Horak I R, Latyshko N V, Hudkova O O, Tokarchuk K O, Kishko T O, Yusova O I, Drobot L B, Tykhomyrov A A
Palladin Institute of Biochemistry, NAS of Ukraine, Kyiv 01054, Ukraine.
Exp Oncol. 2022 May;44(1):31-38. doi: 10.32471/exp-oncology.2312-8852.vol-44-no-1.17241.
Cell surface plasmin is involved in tumor growth and metastatic dissemination by regulating cancer cells adhesion, migration and invasion. Plasmin-induced cell detachment is accompanied by an increased rate of reactive oxygen species (ROS) generation and cell death. However, cancer cells acquire the ability to develop adaptive mechanisms to resist ROS-mediated apoptosis.
To establish the role of adaptor protein Ruk/CIN85 in the control of viability and redox balance in breast adenocarcinoma cells exposed to plasmin(ogen).
Mouse 4T1 cells with the stable overexpression of adaptor protein Ruk/CIN85 (RukUp subline) and corresponding control (Mock subline) were treated with Glu-plasminogen (1-100 nM). Plasminogen to plasmin conversion was monitored spectrophotometrically by cleavage of the specific chromogenic substrate S2251. Specific uPA inhibitor BC11 was used to verify the uPA-mediated mechanism of plasminogen pericellular activation by 4T1 cells. Cell survival rate was assessed by MTT-test and cell proliferation was estimated by colony formation assay. Enzymatic activities of catalase, glutathione peroxidase, superoxide dismutase, as well as hydrogen peroxide (H2O2) levels were measured by spectrophotomertric and fluorometric assays. The intracellular ROS generation was monitored by flow cytometry using H2DCF-DA fluorescent probe.
Plasminogen was shown to be converted into an active proteinase plasmin on the surface of carcinoma cells in uPA-dependent manner. Plasmin(ogen) suppressed proliferation and affected survival of both studied 4T1 sublines. However, RukUp cells displayed higher resistance to plasmin(ogen)-induced cytotoxicity than Mock cells. Plasmin(ogen) promoted significant elevation in ROS generation rate in cells with the basal level of Ruk/CIN85 expression. In contrast, RukUp cells appear to be more effective in counteracting prooxidant changes due to the activation of some enzymes of the glutathione system, in particular glutathione peroxidase, and a concomitant decrease of H2O2 accumulation.
Adaptor protein Ruk/CIN85 is involved in the regulation of redox homeostasis in cancer cells to maintain levels of ROS, thus promoting redox adaptation in cancer cells exposed to plasmin(ogen). Thus, Ruk/CIN85 may represent one of the relevant targets in order to diminish the resistance of cancer cells to ROS-mediated apoptosis.
细胞表面纤溶酶通过调节癌细胞的黏附、迁移和侵袭参与肿瘤生长和转移扩散。纤溶酶诱导的细胞脱离伴随着活性氧（ROS）生成速率的增加和细胞死亡。然而，癌细胞获得了发展适应性机制以抵抗ROS介导的细胞凋亡的能力。
确定衔接蛋白Ruk/CIN85在暴露于纤溶酶（原）的乳腺腺癌细胞的活力控制和氧化还原平衡中的作用。
用谷氨酸纤溶酶原（1-100 nM）处理稳定过表达衔接蛋白Ruk/CIN85的小鼠4T1细胞（RukUp亚系）和相应对照（Mock亚系）。通过特异性显色底物S2251的裂解，用分光光度法监测纤溶酶原向纤溶酶的转化。使用特异性尿激酶型纤溶酶原激活物（uPA）抑制剂BC11来验证4T1细胞uPA介导的纤溶酶原细胞周围激活机制。通过MTT试验评估细胞存活率，通过集落形成试验估计细胞增殖。通过分光光度法和荧光法测定过氧化氢酶、谷胱甘肽过氧化物酶、超氧化物歧化酶的酶活性以及过氧化氢（H2O2）水平。使用H2DCF-DA荧光探针通过流式细胞术监测细胞内ROS的生成。
纤溶酶原在癌细胞表面以uPA依赖的方式转化为活性蛋白酶纤溶酶。纤溶酶（原）抑制了两个研究的4T1亚系的增殖并影响其存活。然而，RukUp细胞对纤溶酶（原）诱导的细胞毒性的抗性高于Mock细胞。在Ruk/CIN85表达处于基础水平的细胞中，纤溶酶（原）促进了ROS生成速率的显著升高。相反，由于谷胱甘肽系统的一些酶，特别是谷胱甘肽过氧化物酶的激活以及H2O2积累的同时减少，RukUp细胞在对抗促氧化剂变化方面似乎更有效。
衔接蛋白Ruk/CIN85参与癌细胞氧化还原稳态的调节以维持ROS水平，从而促进暴露于纤溶酶（原）的癌细胞的氧化还原适应。因此，Ruk/CIN85可能是降低癌细胞对ROS介导的细胞凋亡抗性的相关靶点之一。
