Cisplatin Enhanced The Ability Of The Apoptosis Induction Of TRAIL To Cervical Cancer Cells By Upregualation Of Death Receptor 4,5 In Vitro

Objiective:Cervical cancer is the main malignant disease which has threatened women's health and life worldwide, and only second to breast cancer to be the chief cause of cancer-related death. Chemotherapy remains the major management for patients with advanced cervical cancer or recurrent cervical cancer. But it will cause serious side reaction at the same time if using a great deal of chemotherapy medicine for a long time. Seeking a kind of Chemotherapy method with high potency and low toxicity, especially for treatment of advanced cervical cancer, is constantly the problem clinicians explored hardly. TRAIL ( TNF-related apoptosis-inducing ligand)also named Apo2L, is a member of TNF family discovered recently. The ability of TRAIL to selectively induce many tumor cells apoptosis with no influence on proliferation and differentiation of normal cells makes it have potential application value on cancer therapy and be possible to be used for a new type of Chemotherapy medicine with no adverse effects and toxicities. Our research aims at the feasibility of TRAIL using to cervical cancer treatment through culture of the cervical cancer cell lines Hela and Caski in vitro. We also discuss the effect of TRAIL and cisplatin (CDDP) on growth of cervical cancer cells and its mechanism, which can provides a pre-clinical evidence of using TRAIL to cervical cancer.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).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 4.5×10~4cells/ml (100μl / well) in culture medium. After having stayed over in 24h, the cells were divided into 4 groups and their concentration of the drugs were as follows: (1) basic PRMI 1640 media added with cisplatin(0.5,1.0,2.0mg/L) or with TRAIL(10,20,50,100,200ng/ml) respectively for 24h or 48h; (2) with TRAIL(100ng/ml) and CDDP(1.0mg/L) collectively for 24h; (3)with TRAIL(100ng/ml) or CDDP(1.0mg/L) first for 12h, then added CDDP(1.0mg/L) or TRAIL(100ng/ml) for 12h; (4) Blank control group: added with basic media only for 24h. Each concentration of all the groups had 6 wells. After a time, 10 c MTT (5mg/ml) was add in every well for 4h at 37℃. Then the liquid was aspirated, 200μl DMSO was added into each well. 96-well culture plates were put on oscillator for 8-10min and absorbance (A) of cells in each well was detected with spectrophotometer when wavelength was 490nm. Each assay was performed 3 times independently.3 Expressions of TRAIL recepetors DR4,DR5,DcR1,DcR2 by flow cytometer(FCM): Hela,Caski cells in logarithmic phase were seeded in culture flasks at a density of 4.5×10~4cells/ml in culture medium. After having stayed over in 24h, the cells were divided into experimental group(basic PRMI 1640 media added with 1.0mg/L CDDP) and control group(basic PRMI 1640 only), after 24h the cells were collected and then tested expressions of TRAIL recepetors DR4,DR5,DcR1,DcR2 by FCM through fluorescence index(FI). Each assay was performed 3 times and the mean data are available.4 Expressions of TRAIL receptors DR4,DR5,DcR1,DcR2 before and after CDDP treatment by semi-quantity reverse transcription-polymerase chain reaction (RT-PCR).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 24h, the cells were divided into experimental group (basic PRMI 1640 media added with 1.0mg/L CDDP) and control group (basic PRMI 1640 only), after 8h the cells were collected.Then step by step,to have cells cleavage and total RNA were isolated, cDNA synthesis, PCR amplification and agarose gel electrophoresis.At last,to detect the absorbance ratios of electrophoresis belts gene amplification products of TRAIL receptors DR4,DR5,DcR1,DcR2 andβ-actin by gel imaging system. 5 Statistical analysis: Measurement data were presented as±s, Statistical analysis was performed by t-test and one-way ANOVA. P<0.05 was considered statistically significant.Results: 1 Cell morphology observation: Observation by inverted microscope: Growth behavior of cells in control group were in good condition. 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 TRAIL and CDDP: Inhibitory rates of Hela and Caski cells after 24,48h TRAIL or CDDP treatments had a time-and-dose dependent effect. Comparing with control group, the difference of each group had marked significance (P<0.05). 3 Growth inhibition of Hela and Caski cells after TRAIL combined with CDDP treatments increased, showed a synergic cytotoxic effect. Inhibitory rates of Hela cells combination treatments rose from13.34% (with TRAIL only) to 69.44%; and of Caski cells were 35.44% to 89.66%. 4 Two groups with CDDP-sequential TRAIL and TRAIL-sequential CDDP had different effects on growth inhibition of Hela and Caski cells, former inhibitory rates of Hela and Caski cells were 60.75% and 79.88%, the latter were 35.22%和55.73%. The difference had marked significance inside the two experimental group (P<0.01). 5 Positive expressions of 4 TRAIL receptors by flow cytometer (FCM). Results from FCM: FI of DR4,DR5 were much higher than of DcR1,DcR2,but there was no significant difference between Hela and Caski cells. Expressions of DR4, DR5 were upregulated obviously after 24h CDDP treatment. FI of Hela rose from 1.80 to 2.20 for DR4and from 1.82 to 2.44 for DR5. While, FI of Caski rose from 2.07 to 2.35 for DR4 and from 2.14 to 2.55 for DR5. Comparing with control group, the difference marked significantly (P<0.05).But the difference of expressions of DcR1,DcR2 between the two cell was no statistically significant. 6 Results from RT-PCR: absorbance ratios of electrophoresis belts gene amplification products of DR4,DR5 in Hela andCaski cells were ascended markedly. Comparing with control group, DR4 were 1.25 and 1.40 times in Hela and Caski cells respectively, DR5 were 1.36 and 1.57 times mean time,P<0.05.The difference of expressions of DcR1, DCR2 was no statistically significant(P>0.05 ).Conclusion: 1 The cervical cancer cell line Hela and Caski were sensitive to TRAIL.2 CDDP could increase the sensitivity of Hela and Cask cells to TRAIL, and combined with TRAIL showed a synergic cytotoxic effect.3 Cisplatin enhanced the ability of apoptosis induction of TRAIL to cervical cancer cells by upregualation of Death receptor 4,5. 4 additional studies are needed with the mechanism and signaling pathway of the upregualation of Death receptor by CDDP.5 It is not clear that decoy receptor (DCR) play a role in the CDDP and TRAIL synergic effect.