Caspase-cleaved Fragment Research Articles

Enhancing the Conformational Stability of the cl-Par-4 Tumor Suppressor via Site-Directed Mutagenesis.

Intrinsically disordered proteins play important roles in cell signaling, and dysregulation of these proteins is associated with several diseases. Prostate apoptosis response-4 (Par-4), an approximately 40 kilodalton proapoptotic tumor suppressor, is a predominantly intrinsically disordered protein whose downregulation has been observed in various cancers. The caspase-cleaved fragment of Par-4 (cl-Par-4) is active and plays a role in tumor suppression by inhibiting cell survival pathways. Here, we employed site-directed mutagenesis to create a cl-Par-4 point mutant (D313K). The expressed and purified D313K protein was characterized using biophysical techniques, and the results were compared to that of the wild-type (WT). We have previously demonstrated that WT cl-Par-4 attains a stable, compact, and helical conformation in the presence of a high level of salt at physiological pH. Here, we show that the D313K protein attains a similar conformation as the WT in the presence of salt, but at an approximately two times lower salt concentration. This establishes that the substitution of a basic residue for an acidic residue at position 313 alleviates inter-helical charge repulsion between dimer partners and helps to stabilize the structural conformation.

Serological Markers of Intestinal Barrier Impairment do not Correlate With Duration of Diabetes and Glycated Hemoglobin in Adult Patients With Type 1 and Type 2 Diabetes Mellitus

Growing evidence suggests that diabetes mellitus is associated with impairment of the intestinal barrier. However, it is not clear so far if the impairment of the intestinal barrier is a consequence of prolonged hyperglycemia or the consequence of external factors influencing the gut microbiota and intestinal mucosa integrity. Aim of the study was to perform an estimation of relationship between serological markers of impairment of the intestinal barrier: intestinal fatty acid-binding protein (I-FABP), cytokeratin 18 caspase-cleaved fragment (cCK-18), and soluble CD14 (sCD14) and markers of prolonged hyperglycemia, such as the duration of diabetes mellitus and glycated hemoglobin (HbA1c) via a correlation analysis in patients with diabetes mellitus. In 40 adult patients with type 1 diabetes mellitus and 30 adult patients with type 2 diabetes mellitus the estimation has been performed. Statistically significant positive correlation was found between cCK-18 and HbA1c (r=0.5047, p=0.0275) in patients with type 1 diabetes mellitus with fading insulitis (T1D). In patients with type 1 diabetes mellitus with ongoing insulitis (T1D/INS) and in patients with type 2 diabetes mellitus (T2D), no statistically significant positive correlations were found between serological markers of intestinal barrier impairment (I-FABP, cCK-18, sCD14) and duration of diabetes or levels of HbA1c. Similarly, in cumulative cohort of patients with T1D/INS and patients with T1D we revealed statistically positive correlation only between HbA1c and cCK-18 (r=0.3414, p=0.0311). Surprisingly, we found statistically significant negative correlation between the duration of diabetes mellitus and cCK-18 (r=-0.3050, p=0.0313) only in cumulative group of diabetic patients (T1D, T1D/INS, and T2D). Based on our results, we hypothesize that the actual condition of the intestinal barrier in diabetic patients is much more dependent on variable interactions between host genetic factors, gut microbiota, and environmental factors rather than effects of long-standing hyperglycemia (assessed by duration of diabetes mellitus or HbA1c).

The antiviral action of the RIG-I induced pathway of apoptosis (RIPA) is enhanced by its ability to degrade Otulin, which deubiquitinates IRF3.

Mammalian innate immune response to virus infection is meditated by many cell-intrinsic pathways. RNA viruses, such as Sendai virus, which replicate in the cytoplasm, trigger the RIG-I-like receptor pathway, which activates the transcription factor, IRF3. Activated IRF3 translocates to the nucleus and induces transcription of the genes which encode interferons, the major antiviral cytokines. Interestingly, IRF3 activates another interferon-independent antiviral pathway, called RIG-I induced pathway of apoptosis (RIPA). For activating RIPA, IRF3 translocates from the cytoplasm to mitochondria. RIPA requires linear polyubiquitination of IRF3 by the enzyme complex, LUBAC; ubiquitinated IRF3 binds to Bax and translocates it to mitochondria causing the release of Cytochrome C, activation of caspases and apoptosis of the infected cell. Here, we report that Otulin, the deubiquitinase that removes linear polyubiquitin chains, inhibits RIPA by deubiquitinating IRF3. Ablation of Otulin expression enhanced RIPA and its overexpression inhibited RIPA. In virus-infected cells, to overcome Otulin-mediated inhibition, RIPA actively degrades Otulin. This degradation required sequential actions of RIPA-activated Caspase 3 and proteasomes. Caspase 3 cleaved Otulin at D31; the D31A mutant was not cleaved at all. The caspase-cleaved fragment was totally degraded by proteasomes, which was preceded by its K48-linked ubiquitination. Mass spectrometric analysis of Otulin identified K64 and K197 as the ubiquitinated residues. Otulin interacted with LUBAC after virus infection and the E3-ubiquitin ligase, HOIP, a component of LUBAC, ubiquitinated Otulin to trigger its proteasome-mediated degradation. To assess the impact of Otulin degradation on RIPA-mediated antiviral action, we expressed, in Otulin-ablated cells, a non-degradable mutant of Otulin, in which D31, K64 and K197 had been mutated. The cells expressing the Otulin mutant were less susceptible to virus-induced apoptosis, because RIPA was less active, and consequently virus replication was more robust. Thus, our study has revealed an important positive feedback loop of RIPA.

