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Synergistic Effect and Molecular Mechanism of Homoharringtonine and Bortezomib on SKM-1 Cell Apoptosis.

Zhang J, Chen B, Wu T, Wang Q, Zhuang L, Zhu C, Fan N, Qing W, Ma Y, Xu X - PLoS ONE (2015)

Bottom Line: Simultaneous exposure to HHT and Bortezomib (10.4:1) resulted in a significant reduction of cell proliferation in SKM-1 cells (P < 0.05).HHT and Bortezomib synergistically inhibit SKM-1 cell proliferation and induce apoptosis in vitro.Inhibition of Akt and NF-κB pathway signaling contribute to molecular mechanism of HHT and Bortezomib. miR-3151 abundance is implicated in SKM-1 cell viability, cell proliferation and p53 protein expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT

Background: Myelodysplastic syndromes (MDS) are clonal marrow stem-cell disorders with a high risk of progression to acute myeloid leukemia (AML). Treatment options are limited and targeted therapies are not available for MDS. In the present study, we investigated the cytotoxicity and the molecular mechanism of Homoharringtonine (HHT) and Bortezomib towards high-risk MDS cell line SKM-1 in vitro and the role of miR-3151 was first evaluated in SKM-1 cells.

Methods: SKM-1 cells were treated with different concentrations of HHT or Bortezomib, and cell viability was analyzed with CCK-8 assay. The influence on cell proliferation, cell cycle distribution and the percentage of apoptosis cells were analyzed by flow cytometry. Calcusyn software was used to calculate combination index (CI) values. Western blot was used to analysis phosphorylation of Akt and nuclear NF-κB protein expression in SKM-1 cells. Mature miR-3151 level and p53 protein level were detected after HHT or Bortezomib treatment. The cell proliferation and p53 protein level were reassessed in SKM-1 cells infected with lentivirus to overexpress miR-3151.

Results: Simultaneous exposure to HHT and Bortezomib (10.4:1) resulted in a significant reduction of cell proliferation in SKM-1 cells (P < 0.05). Cell cycle arrest at G0/G1 and G2/M phase was observed (P < 0.05). HHT and Bortezomib synergistically induced cell apoptosis by regulating members of caspase 9, caspase 3 and Bcl-2 family (P < 0.01). The mechanisms of the synergy involved Akt and NF-κB signaling pathway inhibition, downregulation of mature miR-3151 and increment of downstream p53 protein level. Overexpression of miR-3151 promoted cell proliferation and inhibited p53 protein expression in SKM-1 (P < 0.01).

Conclusions: HHT and Bortezomib synergistically inhibit SKM-1 cell proliferation and induce apoptosis in vitro. Inhibition of Akt and NF-κB pathway signaling contribute to molecular mechanism of HHT and Bortezomib. miR-3151 abundance is implicated in SKM-1 cell viability, cell proliferation and p53 protein expression.

No MeSH data available.


Related in: MedlinePlus

HHT and Bortezomib inhibited the cell viability of SKM-1 cells.(A) Time- and dose–response curves of HHT from 0.01 nM to 1000 nM. (B) Time- and dose–response curves of HHT from 2 nM to 100 nM. (C) Time- and dose–response curves of Bortezomib from 0.01 nM to 1000 nM. (D) Time- and dose–response curves of Bortezomib from 0.25 nM to 10 nM. (E) CI values for HHT and Bortezomib combination treatments at the molar ratio of 10.4:1. (F) Celluar viability of SKM-1 or normal PBMC cells simultaneously treated with 46.20 nM HHT and 4.46 nM Bortezomib.
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pone.0142422.g001: HHT and Bortezomib inhibited the cell viability of SKM-1 cells.(A) Time- and dose–response curves of HHT from 0.01 nM to 1000 nM. (B) Time- and dose–response curves of HHT from 2 nM to 100 nM. (C) Time- and dose–response curves of Bortezomib from 0.01 nM to 1000 nM. (D) Time- and dose–response curves of Bortezomib from 0.25 nM to 10 nM. (E) CI values for HHT and Bortezomib combination treatments at the molar ratio of 10.4:1. (F) Celluar viability of SKM-1 or normal PBMC cells simultaneously treated with 46.20 nM HHT and 4.46 nM Bortezomib.

Mentions: SKM-1 cells were plated into 96-well plates and treated with increasing concentrations of HHT (0.01, 0.1, 1, 10, 100 and 1000 nM) or Bortezomib (0.01, 0.1, 1, 10, 100 and 1000 nM) for 24 h, 48 h and 72 h. The CCK-8 assay showed a time and dose-dependent cell viability inhibition by HHT and Bortezomib (Fig 1A, 1B, 1C and 1D, P < 0.01). The IC50 values for HHT in SKM-1 cells at 24 h, 48 h and 72 h were 43.65 ± 4.37 nM, 23.10 ± 3.38 nM and 17.62 ± 6.89 nM, respectively. The IC50 values for Bortezomib in SKM-1 cells at 24 h, 48 h and 72 h were 34.21 ± 5.30 nM, 4.46 ± 4.26 nM and 2.116 ± 8.05 nM, respectively. The dose–effect curves for HHT + Bortezomib were determined by Calcusyn analyses. We found that simultaneous exposure to HHT and Bortezomib for 48 h resulted in a strong synergistic inhibition of cell viability in SKM-1 cells. The best synergistic effect was observed when the molar ratio was 10.4: 1 (HHT: Bortezomib). The CI value at the median effective dose (ED50) was 0.76222 (Fig 1E). The inhibition of cell growth in SKM-1 was more significant than normal PBMC cells simultaneously treated with 46.20 nM HHT and 4.46 nM Bortezomib (Fig 1F).


