Wilson Michael J, Lind Jeremy, Sinha Akhouri A
Research Service, Minneapolis VA Medical Center, University of Minnesota, Minneapolis, MN 55417, United States; Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55417, United States; Masonic Comprehensive Cancer Center, University of Minnesota, Minneapolis, MN 55417, United States.
Research Service, Minneapolis VA Medical Center, University of Minnesota, Minneapolis, MN 55417, United States; Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55417, United States.
Exp Mol Pathol. 2015 Aug;99(1):39-43. doi: 10.1016/j.yexmp.2015.04.008. Epub 2015 Apr 22.
Induction of new proteinase activities that may process growth factors, modify cell surface receptors, cleave extracellular matrix proteins, etc. is considered fundamental in carcinogenesis. The purpose of this study was to characterize a novel proteinase activity induced in sex accessory gland cancers (about 70% in seminal vesicles) of adult male Lobund-Wistar rats by a single injection of N-nitroso-N-methylurea (NMU; 25mg/kg) followed by implanted testosterone propionate (45mg in silastic tubing every 2months) treatment for 10-14months. A 28kDa proteinase activity was detected in tumor extracts using SDS-gelatin gel zymography with incubations done without CaCl2. Its activity was stimulated 15 fold by heparin (optimal activity 1.5-3.0μg/lane) added to the tissue extract-SDS sample buffer prior to electrophoresis. No 28kDa heparin-stimulated proteinase (H-SP) was found in the dorsal, lateral and anterior (coagulating gland) prostate lobes or seminal vesicles of untreated adult rats, but there was a 26-30kDa Ca(2+)-independent proteinase activity in the ventral prostate that showed limited heparin stimulation. The 28kDa H-SP was completely inhibited by 1.0mM 4-(2-aminoethyl)benzenesulfonylfluoride (AESBF) indicating that it was a serine-type proteinase. Other types of proteinase inhibitors were without effect, including serine proteinase inhibitors benzamidine, tranexamic acid and ε-aminocaproic acid. Proteinase activities of about 28kDa were found with casein, fibrinogen or carboxymethylated transferrin as substrate, however, these activities were not stimulated by heparin. Similar levels of activities of the 28kDa H-SP were found in primary tumors and their metastases, but little/no activity was detected in serum, even from rats with large tumor volume and metastases. These data demonstrate overexpression of a heparin-stimulated 28kDa serine proteinase in the primary tumors of sex accessory gland cancers and their metastases. This proteinase either does not leak into or is inactivated in the blood. The role of this proteinase remains to be determined, but its possible interaction with extracellular glycosaminoglycans could focus its proteolytic activity in the tumor microenvironment and affect tumor growth.
诱导可能加工生长因子、修饰细胞表面受体、裂解细胞外基质蛋白等的新蛋白酶活性被认为是致癌过程中的关键因素。本研究的目的是对成年雄性Lobund-Wistar大鼠性腺癌（精囊癌约占70%）中诱导产生的一种新型蛋白酶活性进行表征。通过单次注射N-亚硝基-N-甲基脲（NMU；25mg/kg），随后植入丙酸睾酮（每2个月在硅橡胶管中植入45mg），持续治疗10 - 14个月。使用不添加CaCl₂进行孵育的SDS - 明胶凝胶酶谱法在肿瘤提取物中检测到一种28kDa的蛋白酶活性。在电泳前向组织提取物 - SDS样品缓冲液中添加肝素（最佳活性为1.5 - 3.0μg/泳道）可使其活性提高15倍。在未处理的成年大鼠的背侧、外侧和前部（凝固腺）前列腺叶或精囊中未发现28kDa的肝素刺激蛋白酶（H - SP），但在腹侧前列腺中有26 - 30kDa的不依赖Ca²⁺的蛋白酶活性，其肝素刺激作用有限。28kDa的H - SP被1.0mM的4 -（2 - 氨基乙基）苯磺酰氟（AESBF）完全抑制，表明它是一种丝氨酸型蛋白酶。其他类型的蛋白酶抑制剂无效，包括丝氨酸蛋白酶抑制剂苯甲脒、氨甲环酸和ε - 氨基己酸。以酪蛋白、纤维蛋白原或羧甲基化转铁蛋白为底物时，发现了约28kDa的蛋白酶活性，然而，这些活性不受肝素刺激。在原发性肿瘤及其转移灶中发现了相似水平的28kDa H - SP活性，但在血清中几乎未检测到活性，即使是肿瘤体积大且有转移的大鼠血清中也是如此。这些数据表明肝素刺激的28kDa丝氨酸蛋白酶在性腺癌的原发性肿瘤及其转移灶中过表达。这种蛋白酶要么不泄漏到血液中，要么在血液中失活。这种蛋白酶的作用尚待确定，但其与细胞外糖胺聚糖的可能相互作用可能使其蛋白水解活性集中在肿瘤微环境中并影响肿瘤生长。
