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The flavonoid apigenin downregulates CDK1 by directly targeting ribosomal protein S9.

Iizumi Y, Oishi M, Taniguchi T, Goi W, Sowa Y, Sakai T - PLoS ONE (2013)

Bottom Line: Similar to treatment with apigenin, knockdown of RPS9 inhibited the growth of human colon cancer cells at the G2/M phase by downregulating cyclin-dependent kinase 1 (CDK1) expression at the promoter level.Furthermore, knockdown of RPS9 suppressed G2/M arrest caused by apigenin.These results suggest that apigenin induces G2/M arrest at least partially by directly binding and inhibiting RPS9 which enhances CDK1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, Japan.

ABSTRACT
Flavonoids have been reported to inhibit tumor growth by causing cell cycle arrest. However, little is known about the direct targets of flavonoids in tumor growth inhibition. In the present study, we developed a novel method using magnetic FG beads to purify flavonoid-binding proteins, and identified ribosomal protein S9 (RPS9) as a binding partner of the flavonoid apigenin. Similar to treatment with apigenin, knockdown of RPS9 inhibited the growth of human colon cancer cells at the G2/M phase by downregulating cyclin-dependent kinase 1 (CDK1) expression at the promoter level. Furthermore, knockdown of RPS9 suppressed G2/M arrest caused by apigenin. These results suggest that apigenin induces G2/M arrest at least partially by directly binding and inhibiting RPS9 which enhances CDK1 expression. We therefore raise the possibility that identification of the direct targets of flavonoids may contribute to the discovery of novel molecular mechanisms governing tumor growth.

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Apigenin directly binds to RPS9.(A) Apigenin-binding proteins were purified from whole cell extracts of HT-29 cells with apigenin-fixed (+) or empty (-) beads, and detected by silver staining. Mass spectrometry analysis identified RPS9 as an apigenin-binding protein. Inp: Whole cell extracts of HT-29 cells. (B) Confirmation of mass spectrometry analysis by immunoblotting with an anti-RPS9 antibody. Inp: Whole cell extracts of HT-29 cells. (C) Competition binding assay. The indicated concentrations of free apigenin were added to HT-29 cell extracts. After 1 hr, the extracts were incubated with apigenin-fixed (+) or empty (-) beads. Apigenin-binding proteins were purified and detected by silver staining. Inp: Whole cell extracts of HT-29 cells. (D) Purified recombinant His-RPS9 was incubated with apigenin-fixed (+) or empty (-) beads, and bound His-RPS9 was detected by immunoblotting with anti-RPS9 and anti-His antibodies. Inp: Purified recombinant His-RPS9.
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pone-0073219-g002: Apigenin directly binds to RPS9.(A) Apigenin-binding proteins were purified from whole cell extracts of HT-29 cells with apigenin-fixed (+) or empty (-) beads, and detected by silver staining. Mass spectrometry analysis identified RPS9 as an apigenin-binding protein. Inp: Whole cell extracts of HT-29 cells. (B) Confirmation of mass spectrometry analysis by immunoblotting with an anti-RPS9 antibody. Inp: Whole cell extracts of HT-29 cells. (C) Competition binding assay. The indicated concentrations of free apigenin were added to HT-29 cell extracts. After 1 hr, the extracts were incubated with apigenin-fixed (+) or empty (-) beads. Apigenin-binding proteins were purified and detected by silver staining. Inp: Whole cell extracts of HT-29 cells. (D) Purified recombinant His-RPS9 was incubated with apigenin-fixed (+) or empty (-) beads, and bound His-RPS9 was detected by immunoblotting with anti-RPS9 and anti-His antibodies. Inp: Purified recombinant His-RPS9.

Mentions: To identify the direct target of apigenin, apigenin-binding proteins were purified using magnetic FG beads with epoxy linkers [23,24]. A system was developed in which flavonoids were conjugated onto the beads with potassium carbonate. Phenolic hydroxyl groups of flavonoids covalently bind to epoxy groups of the beads in the system. Apigenin was covalently conjugated to the beads using this system (Figure S1), and apigenin-fixed beads were incubated with whole cell extracts of HT-29 cells. One major apigenin-binding protein was purified from HT-29 cell extracts. This protein was identified as ribosomal protein S9 (RPS9) by mass spectrometry (Figure 2A). Identification of this protein was confirmed by immunoblotting (Figure 2B). Binding of RPS9 was selectively competed with increasing concentrations of apigenin (Figure 2C), suggesting that RPS9 specifically interacted with apigenin. Purified recombinant histidine-tagged RPS9 (His-RPS9) also bound to apigenin-fixed beads, suggesting that this interaction was direct (Figure 2D). Since RPS9 is an RNA-binding protein, we further examined whether RNA was required for this interaction. As shown in Figure S2, apigenin bound to RPS9 in the presence of RNase A. These results indicate that apigenin directly binds to RPS9.


The flavonoid apigenin downregulates CDK1 by directly targeting ribosomal protein S9.

