Effect of Rho-associated kinase inhibitor on cryopreservation of hES cells
ABSTRACT: After thawing, the mechanism of apoptosis advancing in human ES cells was invesitigated mainly by Gene conpath analysis. As time passed after thawing, nuclear fragmented cells increased more and more in cR-/mR- and cR+/mR-, but in cR+/mR+ and cR-/mR+, thawed cells formed cluster markedly, which was seen among three cell lines. Flow cytometry using TUNEL assay supported final colony formation ratio, which defined that such cell death was due to apoptosis. Gene map analysis 12 hours later after thawing of cR-/mR- showed activation of IL-1β & its receptor IL-1R and TGF-β & its receptor ACVR1C required for activation of caspase-8, initiation of caspase-8 and 10 and marked activation of ARHGDIB promoting actin reorganization comparing to cR+/mR+. Recovered cell lines in any condition did not lose proliferation and pluripotency regardless of ROCK inhibitor treatment. These results showed apoptotic events after thawing were more marked in cR- and advanced through autoenhanced cascaded mechanism followed by initiation of self-cytokain activity, and were suppressed by ROCK inhibitor Y-27632. To investigate the protective mechanism caused by a novel cryo-technique of human embryonic stem (hES) cells using ROCK inhibitor Y-27632 against the cell death due to cryopreservation and thawing with Gene map analysis in addition to estimations of survival rate, apoptosis, undifferentiated states and pluripotency, the dissociated hES cells were cryopreserved in a freezing container in the following four conditions: addition of 10µM of Y-27632 to 1: cryoprotection solution and post-thawing medium (cR+/mR+), 2: only addition to cryoprotection (cR+/mR-), 3: only addition to post-thawing medium (cR-/mR+), 4: no addition to any medium (cR-/mR-).
Project description:After thawing, the mechanism of apoptosis advancing in human ES cells was invesitigated mainly by Gene conpath analysis. As time passed after thawing, nuclear fragmented cells increased more and more in cR-/mR- and cR+/mR-, but in cR+/mR+ and cR-/mR+, thawed cells formed cluster markedly, which was seen among three cell lines. Flow cytometry using TUNEL assay supported final colony formation ratio, which defined that such cell death was due to apoptosis. Gene map analysis 12 hours later after thawing of cR-/mR- showed activation of IL-1β & its receptor IL-1R and TGF-β & its receptor ACVR1C required for activation of caspase-8, initiation of caspase-8 and 10 and marked activation of ARHGDIB promoting actin reorganization comparing to cR+/mR+. Recovered cell lines in any condition did not lose proliferation and pluripotency regardless of ROCK inhibitor treatment. These results showed apoptotic events after thawing were more marked in cR- and advanced through autoenhanced cascaded mechanism followed by initiation of self-cytokain activity, and were suppressed by ROCK inhibitor Y-27632. Overall design: To investigate the protective mechanism caused by a novel cryo-technique of human embryonic stem (hES) cells using ROCK inhibitor Y-27632 against the cell death due to cryopreservation and thawing with Gene map analysis in addition to estimations of survival rate, apoptosis, undifferentiated states and pluripotency, the dissociated hES cells were cryopreserved in a freezing container in the following four conditions: addition of 10µM of Y-27632 to 1: cryoprotection solution and post-thawing medium (cR+/mR+), 2: only addition to cryoprotection (cR+/mR-), 3: only addition to post-thawing medium (cR-/mR+), 4: no addition to any medium (cR-/mR-).
Project description:Rho-associated protein kinase (ROCK) plays crucial roles in the proliferation and migration of different types of cells. ROCK inhibitor Y-27632 was previously reported to inhibit melanoma cell growth, and ROCK signaling was suggested to be a therapeutic target for treating melanoma. However, the negative effect of Y-27632 on melanoma cells was mainly seen in studies on murine B16 melanoma cells. Here, we reported that ROCK inhibitor actually promoted human melanoma cell growth and migration in vitro. Y-27632 increased the growth and migration of BRAF-mutated melanoma cells but had a negative effect on wild-type melanoma cells or primary melanocytes. We discovered that Y-27632 enhanced the growth of BRAF-mutated melanoma cells through increased ATK and ERK activity. The in vivo study further confirmed the in vitro finding. These data suggested that the effect of ROCK inhibitor on melanoma cells is cell-context dependent, and the application of ROCK inhibitor in the treatment of melanoma requires further study.
Project description:Expression of programmed cell death ligand (PD-L1) is associated with poor prognosis in breast cancer. Understanding the regulation of PD-L1 expression in breast cancer could provide a new strategy for breast cancer treatment. Here, we demonstrate that moesin (MSN) phosphorylation by Rho-associated protein kinase (ROCK) stabilizes PD-L1 protein levels. Our results indicate that phosphorylated MSN may compete with the E3 ubiquitin ligase SPOP for binding PD-L1. ROCK inhibition via the Y-27632 inhibitor or MSN silencing decreased PD-L1 expression, resulting in T-cell activation both in vitro and in vivo. Administration of Y-27632 into immunocompetent Balb/c mice bearing breast tumors suppressed tumor progression and enhanced CD4+ and CD8+ T-cell infiltration. RNA-seq analysis of Y-27632-treated mouse tumors revealed that ROCK inhibition upregulated several immune response genes. However, the combination of Y-27632 and an anti-PD-1 antibody did not show additive or synergistic effects due to reduced PD-L1 in the presence of Y-27632. Our study unravels a previously unappreciated mechanism of PD-L1 regulation through the ROCK-MSN pathway. Moreover, we found that ROCK inhibitors could be combined with breast cancer immunotherapy.
