Short Chemical Ischemia Triggers Phosphorylation of eIF2α and Death of SH-SY5Y Cells but not Proteasome Stress and Heat Shock Protein Response in both SH-SY5Y and T98G Cells.

Both translation arrest and proteasome stress associated with accumulation of ubiquitin-conjugated protein aggregates were considered as a cause of delayed neuronal death after transient global brain ischemia; however, exact mechanisms as well as possible relationships are not fully understood. The aim of this study was to compare the effect of chemical ischemia and proteasome stress on cellular stress responses and viability of neuroblastoma SH-SY5Y and glioblastoma T98G cells. Chemical ischemia was induced by transient treatment of the cells with sodium azide in combination with 2-deoxyglucose. Proteasome stress was induced by treatment of the cells with bortezomib. Treatment of SH-SY5Y cells with sodium azide/2-deoxyglucose for 15 min was associated with cell death observed 24 h after treatment, while glioblastoma T98G cells were resistant to the same treatment. Treatment of both SH-SY5Y and T98G cells with bortezomib was associated with cell death, accumulation of ubiquitin-conjugated proteins, and increased expression of Hsp70. These typical cellular responses to proteasome stress, observed also after transient global brain ischemia, were not observed after chemical ischemia. Finally, chemical ischemia, but not proteasome stress, was in SH-SY5Y cells associated with increased phosphorylation of eIF2α, another typical cellular response triggered after transient global brain ischemia. Our results showed that short chemical ischemia of SH-SY5Y cells is not sufficient to induce both proteasome stress associated with accumulation of ubiquitin-conjugated proteins and stress response at the level of heat shock proteins despite induction of cell death and eIF2α phosphorylation.

Differential impact of bortezomib on HL-60 and K562 cells.

Bortezomib (PS-341, or Velcade), reversible inhibitor of 20S proteasome approved for the treatment of multiple myeloma and mantle cell lymphoma, exhibited a cytotoxic effect toward other malignancies including leukaemia. In this study, we have documented that incubation of both HL-60 and K562 leukaemia cells with nanomolar concentrations of bortezomib is associated with the death of HL-60 cells observed within 24 hours of incubation with bortezomib and the death of K562 cells that were observed after 72 hours of incubation with bortezomib. The relative resistance of K562 cells to bortezomib correlated well with significantly higher expression of HSP27, HSP70, HSP90α, HSP90ß and GRP75 in these cells. Incubation of both HL-60 and K562 cells with bortezomib induced a cleavage of HSP90ß as well as expression of HSP70 and HSP90ß but bortezomib did not affect levels of HSP27, HSP90α, GRP75 and GRP78. The death of both types of cells was accompanied with proteolytic activation of caspase 3 that was observed in HL-60 cells and proteolytic degradation of procaspase 3 in K562 cells. Our study has also pointed to essential role of caspase 8 in bortezomib-induced cleavage of HSP90ß in both HL-60 and K562 cells. Finally, we have shown that bortezomib induced activation of caspase 9/caspase 3 axis in HL-60 cells, while the mechanism of death of K562 cells remains unknown.

ABT-737 accelerates butyrate-induced death of HL-60 cells. Involvement of mitochondrial apoptosis pathway.

The aim of presented study was to determine effect of NaBu in combination with ABT-737 on cell survival of leukemic cell line HL-60. In addition, analysis of molecular mechanism of NaBu action with a focus on mitochondrial apoptosis was performed. Both NaBu and ABT-737 are inducing death of HL-60 cells with different kinetics. ABT-737-induced cell death is fast while NaBu-induced death preceded by cell cycle arrest in G2 phase is rather slow. Cell viability preceded by cell cycle arrest in G2 phase was significantly decreased after 48 hours of incubation with 2 and 5 mmol/l of NaBu while it was significantly decreased after 24 hours of incubation with 1 µmol/l of ABT-737 combined with 2 and 5 mmol/l of NaBu. Incubation of HL-60 cells with NaBu was associated with increased level of pro-apoptotic protein BIMEL and decreased levels of anti-apoptotic proteins of Bcl-2 family as well as GRP78 involved in ER stress signalling. It seems that ABT-737 accelerates NaBu-induced death of HL-60 cells due to mitochondrial apoptosis resulting from ABT-737-mediated inhibition of functions and NaBu-induced decrease of the levels of anti-apoptotic Bcl-2 family proteins as well as due to accelerated decrease of GRP78 observed after the treatment of cells with combination of NaBu and ABT-737. The effect of combination of both drugs on survival of HL-60 cells seems to be synergistic at high concentrations of NaBu (2 and 5 mmol/) while it is rather antagonistic at concentrations of NaBu less than 1 mmol/l. Finally, it might be assumed that NaBu is capable to induce cell death with mechanisms independent from mitochondrial apoptosis.