Serum caspase-cleaved cytokeratin-18 fragment as a prognostic biomarker in hematological patients with febrile neutropenia

The study aim was to determine the benefit of the measurement of serum caspase-cleaved cytokeratin-18 (CK-18) fragment as a prognostic marker of febrile neutropenia (FN) in hematological patients. The study population consisted of 86 consecutive patients with FN who received intensive chemotherapy for hematological malignancy at the adult hematology ward of Kuopio University Hospital. Twenty-three patients (27%) had acute myeloid leukemia, and 63 patients (73%) were autologous stem cell transplant recipients. Serum caspase-cleaved CK-18 fragment M30, C-reactive protein (CRP) and procalcitonin (PCT) were measured at the onset of FN (d0), on day 1 (d1), and on day 2 (d2). Eight patients (9%) developed severe sepsis, including three patients with septic shock. Eighteen patients (21%) had a blood culture-positive infection. Serum CK-18 fragment peaked on the first day after fever onset in patients with severe sepsis. Higher CK-18 level was associated with severe sepsis, intensive care unit treatment, and fatal outcome, but not with blood culture positivity. In ROC curve analysis, d1 serum CK-18 fragment predicted severe sepsis with an area under the curve (AUC) of 0.767, CRP with an AUC of 0.764, and PCT with an AUC of 0.731. On d2, the best predictive capacity was observed for CRP with an AUC of 0.832. The optimal cutoff of caspase-cleaved CK-18 fragment M30 for predicting severe sepsis was 205 U/L on d1. In hematological patients, serum CK-18 fragment was found to be a potential prognostic marker of severe sepsis at early stages of FN.

Blood BTEXS and heavy metal levels are associated with liver injury and systemic inflammation in Gulf states residents

IntroductionExposures to volatile organic compounds and metals have previously been associated with liver diseases including steatohepatitis, although more data are needed. Benzene, toluene, ethylbenzene, xylenes, styrene (BTEXS) and metals were measured in blood samples collected between May 2012–July 2013 from volunteers participating in home visits for the Gulf Long-term Follow-up (GuLF) Study. This cross-sectional analysis evaluates associations of exposure biomarkers with serum liver injury and adipocytokine biomarkers in a sample of 214 men. MethodsAdult nonsmoking men without a history of liver disease or heavy alcohol consumption were included. The serologic disease biomarkers evaluated were the hepatocellular injury biomarker, cytokeratin 18 [whole (CK18 M65) and caspase-cleaved fragment (CK18 M30)]; and adipocytokines. Confounder-adjusted beta coefficients were determined using linear regression models for the overall sample (primary endpoints) and for obesity-classified sub-groups (secondary endpoints). A product interaction term between the exposure of interest and a dichotomized indicator of obesity was included to determine the disease modifying effects of obesity on the biomarker associations. ResultsThe study sample was 57% white and 51% obese. In the overall sample, lead was positively associated with CK18 M30 (β = 21.7 ± 6.0 (SE), p = 0.0004); IL-1β (β = 32.8 ± 5.2, p < 0.0001); IL-6 (β = 72.8 ± 18.3, p = 0.0001); and IL-8 (β = 140.8 ± 42.2, p = 0.001). Cadmium exposures were associated with increased IL-1β (β = 77.8 ± 26.3, p = 0.003) and IL-8 (β = 419.5 ± 201.2, p = 0.04). There were multiple significant interactions between obesity and exposure to lead, cadmium, benzene and toluene in relation to outcome biomarkers. Among obese participants (n = 108), benzene, lead, and cadmium were each positively associated with CK18 M30, IL-1β, IL-6, and IL-8. In obese subjects, lead was also inversely associated with leptin, and toluene was positively associated with IL-1β. ConclusionFor the overall sample, heavy metal exposures were associated with liver injury (lead only) and/or systemic inflammation (lead and cadmium). Obesity modified the associations between BTEXS and heavy metal exposures on several of the outcome variables. In the obesity subgroup, liver injury was positively associated with lead, cadmium and benzene exposures; systemic inflammation was increased with lead, cadmium, benzene, and toluene exposures; and leptin was inversely associated with lead exposures. The cross-sectional design of this study makes it difficult to determine causality, and all results should be interpreted cautiously. Nonetheless, the potential impact of exposures to lead, cadmium, benzene and toluene in steatohepatitis, an obesity-associated inflammatory liver disease, warrants further investigation.

