineiro, Praça Manoel Terra 330, Uberaba, MG, CEP 38025-050, Brazil Tel: + 55 34 3318 5463; fax: + 55 34 3318 5462; e-mail: [email protected] Received 19 May 2015 Revised form accepted 19 June 2015
proteins play a role in the organization of the actin cytoskeleton, formation of stress fibers, and focal adhesion [7], and are associated with the pathogenesis and progression of human tumors [8]. Proteins belonging to the family of Rho GTPases may be divided into six subfamilies, which comprise 23 members [9,10]. The members of the Rho GTPases family play different regulatory roles affecting the cytoskeletal architecture of actin; thus, their participation in various aspects of cancer progression and tumorigenesis has become increasingly evident [11]. Rho GTPases are overexpressed in several types of human tumors and contribute toward local proliferation and metastasis [10,12]. HA-1077 and Y-27632 have been used as selective inhibitors of ROCKs competing with ATP [5]. The HA-1077 [1-(5-isoquinolinylsulfonyl) homopiperazine dihydrochloride, fasudil dihydrochloride] is an inhibitor of ROCKs that has been well studied. As the HA-1077 acts in inhibiting ROCKs, some studies have reported their antitumor activity [10,13,14]. In MDA-MB-231 (human breast carcinoma) and HT1080 (human fibrosarcoma) cells, fasudil and fasudil-OH inhibit cell migration [4]. Y-27632 has been used widely as an inhibitor of DOI: 10.1097/CAD.0000000000000267
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924 Anti-Cancer Drugs 2015, Vol 26 No 9
ROCKs to explore a variety of roles of ROCKs in pathophysiological processes [15], including cancer [4,16]. This study aimed to test the effect of treatment with HA-1077 on cell migration and invasion of OSCC using the SCC-4 cell line.
did not migrate from the upper chamber were removed with a cotton swab and the cells that migrated through the membrane and the cells that invaded the Matrigel-coated membrane were fixed with methanol for 30 s and stained with Instant Prov as recommended by the manufacturer (Newprov, Pinhais, Paraná, Brazil).
Materials and methods Cell culture
Morphometry
Cell lines of human oral carcinoma SCC-4 were obtained from the American Type Culture Collection. These cells were cultured in Dulbecco’s modified Eagle medium 1 : 1 HAM F12 medium, containing 10% fetal bovine serum, 400 ng/ml hydrocortisone, 100 U/ml penicillin, and 100 μg/ml streptomycin, in an incubator at 37°C in a humidified 5% CO2/95% air atmosphere.
The cells that migrated through the membrane and the cells that invaded the membrane coated with Matrigel were counted in the whole membrane area using a × 20 objective (Axio Vert.A1; Zeiss).
Evaluation of actin cytoskeleton
For morphological analysis, control and treated SCC-4 cells (1 × 105/well) were cultured in six-well plates containing glass coverslips. Cells were treated with HA-1077 (Sigma-Aldrich, St Louis, Missouri, USA) at concentrations of 1, 50, and 100 μmol/l for 6 h, and also HA-1077 50 μmol/l with or without inhibitors Y-27632 30 μmol/l (Calbiochem, San Diego, California, USA) and Toxina A 2 μg/ml (List Biological Labs, Campbell, California, USA), 30 μml/l Y-27632, and Toxin A 2 μg/ml (a specific inhibitor of Rho GTPases) for 24 h (the same period of time used for migration and invasion assays). After incubation, cells were fixed with 4% paraformaldehyde for 1 h and incubated with 0.2% Triton X-100 for 5 min, 3% serum albumin bovine for 20 min, rhodamine-conjugated phalloidin (Molecular Probes, Eugene, Oregon, USA) 1 : 100 for 30 min, and 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) 1 : 100 for 15 min. The slides were mounted with Vectashield (Vector Laboratories, Burlingame, California, USA). Images from the control cells and cells treated with inhibitors were obtained randomly using a laser scanning confocal microscope (Zeiss, Goettingen, Germany). Migration and invasion assays
For the migration assay, control SCC-4 cells and cells treated with HA-1077 (1 × 105 cells/well) were placed in the top chamber plate BioCoat BD 24-well plate, 8.0 μm (BD Bioscience, Bedford, Massachusetts, USA). Three concentrations of HA-1077, 25, 50, and 100 μmol/l, were tested in triplicate. For the invasion assay, control SCC-4 cells and cells treated with HA-1077 50 μmol/l (1 × 105 cells/well) were placed in the top chamber plate BD Matrigel Invasion Chamber 24-well plate, 8.0 μm (BD Bioscience), in triplicate. Medium containing 20% FSB was placed in the lower chamber. Cells were maintained in a humidified atmosphere incubator with 5% CO2/95% air at 37°C for 24 h. Cells that
Statistical analysis
The results were analyzed using SPSS software 16.0 (SPSS Inc., Chicago, Illinois, USA) and graphics were performed using GraphPad Prism (GraphPad, San Diego, California, USA). The variables were analyzed using the homogeneity test of Levene variances. The results of cell migration and cell invasion assays were analyzed using ANOVA/Tukey’s post-test and the t-test, respectively. Differences were considered significant when P less than 0.05.
