We observed that short-term (24?h) cell exposure to 6BIO induced a dose-dependent upregulation of molecular chaperones genes (and genes (Fig.?3C1), as well as in mild upregulation of the Nqo1 protein (Fig.?3C2). Open in a separate window Figure 2 Treatment of human skin fibroblasts with 6BIO activated proteostatic modules. rate of cell cycling, it significantly suppressed cellular senescence-related accumulation of biomolecular damage. Taken together, our presented findings L-Tryptophan suggest that 6BIO is a novel activator Cxcl12 of antioxidant responses and of the proteostasis network in normal human cells; moreover, and given the low levels of biomolecules damage in 6BIO treated senescing cells, this compound likely exerts anti-tumor properties. Introduction Organismal ageing is an inevitable and irreversible consequence of life promoted by both genetic and environmental factors1,2. Specifically, ageing is defined as a time-dependent decline of stress resistance and functional capacity, associated with increased probability of morbidity and mortality. These effects relate to (among others) age-related gradual accumulation of damaged biomolecules (including proteins) which eventually result in the disruption of cellular homeodynamics. Accordingly, ageing is the primary risk factor for major human pathologies, including cancer, diabetes, cardiovascular disorders and neurodegenerative diseases2. Proteome quality control is critical for cellular functionality and it is assured by the curating activity of the proteostasis network (PN) and of antioxidant responses. Central to PN functionality are the two main proteolytic systems, namely the Ubiquitin-Proteasome System (UPS) and the Autophagy-Lysosome Pathway (ALP) along with the armada of the molecular chaperones3,4. UPS degrades both normal short-lived ubiquitinated proteins, as well as non-repairable misfolded, unfolded or damaged proteins3,5,6, whereas ALP is mostly involved in the degradation of long-lived proteins, aggregated ubiquitinated proteins and in the recycling of damaged organelles7C9. On the other hand, molecular chaperones are mostly responsible for the correct folding of proteins and for the prevention of protein aggregation. Moreover, they either refold unfolded and misfolded proteins or drive them for degradation through the two aforementioned degradation machineries10,11. Proteome quality control also depends on the activity of the Nrf2 (Nuclear factor erythroid 2-related factor 2)/Keap1 (Kelch-like ECH-associated protein 1) signalling pathway which regulates cellular responses to oxidative and electrophilic stress. Nrf2 is a key transcription factor regulating the expression of a wide array of phase II and antioxidant enzymes; under normal conditions Nrf2 is inhibited in the cytoplasm by Keap112. The UPS functionality, as well as the antioxidant responses signalling, decline during cellular senescence or ageing13C17 indicating that they are involved in the appearance and, likely, the progression of ageing phenotypes. On the other hand, activation of UPS and of stress responsive pathways has been linked to prolonged efficient removal of damaged and/or dysfunctional polypeptides, exerting thus anti-ageing effects18C21. It is nowadays evident that both healthspan (the disease-free period of life) and/or lifespan (maximum longevity) can be prolonged by genetic, dietary (e.g. caloric restriction) and/or pharmacological interventions suggesting that animals have the latent potential to live longer than they normally do1,2,22. As genetic interventions or prolonged caloric restriction cannot be applied in humans, many studies have been devoted to the identification of natural products (NPs) that can prolong healthspan and/or lifespan. It is well established that NPs represent an extraordinary inventory of high diversity structural scaffolds that can be used as pharmacological modulators of age-related signalling pathways. These pathways may be involved in ageing regulation L-Tryptophan by dampening signalling from nutrient sensing pathways, thus mimicking the systemic effects of caloric restriction or by activating the stress responsive pathways1. Nevertheless, and despite encouraging findings in relation of NPs potential bioactivity towards the delay of cellular senescence and/or ageing; these data along with the targets and bioactive lead molecules will be reported elsewhere. Our herein presented study was focused on bioactive indirubins and specifically a hemi-synthetic cell-permeable indirubin derivative, namely 6-bromoindirubin-3-oxime (6BIO). Indirubins belong to the L-Tryptophan family of bis-indole alkaloids isolated from indigo dye-producing edible plants and gastropod mollusks23. Indirubins and their analogues have been described as potent inhibitors of Cyclin-dependent kinases (CDKs)24, as well as of Glycogen synthase kinase-3 (Gsk-3)25. Gsk-3 is a multifunctional ubiquitously L-Tryptophan expressed serine/threonine kinase that has been functionally involved in diverse cellular processes, including (among others) glycogen synthesis, proliferation, development and apoptosis26C28. In mammalian tissues, Gsk-3 exists as two isoforms (Gsk-3 and Gsk-3) that share 98% homology of their kinase domains, while differing substantially in their L-Tryptophan N-terminal and C-terminal sequences29. Accordingly, Gsk-3 has been involved in several age-related conditions and diseases including inflammation, diabetes, neurodegenerative disorders and cancer28,30C32. Nevertheless, the role of either Gsk-3 or 6BIO in normal human cells senescence remains largely unknown. We report herein that 6BIO is a novel modulator of antioxidant responses and PN activity in human fibroblasts. Moreover, prolonged incubation of cells with 6BIO decreased the rate of.