Garcinol regulates EMT and Wnt signaling pathways in vitro and in vivo, leading to anticancer activity against breast cancer cells

Abstract

Anticancer properties of Garcinia indica-derived garcinol are just beginning to be elucidated. We have earlier reported its cancer cell-specific induction of apoptosis in breast cancer cells, which was mediated through the downregulation of NF-κB signaling pathway. To gain further mechanistic insight, here, we show for the first time that garcinol effectively reverses epithelial-to-mesenchymal transition (EMT), that is, it induces mesenchymal-to-epithelial transition (MET) in aggressive triple-negative MDA-MB-231 and BT-549 breast cancer cells. This was associated with upregulation of epithelial marker E-cadherin and downregulation of mesenchymal markers vimentin, ZEB-1, and ZEB-2. We also found that garcinol upregulates the expression of miR-200 and let-7 family microRNAs (miRNAs), which provides a molecular mechanism for the observed reversal of EMT to MET. Transfection of cells with NF-κB p65 subunit attenuated the effect of garcinol on apoptosis induction through reversal of MET to EMT. Forced transfection of p65 and anti-miR-200s could also reverse the inhibitory effect of garcinol on breast cancer cell invasion. Moreover, treatment with garcinol resulted in increased phosphorylation of β-catenin concomitant with its reduced nuclear localization. The results were also validated in vivo in a xenograft mouse model where garcinol was found to inhibit NF-κB, miRNAs, vimentin, and nuclear β-catenin. These novel findings suggest that the anticancer activity of garcinol against aggressive breast cancer cells is, in part, due to reversal of EMT phenotype, which is mechanistically linked with the deregulation of miR-200s, let-7s, NF-κB, and Wnt signaling pathways.

Figure 1

Chemical structure of garcinol (A) and effect of garcinol on the expression of EMT markers (B). Cells were treated with 25 μmol/L garcinol for 48 hours, and the expression of epithelial marker, E-cadherin and mesenchymal markers, vimentin, ZEB1, and ZEB2 was evaluated by real-time reverse transcription PCR. *, P < 0.05 and **, P < 0.01 versus respective vehicle controls.

Figure 2

Effect of garcinol on the expression of miR-200 family in MDA-MB-231 (A) and BT-549 (C), and on let-7 family in MDA-MB-231 (B) and BT-549 (D) breast cancer cells. Cells were treated with 25 μmol/L garcinol for 48 hours, and analysis was done by real-time reverse transcription PCR. *, P < 0.05 and **, P < 0.01 versus respective vehicle controls.

Figure 3

A, garcinol downregulated NF-κB expression. Overexpression of NF-κB reversed the effect of garcinol (G) in B, mesenchymal markers vimentin, ZEB1, and ZEB2, and garcinol-induced apoptosis in both the cell lines tested (MDA-MB-231 and BT-549; C).*, P < 0.05 and **, P < 0.01 versus respective vehicle controls. OD, optical density.

Figure 4

A, garcinol dose dependently inhibited the invasion of MDA-MB-231 cells. B and C, effect of NF-κB p65 and anti-miR-200 transfection on garcinol-induced inhibition of invasion of MDA-MB-231 cells. C, control (vehicle treatment); G, cells treated with 25 μmol/L garcinol; G + p65, cells transfected with NF-κB p65 followed by 25 μmol/L garcinol treatment, G + miR-200s, cells transfected with anti-miR-200s (anti-miR-200a + anti-miR-200b + anti-miR-200c) followed by garcinol treatment. Bar graphs represent quantitation of the degree of invasiveness as determined by fluorescence values under each condition. *, P < 0.05 and **, P < 0.01 versus vehicle control. AFU, arbitrary fluorescence units.

Figure 5

Effect of garcinol on Wnt signaling. MDA-MB-231 cells were treated with 25 μmol/L garcinol for 48 hours and the expression of various Wnt signaling factors was evaluated by Western blotting. C, control (vehicle-treated) cells; G, garcinol-treated cells. β-Actin was used as the internal loading control.

Figure 6

Garcinol inhibits NF-κB/EMT/miRNAs/Wnt signaling in vivo. MDA-MB-231 cells were injected in SCID mice subcutaneously and mice were randomized into 2 groups: control and garcinol-treated (n = 6 in each group). Tumor remnants were used to isolate proteins, RNA, and for IHC analyses. A, downregulation of NF-κB, mesenchymal marker vimentin, and nuclear β-catenin in tumor remnants as determined by Western blot analysis. C, control tumor; G, tumor from garcinol-treated animal. B, induction of several miR-200 and let-7 family miRNAs in tumors of garcinol-administered mice. C, IHC staining for proliferation marker Ki-67, vascular density marker CD31, epithelial marker E-cadherin, mesenchymal marker vimentin, and β-catenin in representative tumor samples. CD31 stains are shown at ×20 magnification, whereas all others are shown at ×40 magnification. *, P < 0.05 in treated tumors against vehicle control.