Natural triterpenic diols promote apoptosis in astrocytoma cells through ROS-mediated mitochondrial depolarization and JNK activation

Abstract

Background: Triterpene alcohols and acids are multifunctional compounds widely distributed throughout the plant kingdom that exhibit a variety of beneficial health properties, being synthetic analogs of oleanolic acid under clinical evaluation as anti-tumoral therapeutic agents. However, the antineoplastic activity of two natural occurring triterpenoid alcohols extracted from olive oil, erythrodiol (an intermediate from oleanolic acid), and its isomer, uvaol, has barely been reported, particularly on brain cancer cells. Astrocytomas are among the most common and aggressive type of primary malignant tumors in the neurological system lacking effective treatments, and in this study, we addressed the effect of these two triterpenic diols on the human 1321N1 astrocytoma cell line.

Principal findings: Erythrodiol and uvaol effectively affected cell proliferation, as well as cell cycle phases and induced 1321N1 cell death. Both triterpenes successfully modulated the apoptotic response, promoting nuclear condensation and fragmentation. They caused retraction and rounding of cultured cells, which lost adherence from their supports, while F-actin and vimentin filaments disappeared as an organized cytoplasmic network. At molecular level, changes in the expression of surface proteins associated with adhesion or death processes were also observed. Moreover, triterpene exposure resulted in the production of reactive oxygen species (ROS) with loss of mitochondrial transmembrane potential, and correlated with the activation of c-Jun N-terminal kinases (JNK). The presence of catalase reversed the triterpenic diols-induced mitochondrial depolarization, JNK activation, and apoptotic death, indicating the critical role of ROS in the action of these compounds.

Conclusions: Overall, we provide a significant insight into the anticarcinogenic action of erythrodiol and uvaol that may have a potential in prevention and treatment of brain tumors and other cancers.

Figure 1. Molecular structure of uvaol and erythrodiol.

Figure 2. Erythrodiol and uvaol induce detachment on 1321N1 astrocytoma cells.

A, Morphological appearance of the cells exposed to different doses of erythrodiol or uvaol for 6 h (upper panels) or to 25 µM erythrodiol or 50 µM uvaol for different times (lower panels). Cells were visualized under a phase contrast microscope Nikon Eclipse TS100 (×20). B, Cells were treated with different doses of erythrodiol or uvaol for 18 h. The expression of CD44, ICAM and VCAM was determined by flow cytometry. Histograms represent one experiment out of three. Solid gray curves represent unspecific binding; empty black curves, cells cultured in the absence of treatment (control); and empty gray curves, triterpene-treated cells.

Figure 3. Erythrodiol and uvaol induce morphological changes in the cytoskeleton.

A, Cells were treated with different doses of erythrodiol or uvaol for 6 h. Then, cells were stained with FITC-phalloidin (green, b, e, h, k and n) or anti-vimentin Ab (red, c, f, i, l and o) and visualized under a fluorescent microscope (×60). Cells were seeded on standard conditions (B) or in poly-l-lysine treated coverslips (C). After 18 h of exposure to 25 µM of triterpenes, floating cells (B lower panels) and attached cells (B upper panels and C) were fixed and stained with DAPI. Cells were visualized using a Nikon Eclipse 80i fluorescent microscope (×60). The cellular morphology was observed using Nomarski optics. The floating population from the adherent condition was too low to be evaluated.

Figure 4. Effects of triterpenic diols on cell growth and apoptosis in 1321N1 cells.

A, The cells were exposed to different doses of erythrodiol or uvaol for 18 h in the presence of FCS and proliferation was determined by a [³H]-thymidine uptake assay. Data are expressed as the percentage of radioactivity incorporated in FCS-stimulated cells in the absence of triterpenes (327.510±3.889 dpm). B, C, Cells treated as above, but without FCS, were fixed in 70% ethanol and stained with PI (B) or stained with annexin-V-PE (C) and analyzed by flow cytometry. The numerical values are presented as the mean±S.D. of three independent experiments. Percentages in B indicate the number of cells in the sub–G0-G1 phase of the cell cycle. *p<0.05, **p<0.01, ***p<0.001 vs control untreated cells.

