Cell Death Markers Research Articles

Heterogeneous Formation of DNA Double-Strand Breaks and Cell-Free DNA in Leukemia T-Cell Line and Human Peripheral Blood Mononuclear Cells in Response to Topoisomerase II Inhibitors

Background: Improving precision medicine in chemotherapy requires highly sensitive and easily applicable diagnostic tools. In addition, non-invasive molecular real-time monitoring of cytotoxic response is highly desirable. Here, we employed the kinetics of DNA double-strand breaks (DSB) and cell-free DNA (cfDNA) in a cell model of topoisomerase II-inhibitors in T cell leukemia (Jurkat cells) compared to normal cells (peripheral blood mononuclear cells, PBMCs). Methods: We applied automated microscopy to quantify immuno-stained phosphorylated H2AX (γH2AX) as a marker for either DNA damage response (DDR) or cell death and quantitative PCR-based analysis of nuclear and mitochondrial cfDNA concentrations. Results: Jurkat cells displayed a DDR to cytotoxic drug treatment significantly earlier than PBMCs, and etoposide (ETP) induced DSB formation faster than doxorubicin (DOX) in both Jurkat and PBMCs. Jurkat cells exhibited an earlier cytotoxic response compared to PBMC, with a significantly increased mitochondrial cfDNA formation after 2 h of DOX application. In PBMCs, increased cell death was detected after 4 h of incubation with ETP, whereas DOX treatment was less effective. Conclusions: Both automated microscopy and mitochondrial cfDNA quantification analysis indicate that (malignant) Jurkat cells are more sensitive to DOX than (healthy) PBMC. Our real-time approach can improve DDR inducing drug selection and adaptation in cancer therapy and aids in decisions for optimal patient biosampling.

Recompensation of Liver Cirrhosis by TIPS Reduces Epithelial Cell Death Markers, Translating Into Improved Clinical Outcome.

Portal hypertension is the main pathophysiological driver of decompensation in patients with liver cirrhosis. Epithelial cell death markers, m30 and m65, correlate with hepatic injury and predict outcomes across various stages of liver disease. We aim (i) to evaluate whether portal hypertension itself contributes to liver outcome-relevant epithelial injury, and (ii) to analyse the capacity of m30/m65 to predict outcome in patients receiving a transjugular intrahepatic portosystemic shunt (TIPS) for refractory ascites. Sixty-six patients undergoing TIPS placement for refractory ascites and 20 patients with compensated cirrhosis as controls were prospectively enrolled in this monocentric cohort study. Epithelial cell death markers were analysed pre-TIPS, as well as 1-3 and 6-9 months post-TIPS. The capacity of baseline levels of m30/m65 in predicting six-month transplant-free survival rates was analysed by multivariable Cox proportional hazards regression. Levels of m30 and m65 were higher in patients with decompensated cirrhosis (pre-TIPS) compared with compensated cirrhosis (controls). Following correction of portal hypertension by TIPS and recompensation, both markers decreased over time, reaching levels comparable to patients with compensated cirrhosis. On multivariable analysis, pre-TIPS baseline levels of m30 and m65 were not predictive for six-month survival. Correction of portal hypertension via TIPS reduces levels of epithelial cell death markers, indicating that portal hypertension is a driver of outcome-relevant, hepatic cell death in patients with decompensated cirrhosis. Baseline m30/m65 values do not affect six-month survival rates, which suggests that TIPS placement overcomes the unfavourable spontaneous prognosis otherwise indicated by elevated baseline m30/65 levels.

Silver nanoparticle induced immunogenic cell death can improve immunotherapy

Cancer immunotherapy is often hindered by an immunosuppressive tumor microenvironment (TME). Various strategies are being evaluated to shift the TME from an immunologically ‘cold’ to ‘hot’ tumor and hereby improve current immune checkpoint blockades (ICB). One particular hot topic is the use of combination therapies. Here, we set out to screen a variety of metallic nanoparticles and explored their in vitro toxicity against a series of tumor and non-tumor cell lines. For silver nanoparticles, we also explored the effects of core size and surface chemistry on cytotoxicity. Ag-citrate-5 nm nanoparticles were found to induce high cytotoxicity in Renca cells through excessive generation of reactive oxygen species (ROS) and significantly increased cytokine production. The induced toxicity resulted in a shift of the immunogenic cell death (ICD) marker calreticulin to the cell surface in vitro and in vivo. Subcutaneous Renca tumors were treated with anti-PD1 or in combination with Ag-citrate-5 nm. The combination group resulted in significant reduction in tumor size, increased necrosis, and immune cell infiltration at the tumor site. Inhibition of cytotoxic CD8 + T cells confirmed the involvement of these cells in the observed therapeutic effects. Our results suggest that Ag-citrate-5 nm is able to promote immune cell influx and increase tumor responsiveness to ICB therapies.

