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CYLD Regulates Noscapine Activity in Acute Lymphoblastic Leukemia via a Microtubule-Dependent Mechanism.

Yang Y, Ran J, Sun L, Sun X, Luo Y, Yan B - Theranostics (2015)

Bottom Line: Here we demonstrate that cylindromatosis (CYLD), a microtubule-associated tumor suppressor protein, modulates the activity of noscapine both in cell lines and in primary cells of acute lymphoblastic leukemia (ALL).Examination of cellular microtubules as well as in vitro assembled microtubules shows that CYLD enhances the effect of noscapine on microtubule polymerization.These findings thus demonstrate CYLD as a critical regulator of noscapine activity and have important implications for ALL treatment.

View Article: PubMed Central - PubMed

Affiliation: 1. State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.

ABSTRACT
Noscapine is an orally administrable drug used worldwide for cough suppression and has recently been demonstrated to disrupt microtubule dynamics and possess anticancer activity. However, the molecular mechanisms regulating noscapine activity remain poorly defined. Here we demonstrate that cylindromatosis (CYLD), a microtubule-associated tumor suppressor protein, modulates the activity of noscapine both in cell lines and in primary cells of acute lymphoblastic leukemia (ALL). Flow cytometry and immunofluorescence microscopy reveal that CYLD increases the ability of noscapine to induce mitotic arrest and apoptosis. Examination of cellular microtubules as well as in vitro assembled microtubules shows that CYLD enhances the effect of noscapine on microtubule polymerization. Microtubule cosedimentation and fluorescence titration assays further reveal that CYLD interacts with microtubule outer surface and promotes noscapine binding to microtubules. These findings thus demonstrate CYLD as a critical regulator of noscapine activity and have important implications for ALL treatment.

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CYLD depletion attenuates noscapine-induced apoptosis in primary leukemia cells. (A) Immunoblots for CYLD and β-actin expression in control and CYLD siRNA-treated primary leukemia cells. (B) Experiments were performed as in A, and CYLD protein level was determined by densitometric analysis of the blots. (C) Fluorescence/phase-contrast images of primary leukemia cells transfected with control or CYLD siRNAs followed by treatment with 2 μM noscapine for 36 hours. Cells were stained with Alexa Fluor 488-conjugated annexin V (green). (D) Experiments were performed as in C, and the percentage of annexin V-positive cells was quantified. (E) Primary leukemia cells were transfected with control or CYLD siRNAs followed by treatment with 2 μM noscapine for 24 hours. Cells were then stained with PI and Alexa Fluor 488-conjugated annexin V, and analyzed by flow cytometry. (F) Experiments were performed as in E, and the percentage of annexin V-positive cells was quantified. **, p < 0.01; ***, p < 0.001.
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Figure 2: CYLD depletion attenuates noscapine-induced apoptosis in primary leukemia cells. (A) Immunoblots for CYLD and β-actin expression in control and CYLD siRNA-treated primary leukemia cells. (B) Experiments were performed as in A, and CYLD protein level was determined by densitometric analysis of the blots. (C) Fluorescence/phase-contrast images of primary leukemia cells transfected with control or CYLD siRNAs followed by treatment with 2 μM noscapine for 36 hours. Cells were stained with Alexa Fluor 488-conjugated annexin V (green). (D) Experiments were performed as in C, and the percentage of annexin V-positive cells was quantified. (E) Primary leukemia cells were transfected with control or CYLD siRNAs followed by treatment with 2 μM noscapine for 24 hours. Cells were then stained with PI and Alexa Fluor 488-conjugated annexin V, and analyzed by flow cytometry. (F) Experiments were performed as in E, and the percentage of annexin V-positive cells was quantified. **, p < 0.01; ***, p < 0.001.

Mentions: To gain mechanistic insight into the role of CYLD in regulating noscapine activity, we studied the effect of CYLD on downstream cellular events triggered by noscapine. Primary leukemia cells, from #3 ALL patient shown in Figure 1A, were transfected with control or CYLD siRNAs and then treated with noscapine. We found that CYLD expression was dramatically knocked down by the siRNAs (Figures 2A and 2B). Fluorescence microscopy showed that depletion of CYLD remarkably reduced the accumulation of annexin V-positive cells induced by noscapine (Figures 2C and 2D). Flow cytometric analysis of control or CYLD siRNA-transfected primary leukemia cells further demonstrated that both early (annexin V-positive and PI-negative) and late (annexin V-positive and PI-positive) apoptosis induced by noscapine were attenuated by CYLD depletion (Figures 2E and 2F). These data indicate that CYLD is important for the ability of noscapine to induce apoptosis in ALL cells.


