Horak I.R., Latyshko N.V., Hudkova O.O., Tokarchuk K.O., Kishko T.O., Yusova O.I., Drobot L.B., Tykhomyrov A.A.*

Summary. Background: 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. Aim: 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). Materials and Methods: 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. Results: 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. Conclusion: 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.

DOI: 10.32471/exp-oncology.2312-8852.vol-44-no-1.17241

Submitted: October 20, 2021.
*Correspondence: E-mail:
Abbreviations used: CAT — catalase; FBS — fetal bovine serum; GPx — glutathione peroxidase; H2DCF-DA — 2΄,7΄-dichlorodihydrofluorescein diacetate; NADPH — nicotinamide adenine dinucleotide phosphate; PAR — protease-activated receptor; Pg — plasminogen; ROS — reactive oxygen species; Ruk/CIN85 — regulator for ubiquitous kinase/Cbl-interacting protein of 85K; SOD — superoxide dismutase; uPA — urokinase-type plasminogen activator.

Proteins of plasminogen (Pg)/plasmin system play crucial roles in the control of tumor-associated processes including cell proliferation, apoptosis, survival, metabolic and morphological plasticity, adhesion, and migration [1]. Plasmin (EC, which is abundantly converted from its zymogen Pg on the surface of cancer cells, contributes to the invasive and metastatic capacities of these cells [2]. It is well documented that plasmin-mediated cell detachment induces apoptosis/anoikis in normal cells [3]. However, many types of cancer cells develop specific mechanisms to abrogate anoikis and survive after losing adhesive contacts, which contribute to their invasiveness and metastasis [4]. Therefore, the study of the molecular mechanisms that ensure the survival of cancer cells during plasmin exposure is necessary for the development of new anticancer drugs to prevent tumor metastasis.

The mechanisms involved in the regulation of redox balance in cancer cells are an integral part of the signaling networks that control the cell cycle, proliferation, and migration. Compared with normal cells, cancer cells produced increased levels of reactive oxygen species (ROS) that are recognized as key second messengers to drive proliferation, metabolic reprogramming, autophagy, resistance to apoptosis, and other events required for tumor progression. On the other hand, excessive generation of ROS can increase oxidative stress and induce cancer cell death [5]. Plasmin can influence intracellular ROS levels by stimulating protease-activated receptors (PARs) and activating prooxidative processes in detached cells through interrupting integrin-mediated signaling [6, 7]. In this case, activation of antioxidant defense systems, including glutathione-related enzyme system, can maintain ROS at a necessary level to sustain protumorigenic signaling pathways without inducing cancer cell death [8]. Previous studies implicated a tight relationship between ROS production and the expression of urokinase-type Pg activator (uPA) in the control of tumor invasion and metastasis [9]. Nevertheless, the molecular participants, which govern redox balance during plasmin-induced oxidative stress in cancer cells, are still poorly studied. Regulator for ubiquitous kinase/Cbl-interacting protein of 85K (Ruk/CIN85), UniProtKB Q96B97 (human), Q8R550 (mouse) is an adaptor/scaffolding protein encoded by SH3KBP1 gene. It regulates diverse signal transduction to be involved in several important cellular processes, including regulation of activated receptor tyrosine kinases, modulation of cytoskeletal rearrangements, vesicle-mediated transport, epithelial-mesenchymal transition, cell adhesion and migration, programmed cell death [10, 11]. Our previous findings demonstrated that an enhanced level of Ruk/CIN85 contributes to breast cancer cells malignancy [12]. Also, Ruk/CIN85 has been shown to affect redox balance of adenocarcinoma cell lines [13, 14]. Despite this, the exact functions of Ruk/CIN85 in the regulation of redox balance in cancer cells remain largely elusive. Thus, the aim of the present study was to investigate the role of Ruk/CIN85 in the regulation of redox balance in mouse breast adenocarcinoma 4T1 cells exposed to plasmin(ogen).


Cell culture. Mouse breast adenocarcinoma 4T1 cells with stable overexpression of full-length isoform of Ruk/CIN85 (RukUp subline) and corresponding control subline (Mock) were generated using calcium-phosphate transfection of parental cells with a plasmid encoding the full-length isoform of Ruk/CIN85 or with an empty vector as described earlier [15]. Cells were cultured under standard conditions in RPMI-1640 medium supplied with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco). Prior to treatment with Pg, cells were incubated overnight in a serum-free medium.

