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Identification of heat shock protein 32 (Hsp32) as a novel target in acute lymphoblastic leukemia.

Cerny-Reiterer S, Meyer RA, Herrmann H, Peter B, Gleixner KV, Stefanzl G, Hadzijusufovic E, Pickl WF, Sperr WR, Melo JV, Maeda H, Jäger U, Valent P - Oncotarget (2014)

Bottom Line: Hsp32-targeting drugs were found to synergize with imatinib, nilotinib, and bendamustine in producing growth inhibition and apoptosis in Ph+ ALL cells.A siRNA against Hsp32 was found to inhibit growth and survival of ALL cells and to synergize with imatinib in suppressing the growth of ALL cells.In conclusion, Hsp32 is an essential survival factor and potential new target in ALL.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Austria.

ABSTRACT
Heat shock proteins (Hsp) are increasingly employed as therapeutic targets in oncology. We have shown that Hsp32, also known as heme oxygenase-1 (HO-1), serves as survival factor and potential target in Ph+ chronic myeloid leukemia. We here report that primary cells and cell lines derived from patients with acute lymphoblastic leukemia (ALL) express Hsp32 mRNA and the Hsp32 protein in a constitutive manner. Highly enriched CD34+/CD38- ALL stem cells also expressed Hsp32. Two Hsp32-targeting drugs, pegylated zinc protoporphyrine (PEG-ZnPP) and styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP), induced apoptosis and growth arrest in the BCR/ABL1+ cell lines, in Ph- lymphoblastic cell lines and in primary Ph+ and Ph- ALL cells. The effects of PEG-ZnPP and SMA-ZnPP on growth of leukemic cells were dose-dependent. In Ph+ ALL, major growth-inhibitory effects of the Hsp32-targeting drugs were observed in imatinib-sensitive and imatinib-resistant cells. Hsp32-targeting drugs were found to synergize with imatinib, nilotinib, and bendamustine in producing growth inhibition and apoptosis in Ph+ ALL cells. A siRNA against Hsp32 was found to inhibit growth and survival of ALL cells and to synergize with imatinib in suppressing the growth of ALL cells. In conclusion, Hsp32 is an essential survival factor and potential new target in ALL.

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Hsp32-targeting drugs induce apoptosis in ALL cellsA: Primary leukemic cells obtained from patient #17 with Ph+ ALL (imatinib-sensitive phase) and patient #18 with Ph− ALL were incubated in control medium (Co) or medium containing increasing concentrations of either PEG-ZnPP or SMA-ZnPP at 37°C for 24 hours. Thereafter, the numbers of apoptotic cells were determined by light microscopy (upper panels) and AnnexinV staining and flow cytometry (lower panels). B: The lymphoid cells lines BV-173, TOM-1, REH and BL-41 were incubated in control medium (Co) or increasing concentrations of either SMA-ZnPP (left panel) or PEG-ZnPP (right panel) at 37°C for 48 hours. Then, the number (percentage) of apoptotic cells was determined by light microscopy. Asterisk (*): p<0.05. C: A Tunel assay was performed with Z-119 cells after incubation in control medium (Control), PEG-ZnPP (5 μM) or SMA-ZnPP (10 μM) at 37°C for 48 hours. After incubation, cells were analyzed under a fluorescence microscope and photographs taken as described in the text. Original magnification x 40. D: Flow cytometric evaluation of expression of active caspase 3 in two Ph+ALL cell lines (BV-173, TOM-1) and two Ph−ALL cell lines (REH, BL-41) after incubation in control medium or medium containing 10 μM of SMA-ZnPP. Results represent the mean±S.D. of three independent experiments.
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Figure 3: Hsp32-targeting drugs induce apoptosis in ALL cellsA: Primary leukemic cells obtained from patient #17 with Ph+ ALL (imatinib-sensitive phase) and patient #18 with Ph− ALL were incubated in control medium (Co) or medium containing increasing concentrations of either PEG-ZnPP or SMA-ZnPP at 37°C for 24 hours. Thereafter, the numbers of apoptotic cells were determined by light microscopy (upper panels) and AnnexinV staining and flow cytometry (lower panels). B: The lymphoid cells lines BV-173, TOM-1, REH and BL-41 were incubated in control medium (Co) or increasing concentrations of either SMA-ZnPP (left panel) or PEG-ZnPP (right panel) at 37°C for 48 hours. Then, the number (percentage) of apoptotic cells was determined by light microscopy. Asterisk (*): p<0.05. C: A Tunel assay was performed with Z-119 cells after incubation in control medium (Control), PEG-ZnPP (5 μM) or SMA-ZnPP (10 μM) at 37°C for 48 hours. After incubation, cells were analyzed under a fluorescence microscope and photographs taken as described in the text. Original magnification x 40. D: Flow cytometric evaluation of expression of active caspase 3 in two Ph+ALL cell lines (BV-173, TOM-1) and two Ph−ALL cell lines (REH, BL-41) after incubation in control medium or medium containing 10 μM of SMA-ZnPP. Results represent the mean±S.D. of three independent experiments.

