Baicalein Inhibits Progression of Gallbladder Cancer Cells by Downregulating ZFX

<div><p>Baicalein, a widely used Chinese herbal medicine, has multiple pharmacological activities. However, the precise mechanisms of the anti-proliferation and anti-metastatic effects of baicalein on gallbladder cancer (GBC) remain poorly understood. Therefore, the aim of this study was to assess the anti-proliferation and anti-metastatic effects of baicalein and the related mechanism(s) on GBC. In the present study, we found that treatment with baicalein induced a significant inhibitory effect on proliferation and promoted apoptosis in GBC-SD and SGC996 cells, two widely used gallbladder cancer cell lines. Additionally, treatment with baicalein inhibited the metastasis of GBC cells. Moreover, we demonstrated for the first time that baicalein inhibited GBC cell growth and metastasis via down-regulation of the expression level of Zinc finger protein X-linked (ZFX). In conclusion, our studies suggest that baicalein may be a potential phytochemical flavonoid for therapeutics of GBC and ZFX may serve as a molecular marker or predictive target for GBC.</p></div

Baicalein inhibits the proliferation ofgallbladder carcinoma cells <i>in vitro</i> and <i>in vivo</i>.

<p>(A) GBC-SD and SGC996 cells were treated with varying concentrations of baicalein, and the cell proliferation were determined by MTT assay on days 1, 2, and 3. Each value represents the mean ± SD (n = 3). (B and C) Baicalein suppressed colony formation of GBC-SD and SGC996 cells. Cells were treated with baicalein (6, 12, and 24 μmol/L) and were allowed to form colonies in fresh medium for 14 days. The influence of colonies (mean ± SD, n = 3) in colony formation are shown. (D) Baicalein-treated xenograft tumors were much smaller than control tumors. (E) The weight of tumors revealed that xenograft tumor growth in nude mice was significantly slower in baicalein-treated nude tumors than control tumors. The results shown are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.</p

Effect of ZFX on the proliferation, apoptosis, invasive activity and related protein signaling of baicalein-treated GBC-SD cells.

<p>(A and B) ZFX expression in GBC-SD and SGC996 cells induced by baicalein. Cells were treated with 0, 15, 30, 60 and 120 μmol/L baicalein for 48h, then the protein and mRNA expression level of ZFX was determined by Western blot(A) and qPCR(B). (C and D) Cells were transfected with ZFX-GFP plasmid and GFP plasmid, respectively. 72 hours after transfection, cells were treated with 0, 7.5, 15, 30 and 60 μmol/L baicalein for 72h. ZFX-GFP significantly blocked the inhibition of proliferation of GBC-SD cells by baicalein using MTT assay (C). ZFX-GFP significantly blocked the induction of apoptosis of GBC-SD cells by baicalein estimated by the flow cytometry(D). (E and F) 72 hours after transfection, cells were treated with 60 μmol/L baicalein for 24h. ZFX-GFP significantly blocked the anti-migration and invasion effect of GBC-SD cells by baicalein. The cell migration was estimated by wound healing assay(E) and the cell invasion was estimated by transwell assay(F). (G) ZFX-GFP prevented the expression of PARP, the inhibition of MMP2, MMP9 and up-regulated the ratio of Bcl2 /Bax in response to baicalein. 72 hours after transfection, cells were treated with 60 μmol/L baicalein for 48h.The protein levels of PARP, MMP-2, MMP-9,Bcl2, Bax and ZFX were determined by Western blot. The results shown are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.</p

Effect of baicalein on the mobility and invasive potency of human gallbladder carcinoma GBC-SD and SGC996 cells.

<p>Cells were treated with 7.5, 15, 30 and 60μmol/L baicalein for 12 h prior to the migration (A) or invasion (B) assay.(C) Cell monolayers were wounded by scratching with a 200 μl pipette tip. The occupation area in GBC-SD cell monolayer treated with baicalein was dose dependent decreased 24 h after scratch. Baicalein affects the expression of matrix metallopeptidases (MMPs). (D) The mRNA expression of MMP-2 and MMP-9 was detected by qPCR after treated with baicalein for 48h. (E) The protein expression of MMP-2 and MMP-9 was detected by western blot after treated with baicalein for 48h. The results shown are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.</p

Baicalein induces apoptosis in GBC-SD and SGC996 cells.

<p>(A) Apoptotic morphological changes such as abnormal nuclear morphology, reduction in cell number with apoptotic body formation, and cell shrinkage, induced by baicalein (30,60 and 120 μmol/L) treatment for 48 h, were observed by Hoechst 33342 staining in GBC-SD and SGC996 cell lines. (B) Cells were incubated with baicalein (30,60 and 120 μmol/L) for 48 h, followed by staining with annexin-V/PI. Cell apoptosis was estimated by the flow cytometry. (C) Baicalein induces the activation of apoptosis-related proteins in GBC-SD and SGC996 cells. After treatment with baicalein (0, 15, 30, 60, and 120μmol/L) for 48 h, cell lysates were prepared and western blot analysis was performed against Bcl-2, Bax, pro-caspase-3, P53 and PARP. β-Actin was used as a loading control. The results shown are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.</p

Chemical structure of baicalein.

<p>The molecular formula of baicalein is C15H10O5and its molecular weight is 270.24.</p