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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

Simultaneous exposure to HHT and Bortezomib resulted in cell cycle arrest.(A) SKM-1 cells treated with 46.20 nM HHT, 4.46 nM Bortezomib or 46.20 nM HHT + 4.46 nM Bortezomib simultaneously for 72 h. (B) SKM-1 cells treated with HHT (11.55, 23.10, 46.20 nM) or Bortezomib (1.12, 2.23, 4.46 nM) for 72 h. Concurrently, simultaneous treatment experiments were conducted at molar ratio of 10.4: 1. Bar graph of cell cycle distribution. *P < 0.05, #P < 0.01 vs untreated control.
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pone.0142422.g003: Simultaneous exposure to HHT and Bortezomib resulted in cell cycle arrest.(A) SKM-1 cells treated with 46.20 nM HHT, 4.46 nM Bortezomib or 46.20 nM HHT + 4.46 nM Bortezomib simultaneously for 72 h. (B) SKM-1 cells treated with HHT (11.55, 23.10, 46.20 nM) or Bortezomib (1.12, 2.23, 4.46 nM) for 72 h. Concurrently, simultaneous treatment experiments were conducted at molar ratio of 10.4: 1. Bar graph of cell cycle distribution. *P < 0.05, #P < 0.01 vs untreated control.

Mentions: We further examined the effects of HHT (11.55, 23.10, 46.20 nM) and Bortezomib (1.12, 2.23, 4.46 nM) on SKM-1 cell proliferation and cell cycle distribution using BrdU/7AAD double staining flow cytometry. Similar to results from the cell viability assay, simultaneous exposure to HHT and Bortezomib (10.4: 1) for 72 h resulted in a significant reduction of cell proliferation in SKM-1 cells (Fig 2, P<0.05). Cell cycle arrest at G0/G1 and G2/M phase was observed in SKM-1 cells incubated with different concentrations of HHT or Bortezomib for 72 h, which was accompanied by a decreased proportion at S phase (Fig 3, P<0.05). It was more obvious when HHT: Bortezomib was 10.4:1. Collectively, these results indicated that HHT and Bortezomib synergistically inhibited the growth of SKM-1 cells in vitro.


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)

Simultaneous exposure to HHT and Bortezomib resulted in cell cycle arrest.(A) SKM-1 cells treated with 46.20 nM HHT, 4.46 nM Bortezomib or 46.20 nM HHT + 4.46 nM Bortezomib simultaneously for 72 h. (B) SKM-1 cells treated with HHT (11.55, 23.10, 46.20 nM) or Bortezomib (1.12, 2.23, 4.46 nM) for 72 h. Concurrently, simultaneous treatment experiments were conducted at molar ratio of 10.4: 1. Bar graph of cell cycle distribution. *P < 0.05, #P < 0.01 vs untreated control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142422.g003: Simultaneous exposure to HHT and Bortezomib resulted in cell cycle arrest.(A) SKM-1 cells treated with 46.20 nM HHT, 4.46 nM Bortezomib or 46.20 nM HHT + 4.46 nM Bortezomib simultaneously for 72 h. (B) SKM-1 cells treated with HHT (11.55, 23.10, 46.20 nM) or Bortezomib (1.12, 2.23, 4.46 nM) for 72 h. Concurrently, simultaneous treatment experiments were conducted at molar ratio of 10.4: 1. Bar graph of cell cycle distribution. *P < 0.05, #P < 0.01 vs untreated control.
Mentions: We further examined the effects of HHT (11.55, 23.10, 46.20 nM) and Bortezomib (1.12, 2.23, 4.46 nM) on SKM-1 cell proliferation and cell cycle distribution using BrdU/7AAD double staining flow cytometry. Similar to results from the cell viability assay, simultaneous exposure to HHT and Bortezomib (10.4: 1) for 72 h resulted in a significant reduction of cell proliferation in SKM-1 cells (Fig 2, P<0.05). Cell cycle arrest at G0/G1 and G2/M phase was observed in SKM-1 cells incubated with different concentrations of HHT or Bortezomib for 72 h, which was accompanied by a decreased proportion at S phase (Fig 3, P<0.05). It was more obvious when HHT: Bortezomib was 10.4:1. Collectively, these results indicated that HHT and Bortezomib synergistically inhibited the growth of SKM-1 cells in vitro.

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