Kallikrein 6在卵巢癌中的表达、调控及临床意义
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摘要
【目的】卵巢癌是常见妇科恶性肿瘤之一，由于起病隐匿，缺乏早期诊断的有效指标，晚期治疗效果差，严重威胁妇女健康。目前有关卵巢癌发生的确切机制尚未明确，流行病学研究发现，内分泌因素、遗传因素、不良的营养和饮食习惯等是卵巢肿瘤重要的危险因素。因此，积极借助最新分子生物学技术，从基因水平上探讨卵巢癌的发牛机制及卵巢癌相关基因的功能，寻找有关卵巢癌早期诊断、预后监测的新指标是目前卵巢癌研究中至关重要的课题。KLK基因家族是近来新发现的一类与性激素相关的肿瘤标记家族，KLK6基因是该家族成员之一，编码的蛋白hK6是由223个氨基酸组成的丝氨酸蛋白酶，具有胰蛋白酶样活性，与卵巢癌关系密切。本课题通过雌激素调控体外培养的卵巢癌细胞株，观察雌激素对KLK6基因以及卵巢癌细胞牛物学性状的影响，以初步探讨KLK6基因在卵巢癌发生发展中的作用及功能；同时通过免疫组化SP法检测KLK6基因编码的蛋白hK6在卵巢癌中的表达及与临床病理特征、预后的关系，探讨其临床意义。
【方法】1．四种不同浓度(1×10~(-10)、1×10~(-9)、1×10~(-8)、1×10~(-7)mol／L)的17β-E_2作用于体外培养的卵巢癌H08910细胞株72小时后，采用实时荧光定量PCR和流式细胞仪分别检测KLK6 mRNA和蛋白的表达变化，流式细胞仪和MTT法检测调控前后H08910细胞增殖周期和细胞增殖活力的改变，初步探讨KLK6基因的功能及其在卵巢癌发生发展中的作用。
2．通过免疫组化SP法检测hK6蛋白在19例良性卵巢上皮瘤、11例交界性卵巢上皮瘤和45例卵巢上皮癌中的表达，以及该蛋白的表达与患者年龄、原发肿瘤大小、组织分型、分化程度、临床分期、淋巴结转移等临床病理特征和病人术后生存状况的关系，探讨其临床意义。
【结果】1．不同浓度(1×10~(-10)、1×10~(-9)、1×10~(-8)、1×10~(-7)mol／L)的17β-E_2作用72小时后，H08910细胞KLK6 mRNA水平(3.83±0.41、4.14±0.49、6.26±0.38、7.28±1.82)和蛋白水平(10.62±0.35、10.89±0.12、11.88±0.28、12.07±0.15)均明显高于乙醇对照组(P＜0.01)；随着E_2浓度增加，各试验组H08910细胞G_0／G_1期百分率(67.72％、66.98％、66.31％、65.19％)呈下降趋势，S期(17.70％、18.27％、18.55％、19.11％)和G2／M期百分率(14.58％、14.75％、15.14％、15.70％)呈上升趋势；同时，各试验组H08910细胞光吸收度A值(0.7713±0.0151、0.8487±0.0224、0.8577±0.0163、0.8953±0.0310)也逐渐升高，与乙醇对照组(0.4593±0.0638)相比，差异有显著统计学意义(P＜0.01)。
2．hK6蛋白在卵巢良性、交界性及恶性卵巢肿瘤组织中的阳性表达率分别为15.8％，27.3％和60.0％，其在卵巢癌中的阳性表达率明显高于其在良性和交界性肿瘤中的表达，差异有统计学意义(P＜0.01)。hK6的阳性表达与患者的年龄、原发灶大小以及卵巢癌组织学类型无关；中低分化卵巢癌中的阳性表达率(68.4％)高于高分化者(14.3％)，差异有统计学意义(P=0.023＜0.05)；卵巢癌晚期(Ⅲ期)的阳性表达率(76.7％)明显高于早期(Ⅰ／Ⅱ期)阳性表达率(26.7％，P=0.001＜0.01)；有淋巴结转移者的阳性表达率(77.8％)明显高于无淋巴结转移者(33.3％，P=0.003＜0.01)；术后3年内复发／转移／死亡的阳性表达率(75.0％)高于术后3年内病情稳定者(42.9％，p=0.028＜0.05)。
结论：KLK6基因在卵巢癌细胞株中的表达受雌激素上调，同时促进卵巢癌细胞的增殖，可能是卵巢癌发展的促进因子，为卵巢癌防治提供了新的思路。该基因编码的蛋白hK6在卵巢癌组织中的表达明显高于其在良性和交界性卵巢肿瘤中的表达，且其在晚期、分化差、有淋巴结转移、预后差的卵巢癌中高表达，提示可能是一种新的反映卵巢癌恶性程度和预后的肿瘤标记物。
[OBJECTIVE] Ovarian cancer is one of the common malignant tumors in thedepartment of gynecology. Because of the onste delitescence and the lack of tumor markers for early diagnosis, now ovarian cancer is threatening the women's health seriously. At present, the pathogenesis of ovarian cancer is still unknown. The epidemiologic study shows that the risk factors for ovarian cancer include the endocrine factor, the hereditary factor, the bad eating habit and so on.. The search for tumor markers for the early detection and outcome prediction of ovarian carcinoma is one of the critical subjects in the study of ovarian cancer. Meanwhile,it is the key to decrease the fatality rate of the patients to find out the the pathogenesis of ovarian cancer and the role of the cancer associated genes by the latest molecular biology techniques. Human tissue kallikrein(KLK) gene family is one lately discovered tumor markers family that associated with the steroid hormone. KLK6 gene is one member of this family and encodes for human kallikrein 6 (hK6). The hK6 protein, a trypsin-like serine protease,consists of 223 amino acids and is closely related with ovarain cancer. One of the purpose for this reseach is to initially approach the role of KLK6 gene during the development of ovarian cancer by the estrogen regulation test. The other is to find a new tumor marker for ovarian cancer by examining the expression of hK6 in the benign, borderline and malignant ovarian neoplasm through immunohistochemistry .
