γ-分泌酶抑制剂对顺铂耐药上皮性卵巢癌侵袭转移能力的影响
发布时间：2018-08-30 11:12
【摘要】：目的：上皮性卵巢癌（epithelial ovarian cancer, EOC）为最常见的卵巢恶性肿瘤，不易早期诊断，复发及死亡率高。卵巢癌目前标准治疗方法主要为肿瘤细胞减灭术及以铂类为基础的化疗，但约70-80%的患者最终会出现化疗耐药，引起肿瘤的复发及转移，为卵巢癌治疗失败及患者死亡的重要原因，故其5年生存率一直徘徊在30%左右。因此，探讨化疗耐药肿瘤的侵袭转移机制，寻找有效的干预靶点，为EOC患者延长生存期和改善预后的迫切需求。 近年来研究发现，上皮间质转化（epithelial-mesenchymal transition,EMT）不仅参与胚胎发育及创伤修复，在肿瘤的侵袭转移过程中也发挥了重要作用。EMT是指特定的生理或病理情况下，上皮细胞丢失上皮表型的特性转化为间质表型细胞过程。发生EMT的细胞极性丢失，细胞间粘附力下降，运动能力增强，细胞形态延伸，抗凋亡能力增强，从而促进肿瘤侵袭转移。EMT与多种细胞因子、转录因子及信号通路相关。EMT过程伴随着Notch、Wnt、Hedgehog等多种信号通路的活化；转录因子Snail和Slug等表达上调；上皮表型标志物E-cadherin等表达下调，间质表型标志物vimentin等表达上调。此外，发生EMT的肿瘤细胞可获得自我更新等干细胞样特性，这可能与肿瘤细胞发生化疗抵抗相关。 多年研究表明，Notch信号通路在多种实体肿瘤及血液系统肿瘤中均异常表达，且与肿瘤的发生发展密切相关。新近研究表明，Notch信号通路活化可诱导多种肿瘤细胞发生EMT，促进肿瘤的转移和复发。γ-分泌酶为激活Notch信号通路的核心环节，而成为肿瘤治疗的研究热点。本实验通过体外培养人卵巢浆液性囊腺癌细胞株SKOV3及其顺铂耐药株SKOV3/DDP，检测其Notch1mRNA表达量的差异，并进一步研究γ分泌酶抑制剂DAPT作用于SKOV3/DDP细胞后，E-cadherin及vimentin的表达情况，以及其迁移及穿膜能力的改变。旨在从分子水平探讨DAPT能否通过抑制Notch信号通路，从而抑制化疗耐药EOC的侵袭转移能力，，为化疗耐药EOC的治疗策略提供实验依据。 方法：用含10%胎牛血清的RPMI-1640培养基，在37℃，饱和湿度5%CO2的恒温培养箱中常规培养人卵巢浆液性囊腺癌细胞株SKOV3、及其顺铂耐药株SKOV3/DDP。取对数生长期细胞进行实验。 1倒置显微镜观察SKOV3细胞及SKOV3/DDP细胞的形态并拍照。 2实时荧光定量PCR(PT-PC)检测SKOV3细胞及SKOV3/DDP细胞Notch1mRNA表达水平。 3四甲基偶氮唑蓝(MTT)法：对SKOV3/DDP细胞以不同浓度的DAPT(25，50，75μmol/L)作用不同时间(24h、48h、72h)，以加入DAPT的溶剂0.375%DMSO为对照组。MTT法检测DAPT对细胞生长的影响，筛选出药物作用的最适浓度及时间。 4蛋白印迹法（Western blotting）：检测SKOV3细胞及不同浓度的DAPT(0，25，50，75μmol/L)作用于SKOV3/DDP细胞48h后，上皮标记物E-cadherin和间质标记物vimentin的表达水平。 5划痕实验：常规培养SKOV3/DDP细胞使其融合形成单细胞层，无血清培养基同步化24h后，按实验分组加入不同干预（实验组：50μmol/LDAPT；对照组：0.25%DMSO），在培养板底部中央呈“一”字形划痕，分别于给药后0h、24h后倒置显微镜（40×）下观察照相，计算24h内划痕愈合的面积评估细胞的迁移能力。 6Transwell实验：以50μmol/L DAPT作用SKOV3/DDP细胞为实验组，以加入0.25%DMSO为对照组，连续培养24小时后取出Transwell侵袭小室，用95%乙醇固定，经结晶紫染色，在显微镜（200×）下拍照并计数移至微孔膜下层的细胞评估细胞的迁移和侵袭能力。 7应用SPSS16.0软件进行统计学分析。 结果： 1SKOV3细胞与SKOV3/DDP细胞：倒置显微镜下观察，SKOV3细胞呈椭圆形、圆形或立方形，呈铺路石样排列，胞间连接紧密，为典型上皮样细胞形态；SKOV3/DDP细胞呈长梭形，细胞排列较松散，胞间连接不紧密，呈间质细胞样形态；Western blotting检测结果示：SKOV3/DDP细胞的E-cadherin表达量低于SKOV3细胞（0.054±0.012VS0.718±0.125），而Vimentin表达量高于SKOV3细胞（3.232±0.267VS0.517±0.059），差别具有统计学意义（P＜0.01）； RT-PCR结果示SKOV3/DDP细胞Notch1mRNA的表达量为SKOV3细胞的3.952倍。 2MTT结果示，随着DAPT浓度增加和作用时间的延长，SKOV3/DPP细胞的生长抑制率明显升高，差异均有统计学意义（均P＜0.01）。 3Western blotting结果示不同浓度的DAPT（25、50、75μmol/L）作用于SKOV3/DDP细胞48h后，其上皮标记物E-cadherin表达较对照组明显增加（0.207±0.020、0.295±0.011、0.429±0.024VS0.054±0.012）（P＜0.01），且随DAPT作用浓度增加而升高，各组间差异显著（P＜0.01）；间质标记物vimentin表达较对照组明显降低（2.502±0.170、1.873±0.177、1.624±0.057VS3.232±0.266）（P＜0.01），但50μmol/L与75μmol/L浓度组表达差异无统计学意义（P＞0.05）。 4以浓度为50μmol/L的DAPT作用于SKOV3/DDP细胞24h后，划痕试验结果：处理组的划痕愈合面积显著小于对照组（14.31±3.59VS52.91±10.59）（P＜0.01）；Transwell实验结果示处理组穿膜细胞个数明显少于对照组（迁移实验13±2VS37±2，侵袭实验9±1VS27±3），差异均有统计学意义（P＜0.01）。 结论： SKOV3/DDP细胞化疗耐药过程中获得了EMT表型，γ-分泌酶抑制剂DAPT可通过靶向作用于Notch信号通路，部分逆转EMT，抑制人卵巢癌顺铂耐药细胞的增殖、粘附及侵袭转移能力，有望成为有效治疗耐药复发转移EOC的新选择。
