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Suppression of NF-κB and NF-κB-Regulated Gene Expression by Apigenin through IκBα and IKK Pathway in TRAMP Mice.

Shukla S, Shankar E, Fu P, MacLennan GT, Gupta S - PLoS ONE (2015)

Bottom Line: Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation.The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding.Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it's phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Case Western Reserve University, Cleveland, Ohio, United States of America; The Urology Institute, University Hospitals Case Medical Center, Cleveland, Ohio, United States of America.

ABSTRACT
Aberrant Nuclear Factor-κappaB (NF-κB) activation due to rapid IκBα turnover and high basal IκBα kinase (IKK) activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans) inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it's phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.

No MeSH data available.


Related in: MedlinePlus

Effect of apigenin intake on proliferation and NF-κB-regulated genes in the dorsolateral prostate of TRAMP mice.(A) Protein expression of PCNA, COX-2, VEGF, cyclin D1 as determined by Western blot analysis. A significant decrease in PCNA, COX-2, VEGF, cyclin D1 level is observed after apigenin intake. Actin as loading control. (B) IHC of PCNA in the dorsolateral prostate from control and apigenin-fed TRAMP mice. A marked decrease in proliferation as assessed by PCNA expression is observed after apigenin intake. (C) Proliferation labeling index was calculated as number of PCNA positive cells x 100 / total number of cells counted under x40 magnification in four randomly selected areas in each sample. Values represent Mean + SE, **P<0.05, compared to control. Details are described in ‘materials and methods’ section.
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pone.0138710.g005: Effect of apigenin intake on proliferation and NF-κB-regulated genes in the dorsolateral prostate of TRAMP mice.(A) Protein expression of PCNA, COX-2, VEGF, cyclin D1 as determined by Western blot analysis. A significant decrease in PCNA, COX-2, VEGF, cyclin D1 level is observed after apigenin intake. Actin as loading control. (B) IHC of PCNA in the dorsolateral prostate from control and apigenin-fed TRAMP mice. A marked decrease in proliferation as assessed by PCNA expression is observed after apigenin intake. (C) Proliferation labeling index was calculated as number of PCNA positive cells x 100 / total number of cells counted under x40 magnification in four randomly selected areas in each sample. Values represent Mean + SE, **P<0.05, compared to control. Details are described in ‘materials and methods’ section.

Mentions: Next, to gain insight into the NF-κB-mediated mechanism(s) underlying in vivo anticancer efficacy of apigenin, we determined the expression of various NF-κB responsive genes including COX-2, VEGF, cyclin D1, Bcl2 and Bcl-xL along with assessment of proliferation and apoptotic response. We assessed proliferation through a ubiquitous molecular maker of proliferation i.e. proliferating cell nuclear antigen (PCNA) which interacts with the cyclin-cdk complex during cell cycle progression and is absent in the resting G0-phase cells. As shown in Fig 5A, apigenin feeding to TRAMP mice resulted in marked decrease in the protein expression of PCNA, COX-2, VEGF, and cyclin D1, compared to the control group. IHC analysis indicated that apigenin treatment inhibited PCNA expression in the prostate of TRAMP mice thereby reducing the proliferative index (Fig 5B and 5C).


Suppression of NF-κB and NF-κB-Regulated Gene Expression by Apigenin through IκBα and IKK Pathway in TRAMP Mice.

Shukla S, Shankar E, Fu P, MacLennan GT, Gupta S - PLoS ONE (2015)

Effect of apigenin intake on proliferation and NF-κB-regulated genes in the dorsolateral prostate of TRAMP mice.(A) Protein expression of PCNA, COX-2, VEGF, cyclin D1 as determined by Western blot analysis. A significant decrease in PCNA, COX-2, VEGF, cyclin D1 level is observed after apigenin intake. Actin as loading control. (B) IHC of PCNA in the dorsolateral prostate from control and apigenin-fed TRAMP mice. A marked decrease in proliferation as assessed by PCNA expression is observed after apigenin intake. (C) Proliferation labeling index was calculated as number of PCNA positive cells x 100 / total number of cells counted under x40 magnification in four randomly selected areas in each sample. Values represent Mean + SE, **P<0.05, compared to control. Details are described in ‘materials and methods’ section.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4574560&req=5

pone.0138710.g005: Effect of apigenin intake on proliferation and NF-κB-regulated genes in the dorsolateral prostate of TRAMP mice.(A) Protein expression of PCNA, COX-2, VEGF, cyclin D1 as determined by Western blot analysis. A significant decrease in PCNA, COX-2, VEGF, cyclin D1 level is observed after apigenin intake. Actin as loading control. (B) IHC of PCNA in the dorsolateral prostate from control and apigenin-fed TRAMP mice. A marked decrease in proliferation as assessed by PCNA expression is observed after apigenin intake. (C) Proliferation labeling index was calculated as number of PCNA positive cells x 100 / total number of cells counted under x40 magnification in four randomly selected areas in each sample. Values represent Mean + SE, **P<0.05, compared to control. Details are described in ‘materials and methods’ section.
Mentions: Next, to gain insight into the NF-κB-mediated mechanism(s) underlying in vivo anticancer efficacy of apigenin, we determined the expression of various NF-κB responsive genes including COX-2, VEGF, cyclin D1, Bcl2 and Bcl-xL along with assessment of proliferation and apoptotic response. We assessed proliferation through a ubiquitous molecular maker of proliferation i.e. proliferating cell nuclear antigen (PCNA) which interacts with the cyclin-cdk complex during cell cycle progression and is absent in the resting G0-phase cells. As shown in Fig 5A, apigenin feeding to TRAMP mice resulted in marked decrease in the protein expression of PCNA, COX-2, VEGF, and cyclin D1, compared to the control group. IHC analysis indicated that apigenin treatment inhibited PCNA expression in the prostate of TRAMP mice thereby reducing the proliferative index (Fig 5B and 5C).

Bottom Line: Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation.The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding.Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it's phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Urology, Case Western Reserve University, Cleveland, Ohio, United States of America; The Urology Institute, University Hospitals Case Medical Center, Cleveland, Ohio, United States of America.

ABSTRACT
Aberrant Nuclear Factor-κappaB (NF-κB) activation due to rapid IκBα turnover and high basal IκBα kinase (IKK) activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans) inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it's phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.

No MeSH data available.


Related in: MedlinePlus