Project description:The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632-induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632-induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.
Project description:Disease progression in amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motoneurons and their axons which results in a progressive muscle weakness and ultimately death from respiratory failure. The only approved drug, riluzole, lacks clinical efficacy so that more potent treatment options are needed. We have identified rho kinase (ROCK) as a target, which can be manipulated to beneficially influence disease progression in models of ALS. Here, we examined the therapeutic potential of the ROCK inhibitor Y-27632 in both an in vitro and in an in vivo paradigm of motoneuron disease. Application of Y-27632 to primary motoneurons in vitro increased survival and promoted neurite outgrowth. In vivo, SOD1(G93A) mice were orally treated with 2 or 30 mg/kg body weight of Y-27632. The 2 mg/kg group did not benefit from Y-27632 treatment, whereas treatment with 30 mg/kg resulted in improved motor function in male mice. Female mice showed only limited improvement and overall survival was not modified in both 2 and 30 mg/kg Y-27632 groups. In conclusion, we provide evidence that inhibition of ROCK by Y-27632 is neuroprotective in vitro but has limited beneficial effects in vivo being restricted to male mice. Therefore, the evaluation of ROCK inhibitors in preclinical models of ALS should always take gender differences into account.
Project description:We previously demonstrated that the lifespan of primary human keratinocytes could be extended indefinitely by culture in the presence of the Rho kinase (ROCK) inhibitor Y-27632. This technique has proven to be very useful in diverse areas of basic and clinical research.In this follow-up study we determine whether the continual presence of Y-27632 is required for sustained proliferation. We also test whether different ROCK inhibitors can be used for this technique and whether it can also promote indefinite proliferation of animal keratinocytes. We measure keratinocyte gene expression, proliferation, behaviour and lifespan in the presence and absence of Y-27632.We demonstrate that the extension of lifespan observed by culture of keratinocytes in the presence of fibroblast feeders and a ROCK inhibitor is reversible and that cells senesce gradually when the inhibitor is removed from the medium. Conversely, keratinocytes that are close to the end of their replicative life span can be revived by ROCK inhibition. We demonstrate that different inhibitors of ROCK can also efficiently extend the lifespan of human keratinocytes and that ROCK inhibition extends the lifespan of animal keratinocytes derived from mouse and bovine epithelia. Gene expression analysis of human epidermal keratinocytes cells grown in the presence of Y-27632 demonstrates that ROCK inhibition primarily inhibits keratinocyte differentiation. Live-imaging of keratinocytes cultured with ROCK inhibitors show that the effect of ROCK inhibition on cellular proliferation is immediate and ROCK inhibited cells proliferate rapidly without differentiation or stratification.ROCK inhibition rapidly and conditionally induces indefinite proliferation of keratinocytes. This method has far-reaching applications for basic research, as well as for regenerative and personalized medicine.
Project description:Clear cell renal cell carcinoma (CC-RCC) is the most lethal of all genitourinary cancers. The functional loss of the von Hippel-Lindau (VHL) gene occurs in 90% of CC-RCC, driving cancer progression. The objective of this study was to identify chemical compounds that are synthetically lethal with VHL deficiency in CC-RCC. An annotated chemical library, the library of pharmacologically active compounds (LOPAC), was screened in parallel on VHL-deficient RCC4 cells and RCC4VHL cells with re-introduced VHL. The ROCK inhibitor, Y-27632, was identified and validated for selective targeting of VHL-deficient CC-RCC in multiple genetic backgrounds by clonogenic assays. Downregulation of ROCK1 by small interfering RNA (siRNA) selectively reduced the colony-forming ability of VHL-deficient CC-RCC, thus mimicking the effect of Y-27632 treatment, whereas downregulation of ROCK2 had no effect. In addition, two other ROCK inhibitors, RKI 1447 and GSK 429286, selectively targeted VHL-deficient CC-RCC. CC-RCC treatment with ROCK inhibitors is cytotoxic and cytostatic based on bromodeoxyuridine (BrdU) assay, propidium iodide (PI) staining and growth curves, and blocks cell migration based on transwell assay. On the one hand, knockdown of hypoxia-inducible factor (HIF) ? in the VHL-deficient CC-RCC had a protective effect against Y-27632 treatment, mimicking VHL reintroduction. On the other hand, CC-RCCVHL cells were sensitized to Y-27632 treatment in hypoxia (2% O2). These results suggest that synthetic lethality between ROCK inhibition and VHL deficiency is dependent on HIF activation. Moreover, HIF1? or HIF2? overexpression in CC-RCCVHL cells is sufficient to sensitize them to ROCK inhibition. Finally, Y-27632 treatment inhibited growth of subcutaneous 786-OT1 CC-RCC tumors in mice. Thus, ROCK inhibitors represent potential therapeutics for VHL-deficient CC-RCC.