[Abstract]:Objective: Epithelial ovarian cancer (EOC) is the most common malignant tumor of the ovary, which is difficult to be diagnosed early, has high recurrence and mortality rate. The 5-year survival rate of patients with ovarian cancer has been hovering around 30% because metastasis is an important cause of treatment failure and death of patients with ovarian cancer.
Recent studies have shown that epithelial-mesenchymal transition (EMT) is not only involved in embryonic development and wound repair, but also plays an important role in tumor invasion and metastasis. EMT is associated with a variety of cytokines, transcription factors and signaling pathways. The process of EMT is accompanied by the activation of Notch, Wnt, Hedgehog and other signaling pathways. The expression of E-cadherin was down-regulated and that of vimentin was up-regulated. In addition, tumor cells with EMT could acquire stem cell-like characteristics such as self-renewal, which may be related to chemotherapeutic resistance of tumor cells.
Recent studies have shown that Notch signaling pathway is abnormally expressed in a variety of solid tumors and hematological tumors, and is closely related to the occurrence and development of tumors. In this study, the expression of Notch1 mRNA in human ovarian serous cystadenocarcinoma cell line SKOV3 and its cisplatin-resistant strain SKOV3/DDP was detected, and the expression of E-cadherin and vimentin in SKOV3/DDP cells treated with gamma-secretase inhibitor DAPT was further investigated. The purpose of this study is to investigate whether DAPT can inhibit the invasion and metastasis of chemotherapeutic resistant EOC by inhibiting Notch signaling pathway at molecular level, and to provide experimental evidence for the treatment strategy of chemotherapeutic resistant EOC.
METHODS: Human ovarian serous cystadenocarcinoma cell line SKOV3 and its cisplatin-resistant strain SKOV3/DDP were cultured in RMPI-1640 medium containing 10% fetal bovine serum in a constant temperature incubator with saturated humidity of 5% CO2 at 37 C.
1 inverted microscope was used to observe the morphology of SKOV3 cells and SKOV3/DDP cells and take pictures.
2 real-time fluorescence quantitative PCR (PT-PC) was used to detect the expression level of Notch1mRNA in SKOV3 cells and SKOV3/DDP cells.
MTT method: SKOV3/DDP cells were treated with different concentrations of DAPT (25,50,75 micromol/L) for different time (24h,48h,72h) and 0.375% DMSO as the control group. MTT method was used to detect the effect of DAPT on the growth of SKOV3/DDP cells.
