| The morbidity of ovarian malignant tumor is rank of the third in female genital malignant tumor, but the mortality the first. The five-year-survival is nearly 20% to 30%.The cytoreductive surgery and multipl courses of chemotherapy is the basic treatment principle of ovarian cancer at present. Chemotherapy plays a very important role,but the tumor cells can show drug resistance which influences the curative effect and lead to the failure of chemotherapy and tumor recurrence. How to overcome the muftidrug resistance,that is to reverse the drug resistance and to find the reversing drugs of low toxity ,has become an important problem for the chemotherapy curative effect. It is also concerned by scientists home and abroad. 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 and the inhibition of apoptosis may cause the resistance of tumor cells to chemotherapy. Apoptosis is an active cell death under the control of the apoptosis-related genes. The products of genes, which suppress the undergoing apoptosis, play an important role in the resistance of tumor cells to chemotherapy. NF-κB is a transcriptional factor. It has been demonstrated that some apoptosis-inducing factors elicit tumor cells committing suicide, and at the same time they also stimulate NF-κB, which will start the transcription of some certain genes .The products of these genes may hamper the signaling pathway, which will lead to the apoptosis of cells. In recent studies, it has been indicated that some anti-tumor drugs can activate NF-κB, which may decrease their activity in inducing apoptosis as they induce the apoptosis of tumor cells. So, it is a meaning reseach on exploring the role of NF-κB in the generation of drug-resistance of ovarian cancer and the effect on reversal of the resistance.Objective:To observe the expression of NF-κB p65 protein in human ovarian cancer cell line COC1 and COC1/DDP which is resistant to cisplatin, the effects of suppression of NF-κB on the apoptosis of COC1/DDP cells and the Xenografts in Nude Mice, and to investigate the function and mechanism of NF-κB in the drug resistance of ovarian cancer,to investigate the role of the suppression of it in the therapy of ovarian cancer resistant to chemical drugs.Methods:1. Rearch of the role of NF-κB in cisplatin-resistant human ovarian cancer cell line COC1/DDPMTT was used to examin the resistance index of COC1/DDP cells to cisplatin. Immunohistochemical staining (SABC) was used to detect the NF-κB p65 translocated into cell nucleus in COC1 and COC1/DDP cells. Westernblot was performed to examine the difference of NF-κB p65 activity between the nucleus of COC1 and COC1/DDP cells.2. The effect of inhibition of NF-κB on the cisplatin-resistance of human ovarian cancer cell line COC1/DDP.Westernblot was used to testify whether PDTC could suppress the expression of NF-κB in the nucleus of COC1/DDP.TO treat COC1/DDP cells with MTT to examine the growth suppression of PDTC according to various concentration and time on COC1/DDP cells.The concentration of cisplation was assigned as people peak serum concentration,and the time is the same with PDTC's .FCM was used to examine the apoptosis of COC1/DDP treated by DDP group,PDTC group and PDTC+DDP group,and contrast them and that of COC1+DDP group.3. The mechanism of effect of inhibition of NF-κB on cisplatin-resistance of COC1/DDP cell.RT-PCR and Westernblot were used to examine the mRNA and protein expression level of survivin and Bcl-2 in COC1/DDP cells before and after PDTC.4. The effect of inhibition of NF-κB on the sensitivity of human ovarian cancer cell line COC1/DDP xenografts in nude mice to cisplatin. The transplanted model of human ovarian cancer cell line COC1/DDP in nude mice was built by inoculating the COC1/DDP cells into the mice subcutaneously. Cisplatin , PDTC and DDP+PDTC were used to treat the xenografts of nude mice. To observe the effects of drugs on the xenografts of nude mice,the volume and the weight of xenografts of groups are taken as the observation index. To observe whether PDTC or cisplatin is poisonous to the organs of nude mice,the liver and kidney tissues of mice in the four groups were made into slices to be checked by pathologists.Results: 1,COC1/DDP is still resistant to cisplatin for its RI is 5.44. The expression of NF-κB p65 was detected 67.4% in the nucleus of COC1/DDP,but 18.2% of COC1 by Immunohistochemical staining.There is significant difference between the two cell lines(P<0.05).By westernblot ,the OD(optical density) value of NF-κB p65 protein strap in the nucleus of COC1/DDP is (1436.75±227.59),which is higher than (674.38±104.21),that of COC1.There is significant difference between the two cell lines(P<0.05).2,The OD value of NF-κB p65 protein strap declined from (1436.75±227.59) to (536.41±97.83) after PDTC was used on COC1/DDP by westernblot,and the difference is significnant (P<0.05).The minimal dose of PDTC on COC1/DDP cells is 25μM and the time is 48 hours by MTT. So 2.5μg/ml and 48 hours was used as the working conditions of cisplatin in the next experiments.FCM was used to find the apoptosis rate of control group,DDP group,PDTC group and DDP+PDTC group is (0.05±2.31)%,(6.07±3.25)%,(5.09±2.11)% and (13.76±4.71)% respectively.The apoptosis rate of DDP+PDTC group is higher than other three groups(P<0.05).But the apoptosis rate of all groups of COC1/DDP is less than the rate (38.49±3.87)% of COC1+DDP significantly(P<0.05).3,By RT-PCR,the ratio of OD value of Survivin andβ-actin in COC1/DDP cells before and after PDTC are (3.26±1.43)and (1.20±0.42) respectively,and the difference is significant(P<0.05).That of Bcl-2 andβ-actin are (4.18±1.51) and (2.62±1.10) respectively, and the difference is significan(tP<0.05)too.By westernblot,the OD value of Survivin protein strap in the COC1/DDP cells before and after PDTC are (353.82±80.74) and (125.20±53.18) respectively,and the difference is significant (P<0.05).That of Bcl-2 protein strap are (564.23±77.25) and (218.19±64.11) respectively,and the difference is significant (P<0.05) too.4. The volume of xenografts in nude mice of the control group, DDP group, PDTC group and DDP+PDTC group are (638.27±88.14)mm3,(571.23±79.25)mm3,(524.25±117.94)mm3 and (247.64±93.08)mm3 respectively. The weight of xenografts in nude mice of the control group, DDP group, PDTC group and DDP+PDTC group are (2.78±0.67)g,(2.35±0.30)g,(2.41±0.46)g and (1.64±0.57)g respectively. The weight of xenograft of DDP+PDTC group is lower significantly than the other groups(P<0.05), but the control group, DDP group and PDTC group is no significant difference among them (P>0.05). The inhibition rate of xenografts of DDP+PDTC group is 41.01%, and it is higher significantly than 15.45% of DDP group and 13.31% of PDTC group(P<0.05).No abnormal pathological changes were seen in the cells in tissue splice made from the liver and kidney tissues of nude mice of four groups.Conclusions:1,Acquired drug-resistance of ovarian cancer is closely related with the increase of expression of NF-κB.2,Inhibition of NF-κB may increase the sensitivity of cisplatin-resistant COC1/DDP to cisplatin.3,The mechanism that inhibition of NF-κB may increase the sensitivity of cisplatin-resistant COC1/DDP to cisplatin is related with the inhibited expression of the antapoptosis genes,Survivin and Bcl-2.4,PDTC is the chemosensitizer of cisplatin on the human ovarian cell line COC1/DDP,and no poisonous effects of PDTC(10 mg/kg) are found in the nude mice experiment.5,NF-κB is hopeful to become a new target for the research of drug-resistant ovarian cancer.Developing specific and safe NF-κB inhibitor will be useful to treat drug-resistant ovarian cancer.
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