Flavonoids Luteolin and Quercetin Inhibit RPS19 and contributes to metastasis of cancer cells through c-Myc reduction

Abstract

Flavonoids luteolin and quercetin can inhibit growth and metastasis of cancer cells. In our previous report, luteolin and quercetin was shown to block Akt/mTOR/c-Myc signaling. Here, we found luteolin and quercetin reduced protein level and transactivation activity of RPS19 in A431-III cells, which is isolated from parental A431 (A431-P) cell line. Further investigation the inhibitory mechanism of luteolin and quercetin on RPS19, we found c-Myc binding sites on RPS19 promoter. The Akt inhibitor LY294002, mTOR inhibitor rapamycin and c-Myc inhibitor 10058-F4 significantly suppressed RPS19 expression and transactivation activities. Overexpression and knockdown of c-Myc in cancer cells show RPS19 expression was regulated by c-Myc. Furthermore, Knockdown and overexpression of RPS19 was used to analyze of the function of RPS19 in cancer cells. The epithelial-mesenchymal transition (EMT) markers and metastasis abilities of cancer cells were also regulated by RPS19. These data suggest that luteolin and quercetin might inhibit metastasis of cancer cells by blocking Akt/mTOR/c-Myc signaling pathway to suppress RPS19-activated EMT signaling.

Keywords: EMT; Luteolin; Quercetin; RPS19; c-Myc.

Fig. 1

Protein levels of ribosomal protein S19 (RPS19) in A431-P and A431-III cells. A. Protein levels of RPS19 in A431-P and A431-III cells were measured by a Western blot experiment. B. The protein levels of RPS19 in A431-P and A431-III cells was analyzed by ImageJ software. Results from three independent experiments are expressed as the mean ± standard deviation (SD). * Indicates a significant difference compared to the control (p < 0.05).

Fig. 2

Ribosomal protein S19 (RPS19) expression was inhibited by Qu, Qu through Akt/mammalian target of rapamycin (mTOR) signaling. A. The protein level of RPS19 in A431-III cells was measured by Western blotting after treatment with DMSO, 10 and 20 μM of Lu, or 20 and 40 μM of Qu for 24 h. B. Protein level changes of RPS19 after treatment with Lu or Qu were analyzed by ImageJ software. C. Protein levels of RPS19 in A431-III cells were measured by Western blotting after treatment with 5 and 10 nM of rapamycin or 5 and 10 μM of LY294002 for 24 h. D. Protein level changes of RPS19 after treatment with rapamycin or LY294002 were analyzed with ImageJ software. Data represent the mean (SD) of three different experiments. Statistical significance was determined by a one-way ANOVA with Tukey's test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 3

c-Myc regulates ribosomal protein S19 (RPS19) expression. A. A431-P cells were transfected with 1 μg of pcDNA3-HA as the control, and 0.5 and 1.0 μg of pcDNA3-cMyc-HA. Protein levels of RPS19 was measured by Western blotting after 24 h. B. Protein level changes of RPS19 after transfection with the control or pcDNA3-cMyc-HA were analyzed with ImageJ software. C. A431-III cells were treated with DMSO, or 100, 200, and 400 μM of 10058-F4 for 24 h, and protein levels of RPS19 were measured by Western blotting. D. Protein level changes of RPS19 after treatment with DMSO or 10058-F4 were analyzed by ImageJ software. Data represent the mean (SD) of three different experiments. Statistical significance was determined by a one-way ANOVA with Tukey's test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 4

cMyc activates transactivation of ribosomal protein S19 (RPS19) in A431-P cells. A. The RPS19 promoter contains six potential E-box motifs. B. Transactivation of RPS19 was measured by a luciferase assay after transfection of A431-P cells with pcDNA3-HA (control), or 0.5 and 1.0 μg of pcDNA3-cMyc-HA for 24 h in A431-P cells. C. Transactivation of RPS19 was measured by transfection of A431-III cells with pcDNA3-HA (control), 0.25 and 0.5 μg of pcDNA3-cMyc-HA, and combined treated with or without 10058-F4 A431-III cells for 24 h in A431-III cells. Statistical significance between the groups was determined from three separate experiments by an unpaired Student's t-test. Results from three independent experiments are expressed as the mean ± standard deviation (SD). Statistical significance was determined by a one-way ANOVA with Tukey's test (*P < 0.05, **P < 0.01, and ***P < 0.001). that RPS19 expression might be reduced by both Lu and Qu through the Akt/mTOR signaling pathway.

Fig. 5

Transactivation activity of ribosomal protein S19 (RPS19) is reduced by luteolin and quercetin through reduction of Akt/mTOR/cMyc signaling. Transactivation of RPS19 was measured by treatment of A431-III cells with 10 and 20 μM of luteolin (A), 20 and 40 μM of quercetin (B), 5 and 10 μM of LY294002 (C), 200 and 400 μM of 10058-F4 (D) for 24 h. Data represent the mean (SD) of three different experiments. Statistical significance was determined by a one-way ANOVA with Tukey's test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 6

Ribosomal protein S19 (RPS19) activates EMT signaling. Protein levels of RPS19, E-cadherin, and vimentin were measured by Western blotting after overexpression of RPS19 in A431-P cells (A) and RPS19 shRNA knockdown in A431-III cells (B). Protein levels of E-cadherin and vimentin from Western blotting was analyzed by ImageJ software. Data represent the mean (SD) of three different experiments. Statistical significance was determined by a one-way ANOVA with Tukey's test (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 7

Ribosomal protein S19 (RPS19) increases the migratory and invasive abilities of cancer cells. A wound-healing experiment was used to analyze cell migration after RPS19 overexpression in A431-P cells (A, C) and RPS19 shRNA knockdown in A431-III cells (B, D). A trans-well assay was used to analyze the invasive ability with RPS19 overexpression in A431-P cells (E, G) and RPS19 shRNA knockdown in A431-III cells (F, H). Migration and invasion cells were calculated and analyzed by ImageJ software. Statistical significance between the groups was determined from three separate experiments with an unpaired Student's t-test. Results from three independent experiments are expressed as the mean ± standard deviation (SD). Statistical significance was determined by a one-way ANOVA with Tukey's test (*P < 0.05, **P < 0.01, and ***P < 0.001).