The cleavage of XIAP and the down regulation of Bcl xL and survivin observed after 8 h of treatment with TRAIL and SAHA was stronger than after 16 h with TRAIL alone. Similar results were selleck chemicals obtained with TRAIL and NaB. Moreo ver, the kinetic of inactivation of anti apoptotic proteins is similar to that of caspases activation by proteolytic cleav ages. We have previously shown that the down regulation of XIAP, Bcl xL and RIP induced by TRAIL and chemothera peutic drugs was caspases dependent. To analyse if the reduction of the anti apoptotic proteins XIAP, Bcl xL, survivin, and RIP induced by co treatments with TRAIL and HDACIs is likewise caspases dependent, SH EP cells were treated with TRAIL and NaB, SAHA or TSA in the presence of the pan caspases inhibitor zVAD.
As shown in figure 5d, the cleavage of XIAP, and the inactivation of Bcl xL, survivin, and RIP were protected by the addition of zVAD. Similarly, the activation of Bid and cas pase 3 induced by TRAIL and HDACIs were protected by zVAD. These results indicate that sensitisation to TRAIL by low doses of HDACIs increases the caspases dependent cleavages and inactivation of anti apoptotic proteins. In addition, the amount of BimEL was increased in the presence of zVAD indicating that the down regula tion observed by combined treatment was also mediated by caspases dependent cleavage. Down regulation of survivin sensitise NB cells to HDACIs Survivin is over expressed in most human cancers includ ing NB and was shown to be involved in inhibition of apoptosis in tumour cells.
In addition, down regula tion of survivin by siRNAs was shown to sensitise NB cells to TRAIL induced apoptosis. As survivin steady state level is reduced by co treatment with TRAIL and HDACIs, we explored its participation in HDACIs mediated sensiti sation to TRAIL. In this aim, survivin expression, was down regulated in SH EP cells by RNA interference using survivin siRNAs. As shown in figure 6a, survivin expression level was significantly reduced by survivin siRNA as compared to cells transfected with lipofectamine2000 or control siRNA. The effect of sur vivin silencing was analysed by proliferation assay. Results show that reduction of survivin expression with 100 nM siRNAs sensitised NB cells to TRAIL alone, to low doses of HDACIs as well as to combined treatments with weak doses of TRAIL and HDACIs.
Similar results were observed with 25 nM of survivin siRNAs. These results Entinostat indicate that down regulation of survivin induced by HDACIs and TRAIL may account at least in part to the sensitising effect of HDACIs to TRAIL induced cell death. Discussion The present study demonstrates that simultaneous admin istration of TRAIL and subtoxic doses of HDACIs strongly potentiates the triggering of apoptotic cascade in NB cells.