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Sensitization of chemo-resistant human chronic myeloid leukemia stem-like cells to Hsp90 inhibitor by SIRT1 inhibition.

Kim HB, Lee SH, Um JH, Kim MJ, Hyun SK, Gong EJ, Oh WK, Kang CD, Kim SH - Int. J. Biol. Sci. (2015)

Bottom Line: The current investigation was undertaken to examine the effectiveness of the combination treatment of Hsp90 inhibitor and SIRT1 inhibitor in inhibiting the growth of chemo-resistant stem-like cells isolated from human chronic myeloid leukemia K562 cells.Inhibition of SIRT1 by use of SIRT1 siRNA or SIRT1 inhibitors (amurensin G and EX527) effectively potentiated sensitivity of Hsp90 inhibitors (17-AAG and AUY922) in CD44(high) K562 stem-like cells expressing high levels of CSC-related molecules including Oct4, CD34, β-catenin, c-Myc, mutant p53 (mut p53), BCRP and P-glycoprotein (P-gp) as well as CD44.Our data suggest that combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be an effective therapeutic approach to target CSCs that are resistant to current therapies.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Biochemistry, Pusan National University School of Medicine, Yangsan 626-870, Korea.

ABSTRACT
Development of effective therapeutic strategies to eliminate cancer stem-like cells (CSCs), which play a major role in drug resistance and disease recurrence, is critical to improve cancer treatment outcomes. The current investigation was undertaken to examine the effectiveness of the combination treatment of Hsp90 inhibitor and SIRT1 inhibitor in inhibiting the growth of chemo-resistant stem-like cells isolated from human chronic myeloid leukemia K562 cells. Inhibition of SIRT1 by use of SIRT1 siRNA or SIRT1 inhibitors (amurensin G and EX527) effectively potentiated sensitivity of Hsp90 inhibitors (17-AAG and AUY922) in CD44(high) K562 stem-like cells expressing high levels of CSC-related molecules including Oct4, CD34, β-catenin, c-Myc, mutant p53 (mut p53), BCRP and P-glycoprotein (P-gp) as well as CD44. SIRT1 depletion caused significant down-regulation of heat shock factor 1 (HSF1)/heat shock proteins (Hsps) as well as these CSC-related molecules, which led to the sensitization of CD44(high) K562 cells to Hsp90 inhibitor by SIRT1 inhibitor. Moreover, 17-AAG-mediated activation of HSF1/Hsps and P-gp-mediated efflux, major causes of Hsp90 inhibitor resistance, was suppressed by SIRT1 inhibitor in K562-CD44(high) cells. Our data suggest that combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be an effective therapeutic approach to target CSCs that are resistant to current therapies.

No MeSH data available.


Related in: MedlinePlus

Inhibition of protein levels of CSC-related molecules and Hsps, and activation of pro-apoptotic cascade by amurensin G, and suppression of mRNA levels of 17-AAG-mediated induction of Hsp70/Hsp27 and ABCB1/ABCG2 genes by amurensin G in CD44high K562 cells. A: Cells were pretreated with amurensin G (5 μg/ml) for 6 h, and treated with 17-AAG (1 or 10 μM) for additional 24 h, and the indicated proteins were detected by western blot analysis. CF; cleavage fragments. B: Relative mRNA levels of Hsp70/Hsp27 and ABCB1/ABCG2 genes in the cells treated with 17-AAG alone and co-treated with 17-AAG and amurensin G were determined by real time RT-PCR. Each bar represented the mean ± S.D. of triplicate experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control, #p < 0.05, ##p < 0.01 and ###p < 0.001, 17-AAG alone vs. combination with amurensin G.
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Figure 4: Inhibition of protein levels of CSC-related molecules and Hsps, and activation of pro-apoptotic cascade by amurensin G, and suppression of mRNA levels of 17-AAG-mediated induction of Hsp70/Hsp27 and ABCB1/ABCG2 genes by amurensin G in CD44high K562 cells. A: Cells were pretreated with amurensin G (5 μg/ml) for 6 h, and treated with 17-AAG (1 or 10 μM) for additional 24 h, and the indicated proteins were detected by western blot analysis. CF; cleavage fragments. B: Relative mRNA levels of Hsp70/Hsp27 and ABCB1/ABCG2 genes in the cells treated with 17-AAG alone and co-treated with 17-AAG and amurensin G were determined by real time RT-PCR. Each bar represented the mean ± S.D. of triplicate experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control, #p < 0.05, ##p < 0.01 and ###p < 0.001, 17-AAG alone vs. combination with amurensin G.