Results Analysis of the actin cytoskeleton
The morphology of the SCC-4 control cells was a welldeveloped polyhedral, cytoplasm, actin cytoskeleton and an evident cell cortex could be observed (Figs 1a, 2a and 3a). Cells treated with HA-1077 (1, 50, and 100 μmol/l) for 6 h showed morphological changes in cytoskeletal organization and in a concentration-dependent manner. The higher the concentration of HA-1077, the more obvious the changes observed: rounded morphology, underdeveloped cytoplasm, reduced polymerized actin, and cell cortex not evident (Fig. 1). The effect of HA-1077 treatment at 50 μmol/l for 6 h was pronounced, and an altered cell morphology was detected: irregular contours cell, cytoplasm showed a slight decrease in polymerized actin, and cell cortex not evident (Figs 2b and 3b). Y-27632 30 μmol/l treatment led to a marked reduction of F-actin (Fig. 2c). Cells treated with the association of HA-1077 50 μmol/l and Y-27632 30 μmol/l presented irregular cell contours and cell cortex was not evident (Fig. 2d). The morphology of SCC-4 cells treated with Toxin A 2 μg/ml was altered (Fig. 3c). HA-1077 50 μmol/l associated with Toxin A 2 μg/ml cells had a rounded morphology and cytoplasmic extensions, and a disorganized actin cytoskeleton (Fig. 3d). Effect of HA-1077 on cell migration and invasion
To evaluate the effect of HA-1077 on cell migration, SCC-4 cells were treated with three concentrations, 25, 50, and 100 μmol/l, that inhibited the number of SCC-4
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HA-1077 inhibits cell migration/invasion Carboni et al. 925
Fig. 1
(a)
(b)
(c)
(d)
50 μm
Effect of HA-1077 on actin cytoskeleton of the SCC-4 cell line. Confocal analysis of the actin cytoskeleton in a single section in the central region of the cells. F-actin stained with phalloidin conjugated to rhodamine. Control cells (a); cells treated with HA-1077: 1 μmol/l (b), 50 μmol/l (c), and 100 μmol/l (d) for 6 h.
cells that migrated [F(3,11) = 85.020, P < 0.0001] at all concentrations. The number of migratory cells was significantly reduced after treatment with HA-1077: 25 μmol/l (519.7 ± 14.3 cells), 50 μmol/l (484.7 ± 4.7 cells), and 100 μmol/l (416.3 ± 9.4 cells) compared with SCC-4 cells’ control (625.7 ± 27.6 cells). The inhibitory effect of HA-1077 was dose dependent as the number of SCC-4 cells that migrated at 100 μmol/l was statistically different
from that at 25 μmol/l (P < 0.0001) and 50 μmol/l (P < 0.01). Despite the difference between 25 and 50 μmol/l, this was not statistically (P > 0.05) (Fig. 4a). The analysis of the involvement of treatment with HA-1077 on cell invasion showed that the number of SCC-4 cells treated with 50 μmol/l HA-1077 (305.3 ± 80.23 cells) was reduced compared with the number of
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926 Anti-Cancer Drugs 2015, Vol 26 No 9
Fig. 2
(a)
(b)
50 μm 50 μm (c)
(d)
50 μm
50 μm
Effect of Y-27632 associated or not with HA-1077 on actin cytoskeleton organization in SCC-4 cells. Three-dimensional confocal analysis. F-actin stained with phalloidin conjugated to rhodamine and nuclei stained with DAPI. (a) control cells, (b) cells treated with HA-1077 50 μmol/l, (c) cells treated with Y-27632 30 μmol/l, (d) cells treated with HA-1077 50 μmol/l and Y-27632 30 μmol/l for 24 h.
SCC-4 control (492.7 ± 33.2 cells) cells that invaded through Matrigel [t(5) = 8.398, P < 0.0001] (Fig. 4b).
