Department of Tumor Biology, Institute for Cancer Research, Radiumhospitalet, Oslo University Hospital, Oslo, Norway.
BMC Cancer. 2010 Jan 15;10:17. doi: 10.1186/1471-2407-10-17.
High activity of cysteine proteases such as legumain and the cathepsins have been shown to facilitate growth and invasion of a variety of tumor types. In breast cancer, several recent studies have indicated that loss of the cysteine protease inhibitor cystatin E/M leads to increased growth and metastasis. Although cystatin E/M is normally expressed in the skin, its role in cysteine protease regulation and progression of malignant melanoma has not been studied.
A panel of various non-melanoma and melanoma cell lines was used. Cystatin E/M and C were analyzed in cell media by immunoblotting and ELISA. Legumain, cathepsin B and L were analyzed in cell lysates by immunoblotting and their enzymatic activities were analyzed by peptide substrates. Two melanoma cell lines lacking detectable secretion of cystatin E/M were transfected with a cystatin E/M expression plasmid (pCST6), and migration and invasiveness were studied by a Matrigel invasion assay.
Cystatin E/M was undetectable in media from all established melanoma cell lines examined, whereas strong immunobands were detected in two of five primary melanoma lines and in two of six lines derived from patients with metastatic disease. Among the four melanoma lines secreting cystatin E/M, the glycosylated form (17 kD) was predominant compared to the non-glycosylated form (14 kD). Legumain, cathepsin B and L were expressed and active in most of the cell lines, although at low levels in the melanomas expressing cystatin E/M. In the melanoma lines where cystatin E/M was secreted, cystatin C was generally absent or expressed at a very low level. When melanoma cells lacking secretion of cystatin E/M were transfected with pCST6, their intracellular legumain activity was significantly inhibited. In contrast, cathepsin B activity was not affected. Furthermore, invasion was suppressed in cystatin E/M over-expressing melanoma cell lines as measured by the transwell Matrigel assay.
These results suggest that the level of cystatin E/M regulates legumain activity and hence the invasive potential of human melanoma cells.
高活性的半胱氨酸蛋白酶，如组织蛋白酶和组织蛋白酶，已被证明有利于多种肿瘤类型的生长和侵袭。在乳腺癌中，最近的几项研究表明，半胱氨酸蛋白酶抑制剂胱抑素 E/M 的缺失导致生长和转移增加。尽管胱抑素 E/M 在皮肤中正常表达，但它在半胱氨酸蛋白酶调节和恶性黑色素瘤进展中的作用尚未得到研究。
使用了一系列不同的非黑色素瘤和黑色素瘤细胞系。通过免疫印迹和 ELISA 分析细胞培养基中的胱抑素 E/M 和 C。通过免疫印迹分析细胞裂解物中的组织蛋白酶 B 和 L，并通过肽底物分析其酶活性。两个缺乏可检测的胱抑素 E/M 分泌的黑色素瘤细胞系用胱抑素 E/M 表达质粒（pCST6）转染，并通过 Matrigel 侵袭测定研究迁移和侵袭。
在所有检查的黑色素瘤细胞系的培养基中均未检测到胱抑素 E/M，而在五个原发性黑色素瘤系中的两个和六个转移性疾病患者衍生的系中的两个中检测到强免疫带。在四个分泌胱抑素 E/M 的黑色素瘤系中，糖基化形式（17 kD）比非糖基化形式（14 kD）更为主要。大多数细胞系表达并激活了组织蛋白酶 B 和 L，但在表达胱抑素 E/M 的黑色素瘤中水平较低。在胱抑素 E/M 分泌的黑色素瘤系中，胱抑素 C 通常不存在或表达水平非常低。当缺乏胱抑素 E/M 分泌的黑色素瘤细胞用 pCST6 转染时，其细胞内组织蛋白酶 B 活性显著受到抑制。相反，组织蛋白酶 B 活性不受影响。此外，在跨膜 Matrigel 测定中，胱抑素 E/M 过表达的黑色素瘤细胞系的侵袭受到抑制。
这些结果表明，胱抑素 E/M 的水平调节组织蛋白酶 B 的活性，从而调节人黑色素瘤细胞的侵袭潜力。
