| Ovarian cancer is one of the most frequent malignancies in women, and the leading cause of death from gynecological cancer. Optimal cytoreductive surgery and neoadjuvant chemotherapy based on platinum are considered the golden standard for the management of ovarian cancer. Despite greater than 70% of patients achieved good initial response to therapy with platinum agents, the long-term survival remains poor, as the five-year survival rate for ovarian cancer is less 30%. The major obstacles for ovarian cancer therapy are the acquired and intrinsic resistance to platinum-based chemotherapy in patients. The drug-resistant patients more tend to high relapses, high metastases and high mortality eventually. The molecules mechanism of the drug-resistance in tumor cells remains to be clarified. Investigate the effective ways to overcome platinum resistance in tumors will be beneficial to the large numbers of ovarian cancer patients.It is generally agreed that DNA is the cytotoxic target for platinum agents. Platinum agents generally work by forming intra- or inter-strand cross-links in DNA that begins the process of cell cycle arrest and results in tumor cell apoptosis. However, when the DNA is damaged, DNA repair mechanisms are triggered resulting in improved cell survival. It is found that the significantly higher DNA repair capability in chemo-resistant tumor cells which may lead to increased cell viability, and hence resistance to platinum agents. DNA repair pathway has been proposed to be the important mechanism in development of platinum resistance. New chemo-resistant reversal strategies targeting at DNA repair genes is considered to be a promising approach to antitumor therapy.APE1/Ref-1 is a multifunctional protein that is not only an essential enzyme in base excision repair pathway, but also acts as a major redox-signaling factor that has a wide variety of important cellular functions including transcription factor regulation, oxidative signaling,apoptosis and cell cycle control. It has been identified that APE1/Ref-1 plays an important role in DNA repair pathway and regulation of apoptosis signal. In addition, clinical data indicated that APE1/Ref-1 play a pivotal role in carcinogenesis and progression in several tumors. APE1/Ref-1 appears to form a unique link among the DNA BER pathway, cancer, transcription factor regulation, oxidative signaling, and cell-cycle control. It is reasonable to postulate that APE1/Ref-1 may contribute to the molecular mechanism of resistance to platinum chemotherapy.In my previous work, altered levels and subcellular APE1/Ref-1 expression was found in platinum resistant ovarian cancer tissues and ovarian cancer cell lines. It suggested that APE1/Ref-1 may perform a significant role in platinum resistance in ovarian cancer. In our another study, we found that The cisplatin-resistant CP70 cells treated with APE1-siRNA had significant enhancement in cisplatin sensitivity and the effect of APE1 siRNA on cisplatin sensitivity may be partly due to the modulation of the cell cycle, as a result of cellular response to DNA damage.To study the drug resistance mechanism in which APE1/Ref-1 might be involved, we used a case-matched method and paired 30 pairs of platinum sensitive and platinum resistant patients as the object of study. The immunohistochemical method was used to evaluate the level of APE1/Ref-1, MDR1 and p53 proteins expression in different platinum sensitive ovarian cancer patients. According to the APE1/Ref-1features of functional domains, we used DNA recombinant technique to construct four types of different APE1/Ref-1 gene coding region recombinant plasmids. The biological effect and cisplatin sensitivity were observed after platinum sensitive A2780 cell lines transfected with different APE1/Ref-1 recombinant plasmids. Preliminary investigation was carried to find out the possible mechanism which two functions of APE1/Ref-1were involved in platinum resistance. Our study provided the direct evidence of the correlation between APE1/Ref-1 and platinum resistance in ovarian