[METHODS] 1. To culture the ovarian cancer HO8910 cell line with 17-βE_2 at different concentration(1×10~(-10)、1×10~(-9)、1×10~(-8)、1×10~(-7) mol/L) for 72 hours ,then measure the expression of KLK6 mRNA and protein by real-time fluorensence quantitive PCR and flow cytometry respectively and detect the cell proliferation and cell growth cycle by MTT and flow cytometry.
2.To examine the expression of hK6 during the 19 cases of benign , 11 cases of borderline and 45 cases of malignant ovarian neoplasms by immunohistochemistry and statistically analyze whether the expression of hK6 correlate with the clinicopathologic variables and prognosis in patients of ovarian cancer.
[RESULTS]1. The expression of KLK6 mRNA(3.83±0.41、4.14±0.49、6.26±0.38、7.28±1.82) and protein(10.62±0.35、10.89±0.12、11.88±0.28, 12.07±0.15) in the test group HO8910 cells'(1×10~(-10)、1×10~(-9)、1×10~(-8)、1×10~(-7)mol/L) was obviously higher than the ethanol control group(P<0.01 ).And MTT test shows the cell absorption value (0.7713±0.0151、0.8487±0.0224、0.8577±0.0163、0.8953±0.0310)in the test group HO8910 cells was increased significantly,compared to the ethanol control group(0.4593±0.0638)(p<0.01);while the cell percentage in G_0/G_1 phase of cell cycle (67.72%、66.98%、66.31%、65.19%) was decreased and the percentagein S phase(17.70%、18.27%、18.55%、19.11%) and G_2/M phase (14.58%、14.75%、15.14%、15.70%) increased.
2. The expression of hK6 in ovarian cancer tissues (60.0%) was significantly higher than it in the benign(15.8%) and borderline(27.3%) ovarian neoplasm tissues (p<0.01);the expression of hK6 in higher-grade ovarian cancer tissues (68.4%) was higher than it in low-grade(14.3%)(p<0.05);the expression of hK6 in late -stage (stage III) (76.7%) was obviously higher than it in early-stage(stage I or II) (26.7%)(p<0.01); the expression of hK6 was significantly higher in node metastasis (77.8%)than no-metastasis(33.3%)(p<0.01);the expression of hK6 in the cancer tissues that the patients died or their pathogenetic condition recurred or their tumor metastasis during 3 years after their surgery was higher (75.0%) than it in the cancer tissues that the pathogenetic condition of the patients was stable( 42.9%)(p<0.05).
CONCLUSION: 1. The expression of KLK6 mRNA and protein was up-regulated in HO8910 cell under the estrogen stimulation. Meanwhile the cell proliferation was enhanced.KLK6 maybe act as a tumor enhancing factor during the development of ovarian cancer.
2.The expression of hK6 in ovarian cancer tissues was higher than it in the benign and borderline ovarian neoplasm tissues.;the expression of hK6 was higher in the ovarian cancer tissues with late -stage, higher-grade ,node metastasis and that the prognosis of the patients poor. And hK6 was maybe a new ovarian tumor markers that can predict the prognosis of the patients.
【方法】1．四种不同浓度(1×10~(-10)、1×10~(-9)、1×10~(-8)、1×10~(-7)mol／L)的17β-E_2作用于体外培养的卵巢癌H08910细胞株72小时后，采用实时荧光定量PCR和流式细胞仪分别检测KLK6 mRNA和蛋白的表达变化，流式细胞仪和MTT法检测调控前后H08910细胞增殖周期和细胞增殖活力的改变，初步探讨KLK6基因的功能及其在卵巢癌发生发展中的作用。
2．通过免疫组化SP法检测hK6蛋白在19例良性卵巢上皮瘤、11例交界性卵巢上皮瘤和45例卵巢上皮癌中的表达，以及该蛋白的表达与患者年龄、原发肿瘤大小、组织分型、分化程度、临床分期、淋巴结转移等临床病理特征和病人术后生存状况的关系，探讨其临床意义。
【结果】1．不同浓度(1×10~(-10)、1×10~(-9)、1×10~(-8)、1×10~(-7)mol／L)的17β-E_2作用72小时后，H08910细胞KLK6 mRNA水平(3.83±0.41、4.14±0.49、6.26±0.38、7.28±1.82)和蛋白水平(10.62±0.35、10.89±0.12、11.88±0.28、12.07±0.15)均明显高于乙醇对照组(P＜0.01)；随着E_2浓度增加，各试验组H08910细胞G_0／G_1期百分率(67.72％、66.98％、66.31％、65.19％)呈下降趋势，S期(17.70％、18.27％、18.55％、19.11％)和G2／M期百分率(14.58％、14.75％、15.14％、15.70％)呈上升趋势；同时，各试验组H08910细胞光吸收度A值(0.7713±0.0151、0.8487±0.0224、0.8577±0.0163、0.8953±0.0310)也逐渐升高，与乙醇对照组(0.4593±0.0638)相比，差异有显著统计学意义(P＜0.01)。
2．hK6蛋白在卵巢良性、交界性及恶性卵巢肿瘤组织中的阳性表达率分别为15.8％，27.3％和60.0％，其在卵巢癌中的阳性表达率明显高于其在良性和交界性肿瘤中的表达，差异有统计学意义(P＜0.01)。hK6的阳性表达与患者的年龄、原发灶大小以及卵巢癌组织学类型无关；中低分化卵巢癌中的阳性表达率(68.4％)高于高分化者(14.3％)，差异有统计学意义(P=0.023＜0.05)；卵巢癌晚期(Ⅲ期)的阳性表达率(76.7％)明显高于早期(Ⅰ／Ⅱ期)阳性表达率(26.7％，P=0.001＜0.01)；有淋巴结转移者的阳性表达率(77.8％)明显高于无淋巴结转移者(33.3％，P=0.003＜0.01)；术后3年内复发／转移／死亡的阳性表达率(75.0％)高于术后3年内病情稳定者(42.9％，p=0.028＜0.05)。
结论：KLK6基因在卵巢癌细胞株中的表达受雌激素上调，同时促进卵巢癌细胞的增殖，可能是卵巢癌发展的促进因子，为卵巢癌防治提供了新的思路。该基因编码的蛋白hK6在卵巢癌组织中的表达明显高于其在良性和交界性卵巢肿瘤中的表达，且其在晚期、分化差、有淋巴结转移、预后差的卵巢癌中高表达，提示可能是一种新的反映卵巢癌恶性程度和预后的肿瘤标记物。
