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Molecular targeting of the oncoprotein PLK1 in pediatric acute myeloid leukemia: RO3280, a novel PLK1 inhibitor, induces apoptosis in leukemia cells.

Wang NN, Li ZH, Zhao H, Tao YF, Xu LX, Lu J, Cao L, Du XJ, Sun LC, Zhao WL, Xiao PF, Fang F, Su GH, Li YH, Li G, Li YP, Xu YY, Zhou HT, Wu Y, Jin MF, Liu L, Ni J, Wang J, Hu SY, Zhu XM, Feng X, Pan J - Int J Mol Sci (2015)

Bottom Line: PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001).RO3280 treatment regulated several apoptosis-associated genes.These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou 215003, China. wangnn90s@163.com.

ABSTRACT
Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.

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RO3280 induced DNA fragmentation and cleavage of apoptotic markers in acute leukemia cells. (A) Micrographs following Hoechst 33342 staining of cells treated with RO3280 (50 and 100 nM) for 24 h. This demonstrates the induction of DNA fragmentation and abnormal nuclear cell formation; (B) The abnormal nuclear cells were quantified and increased significantly with RO3280 treatment compared with mock treatment in both HL-60 and NB4 cells. **p < 0.01; and (C) Western blot analysis of PARP, caspase-3, and caspase-9. After 24 h treatment with 50 or 100 nM RO3280, cleaved PARP and caspase-9 were detected in lysates from NB4 and HL-60 cells.
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ijms-16-01266-f005: RO3280 induced DNA fragmentation and cleavage of apoptotic markers in acute leukemia cells. (A) Micrographs following Hoechst 33342 staining of cells treated with RO3280 (50 and 100 nM) for 24 h. This demonstrates the induction of DNA fragmentation and abnormal nuclear cell formation; (B) The abnormal nuclear cells were quantified and increased significantly with RO3280 treatment compared with mock treatment in both HL-60 and NB4 cells. **p < 0.01; and (C) Western blot analysis of PARP, caspase-3, and caspase-9. After 24 h treatment with 50 or 100 nM RO3280, cleaved PARP and caspase-9 were detected in lysates from NB4 and HL-60 cells.

Mentions: To determine if RO3280 induces apoptosis in leukemia cells, we assessed Annexin V staining, cell cycle disorder and caspase activation in leukemia cells after treatment. Cells treated with RO3280 for 24 h showed higher Annexin V staining compared with untreated cells (Figure 3). This indicates that RO3280 induces apoptosis in leukemia cells. Cell cycle disorder was determined by a cell cycle assay, which showed that RO3280 significantly induces cell cycle disorder in nine leukemia cell lines (Figure 4). Hoechst 33342 staining showed that 24 h RO3280 treatment induced DNA fragmentation and the formation of abnormal nuclear cells (Figure 5A). When either HL-60 or NB4 cells were treated with RO3280, there was a significant increase in abnormal nuclear cell formation compared with control DMSO treated cells (Figure 5B). Moreover, to clearly demonstrate that RO3280 causes apoptosis in leukemia cells, we assessed the following well-recognized markers of apoptosis: PARP, caspase-3, and caspase-9. After 24 h of treatment with RO3280 (50 or 100 nM), cleaved PARP and caspase-9 were observed in NB4 and HL-60 cells (Figure 5C). These results are consistent with the Annexin V staining and cell cycle analysis, demonstrating that RO3280 induces apoptosis in leukemia cells. This further suggests that RO3280 may have promising antitumor therapeutic applications.


Molecular targeting of the oncoprotein PLK1 in pediatric acute myeloid leukemia: RO3280, a novel PLK1 inhibitor, induces apoptosis in leukemia cells.

Wang NN, Li ZH, Zhao H, Tao YF, Xu LX, Lu J, Cao L, Du XJ, Sun LC, Zhao WL, Xiao PF, Fang F, Su GH, Li YH, Li G, Li YP, Xu YY, Zhou HT, Wu Y, Jin MF, Liu L, Ni J, Wang J, Hu SY, Zhu XM, Feng X, Pan J - Int J Mol Sci (2015)

RO3280 induced DNA fragmentation and cleavage of apoptotic markers in acute leukemia cells. (A) Micrographs following Hoechst 33342 staining of cells treated with RO3280 (50 and 100 nM) for 24 h. This demonstrates the induction of DNA fragmentation and abnormal nuclear cell formation; (B) The abnormal nuclear cells were quantified and increased significantly with RO3280 treatment compared with mock treatment in both HL-60 and NB4 cells. **p < 0.01; and (C) Western blot analysis of PARP, caspase-3, and caspase-9. After 24 h treatment with 50 or 100 nM RO3280, cleaved PARP and caspase-9 were detected in lysates from NB4 and HL-60 cells.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4307303&req=5

ijms-16-01266-f005: RO3280 induced DNA fragmentation and cleavage of apoptotic markers in acute leukemia cells. (A) Micrographs following Hoechst 33342 staining of cells treated with RO3280 (50 and 100 nM) for 24 h. This demonstrates the induction of DNA fragmentation and abnormal nuclear cell formation; (B) The abnormal nuclear cells were quantified and increased significantly with RO3280 treatment compared with mock treatment in both HL-60 and NB4 cells. **p < 0.01; and (C) Western blot analysis of PARP, caspase-3, and caspase-9. After 24 h treatment with 50 or 100 nM RO3280, cleaved PARP and caspase-9 were detected in lysates from NB4 and HL-60 cells.
Mentions: To determine if RO3280 induces apoptosis in leukemia cells, we assessed Annexin V staining, cell cycle disorder and caspase activation in leukemia cells after treatment. Cells treated with RO3280 for 24 h showed higher Annexin V staining compared with untreated cells (Figure 3). This indicates that RO3280 induces apoptosis in leukemia cells. Cell cycle disorder was determined by a cell cycle assay, which showed that RO3280 significantly induces cell cycle disorder in nine leukemia cell lines (Figure 4). Hoechst 33342 staining showed that 24 h RO3280 treatment induced DNA fragmentation and the formation of abnormal nuclear cells (Figure 5A). When either HL-60 or NB4 cells were treated with RO3280, there was a significant increase in abnormal nuclear cell formation compared with control DMSO treated cells (Figure 5B). Moreover, to clearly demonstrate that RO3280 causes apoptosis in leukemia cells, we assessed the following well-recognized markers of apoptosis: PARP, caspase-3, and caspase-9. After 24 h of treatment with RO3280 (50 or 100 nM), cleaved PARP and caspase-9 were observed in NB4 and HL-60 cells (Figure 5C). These results are consistent with the Annexin V staining and cell cycle analysis, demonstrating that RO3280 induces apoptosis in leukemia cells. This further suggests that RO3280 may have promising antitumor therapeutic applications.

Bottom Line: PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001).RO3280 treatment regulated several apoptosis-associated genes.These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology and Oncology, Children's Hospital of Soochow University, Suzhou 215003, China. wangnn90s@163.com.

ABSTRACT
Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.

Show MeSH
Related in: MedlinePlus