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Assessment of the effect of sphingosine kinase inhibitors on apoptosis,unfolded protein response and autophagy of T-cell acute lymphoblastic leukemia cells; indications for novel therapeutics.

Evangelisti C, Evangelisti C, Teti G, Chiarini F, Falconi M, Melchionda F, Pession A, Bertaina A, Locatelli F, McCubrey JA, Beak DJ, Bittman R, Pyne S, Pyne NJ, Martelli AM - Oncotarget (2014)

Bottom Line: Sphingosine kinases play a fundamental role in many signaling pathways associated with cancer, suggesting that proteins belonging to this signaling network represent potential therapeutic targets.Interestingly, we observed a synergistic effect of SKi with the classical chemotherapeutic drug vincristine.In addition, we reported that SKi affected signaling cascades implicated in survival, proliferation and stress response of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, Bologna, Italy.

ABSTRACT
Sphingosine 1-phosphate (S1P) is a bioactive lipid that is formed by the phosphorylation of sphingosine and catalysed by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). Sphingosine kinases play a fundamental role in many signaling pathways associated with cancer, suggesting that proteins belonging to this signaling network represent potential therapeutic targets. Over the last years, many improvements have been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL); however, novel and less toxic therapies are still needed, especially for relapsing and chemo-resistant patients. Here, we analyzed the therapeutic potential of SKi and ROMe, a sphingosine kinase 1 and 2 inhibitor and SK2-selective inhibitor, respectively. While SKi induced apoptosis, ROMe initiated an autophagic cell death in our in vitro cell models. SKi treatment induced an increase in SK1 protein levels in Molt-4 cells, whereas it activated the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) pathway in Jurkat and CEM-R cells as protective mechanisms in a sub-population of T-ALL cells. Interestingly, we observed a synergistic effect of SKi with the classical chemotherapeutic drug vincristine. In addition, we reported that SKi affected signaling cascades implicated in survival, proliferation and stress response of cells. These findings indicate that SK1 or SK2 represent potential targets for treating T-ALL.

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SKi triggers apoptosis in Molt-4 cell line(A) Flow cytometric analysis of Annexin-V-FITC/PI stained Molt-4 cells treated with SKi (6.9 μM) for 6, 16, 24 and 40 h documented a time-dependent increase in apoptotic cells with respect to untreated cells. (B) After SKi treatment, cells were collected, lysed and analyzed by western blotting for cleaved caspase-9, -3, and -8. (C) Molt-4 cells were treated with SKi (6.9 μM) for the indicated time, then processed for TEM analysis that documented apoptosis induction. Arrows point to condensed apoptotic chromatin. Scale bar 2 μm. (D) Western blot analysis for the autophagic marker LC3 and p-mTOR (Ser2448) in cells treated with SKi for the indicated times. (E) Western blot analysis for SK1 expression in T-ALL cell lines upon SKi treatment for 40 h. In (B) (D) and (E) 50 μg of protein was loaded for each lane. β-tubulin was used as a loading control.
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Figure 2: SKi triggers apoptosis in Molt-4 cell line(A) Flow cytometric analysis of Annexin-V-FITC/PI stained Molt-4 cells treated with SKi (6.9 μM) for 6, 16, 24 and 40 h documented a time-dependent increase in apoptotic cells with respect to untreated cells. (B) After SKi treatment, cells were collected, lysed and analyzed by western blotting for cleaved caspase-9, -3, and -8. (C) Molt-4 cells were treated with SKi (6.9 μM) for the indicated time, then processed for TEM analysis that documented apoptosis induction. Arrows point to condensed apoptotic chromatin. Scale bar 2 μm. (D) Western blot analysis for the autophagic marker LC3 and p-mTOR (Ser2448) in cells treated with SKi for the indicated times. (E) Western blot analysis for SK1 expression in T-ALL cell lines upon SKi treatment for 40 h. In (B) (D) and (E) 50 μg of protein was loaded for each lane. β-tubulin was used as a loading control.

Mentions: In order to determine whether the decreased viability was related to apoptosis, Molt-4 cells were treated with a SKi concentration equivalent to the IC50 (6.9 μM) for 6, 16, 24 and 40 h, after which the cells were stained with Annexin-V and propidium iodide (PI) and analyzed by flow cytometry. Cells underwent apoptosis after 6 h of SKi treatment as demonstrated by the increasing presence of Annexin-V+/PI− and Annexin-V+/PI+ cells which reflect early and late apoptosis, respectively (Figure 2A). The presence of low levels of PI-positive only cells after 40 h of treatment demonstrated that cell death was mainly due to apoptosis.