[OBJECTIVE] Ovarian cancer is one of the common malignant tumors in thedepartment of gynecology. Because of the onste delitescence and the lack of tumor markers for early diagnosis, now ovarian cancer is threatening the women's health seriously. At present, the pathogenesis of ovarian cancer is still unknown. The epidemiologic study shows that the risk factors for ovarian cancer include the endocrine factor, the hereditary factor, the bad eating habit and so on.. The search for tumor markers for the early detection and outcome prediction of ovarian carcinoma is one of the critical subjects in the study of ovarian cancer. Meanwhile,it is the key to decrease the fatality rate of the patients to find out the the pathogenesis of ovarian cancer and the role of the cancer associated genes by the latest molecular biology techniques. Human tissue kallikrein(KLK) gene family is one lately discovered tumor markers family that associated with the steroid hormone. KLK6 gene is one member of this family and encodes for human kallikrein 6 (hK6). The hK6 protein, a trypsin-like serine protease,consists of 223 amino acids and is closely related with ovarain cancer. One of the purpose for this reseach is to initially approach the role of KLK6 gene during the development of ovarian cancer by the estrogen regulation test. The other is to find a new tumor marker for ovarian cancer by examining the expression of hK6 in the benign, borderline and malignant ovarian neoplasm through immunohistochemistry .
[METHODS] 1. To culture the ovarian cancer HO8910 cell line with 17-βE_2 at different concentration(1×10~(-10)、1×10~(-9)、1×10~(-8)、1×10~(-7) mol/L) for 72 hours ,then measure the expression of KLK6 mRNA and protein by real-time fluorensence quantitive PCR and flow cytometry respectively and detect the cell proliferation and cell growth cycle by MTT and flow cytometry.
2.To examine the expression of hK6 during the 19 cases of benign , 11 cases of borderline and 45 cases of malignant ovarian neoplasms by immunohistochemistry and statistically analyze whether the expression of hK6 correlate with the clinicopathologic variables and prognosis in patients of ovarian cancer.
[RESULTS]1. The expression of KLK6 mRNA(3.83±0.41、4.14±0.49、6.26±0.38、7.28±1.82) and protein(10.62±0.35、10.89±0.12、11.88±0.28, 12.07±0.15) in the test group HO8910 cells'(1×10~(-10)、1×10~(-9)、1×10~(-8)、1×10~(-7)mol/L) was obviously higher than the ethanol control group(P<0.01 ).And MTT test shows the cell absorption value (0.7713±0.0151、0.8487±0.0224、0.8577±0.0163、0.8953±0.0310)in the test group HO8910 cells was increased significantly,compared to the ethanol control group(0.4593±0.0638)(p<0.01);while the cell percentage in G_0/G_1 phase of cell cycle (67.72%、66.98%、66.31%、65.19%) was decreased and the percentagein S phase(17.70%、18.27%、18.55%、19.11%) and G_2/M phase (14.58%、14.75%、15.14%、15.70%) increased.
2. The expression of hK6 in ovarian cancer tissues (60.0%) was significantly higher than it in the benign(15.8%) and borderline(27.3%) ovarian neoplasm tissues (p<0.01);the expression of hK6 in higher-grade ovarian cancer tissues (68.4%) was higher than it in low-grade(14.3%)(p<0.05);the expression of hK6 in late -stage (stage III) (76.7%) was obviously higher than it in early-stage(stage I or II) (26.7%)(p<0.01); the expression of hK6 was significantly higher in node metastasis (77.8%)than no-metastasis(33.3%)(p<0.01);the expression of hK6 in the cancer tissues that the patients died or their pathogenetic condition recurred or their tumor metastasis during 3 years after their surgery was higher (75.0%) than it in the cancer tissues that the pathogenetic condition of the patients was stable( 42.9%)(p<0.05).
CONCLUSION: 1. The expression of KLK6 mRNA and protein was up-regulated in HO8910 cell under the estrogen stimulation. Meanwhile the cell proliferation was enhanced.KLK6 maybe act as a tumor enhancing factor during the development of ovarian cancer.
2.The expression of hK6 in ovarian cancer tissues was higher than it in the benign and borderline ovarian neoplasm tissues.;the expression of hK6 was higher in the ovarian cancer tissues with late -stage, higher-grade ,node metastasis and that the prognosis of the patients poor. And hK6 was maybe a new ovarian tumor markers that can predict the prognosis of the patients.