Discussion In this study, we used the cell line SCC4, the cell line originating from human tongue squamous cell carcinoma. Cell lines are used widely in research as a model for the study of molecular mechanisms and the identification of
diagnostic and prognostic markers for disorders, particularly in cancer studies [17]. This study shows an inhibitory effect of HA-1077 on cell migration and invasion, suggesting that the use of HA-1077 can be a potential therapy for OSCC to prevent metastasis; the Rho/ROCKs transduction pathway probably plays an important role in this process as HA-1077 and Toxin A treatments affect F-actin in the cells.
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HA-1077 inhibits cell migration/invasion Carboni et al. 927
Fig. 3
(b)
(a)
50 μm (c)
50 μm (d)
50 μm
50 μm
Effect of HA-1077 on actin cytoskeleton organization SCC-4 cells. Three-dimensional confocal analysis. F-actin stained with phalloidin conjugated to rhodamine and nuclei stained with DAPI. (a) Control cells, (b) cells treated with HA-1077 50 μmol/l, (c) cells treated with Toxin A 2 μg/ml, (d) cells treated with HA-1077 50 μmol/l and Toxin A 2 μg/ml for 24 h.
The acquisition of an invasive phenotype and migration capacity are important steps in the development and metastasis of tumors [18]. The Rho/ROCKs pathways are related to the control of cell motility [8]. In this study, migration assays were performed on cell lines SCC4 treated with HA-1077, which is a known inhibitor of ROCKs proteins, and inhibits cell migration and invasion
of cells through the membrane coated with the Matrigel, which mimics basal membrane. ROCKs participate in cytoskeletal organization of actin, cell migration, and cell cortex maintenance [19]. We showed that the SCC-4 cells treated with HA-1077 and/or Y-27632 undergo changes in their morphology because of
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928 Anti-Cancer Drugs 2015, Vol 26 No 9
inhibition of proliferation and expression of α smooth muscle actin; changes in the production of collagen type I were described [22]. MC57 BRAK, fibrosarcoma cell line, injected into mice treated with fasudil suppressed the growth of fibrosarcoma in vivo [25].
Fig. 4
Number of migrating cells
(a) 800
∗∗∗
∗∗∗ ∗∗∗ ∗
600 400 200 0 HA25
C (b) Number of invading cells
∗∗∗
HA50
H100
∗∗
600
400
In head and neck SCC, microRNA-138 overexpression has a negative correlation with downregulation of RhoC and reduces cell motility [26]. In tongue SCC, knockdown of micro RNA-138 causes RhoC and ROCK II overexpression, leading to an elongated morphology, increased stress fibers, and consequently increased cellular migration and invasion [27]. Fasudil inhibits proliferation in a dose-dependent manner, and induces apoptosis in HepG2 and Huh7 (cell lines of hepatocellular carcinoma) [14]. In the extracellular matrix of endometriotic stromal cells, HA-1077 inhibits the expression and proliferation of ROCK1/ROCK2, and induces apoptosis and contractility [28].
200
0 C
HA50
Effect of HA-1077 on SCC-4 cell migration and cell invasion. (a) Effect of HA-1077 on the migration: SCC-4 control cells (C) and SCC-4 cells treated with HA-1077: 25, 50, and 100 μmol/l. (b) Effect of HA-1077 on the cell invasion: control cells (c) and SCC-4 cells treated with 50 μmol/l. *P < 0.05, **P < 0.001, ***P < 0.0001.
