Q-VD-OPH and Q-LEHD-OPH were from MP Biomedicals (Aurora, OH, USA), Ac-DEVD-CHO was purchased from Promega Co. β-elemene was purchased from Zhejiang Institute for Food and Drug Control (Hangzhou, China). Anti-actin antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-capase-3, anti-cleaved caspase-3, anti-caspase-7, anti-cleaved caspase-7, anti-caspase-9, anti-cleaved caspase-9, anti-PARP, anti-cleaved PARP, and anti-survivin antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

U251 and A172 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in recommended growth media. U251 cells were transfected with pMEG06-3HA-survivin (U251-survivin) or pMEG06-3HA vector as control (U251-HA). Stable cell lines were established by G418 selection (0.6 mg/ml, Sigma-Aldrich).

pMEG06-3HA-survivin plasmid, which contains the ORF of survivin gene, a 3HA-Tag and the N-terminal, was purchased from FuGENE Gene Co. Total cDNA was generated by reverse-transcription from U251 mRNA using RT-kits (Takara), and the ORF of HBXIP was amplified by PCR, the specific primers for PCR were as follows: 5′-GAC GAATTCATGGAGGCGACCTTGGAGCA-3′ (forward) and 5′-GATCTCGAGTCAAGAGGCCATTTTGTGCA-3′ (reverse) (23). The resultant cDNA fragments were ligated into pCMV-Tag2B vector, which contains a FLAG-Tag and the N-terminal, for mammalian cell expression.

To determine apoptosis, U251 and A172 cells were stained with annexin V and propidium iodide (PI) using a Vybrant Apoptosis Assay kit (Invitrogen, Carlsbad, CA). In brief, cells were collected by trypsinization and washed with ice-cold phosphate-buffered saline (PBS). Then cells were resuspended in 500 μl binding buffer, and incubated with 5 μl of annexin V-FITC and 10 μl of the 100 μg/ml PI working solution at room temperature for 5 min in the dark. Stained cells were analyzed for apoptosis by flow cytometry. FITC was detected at 518 nm and PI was detected at 620 nm.

For TUNEL assay, a DNA Fragmentation Detection kit, Fluorescent-TdT Enzyme (Cabiochem, San Diego, CA), was used to detect DNA fragmentation. In brief, cells were treated with β-elemene, harvested, and resuspended in 80% ethanol at 4°C overnight. The next day cells were washed with Tris-buffered saline (TBS) and incubated with 20 μg/ml proteinase K for 5 min at room temperature. Cells were then washed three times with PBS, incubated with 100 μl TdT equilibration buffer at room temperature for 30 min, and incubated in the dark with 60 μl of TdT-labeling reaction mixture at 37°C for 60 min. Finally, pellet cells were washed three times with PBS and analyzed by a flow cytometer. The emission wavelength of fluorescein is 517 nm.

Total RNA was extracted using TRIzol (Invitrogen), and after DNase I treatment, cDNAs were synthesized by using the cDNA reverse-transcription kit (Takara). Survivin and GAPDH mRNA was detected by RT-PCR, the specific primers for RT-PCR were as follows: survivin: 5′-ACCAGG TGAGAAGTGAGGGA-3′ (forward) and 5′-AACAGTAGAGGAGCCAGGGA-3′ (reverse) (24); GAPDH: 5′-CATGG GTTCAACATGCCAAGTGGT-3′ (forward) and 5′-TCCACGGCAGCATTAATCACAGGA-3′ (reverse).

For immunoprecipitation, after transfection, cells were lysed in 1 ml of 1% Nonidet P-40, 25 mM Tris-HCl, 150 mM NaCl, 10 mM EDTA, pH 8.0, a 1:50 dilution of a protease inhibitor mixture (Sigma) for 30 min on ice. Cell lysates were centrifuged at 14,000 g for 5 min to pellet cell debris and incubated with primary antibodies overnight at 4°C with rotation followed by the addition of protein A-agarose (60 μl of 50% slurry) for 1 h at 4°C with rotation to capture antibody-antigen complex. The antibody-antigen complex was washed, and samples were mixed with SDS-PAGE sample buffer, boiled for 5 min, electrophoresed on an SDS-polyacrylamide gel, and electro-blotted onto a nitrocellulose membrane (Millipore, Bedford, MA). For western blot detection, proteins were resolved on SDS-PAGE, transferred to a nitrocellulose membrane. The membrane was blocked in PBS containing 5% skimmed milk powder and 0.02% Tween-20, and then probed with specific antibodies at 4°C overnight. After washing, the membrane was incubated with a 1:20,000 dilution of peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG in PBS containing 5% skimmed milk powder. The blot was then developed using the ECL detection kit (Amersham Biosciences) to produce a chemiluminescence signal, which was captured on X-ray film.

