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SW620 cells were seeded at different densities: 50 × 103 (a), and 250 × 103 cells/cm2 (b). The lower seeding density (a) shows rounder cells with few cell-cell contacts. The greater seeding density allows for more cell-cell contact, creating a more epithelial state. Scale Bar = 10 μm.
Source publication
The concept of using tissue density as a mechanism to diagnose a tumor has been around for centuries. However, this concept has not been sufficiently explored in a laboratory setting. Therefore, in this paper, we observed the effects of cell density and extracellular matrix (ECM) density on colon cancer invasion and proliferation using SW620 cells....
Citations
... [23,24]. Similarly, the use of y-27632 has been shown to enhance the cloning efficiency of prostate stem cells and promote the proliferation of limbal epithelial cells and keratinocytes, as well as induce the invasiveness of colon cancer cells in a density-dependent manner [25][26][27][28]. These outcomes suggest that the precise role of RhoA/ROCK and y-27632 may be unclear and cell-and cancer-type specific. ...
... The RhoA/ROCK pathway has many downstream targets, including myosin lightchain phosphatase 1 (MYPT1), myosin regulatory light chain (MLC), and LIM kinases 1 and 2 (LIMK1 and LIMK2) that work together to alter cytoskeletal properties that can affect cellular motility, adhesion, and proliferation [18][19][20]. However, there is conflicting evidence about how inhibition of this pathway plays a role in maintaining and altering epithelial homeostasis [21][22][23][24][25][26][27][28]. To better understand this pathway, Xenopus laevis embryonic skin was used as a model of ciliated epithelium development and treated with the ROCK inhibitor, y-27632. ...
... Inhibition of RhoA/ROCK signaling has previously been shown to have both proand antitumorigenic outcomes [23][24][25][26][27][28]. Here, we show that the inhibition of ROCK with y-27632 caused epithelial outgrowths in Xenopus embryonic skin as early as 48 h posttreatment along the edges of Xenopus embryonic skin, which can serve as a model for ciliated epithelium. ...
Lung epithelial development relies on the proper balance of cell proliferation and differentiation to maintain homeostasis. When this balance is disturbed, it can lead to diseases like cancer, where cells undergo hyperproliferation and then can undergo migration and metastasis. Lung cancer is one of the deadliest cancers, and even though there are a variety of therapeutic approaches, there are cases where treatment remains elusive. The rho-associated protein kinase (ROCK) has been thought to be an ideal molecular target due to its role in activating oncogenic signaling pathways. However, in a variety of cases, inhibition of ROCK has been shown to have the opposite outcome. Here, we show that ROCK inhibition with y-27632 causes abnormal epithelial tissue development in Xenopus laevis embryonic skin, which is an ideal model for studying lung cancer development. We found that treatment with y-27632 caused an increase in proliferation and the formation of ciliated epithelial outgrowths along the tail edge. Our results suggest that, in certain cases, ROCK inhibition can disturb tissue homeostasis. We anticipate that these findings could provide insight into possible mechanisms to overcome instances when ROCK inhibition results in heightened proliferation. Also, these findings are significant because y-27632 is a common pharmacological inhibitor used to study ROCK signaling, so it is important to know that in certain in vivo developmental models and conditions, this treatment can enhance proliferation rather than lead to cell cycle suppression.
... A similar ambivalence towards migration after treatment with ROCK inhibitors has been described in previous studies of other cell types most notably cancer cells. Here extensive research has displayed both, a decrease in motility for example in ovarian, breast and lung cancer cells [31][32][33], as well as an increase in some cancer types such as skin, colon and breast carcinoma cells [34][35][36][37]. However, the pro-migratory effects of ROCK inhibition is not limited to cancer cells but also exhibited in a multitude of non-neoplastic cell types e.g. ...
Background:
The Rho-kinase ROCK II plays a major role in the activation of hepatic stellate cells (HSC), which are the key profibrotic and contractile cells contributing to the development of chronic liver disease. Inhibition of ROCK II ultimately blocks the phosphorylation of the myosin light chain (MLC) and thus inhibits stress fibre assembly and cell contraction. We investigated the effects of the ROCK inhibitors Y-33075 as well as Y-27632 in murine and human hepatic stellate cells.
