The U251 human glioma cell line was obtained from the Chinese Academy of Sciences Cell Bank. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Hyclone; GE Healthcare Life Sciences, Chicago, IL, USA), supplemented with 10% (FBS) at 37°C with 5% CO₂. Allicin (purity ≥90%) was purchased from Shanghai Harvest Pharmaceutical Co., Ltd. (Shanghai, China).

Glioma cells were cultured at 5,000–8,000 cells/well in 96-well plates overnight. Cells were treated with 15, 30, 60 or 90 µg/ml of Allicin for 20 h. Subsequently, 5 mg/ml MTT (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was added for additional 4 h at 37°C. Optical density (OD) was assessed by using a TECAN microplate reader at 490 nm.

Cell viability was measured by MTT assay. Cells were seeded at 5×10³ cells/well into a 96-well plate and incubated with Allicin at various concentrations (15, 30, 60 and 90 µg/ml) for different periods (24, 48 and 72 h). The OD was assessed by using a TECAN microplate reader at 490 nm. For the colony formation assay, cells were cultured in a 6 cm dish (0.5×10³ cells/well) and incubated at 37°C. Following this, different concentrations of Allicin were added for 24 h. The medium was then removed and cells were cultured for another 12 days. The colonies formed were fixed with 10% formalin for 10 min. Giemsa staining was used to stain the samples obtained for 30 min in room temperature and the number of colonies (>50 cells) was counted by using upright light microscope (Leica DM2000; Leica Microsystems, GmbH, Wetzlar, Germany).

U251 cells were treated with different concentrations Allicin (30 and 60 µg/ml) for 48 h, and then washed with ice-cold PBS. The cell lysates were centrifuged at 6,037 × g for 15 min at 4°C and the total protein was extracted using radioimmunoprecipitation buffer (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with protease inhibitors, then subsequently separated via 4–10% Tris glycin/SDS-PAGE. A bicinchoninic acid protein assay kit was used to determine the concentration of protein. The total quantity of protein loaded onto each lane was 40 µg; separated proteins were electrotransferred to ECL nitrocellulose membranes (IPFL00010; EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk (Wuhan Boster Biological Technology, Ltd., Wuhan, China) at room temperature for 1 h and incubated separately with mouse anti-human Fas (1:1,000 dilution, cat. no. 8023S; Cell Signaling Technology, Inc., Danvers, MA, USA), mouse anti-human FasL (1:1,000 dilution, cat. no. 4273; Cell Signaling Technology, Inc.), mouse anti-human Bcl-2 (1:500 dilution, cat. no. sc-509; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit anti-human Bcl-2-associated X protein (Bax; 1:500 dilution, cat. no. sc-526; Santa Cruz Biotechnology, Inc.), rabbit anti-human caspase-3 (1:1,000 dilution, cat. no. 9665; Cell Signaling Technology, Inc.) and mouse anti-human β-actin (1:500 dilution, cat. no. 376421; Santa Cruz Biotechnology, Inc.) at 4°C overnight, and then incubated with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution, cat. no. ab150077; Abcam, Cambridge, MA, USA) for 1 h. The protein levels were visualized using an enhanced chemiluminescence kit (BeyoECL Plus, cat. no. P0018; Beyotime Institute of Biotechnology, Haimen, China) and determined by a Gel Doc 2000 imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Total U251 cell RNA treated with different concentration of Allicin (0, 30 or 60 µg/ml) were isolated using TRIzol reagent (Thermo Fisher Scientific, Inc.). PCR was performed with ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The thermocycling condition were as follows: 95°C for 30 sec, then followed by 40 cycles at 95°C for 5 sec, 60°C for 30 sec, and 72°C for 15 sec. The PCR primers were as follows: Fas sense, 5′-TTCTGCCATAAGCCCTGTC-3′ and antisense, 5′-TTGGTGTTGCTGGTGAGT-3′ (amplification fragment 320 bp); FasL sense, 5′-TTCAGCTCTTCCACCTACAG-3′ and antisense, 5′-ACATTCTCGGTGCCTGTAAC-3′ (amplification fragment 599 bp); caspase-3 sense, 5′-GACAGACAGTGGAAGCGACTGGAT-3′ and antisense, 5′-GCATGGCACAAAGCGACTGGAT-3′; Bcl-2 sense, 5′-CGCCCTGTGGATGACTGAGTA-3′ and antisense, 5′-GGGCCGTACAGTTCCACAAAG-3′; Bax sense, 5′-CCCTTTTGCTTCAGGGTTTCATCCA-3′ and antisense, 5′-CTTGAGACACTCGCTCAGCTTCTTG-3′; U6 sense, 5′-TGCGGGTGCTCGCTTCGGCAGC-3′ and antisense, 5′-CCAGTGCAGGGTCCGAGGT-3′. Fas/FasL, caspase-3, Bcl-2 and Bax mRNA expression levels were determined using a SYBR PrimeScript RT-PCR kit (Takara, Bio, Inc., Otsu, Japan) and normalized to U6 mRNA. The relative expression levels were analyzed using the 2^−ΔΔCq method (12).

