Proteomic tracking of serum protein isoforms as screening biomarkers of ovarian cancer
Proteomic tracking of serum protein isoforms as screening biomarkers of ovarian cancer
复制标题
DOI：
10.1002/pmic.200401321
复制
发表时间：
2005-11-01
期刊：
影响因子：
3.4
通讯作者：
Rice, GE
中科院分区：
文献类型：
--
作者：
Ahmed, N;Oliva, KT;Rice, GE
上皮性卵巢癌是女性癌症死亡的第四大原因。由于该疾病无症状且生存率低，筛查卵巢癌的特定生物标志物是一项重要的健康优先事项。卵巢癌患者血清中差异表达的蛋白质有可能用作癌症特异性生物标志物。在这项研究中，使用蛋白质组学方法筛选了 24 份来自患有高级别卵巢癌的女性的血清样本，并与由 11 名健康女性组成的对照组进行比较。 Affigel-Blue 处理的血清样品通过线性 (pH 4-7) 或窄范围 (PH 5.5-6.7) IEF 条进行第一维处理。通过 8-16% 梯度 SDS-PAGE 解析第一维分离的蛋白质。蛋白质斑点通过 SYPRO Ruby 染色可视化，通过 FX 成像仪成像，并通过 PDQuest 软件进行比较和分析。通过在第一维 4-7 的线性 PH 条带中解析蛋白质，22 个蛋白质点在正常人和卵巢癌患者之间一致表达差异。通过 MALDI-TOF MS 和蛋白质印迹鉴定，其中 6 个蛋白点在 3 级卵巢癌患者中显着上调 (p < 0.05)，被鉴定为触珠蛋白前体的亚型。当血清蛋白在狭窄的 pH 范围试纸 (5.5-6.7) 上解析时，正常和 3 级卵巢癌患者之间有 23 个斑点一致表达差异。其中，通过 MALDI-TOF MS 和蛋白质印迹鉴定出 4 个在 3 级卵巢癌患者中显着下调的蛋白点 (p < 0.05)，为转铁蛋白前体的亚型。从诊断为 2/3 级卵巢癌的女性 (n = 7) 获得的腹膜肿瘤液中也发现血清触珠蛋白和转铁蛋白表达增加。采用一维Western blotting方法检测不同病理级别卵巢癌女性血清中结合珠蛋白和转铁蛋白表达的变化。对患有良性、交界性、1 级、2 级和 3 级癌症的女性采集的血清样本（每组中触珠蛋白 n = 4，转铁蛋白 n = 5）进行分析，并与正常健康女性的血清进行比较。 3 级卵巢癌患者的平均血清触珠蛋白表达是对照受试者的四倍（p < 0.05）。另一方面，3级卵巢癌患者的转铁蛋白表达比正常健康女性降低两倍(p < 0.05)。癌症患者 (n = 7) 的血清中触珠蛋白表达在化疗（六个周期的紫杉醇/卡铂）后下降。随着触珠蛋白的减少，转铁蛋白的表达在四名患者中保持恒定，但在该研究中七名患者中的三名增加。化疗前后血清触珠蛋白表达量的变化与CA 125水平的变化相关。总之，与卵巢癌女性相比，对正常女性血清中差异表达的蛋白质进行蛋白质组学分析可以极大地促进一组生物标志物的发现，这些生物标志物可能有助于以更高的特异性检测卵巢癌。
Epithelial ovarian cancer is the fourth leading cause of cancer death among women. Due to the asymptomatic nature and poor survival characteristic of the disease, screening for specific biomarkers for ovarian cancer is a major health priority. Differentially expressed proteins in the serum of ovarian cancer patients have the potential to be used as cancer-specific biomarkers. In this study, proteomic methods were used to screen 24 serum samples from women with high-grade ovarian cancer and compared to a control group of 11 healthy women. Affigel-Blue treated serum samples were processed either by linear (pH 4-7) or narrow range (PH 5.5-6.7) IEF strips for the first dimension. Proteins separated in first dimension were resolved by 8-16% gradient SDS-PAGE. Protein spots were visualized by SYPRO Ruby staining, imaged by FX-imager and compared and analyzed by PDQuest software. Twenty-two protein spots were consistently differentially expressed between normal and ovarian cancer patients by resolving proteins in a linear PH strip of 4-7 for the first dimension. Six of the protein spots, significantly up-regulated in grade 3 ovarian cancer patients (p < 0.05), were identified by MALDI-TOF MS and Western blotting as the isoforms of haptoglobin precursor. When serum proteins were resolved on narrow pH range strips (5.5-6.7), 23 spots were consistently differentially expressed between normal and grade 3 ovarian cancer patients. Of these, 4 protein spots significantly down regulated in grade 3 ovarian cancer patients (p < 0.05) were identified by MALDI-TOF MS and Western blotting, as isoforms of transferrin precursor. Increased expression of serum haptoglobin and transferrin was also identified in peritoneal tumor fluid obtained from women diagnosed with grade 2/3 ovarian cancer (n = 7). Changes in the expression of haptoglobin and transferrin in the serum of women with different pathological grades of ovarian cancer was examined by one-dimensional Western blotting method. Serum samples collected from women suffering from benign, borderline, grade 1, grade 2 and grade 3 cancer (n = 4 for haptoglobin and n = 5 for transferrin in each group) were analyzed and compared to the serum of normal healthy women. The mean serum haptoglobin expression in grade 3 ovarian cancer patients was fourfold higher than in the control subjects (p < 0.05). On the other hand, transferrin expression in grade 3 ovarian cancer patients was decreased by twofold than in normal healthy women (p < 0.05). Haptoglobin expression in the serum of cancer patients (n = 7) decreased following chemotherapy (six cycles of taxol/carboplatin). Concomitant with the decrease of haptoglobin, transferrin expression remained constant in four patients, but increased in three out of seven patients included in the study. Changes in serum expression of haptoglobin correlated with the change of CA 125 levels before and after chemotherapy. In conclusion, proteomic profiling of differentially expressed proteins in the sera of normal women compared to women with ovarian cancer can greatly facilitate the discovery of a panel of biomarkers that may aid in the detection of ovarian cancer with greater specificity.