双功能酶APE1/Ref-1参与卵巢癌铂类耐药的机制研究
摘要
卵巢癌是严重威胁妇女健康的常见恶性肿瘤,死亡率在女性生殖器恶性肿瘤中占第1位。目前理想的肿瘤细胞减灭术和术后以铂类为主的联合化疗是卵巢癌的标准治疗方案。虽然70%以上的卵巢癌患者对以铂类为基础的化疗能获得临床缓解,但卵巢癌患者预后不佳,5年生存率不到30%。肿瘤细胞原发性或继发性的化疗耐药使肿瘤复发转移的主要原因,严重影响卵巢癌治疗效果及生存率,已经成为目前卵巢癌治疗难以突破的瓶颈[1]。肿瘤细胞的化疗耐药机制目前尚未阐明,寻求有效的途径克服卵巢癌化疗耐药是提高卵巢癌化疗疗效的关键环节。
铂类药物的主要靶点为DNA,通过和细胞内DNA形成链内和链间加合物,从而阻断DNA复制转录,造成细胞死亡。因此肿瘤细胞的DNA修复机制可能在肿瘤细胞的化疗耐药中发挥重要作用。有证据表明,耐药的肿瘤细胞中普遍出现了DNA损伤修复能力提高[2-4],肿瘤细胞对铂类所致DNA损伤的修复能力(DNA repair capability, DRC)增强很可能是癌细胞对铂类药物产生耐药的最重要机制[5]。因此以DNA损伤修复基因为靶点的肿瘤靶向治疗可能会逆转肿瘤细胞的化疗耐药。
现有的研究表明,双功能酶APE1/Ref-1不仅具有核酸内切酶活性,在碱基切除修复途径(Base excision repair, BER)中发挥关键限速酶作用,而且还能通过其氧化还原功能调控多种重要转录因子的活性,在凋亡信号通路中发挥重要作用。APE1/Ref-1是联系DNA损伤修复、氧化应激、凋亡通路及细胞周期调控等多种重要生物学反应的桥梁分子。此外,还有多项研究表明,APE1/Ref-1的表达和定位与多种肿瘤的发生发展及其放化疗敏感性相关。因此我们有充分的理由相信,APE1/Ref-1是非常有潜力的肿瘤治疗靶点[6-7]。
在我们的前期工作中,我们发现APE1/Ref-1在铂类耐药的卵巢癌组织和耐药的卵巢癌细胞中均发生表达水平增高和细胞定位改变,提示APE1/Ref-1在胞核和胞浆中发挥作用不同,可能是铂类耐药的重要环节。在我们的另一项研究中,曾以卵巢癌顺铂耐药细胞株CP70为研究对象,用APE1 siRNA特异性下调APE1/Ref-1表达后,CP70细胞对顺铂的敏感性显著增强,并与细胞周期阻滞有关[138-139]。
为了进一步验证APE1/Ref-1在卵巢癌铂类耐药形成中发挥的作用,本课题首先以卵巢癌铂类敏感和铂类耐药的配对临床资料为研究对象,用免疫组化分别对APE1/Ref-1、MDR1和p53基因表达水平进行检测,进一步验证APE1/Ref-1与卵巢癌铂类耐药及耐药相关基因之间的关系。随后我们将根据APE1/Ref-1两大功能相互独立发挥作用的结构特点,分别构建不同功能结构域的APE1/Ref-1重组质粒。然后以卵巢癌顺铂敏感细胞株A2780细胞为研究对象,观察APE1不同重组质粒对卵巢癌细胞的生物学效应及铂类敏感性的影响,初步探讨APE1/Ref-1的不同功能状态参与卵巢癌铂类耐药的机制。本研究将为APE1参与卵巢癌铂类耐药提供更有力的直接证据,为进一步阐明APE1/Ref-1参与卵巢癌铂类耐药的机制奠定基础,具有重要的理论意义和临床应用前景。
研究目的
1.从临床资料病理组织水平验证APE1/Ref-1表达与卵巢癌铂类敏感性的相关性;
2.构建不同功能结构域的APE1/Ref-1重组质粒,为研究APE1/Ref-1与铂类耐药的相关性提供有效手段;
3.探讨APE1/Ref-1不同功能状态对卵巢癌生物学行为及顺铂敏感性的影响,为进一步阐明APE1/Ref-1参与卵巢癌铂类耐药的机制奠定基础。
研究方法
1.以卵巢癌铂类敏感和铂类耐药的配对临床资料为研究对象,用免疫组化分别对临床病理组织标本中APE1/Ref-1、MDR1和p53基因表达水平进行检测,进一步验证APE1/Ref-1与卵巢癌铂类耐药及耐药相关基因之间的关系。
2.利用重组质粒技术,针对APE1/Ref-1不同功能结构域构建APE1-pEGFP-N1、Redox-pEGFP-N1、Repair-pEGFP-N1和NLS- Repair-pEGFP-N1四种APE1不同结构域的重组质粒。