引文
1．蔡刚，李闻捷，沈茜．实时逆转录聚合酶链反应绝对定量实验优化的研究[J]．上海医学检验杂志，2003，18(6)：343-346．
2．胡成进，沈茜，陈英剑等。乳腺癌KLK6基因表达的实时荧光定量法的建立及初步应用。中华检验医学杂志，2005，28：610-612．
3. Clements JA, Willemsen NW, Myers SA, et al. The Tissue Kallikrein Family of Serine Proteases: Functional Roles in Human Disease and Potential as Clinical Biomarkers. Critical Reviews in Clinical Laboratory Sciences, 2004, 41(3): 265-312.
4. Borgofio CA, Diamandis EP. The emerging roles of human tissue kallikrein in cancer. Nature reviews, 2004, 4(11): 876-890.
5. Yousef GM, Luo LY, Scherer SW, et al. Molecular characterization of zyme/protease M/neurosin (PRSS9), a hormonally regulated kallikrein-like serine protease. Genomics. 1999, 1; 62(2): 251-259.
6. Diamandis EP, Yousef GM, Petraki C, et al. Human kallikrein 6 as a biomarker of alzheimer's disease. Clin Biochem. 2000 Nov; 33(8): 663-667.
7. Blaber SI, Scarisbrick IA, Bernett MJ, et al. Enzymatic properties of rat myelencephalon-specific protease. Biochemistry 2002; 41: 1165-73.
8. Iwata A, Maruyama M, Akagi T, et al. Alpha-synuclein degradation by serine protease neurosin: Implication for pathogenesis of synucleinopathies. Hum Mol Genet 2003; 12: 2625-35.
9. Komatsu N, Saijoh K, Toyama T, et al. Multiple tissue kallikrein mRNA and protein expression in normal skin and skin diseases. Br J Dermatol. 2005 Aug; 153(2): 274-281.
10. Yousef GM, Borgono CA, White NM, et al. In silico analysis of the human kallikrein Gene 6. Tumor Biol. 2004; 25 (5-6): 282-289.
11. Yousef GM, Yacoub GM, Polymeris ME, et al. Kallikrein gene downregulation in breast cancer. Br J Cancer 2004; 90: 167-72.
12. Judith A, Clements, Nicole M, Willemsen, et al. The tissue kallikrein family of serine proteases: functional roles in human disease and potential as clinical biomarkers. Critical reviews in clinical laboratory sciences, 2004, 41: 265—312
13 甑林林，朱旬，郑伟等。三苯氧胺耐药乳腺癌细胞中表皮生长因子受体信号通路研究。中华普外科杂志，2006，21(6)：446—448。
14 任玉萍，吴毅平。胰岛素样生长因子—Ⅰ与乳腺癌。医学分子生物学杂志，2006，3(4)：309—312。
15 Naranishi Y, Kodama J, Yo sh inouch iM, et al. The expression of vascular endothelial growth factor and transforming growth facto r-beta associates with angiogenesis in epithelial ovarian cancer. Int J Gynecol, Pathol, 1997, 16: 256—262.
16 张焱，程云英。雌激素调节卵巢癌细胞增殖与分泌转化生长因子的关系。江苏医药杂志，2004，30(11)：824—826。
17. Prezas P, Arlt M J, Viktorov P, et al. Overexpression of the human tissue kallikrein genes KLK4, 5, 6, and 7 increases the malignant phenotype of ovarian cancer cells. Biol Chem, 2006, 387(6): 807-811.
18. Nagahara H, Mimori K, Utsunomiya T, et al. Clinicopathologic and biological significance of kallikrein 6 overexpression in human gastric cancer. Clin Cancer Res, 2005, 11(19 Pt 1): 6800-6806.
19. Ni X, Zhang W, Huang KC, et al. Characterisation of human kallikrein 6/protease M expression in ovarian cancer. Br J Cancer. 2004 16; 91(4): 725-731.
20．柴芸，谢幸等，激素替代治疗与卵巢癌，中国实用妇科与产科杂志，2003，19(10)：637-639．
21．杨永彬，徐伟，卵巢癌肿瘤标志物研究进展，国外医学妇产科学分册2004，(4)：229—232．
22. Polascik TJ, Oesterling JE, Partin AW. Prostate specific antigen: A decade of discovery—what we have learned and where we are going. J Urol 1999; 162: 293-306.
23. Stephan C, Jung K, Diamandis EP, et al. Prostate-specific antigen, its molecular forms, and other kallikrein markers for detection of prostate cancer. Urology, 2002; 59: 2-8.
24. Becker C, Piironen T, Pettersson K, et al. Testing in serum for human glandular kallikrein 2, and free and total prostate specific antigen in biannual screening for prostate cancer. J Urol, 2003; 170: 1169-74.
25. Yu H, Levesque MA, Clark GM, et al. Prognostic value of prostate-specific antigen for women with breast cancer: A large United States cohort study. Clin Cancer Res 1998; 4: 1489-97
26. Yousef GM, Polymeris ME, Yacoub GM, et al. Parallel overexpression of seven kallikrein genes in ovarian cancer.Cancer Res, 2003, 63(9): 2223—2227.
27. Gomis-Ruth FX, Bayes A, Sotiropoulou G, et al. The structure of human prokallikrein 6 reveals a novel activation mechanism for the kallikrein family. J Biol Chem. 2002 Jul 26;277(30):27273-81. Epub 2002 May 16.
28. Anisowicz, A., Sotiropoulou, G., Stenman, G.,et al. A novel protease homolog differentially expressed in breast and ovarian cancer. Mol Med. 1996 Sep;2(5):624-36.
29. Yamashiro K, Tsuruoka N, Kodama S, et al.. Molecular cloning of a novel trypsin-like serine protease (neurosin) preferentially expressed in brain. Biochim Biophys Acta. 1997 Jan 3; 1350(1):11-14.