Pg to plasmin conversion. Glu-Pg  was purified from the fresh donor’s blood plasma with the use of affinity chromatography on Lys-sepharose column by elution with 0.2 M 6-aminohexanoic acid as described earlier [16]. Pg (0.1 µM) was added to the intact cells or cells pre-incubated for 1 h with the uPA inhibitor BC11 (1, 10, or 20 µM) grown in a 96-well plate (5•103 in 0.1 mL per well). Then, chromogenic substrate S2251 (0.3 mM) was added and mixed. The absorbance was monitored at 405/492 nm wavelength for 150 min by microreader Titertek Multiskan MC (Finland).

Colony formation assay. Cells were seeded onto a 12-well plate (500 cells per well) in a serum-free medium, treated with 10–100 nM of Pg for 24 h, and then cultured for another 12 days in a complete medium. Then, colonies were fixed with methanol, stained with crystal violet dye, and counted.

Cell viability assay. Cells viability in the presence of Pg (10–100 nM) was estimated with the use of MTT-assay as described previously [17]. Briefly, cells were seeded onto a 96-well plate (5•103 cells/well) and incubated with 10–100 nM of Pg for 24 h at 37 oC. Having MTT reagent added (0.4 mg/ml), plates were incubated at 37 oC for 1 h and centrifuged in a cytospin centrifuge to sediment detached cells. The formazan crystals formed were dissolved in DMSO and measured spectrophotometrically at a wavelength of 570/630 nm with the use of absorbance microplate reader µQuant (BioTEK).

ROS generation. Pg (1, 10, or 50 nM) was added to 4T1 cells (5 • 105 in each sample) resuspended in FBS-free DMEM. After that, the cells were immediately loaded with 2΄,7΄-dichlorodihydrofluorescein diacetate (H2DCF-DA) (12.5 µM) and incubated for 1 h at 37 oC in the dark chamber. Then, the cells were diluted with 5-fold ice-cold PBS and kept on ice until analyzed. DCF fluorescence was monitored using a flow cytometer Coulter Epics XL (Beckman Coulter, USA). The medians of the DCF fluorescence intensity were obtained from 10,000 cells in each sample using 480 nm excitation and 540 nm emission settings. Values of autofluorescence signal from untreated cells were chosen for threshold value setting. DCF-positive cell populations were gated separately based on their side-scatter properties. By using the same settings, the fluorescent intensity was obtained from each experimental group. Fluorescent levels were expressed as the percentage increase over the control. The results were analyzed and presented with the use of “FCS Express V3” software (De Novo Software, USA).

Enzymatic activities and H2O2 levels. In order to evaluate activities of antioxidant enzymes, including catalase (CAT, EC, glutathione peroxidase (GPx, EC, Cu,Zn- and Mn-superoxide dismutase (Zn,Cu- and Mn-SOD, EC, as well as levels of total protein and H2O2, cell cytoplasmic extracts were obtained from cultured cells of 70–80% confluence. The extraction was carried out as described elsewhere [14]. The hypotonic buffer (0.4% NP-40, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM Na3VO4, 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL of leupeptin, and 1 µg/mL pepstatin) was used for the procedure. The Pierce BCA assay was used to assess total protein yields in the cell lysates. Levels of H2O2 and activities of CAT, GPx, and SOD were measured with the use of spectrophotometric and fluorometric assays according to the procedures described in [14] in details.

Statistical analysis. Results are representative of at least three independent experiments and presented as Mean ± standard error of mean. The significance of intergroup differences was estimated by the analysis of variance (ANOVA) followed by Bonferroni or Fisher post-hoc tests. The difference was considered significant at P < 0.05.


Pg to plasmin conversion by 4T1 cells was shown even in the absence of exogenous activators, though cells with an increased level of Ruk/CIN85 expression did not differ significantly from the control cell line in the parameters of zymogen activation (Fig. 1).