reorganization of the actin cytoskeleton. According to some studies, it was shown that fasudil induces morphological changes in the cell lines MDA-MB-231 breast cancer cells, lung cancer HT1080 [4], and U251 and T98G glioblastoma cells [20]. In addition, Y-27632 causes morphological changes in PC3 prostate cancer cells [21] and pancreatic stellar cells [22]. Rho GTPases are overexpressed in several types of human tumors, and contribute toward local proliferation and metastasis [10,12]. RhoA, RhoB, and RhoC GTPases have different cellular functions; RhoA and RhoC are overexpressed in tumors and are involved in carcinogenesis and tumor progression [8]. Rather, RhoB is often deleted in human tumors and its expression is correlated inversely with tumor aggressiveness [23]. In human glaucomatous trabecular meshwork cells (GTM3), inhibition of ROCKs by Y-27632 and Y-39983 treatments decreases the formation of stress fibers [24]. In agreement with our studies, a loss of stress fibers, disorganization, and contraction of the actin cytoskeleton were observed. In a culture of Wistar rat pancreatic cells, RhoA activity, ROCK-1 and ROCK-2, was evaluated using the inhibitor Y-27632 and change in cell morphology and stress fiber formation was evaluated using the fasudil and Y-27632,
In human esophageal SCC cell lines, Y-27632 has led to morphological changes and reduction in cell motility [29]. In our study, we showed that inhibition of ROCKs led to morphological changes and inhibition of cell migration/ invasion. Rho, Rac, and Cdc42 can interact with different proteins in a GTP-dependent manner, indicating that each regulates numerous distinct signal transduction pathways [10]. In our study, it was found that ROCKs play an important role in the signaling pathway regulating cell migration because we observed that there was a decrease in cell migration and invasion. RhoA and RhoC GTPases promote proliferation and cell motility [27,30]. The overexpression of RhoA, RhoC, and ROCK is associated with more aggressive bladder cancer [31]. The invasion of cancer cells into the surrounding tissues is a key event in the formation of metastasis and migration depends on the capacity of these cells. The mesenchymal migration is facilitated by proteases to degrade the ECM around. The ameboid migration depends on the formation of protrusions and cell contraction. Both movements require cell polarization to direct migration [8]. RhoA and RhoC promote cell migration and invasion, and their expression in cancer cells increases [8]. RhoA is overexpressed and RhoA activity is increased in hepatocarcinoma compared with nontumor tissue [32,33]. RhoC is crucial for metastatic melanoma cells [34]. The RhoA and RhoC proteins showed increased levels in the esophageal squamous cell carcinoma line. RhoA promoted greater tumor growth, whereas RhoC induced distant metastases. Ki-67 levels, FAK, MMP-1, and MMP-9 were also assessed and correlated with increased cell proliferation and migration [30]. The RhoC protein is overexpressed in the head and neck SCC cell line
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HA-1077 inhibits cell migration/invasion Carboni et al. 929
compared with normal epithelium, and it is correlated with lymph node metastasis [35]. RhoB action on cancer cells promotes inhibition of signal transduction pathways involved in oncogenesis, survival, and the induction of apoptosis [23]. RhoB immunoexpression is detected in normal epithelium, in welldifferentiated, and in situ head and neck SCC; however, it becomes weakly detectable in invasive carcinomas and poorly differentiated head and neck SCC [36], and lung cancer [37]. Proliferation, cell migration, and invasion were inhibited by siRNAs RhoA in the tongue SCC line (SCC-4, and CAL-27) [38]. Persistent Rac1 activated by epidermal growth factor receptor/Vav2 signaling is important for cell motility [39]. Although this study did not identify which proteins were affected by the HA-1077 treatment, it shows an inhibitory effect of HA-1077 on cell migration and invasion, suggesting that HA-1077 can be a potential therapy for OSCC. Further studies should evaluate the effect of HA-1077 on other biological processes that occur in carcinogenesis.
Acknowledgements The authors are grateful for the financial support of Fundação de Amparo à Pesquisa do Estado de Minas Gerais – FAPEMIG.
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Conflicts of interest
There are no conflicts of interest. 27
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HA-1077 inhibits cell migration/invasion of oral squamous cell carcinoma Simone de Sales Costa Moreira Carbonia, Nathália Alves Rodrigues Limaa, Nanci Mendes Pinheiroa, Beatriz Martins Tavares-Murtab and Virgínia Oliveira Cremaa Oral squamous cell carcinoma (OSCC) is the most malignant lesion occurring in the head and neck. The Rho-kinases (ROCKs), effectors of Rho proteins, are involved in actin cytoskeletal organization, cell migration, and maintenance cortex. The HA-1077 inhibits the ROCKs. This study aimed to evaluate the effect of treatment with HA-1077 on cell motility in SCC-4 cells, a cell line originating from human OSCC. F-actin of SCC-4 cells treated or not with HA-1077 (1, 50 and 100 μmol/l), and also HA-1077 50 μmol/l and/or inhibitors Y-27632 30 μmol/l was stained with rhodamine-conjugated phalloidin and analyzed by confocal microscopy. Approximately 1 × 105 cells/well, control and treated with HA-1077 (25, 50, and 100 μmol/l) were added to the migration plate assay. In addition, 1 × 105 cells/well, control and treated with HA-1077 50 μmol/l, were tested by invasion assays (plate coated with Matrigel). The inhibition of ROCKs with HA-1077 and/or Y-27632 leads to morphological changes, affecting the organization of the actin. The inhibitory effect of HA-1077 (P < 0.0001) was dose dependent as the number of cells