30. Little SP, Dixon EP, Norris F, et al Zyme, a novel and potentially amyloidogenic enzyme cDNA isolated from Alzheimer's disease brain. J Biol Chem. 1997 Oct 3;272(40):25135-25142.
31. Constantina D. Petraki, Vassiliki N. Karavana, Pavlos T. et al. The spectrum of human Kallikrein 6 (Zyme/Protease M/Neurosin) expression in human tissues as assessed by immunohistochemistry. Histochemistry & Cytochemistry. 2001 49(11): 1431-1441.
32. Ghosh MC, Grass L, Soosaipillai A, et al. Human kallikrein 6 degrades extracellular matrix proteins and may enhance the metastatic potential of tumour cells. Tumour Biol. 2004 Jul-Aug;25(4): 193-199.
33. Hoffman BR, Katsaros D, Scorilas A,et al. Immunofluorometric quantitation and histochemical localisation of kallikrein 6 protein in ovarian cancer tissue: a new independent unfavourable prognostic biomarker. Br J Cancer. 2002 Sep 23;87(7):763-771.
34. Diamandis EP, Scorilas A, Fracchioli S, et al. Human kallikrein 6 (hK6):a new potential serum biomarker for diagnosis and prognosis of ovarian carcinoma. J Clin Oncol. 2003 15;21(6):1035-1043。
35. Werle E Zur Kenntnis des haushalts des Kallikreins. Biochem. 1934; Z 269:415-434
36. Clements JA. The molecular biology of the kallikreins and their roles in inflammation. In: Farmer SG (ed) The Kinin System. Academic Press, San Diego, CA, 1997; vol 5:71-97
37. Diamandis EP, Yousef GM, Luo LY, et al. The new human kallikrein gene family: implications in carcinogenesis. Trends Endocrinol Metab. 2000;11:54-60
38. Yousef GM, Diamandis EP. The new human tissue kallikrein gene family: Structure, function, and association to disease. Endocr Rev 2001; 22: 184-204.
39. Clements J, Hooper J, Dong Y, et al. The expanded human kallikrein (KLK) gene family: Genomic organisation, tissue-specific expression and potential functions. Biol Chem 2001; 382:5-14
40. Rehault S, Monget P, Mazerbourg S,et al. Insulin-like growth factor binding proteins(IGFBPs) as potential physiological substrates for human kallikreins hK2 and hK3. Eur. J. Biochem. 2001,268(10): 2960-2968。
41. Takayama, T. K., McMullen, B. A., Nelson, P. S., Matsumura, M. & Fujikawa, K. Characterization of hK4 (prostase), a prostate-specific serine protease: activation of the precursor of prostate specific antigen (pro-PSA) and single-chain urokinase-type plasminogen activator and degradation of prostatic acid phosphatase. Biochemistry. 2001, 40: 15341-15348.
42. Frenette, G., Tremblay, R. R., Lazure, C. & Dube, J. Y. Prostatic kallikrein hK2, but not prostate-specific antigen (hK3), activates single-chain urokinase-type plasminogen activator. Int. J. Cancer 1997, 71: 897-899.
43. 86. Lai, L. C., Erbas, H., Lennard, T. W. & Peaston, R. T. Prostate-specific antigen in breast cyst fluid: possible role of prostate-specific antigen in hormone-dependent breast cancer. Int. J. Cancer 66, 743-746 (1996).
44 Denmeade SR, Litvinov I, Sokoll LJ, et al. Prostate-specific antigen (PSA) protein does not affect growth of prostate cancer cells in vitro or prostate cancer xenografts in vivo. Prostate, 2003, 56(1):45-53.
45. Goyal J, Smith KM, Cowan JM, et al. The role for NES1 serine protease as a novel tumor suppressor. Cancer Res 1998, 58(21):4782-4786
46. Jin E, Fujiwara M, Pan X, et al. Protease-activated receptor (PAR)-1 and PAR-2 participate in the cell growth of alveolar capillary endothelium in primary lung adenocarcinomas. Cancer, 2003, 97(3):703-713
47. Derynck,R.,Akhurst, R.J,Balmain A, et al. TGF-βsignaling in tumor suppression and cancer progression. Nature Genet.2001,29(2):117-129
48. Fortier AH, Holaday JW, Liang H, et al. Recombinant prostate specific antigen inhibits angiogenesis in vitro and in vivo. Prostate, 2003, 56(3): 212-219
49. Bayes A, Tsetsenis T, Ventura S, et al. Human kallikrein 6 activity is regulated via anautoproteolytic mechanism of activation/inactivation. Biol.Chem.,2004, 385(6):517-524.
50. Kwiatkowski MK, Recker F, Piironen T, et al. In prostatism patients the ratio of human glandular kallikrein to free PSA improves the discrimination between prostate cancer and benign hyperplasia within the diagnostic "gray zone" of total PSA 4 to 10 ng/mL. Urology 1998; 52: 360-5.
51. Nakamura T, Scorilas A, Stephan C, et al. The usefulness of serum human kallikrein 11 for discriminating between prostate cancer and benign prostatic hyperplasia. Cancer Res 2003; 63:6543-65460
52. Yousef GM, Stephan C, Scorilas A, et al. Differential expression of the human kallikrein gene 14 (KLK14) in normal and cancerous prostatic tissues. The Prostate 2003; 56: 287-92.
