24 Hour Mechanical Stretch Increases Cytoplasmic SPARC Expression in Cultured C2C12 Myotubes.: 387 Board #238 May 27, 11

Recent identification of chemotherapy and radiation resistance genes has led to advancements in early detection and treatment methods in many forms of cancer. Secreted protein, acidic and rich in cysteine (SPARC), has been identified as a resistance-reversal gene in multiple colorectal cancer models. Secreted by skeletal muscle, SPARC has been shown to be transiently increased in the plasma and muscle tissue for 6 hours after a single bout of exercise, giving further evidence to the importance of regular exercise in cancer prevention. PURPOSE: The purpose of this study was to investigate the effect of mechanical stretch on C2C12 myotube SPARC expression as an in-vitro model of exercise to confirm increases in SPARC expression with acute bouts of exercise. METHODS: In a pilot study, C2C12 myocytes were obtained from ATCC (CRL-1772) and cultured in growth medium (DMEM with 10% FBS and 1%P/S) and grown to confluency at 37°C and 5% CO2. After fourth passage, cultures were seeded onto uniaxial stretchers coated with Type I rat tail collagen and grown to confluent monolayers. Myotube differentiation media (DMEM with 10% horse serum and 1% P/S) was added to monolayers to induce myotube differentiation. At ~90% myotube population, cells were subjected to serum free media to quiescence and subsequently applied to a 5% stretch for 24 hours. Cells were extracted from stretchers and cytoplasmic and nuclear extracts were obtained. Control cultures were grown and differentiated on 100mm plates. Cytoplasmic extracts were probed for SPARC expression using Western blot technique and analyzed using ImageJ analysis software. RESULTS: Stretched myotubes showed a significant increase in cytoplasmic SPARC expression (p<0.001) after 24 hour stretch. CONCLUSION: Increased cytoplasmic SPARC in C2C12 myotubes after 24 hour mechanical stretch confirms previous studies of the influence of exercise on SPARC expression. Future investigations will observe SPARC resistance-reversal mechanisms in cultured colon tumor cell lines and clinical implications in animal models of colorectal cancer.