Targeting Multiple Homeostasis-Maintaining Systems by Ionophore Nigericin Is a Novel Approach for Senolysis

Within the present study we proposed a novel approach for senolysis based on the simultaneous disturbance of the several homeostasis-maintaining systems in senescent cells including intracellular ionic balance, energy production and intracellular utilization of damaged products. Of note, we could not induce senolysis by applying ouabain, amiloride, valinomycin or NH4Cl—compounds that modify each of these systems solely. However, we found that ionophore nigericin can disturb plasma membrane potential, intracellular pH, mitochondrial membrane potential and autophagy at once. By affecting all of the tested homeostasis-maintaining systems, nigericin induced senolytic action towards stromal and epithelial senescent cells of different origins. Moreover, the senolytic effect of nigericin was independent of the senescence-inducing stimuli. We uncovered that K+ efflux caused by nigericin initiated pyroptosis in senescent cells. According to our data, the higher sensitivity of senescent cells compared to the control ones towards nigericin-induced death was partially mediated by the lower intracellular K+ content in senescent cells and by their predisposition towards pyroptosis. Finally, we proposed an interval dosing strategy to minimize the negative effects of nigericin on the control cells and to achieve maximal senolytic effect. Hence, our data suggest ionophore nigericin as a new senotherapeutic compound for testing against age-related diseases

Oncogene-Induced Senescence Is a Crucial Antitumor Defense Mechanism of Human Endometrial Stromal Cells

Being the major cellular component of highly dynamic tissue, endometrial stromal cells (EnSCs) are exposed to cycles of proliferation upon hormonal stimulation, which might pose risks for the accumulation of mutations and malignization. However, endometrial stromal tumors are rare and uncommon. The present study uncovered defense mechanisms that might underlie the resistance of EnSCs against oncogenic transformation. All experiments were performed in vitro using the following methods: FACS, WB, RT-PCR, IF, molecular cloning, lentiviral transduction, and CRISPR/Cas9 genome editing. We revealed that the expression of the mutant HRASG12V leads to EnSC senescence. We experimentally confirmed the inability of HRASG12V-expressing EnSCs to bypass senescence and resume proliferation, even upon estrogen stimulation. At the molecular level, the induction of oncogene-induced senescence (OIS) was accompanied by activation of the MEK/ERK, PI3K/AKT, p53/p21WAF/CIP/Rb, and p38/p16INK4a/Rb pathways; however, inhibiting either pathway did not prevent cell cycle arrest. PTEN loss was established as an additional feature of HRASG12V-induced senescence in EnSCs. Using CRISPR-Cas9-mediated PTEN knockout, we identified PTEN loss-induced senescence as a reserve molecular mechanism to prevent the transformation of HRASG12V-expressing EnSCs. The present study highlights oncogene-induced senescence as an antitumor defense mechanism of EnSCs controlled by multiple backup molecular pathways

Mesenchymal Stem/Stromal Cells in Three-Dimensional Cell Culture: Ion Homeostasis and Ouabain-Induced Apoptosis

This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell structures (spheroids) and investigates the conditions for apoptosis induction in 3D eMSCs. Detached from the monolayer culture, (2D) eMSCs accumulate Na+ and have dissipated transmembrane ion gradients, while in compact spheroids, eMSCs restore the lower Na+ content and the high K/Na ratio characteristic of functionally active cells. Organized as spheroids, eMSCs are non-proliferating cells with an active Na/K pump and a lower K+ content per g cell protein, which is typical for quiescent cells and a mean lower water content (lower hydration) in 3D eMSCs. Further, eMSCs in spheroids were used to evaluate the role of K+ depletion and cellular signaling context in the induction of apoptosis. In both 2D and 3D eMSCs, treatment with ouabain (1 µM) results in inhibition of pump-mediated K+ uptake and severe K+ depletion as well as disruption of the mitochondrial membrane potential. In 3D eMSCs (but not in 2D eMSCs), ouabain initiates apoptosis via the mitochondrial pathway. It is concluded that, when blocking the Na/K pump, cardiac glycosides prime mitochondria to apoptosis, and whether a cell enters the apoptotic pathway depends on the cell-specific signaling context, which includes the type of apoptotic protein expressed

Mesenchymal Stem/Stromal Cells in Three-Dimensional Cell Culture: Ion Homeostasis and Ouabain-Induced Apoptosis