53. Stephan C, Yousef GM, Scorilas A, et al. Quantitative analysis of kallikrein 15 gene expression in prostate tissue. J Urol 2003; 169: 361—4
54. Yousef GM, Scorilas A, Chang A, et al.Down-regulation of the human kallikrein gene 5 (KLK5) in prostate cancer tissues, Prostate,2002, 51(2): 126-132
55. Nakamura T., Stephan C, Scorilas A,et al. Quantitative analysis of hippostasin/KLK11 gene expression in cancerous and noncancerous prostatic tissues, Urology, 2003, 61(5): 1042-1046
56. Yousef GM, Scorilas A, Kyriakopoulou LG,et al, Human kallikrein gene 5 (KLK5) expression by quantitative PCR: an independent indicator of poor prognosis in breast cancer, Clin. Chem, 2002,48(8) : 1241-1250.
57. Yousef GM, Scorilas A, Nakamura T, et al, The prognostic value of the human kallikrein gene 9 (KLK9) in breast cancer, Breast Cancer Res. Treat. 2003, 78(2): 149-158.
58 .Talieri M, Diamandis EP, Gourgiotis D, et al, Expression analysis of the human kallikrein 7 (KLK7) in breast tumors: a new potential biomarker for prognosis of breast carcinoma, Thromb. Haemost. 2004, 91(1): 180-186.
59. Luo LY, Diamandis EP, Look MP, et al, Higher expression of human kallikrein 10 in breast cancer tissue predicts tamoxifen resistance, Br J. Cancer, 2002, 86(11): 1790-1796.
60. Shvartsman HS, Lu KH, Lee J, et al. Overexpression of kallikrein 10 in epithelial ovarian carcinomas. Gynecol Oncol 2003, 90(1): 44-50.
61. Luo LY, Yousef G and Diamandis EP. Human tissue kallikrein and testicular cancer. APMIS, 2003., 111(1): 225-233
62. Santin AD, Cane' S, Bellone S, et al.The serine protease stratum corneum chymotryptic enzyme (kallikrein 7)is highly overexpressed in squamous cervical cancer cells, Gynecol. Onco1.,2004, 94(2): 283-288.
63. Cane S, Bignotti E, Bellone S, et al. The novel serine protease tumor associated differentially expressed gene-14 (KLK8/Neuropsin/Ovasin) is highly overexpressed in cervical cancer. Am. J. Obstet. Gynecol. 2004, 190(1): 60-66.
64. Bhattacharjee A, Richards WG, Staunton J, et al. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci USA 2001,98(24): 13790-13795.
65. Iacobuzio-Donahue CA, Ashfaq R, Maitra A, et al. Highly expressed genes in pancreatic ductal adenocarcinomas: a comprehensive characterization and comparison of the transcription profiles obtained from three major technologies. Cancer Res, 2003, 63 (24): 8614-22
66. Yousef GM, Borgono CA, Popalis C, et al.In-silico analysis of kallikrein gene expression in pancreatic and colon cancers, Anticancer Res,2004,24(1): 43-51.
67. Kazuhiko O, Tohru U, Koshi M,et al. Clinical Significance of Human Kallikrein Gene 6 Messenger RNA Expression in Colorectal Cancer.Clinical Cancer Research, 2005,11(4):2889-2893.
2．胡成进，沈茜，陈英剑等。乳腺癌KLK6基因表达的实时荧光定量法的建立及初步应用。中华检验医学杂志，2005，28：610-612．
3. Clements JA, Willemsen NW, Myers SA, et al. The Tissue Kallikrein Family of Serine Proteases: Functional Roles in Human Disease and Potential as Clinical Biomarkers. Critical Reviews in Clinical Laboratory Sciences, 2004, 41(3): 265-312.
4. Borgofio CA, Diamandis EP. The emerging roles of human tissue kallikrein in cancer. Nature reviews, 2004, 4(11): 876-890.
5. Yousef GM, Luo LY, Scherer SW, et al. Molecular characterization of zyme/protease M/neurosin (PRSS9), a hormonally regulated kallikrein-like serine protease. Genomics. 1999, 1; 62(2): 251-259.
6. Diamandis EP, Yousef GM, Petraki C, et al. Human kallikrein 6 as a biomarker of alzheimer's disease. Clin Biochem. 2000 Nov; 33(8): 663-667.
7. Blaber SI, Scarisbrick IA, Bernett MJ, et al. Enzymatic properties of rat myelencephalon-specific protease. Biochemistry 2002; 41: 1165-73.
8. Iwata A, Maruyama M, Akagi T, et al. Alpha-synuclein degradation by serine protease neurosin: Implication for pathogenesis of synucleinopathies. Hum Mol Genet 2003; 12: 2625-35.
9. Komatsu N, Saijoh K, Toyama T, et al. Multiple tissue kallikrein mRNA and protein expression in normal skin and skin diseases. Br J Dermatol. 2005 Aug; 153(2): 274-281.
10. Yousef GM, Borgono CA, White NM, et al. In silico analysis of the human kallikrein Gene 6. Tumor Biol. 2004; 25 (5-6): 282-289.
11. Yousef GM, Yacoub GM, Polymeris ME, et al. Kallikrein gene downregulation in breast cancer. Br J Cancer 2004; 90: 167-72.
12. Judith A, Clements, Nicole M, Willemsen, et al. The tissue kallikrein family of serine proteases: functional roles in human disease and potential as clinical biomarkers. Critical reviews in clinical laboratory sciences, 2004, 41: 265—312
13 甑林林，朱旬，郑伟等。三苯氧胺耐药乳腺癌细胞中表皮生长因子受体信号通路研究。中华普外科杂志，2006，21(6)：446—448。
14 任玉萍，吴毅平。胰岛素样生长因子—Ⅰ与乳腺癌。医学分子生物学杂志，2006，3(4)：309—312。
15 Naranishi Y, Kodama J, Yo sh inouch iM, et al. The expression of vascular endothelial growth factor and transforming growth facto r-beta associates with angiogenesis in epithelial ovarian cancer. Int J Gynecol, Pathol, 1997, 16: 256—262.
