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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 expression correlates with noscapine activity both in cell lines and in primary cells of leukemia. (A) Immunoblots showing CYLD and β-actin expression in primary leukemia cells from 7 ALL patients. (B) Quantitative real-time RT-PCR analysis of CYLD mRNA expression in primary leukemia cells. (C) Primary leukemia cells were treated with 2 μM noscapine for 48 hours, and the percentage of apoptotic cells were quantified by nuclear staining. (D-F) Correlation analysis between the IC50 value of noscapine (D), paclitaxel (E), or vinblastine (F) and CYLD protein or mRNA level in primary leukemia cells. CYLD protein level was determined by densitometric analysis of the blots shown in A. (G) Immunoblots for CYLD and β-actin expression in 6 different leukemia cell lines. (H) Correlation analysis between the IC50 value of noscapine and CYLD protein level in 6 leukemia cell lines. (I) Leukemia cell lines were treated with 2 μM noscapine for 48 hours, and the percentage of apoptotic cells were quantified by nuclear staining.
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Figure 1: CYLD expression correlates with noscapine activity both in cell lines and in primary cells of leukemia. (A) Immunoblots showing CYLD and β-actin expression in primary leukemia cells from 7 ALL patients. (B) Quantitative real-time RT-PCR analysis of CYLD mRNA expression in primary leukemia cells. (C) Primary leukemia cells were treated with 2 μM noscapine for 48 hours, and the percentage of apoptotic cells were quantified by nuclear staining. (D-F) Correlation analysis between the IC50 value of noscapine (D), paclitaxel (E), or vinblastine (F) and CYLD protein or mRNA level in primary leukemia cells. CYLD protein level was determined by densitometric analysis of the blots shown in A. (G) Immunoblots for CYLD and β-actin expression in 6 different leukemia cell lines. (H) Correlation analysis between the IC50 value of noscapine and CYLD protein level in 6 leukemia cell lines. (I) Leukemia cell lines were treated with 2 μM noscapine for 48 hours, and the percentage of apoptotic cells were quantified by nuclear staining.

Mentions: To test the potential role of CYLD in regulating the sensitivity of microtubule-targeting drugs in leukemia cells, we obtained primary leukemia cells from seven patients suffering from ALL. We examined CYLD protein level in these cells by immunoblotting and CYLD mRNA level by quantitative real-time PCR. We found that CYLD protein and mRNA levels varied significantly among patients (Figures 1A and 1B). The efficacy of noscapine to induce apoptosis in these cells was then analyzed by nuclear staining. We found significant differences between the patients with a high or low expression of CYLD in the apoptotic efficacy induced by noscapine; leukemia cells isolated from the patient (#3) who had the highest CYLD expression showed the highest apoptosis in response to noscapine treatment (Figure 1C).


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 expression correlates with noscapine activity both in cell lines and in primary cells of leukemia. (A) Immunoblots showing CYLD and β-actin expression in primary leukemia cells from 7 ALL patients. (B) Quantitative real-time RT-PCR analysis of CYLD mRNA expression in primary leukemia cells. (C) Primary leukemia cells were treated with 2 μM noscapine for 48 hours, and the percentage of apoptotic cells were quantified by nuclear staining. (D-F) Correlation analysis between the IC50 value of noscapine (D), paclitaxel (E), or vinblastine (F) and CYLD protein or mRNA level in primary leukemia cells. CYLD protein level was determined by densitometric analysis of the blots shown in A. (G) Immunoblots for CYLD and β-actin expression in 6 different leukemia cell lines. (H) Correlation analysis between the IC50 value of noscapine and CYLD protein level in 6 leukemia cell lines. (I) Leukemia cell lines were treated with 2 μM noscapine for 48 hours, and the percentage of apoptotic cells were quantified by nuclear staining.
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Figure 1: CYLD expression correlates with noscapine activity both in cell lines and in primary cells of leukemia. (A) Immunoblots showing CYLD and β-actin expression in primary leukemia cells from 7 ALL patients. (B) Quantitative real-time RT-PCR analysis of CYLD mRNA expression in primary leukemia cells. (C) Primary leukemia cells were treated with 2 μM noscapine for 48 hours, and the percentage of apoptotic cells were quantified by nuclear staining. (D-F) Correlation analysis between the IC50 value of noscapine (D), paclitaxel (E), or vinblastine (F) and CYLD protein or mRNA level in primary leukemia cells. CYLD protein level was determined by densitometric analysis of the blots shown in A. (G) Immunoblots for CYLD and β-actin expression in 6 different leukemia cell lines. (H) Correlation analysis between the IC50 value of noscapine and CYLD protein level in 6 leukemia cell lines. (I) Leukemia cell lines were treated with 2 μM noscapine for 48 hours, and the percentage of apoptotic cells were quantified by nuclear staining.
Mentions: To test the potential role of CYLD in regulating the sensitivity of microtubule-targeting drugs in leukemia cells, we obtained primary leukemia cells from seven patients suffering from ALL. We examined CYLD protein level in these cells by immunoblotting and CYLD mRNA level by quantitative real-time PCR. We found that CYLD protein and mRNA levels varied significantly among patients (Figures 1A and 1B). The efficacy of noscapine to induce apoptosis in these cells was then analyzed by nuclear staining. We found significant differences between the patients with a high or low expression of CYLD in the apoptotic efficacy induced by noscapine; leukemia cells isolated from the patient (#3) who had the highest CYLD expression showed the highest apoptosis in response to noscapine treatment (Figure 1C).

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