Systemic inflammation is associated with circulating cell death released keratin 18 fragments in colorectal cancer

ABSTRACT Systemic inflammation is a stage-independent marker of poor prognosis in colorectal cancer (CRC), activated in a complex, multifactorial process. It has been proposed that one of the main factors driving systemic inflammation may be tumor necrosis. Keratin 18 (KRT18) fragments are released from dead cells and their serum levels are markers for apoptotic and necrotic cell death. In CRC, high KRT18 levels associate with advanced disease, but their relationship with tumor necrosis and systemic inflammation is unknown. In this study, serum total soluble KRT18 (tKRT18) and apoptosis-related, caspase-cleaved fragment (aKRT18) levels were measured preoperatively from 328 CRC patients, and their difference was calculated to assess necrosis related KRT18 (nKRT18) levels. The relationships of these markers with tumor necrosis, clinicopathologic features, systemic inflammation markers (C-reactive protein, albumin, and 13 cytokines), and survival were analyzed. High serum tKRT18, aKRT18, and nKRT18 levels showed association with a higher extent of tumor necrosis, distant metastasis, and increased levels of several markers of systemic inflammation, including CXCL8. High serum tKRT18 (multivariable HR 1.94, 95% CI 1.28–2.95, p = .002) and nKRT18 (multivariable HR 1.87, 95% CI 1.24–2.82, p = .003) levels were associated with poor overall survival independent of potential confounding factors. Our results show that tumor necrosis in CRC contributes to serum levels of KRT18 fragments, and both necrosis and KRT18 levels associate with systemic inflammation. Moreover, we show that serum tKRT18 and nKRT18 levels have independent prognostic value in CRC. Our observations confirm the link between cell death and systemic inflammation.

Urinary acrolein metabolite levels in severe acute alcoholic hepatitis patients.

Alcohol-associated liver disease (ALD) remains a major health concern worldwide. Alcohol consumption gives rise to reactive/toxic acrolein, a pathogenic mediator of liver injury in experimental ALD. Elevated acrolein adducts and metabolites are detectable in blood and urine. This study evaluates the major urinary acrolein metabolite, 3-hydroxypropylmercapturic acid (HPMA), in patients with acute alcoholic hepatitis (AAH) and examines its association with disease severity and markers of hepatic inflammation and injury. Urine HPMA was significantly higher in patients with severe [model for end-stage liver disease (MELD) ≥ 20] AAH compared with nonsevere AAH (MELD ≤ 19) or non-alcohol-consuming controls, suggesting that urine HPMA is a novel noninvasive biomarker in severe AAH. The association between HPMA and MELD in patients with AAH was nonlinear. In patients with nonsevere AAH, there was a positive trend, although not significant, whereas in severe AAH the association was negative, indicative of extensive injury and glutathione depletion. Consistent with the multifactorial etiology of ALD, our data identified strong combined effects of HPMA and proinflammatory cytokines on hepatocyte cell death, thereby supporting the pathogenic role of acrolein in liver injury. HPMA, together with IL-1β, showed robust associations with cytokeratin 18 caspase-cleaved fragment (CK18-M30; adjusted R2 = 0.812, P = 0.016) and cytokeratin 18 full-length protein (CK18-M65; adjusted R2 = 0.670, P = 0.048); similarly, HPMA, with IL-8, correlated with CK18-M30 (adjusted R2 = 0.875, P = 0.007) and CK18-M65 (adjusted R2 = 0.831, P = 0.013). The apoptosis index (CK18-M30:CK18-M65 ratio) strongly correlated with HPMA, together with IL-1β (adjusted R2 = 0.777, P = 0.022) or tumor necrosis factor-α (TNFα; adjusted R2 = 0.677, P = 0.046). In patients with severe AAH, IL-1β, IL-8, and TNFα are the predominant proinflammatory cytokines that interact with HPMA and play important mediating roles in influencing the extent/pattern of liver cell death. NEW & NOTEWORTHY This is the first study to examine the urinary acrolein metabolite 3-hydroxypropylmercapturic acid (HPMA) in alcoholic liver disease. HPMA was higher in patients with severe acute alcoholic hepatitis (AAH) compared with controls or nonsevere AAH and may be a novel selective, noninvasive biomarker for severe AAH. Consistent with the multifactorial etiology of alcohol-associated liver disease, we identified strong combined effects of HPMA and proinflammatory cytokines (IL-1β, IL-8, and TNFα) on the extent/pattern of liver cell death, thereby supporting the pathogenic role of acrolein.

