ROCK (Rho-associated coiled-coil forming protein kinase) (IC50 ~5 µM)

In human lung cancer cells, PT-262 (5–40 μM; 24 hours) causes cytotoxicity and growth inhibition [1]. Lung cancer cells are exposed to PT-262 (2–20 μM; 4–24 hours) which causes caspase-3 activation, mitochondrial malfunction, and apoptosis [1]. PT-262 (10–20 μM; 24 hours) suppresses the phosphorylation of CDC2 protein and causes p53 to accumulate in the G2/M phase in lung cancer cells with and without p53 [1]. In lung cancer cells, PT-262 (0-10 μM; 24 hours) suppresses ERK phosphorylation [1]. In lung cancer A549 cells, PT-262 (2 μM; 24 h) causes cytoskeletal alterations and cell elongation [2]. Cell migration is strongly inhibited by PT-262 (2-10 μM; 6 hours) in a concentration-dependent manner [2].

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Treatment with 1–20 µM PT-262 for 24 h induced concentration-dependent cytotoxicity in human lung cancer A549 cells (IC50 ~5 µM).[1]
PT-262 (5–40 µM, 24 h) reduced cell viability in A549 cells in a concentration-dependent manner; IC50 ~5 µM.[1]
PT-262 (10 µM, 24 h) almost completely blocked cell proliferation in A549 cells.[1]
PT-262 (2–20 µM, 24 h) induced active forms of caspase‑3 (12 and 17 kDa) and caused loss of mitochondrial membrane potential in A549 cells.[1]
PT-262 (2–10 µM, 24 h) increased apoptotic cell number in A549 cells, with disruption of F‑actin and β‑tubulin cytoskeleton.[1]
PT-262 (2–10 µM, 24 h) inhibited ERK1/2 phosphorylation in a concentration-dependent manner in A549 cells; IC50 for ERK phosphorylation inhibition ~5 µM.[1]
Combination of PT-262 (8 µM, 24 h) with the MEK1/2 inhibitor PD98059 (50 µM, 2 h pre‑treatment) additively increased cytotoxicity in A549 cells (p<0.05).[1]
PT-262 (2–20 µM, 24 h) did not alter p53 or p21 protein expression in p53‑wild type A549 cells.[1]
PT-262 (2–20 µM, 24 h) decreased cell viability in a concentration-dependent manner in p53‑null H1299 lung cancer cells (IC50 ~5 µM).[1]
PT-262 (10 µM, 24 h) significantly decreased G1 fractions and increased G2/M fractions in both A549 and H1299 cells.[1]
PT-262 (10–20 µM, 24 h) reduced protein levels of cyclin B1 and phospho‑CDC2 at Thr14, Tyr15, and Thr161 in A549 cells (and similarly in H1299 cells), without markedly altering total CDC2 or cyclin D1 levels.[1]

Cytotoxicity assay (MTT): Cells plated in 96‑well plates at 1×10⁴ cells/well for 16–20 h, treated with or without PT-262 for 24 h, washed with PBS, re‑cultured in complete medium for 2 days, then incubated with 0.5 mg/ml MTT reagent for 4 h. Formazan dissolved in DMSO and absorbance measured at 565 nm.[1]
Cell number analysis: Cells plated at 5×10⁵ per 100‑mm dish for 16–20 h, treated with 0–10 µM PT-262 for 24 h, washed with PBS, re‑cultured for 1–5 days, then counted by hemocytometer.[1]
Apoptosis assay (morphology and staining): Cells grown on coverslips, treated with PT-262, fixed with 4% paraformaldehyde, stained with BODIPY FL phallacidin (F‑actin) and anti‑β‑tubulin Cy3, nuclei stained with Hoechst 33258 (2.5 µg/ml). Apoptotic nuclei counted under fluorescence microscope (500 cells counted per experiment).[1]
Mitochondrial membrane potential: Cells treated, trypsinized, fixed in 70% ethanol, incubated with 500 nM DiOC6 at 37°C for 30 min, then analyzed by flow cytometry.[1]
Western blot: Cells lysed in ice‑cold whole cell extract buffer with protease inhibitors. Equal protein amounts subjected to 10–12% SDS‑PAGE, transferred to PVDF membranes, hybridized with primary antibodies (anti‑phospho‑ERK, anti‑ERK‑2, anti‑caspase‑3, anti‑cyclin B1, anti‑cyclin D1, anti‑CDC2, anti‑phospho‑CDC2 at Thr14/Tyr15/Thr161, anti‑p53, anti‑p21), followed by HRP‑conjugated secondary antibody, visualized by enhanced chemiluminescence.[1]
Cell cycle assay: Cells plated at 1×10⁶ per 60‑mm dish, treated with 0–10 µM PT-262 for 24 h, fixed in 70% ethanol, stained with 4 µg/ml propidium iodide containing 1% Triton X‑100 and 100 µg/ml RNase for 30 min, filtered, analyzed by flow cytometry (10,000 cells per sample).[1]

Cell Viability Assay[1]
Cell Types: A549 Cell
Tested Concentrations: 5-40 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Cell viability diminished in A549 cells in a concentration-dependent manner. The IC50 value for human normal lung fibroblasts is >20 μM.

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Cell apoptosis analysis [1]
Cell Types: A549 cells
Tested Concentrations: 2-20 μM
Incubation Duration: 4-24 h
Experimental Results: After 8-24 h treatment with 10 μM, the number of apoptotic cells increased. Active forms of caspase-3 (12 and 17 kD) were induced after 24 h of treatment with 2-20 μM.

