scholarly journals ROCK inhibitors beneficially alter the spatial configuration of TGFβ2-treated 3D organoids from a human trabecular meshwork (HTM)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Chiaki Ota ◽  
Yosuke Ida ◽  
Hiroshi Ohguro ◽  
Fumihito Hikage
Keyword(s):  
Trabecular Meshwork ◽  
Kinase Inhibitors ◽  
Spatial Configuration ◽  
Coiled Coil ◽  
Protein Kinase Inhibitors ◽  
Molecular Pharmacology ◽  
Transendothelial Electrical Resistance ◽  
Beneficial Effects ◽  
2D And 3D ◽  
Aqueous Outflow

AbstractTo elucidate molecular pharmacology of Rho-associated coiled-coil containing protein kinase inhibitors (ROCK-i, Ripasudil and Y27632) on their efficiency for aqueous outflow, 2D or 3D cultures of a human trabecular meshwork (HTM) were prepared in the presence of TGFβ2. Those were examined by transendothelial electrical resistance (TEER, 2D), electronic microscopy (EM, 2D and 3D), expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, and fibronectin (FN) by immunolabeling and/or quantitative PCR (3D), and solidity of 3D organoids by a micro-squeezer. TGFβ2 significantly increased the TEER values in 2D cultures, and the ECM expression indicated that the 3D organoids assumed a more densely packed shape. ROCK-i greatly reduced the TGFβ2-induced enhancement of TEER and the immunolabeled ECM expression of the 3D organoids. In contrast, the mRNA expression of COL1 was increased, and those of COL4 and FN were unchanged. EM revealed that TGFβ2 caused the HTM cells to become more compact and abundant ECM deposits within the 3D organoids were observed. These were significantly inhibited by ROCK-i. The dense solids caused by the presence of TGFβ2 were significantly suppressed by ROCK-i. Current study indicates that ROCK-i cause beneficial effects toward the spatial configuration of TGFβ2-induced HTM 3D organoids.

scholarly journals Diverse effects of pan-ROCK and ROCK2 inhibitors on 2 D and 3D cultured human trabecular meshwork (HTM) cells treated with TGFβ2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Megumi Watanabe ◽  
Yosuke Ida ◽  
Hiroshi Ohguro ◽  
Chiaki Ota ◽  
Fumihito Hikage
Keyword(s):  
Trabecular Meshwork ◽  
Smooth Muscle Actin ◽  
Three Dimensional ◽  
Coiled Coil ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Muscle Actin ◽  
Transendothelial Electrical Resistance ◽  
Gene Expressions ◽  
Rock Inhibitor ◽  
2D And 3D

AbstractA pan-ROCK-inhibitor, ripasudil (Rip), and a ROCK2 inhibitor, KD025, were used To study the effects of Rho-associated coiled-coil containing protein kinase (ROCK)1 and 2 on two-dimensional (2D) and three-dimensional (3D) cultures of a TGFβ2-treated human trabecular meshwork (HTM) cells. In the presence of 5 ng/mL TGFβ2, the effects of these inhibitors were characterized by transendothelial electrical resistance (TEER), FITC-dextran permeability, and the size and stiffness of 3D sphenoids, the expression of extracellular matrix (ECM) including collagen1, 4 and 6, and fibronectin, α-smooth muscle actin, a tissue inhibitor of metalloproteinase (TIMP)1–4, and matrix metalloproteinase (MMP)2, 9 and 14. TGFβ2 caused a significant increase in the TEER values, and decrease in FITC-dextran permeability, as well as a decrease in the sizes and stiffness of the 3D sphenoids. In the presence of ROCK inhibitors, the TGFβ2-induced effects of the TEER and FITC-dextran permeability were inhibited, especially by KD025. Rip induced a significant increase in sizes and a decrease in the stiffness of the TGFβ2-treated 3D sphenoids, although the effects of KD025 were weaker. Gene expressions of most of the ECMs, TIMP2 and MMP9 of 2D and 3D HTM cells were significantly up-regulated by TGFβ2. Those were significantly and differently modulated by Rip or KD025.

