Dietary Flavonoids Luteolin and Quercetin Inhibit Migration and Invasion of Squamous Carcinoma through Reduction of Src/Stat3/S100A7 Signaling

Abstract

Flavonoids are well-known antioxidants and have shown the ability to prevent tumor formation and recurrence. Especially in dietary flavonoids, they have provided convenience and consistence of intake for long-term prevention of tumor formation. Previous reports suggested that S100 calcium-binding protein A7 (S100A7) might activate epithelial-mesenchymal transition (EMT) signaling and promote the metastasis of tumor cells; however, the regulatory signaling was unclear. In this study, we found that S100A7 was highly expressed in cancer cells and could be reduced by luteolin (Lu) and quercetin (Qu) through Src/Stat3 signaling. We found that the protein levels of S100A7, phosphorylated Src (p-Src), and p-Stat3 were increased in A431-III cells. Flavonoids Lu and Qu reduce protein levels of p-Src, p-Stat3 and S100A7 in A431-III cells. Treatment of A431-III cells with Src inhibitor SU6656 and Stat3 inhibitor S3I-201 also reduced the protein levels of S100A7. Transactivation activity of 5'-upstream regions of S100A7 was activated by Stat3 but was reduced by treatment with Lu, Qu, SU6656 and S3I-201. The treatment also reduced the migratory and invasive abilities of A431-III cells. In a further analysis of EMT markers, the protein level of E-cad increased and that of Twist decreased after treatment with the inhibitors and flavonoids. Overexpression of S100A7 decreased the protein level of E-cad and increased the Twist level, whereas knockdown of S100A7 had the opposite effects. Treatment with S3I-201, Lu and Qu, compared to the control, were found to decrease metastasis of tumor cells in zebrafish larvae. These results suggest that Lu and Qu may inhibit Src/Stat3/S100A7 signaling to reduce tumorigenesis of cancer cells.

Keywords: S100A7; Src; Stat3; luteolin; metastasis; quercetin.

Figure 1

S100A7 is more-highly expressed in cervical cancer patients and A431-III cells accompanied by activation of Src/Stat3 signaling. (A) The mRNA levels of S100A7 in A431-P and A431-III cells, analyzed by microarray. (B) The protein levels of S100A7, Src, phosphorylated (p)-Src, Stat3 and p-Stat3 in A431-P and A431-III cells.

Figure 2

Luteolin (Lu) and quercetin (Qu) inhibit S100A7 in A431-III cells by suppressing Src/Stat3 signaling. (A) Cell viability assay of A431-III cells treated with Lu, Qu, Su6656 and S3I-301 using an (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. (B) Western blot analysis of the protein levels in A431-III cells after treatment with 10 and 20 μM of Lu and 20 and 40 μM of Qu. (C) Same as (B), but with 1, 5 and 10 μM of Su6656. (D) Same as (B), but with 100, 200 and 400 μM of S3I-201.

Figure 3

Transactivation activity of S100A7 is regulated by Src/Stat3 signaling. (A) Western blot analysis of the protein level of Stat3 after transfection with pcDNA3-Stat3-HA plasmid in A431-III cells. (B) Transactivation activity of the 5′-upstream regions of S100A7, analyzed by luciferase assay in A431-III cells. Transactivation activity of the 5′-upstream regions of S100A7 was activated by Stat3 (C), but inhibited by luteolin (D), quercetin (E), Su6656 (F), and S3I-201 (G). Statistical significance between groups was analyzed by a one-way ANOVA with Tukey’s test (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Figure 4

Src/Stat3 signaling regulates the migratory ability of A431-III cells, which was reduced by the effectors. A wound-healing assay was conducted, and the cells were observed to migrate into the wound area. (A) A431-III cells were treated with dimethyl sulfoxide (DMSO), 10 and 20 μM of luteolin, 20 and 40 μM of quercetin, 1, 5 and 10 μM of Su6656, and 100, 200 and 400 μM of S3I-201. (B) The numbers of migrating cells treated with luteolin (a), quercetin (b), SU6656 (c) and S3I-201 (d) were measured from (A) and analyzed using ImageJ software (NIH, Bethesda, MA, USA). Statistical significance between groups were analyzed by a one-way ANOVA with Tukey’s test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 5

Src/Stat3 regulates the invasive ability of A431-III cells. (A) A trans-well assay was used to analyze the invasive ability of A431-III cells after pretreatment with 0.1% of DMSO (Aa), 10 μM of Su6656 (Ab), and 400 μM of S3I-201 (Ac) for 24 h prior to seeding. (B) The number of invasive cells were calculated and analyzed by ImageJ software. Statistical significance between groups was analyzed by a one-way ANOVA with Tukey’s test (*** p < 0.001).

Figure 6

Src/Stat3/S100A7 signaling activates the epithelial-mesenchymal transition in A431-III cells. The protein levels of E-cadherin and Twist were analyzed by Western blot after treatment with 10, 20 μM of luteolin and 20, 40 μM of quercetin (A); 1, 5, 10 μM of SU6656 (B); or 100, 200, 400 μM of S3I-201 (C); and after overexpressing S100A7 in A431-P cells (D); or knockdown of S100A7 in A431-III cells by S100A7 shRNAs (E).

Figure 7

Metastasis of A431-III cells were reduced by suppression of Src/Stat3 signaling in zebrafish. (A) A431-III cells were stained with Nile red and microinjected into the pericardiac space of zebrafish larvae at 2 days post-fertilization (dpf). (a) A431-III cells were stained with Nile-Red. (b) The bright field view of 5-dpf zebrafish larvae. Bright field view (c), bright field combined with fluorescent view (d) and fluorescent view (e) of migrative tumor cells enlarged from figure b. (f) Fluorescent view of migrative tumor cells (white arrow) enlarged view from figure e. The number of migrative tumor cells were measured by fluorescent microscope. (B) Measurement of metastatic tumor cell numbers pretreated with 0.1% DMSO (DMSO), 400 μM S3I-201 (S3I-201), 20 μM luteolin, and 40 μM quercetin in zebrafish larvae. Statistical significance between groups were analyzed by a one-way ANOVA with Tukey’s test (** p < 0.01; *** p < 0.001). BF: bright field.