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. 2017 Apr 25;8(17):28906-28921.
doi: 10.18632/oncotarget.15957.

Stellettin B induces apoptosis in human chronic myeloid leukemia cells via targeting PI3K and Stat5

Yali Chen  1   2 , Qianxiang Zhou  1   2 , Lei Zhang  1   2 , Yuxu Zhong  3 , Guanwei Fan  4 , Zhe Zhang  1 , Ran Wang  1 , Meihua Jin  1 , Yuling Qiu  1 , Dexin Kong  1   2
Affiliations

Stellettin B induces apoptosis in human chronic myeloid leukemia cells via targeting PI3K and Stat5

Yali Chen et al. Oncotarget. .

Abstract

Novel agents are still urgently expected for therapy of chronic myeloid leukemia (CML). The in vitro anti-leukemia activity of Stellettin B (Stel B), a triterpenoid we isolated from marine sponge Jaspis stellifera, on human CML K562 and KU812 cells was recently investigated. Stel B inhibited K562 and KU812 cell proliferation with IC50 as 0.035 μM and 0.95 μM respectively. While no obvious cell cycle arrest was observed, apoptosis was induced in K562 cells after Stel B treatment. The Stel B-induced apoptosis might be in mitochondrial pathway, with increase of Bad and Bax, decrease of Bcl-2 and activation of caspase-9. In addition, dose-dependent increase of reactive oxygen species (ROS) and loss of mitochondrial membrane potential (MMP) occurred. Meanwhile, Stel B inhibited phosphorylation of Stat5, expression of 4 PI3K catalytic isoforms, and phosphorylation of the downstream effectors including PDK1 and Akt, suggesting that inhibition against Stat5 and PI3K might be involved in the apoptosis-inducing effect. Combination of Stel B with Imatinib with ratio as IC50 Stel B : 5×IC50 Imatinib led to synergistic effect. Stel B might become a promising candidate for CML therapy alone or together with Imatinib.

