Contributions
Abstract: PB1902
Type: Publication Only
Background
HSP90 belongs to the heat shock protein family, a functional class of chaperone molecules that are transcriptionally upregulated by heat and other stressors. HSP90 facilitates the maturation, stability, activity, and intracellular folding of more than 200 proteins, called 'client proteins'. In cancer cells, HSP90 helps to overcome multiple environmental stresses, including genomic instability/aneuploidy, proteotoxic stress, increased nutrient demands, reduced oxygen levels, and to prevent destruction by the immune system. One of these client proteins of HSP90 is BCR-ABL, the oncoprotein responsible for Chronic Myeloid Leukemia (CML). Cancer cells that depend on this oncoprotein for survival are sensitive to HSP90 inhibition. Hsp90 inhibitors, by preventing nucleotide-dependent cycling interfere with the chaperone activity of HSP90, resulting in targeting of client proteins to proteasome degradation. Alvespimycin (17-DMAG) is an HSP90 inhibitor that has better pharmacokinetic properties and fewer side-effects compared to others benzoquinone ansamycins.
Aims
This work aims to study the effect of alvespimycin in chronic myeloid leukemia cell lines (sensitive and resistant to imatinib) and to explore the role of HSP family in the sensitivity to imatinib.
Methods
In this context, we used 3 CML cells lines: the K562 cells, sensitive to Imatinib, and the K562-RC and K562-RD cells resistant to Imatinib. Cells were incubated in the absence and presence of increasing concentrations of 17-DMAG (from 1 to 1000 nM), in a single dose. The dose-response curves were determined by resazurin assay. Cell death was determined by microscopy (May-Grunwald Giemsa staining) and by flow cytometry (FC), using Annexin V and Propidium Iodide (PI) double staining. The Apostat Probe was used to evaluate caspase expression levels and JC-1 probe to determine the mitochondrial membrane potential, by FC. Cell cycle was evaluated by FC, using PI/RNase assay. The protein expression levels of HSP family were analyzed by western blot.
Results
Our results showed that 17-DMAG induce a reduction in cell lines viability, with an IC50 of 50 nM for K562 and K562-RD cells and lower than 50 nM for the K562-RC cell line, after 48 hours of treatment. This compound induces cell death predominantly by apoptosis, confirmed by morphological analysis, FC and by the increase of JC-1 Monomers/Aggregates ratio. Furthermore, 17-DMAG induces cell cycle arrests in K562 in G0/G1 phase. The HSP protein analysis showed that K562-RC have slightly increased in HSP90 expression comparing with K562 cells.
Conclusion
In conclusion, our results suggest that inhibition of HSP90 by alvespimycin (17-DMAG) could be used as a new potential approach in the treatment of CML, even in case of Imatinib resistance.
This work was supported by Center of Investigation in Environment, Genetics, and Oncobiology (CIMAGO).
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): Chronic myeloid leukemia, Heat shock protein, Treatment
Abstract: PB1902
Type: Publication Only
Background
HSP90 belongs to the heat shock protein family, a functional class of chaperone molecules that are transcriptionally upregulated by heat and other stressors. HSP90 facilitates the maturation, stability, activity, and intracellular folding of more than 200 proteins, called 'client proteins'. In cancer cells, HSP90 helps to overcome multiple environmental stresses, including genomic instability/aneuploidy, proteotoxic stress, increased nutrient demands, reduced oxygen levels, and to prevent destruction by the immune system. One of these client proteins of HSP90 is BCR-ABL, the oncoprotein responsible for Chronic Myeloid Leukemia (CML). Cancer cells that depend on this oncoprotein for survival are sensitive to HSP90 inhibition. Hsp90 inhibitors, by preventing nucleotide-dependent cycling interfere with the chaperone activity of HSP90, resulting in targeting of client proteins to proteasome degradation. Alvespimycin (17-DMAG) is an HSP90 inhibitor that has better pharmacokinetic properties and fewer side-effects compared to others benzoquinone ansamycins.
Aims
This work aims to study the effect of alvespimycin in chronic myeloid leukemia cell lines (sensitive and resistant to imatinib) and to explore the role of HSP family in the sensitivity to imatinib.
Methods
In this context, we used 3 CML cells lines: the K562 cells, sensitive to Imatinib, and the K562-RC and K562-RD cells resistant to Imatinib. Cells were incubated in the absence and presence of increasing concentrations of 17-DMAG (from 1 to 1000 nM), in a single dose. The dose-response curves were determined by resazurin assay. Cell death was determined by microscopy (May-Grunwald Giemsa staining) and by flow cytometry (FC), using Annexin V and Propidium Iodide (PI) double staining. The Apostat Probe was used to evaluate caspase expression levels and JC-1 probe to determine the mitochondrial membrane potential, by FC. Cell cycle was evaluated by FC, using PI/RNase assay. The protein expression levels of HSP family were analyzed by western blot.
Results
Our results showed that 17-DMAG induce a reduction in cell lines viability, with an IC50 of 50 nM for K562 and K562-RD cells and lower than 50 nM for the K562-RC cell line, after 48 hours of treatment. This compound induces cell death predominantly by apoptosis, confirmed by morphological analysis, FC and by the increase of JC-1 Monomers/Aggregates ratio. Furthermore, 17-DMAG induces cell cycle arrests in K562 in G0/G1 phase. The HSP protein analysis showed that K562-RC have slightly increased in HSP90 expression comparing with K562 cells.
Conclusion
In conclusion, our results suggest that inhibition of HSP90 by alvespimycin (17-DMAG) could be used as a new potential approach in the treatment of CML, even in case of Imatinib resistance.
This work was supported by Center of Investigation in Environment, Genetics, and Oncobiology (CIMAGO).
Session topic: 7. Chronic myeloid leukemia – Biology & Translational Research
Keyword(s): Chronic myeloid leukemia, Heat shock protein, Treatment