High Circulating Caspase-Cleaved Keratin 18 Fragments (M30) Indicate Short-Term Mortality in Critically Ill Patients.

Caspase-cleaved fragments of the intermediate filament protein keratin 18 (cytokeratin-18 (CK18)) can be detected in serum as M30 levels and may serve as a circulating biomarker indicating apoptosis of epithelial and parenchymal cells. In order to evaluate M30 as a biomarker in critical illness, we analyzed circulating M30 levels in 243 critically ill patients (156 with sepsis, 87 without sepsis) at admission to the medical intensive care unit (ICU), in comparison to healthy controls (n = 32). M30 levels were significantly elevated in ICU patients compared with healthy controls. Circulating M30 was closely associated with disease severity but did not differ between patients with sepsis and ICU patients without sepsis. M30 serum levels were correlated with biomarkers of inflammation, cell injury, renal failure, and liver failure in critically ill patients. Patients that died at the ICU showed increased M30 levels at admission, compared with surviving patients. A similar trend was observed for the overall survival. Regression analyses confirmed that M30 levels are associated with mortality, and patients with M30 levels above 250.8 U/L displayed an excessive short-term mortality. Thus, our data support the utility of circulating levels of the apoptosis-related keratin fragment M30 as a prognostic biomarker at ICU admission.

Elevated Serum Cytokeratin-18 Levels in Patients with Type 2 Diabetes Mellitus and Nonalcoholic Fatty Liver Disease

Background: Cytokeratin-18 (CK-18) is a caspase-cleaved fragment released by injured hepatocytes, and serum levels of CK-18 were believed to be a marker of hepatic cell damage such as inflammation. Patients with type 2 diabetes (T2DM) were well known to have a high prevalence of the fatty liver condition. However, rare studies had been reported the serum CK-18 level in patients with T2DM and investigated the association between the levels with metabolic biomarkers. Material and Methods: Healthy participants and T2DM patients who followed in the specialized diabetes polyclinic were enrolled. Physical and metabolic factors were collected,and NAFLD was screened by abdominal ultrasound and fatty liver index. Cytokeratin 18 level was detected by commercially available immunoassay. Results: There were 22.8% (29/127) and 35.9%(42/117) participants were diagnosedwith NAFLD in the non-DM group and T2DM group, respectively. Both serum levels of CK-18 M30 and CK-18 M65 were significantly higher in patients with T2DM as compared with those of the non-DM group. Patients with nonalcoholic fatty liver disease (NAFLD) had higher serum CK-18 M30 level whatever withT2DM (205.8±135.1 vs. 108.4±66.19 U/L; P&amp;lt;0.001) or without (177.69±70.53 vs. 87.07±34.57 U/L; P003C0.001). Similarly, serum CK-18 M65 was also higher in patients with NAFLD in theT2DM group (513.9±271.2 vs. 285.4±115.3 U/L; P&amp;lt;0.001)and in the non-DM group (353.5±162.4 vs. 248.51±111.3 U/L; P&amp;lt;0.001).Multi-variate regression analyses demonstrated that fasting plasma glucose was independently and significantly associated with CK-18 M30 (β coefficient: 0.002; P=0.011)) and CK-18 M65 (β coefficient:0.003; P&amp;lt;0.001). These results suggested that CK-18 level was closely connected with diabetes mellitus. Conclusions: Independent of NAFLD, our result suggests that CK-18 level was closely associated with the hyperglycemic milieu. The association between serum CK-18 and T2DM may worth for further investigation. Disclosure T. Lee: None.

An MRM-Based Cytokeratin Marker Assay as a Tool for Cancer Studies: Application to Lung Cancer Pleural Effusions.

The goal of this work was to develop an LC-MRM assay for the quantitative analysis of a set of established and diagnostically important cytokeratin (CK) markers used in cancer diagnosis, prognosis, and therapy monitoring. Second, the potential of this assay in lung cancer diagnosis through pleural effusion (PE) analysis was examined. A multiplexed MRM assay was developed for 17 CKs and their select caspase-cleaved fragments. Isotope-labeled standard peptides were used for high assay specificity and absolute peptide quantitation; with robust standard-flow LC coupled to a latest-generation triple-quadrupole instrument for high sensitivity. The potential clinical applicability was demonstrated by the analysis of 118 PE samples. The MRM assay was evaluated for endogenous detection, linearity, precision, upper and lower limits of quantification, selectivity, reproducibility and peptide stability, and is generally applicable to any epithelial cancer study. A set of 118 patients with known pathologies allowed us to define the range of CK levels in clinical PE samples. Specific CKs were able to differentiate cancer-related PEs from those caused by benign ailments. In addition, they allowed to differentiate between PEs from subjects with small cell lung cancer versus non-small cell lung carcinoma, and to further differentiate the latter into its two subtypes, adenocarcinoma and squamous cell carcinoma. An MRM-based CK assay for carcinoma studies can differentiate between the three lung cancer histological types using less-invasive PE sampling providing potential therapy-guiding information on patients that are inoperable.