This study describes the changes in ion homeostasis of human endometrial mesenchymal stem/stromal cells (eMSCs) during the formation of three-dimensional (3D) cell structures (spheroids) and investigates the conditions for apoptosis induction in 3D eMSCs. Detached from the monolayer culture, (2D) eMSCs accumulate Na+ and have dissipated transmembrane ion gradients, while in compact spheroids, eMSCs restore the lower Na+ content and the high K/Na ratio characteristic of functionally active cells. Organized as spheroids, eMSCs are non-proliferating cells with an active Na/K pump and a lower K+ content per g cell protein, which is typical for quiescent cells and a mean lower water content (lower hydration) in 3D eMSCs. Further, eMSCs in spheroids were used to evaluate the role of K+ depletion and cellular signaling context in the induction of apoptosis. In both 2D and 3D eMSCs, treatment with ouabain (1 µM) results in inhibition of pump-mediated K+ uptake and severe K+ depletion as well as disruption of the mitochondrial membrane potential. In 3D eMSCs (but not in 2D eMSCs), ouabain initiates apoptosis via the mitochondrial pathway. It is concluded that, when blocking the Na/K pump, cardiac glycosides prime mitochondria to apoptosis, and whether a cell enters the apoptotic pathway depends on the cell-specific signaling context, which includes the type of apoptotic protein expressed

Time-Dependent Regulation of IL-2R α-Chain (CD25) Expression by TCR Signal Strength and IL-2-Induced STAT5 Signaling in Activated Human Blood T Lymphocytes.

The expression of the IL-2R α-chain (IL-2Rα) is regulated at the transcriptional level via TCR- and IL-2R-signaling. The question is how to precede in time the activation signals to induce the IL-2Rα expression in native primary T cells. By comparing the effects of selective drugs on the dynamics of CD25 expression during the mitogen stimulation of human peripheral blood lymphocytes, we identified distinct Src- and JAK-dependent stages of IL-2Rα upregulation. PP2, a selective inhibitor of TCR-associated Src kinase, prevents CD25 expression at initial stages of T cell activation, prior to the cell growth. This early IL-2Rα upregulation underlies the T cell competence and the IL-2 responsiveness. We found that the activated with "weak" mitogen, the population of blood lymphocytes has some pool of competent CD25+ cells bearing a high affinity IL-2R. A distinct pattern of IL-2R signaling in resting and competent T lymphocytes has been shown. Based on the inhibitory effect of WHI-P131, a selective drug of JAK3 kinase activity, we concluded that in quiescent primary T lymphocytes, the constitutive STAT3 and the IL-2-induced prolonged STAT5 activity (assayed by tyrosine phosphorylation) is mostly JAK3-independent. In competent T cells, in the presence of IL-2 JAK3/STAT5 pathway is switched to maintain the higher and sustained IL-2Rα expression as well as cell growth and proliferation. We believe that understanding the temporal coordination of antigen- and cytokine-evoked signals in primary T cells may be useful for improving immunotherapeutic strategies

Tetraploidization or autophagy: The ultimate fate of senescent human endometrial stem cells under ATM or p53 inhibition

<p>Previously we demonstrated that endometrium-derived human mesenchymal stem cells (hMESCs) via activation of the ATM/p53/p21/Rb pathway enter the premature senescence in response to oxidative stress. Down regulation effects of the key components of this signaling pathway, particularly ATM and p53, on a fate of stressed hMESCs have not yet been investigated. In the present study by using the specific inhibitors Ku55933 and Pifithrin-α, we confirmed implication of both ATM and p53 in H₂O₂-induced senescence of hMESCs. ATM or p53 down regulation was shown to modulate differently the cellular fate of H₂O₂-treated hMESCs. ATM inhibition allowed H₂O₂-stimulated hMESCs to escape the permanent cell cycle arrest due to loss of the functional ATM/p53/p21/Rb pathway, and induced bypass of mitosis and re-entry into S phase, resulting in tetraploid cells. On the contrary, suppression of the p53 transcriptional activity caused a pronounced cell death of H₂O₂-treated hMESCs via autophagy induction. The obtained data clearly demonstrate that down regulation of ATM or p53 shifts senescence of human endometrial stem cells toward tetraploidization or autophagy.</p

PHA stimulation leads to long-term CD25 expression in human blood T lymphocytes.

