Attenuation of Replication Stress–Induced Premature Cellular Senescence to Assess Anti‐Aging Modalities

Abstract

Described is an in vitro model of premature senescence in pulmonary adenocarcinoma A549 cells induced by persistent DNA replication stress in response to treatment with the DNA damaging drug mitoxantrone (Mxt). The degree of cellular senescence, based on characteristic changes in cell morphology, is measured by laser scanning cytometry. Specifically, the flattening of cells grown on slides (considered the hallmark of cellular senescence) is measured as the decline in local intensity of DNA‐associated DAPI fluorescence (represented by maximal pixels). This change is paralleled by an increase in nuclear area. Thus, the ratio of mean intensity of maximal pixels to nuclear area provides a very sensitive morphometric biomarker for the degree of senescence. This analysis is combined with immunocytochemical detection of senescence markers, such as overexpression of cyclin kinase inhibitors (e.g., p21WAF1) and phosphorylation of ribosomal protein S6 (rpS6), a key marker associated with aging/senescence that is detected using a phospho‐specific antibody. These biomarker indices are presented in quantitative terms defined as a senescence index (SI), which is the fraction of the marker in test cultures relative to the same marker in exponentially growing control cultures. This system can be used to evaluate the anti‐aging potential of test agents by assessing attenuation of maximal senescence. As an example, the inclusion of berberine, a natural alkaloid with reported anti‐aging properties and a long history of use in traditional Chinese medicine, is shown to markedly attenuate the Mxt‐induced SI and phosphorylation of rpS6. The multivariate analysis of senescence markers by laser scanning cytometry offers a promising tool to explore the potential anti‐aging properties of a variety agents. Curr. Protoc. Cytom. 69:9.47.1‐9.47.10. © 2014 by John Wiley & Sons, Inc.

Rhizoma Coptidis and Berberine as a Natural Drug to Combat Aging and Aging-Related Diseases via Anti-Oxidation and AMPK Activation

“…Administration of berberine into cultures of A549 cells undergoing premature senescence reduced the development of senescent phenotype, evidenced by as analysis of cells morphometric features through laser scanning cytometry. The increase in the ratio of mean intensity of maximal pixels to nuclear area is considered to be a very sensitive morphometric biomarker for the degree of senescence (the decline in local intensity of DNA-associated DAPI fluorescence represented by maximal pixels is paralleled by an increase in nuclear area) [ 49 , 50 ]. The characteristic features of cellular morphology combined with immunocytochemical detection of senescence markers, such as overexpression of cyclin kinase inhibitors (e.g., p21WAF1) and phosphorylation of rpS6 are considered to be the most specific biomarkers of senescent cells [ 51 ].…”

“…These biomarker indices are presented in quantitative terms defined as the senescence index (SI), which is the fraction of the marker in test cultures relative to the same marker in exponentially growing control cultures. By using this system, administration of berberine also activated SA-β-gal and induced CDK inhibitor p21WAF1, p16INK4A, and p27KIP1, and through activation of tumor suppressor p53 signaling pathways, attenuated the level of mTOR/rpS6 signaling by lowering the level of phosphorylation of rpS6 and expression of γH2AX [ 47 , 50 ].…”

Aloe-emodin inhibits HER-2 expression through the downregulation of Y-box binding protein-1 in HER-2-overexpressing human breast cancer cells

“…Anthraquinones are a large group of natural aromatic compounds [ 17 ]. Such compounds are historically used as natural dyes, but recent studies demonstrated their medicinal values such as antibacterial, anti-inflammatory, antiviral, anticancer, and antiaging properties [ 18 ]. The anthraquinone emodin inhibits the proliferation of HER-2-overexpressing breast cancer cells [ 19 ] and induces apoptosis of cancer cells [ 20 ].…”

Ribosomal Protein S6: A Potential Therapeutic Target against Cancer?

“…The mTORC1/S6K pathway has been recognized as the key signaling pathway responsible for aging and cellular senescence [ 383 , 384 ]. Since RPS6 is the downstream effector of this mTORC1/S6K pathway, p-RPS6 has been used as a marker for aging and premature senescence [ 385 ].…”