3.利用脂质体法将APE1/Ref-1不同重组质粒分别转染卵巢癌铂类敏感细胞株A2780细胞,荧光显微镜下观察EGFP表达检测转染效率;
4. RT-PCR和western blot分别检测APE1-pEGFP-N1质粒转染后A2780细胞中APE1/Ref-1蛋白水平和mRNA水平;
5. G418抗性筛选,获得稳定转染APE1-pEGFP-N1和空质粒pEGFP-N1的A2780细胞株;
6. MTT法分别绘制卵巢癌A2780细胞分别转染APE1不同重组质粒后生长曲线的变化;
7.平板克隆形成试验检测稳定转染APE1-pEGFP-N1的A2780细胞株的增殖能力变化;
8.流式细胞术检测卵巢癌A2780细胞分别转染APE1不同重组质粒后细胞周期的变化,并计算增殖指数;
9. MTT法检测卵巢癌A2780细胞分别转染APE1不同重组质粒后顺铂敏感性的变化;
10.免疫荧光检测APE1不同重组质粒转染后,A2780细胞中APE1/Ref-1表达及定位的变化。
11.流式细胞术检测APE1不同重组质粒转染对顺铂诱导的A2780细胞凋亡的影响。
研究结果
1.卵巢癌铂类耐药组中APE1高表达和浆表达的比例均明显高于敏感组,两者之间有统计学差异(p<0.05);
2.当APE1出现浆表达时,在耐药组中p53蛋白的阳性率较敏感组明显增高,两者差异有统计学意义(p<0.05);
3. APE1-pEGFP-N1重组质粒转染后,MTT法绘制生长曲线提示A2780细胞生长速度有所提高,克隆形成试验提示A2780单个细胞增殖能力有所提高;
4.细胞周期结果表明,与对照组相比,APE1-pEGFP-N1转染重组质粒转染后,A2780细胞G0/G1期比例降低,G2+S期比例增加,有统计学差异(p<0.05);
5. APE1-pEGFP-N1重组质粒转染组和空质粒转染组相比,增殖指数(PI)有所升高(52.4% vs. 59.5%),提示APE1-pEGFP-N1转染能促进A2780细胞的生长增殖;
6. APE1-pEGFP-N1转染组和Redox-pEGFP-N1转染组增殖指数(PI)分别为60.57%和63.03% ,出现了增殖指数增加的现象;而Repair-pEGFP-N1转染组和NLS-Repair-pEGFP-N1转染组增殖指数分别为41.7%,36.65%,较阴性对照组降低。
7.与阴性对照组和空白对照组相比,APE1-pEGFP-N1和Redox-pEGFP-N1转染组出现G0/G1期比例降低, S期比例增加的现象,而Repair-pEGFP-N1和NLS-Repair-pEGFP-N1转染组出现了G0/G1期比例增高,S期比例降低的现象;
8.在相同的药物浓度下, APE1-pEGFP-N1、Redox-pEGFP-N1、Repair-pEGFP-N1和NLS-Repair-pEGFP-N1四组转染后,顺铂对A2780细胞的抑制率均有所降低,根据IC50值由高到低依次为Redox-pEGFP-N1、NLS-Repair-pEGFP-N1、APE1-pEGFP-N1和Repair-pEGFP-N1(IC50值分别为28.01μM、24.00μM、19.21μM和16.71μM),A2780细胞对顺铂的耐受性分别提高了2.13倍、1.46倍、1.27倍和1.83倍(p<0.05)。
9. APE1-pEGFP-N1转染组和阴性对照组相比,活细胞率略有升高(98.0% vs. 97.2%),凋亡率略有下降(1.1% vs. 1.6%)。Redox-pEGFP-N1转染组凋亡率和坏死率分别为1.7%和0.8%,和阴性对照组相比无明显差异。Repair-pEGFP-N1和NLS-Repair-pEGFP-N1两组中活细胞率分别为93.3%和92.8%,和对照组相比略有降低,而凋亡率略有所增加,分别为4.4%和4.6%。
