Butein inhibits metastatic behavior in mouse melanoma cells through VEGF expression and translation-dependent signaling pathway regulation

Abstract

Background: Melanoma is an aggressive skin cancer and a predominant cause of skin cancer-related deaths. A previous study has demonstrated the ability of butein to inhibit tumor proliferation and invasion. However, the anti-metastatic mechanisms and in vivo effects of butein have not been fully elucidated.

Methods: MTT cell viability assays were used to evaluate the antitumor effects of butein in vitro. Cytotoxic effects of butein were measured by lactate dehydrogenase assay. Anti-migratory effects of butein were evaluated by two-dimensional scratch and transwell migration assays. Signaling transduction and VEGF-releasing assays were measured by Western blotting and ELISA. We also conducted an experimental analysis of the metastatic potential of tumor cells injected into the tail vein of C57BL/6 mice.

Results: We first demonstrated the effect of butein on cell viability at non-cytotoxic concentrations (1, 3, and 10 μM). In vitro, butein was found to inhibit the migration of B16F10 cells in a concentration-dependent manner using transwell and scratch assays. Butein had a dose-dependent effect on focal adhesion kinase, Akt, and ERK phosphorylation in B16F10 cells. Butein efficiently inhibited the mTOR/p70S6K translational inhibition machinery and decreased the production of VEGF in B16F10 cells. Furthermore, the in vivo antitumor effects of butein were demonstrated using a pulmonary metastasis model.

Conclusion: The results of the present study indicate the potential utility of butein in the treatment of melanoma.

Fig. 1

The structure of butein and its effect on melanoma cells viability and cytotoxicity. The structure of butein is shown in a. B16F10 cells were seeded onto plates for 24 hr and starved for another 24 hr with serum-free DMEM. b Quiescent cells were cultivated with DMEM containing 10 % FBS in the absence or presence of various concentrations of butein. After 48 h, cell viability was detected by the MTT assay. c Cytotoxicity was detected by manufacture’s protocol and finally recorded absorbance at 490 nm. Control was taken as basal condition of LDH release. The results are presented as percentages of inhibition (mean ± SEM, n = 4). All P values were obtained from comparisons between control and indicated concentration-treated cells. **, P < 0.01; ***, P < 0.001

Fig. 2

Effect of butein on serum-stimulated B16F10 cell migration. a B16F10 cells (2 × 10⁴) cultured in serum-free DMEM were pretreated with or without butein for 30 minutes, and placed in the upper chamber of a Transwell containing with 0.2 % gelatin-coated filter membrane. DMEM with 10 % FBS was added to the lower chamber. After removal of non-migrated cells and fixation, cells that migrated to the underside filter membrane were stained and quantified by phase-contrast light microscope under a high-power field (HPF; magnification, 100×). Three fields per filter were counted at 100× magnification. The quantitative of migratory cell was shown in b. Data are expressed as mean ± S.E.M. of five independent experiments. *p < 0.05; **p < 0.01 compare with the control (Veh) group

Fig. 3

Effect of butein on serum-stimulated B16F10 cell motility. a B16F10 cells (5 × 10⁴) were seeded onto 6-well plates for 24 hr and starved for another 24 hr with serum-free DMEM. Cells were subjected to injury by scratching with a plastic pipette tip (200 μl). The cells were then treated for 24 hours with or without serum or butein. The quantitative of migratory cell was shown in b. Data are presented as percentages of control (mean ± S.E.M., n = 3). **, P < 0.01 and ***,P < 0.001 versus control group

Fig. 4

Effect of butein on PI3K/Akt/mTOR, ERK and FAK pathways. a B16F10 cells were incubated in the absence or presence of butein (1- to 10 μM) for 12 hrs. Then, the cells were harvested and lysed for the detection of the PI3K/Akt/mTOR, ERK and FAK pathways activation by Western blot. The quantitative densitometry of the relative level of protein phosphorylation (phosphortlation protein/total protein) was performed with Image-Pro Plus and was shown in b. Data are expressed as mean ± S.E.M. of five independent experiments. The protein phosphorylation in all treatment cells were significant lower than control (Veh) group (***p < 0.001 as compare with control (Veh) group)

Fig. 5

Effects of butein on VEGF production in melanoma cells, B16F10. VEGF protein expression was evaluated by ELISA in conditioned medium of B16F10 in the presence or absence of butein at the indicated concentrations. Data are represented as mean ± SEM (n = 6). *P < 0.05 and **P < 0.01 indicates significant differences from the vehicle control

Fig. 6

Effects of butein on experimental lung metastasis. a B16F10 cells (2x10⁶) were injected into the lateral tail vein of C57BL/6 mouse. Twenty days later, the lungs were removed, and surface-visible tumors were counted in the absence and presence of butein treatment. The melanoma metastasis area of lung surface was black and background is dark red. White area was quantified using ImageJ software. The quantitative of lung metastasis colony was shown in b. Corresponding bar graph expressed as percentage of vehicle (n = 8)

Fig. 7

Schematic presentation of butein-mediated signaling transduction modulation