Western blotting: To detect the expression of E-cadherin and vimentin in SKOV3/DDP cells 48 hours after exposure to different concentrations of DAPT (0,25,50,75 micromol/L).
5 Scratch test: SKOV3/DDP cells were cultured routinely and fused to form a single cell layer. After 24 hours of synchronization in serum-free medium, the SKOV3/DDP cells were divided into experimental group (50 micromol/LDAPT) and control group (0.25% DMSO). Scratch was observed under an inverted microscope (40 x) at the bottom of the plate 0 h and 24 h after administration. Photographs were taken to calculate the area of scratch healing within 24h to assess cell migration.
6 Transwell experiment: SKOV3/DDP cells treated with 50 micromol/L DAPT were used as experimental group, and 0.25% DMSO as control group. Transwell invasive cells were taken out after 24 hours of continuous culture, fixed with 95% ethanol, stained with crystal violet, photographed under microscope (200 *) and counted to evaluate the migration and invasiveness of the cells.
7 SPSS16.0 software was used for statistical analysis.
Result:
SKOV3 cells and SKOV3/DDP cells: under inverted microscope, SKOV3 cells were oval, round or cubic, paving stone-like arrangement, tight intercellular junction, typical epithelial-like cell morphology; SKOV3/DDP cells were spindle-shaped, loosely arranged, intercellular junction was not tight, and showed mesenchymal-like morphology; Western blotting detection of nodules; The results showed that the expression of E-cadherin in SKOV3/DDP cells was lower than that in SKOV3 cells (0.054+0.012VS 0.718+0.125), while the expression of Vimentin was higher than that in SKOV3 cells (3.232+0.267VS 0.517+0.059), the difference was statistically significant (P < 0.01); the expression of Notch1 mRNA in SKOV3/DDP cells was 3.952 times that in SKOV3 cells.
The results of 2MTT showed that the growth inhibition rate of SKOV3/DPP cells increased significantly with the increase of DAPT concentration and prolongation of action time (P < 0.01).
Western blotting showed that the expression of E-cadherin in SKOV3/DDP cells treated with different concentrations of DAPT (25,50,75 micromol/L) for 48 hours was significantly higher than that in the control group (0.207.020,0.295.011,0.429.024VS 0.054.012) (P < 0.01), and the expression of E-cadherin was significantly higher than that in the control group (P < 0.01). The expression of vimentin was significantly lower than that of the control group (2.502.170, 1.873.177, 1.624.057VS 3.232.266) (P < 0.01), but there was no significant difference between the 5065507
4 Scratch test showed that the area of scratch healing in the treatment group was significantly smaller than that in the control group (14.31 [3.59] VS 52.91 [10.59]) (P < 0.01); Transwell test showed that the number of penetrating cells in the treatment group was significantly less than that in the control group (13 [2] VS 37 [2] migration test, 9 [1] VS 27 [3] invasion test). The differences were statistically significant (P < 0.01).
Conclusion: SKOV3/DDP cells acquired EMT phenotype during chemotherapeutic resistance, and DAPT, a gamma-secretase inhibitor, can partially reverse EMT by targeting Notch signaling pathway, inhibit the proliferation, adhesion and invasion and metastasis of cisplatin-resistant human ovarian cancer cells. It is expected to be a new choice for effective treatment of drug-resistant recurrence and metastasis of EOC.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31
[Abstract]:Objective: Epithelial ovarian cancer (EOC) is the most common malignant tumor of the ovary, which is difficult to be diagnosed early, has high recurrence and mortality rate. The 5-year survival rate of patients with ovarian cancer has been hovering around 30% because metastasis is an important cause of treatment failure and death of patients with ovarian cancer.
Recent studies have shown that epithelial-mesenchymal transition (EMT) is not only involved in embryonic development and wound repair, but also plays an important role in tumor invasion and metastasis. EMT is associated with a variety of cytokines, transcription factors and signaling pathways. The process of EMT is accompanied by the activation of Notch, Wnt, Hedgehog and other signaling pathways. The expression of E-cadherin was down-regulated and that of vimentin was up-regulated. In addition, tumor cells with EMT could acquire stem cell-like characteristics such as self-renewal, which may be related to chemotherapeutic resistance of tumor cells.
