Modulation of Cellular Senescence in HEK293 and HepG2 Cells by Ultrafiltrates UPla and ULu Is Partly Mediated by Modulation of Mitochondrial Homeostasis under Oxidative Stress

Abstract

Protein probes, including ultrafiltrates from the placenta (UPla) and lung (ULu) of postnatal rabbits, were investigated in premature senescent HEK293 and HepG2 cells to explore whether they could modulate cellular senescence. Tris-Tricine–PAGE, gene ontology (GO), and LC–MS/MS analysis were applied to describe the characteristics of the ultrafiltrates. HEK293 and HepG2 cells (both under 25 passages) exposed to a sub-toxic concentration of hydrogen peroxide (H2O2, 300 μM) became senescent; UPla (10 μg/mL), ULu (10 μg/mL), as well as positive controls lipoic acid (10 μg/mL) and transferrin (10 μg/mL) were added along with H2O2 to the cells. Cell morphology; cellular proliferation; senescence-associated beta-galactosidase (SA-β-X-gal) activity; expression of senescence biomarkers including p16 INK4A (p16), p21 Waf1/Cip1 (p21), HMGB1, MMP-3, TNF-α, IL-6, lamin B1, and phospho-histone H2A.X (γ-H2AX); senescence-related gene expression; reactive oxygen species (ROS) levels; and mitochondrial fission were examined. Tris-Tricine–PAGE revealed prominent detectable bands between 10 and 100 kDa. LC–MS/MS identified 150–180 proteins and peptides in the protein probes, and GO analysis demonstrated a distinct enrichment of proteins associated with “extracellular space” and “proteasome core complex”. UPla and ULu modulated senescent cell morphology, improved cell proliferation, and decreased beta-galactosidase activity, intracellular and mitochondrial ROS production, and mitochondrial fission caused by H2O2. The results from this study demonstrated that UPla and Ulu, as well as lipoic acid and transferrin, could protect HEK293 and HepG2 cells from H2O2-induced oxidative damage via protecting mitochondrial homeostasis and thus have the potential to be explored in anti-aging therapies.

Effectiveness and mechanisms of combined use of antioxidant nutrients in protecting against oxidative stress‐induced neuronal loss and related neurological deficits

“… 63 Therefore, we examined the effectiveness of treatments with melatonin, coenzyme Q10, lecithin, or in combination on the levels of P16 and P21, two key cell cycle inhibitors, and Lamin B1, a nuclear marker. 64 Injection of D‐(+)‐galactose resulted in a significant increase in the levels of P16 and P21 and a significant decrease in the expression level of Lamin B1 in both cortex and hippocampus (Figure 5A,E ). In the cortex and hippocampus, D‐(+)‐galactose‐induced increase in the expression of P16 and P21 was prevented by combined treatment, and D‐(+)‐galactose‐induced increase in the expression of P21 was also attenuated by treatment with melatonin, coenzyme, or lecithin in the cortex and by treatment with melatonin in the hippocampus, whereas D‐(+)‐galactose‐induced increase in the expression of P16 was mitigated by treatment with melatonin or coenzyme Q10, but not lecithin (Figure 5B,C,F,G ).…”

“…Oxidative stress is known for its ability to activate the senescence‐related signaling pathways to exacerbate cellular senescence that is accompanied by cell cycle arrest 63 . Therefore, we examined the effectiveness of treatments with melatonin, coenzyme Q10, lecithin, or in combination on the levels of P16 and P21, two key cell cycle inhibitors, and Lamin B1, a nuclear marker 64 . Injection of D‐(+)‐galactose resulted in a significant increase in the levels of P16 and P21 and a significant decrease in the expression level of Lamin B1 in both cortex and hippocampus (Figure 5A,E).…”

“…63 Therefore, we examined the effectiveness of treatments with melatonin, coenzyme Q10, lecithin, or in combination on the levels of P16 and P21, two key cell cycle inhibitors, and Lamin B1, a nuclear marker. 64…”

Cellular senescence in cancer: molecular mechanisms and therapeutic targets

“…The MMP family members, collagenase‐1 (MMP‐1) and stromelysin‐1 (MMP‐3), show consistent overexpression in senescent cells. 172 , 173 Through secretion of these MMPs, senescent cells can modify the TME by shedding membrane‐associated proteins, rendering them soluble, or degrading signaling molecules and the ECM. These MMPs can cleave IL‐8, MCP‐1, ‐2, and ‐4, 174 and others, such as MMP‐2 and ‐7, can also cleave the additional CXCL/CCL family members that make up the SASP.…”