Evaluation of the cell death markers for aberrated cell free DNA release in high altitude pulmonary edema.

The effect of high altitude (HA, altitude >2500 m) can trigger a maladaptive response in unacclimatized individuals, leading to various HA illnesses such as high altitude pulmonary edema (HAPE). The present study investigates circulating cell free (cf) DNA, a minimally invasive biomarker that can elicit a pro-inflammatory response. Our earlier study observed altered cfDNA fragment patterns in HAPE patients and the significant correlation of these patterns with peripheral oxygen saturation levels. However, the unclear release mechanisms of cfDNA in circulation limit its characterization and clinical utility. The present study not only observed a significant increase in cfDNA levels in HAPE patients (27.03 ± 1.37 ng/ml; n = 145) compared to healthy HA sojourners (controls, 14.57 ± 0.74 ng/ml; n = 65) and highlanders (HLs, 15.50 ± 0.8 ng/ml; n = 34) but also assayed the known cell death markers involved in cfDNA release at HA. The study found significantly elevated levels of the apoptotic marker, annexin A5, and secondary necrosis or late apoptotic marker, High mobility group box 1, in HAPE patients. In addition, we observed ahigher oxidative DNA damage marker, 8-hydroxy-2'-deoxyguanosine, in HAPE compared to controls, suggestive of the role of oxidative DNA status in promoting the inflammatory potential of cfDNA fragments and their plausible role in manifesting HAPE pathophysiology. Extensive in vitro future assays can confirm theimmunogenic role of cfDNA fragments that may act as a danger-associated molecular pattern and associate with markers of cellular stresses in HAPE.

Timing of cartilage articulation following impact injury affects the response of surface zone chondrocytes.

Post-traumatic osteoarthritis develops following an inciting injury to a joint and results in cartilage degeneration. Mechanical loading, including articulation, drives anabolic responses in cartilage clinically, in vivo, and in vitro. Tribological articulation, or sliding of cartilage on a glass counterface, has long been used as an in vitro tool to study cartilage tissue behavior. However, it is unclear if tribological articulation affects chondrocyte fate following injury, and if the timing of articulation impacts the resultant effect. The goal of this study was to investigate the effect of tribological articulation on injured cartilage tissue at two time points: (i) performed immediately after injury and (ii) 24 h after injury. Neonatal bovine femoral cartilage explants were injured using a rapid spring-loaded impactor and subsequently subjected to tribological articulation. Cell death due to impact injury was highest near the articular surface, suggesting a strain-dependent mechanism. Immediate articulation following injury mitigated cell death compared to injury alone or delayed articulation; markers for both general cell death and early-stage apoptosis were markedly decreased in the explants that were immediately slid. Interestingly, mitigation of cell death due to sliding was most predominant at the cartilage surface. Tribological articulation is known to create fluid flow within the tissue, predominantly at the articular surface, which could drive the protective response seen here. Altogether, this work shows that perturbations to the cellular environment immediately following cartilage injury significantly impact chondrocyte fate.

Impact of sample processing delays on plasma markers of inflammation, chemotaxis, cell death, and blood coagulation.

Biosampling studies in critically ill patients traditionally involve bedside collection of samples followed by local processing (ie. centrifugation, aliquotting, and freezing) and storage. However, community hospitals, which care for the majority of Canadian patients, often lack the infrastructure for local processing and storage of specimens. A potential solution is a "simplified" biosampling protocol whereby blood samples are collected at the bedside and then shipped to a central site for processing and storage. One potential limitation of this approach is that delayed processing may alter sample characteristics. To determine whether delays in blood sample processing affect the stability of cytokines (IL-6, TNF, IL-10, IFN-γ), chemokines (IL-8, IP-10, MCP-1, MCP-4, MIP-1α, MIP-1β), cell-free DNA (cfDNA) (released by dying cells), and blood clotting potential in human blood samples. Venous blood was collected into EDTA and citrate sample tubes and stored at room temperature (RT) or 4°C for progressive intervals up to 72 hours, prior to processing. Plasma cytokines and chemokines were quantified using single or multiplex immunoassays. cfDNA was measured using Picogreen DNA Quantification. Blood clotting potential was measured using a thrombin generation assay. Blood samples were collected from 9 intensive care unit (ICU) patients and 7 healthy volunteers. Admission diagnoses for the ICU patients included sepsis, trauma, ruptured abdominal aortic aneurysm, intracranial hemorrhage, gastrointestinal bleed, and hyperkalemia. After pre-processing delays of up to 72 hours at RT or 4°C, no significant changes were observed in plasma cytokines, chemokines, cfDNA, or thrombin formation. Delayed sample processing for up to 72 hours at either RT or 4°C did not significantly affect cytokines, chemokines, cfDNA, or blood clotting potential in plasma samples from healthy volunteers and ICU patients. A "simplified" biosampling protocol is a feasible solution for conducting biosampling research at hospitals without local processing capacity.