Synergistic Effect and Molecular Mechanism of Homoharringtonine and Bortezomib on SKM-1 Cell Apoptosis.

Zhang J, Chen B, Wu T, Wang Q, Zhuang L, Zhu C, Fan N, Qing W, Ma Y, Xu X - PLoS ONE (2015)

HHT and Bortezomib inhibited the cell viability of SKM-1 cells.(A) Time- and dose–response curves of HHT from 0.01 nM to 1000 nM. (B) Time- and dose–response curves of HHT from 2 nM to 100 nM. (C) Time- and dose–response curves of Bortezomib from 0.01 nM to 1000 nM. (D) Time- and dose–response curves of Bortezomib from 0.25 nM to 10 nM. (E) CI values for HHT and Bortezomib combination treatments at the molar ratio of 10.4:1. (F) Celluar viability of SKM-1 or normal PBMC cells simultaneously treated with 46.20 nM HHT and 4.46 nM Bortezomib.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4636319&req=5

pone.0142422.g001: HHT and Bortezomib inhibited the cell viability of SKM-1 cells.(A) Time- and dose–response curves of HHT from 0.01 nM to 1000 nM. (B) Time- and dose–response curves of HHT from 2 nM to 100 nM. (C) Time- and dose–response curves of Bortezomib from 0.01 nM to 1000 nM. (D) Time- and dose–response curves of Bortezomib from 0.25 nM to 10 nM. (E) CI values for HHT and Bortezomib combination treatments at the molar ratio of 10.4:1. (F) Celluar viability of SKM-1 or normal PBMC cells simultaneously treated with 46.20 nM HHT and 4.46 nM Bortezomib.
Mentions: SKM-1 cells were plated into 96-well plates and treated with increasing concentrations of HHT (0.01, 0.1, 1, 10, 100 and 1000 nM) or Bortezomib (0.01, 0.1, 1, 10, 100 and 1000 nM) for 24 h, 48 h and 72 h. The CCK-8 assay showed a time and dose-dependent cell viability inhibition by HHT and Bortezomib (Fig 1A, 1B, 1C and 1D, P < 0.01). The IC50 values for HHT in SKM-1 cells at 24 h, 48 h and 72 h were 43.65 ± 4.37 nM, 23.10 ± 3.38 nM and 17.62 ± 6.89 nM, respectively. The IC50 values for Bortezomib in SKM-1 cells at 24 h, 48 h and 72 h were 34.21 ± 5.30 nM, 4.46 ± 4.26 nM and 2.116 ± 8.05 nM, respectively. The dose–effect curves for HHT + Bortezomib were determined by Calcusyn analyses. We found that simultaneous exposure to HHT and Bortezomib for 48 h resulted in a strong synergistic inhibition of cell viability in SKM-1 cells. The best synergistic effect was observed when the molar ratio was 10.4: 1 (HHT: Bortezomib). The CI value at the median effective dose (ED50) was 0.76222 (Fig 1E). The inhibition of cell growth in SKM-1 was more significant than normal PBMC cells simultaneously treated with 46.20 nM HHT and 4.46 nM Bortezomib (Fig 1F).

Bottom Line: Simultaneous exposure to HHT and Bortezomib (10.4:1) resulted in a significant reduction of cell proliferation in SKM-1 cells (P < 0.05).HHT and Bortezomib synergistically inhibit SKM-1 cell proliferation and induce apoptosis in vitro.Inhibition of Akt and NF-κB pathway signaling contribute to molecular mechanism of HHT and Bortezomib. miR-3151 abundance is implicated in SKM-1 cell viability, cell proliferation and p53 protein expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Hematology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China.

ABSTRACT

Background: Myelodysplastic syndromes (MDS) are clonal marrow stem-cell disorders with a high risk of progression to acute myeloid leukemia (AML). Treatment options are limited and targeted therapies are not available for MDS. In the present study, we investigated the cytotoxicity and the molecular mechanism of Homoharringtonine (HHT) and Bortezomib towards high-risk MDS cell line SKM-1 in vitro and the role of miR-3151 was first evaluated in SKM-1 cells.

Methods: SKM-1 cells were treated with different concentrations of HHT or Bortezomib, and cell viability was analyzed with CCK-8 assay. The influence on cell proliferation, cell cycle distribution and the percentage of apoptosis cells were analyzed by flow cytometry. Calcusyn software was used to calculate combination index (CI) values. Western blot was used to analysis phosphorylation of Akt and nuclear NF-κB protein expression in SKM-1 cells. Mature miR-3151 level and p53 protein level were detected after HHT or Bortezomib treatment. The cell proliferation and p53 protein level were reassessed in SKM-1 cells infected with lentivirus to overexpress miR-3151.

Results: Simultaneous exposure to HHT and Bortezomib (10.4:1) resulted in a significant reduction of cell proliferation in SKM-1 cells (P < 0.05). Cell cycle arrest at G0/G1 and G2/M phase was observed (P < 0.05). HHT and Bortezomib synergistically induced cell apoptosis by regulating members of caspase 9, caspase 3 and Bcl-2 family (P < 0.01). The mechanisms of the synergy involved Akt and NF-κB signaling pathway inhibition, downregulation of mature miR-3151 and increment of downstream p53 protein level. Overexpression of miR-3151 promoted cell proliferation and inhibited p53 protein expression in SKM-1 (P < 0.01).

Conclusions: HHT and Bortezomib synergistically inhibit SKM-1 cell proliferation and induce apoptosis in vitro. Inhibition of Akt and NF-κB pathway signaling contribute to molecular mechanism of HHT and Bortezomib. miR-3151 abundance is implicated in SKM-1 cell viability, cell proliferation and p53 protein expression.

No MeSH data available.


Related in: MedlinePlus