An open-label, phase II study of intravenous anetumab ravtansine, an anti-mesothelin antibody drug conjugate, in pretreated mesothelin-expressing advanced pancreatic cancer.

TPS540 Background: Advanced pancreatic cancer remains one of the deadliest malignancies with median OS of 11 months with the most aggressive therapy regimen. Hence, a need exists for more effective, durable, and targeted therapies. Mesothelin is a membrane associated antigen overexpressed in most pancreatic adenocarcinomas. The biological function of mesothelin remains unknown but it is an internalizing antigen. Given its limited expression in normal tissue and high expression in malignant cells, mesothelin is an excellent target for antibody directed cytotoxic drug delivery. Anetumab ravtansine (AR) is an ADC consisting of a fully human IgG1 antibody directed at mesothelin and conjugated to tubulin-binding toxophore, DM4 (maytansine derivative). Preclinical efforts have shown antiproliferative activity in mesothelin positive cancer cell lines and 75% tumor regression in pancreatic xenograft models (Golfier S, et al. Mol Cancer Ther 13(6):1537-48, 2014). Methods: This is a multicenter, non-randomized, Phase II study evaluating the efficacy of AR in pretreated mesothelin-expressing advanced pancreatic cancer. The study utilizes minimax, Simon 2-stage design testing 5% null hypothesis versus 21% alternative. If no response among the first 20, trial will terminate early. If at least one response among the first 20, trial will accrue additional 10 patients for maximum of 30. Four or more responses out of 30 will reject the null hypothesis. Significance level (alpha) is 0.1 and power is 90%. Probability of stopping early is 36% if the response rate is 5% or lower. Patients will receive AR infusion at 6.5 mg/kg on Day 1 of 21-day cycle. Primary objective: response rate per RECIST 1.1. Secondary objectives: time to progression; toxicity. Eligibility: measurable disease per RECIST 1.1; no more than two prior chemotherapy regimens; 30% of cells with 2+/3+ mesothelin staining per IHC; no history of keratitis or corneal disease. Correlative studies: serum soluble mesothelin levels and apoptotic factors (CK18, caspase-cleaved fragments) at baseline and throughout the study to predict treatment response/outcome. Enrollment is on-going. Clinical trial information: NCT03023722.

Detecting Cleaved Caspase-3 in Apoptotic Cells by Flow Cytometry.

Apoptosis is orchestrated by caspases, a family of cysteine proteases that cleave their substrates on the carboxy-terminal side of specific aspartic acid residues. These proteases are generally present in healthy cells as inactive zymogens, but when stimulated they undergo autolytic cleavage to become fully active. They subsequently cleave their substrates at one or two specific sites, which can result in activation, inactivation, relocalization, or remodeling of the substrate. Consequently, many of the cleaved fragments remain intact during apoptosis and can be detected using substrate-specific antibodies. These fragments are most commonly detected by western blotting, which resolves proteins and their fragments based on molecular mass. However, antibodies that only recognize cleaved fragments can be used to specifically label cells in which caspase cleavage has occurred. It is then possible to quantify these cells by flow cytometry. A number of antibodies that specifically recognize caspase-cleaved fragments have been generated, including antibodies that recognize the cleaved form of caspase-3. This caspase is responsible for the majority of proteolysis during apoptosis, and detection of cleaved caspase-3 is therefore considered a reliable marker for cells that are dying, or have died by apoptosis. This protocol outlines the quantification of apoptosis by flow cytometric detection of cleaved caspase-3.

Serological markers of enterocyte damage and apoptosis in patients with celiac disease, autoimmune diabetes mellitus and diabetes mellitus type 2.