Mentions: Since it has been showed that SIRT1 inhibition hinders BCR-ABL transformation of hematopoietic stem cells and CML disease development, suggesting that SIRT1 facilitates gain of function of CSC properties 33, 34, we determined if depletion of SIRT1 could modulate the up-regulated CSC-related molecules in CD44high K562 cells. When SIRT1 was knock-downed in CD44high K562 cells with increasing amounts of SIRT1 siRNA, the levels of CD44, Oct-4, β-catenin, c-Myc, mut p53, P-gp and BCRP were decreased dose-dependently (Fig. 3A). Since CD44 knockdown was associated with down-regulation of HSF1 35, the levels of HSF1 and its target genes including Hsp 90 and Hsp70 were also determined. As expected, all of them were down-regulated after depletion of SIRT1 in CD44high K562 cells (Fig. 3A). Since it has been known that Hsp90 is required for the activity and stability of the pluripotency transcription factors such as Oct4, Nanog and c-Myc 5, 6, and for stabilization of mut p53 in cancer cells 7, the effect of 17-AAG on the expression of Hsps, CSC-related molecules and proapoptotic proteins in CD44high K562 cells was investigated in the presence or absence of SIRT1 inhibition with siRNA. The expression of CD44, Oct4, mut p53 and c-Myc was not significantly affected by treatment with 17-AAG alone, but was remarkably down-regulated by combined treatment of 17-AAG with SIRT1 siRNA. Moreover, the 17-AAG-stimulated induction of Hsp70/Hsp27 and BCRP/P-gp was suppressed by SIRT1 inhibition, which causes both up-regulation of Bax and cleavage of PARP (Fig. 3B). Similar results were obtained with combination treatment of AUY922 and SIRT1 siRNA (Fig. 3C), or 17-AAG and amurensin G, a potent natural SIRT1 inhibitor 30, in CD44high K562 cells (Fig. 4A). These results were followed by a significant suppression of 17-AAG-mediated mRNA induction of Hsp70, Hsp27, ABCB1 and ABCG2 genes by combined treatment with amurensin G in CD44high K562 cells (Fig. 4B). These results suggested a possibility that the effects of Hsp90 inhibitor can be potentiated by SIRT1 inhibition in CSCs.


Sensitization of chemo-resistant human chronic myeloid leukemia stem-like cells to Hsp90 inhibitor by SIRT1 inhibition.

Kim HB, Lee SH, Um JH, Kim MJ, Hyun SK, Gong EJ, Oh WK, Kang CD, Kim SH - Int. J. Biol. Sci. (2015)