Fig. 1. Urokinase (uPA) inhibitor BC11 differentially affects plasminogen activation rate in 4T1 cells in dependence on Ruk/CIN85 expression: 1 — cells + Pg; 2 — cells + Pg + 1 µM BC11; 3 — cells + Pg + 10 µM BC11; 4 — cells + Pg + 20 µM BC11; 5 — Pg + S2251; 6 — cells + BC11

Moreover, inhibitory analysis with the use of BC11 demonstrated dose-dependent suppression of S2251 cleavage, indicating uPA to be the main activator of Pg conversion. These data are in agreement with the earlier reports that describe enhanced expression of uPA and its receptor uPAR in breast cancers cells that is critical for tissue remodeling and cell migration in the course of breast tumors progression and their metastatic dissemination and is associated with poor prognosis [18, 19].

The effects of plasmin(ogen) on the colony-forming ability of 4T1 cells was analyzed by colony formation assay. As seen from the graph, presented in Fig. 2, a, Pg in the range of 10–100 nM inhibited clonogenic potential of Mock cells more significantly than RukUp cells, having maximal inhibitory effect at 50 nM (p < 0.05).

Fig. 2. 4T1 cells with Ruk/CIN85 overexpression are more resistant to plasmin(ogen) action: a — colonies number; b — representative images of crystal violet-stained colonies; c — representative microphotographs of the colonies; — cells viability (MTT-assay). *p < 0.05 compared to untreated cells, #p < 0.05 compared to Mock treated with equal Pg concentration, n = 4–6

This observation indicates that RukUp cells are more resistant to plasmin(ogen)-induced cytotoxicity compared to Mock cells. In addition, the shape of colonies differed between sublines: in contrast to Mock cells that formed dense round colonies, RukUp cells formed loose colonies with uneven shape characteristic for more malignant cells (Fig. 2, b–c).

The results of the colony formation assay were confirmed by MTT test that showed a dramatic impact of plasmin(ogen) on 4T1 sublines viability (Fig. 2, d). It was observed that treatment with Pg (25–100 nM) for 24 h significantly reduced the rate of cell survival up to 50% compared to untreated cells (Fig. 2). For example, 50 and 100 nM of Pg caused 1.55- and 1.91-fold decrease in viability of 4T1 Mock cells (p < 0.05 compared to intact cells), respectively. However, cells with Ruk/CIN85 overexpression exposed to 50 and 100 nM of Pg demonstrated 1.43- and 1.63-fold decrease in viability, thus being less sensitive to plasmin(ogen)-induced cytotoxicity.

ROS levels were measured using the fluorescent probe, H2DCF-DA, which is currently applied in flow cytometry analysis for direct and reliable relative quantification of intracellular ROS. After cytosolic hydrolysis, H2DCF-DA can be oxidized into 2́ʹ,7ʹ-dichlorofluorescein (DCF), which fluoresces at 520 nm. One of the main advantages of this technique is that it enables the rapid detection of ROS, so that even short-lived oxidative imbalance can be measured [20]. Plasmin(ogen) exposure promoted significant enhancement of ROS generation in 4T1 cells with a basal level of Ruk/CIN85 expression, having the peak of ROS production (approximately 2.5-fold increase as compared to untreated cells, P < 0.05) in the case of 10 nM zymogen concentration (Fig. 3, a, c). In contrast, 4T1 Ruk/CIN85 overexpressing cells were able to maintain the ROS level unchanged during plasmin(ogen) exposure (Fig. 3, b, c).

Fig. 3. 4T1 cells with Ruk/CIN85 overexpression are able to counteract plasmin(ogen)-induced ROS generation (flow cytometry DCF-test): — the effect of plasmin(ogen) on ROS generation in 4T1 Mock subline, typical graphs; — the effect of plasmin(ogen) on ROS generation in 4T1 RukUp subline, typical graphs; c — ROS production, relative units. *p < 0.05 compared to untreated cells, n = 3

