Effects of the proteasome inhibitor bortezomib on osteolytic human prostate cancer cell metastases.

Prostate adenocarcinoma is the most common malignancy diagnosed in males, and bone metastases remain a significant source of morbidity and mortality in this population. The ubiquitin-proteasome cascade is responsible for the degradation of intracellular proteins, and this pathway is thought to play an essential role in the development of malignancies by altering the levels of various proteins involved in the regulation of cell division. Proteasome inhibitors represent a class of chemotherapeutic agents that have been shown to inhibit tumor growth by a number of different mechanisms. Using a murine intratibial injection model, we examined the effects of the proteasome inhibitor bortezomib on the establishment and progression of osteolytic bone lesions induced by human CaP cells (PC-3 cell line). In this study, the intravenous administration of bortezomib (1 mg/kg) did not prevent the initial formation of osteolytic lesions but did appear to inhibit their growth in a time-dependent fashion. In contrast, bortezomib therapy effectively inhibited the establishment and progression of subcutaneous PC-3 tumors, which served as a positive control. These results suggest that proteasome inhibitors such as bortezomib may represent a novel adjunctive therapy for the treatment of osteolytic skeletal metastases, especially when treatment is initiated early during the disease process.

Systemic corticosteroids inhibit bone healing in a rabbit ulnar osteotomy model.

Prolonged systemic administration of corticosteroids causes osteoporosis and increased risk of fracture. Despite this well documented side effect of systemic corticosteroids, the effect of these compounds on fracture healing is not well defined. The goal of this study was to test the hypothesis that systemic corticosteroid therapy adversely affects fracture healing in a rabbit ulnar osteotomy model. Non-critical sized (1 mm) defects were created bilaterally in 18 adult female New Zealand White rabbits. Starting 2 months before operative intervention and continuing for 6 weeks during healing of the osteotomies, a subcutaneous dose of either sterile saline or prednisone (0.15 mg/kg) was administered daily. Serial radiographs of the forelimb were taken immediately postoperatively and weekly beginning the second week postoperatively. After killing at 6 weeks, only 3 of 20 limbs from animals treated with prednisone achieved radiographic union while 13 of 16 control limbs achieved union. The radiographic density of bone in the defect as well as callus size were greater in the control limbs than in the limbs from prednisone-treated animals. DEXA confirmed that the bone mineral content was lower in the ulnae of prednisone-treated rabbits both within the defect and in adjacent ulnar bone. Mechanical data indicated that osteotomies from rabbits chronically treated with prednisone were weaker than in controls. In this rabbit ulnar osteotomy model, chronic prednisone treatment clearly inhibited bone healing.

Parathyroid hormone-related protein analog RS-66271 is an effective therapy for impaired bone healing in rabbits on corticosteroid therapy.

A new class of parathyroid hormone-related protein (PTHrP) analogs has been developed that causes a rapid gain in both trabecular and cortical bone in models of osteopenia. This study investigates the efficacy of the PTHrP analog, RS-66271 ([MAP(1-10)]22-31 hPTHrP(1-34)-NH(2)), as systemic therapy for impaired bone healing in corticosteroid-treated rabbits. A 1 mm defect was created bilaterally in the ulnae of 30 rabbits. Delayed healing was induced by daily injections of prednisone (0.15 mg/kg) beginning 2 months prior to surgery and continuing until killing. Rabbits in the experimental group received daily subcutaneous injections of PTHrP analog RS-66271 (0.01 mg/kg) starting 1 day after surgery. Control animals received subcutaneous normal saline. At the 6 week timepoint, nine of ten ulnae from PTHrP-treated rabbits achieved radiographic union, whereas only two of ten limbs achieved union in control rabbits (p < 0.01). In a separate part of the study, 20 animals (10 control, 10 RS-66271-treated) were killed when radiographic union was achieved bilaterally. In this portion of the study, all limbs in animals treated with PTHrP achieved union by 6 weeks. In the control animals that were allowed to heal for 10 weeks, only 20% of the limbs achieved radiographic union. In addition, ulnae in the PTHrP-analog-treated rabbits showed greater radiographic intensity (20%-40%), larger callus area (209% anteroposterior view, 417% lateral view) (mean area on AP radiographs: control, = 387 +/- 276 mm(2); PTHrP analog, 1195 +/- 408 mm(2)), and greater stiffness (64%) and torque (87%) when compared with controls. RS-66271 was shown to be an effective therapy for preventing impaired bone healing caused by prednisone in a rabbit model.

Generation of melanoma-specific cytotoxic T lymphocytes by dendritic cells transduced with a MART-1 adenovirus.

Dendritic cells (DC) are potent stimulators of primary T cell responses. In this study, we demonstrate that DC, genetically engineered to express the MART-1/Melan-A (MART-1) tumor-associated Ag, express MART-1 mRNA and protein, correctly process and present the HLA-A2.1-restricted immunodominant MART-1 peptide (MART-1(27-35)), and serve as potent stimulators of MART-1-specific CTL in vitro. A replication-defective E1-deleted adenovirus (AdV) was constructed that expresses MART-1 (AdVMART1). Transduced DC produce full length MART-1 mRNA as well as MART-1 protein. AdVMART1 does not significantly down-regulate cell surface class I expression despite having an intact E3 region. Transduction of an HLA-A2-positive/MART-1-negative cell line with AdVMART1 renders these cells sensitive to lysis by CTL specific for the MART-1(27-35) immunodominant peptide. In addition, DC transduced with AdVMART1 stimulated MART-1(27-35)-specific tumor-infiltrating lymphocytes to synthesize IFN-gamma. Finally, AdVMART1-transduced DC were able to generate MART-1(27-35) peptide-specific, class I-restricted CTL in PBL cultures from normal donors. This study supports the use of tumor Ag-engineered DC in genetic immunotherapy.