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Ampelopsin induces apoptosis by regulating multiple c-Myc/S-phase kinase-associated protein 2/F-box and WD repeat-containing protein 7/histone deacetylase 2 pathways in human lung adenocarcinoma cells.

Chen XM, Xie XB, Zhao Q, Wang F, Bai Y, Yin JQ, Jiang H, Xie XL, Jia Q, Huang G - Mol Med Rep (2014)

Bottom Line: Certain changes in apoptotic protein expression were detected following exposure to AMP, including X-linked inhibitor of apoptosis protein release, reduced B-cell lymphoma 2, myeloid cell leukemia 1 and survivin expression levels, increased Bcl-2-associated X protein expression levels and cleaved-poly ADP ribose polymerase expression.The c-Myc/S-phase kinase-associated protein 2 (Skp2) and histone deacetylase (HDAC)1/2 pathways were found to exert an important role in AMP-induced A549 cell apoptosis, as increased levels of c-Myc mRNA and reduced levels of c-Myc/Skp2 and HDAC1 and 2 proteins following AMP treatment were observed.The results suggest that AMP exerts an anticancer effect, which is associated with the degradation of c-Myc, Skp2 and HDAC1 and 2.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Basic Science, Guangzhou Medical University, Guangzhou, Guangdong 510182, P.R. China.

ABSTRACT
Ampelopsin (AMP), a plant flavonoid, has been reported to inhibit cell growth and/or induce apoptosis in various types of tumor. The aim of the present study was to assess the apoptosis-inducing activity of AMP in A549 human lung adenocarcinoma epithelial cells and the associated underlying mechanism. A549 cells were incubated with different concentrations of AMP in culture medium. Cell growth and apoptosis were evaluated by MTT assay and Annexin V/propidium iodide double staining and flow cytometry, respectively. In addition, western blotting and reverse transcription quantitative polymerase chain reaction analysis were used to examine the time-dependent changes in protein expression. Certain changes in apoptotic protein expression were detected following exposure to AMP, including X-linked inhibitor of apoptosis protein release, reduced B-cell lymphoma 2, myeloid cell leukemia 1 and survivin expression levels, increased Bcl-2-associated X protein expression levels and cleaved-poly ADP ribose polymerase expression. The results revealed that AMP was a potent inhibitor of A549 cell proliferation. The c-Myc/S-phase kinase-associated protein 2 (Skp2) and histone deacetylase (HDAC)1/2 pathways were found to exert an important role in AMP-induced A549 cell apoptosis, as increased levels of c-Myc mRNA and reduced levels of c-Myc/Skp2 and HDAC1 and 2 proteins following AMP treatment were observed. The levels of F-box and WD repeat-containing protein 7α (Fbw7α), Fbw7β, Fbw7γ, phosphorylated-(p-)c-Myc (Thr58) and glycogen synthase kinase 3β (GSK3β) proteins involved in c-Myc ubiquitin-dependent degradation were also analyzed. Following exposure to AMP, the expression levels of Fbw7α, Fbw7γ and GSK3β were reduced and p-c-Myc (Thr58) expression levels were increased. The results suggest that AMP exerts an anticancer effect, which is associated with the degradation of c-Myc, Skp2 and HDAC1 and 2. The ability of AMP to induce apoptosis independently of Fbwα and Fbw7γ suggests a possible use in drug-resistant cancer associated with Fbw7 deficiency. Understanding the exact underlying mechanism requires further investigation of the association between c-Myc and Fbw7α/γ reversal, and analysis of whether Thr58 phosphorylation of c-Myc is dependent on GSK3β.

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Effects of ampelopsin on HDAC1 and 2 expression in A549 human pulmonary adenocarcinoma cells. (A) The relative HDAC1 and 2 mRNA levels were detected using quantitative polymerase chain reaction, with GAPDH serving as an internal control. The results are expressed as the mean ± standard error of the mean from five independent experiments. (B) HDAC1 and 2 protein levels assayed by western blotting. In these experiments, cells were exposed to 0, 10, 20 and 30 μM ampelopsin for 48 h. β-actin served as an internal control. *P<0.05 and **P<0.01 vs. the control group. HDAC, histone deacetylase.
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f3-mmr-11-01-0105: Effects of ampelopsin on HDAC1 and 2 expression in A549 human pulmonary adenocarcinoma cells. (A) The relative HDAC1 and 2 mRNA levels were detected using quantitative polymerase chain reaction, with GAPDH serving as an internal control. The results are expressed as the mean ± standard error of the mean from five independent experiments. (B) HDAC1 and 2 protein levels assayed by western blotting. In these experiments, cells were exposed to 0, 10, 20 and 30 μM ampelopsin for 48 h. β-actin served as an internal control. *P<0.05 and **P<0.01 vs. the control group. HDAC, histone deacetylase.

Mentions: Significant downregulation of HDAC1 and 2 mRNA following 30 μM AMP treatment, as compared with the control treatment, was detected by qPCR (Fig. 3A). This result is consistent with the finding that HDAC2 protein expression was downregulated in a dose-dependent manner following exposure to different concentrations of AMP for 48 h (Fig. 3B). These changes may result in a subsequent proportional increase in the levels of histone acetylation, therefore enhancing the expression levels of tumor suppressive proteins.


