Proteasome Stress Triggers Death of SH-SY5Y and T98G Cells via Different Cellular Mechanisms.

Overload or dysfunction of ubiquitin-proteasome system (UPS) is implicated in mechanisms of neurodegeneration associated with neurodegenerative diseases, e.g. Parkinson and Alzheimer disease, and ischemia-reperfusion injury. The aim of this study was to investigate the possible association between viability of neuroblastoma SH-SY5Y and glioblastoma T98G cells treated with bortezomib, inhibitor of 26S proteasome, and accumulation of ubiquitin-conjugated proteins with respect to direct cytotoxicity of aggregates of ubiquitin-conjugated proteins. Bortezomib-induced death of SH-SY5Y cells was documented after 24 h of treatment while death of T98G cells was delayed up to 48 h. Already after 4 h of treatment of both SH-SY5Y and T98G cells with bortezomib, increased levels of both ubiquitin-conjugated proteins with molecular mass more than 150 kDa and Hsp70 were observed whereas Hsp90 was elevated in T98G cells and decreased in SH-SY5Y cells. With respect to the cell death mechanism, we have documented bortezomib-induced activation of caspase 3 in SH-SY5Y cells that was probably a result of increased expression of pro-apoptotic proteins, PUMA and Noxa. In T98G cells, bortezomib-induced expression of caspase 4, documented after 24 h of treatment, with further activation of caspase 3, observed after 48 h of treatment. The delay in activation of caspase 3 correlated well with the delay of death of T98G cells. Our results do not support the possibility about direct cytotoxicity of aggregates of ubiquitin-conjugated proteins. They are more consistent with a view that proteasome inhibition is associated with both transcription-dependent and -independent changes in expression of pro-apoptotic proteins and consequent cell death initiation associated with caspase 3 activation.

Cyclin-dependent kinase 2 inhibitor SU9516 increases sensitivity of colorectal carcinoma cells Caco-2 but not HT29 to BH3 mimetic ABT-737.

Colorectal carcinoma (CRC) that represents one of the major causes for cancer-related death in humans is often associated with over-expression of anti-apoptotic proteins of Bcl-2 family. The aim of presented study was to determine the effect of ABT-737 inhibitor of anti-apoptotic proteins Bcl-2, Bcl-XL and Bcl-w as well as cyclin-dependent kinase 2 (CDK2) inhibitor SU9516 alone and in combination with ABT-737 on survival of colorectal cell lines HT29 and Caco-2. We have shown that both Caco-2 and HT29 cells that are relatively resistant to ABT-737 are also partially sensitive to SU9516, which increased sensitivity of Caco-2 but not HT29 cells to ABT-737. Increased sensitivity of Caco-2 cells to ABT-737 after addition of SU9516 correlated well with SU9516-induced decrease of Mcl-1 expression while we have not observed downregulation of Mcl-1 after the treatment of HT29 cells with SU9516. Instead of this, we have shown that treatment of HT29 cells with SU9516 is associated with decreased expression of tumour suppressor protein p53. Our findings provide a rationale for clinical use of Bcl-2 family inhibitors in combination with CDK2 inhibitors for treatment of Mcl-1-dependent colorectal tumours associated with expression of Bcl-2, Bcl-XL and Bcl-w proteins. In addition, we have shown potential of CDK2 inhibitors for treatment of tumours expressing R273H mutant p53.

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.

Targeting of Bcl-2 family proteins for treatment of acute leukaemia.

Many studies suggest that killing of tumour cell by commonly used therapies (chemotherapy, radiotherapy) is mediated primarily by induction of apoptosis. Therefore, resistance of tumour cells to therapy can be caused by a failure in the ability to initiate apoptosis. Defects in apoptosis signalling pathways are also one of the main features of cancer, and particularly acute leukaemia. Malignant cells constantly resist the effects of cellular stress (e.g. DNA damage, oncogene activation), which would cause death of normal cells through apoptosis. Dysregulation of apoptosis has therefore give rise to tumour growth, disease progression and resistance of malignant cells to chemotherapy. Structural analysis of Bcl-2 family proteins playing a key role in regulation of mitochondrial apoptosis together with studies of their biochemical functions have outlines strategies for generation of drugs, resulting in numerous novel chemical entities with potential to reverse resistance of malignant cells to apoptosis. The use of these therapeutic approaches may in the future represent a new way in cancer therapy with high potential to improve clinical outcome and prognosis of acute leukaemia patients.