Introduction Oral squamous cell carcinoma (OSCC) is an epithelial neoplasm responsible for most malignant lesions occurring in the head and neck [1,2]. It represents about 3% of all malignancies of the body [3]. It is a tumor that leads to invasion and metastasis. The degrees of invasion and metastasis adversely affect the prognosis and treatment of patients. The complications associated with metastasis are the main causes of mortality. Therefore, there is a need to identify molecular pathways that are essential for the invasion and metastasis of cancer cells and to thus develop pharmacologic compounds that can block these pathways effectively [4]. The Rho-kinases (ROCKs), serine threonine protein kinase family belonging to the AGC (kinase linked to G protein), play an important role in many cellular functions through the phosphorylation of a specific substrate [5]. Some in-vitro studies and animal experiments suggest that the interruption pathway Rho/ROCKs affects invasion and metastasis [6]. The ROCKs are the effectors of RhoA, RhoB, and RhoC proteins, which are best characterized; their activation occurs through an activated form of Rho and these 0959-4973 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
migrated at 100 μmol/l was statistically different: 25 μmol/l (P < 0.0001) and 50 μmol/l (P < 0.01). The number of cells treated with HA-1077 50 μmol/l decreased compared with control cells that invaded through Matrigel (P < 0.0001). This study shows an inhibitory effect of HA-1077 on cell migration and invasion, suggesting that the use of HA-1077 can be a potential therapy for OSCC. Anti-Cancer Drugs 26:923–930 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. Anti-Cancer Drugs 2015, 26:923–930 Keywords: cell invasion, cell migration, HA-1077, oral squamous cell carcinoma, Rho GTPases, ROCKs, SCC-4 Departments of aStructural Biology and bPharmacology, Institute of Natural and Biological Sciences, Federal University of Triângulo Mineiro, Uberaba, MG, Brazil Correspondence to Virgínia Oliveira Crema, Department of Structural Biology, Institute of Natural and Biological Sciences, Federal University of Triângulo Mineiro, Praça Manoel Terra 330, Uberaba, MG, CEP 38025-050, Brazil Tel: + 55 34 3318 5463; fax: + 55 34 3318 5462; e-mail: [email protected] Received 19 May 2015 Revised form accepted 19 June 2015
proteins play a role in the organization of the actin cytoskeleton, formation of stress fibers, and focal adhesion [7], and are associated with the pathogenesis and progression of human tumors [8]. Proteins belonging to the family of Rho GTPases may be divided into six subfamilies, which comprise 23 members [9,10]. The members of the Rho GTPases family play different regulatory roles affecting the cytoskeletal architecture of actin; thus, their participation in various aspects of cancer progression and tumorigenesis has become increasingly evident [11]. Rho GTPases are overexpressed in several types of human tumors and contribute toward local proliferation and metastasis [10,12]. HA-1077 and Y-27632 have been used as selective inhibitors of ROCKs competing with ATP [5]. The HA-1077 [1-(5-isoquinolinylsulfonyl) homopiperazine dihydrochloride, fasudil dihydrochloride] is an inhibitor of ROCKs that has been well studied. As the HA-1077 acts in inhibiting ROCKs, some studies have reported their antitumor activity [10,13,14]. In MDA-MB-231 (human breast carcinoma) and HT1080 (human fibrosarcoma) cells, fasudil and fasudil-OH inhibit cell migration [4]. Y-27632 has been used widely as an inhibitor of DOI: 10.1097/CAD.0000000000000267
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924 Anti-Cancer Drugs 2015, Vol 26 No 9
ROCKs to explore a variety of roles of ROCKs in pathophysiological processes [15], including cancer [4,16]. This study aimed to test the effect of treatment with HA-1077 on cell migration and invasion of OSCC using the SCC-4 cell line.
did not migrate from the upper chamber were removed with a cotton swab and the cells that migrated through the membrane and the cells that invaded the Matrigel-coated membrane were fixed with methanol for 30 s and stained with Instant Prov as recommended by the manufacturer (Newprov, Pinhais, Paraná, Brazil).
Materials and methods Cell culture
Morphometry
Cell lines of human oral carcinoma SCC-4 were obtained from the American Type Culture Collection. These cells were cultured in Dulbecco’s modified Eagle medium 1 : 1 HAM F12 medium, containing 10% fetal bovine serum, 400 ng/ml hydrocortisone, 100 U/ml penicillin, and 100 μg/ml streptomycin, in an incubator at 37°C in a humidified 5% CO2/95% air atmosphere.
The cells that migrated through the membrane and the cells that invaded the membrane coated with Matrigel were counted in the whole membrane area using a × 20 objective (Axio Vert.A1; Zeiss).
