α-Hispanolol Induces Apoptosis and Suppresses Migration and Invasion of Glioblastoma Cells Likely via Downregulation of MMP-2/9 Expression and p38MAPK Attenuation

Abstract

α-Hispanolol (α-H) is a labdane diterpenoid that has been shown to induce apoptosis in several human cancer cells. However, the effect of α-H in human glioblastoma cells has not been described. In the present work, we have investigated the effects of α-H on apoptosis, migration, and invasion of human glioblastoma cells with the aim of identifying the molecular targets underlying its mechanism of action. The results revealed that α-H showed significant cytotoxicity against human glioma cancer cell lines U87 and U373 in a concentration- and time-dependent manner. This effect was higher in U87 cells and linked to apoptosis, as revealed the increased percentage of sub-G₁ population by cell cycle analysis and acquisition of typical features of apoptotic cell morphology. Apoptosis was also confirmed by significant presence of annexin V-positive cells and caspase activation. Pretreatment with caspase inhibitors diminishes the activities of caspase 8, 9, and 3 and maintains the percentage of viable glioblastoma cells, indicating that α-H induced cell apoptosis through both the extrinsic and the intrinsic pathways. Moreover, we also found that α-H downregulated the anti-apoptotic Bcl-2 and Bcl-xL proteins and activated the pro-apoptotic Bid and Bax proteins. On the other hand, α-H exhibited inhibitory effects on the migration and invasion of U87 cells in a concentration-dependent manner. Furthermore, additional experiments showed that α-H treatment reduced the enzymatic activities and protein levels of matrix metalloproteinase MMP-2 and MMP-9 and increased the expression of TIMP-1 inhibitor, probably via p38MAPK regulation. Finally, xenograft assays confirmed the anti-glioma efficacy of α-H. Taken together, these findings suggest that α-H may exert anti-tumoral effects in vitro and in vivo through the inhibition of cell proliferation and invasion as well as by the induction of apoptosis in human glioblastoma cells. This research describes α-H as a new drug that may improve the therapeutic efficacy against glioblastoma tumors.

Keywords: apoptosis; caspases; glioblastoma; matrix metalloproteinases; migration; α-hispanolol.

Figure 1

α-H inhibited proliferation and suppressed colony formation in glioblastoma cells. (A) U87 and U373 cells were treated with different concentrations of α-H (1–100 μM) for 24, 48, and 72 h. Cell viability was determined by MTT assay and reported as mean of the cell viability percentage ± S.D. from at least three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 with respect to control. (B) Colony formation of U87 cells treated with α-H (10 μM) was evaluated by clonogenic assay. Representative images of crystal violet-stained cultures are shown on the left. Bar graph shows mean of the colony formation percentage ± S.D. from three independent experiments. **P < 0.01 with respect to vehicle-treated cells.

Figure 2

Effects of α-H on cell cycle and apoptosis in U87 cells. (A) U87 cells were treated with different concentrations of α-H (1, 10, and 25 μM) or vehicle as control for 24 h, then fixed in ethanol, and stained with propidium iodide. DNA content was determined by flow cytometry. Percentage of cells in each phase of the cell cycle (sub-G₁, G₀/G₁, S, and G₂/M) showed in the bar graph were calculated using Flowing software. (B) U87 cells were treated with different concentrations of α-H (1, 10, and 25 μM) or vehicle as control for 24 h. Collected cells were stained with annexin V-FITC and propidium iodide, and then analyzed by flow cytometry. Numbers in the plots represent percentage of the correspondent apoptotic population. Graph bars show percentage of correspondent cellular phenotype. (C) Microphotographs were taken after 24 h of incubation with different concentrations of α-H or vehicle as control. Characteristics of apoptosis such as cell rounding (due to loss of adhesion to substratum) and plasma membrane blebbing are observed in α-H-treated cells (arrows) (scale bars = 40 µm). Data presented are from one representative experiment out of three.

Figure 3

α-H induced apoptosis by regulating caspases and Bcl-2 family in human GBM cells. (A) U87 cells were treated for 24 h with different concentrations of α-H (1, 10, and 25 μM) or vehicle as control in the presence or absence of the indicated caspase inhibitors. Caspase 3, 8, and 9 activities were determined in cell extracts by fluorometry, using specific fluorigenic substrates as described in Materials and Methods. (B) U87 cells were treated for 24 h with 25-μM α-H or vehicle as control in the presence or absence of the caspase inhibitors indicated. Cell viability was determined by MTT assay. Results are reported as mean ± S.D. from three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 with respect to the non-treated cells. ^aP < 0.05, ^bP < 0.01, and ^cP < 0.001 with respect to the α-H (25 μM) treated cells. (C-D) U87 cells were treated for 24 h with different concentrations of α-H (1, 10, and 25 μM) or vehicle as control, and caspases and members of Bcl-2 family protein levels were determined by Western blot. β-actin was used as loading control. A representative experiment of three performed is shown. Bars graphs show densitometry quantification of the bands from three independent experiments. (E) Bax/Bcl-2 ratio was calculated from data obtained by densitometry. *P < 0.05 and **P < 0.01 with respect to the non-treated cells.