Keywords: K562; PI3K; apoptosis; combination; stellettin B.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Potent Effect of Stel B on growth of CML cells
(A) WST-8 assay. K562 cells were cultured in 96-well plates with 0, 0.002, 0.006, 0.018, 0.054, 0.162, 0.486, 1.458 μM of Stel B for 48 h, and cell viability was measured 4 h after addition of WST-8 reagent. (B) WST-8 assay. KU812, U937 and PBMC cells were cultured in 96-well plates with 0, 0.006, 0.054, 0.486, 4.374, 13.122, 39.366 μM of Stel B for 48 h, and cell viability was measured as described in Figure 1A. (C) Soft agar assay. After treatment with 0, 0.009, 0.018 and 0.036 μM of Stel B for 48 h, K562 cells were further grown in soft agar for 10 days, followed by staining with crystal violet. Colonies were counted under a microscope to determine the effect of Stel B on tumorigenicity of K562 cells. (D) Quantification of the colonies formed by K562 cells with or without Stel B treatment in soft agar. Data are expressed as mean ± SD, representative of three independent experiments. *: p < 0.01, ***: p < 0.001, compared with control.
Figure 2
Figure 2. Cell cycle distribution of K562 cells with or without Stel B treatment
Cells were exposed to different concentrations (0, 0.012, 0.015, 0.018, 0.036, 0.054 μM) of Stel B for 48 h. After PI staining, flow cytometry analysis was performed to determine cell cycle distribution. PI: propidium iodide.
Figure 3
Figure 3. Apoptosis of K562 cells induced by Stel B
(A) Flow cytometric analysis of cell apoptosis with Annexin V-FITC/PI double staining. K562 cells were harvested 48 h after treatment by indicated concentrations of Stel B, stained with Annexin V-FITC/PI and subjected to flow cytometry analysis. (B) Quantification of the apoptotic cells in both early and late stage. Data represent mean± SD of three independent experiments. *: p < 0.01, ***: p < 0.001, compared with control.
Figure 4
Figure 4. Effect of Stel B on the expression of apoptosis-related proteins in K562 cells
(A) Expression of the apoptosis-related proteins detected by Western blot in K562 cells after Stel B treatment. K562 cells were treated with indicated concentrations of Stel B for 48 h. The cells were collected to be available for Western blot detection of caspase-9, caspase-3, and PARP, Bcl-2, Bax and Bad. β-actin was used as the loading control. (B) Bar graphs show the relative levels of Procaspase-9, Cleaved-caspase-9, Procaspase-3, Cleaved-caspase-3, and Cleaved PARP (upper panel), as well as Bcl-2, Bax and Bad (lower panel). Data are mean ± SD, representative of three independent experiments. *: p < 0.05, compared with control. (C) Determination of the Bax/Bcl-2 ratio as a well-known apoptosis marker. Data are mean ± SD, representative of three independent experiments. *: p < 0.05, compared with control.
Figure 5
Figure 5. Effect of Stel B on the ROS generation and MMP in K562 cells
(A) Flow cytometric determination of ROS generation in K562 cells with or without Stel B treatment. Cells were treated with indicated concentrations of Stel B for 48 h, followed by DCFH-DA staining for 30 min. The ROS levels were determined by flow cytometer. Data represent mean ± SD of three independent experiments. *: p < 0.05, *: p < 0.01, compared with control. (B) Effect of Stel B on MMP in K562 cells with or without Stel B treatment. Cells were exposed to various concentrations of Stel B for 24 h. MMP (ΔΨm) was measured by the uptake of JC-1 using flow cytometer. Cells containing JC-1 monomers (green) indicate low ΔΨm while those containing JC-1 aggregates (red) indicate high ΔΨm. (C) Quantification of the MMP (% control) in K562 cells with or without Stel B treatment. MMP was calculated as the ratio of the red fluorescence to green fluorescence. Relative MMP represents the MMP of the respective sample over that of control (%). Data represent mean ± SD of three independent experiments. *: p < 0.05, *: p < 0.01, compared with control.
Figure 6
Figure 6. Effect of Stel B on Stat5, PI3K and the downstream effectors in PI3K/Akt pathway in K562 cells
(A) K562 cells were treated with indicated concentrations of Stel B for 48 h. The treated cells were subjected for Western blot analysis of p-Stat5, Stat5, p-Stat3, PI3K-p110α, PI3K-p110β, PI3K-p110δ, PI3K-p110γ, PI3K-p85, p-PDK1, p-Akt, Akt, p-mTOR and p-p70 S6K. β-actin was used as the loading control. (B) Bar graphs show the relative levels of p-Stat5, Stat5 and p-Stat3 (upper panel), PI3K-p110α, PI3K-p110β, PI3K-p110δ, PI3K-p110γ and PI3K-p85 (middle panel), as well as p-PDK1, p-Akt, Akt, p-mTOR and p-p70 S6K (lower panel). Data are mean ± SD, representative of three independent experiments. *: p < 0.05, *: p < 0.01, compared with control (without treatment). (C) K562 cells were treated with Stel B (0, 0.036 μM) in the presence or absence of SH-4-54 (3.2 μM) for 48 h. Signals of p-Stat5, PI3K-p110α, PI3K-p110β, PI3K-p110δ, PI3K-p110γ, PI3K-p85, p-PDK1 and p-Akt were determined by Western blot. β-actin was used as the loading control. (D) Bar graphs show the relative levels of p-Stat5, p-PDK1 and p-Akt (upper panel), PI3K-p110α, PI3K-p110β, PI3K-p110δ, PI3K-p110γ and PI3K-p85 (lower panel). Data are mean ± SD, representative of three independent experiments. *: p < 0.05, *: p < 0.01, compared with mixture.
Figure 7
Figure 7. Synergistic effect of Stel B and Imatinib in K562 cells
(A) Growth inhibitory activity of Stel B and Imatinib on K562 cells determined by MTT assay. K562 cells were exposed with indicated concentrations of Stel B or Imatinib. Cell viability was determined by MTT assay. Data are mean ± SD, representative of three independent experiments. (B, C, D) K562 cells were incubated with various concentrations of Stel B and/or Imatinib (20%, 40%, 60%, 80%, 100% IC50 of each drug) for 48 h, Three fixed ratios of IC50 Stel B: IC50 Imatinib, IC50 Stel B: 5×IC50 Imatinib, 5×IC50 Stel B: IC50 Imatinib were used. Cell viability after different treatments was determined by MTT assay (left). Data are mean ± SD, representative of three independent experiments. Combinational effect was analyzed using CalcuSyn software and the resulting CI-Fa plots are shown (right). The horizontal line of CI = 1, representing additivity, is indicated. Values of drug combinations below the horizontal line indicate synergism. CI: combination index, Fa: Fraction affected.
Figure 8
Figure 8. Effect of the combination of Stel B and Imatinib on tumorigenicity of K562 cells
(A) The cells were treated with Stel B (0.0064 μM) and/or Imatinib (0.1 μM) for 48 h, and grown in soft agar at 37°C. Ten days later, colonies more than 0.1 mm in diameter were counted using Image J software. (B) Quantification of the colonies formed by K562 cells in soft agar after treatment with Stel B and Imatinib alone or in combination. Data are mean ± SD, representative of three independent experiments. *: p < 0.05, compared with mixture (combination).
Figure 9
Figure 9. Effect of the combination of Stel B and Imatinib on apoptosis in K562 cells
(A, B) K562 cells exposed with Stel B (0.0064 μM) and/or Imatinib (0.1 μM) for 48 h, were stained with Annexin V-FITC/PI and analyzed using flow cytometer. Data represent mean ± SD of three independent experiments. *: p < 0.01, compared with mixture (combination). (C) After treatment with Stel B (0.0064 μM) and/or Imatinib (0.1μM) for 48 h, cleavage of caspase-3 and PARP of K562 cells was analyzed by Western blot. β-actin was used as the loading control. (D) Bar graphs show the relative levels of Procaspase-3, Cleaved-caspase-3 and Cleaved PARP. Data are mean ± SD, representative of three independent experiments. *: p < 0.05, compared with mixture.
Figure 10
Figure 10. Effect of Stel B on the growth of multidrug resistant K562/A02 cells
(A) K562/A02 cells in 96-well plates were treated with 0, 0.002, 0.006, 0.018, 0.054, 0.162, 0.486, 1.458, 4.374 μM of Stel B for 48 h. Cell viability was measured by MTT assay. (B) Expression level of multidrug resistance related proteins in K562 and K562/A02 cells. Expression of MDR1 and MRP1 in K562 cells, and K562/A02 cells with or without Stel B (0.62 μM) treatment was analyzed by Western blot. β-actin was used as the loading control. (C) Bar graphs show the relative levels of MDR1 and MRP1. Data are mean ± SD, representative of three independent experiments.
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