Kinase inhibition assays

The inhibition of PKCβII, PKCα, PKCε, or PKCγ activity by Enzastaurin was determined using a filter plate assay format measuring 33P incorporation into myelin basic protein substrate. Reactions were done in 100 μL reaction volumes in 96-well polystyrene plates with final conditions as follows: 90 mM HEPES (pH 7.5), 0.001% Triton X-100, 4% DMSO, 5 mM MgCl2, 100 μM CaCl2, 0.1 mg/mL phosphatidylserine, 5 μg/mL diacetyl glyerol, 30 μM ATP, 0.005 μCi/μL 33ATP, 0.25 mg/mL myelin basic protein, serial dilutions of enzastaurin (1 ~ 2,000 nM), and recombinant human PKCβII, PKCα, PKCε, or PKCγ enzymes (390, 169, 719, or 128 pM, respectively). Reactions were started by addition of the enzyme and incubated at room temperature for 60 mins. They were then quenched with 10% H3PO4, transferred to multiscreen anionic phosphocellulose 96-well filter plates, incubated for 30 to 90 mins, filtered and washed with 4 volumes of 0.5% H3PO4 on a vacuum manifold. Scintillation cocktail was added and plates were read on a Microbeta scintillation counter.

Cell lines

HCT116 colon cancer and U87MG glioblastoma cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below - 20 ℃ for several months.

Reacting condition

4 μM, 48 hrs for both HCT116 colon cancer and U87MG glioblastoma cells; 03 ~ 4 μM for HCT116 colon cancer cells

Applications

In both HCT116 colon cancer and U87MG glioblastoma cells, Enzastaurin induced cell apoptosis. HCT116 colon cancer cells treated with Enzastaurin showed a dose-dependent increase in apoptosis.

Animal models

Athymic nude mice bearing HCT116 colon cancer xenografts

Dosage form

75 mg/kg; p.o.; b.i.d., for 21 days

Applications

In mice bearing HCT116 colon cancer xenografts, Enzastaurin significantly suppressed the growth of HCT116 colon carcinoma. Enzastaurin time-dependently inhibited GSK3βSer9 phosphorylation in HCT116 colon cancer xenograft tissues.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

Enzastaurin is an ATP-competitive, selective oral inhibitor of protein kinase Cβ with IC50 value of 6 nM [1].

Protein kinase C (PKC) is a family of serine-threonine protein kinases that have been proved to play critical roles in the formation and progression of tumor cells. The PKCβ is especially found to contribute to the growth and proliferation of tumors such as diffuse large B-cell lymphoma, multiple myeloma and chronic lymphoid leukemia. The selective PKCβ inhibitor enzastaurin was found to have antiangiogenic activity in tumor model as well as suppress tumor proliferation and induce apoptosis. Besides that, enzastaurin showed the inhibitory effects on phosphorylation of ribosomal protein S6, GSK3β and AKT which are in pathways influenced by PKC. Due to these, enzastaurin was developed as a therapy for cancer [1 and 2].

Enzastaurin at low concentration suppressed cell proliferation of various tumor cells including colon carcinoma (HCT-116), glioblastoma (U87MG), non–small cell lung cancer (A549), melanoma (M14), ovarian cancer (OVCAR-3), breast cancer (MCF-7), leukemia (K562), prostate cancer (PC-3), renal cancer (CAKI-1) and central nervous system cancer (U251). Enzastaurin induced apoptosis of tumor cells at low concentration in a range of 1 to 4 μM and the apoptosis was proved to be caspase-independent. In addition, enzastaurin suppressed the phosphorylation ofGSK3βSer9, ribosomal protein S6Ser240/244 and AKTThr308 time-dependently in tumor cells and affected these pathways [1 and 3].

In mice models, oral administration of enzastaurin at a dose of 75 mg/kg resulted in significant proliferation inhibition of U87MG glioblastoma or HCT116 coloncarcinoma xenografts. In mice bearing human walden strommacro globulinemia xenografts, administration of enzastaurin at dose of 80 mg/kg significantly reduced tumor growth of WM and increased survival [1 and 2].

Reference:
[1] Graff J R, McNulty A M, Hanna K R, et al.  The protein kinase Cβ–selective inhibitor, enzastaurin (LY317615.HCl), suppresses signaling through the AKT pathway, induces apoptosis, and suppresses growth of human colon cancer and glioblastoma xenografts. Cancer Research, 2005, 65(16): 7462-7469.
[2] Moreau A S, Jia X, Ngo H T, et al.  Protein kinase C inhibitor enzastaurin induces in vitro and in vivo antitumor activity in Waldenstr mmacroglobulinemia. Blood, 2007, 109(11): 4964-4972.
[3] Rizvi M A, Ghias K, Davies K M, et al.  Enzastaurin (LY317615), a protein kinase Cβ inhibitor, inhibits the AKT pathway and induces apoptosis in multiple myeloma cell lines. Molecular cancer therapeutics, 2006, 5(7): 1783-1789.