Uhlman Andrew, Folkers Kelly, Liston Jared, Pancholi Harshida, Hinton Ayana
Biology Department, Denison University, 100 W. College St, Granville, OH, 43023, USA.
Cancer Microenviron. 2017 Dec;10(1-3):49-56. doi: 10.1007/s12307-017-0196-7. Epub 2017 Aug 2.
Studies indicate secreted cathepsins are involved in metastasis. V-ATPases, which are necessary for activating intracellular cathepsins, also play a role in metastasis and are targeted to the plasma membrane of metastatic breast cancer cells. We are interested in a connection between cell surface V-ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA-MB231 cells. We investigated whether V-ATPase inhibition would reduce the activity of secreted cathepsin B and cathepsin L. Using cell lysates and conditioned media, we measured cathepsin B and L activity within and outside of the cells. We found different forms of cathepsin B and L were secreted representing the pre-pro, pro and active forms of the proteases. Cathepsin B activity was higher than cathepsin L in conditioned media and in cell lysates. V-ATPase inhibition by concanamycin A decreased cathepsin B activity in conditioned media and significantly decreased cathepsin B activity in cell lysates. Cathepsin L activity showed a slight decrease in cell lysates. Changes in the activity of secreted and intracellular cathepsins following V-ATPase inhibition were supported by changes in the amounts of pro and active forms of cathepsin B in conditioned media and cathepsins B and L in cell lysates. Overall, our data shows that inactive forms of cathepsins B and L are secreted from the MB231 cells and V-ATPase activity is important for the activation of secreted cathepsin B. This indicates a connection between cell surface V-ATPases in metastatic breast cancer cells and the function of secreted cathepsin B.
研究表明，分泌型组织蛋白酶参与转移过程。V-ATP酶是激活细胞内组织蛋白酶所必需的，在转移过程中也发挥作用，并且靶向转移性乳腺癌细胞的质膜。我们对细胞表面V-ATP酶、分泌型组织蛋白酶的激活与MDA-MB231细胞的转移表型之间的联系感兴趣。我们研究了抑制V-ATP酶是否会降低分泌型组织蛋白酶B和组织蛋白酶L的活性。我们使用细胞裂解物和条件培养基，测量了细胞内外组织蛋白酶B和L的活性。我们发现分泌出了不同形式的组织蛋白酶B和L，分别代表蛋白酶的前原、原和活性形式。在条件培养基和细胞裂解物中，组织蛋白酶B的活性高于组织蛋白酶L。通过 concanamycin A抑制V-ATP酶可降低条件培养基中组织蛋白酶B的活性，并显著降低细胞裂解物中组织蛋白酶B的活性。组织蛋白酶L的活性在细胞裂解物中略有下降。条件培养基中组织蛋白酶B的原和活性形式以及细胞裂解物中组织蛋白酶B和L的量的变化支持了V-ATP酶抑制后分泌型和细胞内组织蛋白酶活性的变化。总体而言，我们的数据表明，组织蛋白酶B和L的无活性形式从MB231细胞中分泌出来，V-ATP酶活性对于分泌型组织蛋白酶B的激活很重要。这表明转移性乳腺癌细胞中的细胞表面V-ATP酶与分泌型组织蛋白酶B的功能之间存在联系。