In this experiment, we detected β-elemene-induced apoptosis by annexin V staining and TUNEL assay in human glioma cells. Human glioma U251 and A172 cells were treated with β-elemene at concentrations of 0, 100, 200 and 300 μg/ml for 24 h. Flow cytometry analysis showed a significant increase in the apoptotic population of the cells treated with β-elemene for 24 h when compared with that of the cells without treatment. The most striking increase in the percentage of apoptotic cells occurred at the concentration of 300 μg/ml (Fig. 1A). TUNEL assay further confirmed that apoptosis was induced by β-elemene (Fig. 1B).

Next we tested the activities of caspase-9, -3 and -7 in glioma cells when stimulated with β-elemene. As shown in Fig. 2A and B, β-elemene clearly induced the cleavage of pro-caspase-9, -3, -7 to their active forms in human U251 and A172 glioma cells. Moreover, poly(ADP-ribose) polymerase (PARP), a specific substrate of caspase-9, -3 and -7, was also cleaved into its active form in a dose-dependent manner, indicating that caspase-9, -3, -7-mediated pathways was involved in β-elemene-induced glioma apoptosis. To further prove that, we examined the effect of caspase inhibitors on the β-elemene-induced apoptosis in U251 cells. The results showed that both of pan-caspase inhibitors, Q-VD-OPH and Q-LEHD-OPH, markedly inhibited β-elemene-induced apoptosis. Treatment with a specific caspase-3 inhibitor Ac-DEVD-CHO provided the protection for U251 cells against apoptotic death (Fig. 2C). These results suggested that β-elemene induced caspase-dependent apoptosis of glioma cells.

As anti-apoptotic protein survivin is expressed abundantly in human gliomas and related with poor prognosis, we investigated whether proapoptotic effect of β-elemene can be mediated by regulation of survivin levels. We firstly determined the expression of survivin in glioma cells with or without β-elemene treatment. The results indicated that β-elemene induced a dose- and time-dependent reduction of survivin expression either in mRNA (Fig. 3A and C) or protein levels (Fig. 3B and D), suggesting a potential role for survivin in β-elemene-induced glioma cell death.

Next, we examined whether β-elemene-induced downregulation of survivin was associated with apoptosis in glioma cells. We established stable U251 cell lines with forced expression of HA-tagged survivin and then compared the effect of β-elemene on the U251-HA and U251-sur cells. Transfection efficiency of survivin gene was proved by western blotting (Fig. 4A) and RT-PCR analysis showed that β-elemene treatment did not affect the exogenous HA-tagged survivin expression (Fig. 4B). As expected, the treatment caused a marked decrease in endogenous survivin in both U251-HA and U251-sur cells (Fig. 4B and C), and TUNEL assay showed apoptosis of U251 cells was induced by β-elemene. Remarkably, forced expression of survivin caused a reduction in apoptosis of U251-sur cells when compared with U251-HA cells (Fig. 5A), suggesting that apoptosis of U251 cells induced by β-elemene was overridden by forced expression of survivin.

Since survivin exerts an anti-apoptotic effect through caspase-9, -3 and -7-dependent pathway (25–27), we further evaluated the apoptosis-related proteins in U251-HA and U251-sur cells upon β-elemene stimulation. The cells were treated with 150 μg/ml β-elemene for 24 h and the levels of caspase-9, -3, -7 and PARP were detected by western blotting. Results showed that the forced expression of survivin in U251 cells significantly decreased β-elemene-induced activation of caspase-9, -3, -7, as well as the cleavage of PARP (Fig. 5B).

It has been proved that survivin-HBXIP complexes, but neither survivin nor HBXIP individually, have the capability to bind pro-caspase-9 and inhibit the activation of caspase-9 (23), we hypothesized that in human glioma cells, β-elemene can inhibit the interaction of survivin and HBXIP. To test this hypothesis, we examined if the binding of survivin to HBXIP was affected by β-elemene treatment. Firstly, the ORF of HBXIP gene was cloned into pCMV-Tag2B plasmid to construct pCMV-HBXIP plasmid which can express a FLAG-HBXIP fusion protein in mammalian cells. pMEG-HA-survivin and pCMV-HBXIP were then co-transfected into U251 cells and treated with β-elemene (100 μg/ml). Using co-immunoprecipitation method we clearly showed that survivin was bound to HBXIP protein in glioma cells. More importantly, the interaction between the two molecules was inhibited by β-elemene treatment (Fig. 6A and B). As survivin-HBXIP complex was assumed to be responsible for the retaining and retarding of caspase-9 activity, we suggested that β-elemene-induced impairment in survivin-HBXIP interaction might promote cell apoptosis. Besides, HBXIP level was not obviously affected by β-elemene as shown in Fig. 6C.