It is known that cancer cells tend to have enhanced rates of ROS generation due to aberrant metabolism that contributes to tumor progression and metastasis, while abnormally high ROS levels may lead to apoptosis [21]. Plasmin has been described to induce ROS overproduction in diverse normal and malignant cells via activating PARs that are critical for both ERK1/2- and p38 MAPK-mediated
mitochondrial ROS generation, as well as stimulating PAR-1/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/ROS pathway [7, 22]. Besides, a robust increase in ROS generation may occur as a consequence of defective glucose uptake, diminished pentose phosphate pathway flux, reduced cellular ATP levels caused by detachment from ECM. Thus, oxidative burst in detached cells leads to catastrophic metabolic deviations that impact cell viability [23]. It is important to note that intensities of plasmin(ogen)-induced ROS generation in 4T1 cells are inversely correlated with cell survival rate suggesting that disrupted redox homeostasis could be a major cause of apoptosis/anoikis.

Upon flow cytometric analysis of DCF staining, we found that plasmin(ogen) induced redistribution of cells into the upper right quadrant that corresponds to the population of highly granulated cells with the increased ROS producing capacity (depicted as dot-plots in Fig. 3, а, b). It is important to note that the number of highly granulated RukUp cells was ~6-fold higher than that of Mock cells. It obviously means that plasmin(ogen) exposure results in an increased rate of vesicle formation in cells with Ruk/CIN85 overexpression, but not in cells with basal levels of this adaptor protein. Among all intracellular vesicles, autophagosomes may greatly contribute to elevation of cell granularity. In our recent study, we have demonstrated that Pg treatment induced autophagosomes formation and promoted autophagy flux in lung adenocarcinoma cell line A549 [24]. It has been assumed that autophagy activation might serve as one of the factors of cell resistance against plasmin-induced apoptosis/anoikis. Punctate localization pattern of both endogenous and exogenous Ruk/CIN85 characteristic for proteins involved in membrane trafficking was demonstrated in a number of previous publications. Specifically, Ruk/CIN85 was found to be associated with a subset of COPI-coated vesicles of the Golgi complex [25]. In MCF-7 cells, GFP-Ruk/CIN85 is localized and concentrated in the perinuclear region and in rounded juxtamembrane structures, which were clathrin-positive [26], while in MCF-7 and HEK293 cells transiently transfected with plasmid encoding fusion Ruk/CIN85 with a sensor of H2O2, Hyper, the positive signal was detected in dot-like vesicular structures of different size and localization [27]. It was also shown that in COS-7 cells Ruk/CIN85 was located on the edges of some dark circular areas, part of which was filled with endocytic EGF [28].

To further confirm that enhanced expression of Ruk/CIN85 contributes to the reduction in ROS level in plasmin(ogen)-treated cells, we measured activities of some enzymes of the glutathione-related system and evaluated H2O2 levels after treatment of both 4T1 Mock and 4T1 RukUp cells with 1 nM or 10 nM of Pg for 24 h. We found a strong association of Ruk/CIN85 overexpression with the elevated activity of antioxidant enzymes. We analyzed activities of SOD, CAT, and Gpx as well as hydrogen peroxide (H2O2) content in plasmin(ogen)-exposed 4T1 cells (Fig. 4).

Fig. 4. Plasmin(ogen) differentially affects redox balance in 4T1 cells depending on Ruk/CIN85 expression levels: — SOD activity; b — CAT activity; c — Gpx activity; — H2O2 content. *p < 0.05 compared to Mock cells exposed to the same Pg concentration; #p < 0.05 compared to untreated cells; @p < 0.05 compared to RukUp 1 nM Pg; &p < 0.05 in comparison to Mock 1 nM Pg, n = 4

It should be noticed that SOD is involved in superoxide anion elimination, and thereby it is involved in H2O2 production, which is further detoxified by CAT or Gpx, an enzyme using reduced glutathione as a cofactor. Finally, glutathione reductase then reduces the oxidized glutathione using NADPH to complete the cycle [29].

It was found that treatment of cancer cells with 1 nM of Pg resulted in increased SOD activity by 35% in Mock cells and by 28% in RukUp cells. However, 10 nM of Pg did not affect SOD activity in 4T1 cells when compared to untreated cells (Fig. 4, a).