Mentions: Hsp32 has been described as a survival factor counteracting apoptosis in various neoplastic cells. We next investigated whether the growth-inhibitory effects of Hsp32 inhibitors (SMA-ZnPP and PEG-ZnPP) are associated with induction of apoptosis in ALL cells. We found that both drugs induce apoptosis in primary ALL cells and in the ALL cell lines tested (Figure 3 and Table 2). The apoptosis-producing effects of SMA-ZnPP and PEG-ZnPP on ALL cells were demonstrable by light microscopy (Figure 3A and 3B) as well as in a Tunel assay (Figure 3C). Furthermore, we were able to show by flow cytometry that SMA-ZnPP induces activation of caspase-3 in ALL cells (Figure 3D). In normal bone marrow cells, neither SMA-ZnPP nor PEG-ZnPP were found to induce apoptosis over the dose-range tested (1-40 μM) confirming previous data [29].


Identification of heat shock protein 32 (Hsp32) as a novel target in acute lymphoblastic leukemia.

Cerny-Reiterer S, Meyer RA, Herrmann H, Peter B, Gleixner KV, Stefanzl G, Hadzijusufovic E, Pickl WF, Sperr WR, Melo JV, Maeda H, Jäger U, Valent P - Oncotarget (2014)

Hsp32-targeting drugs induce apoptosis in ALL cellsA: Primary leukemic cells obtained from patient #17 with Ph+ ALL (imatinib-sensitive phase) and patient #18 with Ph− ALL were incubated in control medium (Co) or medium containing increasing concentrations of either PEG-ZnPP or SMA-ZnPP at 37°C for 24 hours. Thereafter, the numbers of apoptotic cells were determined by light microscopy (upper panels) and AnnexinV staining and flow cytometry (lower panels). B: The lymphoid cells lines BV-173, TOM-1, REH and BL-41 were incubated in control medium (Co) or increasing concentrations of either SMA-ZnPP (left panel) or PEG-ZnPP (right panel) at 37°C for 48 hours. Then, the number (percentage) of apoptotic cells was determined by light microscopy. Asterisk (*): p<0.05. C: A Tunel assay was performed with Z-119 cells after incubation in control medium (Control), PEG-ZnPP (5 μM) or SMA-ZnPP (10 μM) at 37°C for 48 hours. After incubation, cells were analyzed under a fluorescence microscope and photographs taken as described in the text. Original magnification x 40. D: Flow cytometric evaluation of expression of active caspase 3 in two Ph+ALL cell lines (BV-173, TOM-1) and two Ph−ALL cell lines (REH, BL-41) after incubation in control medium or medium containing 10 μM of SMA-ZnPP. Results represent the mean±S.D. of three independent experiments.
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Related In: Results  -  Collection