scholarly journals Pan-ROCK and ROCK2 Inhibitors Affect Dexamethasone-Treated 2D- and 3D-Cultured Human Trabecular Meshwork (HTM) Cells in Opposite Manners

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6382
Author(s):  
Megumi Watanabe ◽  
Yosuke Ida ◽  
Masato Furuhashi ◽  
Yuri Tsugeno ◽  
Fumihito Hikage ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Physical Properties ◽  
Trabecular Meshwork ◽  
Electrical Resistance ◽  
Transendothelial Electrical Resistance ◽  
Gene Expressions ◽  
Rock Inhibitor ◽  
Stiffness Analysis ◽  
3D Cell Cultures ◽  
2D And 3D

Effects of a pan-ROCK-inhibitor, ripasudil (Rip), and a ROCK2 inhibitor, KD025 on dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells as a model of steroid-induced glaucoma were investigated. In the presence of Rip or KD025, DEX-treated HTM cells were subjected to permeability analysis of 2D monolayer by transendothelial electrical resistance (TEER) and FITC–dextran permeability, physical properties, size and stiffness analysis (3D), and qPCR of extracellular matrix (ECM), and their modulators. DEX resulted in a significant increase in the permeability, as well as a large and stiff 3D spheroid, and those effects were inhibited by Rip. In contrast, KD025 exerted opposite effects on the physical properties (down-sizing and softening). Furthermore, DEX induced several changes of gene expressions of ECM and their modulators were also modulated differently by Rip and KD025. The present findings indicate that Rip and KD025 induced opposite effects toward 2D and 3D cell cultures of DEX-treated HTM cells.

scholarly journals Application of a MYC degradation screen identifies sensitivity to CDK9 inhibitors in KRAS-mutant pancreatic cancer

2019 ◽  
Vol 12 (590) ◽  
pp. eaav7259 ◽  
Author(s):  
Devon R. Blake ◽  
Angelina V. Vaseva ◽  
Richard G. Hodge ◽  
McKenzie P. Kline ◽  
Thomas S. K. Gilbert ◽  
...  
Keyword(s):  
Protein Stability ◽  
Kinase Inhibitors ◽  
Screening Strategy ◽  
Protein Kinase Inhibitors ◽  
Ductal Adenocarcinoma ◽  
3D Cell Cultures ◽  
Independent Mechanism ◽  
The Stability ◽  
2D And 3D ◽  
Pdac Cell Line

Stabilization of the MYC oncoprotein by KRAS signaling critically promotes the growth of pancreatic ductal adenocarcinoma (PDAC). Thus, understanding how MYC protein stability is regulated may lead to effective therapies. Here, we used a previously developed, flow cytometry–based assay that screened a library of >800 protein kinase inhibitors and identified compounds that promoted either the stability or degradation of MYC in a KRAS-mutant PDAC cell line. We validated compounds that stabilized or destabilized MYC and then focused on one compound, UNC10112785, that induced the substantial loss of MYC protein in both two-dimensional (2D) and 3D cell cultures. We determined that this compound is a potent CDK9 inhibitor with a previously uncharacterized scaffold, caused MYC loss through both transcriptional and posttranslational mechanisms, and suppresses PDAC anchorage-dependent and anchorage-independent growth. We discovered that CDK9 enhanced MYC protein stability through a previously unknown, KRAS-independent mechanism involving direct phosphorylation of MYC at Ser62. Our study thus not only identifies a potential therapeutic target for patients with KRAS-mutant PDAC but also presents the application of a screening strategy that can be more broadly adapted to identify regulators of protein stability.

scholarly journals New Insights Into Pathophysiological Mechanisms Regulating Conventional Aqueous Humor Outflow