Next, we found that RukUp cells are characterized by a 1.6-fold reduction in CAT activity compared to Mock cells. Moreover, studied cells sublines responded differently to Pg treatment: CAT activity in Mock cells was not changed significantly in comparison with intact cells, while CAT activity in RukUp cells treated with 1 nM of Pg was increased significantly compared to both untreated cells (2.2-fold) and Mock cells treated with the same Pg concentration. Surprisingly, treatment of RukUp cells with 10 nM of Pg resulted in a slight decrease in CAT activity (Fig. 4, b). In turn, analysis of Gpx activities revealed no effect of Pg on Mock cells, but in Pg-treated RukUp cells Gpx activity was significantly increased in comparison with untreated cells (by 76% and 55% for 1 nM and 10 nM of Pg, respectively) and with Mock cells exposed to the same concentration of Pg (Fig. 4, c). Thus, it is highlighted that both CAT and Gpx are effectively involved in the elimination of excessive amounts of H2O2 in 4T1 RukUp cells exposed to plasmin(ogen). Finally, it was demonstrated that Pg exposure induced approximately 1.5-fold elevation of H2O2 in Mock cells compared to the intact cells (p < 0.05), while any notable accumulation of H2O2 was not observed in RukUp cells, likely, due to increased CAT and Gpx activities (Fig. 4, d).

ROS signaling is used by cancer cells to drive proliferation, metabolic reprogramming, resistance to apoptosis and other events required for tumor progression, while amplifying oxidative stress can induce cell death. The overproduction of ROS in cancer cells is counterbalanced by a high rate of ROS scavenging that is very important to elicit prosurvival signaling in detached cancer cells [30]. It has been previously shown that the lack of glucose uptake in the detached cells blocks both glycolysis and the pentose phosphate pathway. One of the metabolic functions of the pentose phosphate pathway is to produce NADPH, a crucial cellular reducing agent, which is necessary to provide reduced glutathione for enzymatic ROS quenching by Gpx [29]. We have recently shown that lung adenocarcinoma cells overexpressed a regulatory protein TIGAR (TP53-inducible glycolysis and apoptosis regulator) in the response to Pg exposure [24]. TIGAR is considered as a pro-tumorigenic regulator playing an active role in many cancers to facilitate tumor progression by decreasing the levels of ROS [31]. Having the fructose-2,6-bisphosphatase activity, TIGAR decreases the rate of glycolysis and, in turn, increases the activity of the pentose phosphate pathway. This promotes cells to generate more amounts of NADPH, which is used for the reduction of oxidized glutathione necessary for ROS scavenging and various anabolic processes [32]. Enhanced TIGAR expression may, at least partially, explain an increase in Gpx enzymatic activity in 4T1 RukUp cells exposed to plasmin(ogen). Apart from protecting cells against ROS-induced apoptosis, TIGAR is involved in supporting migration machinery of cancer cells, thus referred to as “motogenic protein” [33, 34]. If TIGAR expression has fallen under the control of Ruk/CIN85 needs to be explored, though the described results clearly indicate that adaptor protein Ruk/CIN85 modulates the activity of antioxidant cellular defence, in particular, enzymes of glutathione-related system.

It has been widely reported that adaptor protein Ruk/CIN85 is involved in the control of redox balance in breast cancer cells [13, 14]. Ruk/CIN85 is able to interact with Tks4, an organizing subunit of NADPH oxidase complex 1, and therefore it may be involved in the control of ROS generation [35]. Our previous studies have revealed that overexpression of Ruk/CIN85 is associated with increased ROS production in breast and colon cancer cells [27, 36]. However, our recent research has demonstrated that 4T1 RukUp cells were characterized by an increase of H2O2 up to 0.8 µM, a concentration that was probably involved in the initiation of cellular signaling [14]. Additionally, it was found that Ruk/CIN85 overexpression in breast cancer cells is strongly associated with increased motility, invasiveness, and drug resistance [12, 17, 37]. Lee & Kim [9] have reported H2O2-induced uPA expression in human hepatoma cells. Taken together, it may be speculated that high levels of Ruk/CIN85 are needed to maintain optimal H2O2 concentration required for pro-invasive and pro-survival signaling in various cancer cells.

