Effects Of PI3K/Akt/mTOR Signalling Pathway On Biologic Behavior Of Diffuse Large B Cell Lymphoma Cell And The Expression Of BCL6

Effects of PI3K/Akt/mTOR signalling pathway on BiologicBehavior of Diffuse Large B Cell Lymphoma Cell and theExpression of BCL6Objective To observe the status of Akt and phospha-Akt(pAkt) inthree diffuse large B cell lyphoma(DLBCL) cell lines, investigatethe effects of Akt activation on cell biologic behavior in DLBCLcell lines, further research the role of Akt in the control of cellcycle progression and the cheraotherapeutic sensitization of thePI3K inhibitor LY294002 .Methods Three DLBCL cell lines ly1, ly8 and ly10 were maintainedin 10%FBS or serum free culture medium. The expression of Akt,pAkt, pS6K1 pGSK3, cyclinD1, p27^Kip1 ,p21^WAF1 , peIF4G, peIF4E and BCL6were detected by Western blotting . LY294002, the inhibitor of PI3K,were used to decrease the level of phospha-Akt, insulin to increasethe level of phospha-Akt and Rapamycin to inhibit the mTOR(mammalian target of Rapamycin) ,and the effects were detectedby Western blotting. The three cell lines were treated withLY294002, doxorubicin combined or sequenced usage of both, andcontrolled by untreatment respectively. Flow cytometry combinedwith PI staining, Annexin V-FITC assay and Brdu incorperationassay were used to analyze the afffections of cell cycle, apotosisand proliferation respectively.Results There were constitutive activation of Akt in three DLBCLcell lines and the levels of phospha-Akt were altered in thedifferent environment. In 10%FBS culture medium phospha-Akt washigher than that in serum free culture medium in ly8 and ly10,however phospha-Akt in ly1 maintained in serum free culture mediumwas a little higher than that in lO%FBS culture medium. When thecell lines ly1,ly8,ly10 were maintained in 10%FBS culture medium,the inhibitor LY294002 can decrease the level of phospha-Aktefficiently in three DLBCL cell lines. The ratio of S phase and proliferation were significantly decreased(P<0．05) ; whileapoptosis was not seen to be increased(P>0．05) . In three DLBCL celllines maintained in 10%FBS culture medium insulin increased thelevel of phospha-Akt efficiently , and Rapamycin decreased thelevel of phospha-S6K1 but increased the level of phospha-Aktsignificantly (P<0．05) ; their usage have no effects on the biologybehaviour of DLBCL cell. When the three cell lines cultureed in 10%FBS culture medium LY294002 can increase the level of pGSK3 andp27^Kip1 (P<0．05) , decreased cyclinD1 (P<0．05) , but have no effects onp21^WAF1 . In the investigation of chemotherapeutic sensitization, thesequenced usage of LY294002 and doxorubicin increased the ratioof apoptosis obviously and there were significant differencebetween the sequenced group and the other four groups (P<0．05) at24h, 48h,72h (ly1) ,48h,72h (ly8) or 24h (ly10) . In ly1 usageof LY294002 decreased the expression of BCL6 significantly at 2h(P<0．05) , and decreased the level of phospha-eIF4Gsignificantly(P<0．05) , but caused eIF4E be phosphorylated;Rapamycin decreased the expression of BCL6 1/2h(P<0．05) and thelevel of phospha-peIF4G at 1/2h(P<0．05) , but caused eIF4E bephosphorylated too.Conclusions LY294002, Rapamycin and insulin can ajust the siganlof PI3K/Akt/mTOR pathway effectively. Activation of Akt may playan important role in development of DLBCL. It is closely relatedto control of cell cycle and proliferation through p27^Kip1 andcyclinD1 , but is not associated with apoptosis. LY294002 may bea potential molecular therapeutic drug targeting Akt signallingpathway in DLBCL. Activated Akt/mTOR pathway can control theexpression of BCL6 through initiation factors eIF4G and eIF4E.