Abstract
Phosphoglucose isomerase/autocrine motility factor (PGI/AMF) is a housekeeping gene product present in all cells, is an essential enzyme of catabolic glycolysis and anabolic gluconeogenesis, and regulates tumor cell growth and metastasis. Because glycolytic enzyme up-regulation of expression contributes to glycolytic flux, leading to increased of cell growth and a resistance to cellular stress of normal fibroblasts whereas down-regulation of PGI/AMF leads to mesenchymal-to-epithelial transition in tumor cells, we examined the involvement of PGI/AMF in overcoming cellular senescence in cancer cells. PGI/AMF cellular expression in HT1080 human fibrosarcoma was down-regulated by small interfering RNA methodology, which resulted in an increased sensitivity to oxidative stress and oxidative stress-induced cellular senescence. Signaling analysis revealed that the senescence pathway involving p21 cyclin-dependent kinase inhibitor was up-regulated in PGI/AMF knockdown cells and that superoxide dismutase is the upstream regulator protein of p21-mediated cellular senescence. A specific inhibitor of PGI/AMF induced cellular senescence and p21 expression in tumor cells exposed to an oxidative stress environment. Taken together, the results presented here suggest that PGI/AMF is involved in oxidative stress-induced cellular senescence and should bring novel insights into the control of cellular growth leading to a new methodology for cancer treatment.
Highlights
Cellular Senescence—To study the possible role of Phosphoglucose isomerase (PGI)/autocrine motility factor (AMF) in cellular senescence, we partially knocked down PGI/AMF in HT1080 fibrosarcoma cells by gene silencing using siRNA duplexes derived from the human PGI/AMF sequence (Fig. 1A; complete silencing of PGI/AMF protein expression results in loss of viability)
Equal amounts of the proteins were a variety of stresses including oxidative stress such as exposure separated on SDS-PAGE gels and transferred to a 0.2-m poly- to H2O2, which induces premature cellular senescence (2– 4, vinylidene fluoride membrane (Osmonics Inc., Minnetonka, 28)
We report that PGI/AMF knockdown induces cellular senescence in tumor cells in response to oxidative stress by investigating SA -gal expression, a well known marker of cellular senescence
Summary
Using the PGI/AMF-siRNA technique, we found that both endogenous and exogenous expressions were suppressed in DCIS.com cells (Fig. 3C) associated with a 2-fold increase in SA -gal-positive cells formation than the control, under oxidative stress condition and under normal conditions (Fig. 3D). Knockdown MCF10A cells, a similar pattern was observed for oxidative stress-induced cellular senescence, but the differences were not statistically significant (Fig. 3F). The effect of PGI/AMF knockdown on cell cycle proteins expression under oxidative stress.
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