| Background and objectiveEsophageal carcinoma is a kind of common malignant tumor in china. It was the fourth in the death rate of malignant tumor in our country. 5-Fluorouracil (5-Fu) is a normal anti-tumor drug in esophageal carcinoma. However, drag resistance does impact the therapeutic effect and prognosis.Nuclear transcription factor-κB (NF-κB) is an important type of nuclear transcription factor. The main form of NF-κB is a dimmer composed of p50 and p65 protein, the former is the part where NF-κB is linked with DNA, and the latter takes part in the primary regulation of gene's transcription and enhances the linkage between p50 and DNA. Inhibition of the NF-κB activity makes tumor cells apoptosis, which improves tumor cells sensitivity to chemotherapy and radiotherapy. There are two NF-κB binding sites in COX-2 promoter. NF-κB can regulate COX-2 genetic transcription and proteinic expression by the binding sites. Ki-67 protein antigen is correlated with cell cleavage and proliferation, and is the sensitive index that reflects cell proliferation. Pentoxifylline (PTX) is a phosphodiesterase inhibition drag. It inhibits phosphorylation and degradation of IκBa, so inhibits the NF-κB activity. Therefore, its effect in tumor therapy has made pentoxifylline be noticed generally. But there was no report about the effect of PTX to EC9706 cell line directly and to NF-κB activity in vitro.This study analyses the effects of PTX to human esophageal carcinoma EC9706 Cell line through observing the cells morphology changes by light microscope, drawing growth curve and MTT assay, and detects the expression of NF-κBp65, COX-2 and Ki-67 protein in EC9706 cells after PTX treatment for 24h through immunocytochemistry, and compares the inhibition effects of 5-Fu and PTX uniting 5-Fu to EC9706 cell line through observing the cells morphology changes by light microscope and MTT assay. This article intends to set an experiment foundation for exploring the new methods to treat esophageal carcinoma.Materials and methodsEC9706 cell line was provided by the Key Laboratory of Henan Tumor Pathology. It was given as a present by national Key Laboratory of molecule oncology in Chinese Academy of Medical Sciences.EC9706 cells were treated with the culture medium that has different concentration of PTX. The cells morphology changes were observe by light microscope, the cells growth state was observe by drawing growth curve, the cells inhibition ratio was detected by MTT assay. The expression of NF-κBp65, COX-2 and Ki-67 protein in EC9706 cells after PTX treatment for 24h was detected through immunocytochemical staining methods. Then, EC9706 cells were treated with the culture medium that has different concentration of 5-Fu and PTX uniting 5-Fu. The cells morphology changes were observe by light microscope, the cells inhibition ratio was compared by MTT assay. The statistical analysis was executed by SPSS10.0 statistical software, using analysis of variance (ANOVA) and t test. The level of significant difference was a=0.05.Results1. To observe the cells morphologic changes by light microscope after PTX treatment for 12～72h: PTX 0.01mg/ml group is similar to control group. The cell population is gradually increasing along with time in PTX 0.1mg/ml,0.5mg/ml,1.0mg/ml groups. But the speed of cell increasing becomes slow; and the morphologic changes can be observed, such as intercellular structure loosing, periplast turbid and so on.2. The cells growth curve after PTX treatment show: The EC9706 cells' proliferation is inhibited in every PTX treated group. Although the EC9706 cell population is gradually increasing along with time in every PTX treated group, the cell population of PTX treated group is fewer than the control group. And the proliferation inhibition is gradually increasing along with the ascending of PTX concentration and the lasting time.3. The inhibition rates of EC9706 cells treated by PTX are detected with MTT assay. The EC9706 cells are treated by different concentration PTX (0.01～1.0mg/ml) for 12h, 24h, 48h and 72h. There is a significant different in all inhibition rates mean(F=40.745, P<0.05) . And there is a significant different between the PTX 