| Background and ObjectivesDDP, a kind of traditional chemiotherapy drugs, has the restriction in clinic for its muti-side effects. Now, it turns from tradition method to combination tharepy. Reachers turn to Tradional Chinese Medine. Emodin is a kind of naturalanthraquinon, which has an anti-carcinoma activity that attracts people’s interest.The mechnisim of anti-carcinoma activity is made up of inhibiting cell proliferation, sticking and metastasis. However, in the process of metastasis, ROS acts as moning signal, which acts on the mitochondrion. Mitochondria membrane emerges disorder, reduces Bcl-2, increase Bax, release Cytochrome C, activate Caspase-2, -3, -9, induce cell apoptosis. Besides damage mitochondria can create ROS further, which can induce apoptosis. ROS plays an important role in the cytotoxic by anthraquinone.ROS is concedered as the most common organism factor in the apopotosis process. The increase ROS can lead to imbanlance of oxidation which is the central of cell apoptosis process. The architecture of emodin is similar to bimenadione and ubiquinone of mitochondrion. Bimenadione is a endogenous ROS activator and ubiquinone is the same in the mitochondrion. Emodin can inhance the ROS of intracellur in a safely and soft way, which can inhance the chemiotharepy. In this study, we intend to uncover the mechanisms of intracellar ROS activator emodin which combined DDP treat human esophageal carcinoma cell line EC-9706.This will provide theoretical foundation of emodin combined chemotherapy drugs.Materials and Methods1. One human esophageal carcinoma cell line, EC-9706 cell was cultured in RPMI-1640 with 10% fetal bovine serum supplemented with 100U/ml penicillin and 100U/ml streptomycin. 2. EC-9706 cell was cultured in the same medium which contained different type of drugs. Emodin group was composed of 30μg/ml Emodin in RPMI-1640 medium with 10% fetal bovine serum. DDP group was made up of 5μg/ml DDP in the same medium.Combined group contained both 30μg/ml Emodin and 5μg/ml DDP in the same medium.Each group cultured the EC-9706 cell with different time including 12h、24h and 48h respectively. The control group was the same to above group except drugs and zero group was not different from control group besides EC-9706 cell in the medium.3. EC-9706 cells were treated with different medium which contained different drugs for 12h、24h and 48h, and untreated cells were taken as control. AO/EB assay was used to evaluate the influence on the change of morphology about EC-9706 cells of each group.4. EC-9706 cells were treated with different medium which contained different drugs for 12h、24h and 48h, and untreated cells were taken as control. MTT assay was used to evaluate the influence of each group on the proliferation and inhibition of the EC-9706 cells. To check whether the two drugs have synergistic effect or not.5. EC-9706 cells were cultured with the different group medium for 24h, then stained by Annexin V– FITC/ PI in double staining method and cell apoptosis was detected by flow cytometry (FCM).6. EC-9706 cells were cultured with the different group medium for 2h, then use the Reactive Oxygen Species Assay Kit to check the intracellular ROS levels which were detected by flow cytometry (FCM). .Results1. AO/EB assay showed that the control group growed well with integrity cell membrane membrane.Cells were full of green fluorescence hardly filled with orange fluorescence.On the contrary,the emodin and DDP of each group were colored different fluorescence.But the combined group was more deeply than the other solo-drug group.This was indicated that the two drugs combined together had more power to induce cells to apoptosis.2. MTT assay showed that EC-9706 cells emerged growth inhibition gradually each in each group with the time went by. The survival rate of EC-9706 cells in Emodin group、DDP group and combind group were 58.35±5.03%, 72.06±2.92% and 47.35±5.78% after 12h, 45.18±4.33%, 56.60±6.41% and 37.29±4.85% after 24h, 40.24±7.71%, 23.06±4.52% and 15.45±2.46% after 48h respectively. Obviously, the cell survival rate in the combined group was sharply lower compared to each drug sololy. The CDI value of each group were 0.94±0.03,0.91±0.04,0.90±0.03 after 12h、24h and 48h later, which implied the two drugs existing synergistic effect.3. FCM showed that the early apoptosis rate of EC-9706 cells in Emodin group、DDP group and combind group were 15.39±3.40%, 12.80±2.41% and 23.09±3.97% after 24h, and the contrast group was only 0.43±1.04%(p<0.05). With the time went by the apoptosis rate elevated gradually. Result showed that the combined group promoted cells to death compare to each drug sololy.4. FCM showed that the intracellar ROS of each group were elevated in different degree. According to the introduction of the ROS assay, EC-9706 cells were treated with different drugs for 2h, and then collected the drug treated cells to check the fluorescence intensity which could reflect the intracellar ROS level in a indirect way. The results showed that the intracellar ROS in each group were 35.57±3.16%, 29.44±2.43% and 43.23±3.68%, with the control group was only 4.73±2.12%. All the above data demonstrated that the combined group elevated the intracellar ROS level obviously, which had statistics significance.Conclusions1. Both Emodin and DDP could inhibit proliferation of human esophageal cancer cell EC-9706 and promote cell apoptosis. The gradual proliferation was displayed in a time-dependent manner. The combined group was more power to inhibit the proliferation of cells, compare to each drug sololy, which showed the two drugs acted together exiting synergistic effect.2. Both Emodin and DDP could elevate the intracellar ROS level comparing to control group. The result showed that the combined group elevated the intracelar ROS level sharply and promoted apoptosis. |
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