Genistein inhibits the growth and regulates the migration and invasion abilities of melanoma cells via the FAK/paxillin and MAPK pathways

Abstract

Genistein is one of the main components of soy-based foods, which are widely known for their many benefits, including anti-cancer, anti-inflammatory, and antioxidant effects. In this study, we investigated the anti-metastasis effects of genistein on B16F10 melanoma cells. Our results showed that genistein strongly inhibited B16F10 cell proliferation and induced apoptosis in time- and concentration-dependent manners. Genistein altered the morphology of B16F10 cells to an elongated shape with slim pseudopodia-like protrusions. Moreover, genistein inhibited the invasion and migration abilities of B16F10 cells in a dose-dependent manner. On one hand, a high concentration of genistein (100 μM) significantly inhibited cell adhesion and migration, as shown by wound healing assays and transwell-migration and invasion assays. Furthermore, the expression levels of p-FAK, p-paxillin, tensin-2, vinculin, and α-actinin were decreased by genistein. As a result, genistein is believed to strongly downregulate the migration and invasion abilities of B16F10 cells via the FAK/paxillin pathway. Moreover, p-p38, p-ERK, and p-JNK levels were also dramatically decreased by treatment with genistein. Finally, genistein significantly decreased the gene expression of FAK, paxillin, vimentin, and epithelial-to-mesenchymal transition-related transcription factor Snail, as shown by real-time PCR (qPCR) analysis. On the other hand, a lower concentration of genistein (12.5 μM) significantly promoted both invasion and migration by activating the FAK/paxillin and MAPK signaling cascades. Taken together, this study showed for the first time that genistein exerts dual functional effects on melanoma cells. Our findings suggest that genistein regulates the FAK/paxillin and MAPK signaling pathways in a highly concentration-dependent manner. Patients with melanoma should therefore be cautious of consuming soy-based foods in their diets.

Keywords: FAK/paxillin pathway; MAPK pathway; genistein; invasion and migration; melanoma cells.

Figure 1

A. Effects of genistein on the viability of B16F10 cells as determined by WST-8 assay. B16F10 cells were incubated with different concentrations of genistein for 24 and 48 h. B. Cell numbers were counted in the 6-well plates after incubation with genistein for 24 and 48 h. C. Long-term colony formation assay of B16F10 cells after treatment with genistein (0, 12.5, 25, 50, and 100 μM). Cells were grown in the absence or presence of genistein at the indicated concentrations for 7 days. Cells were fixed and stained with crystal violet. Data shown are the averages of three replicates; the experiment was repeated three times. *p<0.05, **p<0.01, compared with the DMSO control. Scale bar is 200 μm.

Figure 2. Flow cytometric analysis of B16F10 cell apoptosis after treatment with genistein for 24 and 48 h

A. Influence of the genistein concentration on the ratio of apoptotic cells after treatment for 24 and 48 h. B. The experiment was repeated three times independently. Percentage of total apoptotic cells = Percentage of (Q2+Q3). In each experiment, 10,000 cells were counted (100%). C. TUNEL assay evaluating apoptosis after treatment with genistein for 48 h. Scale bar is 100 μm. *p<0.05, compared with DMSO control; **p<0.01, compared with DMSO control.

Figure 3

A. Morphological changes in B16F10 melanoma cells after treatment with genistein for 24 h. B. Morphological changes in B16F10 melanoma cells after treatment with genistein for 48 h. The pictures were taken under an inverted contrast microscope. Scale bar is 200 μm. C. Flow cytometric analysis of B16F10 cells treated with genistein for 24 and 48 h. Gating of the cells is shown in the plots of the forward versus side scatter of the cells.

Figure 4. Genistein inhibits the adhesion of B16F10 melanoma cells

Cells were incubated with genistein for 24 h. Unattached cells were first removed, and then attached cells were mixed in 4% paraformaldehyde and stained with crystal violet solution for 10 min at room temperature. The OD value was measured at 570 nm using a microplate reader. Percentage of adhered cells was calculated relative to the control cells. The experiment was repeated three times. *p<0.05, compared with DMSO control; ** p<0.01, compared with DMSO control.

Figure 5. Genistein inhibits the mobility of B16F10 cells

Cells were plated into a 6-well plate for confluent monolayer formation in complete medium. Cell monolayers were wounded using a sterile 200 μL micropipette tip, and the remaining cells were incubated in medium containing 0, 12.5, 25, 50, and 100 μM genistein for 24 h. At the indicated time (0, 8, and 24 h) after scraping, the wound areas were photographed A. and the percentage of cell migration inhibition B. was calculated as described in the Materials and Methods. Scale bar is 200 μm. The experiment was repeated three times. **p<0.01, compared with DMSO control for each time point.

Figure 6. A higher concentration of genistein suppressed the migration and invasion of B16F10 cells in vitro

Cells were seeded on membranes incubated with different concentrations of genistein for 24 h. Cells that crossed the membrane and cells on the surface of the lower side of the membrane were stained with crystal violet, photographed under a light microscope at a magnification of ×100, and A. counted C. Cells that penetrated through the Matrigel™ to the lower surface of the filter were stained with crystal violet, photographed under a light microscope at ×100, and B. counted D. Results were obtained from three independent experiments. **p< 0.01 compared with solvent control. Scale bar is 200 μm.

Figure 7. Genistein influences the expression of FAK/paxillin and MAPK pathway proteins in B16F10 cells

A. Total cell lysates from B16F10 cells were prepared after treatment with genistein for 0, 10, 20, 30, and 60 min and 24 h. Next, 40 μg of each cell lysate were loaded onto the gel. (A) After blotting, the membranes were probed with p-FAK and FAK antibodies as described in the Materials and Methods section. B. Integrated band intensities as determined using Image J software. C. Total cell lysates from B16F10 cells were prepared after treatment with genistein for 24 h. Next, 40 μg of the cell lysates were loaded onto the gel. After blotting, the membranes were probed with antibodies against FAK/paxillin pathway proteins as described in the Materials and Methods section. D-H. Integrated intensity band intensities as determined using Image J software. I. The membranes were probed with MAPK pathway antibodies as described in the Materials and Methods section. G-L. Integrated band intensities as determined using Image J software. *p<0.05, **p<0.01, compared with solvent control.

Figure 8. Modulation of p-FAK, FAK, p-paxillin, paxillin, and vimentin by genistein

B16F10 cells were treated with or without 100 μM genistein for 24 h. The cells were then fixed and subjected to immunofluorescence staining with p-FAK, FAK, p-paxillin, paxillin, and vimentin primary antibodies, followed by secondary antibodies (red color), coupled with iFluor 488-conjugated Phalloidin (for F-actin, green color) and DAPI (for nucleus, blue color) staining. Scale bar is 20 μm.

Figure 9. Genistein inhibits the relative mRNA levels of FAK, paxillin,vimentin, and Snail in B16F10 cells (as determined by RT-PCR)

GAPDH were utilized as internal controls, and the relative mRNA levels were calculated according to the 2^−△△Ct method. *p<0.05, **p<0.01, compared with solvent control.

Figure 10. Scheme of the mechanism by which genistein affects melanoma cell survival, proliferation, and migration