In vitro activity: (R)-MG132 is a potent, cell permeable and reversible inhibitor of proteasomes. It can inhibit proteasome activity in lysates of J558L multiple myeloma cells and EMT6 breast cancer cells. In comparison to (S)-MG132, the (R)-MG132 stereoisomer is a more effective inhibitor of chymotrypsin-like (ChTL), trypsin-like (TL), and peptidylglutamyl peptide hydrolyzing proteasome (PGPH) activities. However, (R)-MG132, despite being the most effective inhibitor, having a 5-fold lower IC50 than MG-132 (0.22 μM versus 0.89 μM) does not exert stronger cytostatic/cytotoxic effects against tumor cells.

Kinase Assay: After growing on six-well plates (3×105 cells/well) for 24 h, C6 glioma cells are treated with either PBS (control) or 18.5 μM MG-132 for 3, 6, 12, or 24 h at 37°C. Cells are thoroughly scraped from the culture dishes with a cell scraper and washed with cold PBS. After centrifugation for 10 min at 800×g, the cell pellets are suspended in ice-cold buffer (50 mM Tris-HCl, pH 7.5, 20 μM ATP, 5 mM MgCl2, 1 mM dithiothreitol, and 20% glycerol) and homogenized with a Pyrex glass microhomogenizer (20 strokes). The homogenate is centrifuged at 15 000×g for 10 min at 4°C to obtain supernatant. Protein concentration is determined using protein assay kits. A total of 10 μL (1 μg/μL) of each freshly made supernatant is incubated in a 96-well plate at 37°C for 30 min with 10 μL of 300 μM of Succinyl-LLVY-AMC and 85 μL of assay buffer (20 mM Tris-HCl, pH 7.5, and 20% glycerol). Release of fluorescent AMC is measured with a spectrofluorometer at 440 nm with an excitation wavelength of 380 nm.

Cell Assay: The cytostatic/cytotoxic effects in EMT6 cells are measured using crystal violet staining. Briefly, tumor cells are dispensed into 96-well plates at 1.5 × 103 cells per well and allowed to attach overnight. The following day the investigated agents are added at indicated concentrations. After 24 h of incubation the cells are rinsed with PBS and stained with 0.5% crystal violet in 2% ethanol for 10 min at room temperature. Next, plates are washed four times with tap water and cells are lysed with 1% SDS solution. Absorbance is measured at 595 nm using an ELISA reader. The cytostatic/cytotoxic effects in J588L cells are measured using standard MTT assay. Briefly, tumor cells are dispensed into 96-well plates at 3 × 103 cells per well. The following day the investigated agents are added at indicated concentrations. MTT solution at a concentration of 1 mg/mL is added to each well for the last 4 h of incubation. Then the cells are lysed using resuspension buffer containing SDS (0.2 g/mL) and DMF (0.5 mL/mL) at pH 4.7 and left overnight in the incubator. On the following day the absorbance is measured at 570 nm using an ELISA reader.

Mice: C.B-17/lcr-scid/scidJcl mice are inoculated s.c. with HeLa, CaSki, or C33A (1×107 cells). Tumors are allowed to grow for 1 week. Mice are killed and tumors are removed. Tumors are then cut into 2-mm diameter pieces and s.c. transplanted in C.B-17/lcr-scid/scidJcl mice (n=6 per group). One week after inoculation, mice are treated with i.v. injection of saline (control), MG-132 (1 mg/kg/dose) twice a week for 4 weeks. The volume (V) of tumors is measured before every injection, as estimated using equation V=a×b2/2 where a and b are major and minor axes of the tumor measured by a caliper, respectively. Rat: Male Sprague-Dawley rats (8 weeks old, 180-230 g) are used to establish pressure-overload model. All animals are separated into four groups (10 rats per group): (i) vehicle-treated sham group; (ii) MG-132-treated sham group; (iii) vehicle-treated abdominal aortic banding (AAB) group; and (iv) MG-132-treated AAB group. Under intraperitoneal pentobarbital (50 mg/kg) anesthesia, AAB is created using a 5-0 suture tied twice around the abdominal aorta in which a 21-gauge needle is inserted. The needle is then retracted yielding a 70-80% constriction with an outer aortic diameter of ~0.8 mm. In the sham surgery rats, the same surgery is performed except the aorta is constricted. At Day 3 after the surgery, MG-132-treated rats are intraperitoneally injected with 0.1 mg/kg/day of MG-132 for 8 weeks. All control animals are injected with a corresponding volume of vehicle only (0.1% DMSO).

J Med Chem. 2010;53(4):1509-18; J Biol Chem. 2005;280(31):28394-401; Oncol Rep. 2009;22(1):215-21.