Evaluation of actin cytoskeleton
For morphological analysis, control and treated SCC-4 cells (1 × 105/well) were cultured in six-well plates containing glass coverslips. Cells were treated with HA-1077 (Sigma-Aldrich, St Louis, Missouri, USA) at concentrations of 1, 50, and 100 μmol/l for 6 h, and also HA-1077 50 μmol/l with or without inhibitors Y-27632 30 μmol/l (Calbiochem, San Diego, California, USA) and Toxina A 2 μg/ml (List Biological Labs, Campbell, California, USA), 30 μml/l Y-27632, and Toxin A 2 μg/ml (a specific inhibitor of Rho GTPases) for 24 h (the same period of time used for migration and invasion assays). After incubation, cells were fixed with 4% paraformaldehyde for 1 h and incubated with 0.2% Triton X-100 for 5 min, 3% serum albumin bovine for 20 min, rhodamine-conjugated phalloidin (Molecular Probes, Eugene, Oregon, USA) 1 : 100 for 30 min, and 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) 1 : 100 for 15 min. The slides were mounted with Vectashield (Vector Laboratories, Burlingame, California, USA). Images from the control cells and cells treated with inhibitors were obtained randomly using a laser scanning confocal microscope (Zeiss, Goettingen, Germany). Migration and invasion assays
For the migration assay, control SCC-4 cells and cells treated with HA-1077 (1 × 105 cells/well) were placed in the top chamber plate BioCoat BD 24-well plate, 8.0 μm (BD Bioscience, Bedford, Massachusetts, USA). Three concentrations of HA-1077, 25, 50, and 100 μmol/l, were tested in triplicate. For the invasion assay, control SCC-4 cells and cells treated with HA-1077 50 μmol/l (1 × 105 cells/well) were placed in the top chamber plate BD Matrigel Invasion Chamber 24-well plate, 8.0 μm (BD Bioscience), in triplicate. Medium containing 20% FSB was placed in the lower chamber. Cells were maintained in a humidified atmosphere incubator with 5% CO2/95% air at 37°C for 24 h. Cells that
Statistical analysis
The results were analyzed using SPSS software 16.0 (SPSS Inc., Chicago, Illinois, USA) and graphics were performed using GraphPad Prism (GraphPad, San Diego, California, USA). The variables were analyzed using the homogeneity test of Levene variances. The results of cell migration and cell invasion assays were analyzed using ANOVA/Tukey’s post-test and the t-test, respectively. Differences were considered significant when P less than 0.05.
Results Analysis of the actin cytoskeleton
The morphology of the SCC-4 control cells was a welldeveloped polyhedral, cytoplasm, actin cytoskeleton and an evident cell cortex could be observed (Figs 1a, 2a and 3a). Cells treated with HA-1077 (1, 50, and 100 μmol/l) for 6 h showed morphological changes in cytoskeletal organization and in a concentration-dependent manner. The higher the concentration of HA-1077, the more obvious the changes observed: rounded morphology, underdeveloped cytoplasm, reduced polymerized actin, and cell cortex not evident (Fig. 1). The effect of HA-1077 treatment at 50 μmol/l for 6 h was pronounced, and an altered cell morphology was detected: irregular contours cell, cytoplasm showed a slight decrease in polymerized actin, and cell cortex not evident (Figs 2b and 3b). Y-27632 30 μmol/l treatment led to a marked reduction of F-actin (Fig. 2c). Cells treated with the association of HA-1077 50 μmol/l and Y-27632 30 μmol/l presented irregular cell contours and cell cortex was not evident (Fig. 2d). The morphology of SCC-4 cells treated with Toxin A 2 μg/ml was altered (Fig. 3c). HA-1077 50 μmol/l associated with Toxin A 2 μg/ml cells had a rounded morphology and cytoplasmic extensions, and a disorganized actin cytoskeleton (Fig. 3d). Effect of HA-1077 on cell migration and invasion
To evaluate the effect of HA-1077 on cell migration, SCC-4 cells were treated with three concentrations, 25, 50, and 100 μmol/l, that inhibited the number of SCC-4
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HA-1077 inhibits cell migration/invasion Carboni et al. 925
Fig. 1
(a)
(b)
(c)
(d)
50 μm
Effect of HA-1077 on actin cytoskeleton of the SCC-4 cell line. Confocal analysis of the actin cytoskeleton in a single section in the central region of the cells. F-actin stained with phalloidin conjugated to rhodamine. Control cells (a); cells treated with HA-1077: 1 μmol/l (b), 50 μmol/l (c), and 100 μmol/l (d) for 6 h.