Recent studies have shown that Notch signaling pathway is abnormally expressed in a variety of solid tumors and hematological tumors, and is closely related to the occurrence and development of tumors. In this study, the expression of Notch1 mRNA in human ovarian serous cystadenocarcinoma cell line SKOV3 and its cisplatin-resistant strain SKOV3/DDP was detected, and the expression of E-cadherin and vimentin in SKOV3/DDP cells treated with gamma-secretase inhibitor DAPT was further investigated. The purpose of this study is to investigate whether DAPT can inhibit the invasion and metastasis of chemotherapeutic resistant EOC by inhibiting Notch signaling pathway at molecular level, and to provide experimental evidence for the treatment strategy of chemotherapeutic resistant EOC.
METHODS: Human ovarian serous cystadenocarcinoma cell line SKOV3 and its cisplatin-resistant strain SKOV3/DDP were cultured in RMPI-1640 medium containing 10% fetal bovine serum in a constant temperature incubator with saturated humidity of 5% CO2 at 37 C.
1 inverted microscope was used to observe the morphology of SKOV3 cells and SKOV3/DDP cells and take pictures.
2 real-time fluorescence quantitative PCR (PT-PC) was used to detect the expression level of Notch1mRNA in SKOV3 cells and SKOV3/DDP cells.
MTT method: SKOV3/DDP cells were treated with different concentrations of DAPT (25,50,75 micromol/L) for different time (24h,48h,72h) and 0.375% DMSO as the control group. MTT method was used to detect the effect of DAPT on the growth of SKOV3/DDP cells.
Western blotting: To detect the expression of E-cadherin and vimentin in SKOV3/DDP cells 48 hours after exposure to different concentrations of DAPT (0,25,50,75 micromol/L).
5 Scratch test: SKOV3/DDP cells were cultured routinely and fused to form a single cell layer. After 24 hours of synchronization in serum-free medium, the SKOV3/DDP cells were divided into experimental group (50 micromol/LDAPT) and control group (0.25% DMSO). Scratch was observed under an inverted microscope (40 x) at the bottom of the plate 0 h and 24 h after administration. Photographs were taken to calculate the area of scratch healing within 24h to assess cell migration.
6 Transwell experiment: SKOV3/DDP cells treated with 50 micromol/L DAPT were used as experimental group, and 0.25% DMSO as control group. Transwell invasive cells were taken out after 24 hours of continuous culture, fixed with 95% ethanol, stained with crystal violet, photographed under microscope (200 *) and counted to evaluate the migration and invasiveness of the cells.
7 SPSS16.0 software was used for statistical analysis.
Result:
SKOV3 cells and SKOV3/DDP cells: under inverted microscope, SKOV3 cells were oval, round or cubic, paving stone-like arrangement, tight intercellular junction, typical epithelial-like cell morphology; SKOV3/DDP cells were spindle-shaped, loosely arranged, intercellular junction was not tight, and showed mesenchymal-like morphology; Western blotting detection of nodules; The results showed that the expression of E-cadherin in SKOV3/DDP cells was lower than that in SKOV3 cells (0.054+0.012VS 0.718+0.125), while the expression of Vimentin was higher than that in SKOV3 cells (3.232+0.267VS 0.517+0.059), the difference was statistically significant (P < 0.01); the expression of Notch1 mRNA in SKOV3/DDP cells was 3.952 times that in SKOV3 cells.
The results of 2MTT showed that the growth inhibition rate of SKOV3/DPP cells increased significantly with the increase of DAPT concentration and prolongation of action time (P < 0.01).
Western blotting showed that the expression of E-cadherin in SKOV3/DDP cells treated with different concentrations of DAPT (25,50,75 micromol/L) for 48 hours was significantly higher than that in the control group (0.207.020,0.295.011,0.429.024VS 0.054.012) (P < 0.01), and the expression of E-cadherin was significantly higher than that in the control group (P < 0.01). The expression of vimentin was significantly lower than that of the control group (2.502.170, 1.873.177, 1.624.057VS 3.232.266) (P < 0.01), but there was no significant difference between the 5065507
4 Scratch test showed that the area of scratch healing in the treatment group was significantly smaller than that in the control group (14.31 [3.59] VS 52.91 [10.59]) (P < 0.01); Transwell test showed that the number of penetrating cells in the treatment group was significantly less than that in the control group (13 [2] VS 37 [2] migration test, 9 [1] VS 27 [3] invasion test). The differences were statistically significant (P < 0.01).
Conclusion: SKOV3/DDP cells acquired EMT phenotype during chemotherapeutic resistance, and DAPT, a gamma-secretase inhibitor, can partially reverse EMT by targeting Notch signaling pathway, inhibit the proliferation, adhesion and invasion and metastasis of cisplatin-resistant human ovarian cancer cells. It is expected to be a new choice for effective treatment of drug-resistant recurrence and metastasis of EOC.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R737.31
【共引文献】
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