Impairment of mucosal barrier integrity of small intestine might be causative in immune-mediated gastrointestinal diseases. We tested the markers of epithelial apoptosis - cytokeratin 18 caspase-cleaved fragment (cCK-18), and enterocyte damage - intestinal fatty acid-binding protein (I-FABP) and soluble CD14 (sCD14) in sera of patients with untreated celiac disease (CLD), those on gluten-free diet (CLD-GFD), patients with autoimmune diabetes mellitus (T1D), T1D with insulitis (T1D/INS), and diabetes mellitus type 2 (T2D). We found elevated levels of cCK-18 (P<0.001), I-FABP (P<0.01) and sCD14 (P<0.05) in CLD when compared to healthy controls. However, the levels of cCK-18 (P<0.01) and I-FABP (P<0.01) in CLD-GFD were higher when compared with controls. Interestingly, elevated levels of cCK-18 and I-FABP were found in T2D and T1D (P<0.001), and T1D/INS (P<0.01, P<0.001). Twenty-two out of 43 CLD patients were seropositive for cCK-18, 19/43 for I-FABP and 11/43 for sCD14; 9/30 of T2D patients were positive for cCK-18 and 5/20 of T1D/INS for sCD14, while in controls only 3/41 were positive for cCK-18, 3/41 for I-FABP and 1/41 for sCD14. We documented for the first time seropositivity for sCD14 in CLD and potential usefulness of serum cCK-18 and I-FABP as markers of gut damage in CLD, CLD-GFD, and diabetes.

Decrease of mitochondrial p53 during late apoptosis is linked to its dephosphorylation on serine 20

Following a genotoxic stress, the tumor suppressor p53 translocates to mitochondria to take part in direct induction of apoptosis, via interaction with BCL-2 family members such as BAK and BAX. We determined the kinetics of the mitochondrial translocation of p53 in HCT-116 and PA-1 cells exposed to different genotoxic stresses (doxorubicin, camptothecin, UVB). This analysis revealed an early escalation in the amount of mitochondrial p53, followed by a peak amount and a decrease of mitochondrial p53 at later time points. We show that the serine 20 phosphorylated form of p53 is present at the mitochondria and that the decrease of p53 mitochondrial level during late apoptosis correlates with a decrease of Ser-20 phosphorylation. Moreover, the S20A p53 mutant translocates well to mitochondria after a genotoxic stress but its mitochondrial localization is very low during late apoptosis when compared to wt p53. The S20A mutant also appears to be compromised for interaction with BAK. We propose here that the level of serine 20 phosphorylation is influential on p53 mitochondrial localization during late apoptosis. Additionally, we report the presence of a new ≃45 kDa caspase-cleaved fragment of p53 in the cytosolic and mitochondrial fractions of apoptotic cells.

The dili-sim initiative. Integrated systems pharmacology modeling to explain and predict drug hepatotoxicity

Drugs-induced liver injury (DILI) can occur through a variety of adverse outcome pathways. Understanding and predicting the effects of multiple toxicity pathways as a function of time and exposure are difficult without systematic organization. Quantitative systems modeling can combine multiple drug effects to address this challenge. The DILIsim Initiative is a public-private partnership involving scientists from 14 major pharmaceutical companies and the FDA; it is now entering its fourth year. In addition to financial support, companies provide often unpublished data and perform in kind research to fill gaps in knowledge. The software produced by the initiative, DILIsym®, is a highly specified, mechanistic, hepatic model that utilizes extensive kinetic information among interrelated biological processes to explore the hepatotoxic underpinnings via simulations. Mechanisms currently included in the model are oxidative stress, mitochondrial dysfunction, bile acid transporter inhibition, and lipotoxicity. The Dilisym® software was originally developed to explain and predict interspecies differences in dose dependent hepatotoxicity to help inform first in man dosing. However, the modeling effort has expanded to improve interpretation of traditional and mechanistic serum biomarkers including miR122, CK18 and its caspase-cleaved fragment, and HMGB1. It is now possible to utilize DILIsym® to predict the range of percent hepatocyte loss through necrosis or apoptosis from measurements of these biomarkers in serial serum samples archived from clinical trials. By varying parameters within Dilisym®, it is possible to create simulated patient populations that mimic selected clinic populations in terms of susceptibility to DILI. This approach successfully recently predicted the latency and incidence of serum ALT elevations that were observed in the clinical trials of troglitazone (ClinPharmacolTher. 96(5):589–598, 2014). Systems modeling tools such as DILIsym® will increasingly be used to support decision making throughout the life cycle of new drug candidates. Drugs-induced liver injury (DILI) can occur through a variety of adverse outcome pathways. Understanding and predicting the effects of multiple toxicity pathways as a function of time and exposure are difficult without systematic organization. Quantitative systems modeling can combine multiple drug effects to address this challenge. The DILIsim Initiative is a public-private partnership involving scientists from 14 major pharmaceutical companies and the FDA; it is now entering its fourth year. In addition to financial support, companies provide often unpublished data and perform in kind research to fill gaps in knowledge. The software produced by the initiative, DILIsym®, is a highly specified, mechanistic, hepatic model that utilizes extensive kinetic information among interrelated biological processes to explore the hepatotoxic underpinnings via simulations. Mechanisms currently included in the model are oxidative stress, mitochondrial dysfunction, bile acid transporter inhibition, and lipotoxicity. The Dilisym® software was originally developed to explain and predict interspecies differences in dose dependent hepatotoxicity to help inform first in man dosing. However, the modeling effort has expanded to improve interpretation of traditional and mechanistic serum biomarkers including miR122, CK18 and its caspase-cleaved fragment, and HMGB1. It is now possible to utilize DILIsym® to predict the range of percent hepatocyte loss through necrosis or apoptosis from measurements of these biomarkers in serial serum samples archived from clinical trials. By varying parameters within Dilisym®, it is possible to create simulated patient populations that mimic selected clinic populations in terms of susceptibility to DILI. This approach successfully recently predicted the latency and incidence of serum ALT elevations that were observed in the clinical trials of troglitazone (ClinPharmacolTher. 96(5):589–598, 2014). Systems modeling tools such as DILIsym® will increasingly be used to support decision making throughout the life cycle of new drug candidates.

