Study On The Role Of NF-κB In The Process Of Hepatocarcinoma Apoptosis

Hepatocellular carcinoma (HCC) is one of the commonest human malignant cancers, and usually detected later and developing faster. Chemotherapy is one of the important and common treatments of HCC, which often is one of the vital means of adjunctive therapy and recurrence therapy of postoperation and inoperableness therapy. At present there has been an agreement that the induction of apoptosis by chemotherapeutic drugs is the main mechanism through which tumor cells are killed. Apoptosis is an active cell death under the control of the apoptosis-related genes. However, the inhibition of apoptosis may cause the resistance of tumor cells to chemotherapy, so it reduces the effect of chemotherapeutic drugs. It is obvious that chemotherapeutic drugs activate a series of biological signals, which keep the balance with the apoptosis induced by chemotherapeutic drugs. The interaction of promotive and inhibitive factors of apoptosis determines the cell life and death.NF-κB is a kind of transcription factor that exists extensively, acts diversely and is related to apoptosis; inflammation; immunity react; injury, etc. Recent study indicated that chemotherapeutic drugs, especially 5-Fu, can activate NF-κB when they come into effect. Besides, it is reported that NF-κB has significant relation with the apoptosis of embryonic development. Therefore, people gradually realize that the tumorigeness and innate or acquired drug resistance have relation with the apoptosis regulated by NF-κB. The role and practical application of NF-κB have become the research hot topic, which are greatly potential and significant. The experiment intents to investigate the role that NF-κB and its specific inhibitor plays in the liver cancer cells apoptosis, to explore the reason of liver cancer cells persisting in apoptosis induced by chemotherapy drug and look for the corresponding methods so as to offer the experimental basis to improve treatment efficiency of chemotherapeutic drug.OBJECTIVES:To explore NF-κB and TSLC1 expression in liver carcinoma tissues and its correlation with the pathological stage of cancer tissues. To explore that NF-κB transfers from cytoplasm to nucleus of HCC (SMMC-7721) after activated by 5-Fu. After NF-κB was inhibited by the specific inhibitor of NF-κB, PDTC, the apoptosis rate of SMMC-7721 cells changed during the period.METHODS:By means of immunohistochemical (IHC) EnVision method, the expressions of NF-κB and TSLC1 were compared in HCC tissues from 60 cases and normal liver tissues from 15 cases. The CGIR (cell growth inhibition rate) of SMMC-7721 cells was detected by MTT assay. The transferring of NF-κB from cytoplasm to nucleus was observed by Means of immunofluorescence (IF) assay. After the nucleus protein of SMMC-7721 cells had been extracted, Western blot was performed to detect the change of NF-κB in the nucleus treated 5-Fuor 5-Fuplus PDTC. The apoptosis rates (AR) of SMMC-7721 cells on different conditions were detected by FCM method. RESULTS:The positive expression of NF-κB p65 in the liver cancer group and the control group were 53.3%, 13.3% (significantly different, P<0.05). The positive expression of TSLC1 were 36.7%, 80.0% (significantly different, P<0.05) in the liver cancer group and the control group respectively. The positive expression of TSLC1 and NF-κB was significantly different for different pathological grades (P<0.05).The expression of TSLC1 was closely related to NF-κB (r=-0.397, P<0.05). MTT assay: Administration of 5-Fu combined with PDTC signifycantly improved cytotoxity of 5-Fu to liver cancer cells. The CGIR (cell growth inhibition rate) was only 12.21% when cells were treated with 100μg/ml 5-Fu alone for 24h. The inhibition rate increased to 31.43% when cells were pretreated with 50μM PDTC before exposed to 100μg/ml 5-Fu. The differences between both methods were significant at different concentrations of 5-Fu, (P<0.05). The transferring from cytoplasm to nucleus of NF-κB induced by 5-Fu increased gradually with time. IF assay was performed to detect the change of NF-κB in the nucleus treated 5-Fu plus PDTC in 3h,6h,12h. After the nucleus protein of SMMC-7721 cells had been extracted, Western blot assay was performed to detect the change of NF-κB in the nucleus treated by 5-Fu, PDTC or 5-Fu plus PDTC in 3h, 6h, 12h. Protein luminance were 0.28, 0.59, 0.72; 0.09, 0.12, 0.14 and 0.15, 0.26, 0.32 individually. FCM assay: Apoptotic rate of PDTC group, 5-Fu group and 5-Fu plus PDTC group were 7.32%, 9.42% and 25.63% individually.CONCLUSIONS:The abnormal expression of NF-κB is closely associated with the occurrence and development of HCC. The expression of NF-κB is significantly different for different pathological grades. Its high expression indicates a poor promising. NF-κB is closely related to TSLC1, which indicates that NF-κB is involved in the anti-apoptosis course of liver cancer cell by anti-oncogene TSLC1. NF-κB in SMMC-7721 cells can be activated by the drug such as 5-Fu and transfers from cytoplasm into nucleus, Which can be specially inhibited by PDTC. After specially inhibited, the apoptotic rate increases significantly. It is proved that the activation of NF-κB can efficiently inhibit SMMC-7721 cells from apoptosis. The result notifies that the chemotherapy drugs plus NF-κB specific inhibitor can improve treatment efficiency and provide a new tumor treatment pathway.