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Show All Figures
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Figure 3: Hsp32-targeting drugs induce apoptosis in ALL cellsA: Primary leukemic cells obtained from patient #17 with Ph+ ALL (imatinib-sensitive phase) and patient #18 with Ph− ALL were incubated in control medium (Co) or medium containing increasing concentrations of either PEG-ZnPP or SMA-ZnPP at 37°C for 24 hours. Thereafter, the numbers of apoptotic cells were determined by light microscopy (upper panels) and AnnexinV staining and flow cytometry (lower panels). B: The lymphoid cells lines BV-173, TOM-1, REH and BL-41 were incubated in control medium (Co) or increasing concentrations of either SMA-ZnPP (left panel) or PEG-ZnPP (right panel) at 37°C for 48 hours. Then, the number (percentage) of apoptotic cells was determined by light microscopy. Asterisk (*): p<0.05. C: A Tunel assay was performed with Z-119 cells after incubation in control medium (Control), PEG-ZnPP (5 μM) or SMA-ZnPP (10 μM) at 37°C for 48 hours. After incubation, cells were analyzed under a fluorescence microscope and photographs taken as described in the text. Original magnification x 40. D: Flow cytometric evaluation of expression of active caspase 3 in two Ph+ALL cell lines (BV-173, TOM-1) and two Ph−ALL cell lines (REH, BL-41) after incubation in control medium or medium containing 10 μM of SMA-ZnPP. Results represent the mean±S.D. of three independent experiments.
Mentions: Hsp32 has been described as a survival factor counteracting apoptosis in various neoplastic cells. We next investigated whether the growth-inhibitory effects of Hsp32 inhibitors (SMA-ZnPP and PEG-ZnPP) are associated with induction of apoptosis in ALL cells. We found that both drugs induce apoptosis in primary ALL cells and in the ALL cell lines tested (Figure 3 and Table 2). The apoptosis-producing effects of SMA-ZnPP and PEG-ZnPP on ALL cells were demonstrable by light microscopy (Figure 3A and 3B) as well as in a Tunel assay (Figure 3C). Furthermore, we were able to show by flow cytometry that SMA-ZnPP induces activation of caspase-3 in ALL cells (Figure 3D). In normal bone marrow cells, neither SMA-ZnPP nor PEG-ZnPP were found to induce apoptosis over the dose-range tested (1-40 μM) confirming previous data [29].

Bottom Line: Hsp32-targeting drugs were found to synergize with imatinib, nilotinib, and bendamustine in producing growth inhibition and apoptosis in Ph+ ALL cells.A siRNA against Hsp32 was found to inhibit growth and survival of ALL cells and to synergize with imatinib in suppressing the growth of ALL cells.In conclusion, Hsp32 is an essential survival factor and potential new target in ALL.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Boltzmann Cluster Oncology, Medical University of Vienna, Austria.

ABSTRACT
Heat shock proteins (Hsp) are increasingly employed as therapeutic targets in oncology. We have shown that Hsp32, also known as heme oxygenase-1 (HO-1), serves as survival factor and potential target in Ph+ chronic myeloid leukemia. We here report that primary cells and cell lines derived from patients with acute lymphoblastic leukemia (ALL) express Hsp32 mRNA and the Hsp32 protein in a constitutive manner. Highly enriched CD34+/CD38- ALL stem cells also expressed Hsp32. Two Hsp32-targeting drugs, pegylated zinc protoporphyrine (PEG-ZnPP) and styrene maleic acid-micelle-encapsulated ZnPP (SMA-ZnPP), induced apoptosis and growth arrest in the BCR/ABL1+ cell lines, in Ph- lymphoblastic cell lines and in primary Ph+ and Ph- ALL cells. The effects of PEG-ZnPP and SMA-ZnPP on growth of leukemic cells were dose-dependent. In Ph+ ALL, major growth-inhibitory effects of the Hsp32-targeting drugs were observed in imatinib-sensitive and imatinib-resistant cells. Hsp32-targeting drugs were found to synergize with imatinib, nilotinib, and bendamustine in producing growth inhibition and apoptosis in Ph+ ALL cells. A siRNA against Hsp32 was found to inhibit growth and survival of ALL cells and to synergize with imatinib in suppressing the growth of ALL cells. In conclusion, Hsp32 is an essential survival factor and potential new target in ALL.

Show MeSH
Related in: MedlinePlus