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

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