Lovastatin sensitized human glioblastoma cells to TRAIL-induced apoptosis

Abstract

Synergy study with chemotherapeutic agents is a common in vitro strategy in the search for effective cancer therapy. For non-chemotherapeutic agents, efficacious synergistic effects are uncommon. Here, we have examined two non-chemotherapeutic agents for synergistic effects: lovastatin and Tumor Necrosis Factor (TNF)-related apoptosis-inducing ligand (TRAIL) for synergistic effects; on three human malignant glioblastoma cell lines, M059K, M59J, and A172. Cells treated with lovastatin plus TRAIL for 48 h showed 50% apoptotic cell death, whereas TRAIL alone (1,000 ng/ml) did not, suggesting that lovastatin sensitized the glioblastoma cells to TRAIL attack. Cell cycle analysis indicated that lovastatin increased G0-G1 arrest in these cells. Annexin V study demonstrated that apoptosis was the predominant mode of cell death. We conclude that the combination of lovastatin and TRAIL enhances apoptosis synergistically. Moreover, lovastatin sensitized glioblastoma cells to TRAIL, suggesting a new strategy to treat glioblastoma.

Fig. 1

Anti-proliferation effects on glioblastoma cell lines A172, M059K, and M059J. MTT assays were preformed after 48 h incubation time treated with 0, 500, 1,000 ng/ml TRAIL in normal medium (a) or serum free medium (b). A172 subjected to minor TRAIL-induced cell death while M059J and M059K cells were resistant to TRAIL. Same trend of data were obtained in both normal medium and serum free condition. Experiment set were repeated at least three times with triplicate wells for each condition (mean ± SD)

Fig. 2

Synergistic anti-proliferation effects on glioblastoma cell lines A172, M059J, and M059K. Glioblastoma cells were treated with 0, 5, 20 and 40 μM lovastatin alone or 0 μM lovastatin plus 500 ng/ml TRAIL, 5 μM lovastatin plus 500 ng/ml TRAIL, 20 μM lovastatin plus 500 ng/ml TRAIL and 40 μM lovastatin plus 500 ng/ml TRAIL for 48 h. Percentage of viable glioblastoma cells showed synergistic anti-proliferation effects by combined the two agents. In A172 glioblastoma cells (a), 500 ng/ml TRAIL with 5, 20 and 40 μM lovastatin induced cell death significantly when compared with lovastatin only groups. In M059J (b) and M059K (c), 500 ng/ml TRAIL with 1, 5, 20 and 40 μM lovastatin induced cell death significantly when compared with lovastatin only groups. ANOVA were used for statistics analysis and *P < 0.05, **P < 0.01. Experimental set were repeated for at least three times with triplicate wells for each condition (mean ± SD)

Fig. 3

Lovastatin-induced glioblastoma cells arrested in G₀–G₁ Phase. Propidium Iodide staining for cell cycle analysis were performed after glioblastoma cells were treated with normal medium without lovastatin, serum free medium without lovastatin and 20 μM lovastatin for 48 h. Serum free condition was used as a positive control which is commonly known to induce cell arrest in G₀–G₁ phase. Serum free condition and lovastatin increased G₀–G₁ cell arrest in all glioblastoma cells and reached significant level (except serum free condition in M059J). ANOVA were used for statistics analysis and *P < 0.05, **P < 0.01. Experimental set were repeated for at least three times with triplicate wells for each condition (mean ± SD)

Fig. 4

The synergistic apoptotic effects were quantified by flow cytometry using Annexin V and PI staining. Glioblastoma cells were treated with DMSO (Control), 5 μM lovastatin, 20 μM lovastatin, 500 ng/ml TRAIL, 5 μM lovastatin plus 500 ng/ml TRAIL and 20 μM lovastatin plus 500 ng/ml TRAIL for 48 h. Then cells were stained with Annexin V and PI to determine percentage of apoptotic cell death using flow cytometry. Synergistic apoptotic effects were observed in three glioblastoma cell lines A172 (a), M059J (b) and M059K (c), reached significant level. The portion of apoptotic cell death was indicated in low-right quarter of the flow-cytometry scatter plot and the trend of apoptotic cells between groups. ANOVA were used for statistics analysis and *P < 0.05, **P < 0.01 (compared to control group), ⁺P < 0.05, ⁺⁺P < 0.01 (compared to TRAIL only group). Experimental set were repeated for at least three times (mean ± SD)

Fig. 5

DNA fragmentation was detected in combination of lovastatin and TRAIL. Same treatment in apoptotic cell staining was preformed for DNA fragmentation ELISA detection. Significant level of DNA fragmentation was detected in all glioblastoma cells when combined with lovastatin and TRAIL. ANOVA were used for statistics analysis and *P < 0.05, **P < 0.01 (compared to control group), ⁺P < 0.05, ⁺⁺P < 0.01 (compared to TRAIL only group). Experimental set were repeated for at least three times (mean ± SD)

Fig. 6

The expression of TRAIL receptor mRNA in glioblastoma cells. The cells were treated with normal serum medium, serum free medium, 5 and 20 μM lovastatin with normal serum medium. At the end of the treatment, RNA was isolated for the detection of TRAIL-R1 (DR4) (a) and TRAIL-R2 (DR5) (b) by RT-PCR

Fig. 7

The expression of TRAIL-R1 (DR4) and TRAIL-R2 (DR5) proteins in glioblastoma cells. The cells were treated with normal serum medium, serum free medium, 5 and 20 μM lovastatin with normal serum medium for 48 h. At the end of the treatment, proteins were isolated for the detection of TRAIL-R1 (a, b) and TRAIL-R2 (c, d) by Western blot. Representative Western blots were shown (a, c). The target bands were scanned and normalized to β-tubulin. The index of densities was calculated (b, d). *P = 0.05, **P < 0.01