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Cell cycle analysis [1]
Cell Types: A549 and H1299 Cell
Tested Concentrations: 10-20 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: After 10 μM for 24 hrs (hours), the G1 fraction in A549 and H1299 cells Dramatically diminished, and the G2/M fraction increased. In A549 cells, protein levels of cyclin B1 and phosphorylated CDC2 were diminished at Thr14, Tyr15, and Thr161 in a concentration-dependent manner.

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Western Blot Analysis [1]
Cell Types: A549 cells
Tested Concentrations: 0-10 μM
Incubation Duration: 24 h
Experimental Results: S

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Cytoskeleton staining and confocal microscopy: A549 cells cultured on coverslips were treated with PT-262 for 16-20 h, then fixed in 4% paraformaldehyde, blocked in PBS containing 10% FBS and 0.3% Triton X-100. Actin filaments (F-actin) were stained with BODIPY FL phallacidin (20 U/ml), β-tubulin with Cy3-labeled mouse anti-β-tubulin (1:50), and nuclei with Hoechst 33258 (2.5 µg/ml) for 30 min each. Samples were examined under a confocal laser scanning microscope. Cell length was measured using Leica confocal software. [2]
Bio-atomic force microscopy (Bio-AFM): Cells were analyzed by a confocal microscope combined with Bio-AFM mounted on an inverted microscope. Silicon nitride non-sharpened cantilever (nominal force constant 0.06 N/m) was used. Images were scanned using contact mode with line scan rates 0.5-2 Hz. [2]
Boyden chamber migration assay: Polycarbonate filters (8 µm pore size) were soaked in 0.5 M acetic acid overnight, washed, incubated in 100 µg/ml gelatin for 16 h, air-dried, then incubated in 10 µg/ml fibronectin for 2 h. Test drugs were added to bottom well. Cells on upper surface were removed with cotton swab. Cells on lower surface were counted after hematoxylin staining under light microscope. [2]
Wound healing assay: A549 cell monolayer (>90% confluent) was scraped with a pipette tip to generate 6-7 mm wound. Cells were treated with or without PT-262, washed with PBS, re-cultured in fresh medium. Photographs taken at same wound position at 8 and 24 h. [2]
Western blot analysis: Cells lysed in ice-cold whole cell extract buffer with protease inhibitors. Equal protein amounts were subjected to electrophoresis on 10-12% SDS-polyacrylamide gels, transferred to PVDF membranes, hybridized with primary antibodies (anti-RhoA, anti-ROCK1, anti-phospho-MLC Ser19, anti-MLC, anti-ERK-2), then with HRP-conjugated secondary antibody. Protein bands visualized by enhanced chemiluminescence detection system. [2]
RhoA GTPase activity assay (pull-down): Cells treated with or without PT-262. Cell lysates were incubated with GST-Rhotekin-RBD and glutathione-sepharose beads. Activated GTP-RhoA bound to beads was precipitated and subjected to Western blot using anti-RhoA antibody. Levels of active RhoA normalized to total RhoA. GDPβS and GTPγS used as negative and positive controls. [2]
siRNA transfection: Control siRNA (5'-UUCUCCGAUCGUCUCCGU-3') and RhoA siRNA (5'-CGGAAUGAUGAGCACACACA-3') were transfected into A549 cells using Lipofectamine 2000 according to manufacturer's recommendations. RhoA gene expression analyzed by RT-PCR. [2]

PT-262 showed lower toxicity toward human normal lung fibroblasts (IC50 >20 µM).[1]

[1]. 7-Chloro-6-piperidin-1-yl-quinoline-5,8-dione (PT-262), a novel synthetic compound induces lung carcinoma cell death associated with inhibiting ERK and CDC2 phosphorylation via a p53-independent pathway. Cancer Chemother Pharmacol. 2008 Oct;62(5):799-808.

[2]. 7-Chloro-6-piperidin-1-yl-quinoline-5,8-dione (PT-262), a novel ROCK inhibitor blocks cytoskeleton function and cell migration. Biochem Pharmacol. 2011 Apr 1;81(7):856-65.

PT-262 is a 5,8‑quinolinedione derivative synthesized from 6,7‑dichloroquinoline‑5,8‑dione and piperidine. It induces lung cancer cell death via p53‑independent pathway by inhibiting ERK and CDC2 phosphorylation, leading to G2/M arrest and apoptosis. Unlike other quinolinediones (e.g., LY83583, NSC 663284), PT‑262 does not directly inhibit MEK1/2 (unpublished data) but suppresses ERK phosphorylation through an unknown mechanism. Its unique chloride substituent and piperidine group may contribute to specific electronic configuration for strong protein inhibition.[1]

C14H13CLN2O2

276.72

276.066

86811-36-1

12424014

Brown to reddish brown solid powder

2.7

0

4

1

19

441

0

O=C1C2C=CC=NC=2C(=O)C(Cl)=C1N1CCCCC1

XBHXHCMFAUSIKK-UHFFFAOYSA-N

InChI=1S/C14H13ClN2O2/c15-10-12(17-7-2-1-3-8-17)13(18)9-5-4-6-16-11(9)14(10)19/h4-6H,1-3,7-8H2

7-chloro-6-piperidin-1-ylquinoline-5,8-dione

PT-262 PT262 PT 262

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~361.38 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.03 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)