cancer.OBJECTIVES1. To investigate the correlation of APE1/Ref-1 expression with the platinum resistance in ovarian cancer;2. To construct four types of different APE1/Ref-1 gene coding region recombinant plasmids in order to provide effective means for explore the relationship between APE1/Ref-1 functions and platinum resistance.3. To observe the effect of different APE1/Ref-1 recombinant plasmids on biological behaviors and cisplatin sensitivity in ovarian cancer cells. These findings would provide the groundwork for new understanding about the role of APE1/Ref-1 in the molecular mechanism of resistance to platinum chemotherapy.METHODS1. Using a case-matched design paired for gender, age, and FIGO stage, cell differentiation, 30 pairs of patients were included and assigned into the platinum sensitive group and platinum resistant group. The immunohistochemical method was used to measure the expression of APE1/Ref-1, MDR1 and p53 proteins in ovarian cancer tissues.2. DNA recombinant technique was used to construct four types of different APE1/Ref-1 gene coding region recombinant plasmids: APE1-pEGFP-N1、Redox-pEGFP-N1、Repair-pEGFP-N1 and NLS- Repair-pEGFP-N1 for explore the possible roles and mechanisms of DNA repair and redox function in APE1/Ref-1 gene.3. Cellular transfection were performed to A2780 cells under mediation with liposome. The transfection efficiency was confirmed by fluoroscope.4. RT-PCR and Western blot were used to evaluate the APE1/Ref-1 mRNA and protein level respectively in A2780 cells transfected with APE1-pEGFP-N1 plasmid.5. By G418 screening, stable transfected of A2780 cells with APE1-pEGFP-N1 and empty vector were obtained.6. MTT assays were performed to evaluate the effects of four types of different APE1/Ref-1 recombinant plasmids on cell proliferation.7. Clone-forming assays were carried out to evaluate the effect of different APE1/Ref-1 recombinant plasmids on cell proliferation.8. The effect of different APE1/Ref-1 recombinant plasmids on cell cycle was observed by flow cytometry analysis in transfected A2780 cells.9. MTT assays were performed to determine the cisplatin sensitivity of the A2780 cells transfected with different APE1/Ref-1 recombinant plasmids, and the IC50 values of cisplatin were calculated.10. Indirect immunofluorescence was used to observe subcellular localization of A2780 cells transfected with different APE1/Ref-1 recombinant plasmids;11. Apoptosis of the cisplatin-treated in transfected A2780 cells was examined by AnnexinⅤ/PI analysis using flow cytometry, to study the underlying mechanisms for sensitization of CP70 cells To investigate the apoptotic effect on cisplatin sensitivity in A2780 cells transfected with different APE1/Ref-1 recombinant plasmids.RESULTS1. Ovarian cancer cases of platinum resistant group showed high level of APE1/Ref-1 expression ( n=20/30; 62.5%), while in patients sensitive to platinum chemotherapy APE1/Ref-1 high level expression was found in 12 out of 30 (37.5%) patients (p<0.05). On the other hand, APE1/Ref-1 cytoplasm localization was found in a statistically significant higher percentage in platinum resistant group than in patients sensitive to platinum chemotherapy (n=17/30; 56.7% vs n=9/30; 30.0%,p<0.05).2. The location status of APE1 /p53 was significantly correlated with platinum-based chemotherapy sensitivity. When APE1/Ref-1 were cytoplasm localization, p53 positive rate was obviously higher in cases of platinum resistant group compared with platinum sensitive group (p<0.05).3. The growth curve made by MTT indicated that the proliferation of A2780 cells transfected with APE1-pEGFP-N1 plasmid were speed up. Meanwhile, the clone-forming assays presented that the proliferative activity were increased in A2780 cells transfected with APE1-pEGFP-N1 plasmid.4. Cell cycle analysis by flow cytometry showed that APE1-pEGFP-N1 plasmid resulted in accumulation in G2 and S phase of A2780 cells. While the proportion of G0/G1 phase were decreased in APE1-pEGFP-N1 plasmid transfected A2780 cells(p<0.05).5. Assessing the cell DNA