16 张焱，程云英。雌激素调节卵巢癌细胞增殖与分泌转化生长因子的关系。江苏医药杂志，2004，30(11)：824—826。
17. Prezas P, Arlt M J, Viktorov P, et al. Overexpression of the human tissue kallikrein genes KLK4, 5, 6, and 7 increases the malignant phenotype of ovarian cancer cells. Biol Chem, 2006, 387(6): 807-811.
18. Nagahara H, Mimori K, Utsunomiya T, et al. Clinicopathologic and biological significance of kallikrein 6 overexpression in human gastric cancer. Clin Cancer Res, 2005, 11(19 Pt 1): 6800-6806.
19. Ni X, Zhang W, Huang KC, et al. Characterisation of human kallikrein 6/protease M expression in ovarian cancer. Br J Cancer. 2004 16; 91(4): 725-731.
20．柴芸，谢幸等，激素替代治疗与卵巢癌，中国实用妇科与产科杂志，2003，19(10)：637-639．
21．杨永彬，徐伟，卵巢癌肿瘤标志物研究进展，国外医学妇产科学分册2004，(4)：229—232．
22. Polascik TJ, Oesterling JE, Partin AW. Prostate specific antigen: A decade of discovery—what we have learned and where we are going. J Urol 1999; 162: 293-306.
23. Stephan C, Jung K, Diamandis EP, et al. Prostate-specific antigen, its molecular forms, and other kallikrein markers for detection of prostate cancer. Urology, 2002; 59: 2-8.
24. Becker C, Piironen T, Pettersson K, et al. Testing in serum for human glandular kallikrein 2, and free and total prostate specific antigen in biannual screening for prostate cancer. J Urol, 2003; 170: 1169-74.
25. Yu H, Levesque MA, Clark GM, et al. Prognostic value of prostate-specific antigen for women with breast cancer: A large United States cohort study. Clin Cancer Res 1998; 4: 1489-97
26. Yousef GM, Polymeris ME, Yacoub GM, et al. Parallel overexpression of seven kallikrein genes in ovarian cancer.Cancer Res, 2003, 63(9): 2223—2227.
27. Gomis-Ruth FX, Bayes A, Sotiropoulou G, et al. The structure of human prokallikrein 6 reveals a novel activation mechanism for the kallikrein family. J Biol Chem. 2002 Jul 26;277(30):27273-81. Epub 2002 May 16.
28. Anisowicz, A., Sotiropoulou, G., Stenman, G.,et al. A novel protease homolog differentially expressed in breast and ovarian cancer. Mol Med. 1996 Sep;2(5):624-36.
29. Yamashiro K, Tsuruoka N, Kodama S, et al.. Molecular cloning of a novel trypsin-like serine protease (neurosin) preferentially expressed in brain. Biochim Biophys Acta. 1997 Jan 3; 1350(1):11-14.
30. Little SP, Dixon EP, Norris F, et al Zyme, a novel and potentially amyloidogenic enzyme cDNA isolated from Alzheimer's disease brain. J Biol Chem. 1997 Oct 3;272(40):25135-25142.
31. Constantina D. Petraki, Vassiliki N. Karavana, Pavlos T. et al. The spectrum of human Kallikrein 6 (Zyme/Protease M/Neurosin) expression in human tissues as assessed by immunohistochemistry. Histochemistry & Cytochemistry. 2001 49(11): 1431-1441.
32. Ghosh MC, Grass L, Soosaipillai A, et al. Human kallikrein 6 degrades extracellular matrix proteins and may enhance the metastatic potential of tumour cells. Tumour Biol. 2004 Jul-Aug;25(4): 193-199.
33. Hoffman BR, Katsaros D, Scorilas A,et al. Immunofluorometric quantitation and histochemical localisation of kallikrein 6 protein in ovarian cancer tissue: a new independent unfavourable prognostic biomarker. Br J Cancer. 2002 Sep 23;87(7):763-771.
34. Diamandis EP, Scorilas A, Fracchioli S, et al. Human kallikrein 6 (hK6):a new potential serum biomarker for diagnosis and prognosis of ovarian carcinoma. J Clin Oncol. 2003 15;21(6):1035-1043。
35. Werle E Zur Kenntnis des haushalts des Kallikreins. Biochem. 1934; Z 269:415-434
36. Clements JA. The molecular biology of the kallikreins and their roles in inflammation. In: Farmer SG (ed) The Kinin System. Academic Press, San Diego, CA, 1997; vol 5:71-97
37. Diamandis EP, Yousef GM, Luo LY, et al. The new human kallikrein gene family: implications in carcinogenesis. Trends Endocrinol Metab. 2000;11:54-60
38. Yousef GM, Diamandis EP. The new human tissue kallikrein gene family: Structure, function, and association to disease. Endocr Rev 2001; 22: 184-204.
39. Clements J, Hooper J, Dong Y, et al. The expanded human kallikrein (KLK) gene family: Genomic organisation, tissue-specific expression and potential functions. Biol Chem 2001; 382:5-14
40. Rehault S, Monget P, Mazerbourg S,et al. Insulin-like growth factor binding proteins(IGFBPs) as potential physiological substrates for human kallikreins hK2 and hK3. Eur. J. Biochem. 2001,268(10): 2960-2968。
41. Takayama, T. K., McMullen, B. A., Nelson, P. S., Matsumura, M. & Fujikawa, K. Characterization of hK4 (prostase), a prostate-specific serine protease: activation of the precursor of prostate specific antigen (pro-PSA) and single-chain urokinase-type plasminogen activator and degradation of prostatic acid phosphatase. Biochemistry. 2001, 40: 15341-15348.
