In search of antiaging modalities: Evaluation of mTOR‐ and ROS/DNA damage‐signaling by cytometry

Abstract

This review presents the evidence in support of the IGF-1/mTOR/S6K1 signaling as the primary factor contributing to aging and cellular senescence. Reviewed are also specific interactions between mTOR/S6K1 and ROS-DNA damage signaling pathways. Outlined are critical sites along these pathways, including autophagy, as targets for potential antiaging (gero-suppressive) and/or chemopreventive agents. Presented are applications of flow- and laser scanning- cytometry utilizing phospho-specific Abs, to monitor activation along these pathways in response to the reported antiaging drugs rapamycin, metformin, berberine, resveratrol, vitamin D3, 2-deoxyglucose, and acetylsalicylic acid. Specifically, effectiveness of these agents to attenuate the level of constitutive mTOR signaling was tested by cytometry and confirmed by Western blotting through measuring phosphorylation of the mTOR-downstream targets including ribosomal protein S6. The ratiometric analysis of phosphorylated to total protein along the mTOR pathway offers a useful parameter reporting the effects of gero-suppressive agents. In parallel, their ability to suppress the level of constitutive DNA damage signaling induced by endogenous ROS was measured. While the primary target of each of these agents may be different the data obtained on several human cancer cell lines, WI-38 fibroblasts and normal lymphocytes suggest common downstream mechanism in which the decline in mTOR/S6K1 signaling and translation rate is coupled with a reduction of oxidative phosphorylation and ROS that leads to decreased oxidative DNA damage. The combined assessment of constitutive γH2AX expression, mitochondrial activity (ROS, ΔΨPm), and mTOR signaling provides an adequate gamut of cell responses to test effectiveness of gero-suppressive agents. Described is also an in vitro model of induction of cellular senescence by persistent replication stress, its quantitative analysis by laser scanning cytometry, and application to detect the property of the studied agents to attenuate the induction of senescence. Discussed is cytometric analysis of cell size and heterogeneity of size as a potential biomarker used to asses gero-suppressive agents and longevity.

Synergistic cell death in FLT3-ITD positive acute myeloid leukemia by combined treatment with metformin and 6-benzylthioinosine

“…Metformin has been shown to effectively inhibit ROS production in cells by suppressing oxidative phosphorylation [30, 31]. We examined levels of ROS in the cells and found that the baseline levels differed between them.…”

Cytoprotective activated protein C averts Nlrp3 inflammasome–induced ischemia-reperfusion injury via mTORC1 inhibition

“…45 Intriguingly, mTORC1 regulates mitochondrial quality and ROS, 63 suggesting that inhibition of mTORC1 and mitochondrial ROS by aPC may be mechanistically linked. Because the regulation of mitochondrial ROS and mTORC1 is mutual, 64 additional studies are required to decipher the exact mechanism through which aPC regulates mTORC1 and mitochondrial ROS.…”

Prolonged Growth Hormone/Insulin/Insulin-like Growth Factor Nutrient Response Signaling Pathway as a Silent Killer of Stem Cells and a Culprit in Aging

“…A decrease in autophagy activity leads to accumulation of protein aggregates, which negatively affect cell function and lead to damage and degeneration of mitochondria, thus contributing to aging [28]. …”