10. APE1-pEGFP-N1转染组和顺铂处理组相比,凋亡率略有升高(8.4% vs. 6.6%),坏死率显著降低(28.4% vs. 41.3%)。Redox-pEGFP-N1转染组凋亡率和坏死率和顺铂处理组相比均有所降低,其中坏死率降低更为显著(5.5% vs. 6.6%,24.1% vs. 41.3%)。转染不同APE1重组质粒的四组细胞与顺铂处理组相比,Repair-pEGFP-N1和NLS-Repair-pEGFP-N1两组坏死率降低最为明显,分别为22.3%和23.7%,但凋亡率有所增加,分别为20.5%和13.5%。
结论
1. APE1/Ref-1的表达水平和细胞定位均与卵巢癌铂类耐药相关,其参与卵巢癌铂类耐药的机制可能与P53突变相关。
2.提高APE1/Ref-1表达水平能通过促进A2780细胞DNA合成而对A2780细胞产生刺激增殖作用,并增加A2780细胞对顺铂的耐受性。
3. APE1/Ref-1的不同功能状态对A2780细胞周期及DNA合成有调控作用。APE1/Ref-1表达增高能促进肿瘤细胞中蛋白质合成,而APE1/Ref-1的氧化还原功能在这一过程中扮演重要角色。
4. APE1/Ref-1的氧化还原功能和DNA修复功能都在卵巢癌A2780细胞对顺铂的化疗应激中发挥了一定作用。其中APE1/Ref-1的氧化还原功能对A2780细胞顺铂敏感性影响最为显著,可能与APE1通过氧化还原功能调控下游凋亡相关因子从而对抗顺铂诱导的细胞凋亡有关。而APE1/Ref-1的DNA修复功能也通过DNA修复酶活性在A2780细胞对顺铂的抵抗中发挥一定作用。
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.
OBJECTIVES
1. 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.
METHODS
1. 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.
RESULTS
1. 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.
CONCLUSION
1. 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.
铂类药物的主要靶点为DNA,通过和细胞内DNA形成链内和链间加合物,从而阻断DNA复制转录,造成细胞死亡。因此肿瘤细胞的DNA修复机制可能在肿瘤细胞的化疗耐药中发挥重要作用。有证据表明,耐药的肿瘤细胞中普遍出现了DNA损伤修复能力提高[2-4],肿瘤细胞对铂类所致DNA损伤的修复能力(DNA repair capability, DRC)增强很可能是癌细胞对铂类药物产生耐药的最重要机制[5]。因此以DNA损伤修复基因为靶点的肿瘤靶向治疗可能会逆转肿瘤细胞的化疗耐药。
现有的研究表明,双功能酶APE1/Ref-1不仅具有核酸内切酶活性,在碱基切除修复途径(Base excision repair, BER)中发挥关键限速酶作用,而且还能通过其氧化还原功能调控多种重要转录因子的活性,在凋亡信号通路中发挥重要作用。APE1/Ref-1是联系DNA损伤修复、氧化应激、凋亡通路及细胞周期调控等多种重要生物学反应的桥梁分子。此外,还有多项研究表明,APE1/Ref-1的表达和定位与多种肿瘤的发生发展及其放化疗敏感性相关。因此我们有充分的理由相信,APE1/Ref-1是非常有潜力的肿瘤治疗靶点[6-7]。
在我们的前期工作中,我们发现APE1/Ref-1在铂类耐药的卵巢癌组织和耐药的卵巢癌细胞中均发生表达水平增高和细胞定位改变,提示APE1/Ref-1在胞核和胞浆中发挥作用不同,可能是铂类耐药的重要环节。在我们的另一项研究中,曾以卵巢癌顺铂耐药细胞株CP70为研究对象,用APE1 siRNA特异性下调APE1/Ref-1表达后,CP70细胞对顺铂的敏感性显著增强,并与细胞周期阻滞有关[138-139]。