Methods:
Primary isolated HSC from FVB/NJ mice and the immortalized human HSC line TWNT-4 were culture-activated and incubated with Y-27632 and Y-33075 (10nM to 10μM) for 24h. Protein expression levels were analyzed by Western Blots and transcriptional levels of pro-fibrotic markers and proliferative markers were evaluated using real-time qPCR. Migration was investigated by wound-healing assay. Proliferation was assessed by BrdU assay. Contraction of HSC was measured using 3D collagen matrices after incubation with Y-27632 or Y-33075 in different doses.
Results:
Both Rho-kinase inhibitors, Y-27632 and Y-33075, reduced contraction, fibrogenesis and proliferation in activated primary mouse HSC (FVB/NJ) and human HSC line (TWNT-4) significantly. Y-33075 demonstrated a 10-times increased potency compared to Y-27632. Surprisingly, both inhibitors mediated a substantial and unexpected increase in migration of HSC in FVB/NJ.
Conclusion:
ROCK inhibition by the tested compounds decreased contraction but increased migration. Y-33075 proved more potent than Y27632 in the inhibition of contraction of HSCs and should be further evaluated in chronic liver disease.
... ECM degradation and remodeling caused by bioactive molecules such as MMPs released from cancer cells are crucial not only to normal development but also to tumor invasion and metastasis [42,61,62]. Invadopodia are podosome-like membrane protrusions formed by invasive malignant cells with matrix degradation ability [63]. ...
Objectives
To explore the correlation between tumor endothelial marker 1 (TEM1) and matrix metalloproteinase 2 (MMP-2) in uterine sarcoma and their roles in the progression of uterine sarcoma.
Methods
Uterine leiomyosarcoma (uLMS, n = 25) and uterine leiomyoma (n = 25) specimens were collected from a total of 50 patients. Immunohistochemistry assay was conducted to determine the expression of TEM1, MMP-2 and MMP-9. TEM1 over expression (hTEM1) and low expression (shRNA-TEM1) MES-SA cell lines were established as in vitro uterine sarcoma models. MMP-2 mRNA, protein expression and enzymatic activity were verified using qPCR, Western blot and gelatin zymography respectively. MMP-2 expression was downregulated using MMP-2 siRNA in hTEM1 MES-SA cells to better study the role of MMP-2. The invasive and migratory capacities of hTEM1, shRNA-TEM1, and hTEM1 treated with MMP-2 siRNA MES-SA cells were determined using transwell assays. Extracellular matrix (ECM) remodeling mediated by TEM1 was examined using cell-ECM adhesion and fluorescent gelatin-ECM degradation assays. The immunofluorescence of F-actin was examined to analyze the formation of invadopodia. Subcutaneous and intraperitoneal xenografts were established to validate the role of TEM1 in promoting uterine sarcoma metastasis.
Results
TEM1 and MMP-2 were expressed in 92% (n = 23) and 88% (n = 22) of uterine leiomyosarcoma specimens, respectively. Both TEM1 and MMP-2 were highly expressed in 100% (n = 17) of high stage (III-IV) uterine leiomyosarcoma specimens. In addition, TEM1 expression was positively correlated with MMP-2 expression in uterine leiomyosarcoma. The successful establishment of in vitro uterine sarcoma models was confirmed with qPCR and Western blotting tests. TEM1 promoted the invasion and metastasis of uterine sarcoma in vivo and in vitro. MMP-2 expression and activity were up-regulated in hTEM1 cells but down-regulated in shRNA-TEM1 cells. Importantly, MMP-2 knockdown impaired the invasive and migratory capacity of hTEM1 cells. TEM1 promoted ECM remodeling by increasing cell-ECM adhesion and ECM degradation. TEM1 overexpression also induced the formation of invadopodia.
Conclusion
TEM1 was co-expressed and positively correlated with MMP-2 in uterine leiomyosarcoma specimens. In addition, both TEM1 and MMP-2 were associated with tumor development. TEM1 promoted uterine sarcoma progression by regulating MMP-2 activity and ECM remodeling.
