Transfection of glucosylceramide synthase antisense inhibits mouse melanoma formation

Abstract

MEB4 murine melanoma cells synthesize GM3 as the major ganglioside. Inhibition of GM3 synthesis by a specific glucosylceramide synthase inhibitor resulted in reduced tumorigenicity and metastatic potential of these cells. We used a molecular approach-antisense transfection targeting the glucosylceramide synthase gene-to regulate glycosphingolipid synthesis by MEB4 cells and examine the influence on tumor formation. Antisense transfection inhibited the synthesis of the direct product of glucosylceramide synthase, glucosylceramide, and consequently GM3 ganglioside, by MEB4 cells, reducing the concentration of GM3 in the transfectants by up to 58%. Although neither morphology nor proliferation kinetics of the cultured cells was affected, the inhibition of glycosphingolipid synthesis and reduction of total ganglioside content caused a striking reduction in melanoma formation in mice. Only 1/60 (2%) of mice injected ID with 104 antisense-transfected MA173 cells formed a tumor, compared to 31/60 (52%) of mice receiving MEB4 cells and 7/15 (47%) of mice receiving the MS2 sense-transfected cells (p < 0.001 and p = 0.005, respectively). These findings demonstrate that stable transfection of glucosylceramide synthase antisense reduces cellular glycosphingolipid levels and reduces tumorigenicity, providing further experimental support for an enhancing role of gangliosides in tumor formation.