A caspase activity kit (Beyotime Institute of Biotechnology) was used to detected the activity of caspase-3, −8 and −9. Cells (1×10⁶ cells/well) were added to Allicin (0, 30 and 60 µg/ml) for 24 h. Data was obtained by measuring the enzyme labeling meter with the wavelength of 490 nm via a microplate reader (Infinite F50 Tecan Group, Ltd., Männedorf, Switzerland). The U251 cells were isolated with caspase assay buffer (Nanjing Kaiji Materials, Co., Ltd., Nanjing, China) and then supernatant was collected and centrifuged at 7,546 × g at 4°C for 10 min. The relative activity of caspases was measuring by an enzyme-labeling meter with a microplate reader.

Cells were treated with Allicin for 24 h. The media was then removed and cells were fixed with 4% formaldehyde. Cells were then stained with 200 µM Hoechst 33258 for 10 min at room temperature, and the slides were examined under a fluorescence microscope (Olympus Corporation, Tokyo, Japan).

U251 cells were seeded onto a 6-well plate at 5×10⁵ cells/well for 24 h and treated with different amounts of Allicin (0, 30 and 60 µg/ml) for 48 h. The samples were collected at a concentration of 1×10⁶ cells/ml with 4°C PBS and suspended in 400 µl binding buffer. Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI; BD Pharmingen; BD Biosciences, Franklin Lakes, NJ, USA) were added into the labeled tube and cells were incubated for 20 min. The samples were examined by using flow cytometry (BD Biosciences; Clontech, Palo Alto, CA, USA).

All results were summarized from three independent experiments. Results are expressed as the mean ± standard error. Comparisons of each treatment data with control were carried out for statistical difference by the paired t-test. One way analysis of variance with followed by a Turkey's post-hoc test was used to analyze statistical differences between groups by using the statistical software SPSS 17.0 (SPSS, Inc., Chicago, IL, USA); graphs were produced using by GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

The cytotoxic effect of Allicin (P<0.05; Fig. 1B) on U251 cells was measured by MTT assay. The results demonstrated that Allicin decreased cell survival in a dose-dependent manner, indicating its cytotoxicity. The median IC50 was 41.97 µg/ml; therefore, 30 and 60 µg/ml doses of Allicin were chosen for the present study.

In the present study, Annexin V/PI staining was used to demonstrate the apoptosis-inducing effect of Allicin. The results demonstrated that the rate of apoptosis increased (30 µg/ml for 9.1±3.2% and 60 µg/ml for 51.4±3.8%) at higher Allicin concentrations compared with control cells (3.3±1.5%). Flow cytometry assay demonstrated that the rate of apoptosis increased in response to treatment with Allicin in a dose-dependent manner (P<0.05; Fig. 1C).

U251 cells were treated with Allicin for 24 h, then stained with Hoechst 33258. The morphological changes of apoptosis cells (such as condensation of chromatin and nuclear fragmentation) was detected under a fluorescence microscope (Fig. 2A). The level of apoptosis was increased with the dose of Allicin in U251 cells (Fig. 2B). These results demonstrated that Allicin induced apoptosis in a dose-dependent manner.

Colony formation was used to confirm the proliferation ability by treating with Allicin in U251 glioma cells, which indicated that the inhibition ability of Allicin was irreversible (Fig. 3A). MTT assay was used to detect the effect of Allicin on the growth of glioma cells. Allicin was demonstrated inhibit the viability of glioma cell in a dose- and time-dependent manner (Fig. 3B; P<0.05).

Fas (CD95/APO-1) induces apoptosis by binding to a member of the tumor necrosis factor receptor (TNFR) family (FasL), which is a cell surface receptor protein (13). RT-qPCR was used to measure the expression of Fas and FasL mRNA. The data demonstrated that after treating with different amounts of Alllicin, Fas, FasL and Bax mRNA expression levels were significantly increased, while Bcl-2 expression levels were decreased, in a dose-dependent manner (P<0.05, Fig. 4A). These results were reflected at the protein level (P<0.05; Fig. 4B and C). These results demonstrated that Allicin induces apoptosis a dose-dependent manner.

After treating cells with various amounts of Allicin for 24 h, caspase-3, −8 and −9 enzyme activities were upregulated (P<0.01; Fig. 4D). Therefore, apoptosis signaling pathways which were activated by caspases may be involved in the antitumor effects of Allicin.