0.01mg/ml group and the control group (P<0.05) after 48h. There is a significant different between the PTX 0.1, 0.5, 1.0mg/ml groups and the control group (P< 0.05) at 12h, 24h, 48h and 72h.4. The cell positive rates of expression of NF-κB P65 protein decrease gradually in EC9706 cell after PTX treatment 24h. The cell positive rates are displayed 90.33±1.53%,87.67±1.15%,83.00±2.65%,73.33±3.79%,61.00±2.00% respectively from the control group to all PTX groups. There is a significant difference in them (F=74.437,P<0.01) . There is no difference between the PTX 0.01mg/ml group and control group (P>0.05 ) . The positive rates are significant different among the rest groups (P<0.05) .5. The cell positive rates of expression of COX-2 protein decrease gradually in EC9706 cell after PTX treatment 24h. The cell positive rates are displayed 90.33±1.53%,86.00±2.65%,78.33±3.06%,70.33±5.03%,56.33±6.66% respectively from the control group to all PTX groups. There is a significant difference in them(F=30.966, P<0.01). There is no difference between the PTX 0.01mg/ml group and the control group (P>0.05). The positive rates are evidently different among the rest groups (P<0.05) .6. The cell positive rates of expression of Ki-67 protein decrease gradually in EC9706 cell after PTX treatment 24h. The cell positive rates are displayed 63.33±10.69%,43.00±4.58%,32.33±5.86%,23.33±3.05%,14.67±9.02% respectively from the control group to all PTX groups. There is a significant difference in them (F=20.496, P<0.01) . there is a significant difference between each PTX group and the control group (P<0.05) . There is no difference between the PTX 0.01mg/ml group and the PTX 0.1mg/ml group, the PTX 0.1mg/ml group and the PTX 0.5mg/ml group, the PTX 0.5mg/ml group and the PTX 1.0mg/ml group (P>0.05) .7. The cells morphologic changes have been observed by light microscope after 5-Fu and PTX uniting 5-Fu treatment for 12～72h: The morphologic changes can be observed in 5-Fu groups at 24h, such as cell shrinking, flotation, periplast turbid and so on. The cell population is gradually decreasing and the cellular fragment is gradually increasing along with the ascending of 5-Fu concentration. The morphologic changes can be observed in PTX uniting 5-Fu groups at 12h, and is similar to 5-Fu group at 24h. But the cell population is more decreasing and the cellular fragment is more increasing along with the ascending of PTX uniting 5-Fu concentration.8. The inhibition rates of EC9706 cells treated by 5-Fu and 5-Fu uniting PTX are detected with MTT assay. The EC9706 cells are treated by different concentration 5-Fu and 5-Fu uniting PTX for 12h, 24h, 48h and 72h. There is a significant different in all inhibition rates mean at the same time ( P<0.01) . There is a significant different between the union group I -IV and the 5-Fu 25μg/ml group, between the union group VI-VIII and the 5-Fu 50μg/ml group at 12h; between the union groupI -IV and the 5-Fu 25μg/ml group, between the union group V -VIII and the 5-Fu 50μg/ml group at 24h; between the union group II -IV and the 5-Fu 25μg/ml group, between the union groupVIII and the 5-Fu 50μg/ml group at 48h; between the union group III-IV and the 5-Fu 25μg/ml group, between the 5-Fu uniting PTX group V-VIII and the 5-Fu 50μg/ml group at 72h (P<0.05) .There is no significant different between 5-Fu uniting PTX group III-IV and 5-Fu 50μg/ml group at 24h, 48h and 72h (P>0.05) .Conclusions1. PTX can inhibit proliferation of EC9706 cells. The effect on inhibition is gradually increasing along with the ascending of PTX concentration and the lasting of time.2. PTX can down-regulates NF-κB P65,COX-2,Ki67 protein expression in EC9706 cells, which indicated that PTX can inhibite the activity of NF-κB P65 and expression of COX-2 in EC9706, and can inhibit the proliferation of tumor cell.3. The 5-Fu's inhibition to EC9706 cells is gradually increasing along with the ascending of PTX concentration under the same 5-Fu concentration; the PTX 0.5mg/ml and 1.0mg/ml uniting 5-Fu25μg/ml groups' inhibition to EC9706 is similar to 5-Fu50μg/ml, which indicated PTX can make the EC9706 cells more sensitive to 5-Fu, and decrease dose of 5-Fu. |
PDF Full Text Request