Inhibition of 3-phosphoinositide-dependent protein kinase 1 (PDK1) can revert cellular senescence in human dermal fibroblasts.

Cellular senescence is defined as a stable, persistent arrest of cell proliferation. Here, we examine whether senescent cells can lose senescence hallmarks and reenter a reversible state of cell-cycle arrest (quiescence). We constructed a molecular regulatory network of cellular senescence based on previous experimental evidence. To infer the regulatory logic of the network, we performed phosphoprotein array experiments with normal human dermal fibroblasts and used the data to optimize the regulatory relationships between molecules with an evolutionary algorithm. From ensemble analysis of network models, we identified 3-phosphoinositide-dependent protein kinase 1 (PDK1) as a promising target for inhibitors to convert the senescent state to the quiescent state. We showed that inhibition of PDK1 in senescent human dermal fibroblasts eradicates senescence hallmarks and restores entry into the cell cycle by suppressing both nuclear factor κB and mTOR signaling, resulting in restored skin regeneration capacity. Our findings provide insight into a potential therapeutic strategy to treat age-related diseases associated with the accumulation of senescent cells.

3-Methyladenine Inhibits Procollagen-1 and Fibronectin Expression in Dermal Fibroblasts Independent of Autophagy.

BACKGROUND: Autophagy is deeply associated with aging, but little is known about its association with the extracellular matrix (ECM). 3-methyladenine (3-MA) is a commonly used autophagy inhibitor. OBJECTIVE: We used this compound to investigate the role of autophagy in dermal ECM protein synthesis. METHODS: Normal human dermal fibroblasts (NHDFs) were treated with 3-MA for 24 h, and mRNA encoding several ECM proteins was analyzed in addition to the protein expression of procollagen-1 and fibronectin. Several phosphoinositide 3-kinase (PI3K) inhibitors, an additional autophagy inhibitor, and small interfering RNA (siRNA) targeting autophagy-related genes were additionally used to confirm the role of autophagy in ECM synthesis. RESULTS: Only 3-MA, but not other chemical compounds or autophagy-related genetargeting siRNA, inhibited the transcription of procollagen-1 and fibronectin-encoding genes. Further, 3-MA did not affect the activation of regulatory Smads, but inhibited the interaction between Smad3 with p300. Moreover, 3-MA treatment increased the phosphorylation of cAMP response element-binding protein (CREB); however, CREB knock-down did not recover 3-MA-induced procollagen-1 and fibronectin downregulation. CONCLUSION: We revealed that 3-MA might inhibit procollagen-1 and fibronectin synthesis in an autophagy-independent manner by interfering with the binding between Smad3 and p300. Therefore, 3-MA could be a candidate for the treatment of diseases associated with the accumulation of ECM proteins.

Silymarin inhibits melanin synthesis in melanocyte cells.

OBJECTIVES: The aim was to search for inhibitors of melanogenesis from natural resources. METHODS: The inhibitory effect of silymarin on melanogenesis in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab, was studied. KEY FINDINGS: Silymarin significantly prevented melanin production in a dose-dependent manner with an IC50 value (concentration producing 50% maximal inhibition) of 28.2 microg/ml, without effects on cell viability. Also, silymarin inhibited L-DOPA oxidation activity of tyrosinase, the rate-limiting melanogenic enzyme, in cell based-systems but it did not directly affect cell-free tyrosinase activity. Furthermore, Western blot analysis indicated that silymarin decreased the expression of tyrosinase protein. CONCLUSIONS: This study suggests that the depigmenting effect of silymarin might be attributable to inhibition of tyrosinase expression and that silymarin may be useful as a natural skin-lightening agent.