ObjectivePrevious work has established that the deacetylase sirtuin-1 (SIRT1) is cleaved by cathepsin B in chondrocytes subjected to proinflammatory stress, yielding a stable but inactive N-terminal (NT) polypeptide (75SIRT1) and a C-terminal (CT) fragment. The present work examined if chondrocyte-derived NT-SIRT1 is detected in serum and may serve as an investigative and exploratory biomarker of osteoarthritis (OA).MethodsWe developed a novel ELISA assay to measure the ratio of NT to CT of SIRT1 in the serum of human individuals and mice subjected to post-traumatic OA (PTOA) or age-dependent OA (ADOA). We additionally monitored NT/CT SIRT1 in mice subject to ADOA/PTOA followed by senolytic clearance. Human chondrosenescent and non-senescent chondrocytes were exposed to cytokines and analysed for apoptosis and NT/CT SIRT1 ratio in conditioned medium.ResultsWild-type mice with PTOA or ADOA of moderate severity exhibited increased serum NT/CT SIRT1 ratio. In contrast, this ratio remained low in cartilage-specific Sirt1 knockout mice despite similar or increased PTOA and ADOA severity. Local clearance of senescent chondrocytes from old mice with post-traumatic injury resulted in a lower NT/CT ratio and reduced OA severity. While primary chondrocytes exhibited NT/CT ratio increased in conditioned media after prolonged cytokine stimulation, this increase was not evident in cytokine-stimulated chondrosenescent cells. Finally, serum NT/CT ratio was elevated in humans with early-stage OA.ConclusionsIncreased levels of serum NT/CT SIRT1 ratio correlated with moderate OA in both mice and humans, stemming at least in part from non-senescent chondrocyte apoptosis, possibly a result of prolonged inflammatory insult.
IntroductionLysosomal cathepsins have been reported to contribute to Osteoarthritis (OA) pathophysiology due to their increase in pro-inflammatory conditions. Given the causal role of cathepsins in OA, monitoring their specific activity could provide means for assessing OA severity. To this end, we herein sought to assess a cathepsin activity-based probe (ABP), GB123, in vitro and in vivo.MethodsProtein levels and activity of cathepsins B and S were monitored by immunoblot analysis and GB123 labeling in cultured primary chondrocytes and conditioned media, following stimuli with tumor necrosis factor alpha (TNFα) and/or Interleukin 1 beta (IL-1β). Similarly, cathepsin activity was examined in sections of intact cartilage (IC) and degraded cartilage (DC) regions of OA. Finally, synovial fluid (SF) and serum from donors with no signs of diseases, early OA, late OA and rheumatoid arthritis (RA) patients were analyzed with GB123 to detect distinct activity levels of cathepsin B and S.ResultsCathepsin activity in cell lysates, conditioned media explants and DC sections showed enhanced enzymatic activity of cathepsins B and S. Further histological analysis revealed that cathepsin activity was found higher in superficial zones of DC than in IC. Examining serum and SF revealed that cathepsin B is significantly elevated with OA severity in serum and SF, yet levels of cathepsin S are more correlated with synovitis and RA.ConclusionsBased on our data, cathepsin activity monitored by ABPs correlated well with OA severity and joint inflammation, directing towards a novel etiological target for OA, which possesses significant translational potential in developing means for non-invasive detection of early signs of OA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0586-5) contains supplementary material, which is available to authorized users.
The C-terminus of SIRT1 can be cleaved by cathepsin B at amino acid H533 to generate a lower-functioning, N-terminally intact 75 kDa polypeptide (75SIRT1) that might be involved in age-related pathologies. However, the mechanisms underlying cathepsin B docking to and cleavage of SIRT1 are unclear. Here, we first identified several 75SIRT1 variants that are augmented with aging correlatively with increased cathepsin B levels in various mouse tissues, highlighting the possible role of this cleavage event in age-related pathologies. Then, based on H533 point mutation and structural modeling, we generated a functionally intact ΔSIRT1 mutant, lacking the internal amino acids 528-543 (a predicted C-terminus loop domain), which exhibits resistance to cathepsin B cleavage and in cell cultures. Finally, we showed that cells expressing ΔSIRT1 under pro-inflammatory stress are more likely to undergo caspase 9- dependent apoptosis than those expressing 75SIRT1. Thus, our data suggest that the 15-amino acid predicted loop motif embedded in the C-terminus of SIRT1 is susceptible to proteolytic cleavage by cathepsin B, leading to the formation of several N-terminally intact SIRT1 truncated variants in various aging mouse tissues.This article has an associated First Person interview with the first author of the paper.
