The Effects Of Creatine And Creatinine On Rates Of Apoptosis In Doxorubicin-treated Myoblasts

Abstract

Doxorubicin (DOX) is a powerful chemotherapy agent that is associated with a number of deleterious side effects, including skeletal muscle dysfunction and skeletal muscle wasting. Although the exact mechanisms behind the observed myotoxicity have yet to be fully understood, the direct effect of DOX can generally be attributed to the generation of reactive oxygen species (ROS) and interference with DNA replication. Conversely, creatine (Cr) supplementation has been shown to have a therapeutic role in several disease states characterized by muscle atrophy, which is a hallmark of DOX treatment. Yet, there has been no investigation into the effects of Cr or creatinine (CrN) on DOX-induced apoptosis. PURPOSE: To investigate the effects of Cr and CrN treatment on DOX-induced apoptosis. METHODS: Rat skeletal muscle cells (RKSMC) were cultured in skeletal muscle growth medium until they reached 90-95% confluency. Cells were then collected and seeded on to a 96-well plate at a density of 10,000 cells/ml containing fresh skeletal muscle growth media and allowed to recover for 24 hours. Cells were then exposed to fresh growth media containing either 1.5 μM of DOX, 10 mM of Cr, 10 mM CrN, 1.5 μM DOX + 10 mM Cr, or 1.5 μM DOX + 10 mM CrN for an additional 24 hours. Rates of apoptosis were then assessed using an Annexin V apoptosis detection kit (BD Pharmagen) and high contrast staining. RESULTS: In the cells treated with DOX, 31±5.9% of imaged cells were undergoing apoptosis, which was significantly higher than the Cr (11.9±3.8%) and the CrN (10.1±4.9%) treated group (P= 0.04 and P= 0.03, respectively). No significant difference in rates of apoptosis was found between Cr+DOX, CrN+DOX, or the DOX treated groups CONCLUSION: Initial evidence from this investigation does not support the use of Cr or CrN to protect against DOX-induced apoptosis.