Ampelopsin induces apoptosis by regulating multiple c-Myc/S-phase kinase-associated protein 2/F-box and WD repeat-containing protein 7/histone deacetylase 2 pathways in human lung adenocarcinoma cells.

Chen XM, Xie XB, Zhao Q, Wang F, Bai Y, Yin JQ, Jiang H, Xie XL, Jia Q, Huang G - Mol Med Rep (2014)

Effects of ampelopsin on HDAC1 and 2 expression in A549 human pulmonary adenocarcinoma cells. (A) The relative HDAC1 and 2 mRNA levels were detected using quantitative polymerase chain reaction, with GAPDH serving as an internal control. The results are expressed as the mean ± standard error of the mean from five independent experiments. (B) HDAC1 and 2 protein levels assayed by western blotting. In these experiments, cells were exposed to 0, 10, 20 and 30 μM ampelopsin for 48 h. β-actin served as an internal control. *P<0.05 and **P<0.01 vs. the control group. HDAC, histone deacetylase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4237074&req=5

f3-mmr-11-01-0105: Effects of ampelopsin on HDAC1 and 2 expression in A549 human pulmonary adenocarcinoma cells. (A) The relative HDAC1 and 2 mRNA levels were detected using quantitative polymerase chain reaction, with GAPDH serving as an internal control. The results are expressed as the mean ± standard error of the mean from five independent experiments. (B) HDAC1 and 2 protein levels assayed by western blotting. In these experiments, cells were exposed to 0, 10, 20 and 30 μM ampelopsin for 48 h. β-actin served as an internal control. *P<0.05 and **P<0.01 vs. the control group. HDAC, histone deacetylase.
Mentions: Significant downregulation of HDAC1 and 2 mRNA following 30 μM AMP treatment, as compared with the control treatment, was detected by qPCR (Fig. 3A). This result is consistent with the finding that HDAC2 protein expression was downregulated in a dose-dependent manner following exposure to different concentrations of AMP for 48 h (Fig. 3B). These changes may result in a subsequent proportional increase in the levels of histone acetylation, therefore enhancing the expression levels of tumor suppressive proteins.

Bottom Line: Certain changes in apoptotic protein expression were detected following exposure to AMP, including X-linked inhibitor of apoptosis protein release, reduced B-cell lymphoma 2, myeloid cell leukemia 1 and survivin expression levels, increased Bcl-2-associated X protein expression levels and cleaved-poly ADP ribose polymerase expression.The c-Myc/S-phase kinase-associated protein 2 (Skp2) and histone deacetylase (HDAC)1/2 pathways were found to exert an important role in AMP-induced A549 cell apoptosis, as increased levels of c-Myc mRNA and reduced levels of c-Myc/Skp2 and HDAC1 and 2 proteins following AMP treatment were observed.The results suggest that AMP exerts an anticancer effect, which is associated with the degradation of c-Myc, Skp2 and HDAC1 and 2.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Basic Science, Guangzhou Medical University, Guangzhou, Guangdong 510182, P.R. China.

ABSTRACT
Ampelopsin (AMP), a plant flavonoid, has been reported to inhibit cell growth and/or induce apoptosis in various types of tumor. The aim of the present study was to assess the apoptosis-inducing activity of AMP in A549 human lung adenocarcinoma epithelial cells and the associated underlying mechanism. A549 cells were incubated with different concentrations of AMP in culture medium. Cell growth and apoptosis were evaluated by MTT assay and Annexin V/propidium iodide double staining and flow cytometry, respectively. In addition, western blotting and reverse transcription quantitative polymerase chain reaction analysis were used to examine the time-dependent changes in protein expression. Certain changes in apoptotic protein expression were detected following exposure to AMP, including X-linked inhibitor of apoptosis protein release, reduced B-cell lymphoma 2, myeloid cell leukemia 1 and survivin expression levels, increased Bcl-2-associated X protein expression levels and cleaved-poly ADP ribose polymerase expression. The results revealed that AMP was a potent inhibitor of A549 cell proliferation. The c-Myc/S-phase kinase-associated protein 2 (Skp2) and histone deacetylase (HDAC)1/2 pathways were found to exert an important role in AMP-induced A549 cell apoptosis, as increased levels of c-Myc mRNA and reduced levels of c-Myc/Skp2 and HDAC1 and 2 proteins following AMP treatment were observed. The levels of F-box and WD repeat-containing protein 7α (Fbw7α), Fbw7β, Fbw7γ, phosphorylated-(p-)c-Myc (Thr58) and glycogen synthase kinase 3β (GSK3β) proteins involved in c-Myc ubiquitin-dependent degradation were also analyzed. Following exposure to AMP, the expression levels of Fbw7α, Fbw7γ and GSK3β were reduced and p-c-Myc (Thr58) expression levels were increased. The results suggest that AMP exerts an anticancer effect, which is associated with the degradation of c-Myc, Skp2 and HDAC1 and 2. The ability of AMP to induce apoptosis independently of Fbwα and Fbw7γ suggests a possible use in drug-resistant cancer associated with Fbw7 deficiency. Understanding the exact underlying mechanism requires further investigation of the association between c-Myc and Fbw7α/γ reversal, and analysis of whether Thr58 phosphorylation of c-Myc is dependent on GSK3β.

Show MeSH
Related in: MedlinePlus