Iizumi Y, Oishi M, Taniguchi T, Goi W, Sowa Y, Sakai T - PLoS ONE (2013)

Apigenin directly binds to RPS9.(A) Apigenin-binding proteins were purified from whole cell extracts of HT-29 cells with apigenin-fixed (+) or empty (-) beads, and detected by silver staining. Mass spectrometry analysis identified RPS9 as an apigenin-binding protein. Inp: Whole cell extracts of HT-29 cells. (B) Confirmation of mass spectrometry analysis by immunoblotting with an anti-RPS9 antibody. Inp: Whole cell extracts of HT-29 cells. (C) Competition binding assay. The indicated concentrations of free apigenin were added to HT-29 cell extracts. After 1 hr, the extracts were incubated with apigenin-fixed (+) or empty (-) beads. Apigenin-binding proteins were purified and detected by silver staining. Inp: Whole cell extracts of HT-29 cells. (D) Purified recombinant His-RPS9 was incubated with apigenin-fixed (+) or empty (-) beads, and bound His-RPS9 was detected by immunoblotting with anti-RPS9 and anti-His antibodies. Inp: Purified recombinant His-RPS9.
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Related In: Results  -  Collection

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pone-0073219-g002: Apigenin directly binds to RPS9.(A) Apigenin-binding proteins were purified from whole cell extracts of HT-29 cells with apigenin-fixed (+) or empty (-) beads, and detected by silver staining. Mass spectrometry analysis identified RPS9 as an apigenin-binding protein. Inp: Whole cell extracts of HT-29 cells. (B) Confirmation of mass spectrometry analysis by immunoblotting with an anti-RPS9 antibody. Inp: Whole cell extracts of HT-29 cells. (C) Competition binding assay. The indicated concentrations of free apigenin were added to HT-29 cell extracts. After 1 hr, the extracts were incubated with apigenin-fixed (+) or empty (-) beads. Apigenin-binding proteins were purified and detected by silver staining. Inp: Whole cell extracts of HT-29 cells. (D) Purified recombinant His-RPS9 was incubated with apigenin-fixed (+) or empty (-) beads, and bound His-RPS9 was detected by immunoblotting with anti-RPS9 and anti-His antibodies. Inp: Purified recombinant His-RPS9.
Mentions: To identify the direct target of apigenin, apigenin-binding proteins were purified using magnetic FG beads with epoxy linkers [23,24]. A system was developed in which flavonoids were conjugated onto the beads with potassium carbonate. Phenolic hydroxyl groups of flavonoids covalently bind to epoxy groups of the beads in the system. Apigenin was covalently conjugated to the beads using this system (Figure S1), and apigenin-fixed beads were incubated with whole cell extracts of HT-29 cells. One major apigenin-binding protein was purified from HT-29 cell extracts. This protein was identified as ribosomal protein S9 (RPS9) by mass spectrometry (Figure 2A). Identification of this protein was confirmed by immunoblotting (Figure 2B). Binding of RPS9 was selectively competed with increasing concentrations of apigenin (Figure 2C), suggesting that RPS9 specifically interacted with apigenin. Purified recombinant histidine-tagged RPS9 (His-RPS9) also bound to apigenin-fixed beads, suggesting that this interaction was direct (Figure 2D). Since RPS9 is an RNA-binding protein, we further examined whether RNA was required for this interaction. As shown in Figure S2, apigenin bound to RPS9 in the presence of RNase A. These results indicate that apigenin directly binds to RPS9.

Bottom Line: Similar to treatment with apigenin, knockdown of RPS9 inhibited the growth of human colon cancer cells at the G2/M phase by downregulating cyclin-dependent kinase 1 (CDK1) expression at the promoter level.Furthermore, knockdown of RPS9 suppressed G2/M arrest caused by apigenin.These results suggest that apigenin induces G2/M arrest at least partially by directly binding and inhibiting RPS9 which enhances CDK1 expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular-Targeting Cancer Prevention, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, Japan.

ABSTRACT
Flavonoids have been reported to inhibit tumor growth by causing cell cycle arrest. However, little is known about the direct targets of flavonoids in tumor growth inhibition. In the present study, we developed a novel method using magnetic FG beads to purify flavonoid-binding proteins, and identified ribosomal protein S9 (RPS9) as a binding partner of the flavonoid apigenin. Similar to treatment with apigenin, knockdown of RPS9 inhibited the growth of human colon cancer cells at the G2/M phase by downregulating cyclin-dependent kinase 1 (CDK1) expression at the promoter level. Furthermore, knockdown of RPS9 suppressed G2/M arrest caused by apigenin. These results suggest that apigenin induces G2/M arrest at least partially by directly binding and inhibiting RPS9 which enhances CDK1 expression. We therefore raise the possibility that identification of the direct targets of flavonoids may contribute to the discovery of novel molecular mechanisms governing tumor growth.

Show MeSH
Related in: MedlinePlus