为了进一步验证APE1/Ref-1在卵巢癌铂类耐药形成中发挥的作用,本课题首先以卵巢癌铂类敏感和铂类耐药的配对临床资料为研究对象,用免疫组化分别对APE1/Ref-1、MDR1和p53基因表达水平进行检测,进一步验证APE1/Ref-1与卵巢癌铂类耐药及耐药相关基因之间的关系。随后我们将根据APE1/Ref-1两大功能相互独立发挥作用的结构特点,分别构建不同功能结构域的APE1/Ref-1重组质粒。然后以卵巢癌顺铂敏感细胞株A2780细胞为研究对象,观察APE1不同重组质粒对卵巢癌细胞的生物学效应及铂类敏感性的影响,初步探讨APE1/Ref-1的不同功能状态参与卵巢癌铂类耐药的机制。本研究将为APE1参与卵巢癌铂类耐药提供更有力的直接证据,为进一步阐明APE1/Ref-1参与卵巢癌铂类耐药的机制奠定基础,具有重要的理论意义和临床应用前景。
研究目的
1.从临床资料病理组织水平验证APE1/Ref-1表达与卵巢癌铂类敏感性的相关性;
2.构建不同功能结构域的APE1/Ref-1重组质粒,为研究APE1/Ref-1与铂类耐药的相关性提供有效手段;
3.探讨APE1/Ref-1不同功能状态对卵巢癌生物学行为及顺铂敏感性的影响,为进一步阐明APE1/Ref-1参与卵巢癌铂类耐药的机制奠定基础。
研究方法
1.以卵巢癌铂类敏感和铂类耐药的配对临床资料为研究对象,用免疫组化分别对临床病理组织标本中APE1/Ref-1、MDR1和p53基因表达水平进行检测,进一步验证APE1/Ref-1与卵巢癌铂类耐药及耐药相关基因之间的关系。
2.利用重组质粒技术,针对APE1/Ref-1不同功能结构域构建APE1-pEGFP-N1、Redox-pEGFP-N1、Repair-pEGFP-N1和NLS- Repair-pEGFP-N1四种APE1不同结构域的重组质粒。
3.利用脂质体法将APE1/Ref-1不同重组质粒分别转染卵巢癌铂类敏感细胞株A2780细胞,荧光显微镜下观察EGFP表达检测转染效率;
4. RT-PCR和western blot分别检测APE1-pEGFP-N1质粒转染后A2780细胞中APE1/Ref-1蛋白水平和mRNA水平;
5. G418抗性筛选,获得稳定转染APE1-pEGFP-N1和空质粒pEGFP-N1的A2780细胞株;
6. MTT法分别绘制卵巢癌A2780细胞分别转染APE1不同重组质粒后生长曲线的变化;
7.平板克隆形成试验检测稳定转染APE1-pEGFP-N1的A2780细胞株的增殖能力变化;
8.流式细胞术检测卵巢癌A2780细胞分别转染APE1不同重组质粒后细胞周期的变化,并计算增殖指数;
9. MTT法检测卵巢癌A2780细胞分别转染APE1不同重组质粒后顺铂敏感性的变化;
10.免疫荧光检测APE1不同重组质粒转染后,A2780细胞中APE1/Ref-1表达及定位的变化。
11.流式细胞术检测APE1不同重组质粒转染对顺铂诱导的A2780细胞凋亡的影响。
研究结果
1.卵巢癌铂类耐药组中APE1高表达和浆表达的比例均明显高于敏感组,两者之间有统计学差异(p<0.05);
2.当APE1出现浆表达时,在耐药组中p53蛋白的阳性率较敏感组明显增高,两者差异有统计学意义(p<0.05);
3. APE1-pEGFP-N1重组质粒转染后,MTT法绘制生长曲线提示A2780细胞生长速度有所提高,克隆形成试验提示A2780单个细胞增殖能力有所提高;
4.细胞周期结果表明,与对照组相比,APE1-pEGFP-N1转染重组质粒转染后,A2780细胞G0/G1期比例降低,G2+S期比例增加,有统计学差异(p<0.05);
5. APE1-pEGFP-N1重组质粒转染组和空质粒转染组相比,增殖指数(PI)有所升高(52.4% vs. 59.5%),提示APE1-pEGFP-N1转染能促进A2780细胞的生长增殖;
6. APE1-pEGFP-N1转染组和Redox-pEGFP-N1转染组增殖指数(PI)分别为60.57%和63.03% ,出现了增殖指数增加的现象;而Repair-pEGFP-N1转染组和NLS-Repair-pEGFP-N1转染组增殖指数分别为41.7%,36.65%,较阴性对照组降低。
