Studies On Mechanisms Of Metformin’s Inhibition Of BAFF-Dependent Proliferation And Survival Via MTOR-PTEN/Akt Pathway In B Cells

The present study,using cellular and molecular biology methods and techniques including Western blotting,RNA interference,cell counting,MTS assay,trypan blue exclusion,etc.,and employing Raji cells and mouse primary spleen B lymphocytes(Primary B)as experimental objects,investigated molecular mechanisms of metformin inhibition of hsBAFF-promoted proliferation and survival by mTOR/S6K1 via PTEN/Akt-Erk1/2 pathway in B cells.The detailed results were summarized as follows: 1.Metformin inhibits hsBAFF-promoted proliferation and survival via Erk1/2 pathway in B cellsRaji cells and Primary B cells,or Raji cells infected with shRNA Erk1/2 and shRNA GFP,or Raji cells infected with Ad-MKK1-R4 F,Ad-MKK1-K97 M and Ad-GFP were pretreated with different concentrations of metformin(1-5 mM)for 4 h and then stimulated with/without hsBAFF(2.5 μg/ml)for 12 h or 48 h,or pretreated with/without metformin(1.5 mM)for 4 h and then stimulated with/without hsBAFF(2.5 μg/ml)for 12 h or 48 h,or pretreated with/without U0126(5 μM)for 1 h and then stimulated with/without hsBAFF(2.5 μg/ml)for 12 h or 48 h.After that,the expressions of related proteins were determined by Western blotting;Cells counting,MTS assay and trypan blue exclusion were conducted to evaluate cell proliferation and survival,respectively.The results showed that metformin inhibited hsBAFF-stimulated B-cell proliferation and survival in a dose-dependent manner;Metformin inhibited hsBAFF-induced Erk1/2 phosphorylation in B cells;Inhibition of Erk1/2 enhanced metformin’s inhibition of hsBAFF-promoted B-cell proliferation and survival;Metformin inhibits hsBAFF-promoted B-cell proliferation and survival through MKK1-Erk1/2 pathway.The data suggest that metformin inhibits hsBAFF-promoted proliferation and survival via Erk1/2 pathway in B cells.2.Metformin suppresses hsBAFF-promoted proliferation and survival via PTEN/Akt-Erk1/2 pathway in B cellsRaji cells and Primary B cells,Raji cells infected with Ad-PTEN,Ad-dn-Akt and Ad-GFP were pretreated with different concentrations of metformin(1-5 mM)for 4 h and then stimulated with/without hsBAFF(2.5 μg/ml)for 12 h or 48 h,or pretreated with/without metformin(1.5 mM)for 4 h and then Akt inhibitor X(20 μM)or U0126(5 μM)for 1 h,followed by stimulating with/without hsBAFF(2.5 μg/ml)for 12 h or 48 h.After that,the expressions of related proteins were determined by Western blotting;Cells counting and MTS assay were conducted to evaluate cell proliferation and survival,respectively.The results showed that metformin inhibited hsBAFF-induced phosphorylation of PTEN and Akt in B cells;Expreesion of dn-Akt strengthened metformin inhibition of hsBAFF-activated Erk1/2-dependent B-cell proliferation and survival;Overexpression of PTEN potentiated metformin blockage of hsBAFF-induced PTEN/Akt-Erk1/2-dependent B-cell proliferation and survival.The data suggest that metformin inhibits hsBAFF-promoted proliferation and survival viah PTEN/Akt-Erk1/2 pathway in B cells.3.Metformin blocks hsBAFF-promoted proliferation and survival via mTOR/S6K1 signaling in B cellsRaji cells and Primary B cells,Raji cells infected with shRNA mTOR and shRNA GFP were pretreated with different concentrations of metformin(1-5 mM or 1.5 mM)for 4 h and then stimulated with/without hsBAFF(2.5 μg/ml)for 12 h or 48 h,or pretreated with/without metformin(1.5 mM)for 4 h and then rapamycin(100 ng/ml)for 2 h,followed by stimulating with/without hsBAFF(2.5 μg/ml)for 12 h or 48 h.After that,the expressions of related proteins were determined by Western blotting;Cells counting and MTS assay were conducted to evaluate cell proliferation and survival,respectively.The results showed that metformin inhibited hsBAFF-induced S6K1 phosphorylation in B cells;Rapamycin strengthened metformin-inhibited S6K1 and PTEN/Akt-Erk1/2 pathway and thus attenuated hsBAFF-promoted B-cell proliferation and survival;Downregulation of mTOR enhanced metformin inhibition of hsBAFF-promoted B-cell proliferation and survival via PTEN/Akt-Erk1/2 pathway.The data suggest that metformin declines BAFF-promoted proliferation and survival by mTOR/S6K1 via PTEN/Akt-Erk1/2 signaling pathway in B cells.