Inhibition of protein levels of CSC-related molecules and Hsps, and activation of pro-apoptotic cascade by amurensin G, and suppression of mRNA levels of 17-AAG-mediated induction of Hsp70/Hsp27 and ABCB1/ABCG2 genes by amurensin G in CD44high K562 cells. A: Cells were pretreated with amurensin G (5 μg/ml) for 6 h, and treated with 17-AAG (1 or 10 μM) for additional 24 h, and the indicated proteins were detected by western blot analysis. CF; cleavage fragments. B: Relative mRNA levels of Hsp70/Hsp27 and ABCB1/ABCG2 genes in the cells treated with 17-AAG alone and co-treated with 17-AAG and amurensin G were determined by real time RT-PCR. Each bar represented the mean ± S.D. of triplicate experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control, #p < 0.05, ##p < 0.01 and ###p < 0.001, 17-AAG alone vs. combination with amurensin G.
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Figure 4: Inhibition of protein levels of CSC-related molecules and Hsps, and activation of pro-apoptotic cascade by amurensin G, and suppression of mRNA levels of 17-AAG-mediated induction of Hsp70/Hsp27 and ABCB1/ABCG2 genes by amurensin G in CD44high K562 cells. A: Cells were pretreated with amurensin G (5 μg/ml) for 6 h, and treated with 17-AAG (1 or 10 μM) for additional 24 h, and the indicated proteins were detected by western blot analysis. CF; cleavage fragments. B: Relative mRNA levels of Hsp70/Hsp27 and ABCB1/ABCG2 genes in the cells treated with 17-AAG alone and co-treated with 17-AAG and amurensin G were determined by real time RT-PCR. Each bar represented the mean ± S.D. of triplicate experiments. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control, #p < 0.05, ##p < 0.01 and ###p < 0.001, 17-AAG alone vs. combination with amurensin G.
Mentions: Since it has been showed that SIRT1 inhibition hinders BCR-ABL transformation of hematopoietic stem cells and CML disease development, suggesting that SIRT1 facilitates gain of function of CSC properties 33, 34, we determined if depletion of SIRT1 could modulate the up-regulated CSC-related molecules in CD44high K562 cells. When SIRT1 was knock-downed in CD44high K562 cells with increasing amounts of SIRT1 siRNA, the levels of CD44, Oct-4, β-catenin, c-Myc, mut p53, P-gp and BCRP were decreased dose-dependently (Fig. 3A). Since CD44 knockdown was associated with down-regulation of HSF1 35, the levels of HSF1 and its target genes including Hsp 90 and Hsp70 were also determined. As expected, all of them were down-regulated after depletion of SIRT1 in CD44high K562 cells (Fig. 3A). Since it has been known that Hsp90 is required for the activity and stability of the pluripotency transcription factors such as Oct4, Nanog and c-Myc 5, 6, and for stabilization of mut p53 in cancer cells 7, the effect of 17-AAG on the expression of Hsps, CSC-related molecules and proapoptotic proteins in CD44high K562 cells was investigated in the presence or absence of SIRT1 inhibition with siRNA. The expression of CD44, Oct4, mut p53 and c-Myc was not significantly affected by treatment with 17-AAG alone, but was remarkably down-regulated by combined treatment of 17-AAG with SIRT1 siRNA. Moreover, the 17-AAG-stimulated induction of Hsp70/Hsp27 and BCRP/P-gp was suppressed by SIRT1 inhibition, which causes both up-regulation of Bax and cleavage of PARP (Fig. 3B). Similar results were obtained with combination treatment of AUY922 and SIRT1 siRNA (Fig. 3C), or 17-AAG and amurensin G, a potent natural SIRT1 inhibitor 30, in CD44high K562 cells (Fig. 4A). These results were followed by a significant suppression of 17-AAG-mediated mRNA induction of Hsp70, Hsp27, ABCB1 and ABCG2 genes by combined treatment with amurensin G in CD44high K562 cells (Fig. 4B). These results suggested a possibility that the effects of Hsp90 inhibitor can be potentiated by SIRT1 inhibition in CSCs.

Bottom Line: The current investigation was undertaken to examine the effectiveness of the combination treatment of Hsp90 inhibitor and SIRT1 inhibitor in inhibiting the growth of chemo-resistant stem-like cells isolated from human chronic myeloid leukemia K562 cells.Inhibition of SIRT1 by use of SIRT1 siRNA or SIRT1 inhibitors (amurensin G and EX527) effectively potentiated sensitivity of Hsp90 inhibitors (17-AAG and AUY922) in CD44(high) K562 stem-like cells expressing high levels of CSC-related molecules including Oct4, CD34, β-catenin, c-Myc, mutant p53 (mut p53), BCRP and P-glycoprotein (P-gp) as well as CD44.Our data suggest that combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be an effective therapeutic approach to target CSCs that are resistant to current therapies.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Biochemistry, Pusan National University School of Medicine, Yangsan 626-870, Korea.

ABSTRACT
Development of effective therapeutic strategies to eliminate cancer stem-like cells (CSCs), which play a major role in drug resistance and disease recurrence, is critical to improve cancer treatment outcomes. The current investigation was undertaken to examine the effectiveness of the combination treatment of Hsp90 inhibitor and SIRT1 inhibitor in inhibiting the growth of chemo-resistant stem-like cells isolated from human chronic myeloid leukemia K562 cells. Inhibition of SIRT1 by use of SIRT1 siRNA or SIRT1 inhibitors (amurensin G and EX527) effectively potentiated sensitivity of Hsp90 inhibitors (17-AAG and AUY922) in CD44(high) K562 stem-like cells expressing high levels of CSC-related molecules including Oct4, CD34, β-catenin, c-Myc, mutant p53 (mut p53), BCRP and P-glycoprotein (P-gp) as well as CD44. SIRT1 depletion caused significant down-regulation of heat shock factor 1 (HSF1)/heat shock proteins (Hsps) as well as these CSC-related molecules, which led to the sensitization of CD44(high) K562 cells to Hsp90 inhibitor by SIRT1 inhibitor. Moreover, 17-AAG-mediated activation of HSF1/Hsps and P-gp-mediated efflux, major causes of Hsp90 inhibitor resistance, was suppressed by SIRT1 inhibitor in K562-CD44(high) cells. Our data suggest that combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be an effective therapeutic approach to target CSCs that are resistant to current therapies.

No MeSH data available.


Related in: MedlinePlus