Juglone reduces growth and migration of U251 glioblastoma cells and disrupts angiogenesis

Abstract

Accumulating data show that prolylisomerase (Pin1) is overexpressed in human glioblastoma multiforme (GBM) specimens. Therefore, Pin1 inhibitors should be investigated as a new chemotherapeutic drug that may enhance the clinical management of human gliomas. Recently, juglone, a Pin1 inhibitor, was shown to exhibit potent anticancer activity in various tumor cells, but its role in human glioma cells remains unknown. In the present study, we determined if juglone exerts antitumor effects in the U251 human glioma cell line and investigated its potential underlying molecular mechanisms. Cell survival, apoptosis, migration, angiogenesis and molecular targets were identified with multiple detection techniques including the MTT cell proliferation assay, dual acridine orange/ethidium bromide staining, electron microscopy, transwell migration assay, chick chorioallantoic membrane assay, quantitative real-time polymerase chain reaction and immunoblotting. The results showed that 5-20 µM juglone markedly suppressed cell proliferation, induced apoptosis, and enhanced caspase-3 activity in U251 cells in a dose- and time-dependent manner. Moreover, juglone inhibited cell migration and the formation of new blood vessels. At the molecular level, juglone markedly suppressed Pin1 levels in a time-dependent manner. TGF-β1/Smad signaling, a critical upstream regulator of miR-21, was also suppressed by juglone. Moreover, the transient overexpression of Pin1 reversed its antitumor effects in U251 cells and inhibited juglone-mediated changes to the TGF-β1/miR-21 signaling pathway. These findings suggest that juglone inhibits cell growth by causing apoptosis, thereby inhibiting the migration of U251 glioma cells and disrupting angiogenesis; and that Pin1 is a critical target for juglone's antitumor activity. The present study provides evidence that juglone has in vitro efficacy against glioma. Therefore, additional studies are warranted to examine the clinical potential of juglone in human gliomas.

Figure 1.

Juglone-induced reduction in cell viability and apoptosis in U251 cells. (A) U251 cells were treated with 2, 5, 10, 20, 50 and 100 µM juglone for 24, 48 or 72 h. Cell viability was detected by MTT assays. All of the assays were conducted in six replicates (n=6) for each treatment. Data are shown as mean ± SD from three independent experiments. (B) U251 cells after exposure to 20 µM juglone for 24, 48 and 72 h. Representative image of AO/EB staining (upper panel) and transmission electron microscopy (lower panel) of U251 cells treated with 20 µM juglone for 48 h. White arrows indicate apoptotic cells with morphological characteristics (chromatin condensation and nuclear shrinkage, original magnification, ×100). Transmission electron microscopy showed characteristic changes of apoptosis in U251 cells treated with 20 µM juglone for 48 h (chromosomal DNA condensation, segmentation of the nucleus, sunken nucleus membrane and loss of microvilli, original magnification, ×8000). (C) Statistical bar graph of the abundance of apoptotic cells induced by juglone according to AO/EB staining. (D) Juglone enhanced the activity of caspase-3 by 7-amino-methyl coumarin assay. Data are shown as mean ± SD of three independent experiments, *P<0.05 vs. control.

Figure 2.

Juglone inhibits the migration of U251 cells. U251 cells were treated with 5, 10 and 20 µM juglone for 24 h. Cells were subjected to Transwell (A and B) and wound-healing assays (C and D) to evaluate cell migration. Data are presented as mean ± SD from three independent experiments. *P<0.05 vs. control.

Figure 3.

Juglone exerts anti-angiogenic activity. (A) Juglone inhibited new vessel growth in a CAM assay. (B) Quantitative bar graph of (A) showing branch points per field. n=5 independent experiments. (C and D) Julgone significantly suppressed tube formation of HUVECs in Matrigel (magnification, ×100). n=6 independent experiments. (E and F) HUVECs were incubated with juglone (0, 5, 10, or 20 µM) for 24 h and cell lysates were prepared. The expression of (E) VEGF and (F) CD31 in cell lysates was determined by western blot analysis. Anti-GAPDH was used as a loading control. Western blot band is a representative result from three independent experiments. *P<0.05 vs. control.

Figure 4.

Effects of juglone on Pin1, TGF-β1, Smad2/3 and miR-21 expression. U251 cells were treated with 5, 10 and 20 µM juglone for 24 h. (A) Western blot analysis of Pin1 in HUVECs with the indicated treatments. (B and C) Juglone-induced downregulation of TGF-β mRNA and protein expression, as determined by qPCR and western blotting. (D) Juglone promoted a decrease in phospho-Smad2/3, as determined by western blotting. (E) Bar diagram summarizing the effects of juglone on miR-21 expression by qPCR. Data are shown as mean ± SD of three independent experiments, *P<0.05 vs. control.

Figure 5.

Effects of Pin1 overexpression on the antitumor activity of juglone. U251 cells were transfected with 1 µg/ml Pin1 DNA or empty vector (EV, pc-DNA3.1 plasmid) (A) Pin1 expression determined by western blot analysis. Average band density from three independent experiments. *P<0.05 vs. control. (B) U251 cells were treated with 20 µM juglone for 48 h. The number of apoptotic cells as determined by the AO/EB assay. n=6 independent experiments for each condition. (C and D) U251 cells were treated with 10 µM juglone for 24 h. Cells were subjected to Transwell migration assay. (E) TGF-β protein expression, as determined by western blotting. (F) Bar diagram summarizing the effects of juglone on miR-21 expression as determined by qPCR. Data are shown as mean ± SD of three independent experiments. *P<0.05 vs. control, ^#p<0.05 vs. cells treated with juglone.