Project description:Rho-associated kinase (ROCK) plays a critical role in pressure overload-induced left ventricular remodelling. However, the underlying mechanism remains unclear. Here, we reported that TGF-?1-induced ROCK elevation suppressed BMP-2 level and strengthened fibrotic response. Exogenous BMP-2 supply effectively attenuated TGF-?1 signalling pathway through Smad6-Smurf-1 complex activation. In vitro cultured cardiomyocytes, mechanical stretch up-regulated cardiac TGF-?1, TGF-?1-dependent ROCK and down-regulated BMP-2, but BMP-2 level could be reversed through blocking TGF-?1 receptor by SB-431542 or inhibition of ROCK by Y-27632. TGF-?1 could also activate ROCK and suppress endogenous BMP-2 level in a dose-dependent manner. Knock-down BMP-2 enhanced TGF-?1-mediated PKC-? and Smad3 signalling cascades. In contrast, treatment with Y-27632 or SB-431542, respectively suppressed ROCK-dependent PKC-? and Smad3 activation, but BMP-2 was only up-regulated by Y-27632. In addition, BMP-2 silencing abolished the effect of Y-27632, but not SB-431542 on suppression of TGF-?1 pathway. Further experiments showed that Smad6 Smurf1 interaction were required for BMP-2-evoked antagonizing effects. Smad6 overexpression attenuated TGF-?1-induced activation of PKC-? and Smad3, promoted TGF-? RI degradation in BMP-2 knock-down cardiomyocytes, and could be abolished after knocking-down Smurf-1, in which Smad6/Smurf1 complex formation was critically involved. In vivo data showed that pressure overload-induced collagen deposition was attenuated, cardiac function was improved and TGF-?1-dependent activation of PKC-? and Smad3 was reduced after 2 weeks treatment with rhBMP-2(0.5 mg/kg) or Y-27632 (10 mg/kg) in mice that underwent surgical transverse aortic constriction. In conclusion, we propose that BMP-2, as a novel fibrosis antagonizing cytokine, may have potential beneficial effect in attenuating pressure overload-induced cardiac fibrosis.
Project description:Hutchinson-Gilford progeria syndrome (HGPS) constitutes a genetic disease wherein an aging phenotype manifests in childhood. Recent studies indicate that reactive oxygen species (ROS) play important roles in HGPS phenotype progression. Thus, pharmacological reduction in ROS levels has been proposed as a potentially effective treatment for patient with this disorder. In this study, we performed high-throughput screening to find compounds that could reduce ROS levels in HGPS fibroblasts and identified rho-associated protein kinase (ROCK) inhibitor (Y-27632) as an effective agent. To elucidate the underlying mechanism of ROCK in regulating ROS levels, we performed a yeast two-hybrid screen and discovered that ROCK1 interacts with Rac1b. ROCK activation phosphorylated Rac1b at Ser71 and increased ROS levels by facilitating the interaction between Rac1b and cytochrome c. Conversely, ROCK inactivation with Y-27632 abolished their interaction, concomitant with ROS reduction. Additionally, ROCK activation resulted in mitochondrial dysfunction, whereas ROCK inactivation with Y-27632 induced the recovery of mitochondrial function. Furthermore, a reduction in the frequency of abnormal nuclear morphology and DNA double-strand breaks was observed along with decreased ROS levels. Thus, our study reveals a novel mechanism through which alleviation of the HGPS phenotype is mediated by the recovery of mitochondrial function upon ROCK inactivation.
Project description:PURPOSE:The purpose of this study was to investigate the feasibility of poly lactic/glycolic acid (PLGA) as a drug delivery carrier of Rho kinase (ROCK) inhibitor for the treatment of corneal endothelial disease. METHOD:ROCK inhibitor Y-27632 and PLGA were dissolved in water with or without gelatin (W1), and a double emulsion [(W1/O)/W2] was formed with dichloromethane (O) and polyvinyl alcohol (W2). Drug release curve was obtained by evaluating the released Y-27632 by using high performance liquid chromatography. PLGA was injected into the anterior chamber or subconjunctiva in rabbit eyes, and ocular complication was evaluated by slitlamp microscope and histological analysis. RESULTS:Y-27632 incorporated PLGA microspheres with different molecular weights, and different composition ratios of lactic acid and glycolic acid were fabricated. A high molecular weight and low content of glycolic acid produced a slower and longer release. The Y-27632 released from PLGA microspheres significantly promoted the cell proliferation of cultured corneal endothelial cells. The injection of PLGA did not induce any evident eye complication. CONCLUSIONS:ROCK inhibitor-incorporated PLGA microspheres were fabricated, and the microspheres achieved the sustained release of ROCK inhibitor over 7-10 days in vitro. Our data should encourage researchers to use PLGA microspheres for treating corneal endothelial diseases.