Cytosolic Sialidase Neu2 Suppresses FBJ Cell Growth Through Cdk2 Signaling Pathway

Cytosolic sialidase neuraminidase 2 (Neu2) is a kind of glycoside hydrolase enzyme which, localized in the cytoplasm, and cleaves sialic acid residues from the glycoconjugates. To explore the role of Neu2 in pathways where cell growth was regulated by sialylated glycoconjugates, we transfected FBJ-S1 cells with Neu2 cDNA containing plasmid to over-express Neu2 mRNA expression. The Neu2 mRNA expression assessed by RT-PCR and total sialidase activity analysis determined by the use of 4-MU-NeuAC revealed a significant enhancement of Neu2 expression in Neu2 cDNA introduced transfectants. The over-expression of Neu2 induced G2/M arrest as detected by flow cytometry using proidium iodide stained cells. Cell growth retardation in Neu2 over-expressed cells was further confirmed by WST assay. To elucidate the mechanisms of these inhibitory effects, determination of cell cycle and apoptosis related molecules at mRNA was performed. RT-PCR results manifested that the G1/S regulating cyclin dependent kinase (Cdk2) and anti-apoptotic Bcl2 were down-regulated by Neu2 over-expression. To further confirm the above observations, the siRNAs targeting Neu2 were also introduced into FBJ-S1 cells. The results exhibited a profound decrease of Neu2 at both mRNA and enzyme activity levels. In the Neu2-silenced FBJ-S1 cells Cdk2 and Bcl2 mRNA expression was impaired. Silencing of either Cdk2 or Bcl2 revealed that Cdk2 is located at the up-stream of Bcl2 in Bcl2 signaling pathway. In order to find out the sialylated glycoproteins which were targeted and degradated by Neu2 in FBJ-S1 cells, biotinylated lectin blotting was used to detect the change in sialylated glycoproteins. However, the sialylated glycoproteins recognized by MAM and SSA respectively failed to show any significant change between the Neu2 cDNA and vector transfected FBJ-S1 cells.This study indicates the regulatory role of Neu2 in FBJ-S1 cell growth via Cdk2 and Bcl2 signaling pathways. To confirm the cell growth inhibitory effect of Neu2, the Neu2 cDNA was introduced into another highly metastatic FBJ-LL cell line, and two G418 resistant Neu2 cDNA transfected monoclonal FBJ-LL cell lines were obtained. As similar as the results of FBJ-S1 cells, introduction of Neu2 cDNA into FBJ-LL cells induced a significant elevation of Neu2 mRNA expression and about 1.5 times increase of total sialidase activity in these monoclonal transfectants. Neu2 over-expression also caused significant repression on the growth of the two stable transfectants. Different with FBJ-S1 cells, Neu2 over-expression caused a G1 arrest in FBJ-LL cells. The RT-PCR results revealed that Cdk2 and Bcl2 mRNA expression was suppressed in Neu2 over-expressed FBJ-LL cells as same as FBJ-S1 cells, but Cyclin B2 mRNA expression was down-regulated only in Neu2 cDNA stablely transfected FBJ-LL cells. siRNAs targeting Cdk2, Bcl2 and Cyclin B2 (Ccnb2) were introduced into FBJ-S1 cells, and silencing of Cdk2 resulted in the reduction of the Bcl2 and Ccnb2 mRNA expression which implies that Cdk2 is also located at up-stream of Bcl2 and Ccnb2 in FBJ-LL cells. This study confirmed the cell growth regulatory role of Neu2 in FBJ-LL through Cdk2 signaling pathways. Moreover, Bcl2 and Ccnb2 are regulated by Cdk2 which was reduced by Neu2 over-expression in FBJ-LL cells.