Pyroptosis is not likely to play a major role in anthracycline-induced cytotoxicity in H9c2 cardiomyocytes and human cardiac endothelial cells

Abstract Background Although anthracyclines are widely used as effective chemotherapeutic agents, anthracycline-induced cardiotoxicity (AIC) causes significant morbidity and mortality. The precise mechanism of AIC remains elusive, although apoptosis may be involved. Recent research suggests pyroptosis, a programmed lytic cell death pathway, might also contribute to AIC. Although studies have focused on AIC in the myocardium, emerging evidence suggests that AIC may also affect vascular cells. Furthermore, the cytotoxicity of doxorubicin (DOX) or its supposed active metabolite, doxorubicinol (DOXOL), has not been widely investigated in cardiac vascular cells. Purpose This study examines the hypothesis that pyroptosis plays a role in DOX and DOXOL cytotoxicity in H9c2 immortalised rat cardiac myoblasts and two types of human cardiac endothelial cells. Methods In vitro experiments were conducted to ascertain dose-dependent toxicity in H9c2, and cardiac endothelial cells following treatment with DOX or DOXOL. The type of cell death was determined by assessing cell morphology and fluorescent staining with markers of apoptosis (Annexin V) and lytic cell death (Propidium Iodide PI) following DOX or DOXOL treatment in comparison to known inducers of pyroptosis (LPS & Nigericin). Western blot experiments were conducted to study the expression of GSDMD (involved in pyroptosis), and caspase 3 (involved in apoptosis), following DOX or DOXOL. A human monocytic acute myeloid leukaemia cell line (THP-1 cells) was used as a positive control for pyroptosis. Results Dose-dependent cell death following DOX treatment was higher than with DOXOL in both H9c2 and cardiac endothelial cells. DOX caused apoptotic changes in all cell types. In contrast, DOXOL had weaker effects, inducing early apoptosis. No cleavage (activation) of GSDMD or caspase 3 was detected after DOX or DOXOL. Interestingly, in THP-1 cells, DOX induced predominantly apoptotic cell death, whereas DOXOL induced predominantly pyroptotic cell death, assessed both morphologically and by caspase 3 and GSDMD cleavage. Conclusion DOX and DOXOL primarily induce apoptotic cell death in H9c2 cardiomyocytes and cardiac endothelial cells. Interestingly, DOXOL, supposedly the active metabolite, was less cytotoxic than DOX. THP-1 cells undergo apoptotic or pyroptotic cell death with DOX or DOXOL, respectively. In conclusion, anthracycline-induced cytotoxicity is likely to be due to apoptotic injury rather than pyroptosis.

Markers for Cell Death and Purine Metabolites in Patients with and without Delayed Graft Function after Kidney Transplantation

Markers for Cell Death and Purine Metabolites in Patients with and without Delayed Graft Function after Kidney Transplantation

Novel bispecific antibody-drug conjugate targeting PD-L1 and B7-H3 enhances antitumor efficacy and promotes immune-mediated antitumor responses

BackgroundAntibody-drug conjugates (ADCs) offer a promising approach, combining monoclonal antibodies with chemotherapeutic drugs to target cancer cells effectively while minimizing toxicity.MethodsThis study examined the therapeutic efficacy and potential mechanisms of...

Neurological Restorative Effects of (-)-Epicatechin in a Model of Gulf War Illness.