proliferation Index (PI) of cells effected by APE1-pEGFP-N1 plasmid by flow cytometry, the data showed that APE1-pEGFP-N1 accelerated the proliferation of A2780 cells compared with empty vector transfected cells(59.5% vs. 52.4%).6. The cell DNA proliferation Index (PI) of APE1-pEGFP-N1 transfected cells and Redox-pEGFP-N1 transfected cells were 60.57% and 63.03% respectively, showed higher proliferation rate compared with empty vector group. By contrast, the proliferation Index of Repair-pEGFP-N1 and NLS-Repair-pEGFP-N1 transfected cells were lower than empty vector transfected cells(41.7% vs. 56.65%, 36.65% vs. 56.65%).7. Cell cycle analysis by flow cytometry showed that the proportions of G0/G1 phase were decreased and the proportion of S phase were increased in APE1-pEGFP-N1 and Redox-pEGFP-N1 transfected cells. On the other hand, cells transfected with Repair-pEGFP-N1 and NLS-Repair-pEGFP-N1 were both accumulated with G0/G1 phase, in the meantime the proportion of S phase were decreased.8. Exposured to the same concentrations of cisplatin, the inhibiting rate of A2780 cells transfected with APE1-pEGFP-N1, Redox-pEGFP-N1, Repair-pEGFP-N1 and NLS-Repair-pEGFP-N1 plasmids were dropped of different degrees. Under the APE1-pEGFP-N1, Redox-pEGFP-N1, Repair-pEGFP-N1 and NLS-Repair-pEGFP-N1 plasmids transfection, the 50% inhibitory concentration (IC50) to cisplatin were increased by 2.13-fold, 1.46-fold ,1.27-fold and 1.83-fold respectively in A2780 cells. 9. It was found that the viability of cells was slightly increased and the apoptosis rate was weakly decreased in APE1-pEGFP-N1 transfected A2780 cells (98.0% vs. 97.2%, 1.1% vs. 1.6%). However, A2780 cells transfected with Redox-pEGFP-N1 did not change much in survival cells and apoptosis cells. By contrast, the viability of Repair-pEGFP-N1and NLS-Repair-pEGFP-N1 transfected A2780 cells ranging from 93.3% to 92.8% compared with 97.2% for empty vector transfected cells. Meanwhile the apoptosis rate in A2780 cells transfected with Repair-pEGFP-N1and NLS-Repair-pEGFP-N1were 4.4% and 4.6% separately, were obviously improved compared with 1.6% for empty vector transfected cells.10. The apoptosis rate in A2780 cells treated with APE1-pEGFP-N1 and DDP was improved compared to DDP treated cells (8.4% vs. 6.6%), but the necrosis rate was reduced (28.4% vs. 41.3%). In the A2780 cells transfected with Redox-pEGFP-N1, the apoptosis rate and necrosis rate were fall down similarly compared with DDP treated cells (5.5% vs. 6.6%,24.1% vs. 41.3%). In the four types of APE1 recombination plasmids transfected cells, the more noticeable of reduction of necrosis rate were Repair-pEGFP-N1 and NLS-Repair-pEGFP-N1 transfected cells. Meanwhile, the apoptosis rates were lightly increased in Repair-pEGFP-N1 and NLS-Repair- pEGFP-N1 transfected cells.CONCLUSION1. The expression level and the localization of APE1/Ref-1 were correlated with platinum resistance in ovarian cancer. The APE1/Ref-1 expression status and p53 mutation may be attributed to the resistance mechanisms of ovarian cancer cells.2. The elevated APE1/Ref-1 expression level enhanced cell proliferation by stimulating the DNA synthesis in S phase, and resulted in improvement the tolerance of cisplatin in A2780 cells simultaneously.3. Both DNA repair function and redox function of APE1/Ref-1 were involved in the regulation of cell cycle and DNA synthesis in A2780 cells. Our data suggested that the redox function of APE1/Ref-1 play a significant role in the acceleration of protein synthesis caused by improved APE1/Ref-1 expression.4. Under the cisplatin stress, both DNA repair function and redox function of APE1/Ref-1 were taken part in the fight against DDP agent and contributed to more tumor cell survival. The effect of redox function on cisplatin sensitivity improvement was most significantly, which might ascribe to the modulation of apoptosis factors by redox function. Parallelly, the DNA repair function also play a role in forming platinum resistance by AP endonuclease. |
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