Apoptosis Induced By Allicin On Cervical Cancer Cells Through Upregualation Of TRAIL And Caspase-8 In Vitro

Objective: Cervical cancer is one of the most common types of cancer in women. Recently, cervical cancer incidence increases obviously, especially among young women. With the development of Neoadjuvant Chemotherapy and the transmission of medical pattern, chemotherapy of cervical cancer has been an imperative method. In this therapy, natural anti-cancer drugs which can kill tumor cells most but have the least toxicity to normal cells becomes one of the focuses. Allicin is the main bioactive substance from garlic.Some experiments show that Allicin can inhibit tumor growth by inducing tumor cells apoptosis.Our research compares between before and after allicin treatments on human cervical cancer cell lines Hela and Caski in vitro and expressions of TRAIL and caspase-8, activity of caspase-8 enzyme, and then discusses the effect of allicin on human cervical cancer cell lines and its mechanism,which can provides a pre-clinical evidence of adjuvant therapy to maglinant tumors of allicin in clinic.Methods:1 Cell culture: Refrigerated Hela,Caski 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: Hela,Caski 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 7 groups and their culture media were as follows: basic PRMI 1640 media added with allicin,the concentrations of allicin were 6.25,12.5,25,50,75 and 100 mg/L respectively;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 ligand and death receptors 4,5 by flow cytometer(FCM): Hela,Caski cells in logarithmic phase were seeded in culture flasks at a density of 4.5×104cells/ml in culture medium. After having stayed over, the cells were collected and then tested expressions of TRAIL ligand and death receptors 4,5 by FCM.4 Expressions of TRAIL ligand before and after allicin treatment by flow cytometer (FCM):Hela,Caski 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 allicin) and control group(basic PRMI 1640 only) and were tested expressions of TRAIL ligand by FCM through fluorescence index(FI). Each assay was performed 3 times and the mean data are available.5 Caspase-8 activity assay before and after allicin treatment:Hela,Caski 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 allicin) and control group(basic PRMI 1640 only) and were tested OD405 by spectrophotometer.The changes of caspase-8 activity showed by the ratios of ODallicin / ODcontrol.Each assay was performed 3 times and the mean data are available.6 Expressions of TRAIL ligand and caspase-8 before and after allicin treatment by semi-quantity reverse transcription- polymerase chain reaction, (RT-PCR):(1) Took 1×106 cells after 12h allicin teament to have cells cleavage and total RNA were isolated according to instruction of Trizol Reagnt kit. (2) DNA synthesis. (3) PCR amplification. (4) Agarose gel electrophoresis: Detected the absorbance ratios of electrophoresis belts of gene amplification products of TRAIL,Caspase-8 andβ-actin by gel imaging system. Statistical analysis: Expressions of TRAIL,Caspase-8 and absorbance value(OD) were presented as±s, Statistical analysis was performed by an one-way ANOVA, follow by Newman-Keuls test, 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 Hela,Caski cells in control group. Extension cells showed anomalous polygon. When incubated for 48h, with cells shrinking, cell volume was smaller, intercellular space enlarged and apoptosis body formation. (Fig.1) 2.Growth inhibition of cells by allicin: Inhibitory rates of Hela and Caski cells after 24,48,72h allicin treatments had a time-and-dose-dependent effect. (Fig.2,3) Comparing with control group, the difference of each group had marked significance (P<0.05).3.Positive expressions of TRAIL ligand and death receptors 4,5 were detected by flow cytometer(FCM). (Fig.4) 4.Results from FCM: Expressions of TRAIL ligand were upregulated obviously after 48h allicin treatment.FI of Hela rose from 1.96 to 2.34 for TRAIL protein. While, FI of Caski rose from 2.02 to 2.54 for TRAIL protein.Comparing with control group, the differences had marked significance (P<0.05). (Tab9) (Fig.5). 5.Caspase-8 activity assay: caspase-8 activities were upregulated obviously after 48h allicin treatment. The ratio of ODallicin/ODcontrol of Hela was 2.26 times than control. While,the ratio of ODallicin/ODcontrol of Caski was 1.69 times than control. Comparing with control group, the differences marked significancely (P<0.05). (Tab10).6.Results from RT-PCR: Absorbance ratios of electrophoresis belts of gene amplification products of TRAIL,Caspase-8 andβ-actin were ascended markedly. Comparing with control group,they were 1.43,1.47 times for Hela and 1.39,1.55 times for Caski and the differences marked significancely (P<0.05). (Tab10).Conclusion: (1) Allicin could inhibit cell proliferation and enhance apoptosis in cervical cancers cell lines Hela and Caski in vitro. Inhibitory rates were in a dose-and-time-dependent manner. (2) Caski cell was more sensitive to allicin than Hela cell. (3) Hela and Caski were sensitive to TRAIL. (4)Allicin could upregulate TRAIL on both transcriptional and translational level, which might be one of its mechanisms of apoptosis induction though TRAIL apoptosis pathway. (5) Allicin also could upmodulate Caspase-8mRNA on transcriptional level and active caspase-8 protein, which might be another one of its mechanisms of apoptosis induction though Caspase-medicated apoptosis pathway. The conclusion will provide theoretical basis for cervical cancers adjuvant clinical treatments by allicin.