Assessment of the effect of sphingosine kinase inhibitors on apoptosis,unfolded protein response and autophagy of T-cell acute lymphoblastic leukemia cells; indications for novel therapeutics.

Evangelisti C, Evangelisti C, Teti G, Chiarini F, Falconi M, Melchionda F, Pession A, Bertaina A, Locatelli F, McCubrey JA, Beak DJ, Bittman R, Pyne S, Pyne NJ, Martelli AM - Oncotarget (2014)

SKi triggers apoptosis in Molt-4 cell line(A) Flow cytometric analysis of Annexin-V-FITC/PI stained Molt-4 cells treated with SKi (6.9 μM) for 6, 16, 24 and 40 h documented a time-dependent increase in apoptotic cells with respect to untreated cells. (B) After SKi treatment, cells were collected, lysed and analyzed by western blotting for cleaved caspase-9, -3, and -8. (C) Molt-4 cells were treated with SKi (6.9 μM) for the indicated time, then processed for TEM analysis that documented apoptosis induction. Arrows point to condensed apoptotic chromatin. Scale bar 2 μm. (D) Western blot analysis for the autophagic marker LC3 and p-mTOR (Ser2448) in cells treated with SKi for the indicated times. (E) Western blot analysis for SK1 expression in T-ALL cell lines upon SKi treatment for 40 h. In (B) (D) and (E) 50 μg of protein was loaded for each lane. β-tubulin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202168&req=5

Figure 2: SKi triggers apoptosis in Molt-4 cell line(A) Flow cytometric analysis of Annexin-V-FITC/PI stained Molt-4 cells treated with SKi (6.9 μM) for 6, 16, 24 and 40 h documented a time-dependent increase in apoptotic cells with respect to untreated cells. (B) After SKi treatment, cells were collected, lysed and analyzed by western blotting for cleaved caspase-9, -3, and -8. (C) Molt-4 cells were treated with SKi (6.9 μM) for the indicated time, then processed for TEM analysis that documented apoptosis induction. Arrows point to condensed apoptotic chromatin. Scale bar 2 μm. (D) Western blot analysis for the autophagic marker LC3 and p-mTOR (Ser2448) in cells treated with SKi for the indicated times. (E) Western blot analysis for SK1 expression in T-ALL cell lines upon SKi treatment for 40 h. In (B) (D) and (E) 50 μg of protein was loaded for each lane. β-tubulin was used as a loading control.
Mentions: In order to determine whether the decreased viability was related to apoptosis, Molt-4 cells were treated with a SKi concentration equivalent to the IC50 (6.9 μM) for 6, 16, 24 and 40 h, after which the cells were stained with Annexin-V and propidium iodide (PI) and analyzed by flow cytometry. Cells underwent apoptosis after 6 h of SKi treatment as demonstrated by the increasing presence of Annexin-V+/PI− and Annexin-V+/PI+ cells which reflect early and late apoptosis, respectively (Figure 2A). The presence of low levels of PI-positive only cells after 40 h of treatment demonstrated that cell death was mainly due to apoptosis.

Bottom Line: Sphingosine kinases play a fundamental role in many signaling pathways associated with cancer, suggesting that proteins belonging to this signaling network represent potential therapeutic targets.Interestingly, we observed a synergistic effect of SKi with the classical chemotherapeutic drug vincristine.In addition, we reported that SKi affected signaling cascades implicated in survival, proliferation and stress response of cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, Bologna, Italy.

ABSTRACT
Sphingosine 1-phosphate (S1P) is a bioactive lipid that is formed by the phosphorylation of sphingosine and catalysed by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). Sphingosine kinases play a fundamental role in many signaling pathways associated with cancer, suggesting that proteins belonging to this signaling network represent potential therapeutic targets. Over the last years, many improvements have been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL); however, novel and less toxic therapies are still needed, especially for relapsing and chemo-resistant patients. Here, we analyzed the therapeutic potential of SKi and ROMe, a sphingosine kinase 1 and 2 inhibitor and SK2-selective inhibitor, respectively. While SKi induced apoptosis, ROMe initiated an autophagic cell death in our in vitro cell models. SKi treatment induced an increase in SK1 protein levels in Molt-4 cells, whereas it activated the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) pathway in Jurkat and CEM-R cells as protective mechanisms in a sub-population of T-ALL cells. Interestingly, we observed a synergistic effect of SKi with the classical chemotherapeutic drug vincristine. In addition, we reported that SKi affected signaling cascades implicated in survival, proliferation and stress response of cells. These findings indicate that SK1 or SK2 represent potential targets for treating T-ALL.

Show MeSH
Related in: MedlinePlus