| In Vitro | In vitro activity: T56-LIMKi (also known as T5601640) is a potent and selective inhibitor of LIMK2 (LIM kinase 2) with an IC50 of 35.2 μM in a cell (Panc-1 cells) assay. T56-LIMKi inhibits LIMK2 with high specificity, and shows little or no cross-reactivity with LIMK1. T56-LIMKi decreases phosphorylated cofilin (p-cofilin) levels and thus inhibits growth of several cancerous cell lines, including those of pancreatic cancer, glioma and schwannoma. In a nude mouse Panc-1 xenograft model, T56-LIMKi reduced tumor size and p-cofilin levels in the Panc-1 tumors. LIM kinases are important cell cytoskeleton regulators that play a prominent role in cancer manifestation and neuronal diseases. Also LIMKs play a contributory role in several neurodevelopmental disorders and in cancer growth and metastasis. Thus, T56-LIMKi has the potential to be developed as a candidate drug for cancer therapy and neuronal disorders.
Kinase Assay: T56-LIMKi (also known as T5601640) is a potent and selective inhibitor of LIMK2 (LIM kinase 2) with an IC50 of 35.2 μM in a cell (Panc-1 cells) assay.
Cell Assay: NF1 knockout mouse embryonic fibroblasts (MEFs) were prepared from NF1+/- mice, as described previously. MEFs, HeLa, Panc-1, and U-87 cells were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. The human NF1 MPNST cell line ST88-14 was obtained from Dr. Nancy Ratner (University of Cincinnati) and maintained in RPMI/15% FCS. A549 cells were maintained in Nutrient Mixture F-12 Ham containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100μg/ml streptomycin. For biochemical and Western blotting assays, cells were plated at 5×105 cells per 10-cm dish or at 15×104 cells per 6-well plate, and for immumofluorescence at 7500 cells per 18-mm glass coverslip. For growth inhibition assays, cells were plated at 5×103 per 24-well plate. All treatments are specified in the text or figure legends. |
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| In Vivo | T56-LIMKi causes inhibition of cofilin phosphorylation and Panc-1 tumor shrinkage in vivo. Mice treated with T56-LIMKi (60 mg/kg) shows a significant decrease in tumor volume compared to control. Nude CD1-Nu mice (6 weeks old) were housed in barrier facilities on a 12-h light/dark cycle. Food and water were supplied ad libitum. On day zero, 5 × 106 Panc-1 cells in 0.1 ml of PBS were implanted Subcutaneous (s.c.), just above the right femoral joint. S.c. tumors were measured with a caliper, and animal weights were recorded every 4-5 days. Tumor volumes were calculated using the formula: [length × width] × [(length + width)/2]. When tumor volumes reached 0.06–0.07 cm3 (day 0 of treatment), the mice were randomly separated into three groups. Control mice were fed with CMC (vehicle), and T56-LIMKi-treated mice received either 30 or 60 mg/kg of T56-LIMKi daily (by oral administration of 0.1 ml with 0.5% w/v CMC) For imunoblot procedures, tumors were weighed and then homogenized (10% w/v) in lysis buffer as detailed previously. Total amounts of p-cofilin, cofilin and β-tubulin were determined in samples (60 μg protein) of each lysate by SDS-PAGE followed by immunoblotting with the specific antibodies All animal procedures were in accordance with International Laws and Policies. |
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