Y-27632 dihydrochloride

Biological Activity

Y-27632 is a potent, selective inhibitor of Rho-associated protein kinases (ROCK) with IC50 values of 140-220 nM for ROCK1 and ROCK2. Y-27632 inhibited the kinase activity of both ROCK-I and ROCK-II in vitro, and this inhibition was reversed by ATP in a competitive manner. The ROCK family of Rho-associated serine-threonine protein kinases is known to play an important role in Rho-mediated cell adhesion and smooth muscle contraction. It also inhibits PRK2 with an IC50 value of 600 nM. One-hour treatment with 10 µM Y-27632 blocks apoptosis of dissociated cultured human embryonic stem cells, increasing cloning efficiency by 25% and sustaining survival up to 30 passages. Y-27632 was found to increases human embryonic stem cell (hESC) survival.

Product Citations

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  Oncotarget. 2017 Dec 28;9(12):10375-10387.

  The crosstalk between breast carcinoma-associated fibroblasts and cancer cells promotes RhoA-dependent invasion via IGF-1 and PAI-1.
  Y-27632 dihydrochloride purchased from AbMole

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  Nat Protoc. 2018 Feb;13(2):235-247.

  A surgical orthotopic organoid transplantation approach in mice to visualize and study colorectal cancer progression
  Y-27632 dihydrochloride purchased from AbMole

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  Nat Protoc. 2018 Jan;13(1):59-78.

  Measuring mutation accumulation in single human adult stem cells by whole-genome sequencing of organoid cultures
  Y-27632 dihydrochloride purchased from AbMole

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  Cell. 2017 Dec 5;pii: S0092-8674(17)31319-3.

  A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity
  Y-27632 dihydrochloride purchased from AbMole

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  Am J Clin Exp Urol. 2017 Nov 9;5(3):25-33.

  Organoid culture of human prostate cancer cell lines LNCaP and C4-2B
  Y-27632 dihydrochloride purchased from AbMole

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  Mater Sci Eng C Mater Biol Appl. 2017 Sep 1;78:443-451.

  A dual-layer macro/mesoporous structured TiO2 surface improves the initial adhesion of osteoblast-like cells
  Y-27632 dihydrochloride purchased from AbMole

• 

  Nature. 2016 Nov 24;539(7630):560-564.

  Designer matrices for intestinal stem cell and organoid culture
  Y-27632 dihydrochloride purchased from AbMole

• 

  Nat Protoc. 2016 Feb;347-58.

  Organoid culture systems for prostate epithelial and cancer tissue.
  Y-27632 dihydrochloride purchased from AbMole

Customer Product Validations & Biological Datas

	Source	Oncotarget (2017). Figure 6. Y27632 (Abmole Bioscience)
	Method	intraperitoneally
	Cell Lines	SCID mice
	Concentrations	25 mg/kg
	Incubation Time	18 days
	Results	Similar to our in vitro findings, Y27632 significantly increased the expression of PAI-1, confirming the existence of a regulatory mechanism between ROCK and PAI-1 in vivo

	Source	Oncotarget (2017). Figure 5. Y27632 (Abmole Bioscience)
	Method	intraperitoneally
	Cell Lines	SCID mice
	Concentrations	25 mg/kg
	Incubation Time	18 days
	Results	After 18 days of treatment, we did not observe any effect of PQ401, Y27632 or PQ401 combined with Y27632 on primary tumor growth, suggesting that the inhibition of ROCK or IGF-1 receptor did not affect cancer cell proliferation.

	Source	Oncotarget (2017). Figure 4. Y27632 (Abmole Bioscience)
	Method	immunoblotting
	Cell Lines	MDA-MB-231 cell
	Concentrations	10 μM
	Incubation Time	72 h
	Results	Interestingly, PAI-1 expression increased in cells treated with Y27632, suggesting the existence of a regulatory loop between PAI-1 and ROCK

	Source	Oncotarget (2017). Figure 2. Y27632 (Abmole Bioscience)
	Method	Transwell
	Cell Lines	MDA-MB-231 cells
	Concentrations	10 μM
	Incubation Time	72 h
	Results	Y27632 did not affect the scattering and invasion of MDA-MB-231 cultured without CAFs, but significantly reduced the scattering and invasion induced by CAFs, suggesting that CAFs promote the invasion of MDA-MB-231 cells via ROCK1/2.

	Source	Nat Protoc (2018). Figure 1. Y-27632 (Abmole Bioscience)
	Method	i.e.
	Cell Lines	NSG (NOD.Cg-Prkd cscidIl 2rgtm1Wjl/SzJ) mice
	Concentrations	10 μM
	Incubation Time	5–10 min
	Results	Administration of tamoxifen to these mice induces specific activation of the Cre enzyme within the epithelial cells of the small and large intestine, with consequential deletion of both Apc alleles, expression of an oncogenic form of Kras (KrasG12D) and deletion of one of the two Trp53 alleles

	Source	University of California (2017). Figure 7. Y-27632 (Abmole Bioscience)
	Method	Patient-derived organoid culture
	Cell Lines	biopsy tissue
	Concentrations	10 μM
	Incubation Time	30 min
	Results	mCRPC patient-derived organoids are sensitive to olaparib. Organoids from patients with homozygous loss of CHD1 (V5272 and V5372) show increased sensitivity to PARP inhibition.