Figure 5. Effect of triterpenic diols in JNK activation.

Cells were stimulated with 25 µM erythrodiol or 50 µM uvaol at the indicated times (A), or with different doses of erythrodiol or uvaol for 4 h (B), and assayed for an in vitro JNK-kinase assay as described in Materials and Methods. Exposure to 200 U/ml of TNF for 15 min was used as positive control. Results are representative of four separate experiments. Cells were exposed to different doses of SP600125 in the presence of 25 µM erythrodiol or 50 µM uvaol for 18 h. Then, the cells were analyzed by phase-contrast microscopy using a Nikon Eclipse TS100 microscope (×40; C) or labeled with annexin V–PE and analyzed by flow cytometry (D). In the histograms, cells obtained after triterpene treatment in the absence of the inhibitor (open black curves) are compared with cells treated in the presence of the inhibitor (open gray curves). Gray solid curves, resting control cells. *p<0.05, **p<0.01, vs triterpenes treated cells in the absence of the inhibitor.

Figure 6. ROS-dependent JNK activation contributes to triterpenes-stimulated apoptosis.

A, Analysis of ROS production and ΔΨ_m evaluation. Cells were treated with different doses of erythrodiol or uvaol for 30 min: 5 µM (black empty curve), 25 µM (dark grey empty curve) or 50 µM (light gray empty curve), and then stained with DCFH-DA (upper histograms), or treated with 25 µM erythrodiol or 50 µM uvaol for 6 h (black empty curve) or 18 h (gray empty curve) and staining with Rh123 (lower histograms). Intracellular ROS and ΔΨ_m was monitored by flow cytometry or under a fluorescence microscope (×40). B, Cells were preincubated with catalase, treated with 25 µM erythrodiol or 50 µM uvaol for 30 additional min and stained with DCFH-DA (upper histograms) or for 18 h and staining with Rd123 (lower histograms), and analyzed by flow cytometry. C, Cells were preincubated with different doses of catalase, treated with 25 µM erythrodiol or 50 µM uvaol for 4 h and assayed for an in vitro JNK-kinase assay. Exposure to 200 U/ml of TNF for 15 min was used as positive control. D, Cells were preincubated with catalase, treated with 25 µM erythrodiol or 50 µM uvaol for 18 h, stained with annexin-V-PE and analyzed under light microscope (×40) or by flow cytometry. In the histograms, cells obtained after triterpene treatment in the absence of the antioxidant (open black curves) are compared with cells treated in the presence of the antioxidant (open gray curves). In all the histograms, the solid grey curve represent the resting/control cells. The results are representative of three independent experiments.

Figure 7. Effect of triterpenic diols on the expression and activity of death receptor/ligand systems.

A, The expression of TNFR1, CD40, Fas and FasL was determined by flow cytometry, in 1321N1 cell exposed to 25 µM erythrodiol or 50 µM uvaol for 18 h. Histograms represent one experiment out of three. Solid gray curves represent unspecific binding; empty black curves, cells cultured in the absence of treatment (control); and empty gray curves, triterpene-treated cells. B, Cells were treated with 25 µM erythrodiol or 50 µM uvaol, in absence (black empty curve) or presence (grey empty curve) of anti-CD40, anti-Fas or anti FasL antibodies for 18 h. Afterward, the cells were labeled with annexin-V PE and analyzed by flow cytometry.

Figure 8. Effect of triterpenic diols on different tumor cell types.

The indicated cell types were treated without or with 25 µM (black empty curve) or 50 µM (grey empty curve) of erythrodiol or uvaol for 24 h and stained with annexin-V PE. Solid gray curves represent labeling of resting/control cells.