CYLD Regulates Noscapine Activity in Acute Lymphoblastic Leukemia via a Microtubule-Dependent Mechanism.

Yang Y, Ran J, Sun L, Sun X, Luo Y, Yan B - Theranostics (2015)

CYLD depletion attenuates noscapine-induced apoptosis in primary leukemia cells. (A) Immunoblots for CYLD and β-actin expression in control and CYLD siRNA-treated primary leukemia cells. (B) Experiments were performed as in A, and CYLD protein level was determined by densitometric analysis of the blots. (C) Fluorescence/phase-contrast images of primary leukemia cells transfected with control or CYLD siRNAs followed by treatment with 2 μM noscapine for 36 hours. Cells were stained with Alexa Fluor 488-conjugated annexin V (green). (D) Experiments were performed as in C, and the percentage of annexin V-positive cells was quantified. (E) Primary leukemia cells were transfected with control or CYLD siRNAs followed by treatment with 2 μM noscapine for 24 hours. Cells were then stained with PI and Alexa Fluor 488-conjugated annexin V, and analyzed by flow cytometry. (F) Experiments were performed as in E, and the percentage of annexin V-positive cells was quantified. **, p < 0.01; ***, p < 0.001.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4402491&req=5

Figure 2: CYLD depletion attenuates noscapine-induced apoptosis in primary leukemia cells. (A) Immunoblots for CYLD and β-actin expression in control and CYLD siRNA-treated primary leukemia cells. (B) Experiments were performed as in A, and CYLD protein level was determined by densitometric analysis of the blots. (C) Fluorescence/phase-contrast images of primary leukemia cells transfected with control or CYLD siRNAs followed by treatment with 2 μM noscapine for 36 hours. Cells were stained with Alexa Fluor 488-conjugated annexin V (green). (D) Experiments were performed as in C, and the percentage of annexin V-positive cells was quantified. (E) Primary leukemia cells were transfected with control or CYLD siRNAs followed by treatment with 2 μM noscapine for 24 hours. Cells were then stained with PI and Alexa Fluor 488-conjugated annexin V, and analyzed by flow cytometry. (F) Experiments were performed as in E, and the percentage of annexin V-positive cells was quantified. **, p < 0.01; ***, p < 0.001.
Mentions: To gain mechanistic insight into the role of CYLD in regulating noscapine activity, we studied the effect of CYLD on downstream cellular events triggered by noscapine. Primary leukemia cells, from #3 ALL patient shown in Figure 1A, were transfected with control or CYLD siRNAs and then treated with noscapine. We found that CYLD expression was dramatically knocked down by the siRNAs (Figures 2A and 2B). Fluorescence microscopy showed that depletion of CYLD remarkably reduced the accumulation of annexin V-positive cells induced by noscapine (Figures 2C and 2D). Flow cytometric analysis of control or CYLD siRNA-transfected primary leukemia cells further demonstrated that both early (annexin V-positive and PI-negative) and late (annexin V-positive and PI-positive) apoptosis induced by noscapine were attenuated by CYLD depletion (Figures 2E and 2F). These data indicate that CYLD is important for the ability of noscapine to induce apoptosis in ALL cells.

Bottom Line: Here we demonstrate that cylindromatosis (CYLD), a microtubule-associated tumor suppressor protein, modulates the activity of noscapine both in cell lines and in primary cells of acute lymphoblastic leukemia (ALL).Examination of cellular microtubules as well as in vitro assembled microtubules shows that CYLD enhances the effect of noscapine on microtubule polymerization.These findings thus demonstrate CYLD as a critical regulator of noscapine activity and have important implications for ALL treatment.

View Article: PubMed Central - PubMed

Affiliation: 1. State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China.

ABSTRACT
Noscapine is an orally administrable drug used worldwide for cough suppression and has recently been demonstrated to disrupt microtubule dynamics and possess anticancer activity. However, the molecular mechanisms regulating noscapine activity remain poorly defined. Here we demonstrate that cylindromatosis (CYLD), a microtubule-associated tumor suppressor protein, modulates the activity of noscapine both in cell lines and in primary cells of acute lymphoblastic leukemia (ALL). Flow cytometry and immunofluorescence microscopy reveal that CYLD increases the ability of noscapine to induce mitotic arrest and apoptosis. Examination of cellular microtubules as well as in vitro assembled microtubules shows that CYLD enhances the effect of noscapine on microtubule polymerization. Microtubule cosedimentation and fluorescence titration assays further reveal that CYLD interacts with microtubule outer surface and promotes noscapine binding to microtubules. These findings thus demonstrate CYLD as a critical regulator of noscapine activity and have important implications for ALL treatment.

Show MeSH
Related in: MedlinePlus