<p>(A) The representative histograms of nine experiments on PBL from different donors are shown. PBL were cultivated with 10 μg/ml PHA in the absence or presence of 80 μM WHI-P131 (WHI) or 1.0 μM PP2 and after 5, 24 or 48 h were analyzed by flow cytometry. The dot plots have been obtained with Win MDI Version 2.8. In order to identify the axis labels, the additional vertical axis is shown in the upper row of histograms. Y-axis is 4 decades logarithmic scale. <b>1, 2 –</b>The gate was chosen considering the cell size increase during blasttransformation: 1- CTR—control, non-stimulated PBL, 2 –after 48 h of culture with 10 μg/ml PHA. X-axis–forward scatter (FSC), Y-axis–side scatter (SSC). (B) Time-course of CD25+ expression in PHA-induced PBL. 1 –Total number of CD25+ cells, 2—large CD25+ cells, 3—small CD25+ cells. (C) WHI-P131- and PP2-inibitable portions of CD25+ cells in PHA-stimulated PBL. Summary of independent experiments is presented as mean ±SEM (n = 9, p˂0.05). In each experiment, when analyzing the PHA-induced CD25 expression changes, the background CD25 expression in resting cells (CTR) was subtracted. Summary data are presented as mean ± SEM (n = 10, p˂0.05).</p

The inhibitory effect of WHI-P131 and PP2 on CD25 expression in PHA-stimulated human blood T lymphocytes.

<p>(A) The PP2-inhibitable expression of CD25 is timely associated with the initial stages of PBL activation. Cells were cultivated with 10 μg/ml PHA without or with 80 μM WHI-P131 (WHI) or 1.0 μM PP2 for 19 h (middle row) or cells were cultivated with 10 μg/ml PHA for 19 h and thereafter WHI-P131 or PP2 were added for the next 21 h (bottom row). Additional Y-axis is 4 decades logarithmic scale. Representative data on PBL from one donor are shown. (B) Summary data of four independent experiments are shown as mean + SEM (n = 4, p˂0.05). (C) The inhibitory effects of WHI-P131 (WHI) and PP2 on CD25 expression are additive. Cells were cultivated with 10 μg/ml PHA without or with WHI-P131 (WHI, 20 and 50 μM), or PP2 (0.4 and 0.8 μM), or WHI-P131 (20 μM) and PP2 (0.4 μM) were given simultaneously for 24 h. Summary of independent experiments are presented as mean + SEM (n = 4). ⃰, <i>p<</i>0.05. CTR—control, non-stimulated PBL.</p

The growth, proliferation and viability of human blood T lymphocytes, stimulated with PHA or IL-2 in the absence or presence of WHI-P131 and PP2 for 48 h.

<p>The growth, proliferation and viability of human blood T lymphocytes, stimulated with PHA or IL-2 in the absence or presence of WHI-P131 and PP2 for 48 h.</p

Stimulation with non-mitogenic PHA and IL-2 leads to long-term CD25 expression and proliferation in human blood T lymphocytes.

<p>(A) The representative histograms of one experiment on PBL from one donor are shown. PBL were not stimulated (CTR) or stimulated with 0.7 μg/ml (0.7PHA), or 10 μg/ml (10PHA) PHA, or 200 U/ml IL-2 (IL-2) for 48 h. In the same experiment, PBL were preincubated in culture medium with 0.7 μg/ml PHA and 80 μM WHI-P131 (WHI) or 1.0 μM PP2 for 24 h prior to IL-2 stimulation, or drugs were added simultaneously with 0.7 μg/ml PHA for 24 h and thereafter IL-2 was added for the next 24 h. Additional Y-axis is 4 decades logarithmic scale. (B) CD25 expression in resting PBL in response to 0.7 μg/ml PHA (2) and 10 μg/ml PHA (8), or in competent PBL in response to IL-2 in the absence (3) or presence of WHI-P131 (4) or PP2 (5), or in response to IL-2 in PBL preactivated with 0.7 μM PHA in the presence of WHI-P131 (6) or PP2 (7). Summary data of independent experiments on PBL from different donors are shown as mean + SEM (n = 4, p˂0.05). (C) Proliferation of resting PBL in response to 0.7 μg/ml PHA (2) and 10 μg/ml PHA (6, 7), or competent PBL in response to IL-2 in the absence (3) or presence of PP2 (4) or WHI-P131 (5). Bars represent the number of cells in (S+G2+M) phases of cell cycle at 48h after PBL stimulation. Summary data of independent experiments on PBL from different donors is shown as mean + SEM (n = 4, p˂0.05).</p