... What is more, it has been verified that Rho-associated coiled coil-forming protein kinase (ROCK-1 and ROCK-2) participates in smooth muscle contraction and apoptosis [20]. Meanwhile, the Rho/ROCK pathway is a crucial component of axon-guided cell contraction and apoptosis regulation [21]. ...
For this study, we investigate more deeply the effect calcium (Ca) develops on the mechanism underlying fluoride-triggered osteocyte apoptosis. We detected the morphology of osteocytes by HE staining, mitochondrial microstructure by using the transmission electron microscope, and the biochemical indexes related to bone metabolism and the expression of apoptosis-related genes. These results showed that NaF brought out the reduced osteocytes and ruptured mitochondrial outer membrane, with a significantly increased StrACP activity by 10.414 IU/L at the 4th week (P < 0.05), markedly upregulating the mRNA expression of Bax, Cyto-C, Apaf-1, caspase-7, ROCK-1, BMP-2, and BGP (P < 0.01), as well as caspase-6 (P < 0.05), while downregulating Bcl-2 by 61.3% (P < 0.01). Through immunohistochemical analysis, we also found that NaF notably increased the protein expression of ROCK-1 (P < 0.05) and Cyto-C, BMP-2, and BGP (P < 0.01), suggesting that NaF triggered the activation of the mitochondrial apoptotic pathway and Rho/ROCK signaling pathway. Nevertheless, 1% Ca supplementation in diet notably enhanced the mRNA expression of Bcl-2 by 39.3% (P < 0.01), thus blocking the increment of the expression of mitochondrial apoptotic pathway–related genes and ROCK-1. Meanwhile, Ca could attenuate the StrACP activity by 10.741 IU/L at the 4th week (P < 0.05) and protect the integrity of the mitochondrial outer membrane. These findings strongly suggest that 1% Ca abated the mitochondrial apoptosis pathway by increasing the anti-apoptotic gene Bcl-2 expression, and effectively inhibited the hyper-activation of ROCK-1, dually protecting the structural integrity of the mitochondrial outer membrane and maintaining normal cellular metabolic function.
... Finally, Y-27632 has been shown to reduce the metastatic growth of human prostate cancer PC3 cells in immune-compromised mice [498]; to significantly decrease intrahepatic metastasis orthotropic implantation of CBO140C12 HCC tumour fragments into mice liver [499], and to impair the metastatic dissemination of HT29 human colorectal carcinoma cells in an orthotropic mouse model of liver metastasis [500]. However, it is important to underline that ROCK inhibition by Y-27632 has been also shown to increase the proliferation and migration of a series of in vitro and in vivo cancer models [501][502][503][504], those effects being explained by the observation that ROCK activation can be implicated in the negative feedback mechanisms controlling pro-proliferative pathways [505]. ...
Cancer is a pathology characterized by a loss or a perturbation of a number of typical features of normal cell behaviour. Indeed, the acquisition of an inappropriate migratory and invasive phenotype has been reported to be one of the hallmarks of cancer. The cytoskeleton is a complex dynamic network of highly ordered interlinking filaments playing a key role in the control of fundamental cellular processes, like cell shape maintenance, motility, division and intracellular transport. Moreover, deregulation of this complex machinery contributes to cancer progression and malignancy, enabling cells to acquire an invasive and metastatic phenotype. Metastasis accounts for 90% of death from patients affected by solid tumours, while an efficient prevention and suppression of metastatic disease still remains elusive. This results in the lack of effective therapeutic options currently available for patients with advanced disease. In this context, the cytoskeleton with its regulatory and structural proteins emerges as a novel and highly effective target to be exploited for a substantial therapeutic effort toward the development of specific anti-metastatic drugs. Here we provide an overview of the role of cytoskeleton components and interacting proteins in cancer metastasis with a special focus on small molecule compounds interfering with the actin cytoskeleton organization and function. The emerging involvement of microtubules and intermediate filaments in cancer metastasis is also reviewed.
... Factors potentiating the ROCK signaling pathway in lung cancer. The increased stiffness of the extracellular matrix (ECM) in a tumor is one of the factors recognized to activate Rho GTPases, as demonstrated in a 3D culture system with varying levels of tissue or substrate stiffness (12,52). The aim of this ROCK activation is to counterbalance the external force exerted on cells by increasing the contractility of the internal cytoskeletal structure (53). ...