7.与阴性对照组和空白对照组相比,APE1-pEGFP-N1和Redox-pEGFP-N1转染组出现G0/G1期比例降低, S期比例增加的现象,而Repair-pEGFP-N1和NLS-Repair-pEGFP-N1转染组出现了G0/G1期比例增高,S期比例降低的现象;
8.在相同的药物浓度下, APE1-pEGFP-N1、Redox-pEGFP-N1、Repair-pEGFP-N1和NLS-Repair-pEGFP-N1四组转染后,顺铂对A2780细胞的抑制率均有所降低,根据IC50值由高到低依次为Redox-pEGFP-N1、NLS-Repair-pEGFP-N1、APE1-pEGFP-N1和Repair-pEGFP-N1(IC50值分别为28.01μM、24.00μM、19.21μM和16.71μM),A2780细胞对顺铂的耐受性分别提高了2.13倍、1.46倍、1.27倍和1.83倍(p<0.05)。
9. APE1-pEGFP-N1转染组和阴性对照组相比,活细胞率略有升高(98.0% vs. 97.2%),凋亡率略有下降(1.1% vs. 1.6%)。Redox-pEGFP-N1转染组凋亡率和坏死率分别为1.7%和0.8%,和阴性对照组相比无明显差异。Repair-pEGFP-N1和NLS-Repair-pEGFP-N1两组中活细胞率分别为93.3%和92.8%,和对照组相比略有降低,而凋亡率略有所增加,分别为4.4%和4.6%。
10. APE1-pEGFP-N1转染组和顺铂处理组相比,凋亡率略有升高(8.4% vs. 6.6%),坏死率显著降低(28.4% vs. 41.3%)。Redox-pEGFP-N1转染组凋亡率和坏死率和顺铂处理组相比均有所降低,其中坏死率降低更为显著(5.5% vs. 6.6%,24.1% vs. 41.3%)。转染不同APE1重组质粒的四组细胞与顺铂处理组相比,Repair-pEGFP-N1和NLS-Repair-pEGFP-N1两组坏死率降低最为明显,分别为22.3%和23.7%,但凋亡率有所增加,分别为20.5%和13.5%。
结论
1. APE1/Ref-1的表达水平和细胞定位均与卵巢癌铂类耐药相关,其参与卵巢癌铂类耐药的机制可能与P53突变相关。
2.提高APE1/Ref-1表达水平能通过促进A2780细胞DNA合成而对A2780细胞产生刺激增殖作用,并增加A2780细胞对顺铂的耐受性。
3. APE1/Ref-1的不同功能状态对A2780细胞周期及DNA合成有调控作用。APE1/Ref-1表达增高能促进肿瘤细胞中蛋白质合成,而APE1/Ref-1的氧化还原功能在这一过程中扮演重要角色。
4. APE1/Ref-1的氧化还原功能和DNA修复功能都在卵巢癌A2780细胞对顺铂的化疗应激中发挥了一定作用。其中APE1/Ref-1的氧化还原功能对A2780细胞顺铂敏感性影响最为显著,可能与APE1通过氧化还原功能调控下游凋亡相关因子从而对抗顺铂诱导的细胞凋亡有关。而APE1/Ref-1的DNA修复功能也通过DNA修复酶活性在A2780细胞对顺铂的抵抗中发挥一定作用。
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.
OBJECTIVES
1. 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.
METHODS
1. 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.
RESULTS
1. 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.
CONCLUSION
1. 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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