A robust in vitro screening assay to identify NF-kappaB inhibitors for inflammatory muscle diseases.

Specific therapies are not available for inflammatory muscle diseases. We and others have shown that the pro-inflammatory NF-kappaB pathway is highly activated in these conditions. Since NF-kappaB is an important therapeutic target, we decided to utilize an in vitro screening assay to identify potential inhibitors that block TNF-alpha induced NF-kappaB activation in a C2C12 muscle line stably expressing an NF-kappaB luciferase reporter gene. Upon evaluation of multiple anti-inflammatory agents in undifferentiated myoblasts as well as differentiated myotubes , we found different levels of inhibition depending on the state of differentiation. Interestingly, we found that some drugs that are known to inhibit NF-kappaB in immune cells were not effective in muscle cells. Drug toxicity was assessed for using an MTT cell viability assay, and the validity of the luciferase assay was verified by immunostaining for NF-kappaB nuclear translocation in myoblasts. In conclusion, we have determined the optimal assay conditions for detecting potentially valuable NF-kappaB inhibitors for the first time in a muscle cell line that may have significant therapeutic potential for inflammatory muscle diseases.