In summary, we obtained novel data that support the role of adaptor protein Ruk/CIN85 as the main regulator of redox status in breast cancer cells. In addition, we found a plausible link between plasmin(ogen)-induced oxidative stress and activation of the enzymatic antioxidant system in 4T1 breast adenocarcinoma cells depending on the levels of Ruk/CIN85 expression. Based on the results obtained, we also suggest that the content of Ruk/CIN85 in cancer cells can be considered as one of the factors contributing to their acquisition of resistance to ROS-induced apoptosis and, as a result, may play a decisive role in the survival of cancer cells exposed to plasmin(ogen). Thus, Ruk/CIN85 may represent one of the relevant molecular targets regulating the resistance of cancer cells to ROS-mediated apoptosis.


This study was funded under the budget program KPKVK 6541230 “Support for the Development of Priority Areas of Research” of NAS of Ukraine (2020–2021).


The authors declare that they have no conflict of interest regarding the publication of this paper.


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І.Р. Горак, Н.В. Латишко, О.О. Гудкова, К.О. Токарчук, Т.О. Кішко, О.І. Юсова, Л.Б. Дробот, А.О. Тихомиров

Інститут біохімії ім. О.В. Палладіна НАН України, Київ, Україна

Резюме. Стан питання: Плазмін, що утворюється на клітинній поверхні, залучається до контролю пухлинного росту та мета­стазування через регулювання адгезії, міграції та інвазії ракових клітин. Зниження адгезивності клітин, індуковане плазміном, супроводжується посиленням генерування активних форм оксигену (АФО) та клітинною загибеллю. Водночас для ракових клітин характерним є набуття механізмів протидії апоптозу, опосередкованому АФО. Мета: З’ясувати роль адаптерного протеїну Ruk/CIN85 у контролі окисно-відновного балансу та виживання клітин аденокарциноми грудної залози за умови впливу плазмін(оген)у. Матеріали та методи: Мишачі клітини лінії 4Т1 зі стабільною надекспресією адаптерного протеїну Ruk/CIN85 (сублінія RukUp) та відповідні контрольні клітини (сублінія Mock) культивували за наявності плазміногену (1–100 нМ). Перетворення плазміногену на плазмін визначали спектрофотометрично за швидкістю розщеплення специфічного хромогенного субстрату S2251. Специфічний інгібітор uPA ВС11 використовували для дослідження uPA-опосередкованого механізму перицелюлярної активації плазміногену клітинами 4Т1. Рівень виживання клітин оцінювали за допомогою МТТ-тесту, проліферативну активність клітин аналізували методом формування колоній. Ензиматичні активності каталази, глутатіонпероксидази, супероксиддисмутази, а також рівні пероксиду гідрогену (H2O2) вимірювали флюорометрично. Внутрішньоклітинне генерування АФО досліджували за допомогою протокової цитометрії з використанням флуоресцентного зонду H2DCF-DA. Результати: Було показано, що плазміноген перетворюється на активну протеїназу плазмін на поверхні аденокарциномних клітин у uPA-залежний спосіб. Плазмін(оген) пригнічував проліферацію та виживання обох досліджуваних субліній клітин. Однак, клітини RukUp демонстрували більшу резистентність до плазмін(оген)-індукованої цитотоксичності у порівнянні з клітинами Mock. Плазмін(оген) спричиняв значне посилення генерування АФО в клітинах з базальним рівнем експресії Ruk/CIN85. На відміну від контролю, клітини RukUp виявилися здатними більш ефективно протидіяти прооксидантним змінам через активацію деяких ензимів системи глутатіону, головним чином глутатіонпероксидази, та зниження рівня акумуляції H2O2. Висновки: Адаптерний протеїн Ruk/CIN85 залучений до контролю окисно-відновного гомео­стазу в ракових клітинах завдяки підтриманню утворення АФО на рівні, необхідному для адаптації клітин до впливу плазмін(оген)у. Таким чином, скерований вплив на рівень експресії Ruk/CIN85 може бути ефективним підходом, спрямованим на зниження резистентності ракових клітин до АФО-опосередкованого апоптозу.

Ключові слова: рак грудної залози, аденокарциномні клітини грудної залози миші лінії 4Т1, плазміноген/плазмін, Ruk/CIN85, активні форми оксигену (АФО), ензими системи глутатіону.

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