Background: Epothilones, including ixabepilone, have demonstrated antitumor activity in a wide range of chemonaïve and chemo-resistant cancer models. This study was conducted to determine efficacy and toxicity of the weekly schedule (weekly x 3 w/ no dosing in Week 4) compared to the every 3-week schedule in MBC patients.Patients and Methods: Patients with HER2-negative MBC that was measurable, or nonmeasurable with serum CA27.29 (or CA15.3) ≥50, performance status (ECOG 0-2) with no limit on prior chemotherapy were enrolled. Any existing peripheral neuropathy must have been ≤Grade 1. Treatment: Patients were randomly assigned to receive ixabepilone 16 mg/m2 IV over 1-hour on Days 1, 8, and 15 of each 28-day cycle (Arm 1), or 40 mg/m2 IV over 3-hours on Day 1 of each 21-day cycle (Arm 2). Toxicity was assessed and graded at every visit using the NCI CTCAE v3. Disease was assessed every 12 weeks during treatment.Results: 173 patients enrolled to date out of a planned accrual of 176 patients (85 in Arm 1 and 88 in Arm 2); median follow-up was 3.8 and 3.48 months, respectively. Median age [range] was 60 [37-79] in Arm 1 and 57 [39-80] in Arm 2, respectively. ECOG PS 2 was ∼7% in both treatment arms, all others were 0 or 1. Most patients had received prior chemotherapy for MBC (Arm 1/Arm 2: 1 or more prior regimens 74%/73%), and/or hormonal therapy. Prior adjuvant/neoadjuvant therapy had been received by 53% and 73% of patients in Arms 1 and 2, respectively. Prior anthracyclines had been administered to 61% and 76% in Arms 1 and 2, respectively, while taxanes had been administered to 69% and 75%, respectively. Hormone receptor status ER/PR Arm 1/Arm 2: +/+ 56.5%/44%, +/- 15%/18%, and -/- 20%/24%. Dosing: Arm 1 received a mean dose of 14.8 mg/m2, whereas Arm 2 received 37.2 mg/m2 over a median of 3 28-day cycles and 4 21-day cycles, respectively. Mean dose intensity was 93% in both arms. Grade 3-4 treatment-related adverse events (TRAEs) occurred in 22% (Arm 1) and 57% (Arm 2) of patients; serious adverse events occurred in 28% and 36% of Arm 1 and Arm 2 patients, respectively. Grade 3-4 TR neutropenia occurred in only 5% of Arm 1 patients, but was 33% in Arm 2; febrile neutropenia was limited to 1 patient in Arm 2 (Grade 3). Other Grade 3-4 TRAEs in >5% of patients (Arm 1/Arm 2) were: fatigue (5%/16%), neuropathy (5% [4% Grade 3, 1% Grade 4]/14% [∼13% Grade 3, 1% Grade 4]), dehydration (1%/10%), and vomiting (0%/6%). Discontinuations due to TRAEs were 7% in Arm 1 and 14% in Arm 2. Growth factor use was limited to 2 patients (2%) in Arm 2, due to anemia. Most deaths were due to PD.Conclusion: Both schedules have an acceptable safety profile. Preliminary safety results show the every 3-week ixabepilone schedule resulted in more adverse events than the weekly schedule and Grade 3-4 neuropathy occurred less frequently with the weekly dosing. Preliminary ORR, PFS, and OS will be reported.Supported, in part, a research grant from Bristol-Myers Squibb, Princeton, NJ. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6099.
Conclusions: Bovine SF from joints of healthy calves contains active aggrecanases that are capable of cleaving both adult human and immature bovine aggrecan in the IGD and in the CS2 region. Human SF from knees of adult injured patients, has the same ability to cleave in the CS2 region of both immature bovine and adult human aggrecan, but may have less activity against the bovine aggrecan IGD site. The cleavage seen in the IGD and the CS2 region of bovine aggrecan after incubation with JC is a delayed process that may involve additional time for diffusive transport of aggrecanases out of the JC, as well as time needed for induction or activation of the aggrecanases. EVALUATING CATHEPSIN ACTIVITY AND SIRT1 CLEAVAGE IN EXPERIMENTAL OSTEOARTHRITIS
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