	Source	Nat Protoc (2018). Figure 4. Y-27632 dihydrochloride (Abmole Bioscience, USA)
	Method	FACS plots
	Cell Lines	Human liver single-cell
	Concentrations	10 μM
	Incubation Time	7 d
	Results	FACS plots depicting gating strategies for sorting single cells from a human liver organoid culture

	Source	Cell (2017). Figure 1. Y-27632 (Abmole Bioscience)
	Method	PARPi assay
	Cell Lines	BC organoid lines
	Concentrations	< 100 μm
	Incubation Time	48 h
	Results	Inhibition of Rho-associated coiled-coil containing protein kinase (ROCK) has been shown to allow long-term proliferation of tumor epithelial cells in vitro and addition of the specific ROCK inhibitor Y-27632 indeed improved culture conditions.

	Source	Am J Clin Exp Urol (2017) . Figure 1. Y-27632 (Abmole Bioscience, USA)
	Method	Organoid culture
	Cell Lines	LNCaP and C4-2B cells
	Concentrations	10 μM
	Incubation Time	48-72 h
	Results	A few cells presented as an aggregate of two cells or three cells. At day 7, LNCaP cells and C4-2B cells started to present aggregates of multiple cells. At day 14, organoids were formed as aggregates of LNCaP cells and C4-2B cells

	Source	Nature Protocols (2016) . Figure 3. Y-27632 (Abmole Bioscience)
	Method	MTT assay
	Cell Lines	mouse and human luminal cell
	Concentrations	10 μM
	Incubation Time	2–3 d
	Results	Absence of the ROCK inhibitor (Y-27632) will greatly decrease the efficiency of organoid outgrowth.

	Source	Nature (2016). Figure 1. Y-27632 (Abmole Bioscience Inc)
	Method	Cell culture.
	Cell Lines	single cells
	Concentrations	10 μM
	Incubation Time
	Results

	Source	Materials Science and Engineering (2017). Figure 8. Y-27632 (Abmole Bioscience Inc, USA)
	Method	Cytoskeleton and nucleus staining in the absence or presence of ROCK inhibitor (Y-27632)
	Cell Lines	SaOS-2 cells
	Concentrations	10 μM
	Incubation Time	4 h and 24 h
	Results	After inhibition of ROCK by Y-27632, an expected formation of finger-like filopodia was seen for the cells of all groups after 4 h, as shown by white arrows in Fig. 8b, f and j.

	Source	Materials Science and Engineering (2017). Figure 7. Y-27632 (Abmole Bioscience Inc, USA)
	Method	Cytoskeleton and nucleus staining in the absence or presence of ROCK inhibitor (Y-27632)
	Cell Lines	SaOS-2 cells
	Concentrations	10 μM
	Incubation Time	4 h and 24 h
	Results	The attached cells on the three investigated surfaces in the presence or absence (control) of Y-27632 after 4 h and 24 h were summarized in Fig. 7

Protocol

Cell Experiment
Cell lines	cyES cells
Preparation method	For 5-bromo-2′-deoxyuridine (BrdU) incorporation assays, dissociated cyES cells were seeded at a concentration of 500 cells/cm2 onto fresh feeder layers in the presence of the ROCK inhibitors Y-27632 (Wako Pure Chemical Industries, Tokyo, Japan) or Fasudil (Tocris Bioscience, MO, USA) during the first 24 h of culture. The BrdU incorporation assay was performed using colorimetric BrdU Cell proliferation Kit (Roche Diagnostics) according to the manufacturer's instructions. BrdU incorporations were also observed by immunocytochemical (ICC) staining with BrdU Labeling and Detection Kit I (Roche Diagnostics).
Concentrations	0, 0.1, 0.5, 5, 10, 20µM
Incubation time	24 h

Animal Experiment
Animal models	EAC modeling
Formulation	dissolved in 0.9% NaCl
Dosages	0.1, 1, 10 mg/kg/day for 14 days
Administration	i.p.

Conversion of different model animals based on BSA (Value based on data from FDA Draft Guidelines)

Species	Mouse	Rat	Rabbit	Guinea pig	Hamster	Dog
Weight (kg)	0.02	0.15	1.8	0.4	0.08	10
Body Surface Area (m2)	0.007	0.025	0.15	0.05	0.02	0.5
Km factor	3	6	12	8	5	20

Animal A (mg/kg) = Animal B (mg/kg) multiplied by	Animal B Km
	Animal A Km

For example, to modify the dose of resveratrol used for a mouse (22.4 mg/kg) to a dose based on the BSA for a rat, multiply 22.4 mg/kg by the K_m factor for a mouse and then divide by the K_m factor for a rat. This calculation results in a rat equivalent dose for resveratrol of 11.2 mg/kg.

Chemical Information

References

The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation.
Li X, et al. Stem Cells Dev. 2008 Dec;17(6):1079-85. PMID: 19006455.

Effects of rho-associated protein kinase inhibitor Y-27632 on intraocular pressure and outflow facility.
Honjo M, et al. Invest Ophthalmol Vis Sci. 2001 Jan;42(1):137-44. PMID: 11133858.

Pharmacological properties of Y-27632, a specific inhibitor of rho-associated kinases.
Ishizaki T, et al. Mol Pharmacol. 2000 May;57(5):976-83. PMID: 10779382.