Maternal and fetal serum levels of caspase-cleaved fragments of cytokeratin-18 in intrahepatic cholestasis of pregnancy

Objective: We aimed to investigate the relationship between intrahepatic cholestasis of pregnancy (ICP) and caspase-cleaved fragments of cytokeratin-18, also referred to as M30, a marker of apoptosis. Methods: In this case–control study, maternal and umbilical cord blood venous samples were obtained from 21 pregnant women with ICP and 22 healthy pregnant women as a control group. M30 levels were compared among the groups. Results: Maternal serum M30 levels were significantly higher in the severe ICP group than in the control (p < 0.001) and mild ICP groups (p = 0.006). The values were comparable between the mild ICP and the control groups. The umbilical cord serum M30 levels were also significantly greater in the severe ICP group than in the control group (p = 0.001). Conclusions: Changes in M30 levels, as an apoptosis marker, may shed light on the pathogenesis of ICP. Explaining the mechanisms of bile acid (BA)-induced hepatocyte injury may contribute further therapeutic strategies for the treatment of human cholestatic diseases.

Anti-Myeloma Activity of Combined Inhibition of the Proteasome with Carfilzomib (CFZ) and XPO1/CRM1-Dependent Nuclear Export By Selinexor (KPT-330) Via a Novel Mechanism of Intracellular Activation of Caspase 10-Dependent Apoptosis

INTRODUCTIONPreviously, our work highlighted the synergistic activity of combining the selective inhibitor of nuclear export (SINE) selinexor with the irreversible proteasome inhibitor (PI) CFZ in multiple myeloma (MM) patient-derived plasma cells and cell lines, and in a murine xenograft model (Rosebeck et al, Blood 2013:122(21):279). Our studies were the first to suggest a role for both autophagy and apoptosis in response to combined SINE/PI treatment. Importantly, our results served as pre-clinical rationale for a currently-enrolling phase I clinical trial for the treatment of refractory and/or relapsed MM (NCT02199665). To gain insight into the molecular mechanism of synergy, we explored the novel role of caspase 10, which was implicated in our previous work, in the cytotoxic effects of combined KPT-330/CFZ treatment.METHODSMM cell lines were grown in RPMI1640/10% FBS. Subcellular fractionation was performed by three rounds of freeze-thaw in CHAPS lysis buffer. Caspase activity was assayed according to the manufacturer’s instructions (BioVision). Otherwise, our studies used standard cellular and molecular biology techniques.RESULTSFirst, we demonstrate an increase in activity of caspases 10, 8, 9, and 3, but not caspase 1, in response to KPT-330/CFZ treatment. Inhibition of caspase 10 activity prevented activation of caspases 8, 9 and 3, abrogated cleavage of apoptotic substrates, including PARP and NF-κB, and significantly protected MM cells from the cytotoxic effects of SINE/PI treatment.Caspases 8 and 10 are orthologs; caspase 10 is not present in mice and is not well-characterized. In response to PI, caspase 8 is recruited to autophagic membranes via interaction with Sequestosome1 (p62) and LC3, key autophagy components whose membrane localization is enriched upon PI treatment, creating an intracellular death-inducing signaling complex. Therefore, we hypothesized that, given the homology and functional overlap between caspases 8 and 10, the same could be possible of caspase 10. To test this, we performed subcellular fractionation to separate soluble cytoplasmic proteins from detergent-insoluble, membrane-embedded proteins and interrogated the relationship between caspase 10 and p62. In untreated cells we were struck by the presence of active fragments of caspases 10, 8, and 9, but not 3, in the insoluble fractions; full-length pro-caspases were found mostly in the soluble extracts. Selinexor/CFZ enhanced activation of all caspases and nearly the entire population of active caspases was found in the detergent-insoluble fractions. Total p62 levels were prominently induced by SINE/PI treatment and two cleavage fragments that correspond to the size of previously-identified in vitro caspase-cleaved fragments were found exclusively in the insoluble fraction. These data suggested that relocalization of caspase fragments to an intracellular membrane harnesses caspase activity.Next, we used indirect immunofluorescence microscopy to assess the localization of caspases 10 and 8 and p62. Untreated cells showed diffuse staining, whereas selinexor/CFZ-treated cells had punctate staining and co-localization of caspases 10 and 8 with p62. Finally, in untreated cell lysates, GST-p62 pulled down endogenous LC3 and pro-caspase 10, but not its active fragments. Pro-caspase 10/GST-p62 association was dramatically reduced when using lysates from SINE/PI-treated cells, suggesting a priming effect of their association.CONCLUSIONSOur results are the first to demonstrate an intracellular mechanism of caspase 10 activation and suggest a model of synergy wherein selinexor/CFZ increase autophagic membranes embedded with p62 and LC3 that associate with pro-caspase 10 promoting its activation, likely by induced proximity, cleavage of other caspases and targets, and subsequent apoptosis (Figure). The nature of the novel intracellular membrane-embedded aggregate of active caspases requires further investigation. [Display omitted] DisclosuresMcCauley:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Patents & Royalties. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics: Employment. Jakubowiak:Bristol Myers-Squib: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; SkylineDx: Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Biomarkers distinguish apoptotic and necrotic cell death during hepatic ischemia/reperfusion injury in mice.