cells that migrated [F(3,11) = 85.020, P < 0.0001] at all concentrations. The number of migratory cells was significantly reduced after treatment with HA-1077: 25 μmol/l (519.7 ± 14.3 cells), 50 μmol/l (484.7 ± 4.7 cells), and 100 μmol/l (416.3 ± 9.4 cells) compared with SCC-4 cells’ control (625.7 ± 27.6 cells). The inhibitory effect of HA-1077 was dose dependent as the number of SCC-4 cells that migrated at 100 μmol/l was statistically different
from that at 25 μmol/l (P < 0.0001) and 50 μmol/l (P < 0.01). Despite the difference between 25 and 50 μmol/l, this was not statistically (P > 0.05) (Fig. 4a). The analysis of the involvement of treatment with HA-1077 on cell invasion showed that the number of SCC-4 cells treated with 50 μmol/l HA-1077 (305.3 ± 80.23 cells) was reduced compared with the number of
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926 Anti-Cancer Drugs 2015, Vol 26 No 9
Fig. 2
(a)
(b)
50 μm 50 μm (c)
(d)
50 μm
50 μm
Effect of Y-27632 associated or not with HA-1077 on actin cytoskeleton organization in SCC-4 cells. Three-dimensional confocal analysis. F-actin stained with phalloidin conjugated to rhodamine and nuclei stained with DAPI. (a) control cells, (b) cells treated with HA-1077 50 μmol/l, (c) cells treated with Y-27632 30 μmol/l, (d) cells treated with HA-1077 50 μmol/l and Y-27632 30 μmol/l for 24 h.
SCC-4 control (492.7 ± 33.2 cells) cells that invaded through Matrigel [t(5) = 8.398, P < 0.0001] (Fig. 4b).
Discussion In this study, we used the cell line SCC4, the cell line originating from human tongue squamous cell carcinoma. Cell lines are used widely in research as a model for the study of molecular mechanisms and the identification of
diagnostic and prognostic markers for disorders, particularly in cancer studies [17]. This study shows an inhibitory effect of HA-1077 on cell migration and invasion, suggesting that the use of HA-1077 can be a potential therapy for OSCC to prevent metastasis; the Rho/ROCKs transduction pathway probably plays an important role in this process as HA-1077 and Toxin A treatments affect F-actin in the cells.
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HA-1077 inhibits cell migration/invasion Carboni et al. 927
Fig. 3
(b)
(a)
50 μm (c)
50 μm (d)
50 μm
50 μm
Effect of HA-1077 on actin cytoskeleton organization SCC-4 cells. Three-dimensional confocal analysis. F-actin stained with phalloidin conjugated to rhodamine and nuclei stained with DAPI. (a) Control cells, (b) cells treated with HA-1077 50 μmol/l, (c) cells treated with Toxin A 2 μg/ml, (d) cells treated with HA-1077 50 μmol/l and Toxin A 2 μg/ml for 24 h.
The acquisition of an invasive phenotype and migration capacity are important steps in the development and metastasis of tumors [18]. The Rho/ROCKs pathways are related to the control of cell motility [8]. In this study, migration assays were performed on cell lines SCC4 treated with HA-1077, which is a known inhibitor of ROCKs proteins, and inhibits cell migration and invasion
of cells through the membrane coated with the Matrigel, which mimics basal membrane. ROCKs participate in cytoskeletal organization of actin, cell migration, and cell cortex maintenance [19]. We showed that the SCC-4 cells treated with HA-1077 and/or Y-27632 undergo changes in their morphology because of
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928 Anti-Cancer Drugs 2015, Vol 26 No 9
inhibition of proliferation and expression of α smooth muscle actin; changes in the production of collagen type I were described [22]. MC57 BRAK, fibrosarcoma cell line, injected into mice treated with fasudil suppressed the growth of fibrosarcoma in vivo [25].
Fig. 4
Number of migrating cells
(a) 800
∗∗∗
∗∗∗ ∗∗∗ ∗
600 400 200 0 HA25
C (b) Number of invading cells
∗∗∗
HA50
H100
∗∗
600
400
In head and neck SCC, microRNA-138 overexpression has a negative correlation with downregulation of RhoC and reduces cell motility [26]. In tongue SCC, knockdown of micro RNA-138 causes RhoC and ROCK II overexpression, leading to an elongated morphology, increased stress fibers, and consequently increased cellular migration and invasion [27]. Fasudil inhibits proliferation in a dose-dependent manner, and induces apoptosis in HepG2 and Huh7 (cell lines of hepatocellular carcinoma) [14]. In the extracellular matrix of endometriotic stromal cells, HA-1077 inhibits the expression and proliferation of ROCK1/ROCK2, and induces apoptosis and contractility [28].