Lung cancer is one of the most lethal forms of cancer known to man, affecting millions of individuals worldwide. Despite advancements being made in lung cancer treatments, the prognosis of patients with the disease remains poor, particularly among patients with late‑stage lung cancer. The elucidation of the signaling pathways involved in lung cancer is a critical approach for the treatment of the disease. Over the past decades, accumulating evidence has revealed that Rho‑associated kinase (ROCK) is overexpressed in lung cancer and is associated with tumor growth. The present review discusses recent findings of ROCK signaling in the pathogenesis of lung cancer that were conducted in pre‑clinical studies. The significant role of ROCK in cancer cell apoptosis, proliferation, migration, invasion and angiogenesis is discussed. The present review also suggests the use of ROCK as a potential target for the development of lung cancer therapies, as ROCK inhibition can reduce multiple hallmarks of cancer, particularly by decreasing cancer cell migration, which is an initial step of metastasis.
... However, knockdown of myosin light chain kinase was shown to cause a significant increase in the total number of invadopodia and the extent of ECM degradation in squamous cell carcinoma (Jerrell and Parekh, 2019). This is also in line with the finding that the general decrease of actomyosin contractility in response to the ROCK inhibitor Y27632 increases cancer cell invasiveness (Vishnubhotla et al., 2012). ...
Integrin adhesions are a structurally and functionally diverse family of transmembrane, multi-protein complexes that link the intracellular cytoskeleton to the extracellular matrix (ECM). The different members of this family, including focal adhesions (FAs), focal complexes, fibrillar adhesions, podosomes and invadopodia, contain many shared scaffolding and signaling ‘adhesome’ components, as well as distinct molecules that perform specific functions, unique to each adhesion form. In this Hypothesis, we address the pivotal roles of mechanical forces, generated by local actin polymerization or actomyosin-based contractility, in the formation, maturation and functionality of two members of the integrin adhesions family, namely FAs and invadopodia, which display distinct structures and functional properties. FAs are robust and stable ECM contacts, associated with contractile stress fibers, while invadopodia are invasive adhesions that degrade the underlying matrix and penetrate into it. We discuss here the mechanisms, whereby these two types of adhesion utilize a similar molecular machinery to drive very different – often opposing cellular activities, and hypothesize that early stages of FAs and invadopodia assembly use similar biomechanical principles, whereas maturation of the two structures, and their ‘adhesive’ and ‘invasive’ functionalities require distinct sources of biomechanical reinforcement.
... Indeed, the use of ROCK inhibitors such as Y-27632 or Fasudil (HA-1077) decreased the migration and invasion capacities of several cancer cell types 22,23 , among which colon cancer cells 19,24 . However, several other studies reported a pro-cancerous effect of ROCK inhibition by promoting the growth and migration of some cancer cells 25,26 , such as a gain in colon cancer cell proliferation and invasion [27][28][29] . Signalling pathways involved in such deleterious effects are still elusive, and certainly need to be better understood. ...
... In this study, we show that the inhibition of ROCK, using conventional ROCK inhibitors at classical concentrations used both in vitro and in vivo, increases the invasive capacities of SW620 human colon cancer cells, and also those of MDA-MB-231 human breast cancer cells. While ROCK inhibitors are generally used to inhibit cell migration and invasion 7,8,19 , we are not the first to demonstrate pro-invasive effects of ROCK inhibition 25,29 . In www.nature.com/scientificreports/ ...
... B16 melanoma cells, Y-27632 induced invasion via enhanced AKT and ERK signalling pathways 25 . In SW620 colon cancer cells, Y-27632 was also shown to increase invasiveness through 3D matrices composed of collagen I, but the mechanisms involved were not identified 29 . www.nature.com/scientificreports/ ...