Hepatic ischemia/reperfusion (IRP) injury is a significant clinical problem during tumor-resection surgery (Pringle maneuver) and liver transplantation. However, the relative contribution of necrotic and apoptotic cell death to the overall liver injury is still controversial. To address this important issue with a standard murine model of hepatic IRP injury, plasma biomarkers of necrotic cell death such as micro-RNA 122, full-length cytokeratin 18 (FK18), and high-mobility group box 1 (HMGB1) protein and plasma biomarkers of apoptosis such as plasma caspase-3 activity and caspase-cleaved fragment of cytokeratin 18 (CK18) coupled with markers of inflammation (hyperacetylated HMGB1) were compared by histological features in hematoxylin and eosin-stained and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-stained liver sections. After 45 minutes of hepatic ischemia and 1 to 24 hours of reperfusion, all necrosis markers increased dramatically in plasma by 40- to >10,000-fold over the baseline with a time course similar to that of alanine aminotransferase. These data correlated well with histological characteristics of necrosis. Within the area of necrosis, most cells were TUNEL positive; initially (≤3 hours of reperfusion), the staining was restricted to nuclei, but it later spread to the cytosol, and this is characteristic of karyorrhexis during necrotic cell death. In contrast, the lack of morphological evidence of apoptotic cell death and relevant caspase-3 activity in the postischemic liver correlated well with the absence of caspase-3 activity and CK18 (except for a minor increase at 3 hours of reperfusion) in plasma. A quantitative comparison of FK18 (necrosis) and CK18 (apoptosis) release indicated dominant cell death by necrosis during IRP and only a temporary and very minor degree of apoptosis. These data suggest that the focus of future research should be the elucidation of necrotic signaling mechanisms to identify relevant targets, which may be used to attenuate hepatic IRP injury.

PTH-082 Do Serum Markers Of Cell Injury And Death Have Potential To Become Mechanistic Markers In Non-alcoholic Fatty Liver Disease (nafld)?

IntroductionDegree of hepatocellular injury, necrosis/apoptosis and inflammation may be assessed by the serum markers that reflect the pathogenic process. We investigated the correlation of circulating miR-122, High Mobility Group Box-1...

Improving Topical Non-Melanoma Skin Cancer Treatment: In vitro Efficacy of a Novel Guanosine-Analog Phosphonate

Actinic keratosis, a frequent carcinoma in situ of non-melanoma skin cancer (NMSC), can transform into life-threatening cutaneous squamous cell carcinoma. Current treatment is limited due to low complete clearance rates and asks for novel therapeutic concepts; the novel purine nucleotide analogue OxBu may be an option. In order to enhance skin penetration, solid lipid nanoparticles (SLN, 136-156 nm) were produced with an OxBu entrapment efficiency of 96.5 ± 0.1%. For improved preclinical evaluation, we combined tissue engineering with clinically used keratin-18 quantification. Three doses of 10^-3 mol/l OxBu, dissolved in phosphate-buffered saline as well as loaded to SLN, were effective on reconstructed NMSC. Tumour response and apoptosis induction were evaluated by an increase in caspase-cleaved fragment of keratin-18, caspase-7 activation as well as by reduced expression of matrix metallopeptidase-2 and Ki-67. OxBu efficacy was superior to equimolar 5-fluorouracil solution, and thus the drug should be subjected to the next step in preclinical evaluation.