200
0 C
HA50
Effect of HA-1077 on SCC-4 cell migration and cell invasion. (a) Effect of HA-1077 on the migration: SCC-4 control cells (C) and SCC-4 cells treated with HA-1077: 25, 50, and 100 μmol/l. (b) Effect of HA-1077 on the cell invasion: control cells (c) and SCC-4 cells treated with 50 μmol/l. *P < 0.05, **P < 0.001, ***P < 0.0001.
reorganization of the actin cytoskeleton. According to some studies, it was shown that fasudil induces morphological changes in the cell lines MDA-MB-231 breast cancer cells, lung cancer HT1080 [4], and U251 and T98G glioblastoma cells [20]. In addition, Y-27632 causes morphological changes in PC3 prostate cancer cells [21] and pancreatic stellar cells [22]. Rho GTPases are overexpressed in several types of human tumors, and contribute toward local proliferation and metastasis [10,12]. RhoA, RhoB, and RhoC GTPases have different cellular functions; RhoA and RhoC are overexpressed in tumors and are involved in carcinogenesis and tumor progression [8]. Rather, RhoB is often deleted in human tumors and its expression is correlated inversely with tumor aggressiveness [23]. In human glaucomatous trabecular meshwork cells (GTM3), inhibition of ROCKs by Y-27632 and Y-39983 treatments decreases the formation of stress fibers [24]. In agreement with our studies, a loss of stress fibers, disorganization, and contraction of the actin cytoskeleton were observed. In a culture of Wistar rat pancreatic cells, RhoA activity, ROCK-1 and ROCK-2, was evaluated using the inhibitor Y-27632 and change in cell morphology and stress fiber formation was evaluated using the fasudil and Y-27632,
In human esophageal SCC cell lines, Y-27632 has led to morphological changes and reduction in cell motility [29]. In our study, we showed that inhibition of ROCKs led to morphological changes and inhibition of cell migration/ invasion. Rho, Rac, and Cdc42 can interact with different proteins in a GTP-dependent manner, indicating that each regulates numerous distinct signal transduction pathways [10]. In our study, it was found that ROCKs play an important role in the signaling pathway regulating cell migration because we observed that there was a decrease in cell migration and invasion. RhoA and RhoC GTPases promote proliferation and cell motility [27,30]. The overexpression of RhoA, RhoC, and ROCK is associated with more aggressive bladder cancer [31]. The invasion of cancer cells into the surrounding tissues is a key event in the formation of metastasis and migration depends on the capacity of these cells. The mesenchymal migration is facilitated by proteases to degrade the ECM around. The ameboid migration depends on the formation of protrusions and cell contraction. Both movements require cell polarization to direct migration [8]. RhoA and RhoC promote cell migration and invasion, and their expression in cancer cells increases [8]. RhoA is overexpressed and RhoA activity is increased in hepatocarcinoma compared with nontumor tissue [32,33]. RhoC is crucial for metastatic melanoma cells [34]. The RhoA and RhoC proteins showed increased levels in the esophageal squamous cell carcinoma line. RhoA promoted greater tumor growth, whereas RhoC induced distant metastases. Ki-67 levels, FAK, MMP-1, and MMP-9 were also assessed and correlated with increased cell proliferation and migration [30]. The RhoC protein is overexpressed in the head and neck SCC cell line
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HA-1077 inhibits cell migration/invasion Carboni et al. 929
compared with normal epithelium, and it is correlated with lymph node metastasis [35]. RhoB action on cancer cells promotes inhibition of signal transduction pathways involved in oncogenesis, survival, and the induction of apoptosis [23]. RhoB immunoexpression is detected in normal epithelium, in welldifferentiated, and in situ head and neck SCC; however, it becomes weakly detectable in invasive carcinomas and poorly differentiated head and neck SCC [36], and lung cancer [37]. Proliferation, cell migration, and invasion were inhibited by siRNAs RhoA in the tongue SCC line (SCC-4, and CAL-27) [38]. Persistent Rac1 activated by epidermal growth factor receptor/Vav2 signaling is important for cell motility [39]. Although this study did not identify which proteins were affected by the HA-1077 treatment, it shows an inhibitory effect of HA-1077 on cell migration and invasion, suggesting that HA-1077 can be a potential therapy for OSCC. Further studies should evaluate the effect of HA-1077 on other biological processes that occur in carcinogenesis.
Acknowledgements The authors are grateful for the financial support of Fundação de Amparo à Pesquisa do Estado de Minas Gerais – FAPEMIG.
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Conflicts of interest
There are no conflicts of interest. 27
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