Effects of chemotherapy on bone growth and chondrocyte cell death signaling

Sammanfattning: Glucocorticoids (GCs) are widely used in both children and adults to treat common inflammatory diseases, including asthma, rheumatoid arthritis, ulcerative colitis and Crohn’s disease. However, a multitude of undesired side effects have been reported in patients being treated with GCs, such as osteoporosis, obesity, metabolic disturbances, myopathy and decreased linear bone growth (in children). Dexamethasone, a widely used GC, often causes bone growth impairment as an undesired side-effect in treated children. This observation is supported by experimental data showing that dexamethasone alters proliferation/differentiation and abnormally triggers apoptosis within the growth plate, which may play a key role in the pathophysiology of dexamethasone-induced growth retardation. By investigating these mechanisms, we found that dexamethasone activates caspase-8, -9 and -3 in proliferative chondrocytes. In addition, the Akt-PI3K signaling pathway, which plays a key role in the survival and proliferation of growth plate chondrocytes, is also impaired due to dexamethasone-induced inhibition of Akt phosphorylation. The observation of caspase-9 activation from these studies suggests that an intrinsic apoptotic pathway is also activated in chondrocytes. Therefore, we hypothesized that Bax, a pro-apoptotic member of the Bcl-2 family that is known to regulate intrinsic apoptosis, may play a key role in dexamethasone-induced retardation of bone growth. In chondrocytes, dexamethasone induced conformational changes in Bax, dissipation of the mitochondrial membrane potential and resulted in the release of cytochrome c. Further, Bax-siRNA prevented chondrocytes from undergoing apoptosis. Bax activation was also observed in human growth plate cartilage specimens cultured ex vivo in the presence of dexamethasone. Finally, we observed that Bax-deficient mice were protected from dexamethasone-induced bone growth retardation. Collectively, our data reveal a novel role for Bax in dexamethasone-induced bone growth retardation and impaired bone formation. These findings highlight the possibility for new therapeutic approaches to prevent GC-induced growth failure by specifically targeting Bax (Paper- I, II). Proteasome inhibitors (PIs) such as MG262 and bortezomib are a novel class of anticancer drugs. Bortezomib has recently been introduced clinically to treat multiple myeloma and is under clinical trials in children to treat various cancers. Here we show for the first time that systemic administration of PIs specifically impairs the ubiquitin/proteasome system (UPS) in growth plate chondrocytes. Importantly, we found that young mice display severe growth retardation during treatment, as well as 45 days after the cessation of treatment, with clinically relevant amounts of PIs. Dysfunction of the UPS was also accompanied by the induction of apoptosis (p53-, apoptosis-inducing factor (AIF)- and Bax-mediated apoptosis) of stem-like and proliferative chondrocytes in the growth plate. We also provide evidence that AIF serves as a direct target protein for ubiquitin, thus explaining its prominent upregulation upon proteasome inhibition. Suppression of p53 or AIF expression with siRNA partially rescued chondrocytes from PI-induced apoptosis (35 and 41%, respectively). These findings show that PIs may selectively target essential cell populations in the growth plate, causing significant growth failure, and our results could have important implications for the use of PIs in the treatment of childhood cancer (Paper-III, IV).