The acquisition of invasive capacities by carcinoma cells, i.e. their ability to migrate through and to remodel extracellular matrices, is a determinant process leading to their dissemination and to the development of metastases. these cancer cell properties have often been associated with an increased Rho-ROCK signalling, and ROCK inhibitors have been proposed for anticancer therapies. In this study we used the selective ROCK inhibitor, Y-27632, to address the participation of the Rho-ROCK signalling pathway in the invasive properties of SW620 human colon cancer cells. Contrarily to initial assumptions, Y-27632 induced the acquisition of a pro-migratory cell phenotype and increased cancer cell invasiveness in both 3- and 2-dimensions assays. This effect was also obtained using the other ROCK inhibitor Fasudil as well as with knocking down the expression of ROCK-1 or ROCK-2, but was prevented by the inhibition of NaV1.5 voltage-gated sodium channel activity. Indeed, ROCK inhibition enhanced the activity of the pro-invasive NaV1.5 channel through a pathway that was independent of gene expression regulation. In conclusions, our evidence identifies voltage-gated sodium channels as new targets of the ROCK signalling pathway, as well as responsible for possible deleterious effects of the use of ROCK inhibitors in the treatment of cancers.
... It is important to note that pan-ROCK inhibition using Y-27632 has been shown to increase the proliferation and migration of a number of in vitro and in vivo cancer models Vishnubhotla et al., 2012;Yang and Kim, 2014;Chang et al., 2018). These effects may be explained by the observation that ROCK activation can contribute to negative feedback mechanisms that regulate pro-proliferative pathways. ...
As key regulators of cytoskeletal dynamics, Rho GTPases coordinate a wide range of cellular processes, including cell polarity, cell migration, and cell cycle progression. The adoption of a pro-migratory phenotype enables cancer cells to invade the stroma surrounding the primary tumor and move toward and enter blood or lymphatic vessels. Targeting these early events could reduce the progression to metastatic disease, the leading cause of cancer-related deaths. Rho GTPases play a key role in the formation of dynamic actin-rich membrane protrusions and the turnover of cell-cell and cell-extracellular matrix adhesions required for efficient cancer cell invasion. Here, we discuss the roles of Rho GTPases in cancer, their validation as therapeutic targets and the challenges of developing clinically viable Rho GTPase inhibitors. We review other therapeutic targets in the wider Rho GTPase signaling network and focus on the four best characterized effector families: p21-activated kinases (PAKs), Rho-associated protein kinases (ROCKs), atypical protein kinase Cs (aPKCs), and myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs).
... It was demonstrated that the prolonged release of dexamethasone (a steroidal anti-inflammatory drug) provided by dexamethasonecontaining PLGA scaffolds effectively generated differentiation of bone marrow stem cells to osteoblasts or chondrocytes. 9 Furthermore, some bioactive molecules such as chondroitin sulfate, 6 retinoic acid, 10 proangiogenic growth factors (PDGF-BB, FGF2, and VEGF), 11 l-lysine, 12 sphingolipid growth factor (FTY720), 13 and ROCK inhibitor (Y-27632) 14 were used to improve the form and function of the construct or to improve cell adhesion and proliferation for corneal tissue engineering. However, so far, no information was available in the literature about small, drug (with a treatment purpose)-loaded cell scaffolds for the treatment of severe corneal injury. ...
Purpose:
The aim of this study was to design naproxen sodium (NS)-containing, biomimetic, porous poly(lactide-co-glycolide) (PLGA) scaffolds for regeneration of damaged corneal epithelium.
Methods:
NS-incorporated PLGA scaffolds were prepared using the emulsion freeze-drying method and then coated with collagen or poly-l-lysine. Porosity measurements of the scaffolds were performed by the gas adsorption/desorption method and the scaffolds demonstrated highly porous, open-cellular pore structures with pore sizes from 150 to 200 μm.
Results:
The drug loading efficiency of scaffolds was found to be higher than 84%, and about 90%-98% of NS was released at the end of 7 days with a fast drug release rate at the initial period of time and then in a slow and sustained manner. The corneal epithelial cells were isolated from New Zealand white rabbits. The obtained cells were seeded onto scaffolds and continued to increase during the time period of the study, indicating that the scaffolds might promote corneal epithelial cell proliferation without causing toxic effects for at least 10 days.
Conclusions:
The NS-loaded PLGA scaffolds exhibited a combination of controlled drug release and biomimetic properties that might be attractive for use in treatment of corneal damage both for controlled release and biomedical applications.