Medicina ◽  
2013 ◽  
Vol 49 (4) ◽  
pp. 26 ◽  
Author(s):  
Daiva Paulavičiūtė-Baikštienė ◽  
Rūta Baršauskaitė ◽  
Ingrida Janulevičienė
Keyword(s):  
Intraocular Pressure ◽  
Aqueous Humor ◽  
Trabecular Meshwork ◽  
Kinase Inhibitors ◽  
Transforming Growth Factor ◽  
Protein Kinase Inhibitors ◽  
Pathophysiological Mechanisms ◽  
Aqueous Humor Outflow ◽  
Trabecular Meshwork Cells ◽  
Glaucoma Treatment

The aim of the article was to overview the pathophysiology of the conventional outflow pathway, trabecular meshwork, and intraocular pressure and to discuss the options of future glaucoma treatment directed to improvement in aqueous outflow. The literature search in the Medline, Embase, and Cochrane databases from April to May 2012 was performed; a total of 47 articles analyzed. The diminished conventional pathway may be altered by several pathophysiological mechanisms like TM obstruction caused by transforming growth factor-β2, clastic nondeformable cells, macrophages leaking from hypermature cataract, iris pigment, lens capsular fragments after YAG-laser posterior capsulotomy, proteins and their subfragments. It is known that trabecular meshwork contraction reduces outflow, and the actomyosin system is directly linked to this mechanism. New glaucoma drugs are still under investigation, but it is already proven that agents such as latranculin-B are effective in improving aqueous drainage. Selective Rho-associated coiled coilforming protein kinase inhibitors have been shown to cause a significant improvement in outflow facility and may become a new option for glaucoma treatment. Caldesmon negatively regulates actin-myosin interactions and thus increases outflow. Stem cells may replace missing or nonfunctional trabecular meshwork cells and hopefully will bring a new treatment solution. Pathophysiological mechanisms regulating conventional aqueous humor outflow are still not fully understood and require further investigations. Future treatment decisions should be directed to a specific mechanism regulating an elevation in intraocular pressure.

Alkaloids as Anticancer Agents: A Review of Chinese Patents in Recent 5 Years

2020 ◽  
Vol 15 (1) ◽  
pp. 2-13 ◽  
Author(s):  
Hongyu Tao ◽  
Ling Zuo ◽  
Huanli Xu ◽  
Cong Li ◽  
Gan Qiao ◽  
...  
Keyword(s):  
Anticancer Activity ◽  
Anticancer Agents ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Cdk Inhibitors ◽  
Comprehensive Overview ◽  
P53 Expression ◽  
Study Results

Background: In recent years, many novel alkaloids with anticancer activity have been found in China, and some of them are promising for developing as anticancer agents. Objective: This review aims to provide a comprehensive overview of the information about alkaloid anticancer agents disclosed in Chinese patents, and discusses their potential to be developed as anticancer drugs used clinically. Methods: Anticancer alkaloids disclosed in Chinese patents in recent 5 years were presented according to their mode of actions. Their study results published on PubMed, and SciDirect databases were presented. Results: More than one hundred anticancer alkaloids were disclosed in Chinese patents and their mode of action referred to arresting cell cycle, inhibiting protein kinases, affecting DNA synthesis and p53 expression, etc. Conclusion: Many newly found alkaloids displayed potent anticancer activity both in vitro and in vivo, and some of the anticancer alkaloids acted as protein kinase inhibitors or CDK inhibitors possess the potential for developing as novel anticancer agents.

scholarly journals Thiazolidine-diones. Biochemical and biological activity of a novel class of tyrosine protein kinase inhibitors.

1990 ◽  
Vol 265 (36) ◽  
pp. 22255-22261
Author(s):  
J F Geissler ◽  
P Traxler ◽  
U Regenass ◽  
B J Murray ◽  
J L Roesel ◽  
...  
Keyword(s):  
Biological Activity ◽  
Protein Kinase ◽  
Kinase Inhibitors ◽  
Protein Kinase Inhibitors ◽  
Tyrosine Protein Kinase
Sign in / Sign up

Export Citation Format

Share Document