Exercise Alters Ceramide Metabolism in Melanoma

Abstract

IntroductionWe previously demonstrated that exercise enhanced the efficacy of chemotherapy against B16F10 melanoma in mice. Exercise likely impacts both the tumor microenvironment and the tumor cells. Exercise has been demonstrated to modulate ceramide metabolism in healthy tissue, but the relationship between exercise and ceramide in tumors has not been evaluated. Ceramides are essential sphingolipid metabolites involved in cell death and survival pathways. Low content of ceramides in melanoma is a potential mechanism for drug resistance. Thus, increasing ceramides content in tumor cells is likely to increase sensitivity to chemotherapy. We hypothesized that exercise would increase accumulation of ceramide in tumor by regulating ceramide metabolism.MethodsC57BL/6 male mice were subcutaneously injected with B16F10 or BP melanoma cells and randomly assigned into sedentary control (No Ex) and exercise (Ex) groups. After tumor establishment, Ex mice performed treadmill running at 12 meter/min for 45 min for 5 consecutive exercise days with two days off then 5 additional exercise days. Ceramides were measured in tumor homogenates by liquid chromatography‐tandem mass spectrometry (LC/MS). Ceramide metabolizing enzymes were analyzed in tumors by immunofluorescence staining and western blotting. Cultured B16F10 or BP cells were treated with serum collected from corresponding melanoma tumor bearing mouse to examine effect of circulating factors on ceramide regulating enzymes.ResultsSeveral ceramide species were increased in Ex compared to No Ex B16F10 tumors, with the largest exercise‐induced increases in C16‐, dihydro‐C16‐, and C20‐ ceramide. Significantly increased expression of ceramide synthase 6 (CerS6), which generates C16‐ceramide using sphinganine or sphingosine, was observed in Ex B16F10 tumors by immunofluorescence staining and western blotting. Surprisingly, there was a trend toward an increase in the ceramide degrading enzyme acid ceramidase (aCDase) in Ex tumors. This may suggest a feedback loop. In contrast, Ex significantly suppressed expression of sphingosine kinase 1 (Sphk1), which converts sphingosine to the key survival sphingolipid S1P, in B16F10 tumors. Likewise, Sphk1 protein expression decreased in B16F10 cells treated in vitro with serum from Ex. Unlike the findings in B16F10 tumors and cultured B16F10 cells, no change in C16‐Ceramide, CerS6, or Sphk1 was observed in Ex BP tumors and in in vitro treatment of BP cells with Ex mice serum.DiscussionOur data suggests that exercise may contribute to B16F10 tumor cell death in response to chemotherapy by modulating ceramide metabolism toward a pro‐apoptotic ceramide/sphingosine‐1‐phosphate balance. However, these results were not consistent in BP tumors. This raises the important finding that exercise can have different effects between tumors of different patient or mouse origin, even with the same diagnosis (i.e. B16F10 vs BP melanoma models). Future work to determine the extent to which exercise regulates ceramide‐sphingolipid signaling in other melanoma tumor models, and in patients, is necessary. Future work will also determine whether exercise‐induced changes in ceramide levels are responsible for the previously reported exercise‐induced increased chemotherapy efficacy against melanoma tumors.