Kaempferol Inhibits the Invasion and Migration of Renal Cancer Cells through the Downregulation of AKT and FAK Pathways

Abstract

Kaempferol, which is isolated from several natural plants, is a polyphenol belonging to the subgroup of flavonoids. Kaempferol exhibits various pharmacological activities, including anti-inflammatory, antioxidant, antimicrobial, and anticancer activities. In this study, kaempferol can significantly inhibit the invasion and migration of 786-O renal cell carcinoma (RCC) without cytotoxicity. We examined the potential mechanisms underlying its anti-invasive activities on 786-O RCC cells. Western blot was performed, and the results showed that kaempferol attenuates the manifestation of metalloproteinase-2 (MMP-2) protein and activity. The inhibitive effect of kaempferol on MMP-2 may be attributed to the downregulation of phosphorylation of Akt and focal adhesion kinase (FAK). By examining the SCID mice model, we found that kaempferol can safely inhibit the metastasis of the 786-O RCC cells into the lungs by about 87.4% as compared to vehicle treated control animals. In addition, the lung tumor masses of mice pretreated with 2-10 mg/kg kaempferol were reduced about twofold to fourfold. These data suggested that kaempferol can play a promising role in tumor prevention and cancer metastasis inhibition.

Keywords: invasion.; kaempferol; metastasis; renal cell carcinoma.

Figure 1

Effects of kaempferol on cell viability of RCC cells. (A) 786-O and (B) HK-2 cells were treated with kaempferol for 24 h by MTT assay. Results were statistically evaluated through one-way ANOVA with post-hoc Dunnett's test. Results from three repeated and separated experiments were similar.

Figure 2

Effects of kaempferol on the cell invasion and migration of RCC cell lines. (A) 786-O cells were treated with kaempferol for 24 h by Boyden chamber invasion and migration assay. Scale bar, 100 μm. (B) The wound healing assay was conducted as described in the Materials and Methods section after 786-O cells were treated with different concentrations of kaempferol for 6 and 24 h. Scale bar, 50 μm. Results were statistically evaluated by using one-way ANOVA with post hoc Dunnett's test (*: P < 0.05; **: P < 0.01; ***: P < 0.001). Results from three repeated and separated experiments were similar.

Figure 3

Inhibitory effects of kaempferol on the expression and activity of MMP-2. 786-O cells were treated with kaempferol for 24 h. (A) Western blot analysis of MMP-2 with β-actin as an internal control. (B) MMP-2 activity was analyzed by gelatin zymography. Data represent mean ± SD, with that of control being 100%, and the statistical significance of results was analyzed using one-way ANOVA with post-hoc Dunnett's test (*: P < 0.05; **: P < 0.01).

Figure 4

Effects of kaempferol on the expression levels of PI3K/Akt, ERK1/2, and FAK protein. (A) Western blot analysis of PI3K, p-Akt, and total-Akt, with β-actin as an internal control in 786-O cells after 24 h of treatment with kaempferol. (B) Western blotting with anti-p-FAK, total-FAK, p-ERK1/2, total-ERK1/2 antibodies, with anti-β-actin as an internal control. Similar results were obtained from three repeated and independent experiments. Data represent mean ± SD, with that of control being 100%, and the statistical significance of results was analyzed using one-way ANOVA with post-hoc Dunnett's test (**: P < 0.01; ***: P < 0.001).

Figure 5

In vivo antimetastatic effects of kaempferol. 786-O cells were injected into the tail veins of six-week-old female SCID mice. After injection of cells, kaempferol (Kae, 2 and 10 mg/kg/d) and placebo (vehicle alone) were administered oral gavage for 150 days. Mice were sacrificed and then analyzed for representative photographs of (A) lungs, (B) lung weight, (C) the number of lung metastasis (D) and body weight in mice. (E) Histopathology of lung of metastatic tumor-bearing animals. Lungs of the metastasis-induced animals were fixed in neutral buffered formalin, and stained with hematoxylin and eosin. Arrows show areas of metastatic nodules (tumor). Results were statistically evaluated by using one-way ANOVA with post-hoc Dunnett's test (**: P < 0.01; ***: P < 0.001).

Figure 6

Proposed molecular targets in anti-metastatic efficacy of kaempferol. Kaempferol targets FAK and Akt-mediated expression of MMP-2 and inhibits cell invasion,migration, and metastasis of 786-O cells.