Figure 4

Effects of α-H on the motility and invasiveness of U87 cells. (A) Subconfluent U87 cultures were wound scratched and then incubated in media containing varying concentrations of α-H (1, 10, and 25 μM) or vehicle as control for 48 h. Cell migration was evaluated by photomicroscopy. Representative photographs showed the same area at time zero and after 48 h of incubation with or without α-H. Bars graph represents mean ± S.D. of percentage wound closure from three independent experiments. *P < 0.05 and **P < 0.01 with respect to the non-treated cells. (B) Cell invasion was determined using a transwell assay system. U87 cells treated with indicated concentrations of α-H (1, 10, and 25 μM) or vehicle as control were seeded into the upper chamber of the system in serum-free media. The bottom well was filled with complete medium as chemotactic attractant environment. After 48 h, cells migrated to the bottom side of the filter were fixed, crystal violet stained, and evaluated by light microscopy (left panel). Magnification, ×100. Invasion rate is quantified by spectrophotometry after eluting cell staining, as described in Materials and Methods. Values represent the mean percentage of migrated cells ± S.D. from three independent experiments performed in triplicate. *P < 0.05 and **P < 0.01 with respect to the non-treated cells.

Figure 5

α-H inhibited the expression and activity of MMP-2 and MMP-9. U87 cells were treated with different concentrations of α-H (1, 10, and 25 μM) or vehicle as control for 24 h. (A) Conditioned medium was collected, and the activities of MMP-2 and MMP-9 were determined by gelatin zymography assay. A representative experiment is shown of three performed. Bars graphs show densitometry quantification of the bands and represent mean percentage of MMPs activities ± S.D. from three independent experiments. *P < 0.05 and **P < 0.01 with respect to the non-treated cells. (B) MMP-2 and MMP-9 protein levels were determined by Western blot. β-actin was used as loading control. A representative experiment is shown of three performed. Bars graphs show densitometry quantification of the bands and represent means MMPs/β-actin ratio ± S.D. from three independent experiments. *P < 0.05 and **P < 0.01 with respect to the non-treated cells. (C) mRNA expression of MMP-2 and MMP-9 was determined by quantitative PCR. Values represent the means ± S.D. of three independent experiments performed in triplicate. *P < 0.05 and **P < 0.01 with respect to the non-treated cells.

Figure 6

TIMP-1 expression is upregulated after α-H treatment. U87 cells were treated with different concentrations of α-H (1, 10, and 25 μM) or vehicle as control for 24 h. (A) TIMP-1 protein levels were determined by Western blot. β-actin was used as loading control. A representative experiment is shown of three performed. Bars graphs show densitometry quantification of the bands and represent means ± S.D. from three independent experiments. (B) mRNA expression of TIMP-1 was determined by quantitative PCR. Values represent the means ± S.D. of three independent experiments performed in triplicate. (C) MMP-9/TIMP-1 ratio was calculated from data obtained by densitometry. MMP-9 data were from Figure 5B. *P < 0.05 and **P < 0.01 with respect to the non-treated cells.

Figure 7

α-H treatment inhibited p38MAPK signal showing similar effects on MMPs and TIMP-1 expression as a specific p38MAPK inhibitor. (A) U87 cells were treated with different concentrations of α-H (1, 10, and 25 μM) or vehicle as control for 24 h. p-p38 and p38 protein levels were determined by Western blot. β-actin was used as loading control. A representative experiment is shown of three performed. Bars graphs show densitometry quantification of the bands and represent means ± S.D. from three independent experiments. (B) Cells were treated with a specific p38MAPK inhibitor (SB202190, 10 μM) or α-H (25 μM) for 24 h. MMP-9, MMP-2, and TIMP-1 protein levels were determined by Western blot. β-actin was used as loading control. A representative experiment is shown of three performed. Bars graphs show densitometry quantification of the bands and represent means ± S.D. from three independent experiments. (C) mRNA expression of MMP-9, MMP-2, and TIMP-1 was determined by quantitative PCR on U87 cells treated as in B. Values represent the means ± S.D. of three independent experiments performed in triplicate. *P < 0.05 and **P < 0.01 with respect to the non-treated cells.

Figure 8

Inhibitory effects of α-H on U87 xenograft growth. U87 cells (3 × 10⁶ cells, 100-µl PBS) were implanted subcutaneously into the right flanks of NSG mice. When tumors reached a volume of ∼200 mm³, mice were treated i.v. every 3 days with α-H (1 mg/kg) or with vehicle as indicated by black arrows. After 10 days of treatment, the tumor nodules were isolated and weighted. (A) Tumor volume was calculated as described in Material and Methods. Results are the means for groups (n = 5) ± S.D. (B) Representative images of tumor nodules harvested on day 21 from each treatment group. Scale bar = 1 cm. (C) Weight of tumors isolated on day 21. *P < 0.01 with respect to vehicle group (D) Body weight was measured at indicated times. Results are the means for groups (n = 5) ± S.D. (E) Expression levels of MMP-9, TIMP-1, and p-p38 were determined by Western blot on tumor tissues of vehicle control and α-H-treated mice. β-actin was used as loading control. Bar graphs show densitometry quantification of the bands and represent means ± S.D of the different animals. (F) Immunohistochemical analysis of proliferation marker Ki-67 and TUNEL-positive cells in the tumor tissues (left panel). The results shown are representative of randomly selected tumor fields from each specimen. Graphs represent positive cells in IHC staining evaluated as described in Materials and Methods (right panel). *P < 0.05, **P < 0.01, and ***P < 0.001 with respect to vehicle group.