University of Kentucky
Catabolic Response of C2C12 Myotubes Following Doxorubicin Exposure
Institution
University of Kentucky
Faculty Advisor/ Mentor
Michael B. Reid; Jennifer Moylan
Abstract
Doxorubicin, a commonly prescribed chemotherapeutic agent, causes skeletal muscle wasting in cancer patients undergoing chemotherapy. Doxorubicin increases oxidants and decreases skeletal muscle mass in vivo. The purpose of this study was to investigate the cellular response of doxorubicin in vitro. We hypothesized that doxorubicin causes a catabolic response in C2C12 myotubes, increasing ROS and promoting atrophy. Cultured myotubes were exposed to doxorubicin (0.2 µM, 2-48 hrs).Cytosolic oxidant activity was measured using the redox sensitive probe dichlorofluorescin. We used real time PCR and Western blot to measure mRNA and protein for ubiquitin ligases MAFbx/atrogin-1 and MuRF1, the caspase-3 protease, and myofibrillar proteins actin and myosin. Oxidant activity was elevated 13 ± 9 % (2 hrs, n=18, p<0.05). Following doxorubicin (48 hrs) actin (-49 ± 4 %, n=3, p<0.01) and myosin (-40 ± 9 %, n=11, p<0.05) proteins were decreased. Doxorubicin increased MAFbx/atrogin-1 mRNA 16 and 24 hrs (74 ± 8%, n=3, p<0.01; 132 ± 8 %, n=3, p<0.01) following exposure, and elevated protein at 24 hrs (15 ± 4 %, n=13, p<0.05). Doxorubicin did not alter MuRF1 mRNA or protein (data not shown). Caspase-3 precursor and active form were elevated 6 hrs (precursor: 25 ± 7 %, n=3, p<0.05; active: 125 ± 35 %, n=3) and 24 hrs (precursor: 36 ± 6, n=3, p<0.01; active: 87 ± 12 %, n=3, p<0.01) following doxorubicin. Our data suggested that doxorubicin increases oxidants, leading to downstream catabolic signaling.
Catabolic Response of C2C12 Myotubes Following Doxorubicin Exposure
Doxorubicin, a commonly prescribed chemotherapeutic agent, causes skeletal muscle wasting in cancer patients undergoing chemotherapy. Doxorubicin increases oxidants and decreases skeletal muscle mass in vivo. The purpose of this study was to investigate the cellular response of doxorubicin in vitro. We hypothesized that doxorubicin causes a catabolic response in C2C12 myotubes, increasing ROS and promoting atrophy. Cultured myotubes were exposed to doxorubicin (0.2 µM, 2-48 hrs).Cytosolic oxidant activity was measured using the redox sensitive probe dichlorofluorescin. We used real time PCR and Western blot to measure mRNA and protein for ubiquitin ligases MAFbx/atrogin-1 and MuRF1, the caspase-3 protease, and myofibrillar proteins actin and myosin. Oxidant activity was elevated 13 ± 9 % (2 hrs, n=18, p<0.05). Following doxorubicin (48 hrs) actin (-49 ± 4 %, n=3, p<0.01) and myosin (-40 ± 9 %, n=11, p<0.05) proteins were decreased. Doxorubicin increased MAFbx/atrogin-1 mRNA 16 and 24 hrs (74 ± 8%, n=3, p<0.01; 132 ± 8 %, n=3, p<0.01) following exposure, and elevated protein at 24 hrs (15 ± 4 %, n=13, p<0.05). Doxorubicin did not alter MuRF1 mRNA or protein (data not shown). Caspase-3 precursor and active form were elevated 6 hrs (precursor: 25 ± 7 %, n=3, p<0.05; active: 125 ± 35 %, n=3) and 24 hrs (precursor: 36 ± 6, n=3, p<0.01; active: 87 ± 12 %, n=3, p<0.01) following doxorubicin. Our data suggested that doxorubicin increases oxidants, leading to downstream catabolic signaling.