42. Frenette, G., Tremblay, R. R., Lazure, C. & Dube, J. Y. Prostatic kallikrein hK2, but not prostate-specific antigen (hK3), activates single-chain urokinase-type plasminogen activator. Int. J. Cancer 1997, 71: 897-899.
43. 86. Lai, L. C., Erbas, H., Lennard, T. W. & Peaston, R. T. Prostate-specific antigen in breast cyst fluid: possible role of prostate-specific antigen in hormone-dependent breast cancer. Int. J. Cancer 66, 743-746 (1996).
44 Denmeade SR, Litvinov I, Sokoll LJ, et al. Prostate-specific antigen (PSA) protein does not affect growth of prostate cancer cells in vitro or prostate cancer xenografts in vivo. Prostate, 2003, 56(1):45-53.
45. Goyal J, Smith KM, Cowan JM, et al. The role for NES1 serine protease as a novel tumor suppressor. Cancer Res 1998, 58(21):4782-4786
46. Jin E, Fujiwara M, Pan X, et al. Protease-activated receptor (PAR)-1 and PAR-2 participate in the cell growth of alveolar capillary endothelium in primary lung adenocarcinomas. Cancer, 2003, 97(3):703-713
47. Derynck,R.,Akhurst, R.J,Balmain A, et al. TGF-βsignaling in tumor suppression and cancer progression. Nature Genet.2001,29(2):117-129
48. Fortier AH, Holaday JW, Liang H, et al. Recombinant prostate specific antigen inhibits angiogenesis in vitro and in vivo. Prostate, 2003, 56(3): 212-219
49. Bayes A, Tsetsenis T, Ventura S, et al. Human kallikrein 6 activity is regulated via anautoproteolytic mechanism of activation/inactivation. Biol.Chem.,2004, 385(6):517-524.
50. Kwiatkowski MK, Recker F, Piironen T, et al. In prostatism patients the ratio of human glandular kallikrein to free PSA improves the discrimination between prostate cancer and benign hyperplasia within the diagnostic "gray zone" of total PSA 4 to 10 ng/mL. Urology 1998; 52: 360-5.
51. Nakamura T, Scorilas A, Stephan C, et al. The usefulness of serum human kallikrein 11 for discriminating between prostate cancer and benign prostatic hyperplasia. Cancer Res 2003; 63:6543-65460
52. Yousef GM, Stephan C, Scorilas A, et al. Differential expression of the human kallikrein gene 14 (KLK14) in normal and cancerous prostatic tissues. The Prostate 2003; 56: 287-92.
53. Stephan C, Yousef GM, Scorilas A, et al. Quantitative analysis of kallikrein 15 gene expression in prostate tissue. J Urol 2003; 169: 361—4
54. Yousef GM, Scorilas A, Chang A, et al.Down-regulation of the human kallikrein gene 5 (KLK5) in prostate cancer tissues, Prostate,2002, 51(2): 126-132
55. Nakamura T., Stephan C, Scorilas A,et al. Quantitative analysis of hippostasin/KLK11 gene expression in cancerous and noncancerous prostatic tissues, Urology, 2003, 61(5): 1042-1046
56. Yousef GM, Scorilas A, Kyriakopoulou LG,et al, Human kallikrein gene 5 (KLK5) expression by quantitative PCR: an independent indicator of poor prognosis in breast cancer, Clin. Chem, 2002,48(8) : 1241-1250.
57. Yousef GM, Scorilas A, Nakamura T, et al, The prognostic value of the human kallikrein gene 9 (KLK9) in breast cancer, Breast Cancer Res. Treat. 2003, 78(2): 149-158.
58 .Talieri M, Diamandis EP, Gourgiotis D, et al, Expression analysis of the human kallikrein 7 (KLK7) in breast tumors: a new potential biomarker for prognosis of breast carcinoma, Thromb. Haemost. 2004, 91(1): 180-186.
59. Luo LY, Diamandis EP, Look MP, et al, Higher expression of human kallikrein 10 in breast cancer tissue predicts tamoxifen resistance, Br J. Cancer, 2002, 86(11): 1790-1796.
60. Shvartsman HS, Lu KH, Lee J, et al. Overexpression of kallikrein 10 in epithelial ovarian carcinomas. Gynecol Oncol 2003, 90(1): 44-50.
61. Luo LY, Yousef G and Diamandis EP. Human tissue kallikrein and testicular cancer. APMIS, 2003., 111(1): 225-233
62. Santin AD, Cane' S, Bellone S, et al.The serine protease stratum corneum chymotryptic enzyme (kallikrein 7)is highly overexpressed in squamous cervical cancer cells, Gynecol. Onco1.,2004, 94(2): 283-288.
63. Cane S, Bignotti E, Bellone S, et al. The novel serine protease tumor associated differentially expressed gene-14 (KLK8/Neuropsin/Ovasin) is highly overexpressed in cervical cancer. Am. J. Obstet. Gynecol. 2004, 190(1): 60-66.
64. Bhattacharjee A, Richards WG, Staunton J, et al. Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses. Proc Natl Acad Sci USA 2001,98(24): 13790-13795.
65. Iacobuzio-Donahue CA, Ashfaq R, Maitra A, et al. Highly expressed genes in pancreatic ductal adenocarcinomas: a comprehensive characterization and comparison of the transcription profiles obtained from three major technologies. Cancer Res, 2003, 63 (24): 8614-22
66. Yousef GM, Borgono CA, Popalis C, et al.In-silico analysis of kallikrein gene expression in pancreatic and colon cancers, Anticancer Res,2004,24(1): 43-51.
67. Kazuhiko O, Tohru U, Koshi M,et al. Clinical Significance of Human Kallikrein Gene 6 Messenger RNA Expression in Colorectal Cancer.Clinical Cancer Research, 2005,11(4):2889-2893.