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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 causes cell cycle arrest at the G2/M phase by binding to RPS9.(A) HT-29 cells were transfected with siRPS9 or siCtrl. After 72 hr, the cells were treated with various concentrations of apigenin for 24 hr. Cell cycle distribution was analyzed by flow cytometry. The G2/M population in the samples without apigenin was normalized to 1. Only in this panel, comparisons were performed using unpaired Student’s t-test. (B) SW620 cells were treated with the indicated concentrations of apigenin. After 24 hr, the cells were lysed with RIPA buffer. The lysates were analyzed by immunoblotting. (C) SW620 cells were transfected with siRPS9 or siCtrl. After 72 hr, the cells were lysed with RIPA buffer. The lysates were analyzed by immunoblotting. CT: control, Data are means ± SD (n = 3). *P < 0.05, **P < 0.01 relative to siCtrl.
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pone-0073219-g005: Apigenin causes cell cycle arrest at the G2/M phase by binding to RPS9.(A) HT-29 cells were transfected with siRPS9 or siCtrl. After 72 hr, the cells were treated with various concentrations of apigenin for 24 hr. Cell cycle distribution was analyzed by flow cytometry. The G2/M population in the samples without apigenin was normalized to 1. Only in this panel, comparisons were performed using unpaired Student’s t-test. (B) SW620 cells were treated with the indicated concentrations of apigenin. After 24 hr, the cells were lysed with RIPA buffer. The lysates were analyzed by immunoblotting. (C) SW620 cells were transfected with siRPS9 or siCtrl. After 72 hr, the cells were lysed with RIPA buffer. The lysates were analyzed by immunoblotting. CT: control, Data are means ± SD (n = 3). *P < 0.05, **P < 0.01 relative to siCtrl.

Mentions: To clarify the role of RPS9 in apigenin-induced cell cycle arrest, we investigated whether knockdown of RPS9 influenced this cell cycle arrest as previously performed as to other target proteins [36,37]. Apigenin caused G2/M arrest in HT-29 cells transfected with control siRNA, but not in HT-29 cells transfected with RPS9-targeting siRNA (Figure 5A). The results show that RPS9 is required for the G2/M arrest caused by apigenin, suggesting that apigenin induces G2/M arrest by inhibiting RPS9. On the other hand, we examined whether apigenin inhibited nascent protein synthesis, since knockdown of RPS9 partially inhibited nascent protein synthesis [34]. As shown in Figure S4, apigenin decreased one of major nascent proteins indicated by an arrow with different molecular weight from that of CDK1, but not most of proteins. This reduction of nascent protein synthesis indicated by an arrow by apigenin might raise the possibility that apigenin affects protein synthesis through RPS9. We then performed the similar experiments using another human colon cancer cell line As shown in Figure 5B and C, knockdown of RPS9 as well as apigenin downregulated CDK1 in human colon cancer SW620 cells. These results suggest that apigenin downregulates CDK1 by inhibiting RPS9 not only in HT-29 cells but also in other human malignant tumor cells.


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 causes cell cycle arrest at the G2/M phase by binding to RPS9.(A) HT-29 cells were transfected with siRPS9 or siCtrl. After 72 hr, the cells were treated with various concentrations of apigenin for 24 hr. Cell cycle distribution was analyzed by flow cytometry. The G2/M population in the samples without apigenin was normalized to 1. Only in this panel, comparisons were performed using unpaired Student’s t-test. (B) SW620 cells were treated with the indicated concentrations of apigenin. After 24 hr, the cells were lysed with RIPA buffer. The lysates were analyzed by immunoblotting. (C) SW620 cells were transfected with siRPS9 or siCtrl. After 72 hr, the cells were lysed with RIPA buffer. The lysates were analyzed by immunoblotting. CT: control, Data are means ± SD (n = 3). *P < 0.05, **P < 0.01 relative to siCtrl.
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getmorefigures.php?uid=PMC3756953&req=5

pone-0073219-g005: Apigenin causes cell cycle arrest at the G2/M phase by binding to RPS9.(A) HT-29 cells were transfected with siRPS9 or siCtrl. After 72 hr, the cells were treated with various concentrations of apigenin for 24 hr. Cell cycle distribution was analyzed by flow cytometry. The G2/M population in the samples without apigenin was normalized to 1. Only in this panel, comparisons were performed using unpaired Student’s t-test. (B) SW620 cells were treated with the indicated concentrations of apigenin. After 24 hr, the cells were lysed with RIPA buffer. The lysates were analyzed by immunoblotting. (C) SW620 cells were transfected with siRPS9 or siCtrl. After 72 hr, the cells were lysed with RIPA buffer. The lysates were analyzed by immunoblotting. CT: control, Data are means ± SD (n = 3). *P < 0.05, **P < 0.01 relative to siCtrl.
Mentions: To clarify the role of RPS9 in apigenin-induced cell cycle arrest, we investigated whether knockdown of RPS9 influenced this cell cycle arrest as previously performed as to other target proteins [36,37]. Apigenin caused G2/M arrest in HT-29 cells transfected with control siRNA, but not in HT-29 cells transfected with RPS9-targeting siRNA (Figure 5A). The results show that RPS9 is required for the G2/M arrest caused by apigenin, suggesting that apigenin induces G2/M arrest by inhibiting RPS9. On the other hand, we examined whether apigenin inhibited nascent protein synthesis, since knockdown of RPS9 partially inhibited nascent protein synthesis [34]. As shown in Figure S4, apigenin decreased one of major nascent proteins indicated by an arrow with different molecular weight from that of CDK1, but not most of proteins. This reduction of nascent protein synthesis indicated by an arrow by apigenin might raise the possibility that apigenin affects protein synthesis through RPS9. We then performed the similar experiments using another human colon cancer cell line As shown in Figure 5B and C, knockdown of RPS9 as well as apigenin downregulated CDK1 in human colon cancer SW620 cells. These results suggest that apigenin downregulates CDK1 by inhibiting RPS9 not only in HT-29 cells but also in other human malignant tumor cells.

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