The pro-apoptotic effect of chronic contractile activity-induced extracellular vesicles on Lewis Lung Carcinoma cells

Regular exercise reduces tumor growth in vivo and in vitro, but the exact mechanisms have yet to be fully elucidated. We have previously shown that chronic contractile activity (CCA) increases the concentration of skeletal muscle-derived EVs, and these in turn increased mitochondrial biogenesis in myoblasts. Here, we hypothesized that skeletal muscle-EVs derived post-CCA will mediate the anti-tumorigenic effects associated with chronic exercise. C2C12 myoblasts were differentiated into myotubes, electrically paced, and EVs isolated from conditioned media from control and CCA myotubes using differential ultracentrifugation. Lewis lung carcinoma (LLC) cells were treated with the total number of control-EVs or CCA-EVs isolated after each day of contractile activity for 4 days. Permeabilized CCA-EVs with or without proteinase K before co-culture with LLC cells were used as controls. Effect of EV treatment on cell count, viability, apoptosis, senescence, migration, and mitochondrial content was measured. CCA-EV treatment reduced cell count by 18% and cell viability by 6% vs. control-EVs. CCA-EVs increased the incidence of apoptotic hallmarks: DNA fragmentation by 13%, Annexin V+/PI+ cells by 21%, and the expression of pro-apoptotic Bax (by 25%) and Bax/Bcl-2 ratio (by 60%) vs. control-EVs. CCA-EVs increased number of senescent cells by 29%, and senescence markers, HMGB1 (by 49%) and p16 (by 92%) vs. control-EVs. When CCA-EVs were pretreated with Triton X-100 with or without proteinase-K, the increase in apoptosis and senescence was abrogated, confirming the effect is due to intact EVs and likely through EV membrane-proteins. CCA-EVs did not have any effect on cell migration and mitochondrial content vs. control-EVs. This study illustrates for the first time the potential of CCA-induced skeletal muscle-EVs in mediating anti-tumorigenic effects traditionally linked with chronic exercise.