Regulation Of Inducible Nitric Oxide Synthase (NOS2) By Ganglioside GD1a In Murine Osteosarcoma Cell

Inducible nitric oxide synthase (iNOS, NOS2) has been shown to be over expressed in a number of tumors compared to normal tissues and related to tumor cell growth and metastasis. Therefore it potentially represents an exploitable target in future anticancer therapy. Previously, it has been demonstrated that ganglioside GD1a suppresses the metastasis of FBJ virus-induced murine osteosarcoma cells. To elucidate the mechanism, research has been carried out to clarify which molecules possibly related to tumor metastasis are regulated by GD1a, using GD1a lacking FBJ-LL and FBJ-M5 cells, and GD1a-enriched FBJ-S1 and FBJ-LA5-30 cells. The present study has shown that NOS2 expression and activity were inversely regulated by ganglioside GD1a in FBJ cells as revealed by RT-PCR, Western blotting and NO concentration determination. An increase in GD1a in FBJ-LL cells following GM2/GD2 synthase cDNA transfection suppressed NOS2, and a decrease in GDI a by double-stranded RNA (dsRNA) directed toward GM2/GD2 synthase in FBJ-S1 cells augmented NOS2 expression. Addition of GD1a directly to FBJ-LL cells also induced a decrease in NOS2. Taken together it is evident that NOS2 in FBJ cells is under the control of ganglioside GD1a. To explore the possible pathways by which GD1a controls NOS2 expression, several signaling pathway inhibitors were given to the cells, especially MAPK pathway inhibitors, because it has been reported that MAPK pathway is a major pathway that regulates NOS2 expression. Co-treatment of cells with GD1a and ERK1/2 inhibitor U0126 blocked the inhibition of NOS2 by GD1a in FBJ-LL cells. However, co-treatment of cells with GD1a and the other MAPK inhibitors such as SB203580 and SP600125, which are inhibitors of P38 and JNK, respectively, did not change the suppression. Moreover, the expression of NOS2 was increased when FBJ-LL cells were transfected with small interfering RNA (siRNA) directed toward ERK1/2 to down-regulate ERK1/2 expression. Thus, the GD1a signal suggested to be mediated via ERK1/2 pathway in FBJ virus-induced murine osteosarcoma cells. This study also indicates that GD1a may suppress FBJ cells metastasis through NOS2.