Effect Of Allicin On TRAIL-induced Apoptosis In Ovarian Cancer SKOV-3 Cells In Vitro

Objective: Ovarian cancer is one of the most common types of malignant tumor of the female reproductive system. It has tall death rate and worse therapeutic effects. With the development of Neoadjuvant Chemotherapy and the transmission of medical pattern, chemotherapy of ovarian cancer has been an imperative method. In this therapy, anti-cancer drugs which can kill tumor cells most but have the least toxicity to normal cells becomes one of the focuses. TRAIL (TNF-related apoptosis-inducing ligand) is a member of TNF family discovered recently. The ability of TRAIL to selectively induce many tumor cells apoptosis with no influence on normal cells makes it have potential application value on cancer therapy. Allicin is the main bioactive substance from garlic. Some experiments show that Allicin can inhibit tumor growth by inducing tumor cells apoptosis.Our research aims to discuss the effects of Allicin and TRAIL on growth of cervical cancer cells and its mechanism based on ovarian cancer cell lines SKOV-3, which can provide a new thought of chemotherapy for ovarian cancer in clinic.Methods: 1 Cell culture: Refrigerated SKOV-3 cells were anabiosised and grown in 100ml culture flasks in PRMI 1640 supplemented with 10% fetal bovine serum (FBS), 100ug/ml streptomycin and 100IU/ml penicillin. The cells were incubated in 37℃, 5%CO2 and saturated degree of humidity culture box with loosen covers. Cells were digested and subcultured with 0.25% steapsin.2 Cell proliferation: SKOV-3 cells were seeded in 96-well culture plates at a density of 6×104cells/ml(100ul/well) in culture medium. After having stayed over in 24h, the cells were divided into 4 groups and their culture media were as follows: (1)basic PRMI 1640 media added with allicin, the concentrations of allicin were 12.5, 25, 50 and 100mg/L respectively; (2)basic PRMI 1640 media added with TRAIL, the concentrations of TRAIL were 25, 50, 100 and 200ng/ml respectively; (3)with TRAIL(100ng/ml) and allicin (50mg/L) collectively; (4)blank control group: basic media only. Each concentration of all the groups had 6 wells. After 24, 48, and 72h, 10ul MTT (5mg/ml) was add in every well for 4h at 37℃. Then the liquid was aspirated, 100ul DMSO was added into each well. 96-well culture plates were put on oscillator for 8-10min and absorbance (OD) of cells in each well was detected with spectrophotometer when wavelength was 492nm. Each assay was performed 3 times independently.3 Expressions of TRAIL recepetors DR4 and DR5 by flow cytometer(FCM): SKOV-3 cells in logarithmic phase were seeded in culture flasks at a density of 4.5×104cells/ml in culture medium. After having stayed over in 24h, the cells were divided into experimental group(basic PRMI 1640 media added with 50mg/L allicin) and control group(basic PRMI 1640 only), after 24h the cells were collected and then tested expressions of TRAIL recepetors DR4 and DR5 by FCM through fluorescence index(FI). Each assay was performed 3 times and the mean data are available.4 Caspase activity assay before and after allicin treatment: SKOV-3 cells in logarithmic phase were seeded in culture flasks at a density of 4.5×104cells/ml in culture medium. After having stayed over in 48h, the cells were divided into experimental group(basic PRMI 1640 media added with 50mg/L allicin) and control group(basic PRMI 1640 only) and were tested OD405 by spectrophotometer. The changes of Caspase-3 and 8 activity showed by the ratios of ODallicin/ODblank. Each assay was performed 3 times and the mean data are avaliable.5 Expressions of TRAIL receptors DR4, DR5 and Caspase-3, 8 before and after allicin treatment by semi-quantity reverse transcription-polymerase chain reaction (RT-PCR): SKOV-3 cells in logarithmic phase were seeded in culture flasks at a density of 4.5×104cells/ml in culture medium. After 24h, the cells were divided into experimental group (basic PRMI 1640 media added with 50mg/L allicin) and control group(basic PRMI 1640 only), after 8h the cells were collected. Have cells cleavage and total RNA were isolated, cDNA synthesis, PCR amplification and agarose gel electrophoresis. Detect the absorbance ratios of electrophoresis belts gene amplification products of DR4, DR5, Caspase-3, 8 andβ-actin by gel imaging system.Statistical analysis: Expressions of TRAIL receptors DR4, DR5, Caspase-3, 8 and absorbance value (OD) were presented as±s, Statistical analysis was performed by t-test and one-way ANOVA. Enumeration data were analyzed byχ2-test. P<0.05 was considered statistically significant.Results: 1 Cell morphology observation: Observation by inverted microscope: Growth behavior of SKOV-3 cells in control group. Extension cells showed anomalous polygon. When incubated for 24h, with cells shrinking, cell volume was smaller, intercellular space enlarged and apoptosis body formation. 2 Growth inhibition of cells by allicin: Inhibitory rates of SKOV-3 cells after 24, 48, 72h allicin and TRAIL respective and collective treatments had a time-and-dose- dependent effect. Comparing with control group, the difference of each group had marked significance (P<0.05). 3 Positive expressions of TRAIL receptors by flow cytometer (FCM): Expressions of DR4 and DR5 were upregulated obviously after 24h allicin treatment. FI rose from 1.78 to 2.27 for DR4 and from 1.94 to 2.58 for DR5. Comparing with control group, the difference marked significantly (P<0.05). 4 Caspase activity assay: Activities of Caspase-3 and 8 were upregulated obviously after 48h allicin treatment. The ratio of ODallicin/ODblank of Caspase-3, 8 was 2.49, 2.08 times than control, respectively. Comparing with control group, the differences marked significancely (P<0.05). 5 Results from RT-PCR: Absorbance ratios of electrophoresis belts of gene amplification products of DR4, DR5, Caspase-3, 8 andβ-actin were ascended markedly. Comparing with control group, DR4, DR5 were 1.45, 1.52 times respectively, Caspase-3 and 8 were 2.03, 1.55 times respectively. Comparing with control group, the differences marked significancely (P<0.05).Conclusion: (1)The ovarian cancer cell line SKOV-3 was sensitive to Allicin and TRAIL. (2)Allicin could enhance TRAIL-induced apoptosis in ovarian cancer SKOV-3 cells. (3)Allicin could upregulate TRAIL death receptor 4 and 5, which might be one of its mechanisms of enhancing TRAIL- induced apoptosis. (4)Allicin also could upregulate Caspase-3 and 8 mRNA and could activate Caspase-3, 8 protein, which might be another one of its mechanisms of enhancing TRAIL- induced apoptosis.The conclusion will provide theoretical basis for ovarian cancers adjuvant clinical treatments by allicin and TRAIL.