Gulf War Illness (GWI) afflicts US military personnel who served in the Persian Gulf War. Suspect causal agents include exposure to pyridostigmine (PB), permethrin (PM) and N,N-diethyl-m-toluamide (DEET). Prominent symptoms include cognitive deficits, such as memory impairment. In aging animal models, we have documented the beneficial effect of the flavanol (-)-epicatechin (Epi) on hippocampus structure and related function. Using a rat model of GWI, we examined the effects of Epi on hippocampus inflammation, oxidative stress, mitochondrial dysfunction, cell death/survival pathways, and memory endpoints. Male Wistar rats underwent 3 weeks of exposure to either vehicles or DEET, PM, PB, and stress. Subgroups of GWI rats were then allocated to receive orally 15 days of either water (vehicle) or 1 mg/kg/day of Epi treatment. Object recognition tasks were performed to assess memory. Hippocampus samples were analyzed. Epi treatment yields significant improvements in short- and long-term memory versus GWI rats. Hippocampus oxidative stress and pro-inflammatory cytokine levels showed significant increases with GWI that were largely normalized with Epi becoming comparable to controls. Significant increases in markers of hippocampus neuroinflammation and cell death were noted with GWI and were also largely reduced with Epi. Neuronal survival signaling pathways were adversely impacted by GWI and were partially or fully restored by Epi. Markers of mitochondrial function were adversely impacted by GWI and were fully restored by Epi. In conclusion, in an animal model of GWI, Epi beneficially impacts recognized markers of hippocampus neuroinflammation, oxidative stress, cell survival, neurotoxicity and mitochondrial function leading to improved memory.

Rationally designed catalytic nanoplatform for enhanced chemoimmunotherapy via deploying endogenous plus exogenous copper and remodeling tumor microenvironment

Chemodynamic therapy represents a novel tumor therapeutic modality via triggering catalytic reactions in tumors to yield highly toxic reactive oxygen species (ROS). Nevertheless, low efficiency catalytic ability, potential systemic toxicity and inefficient tumor targeting, have hindered the efficacy of chemodynamic therapy. Herein, a rationally designed catalytic nanoplatform, composed of folate acid conjugated liposomes loaded with copper peroxide (CP) and chloroquine (CQ; a clinical drug) (denoted as CC@LPF), could power maximal tumor cytotoxicity, mechanistically via maneuvering endogenous and exogenous copper for a highly efficient catalytic reaction. Despite a massive autophagosome accumulation elicited by CP-powered autophagic initiation and CQ-induced autolysosomal blockage, the robust ROS, but not aberrant autophagy, underlies the synergistic tumor inhibition. Otherwise, this combined mode also elicits an early onset, above all, long-term high-level existence of immunogenic cell death markers, associated with ROS and aberrant autophagy -triggered endoplasmic reticulum stress. Besides, CC@LPF, with tumor targeting capability and selective tumor cytotoxicity, could elicit intratumor dendritic cells (mainly attributed to CQ) and tumor infiltrating CD8+ T cells, upon combining with PD-L1 therapeutic antibody, further induce significant anti-tumor effect. Collectively, the rationally designed nanoplatform, CC@LPF, could enhance tumor chemoimmunotherapy via deploying endogenous plus exogenous copper and remodeling tumor microenvironment.Graphical abstract

Reprogramming tumor microenvironment with precise photothermal therapy by calreticulin nanobody-engineered probiotics

Targeted therapies have revolutionized traditional cancer treatments by precisely targeting tumor cells, enhancing efficacy and safety. Despite this advancement, the proportion of cancer patients eligible for such therapies remains low due to the absence of suitable targets. Here, we investigate whether the translocation of the immunogenic cell death (ICD) marker calreticulin (CALR) from the endoplasmic reticulum (ER) to the cell surface following ICD induction can serve as a target for targeted therapies. To target CALR, a nanobody Nb215 identified from a naïve VHH phage library with high binding affinity to both human and mouse CALR was employed to engineer probiotic EcN 1917. Our results demonstrated that CALR nanobody-modified EcN-215 coupled with the photothermal dye indocyanine green (ICG) was able to exert NIR-II imaging-guide photothermal therapy (PTT). Moreover, PTT with EcN-215/ICG can reshape the tumor microenvironment by enhancing the infiltration of CD45+CD3+ T cells and CD11b+F4/80+ macrophages. Furthermore, the antitumor activity of CALR-targeted EcN-215/ICG is synergistically enhanced by blocking CD47-SIRPα axis. Collectively, our study provides a proof of concept for CALR-targeted therapy. Given that CALR translocation can be induced by various anticancer therapies across numerous tumor cell lines, CALR-targeted therapies hold promise as a novel approach for treating multiple types of cancers.

Machine learning-based cell death marker for predicting prognosis and identifying tumor immune microenvironment in prostate cancer

BackgroundProstate cancer (PCa) incidence and mortality rates are rising, necessitating precise prognostic tools to guide personalized treatment. Dysregulation of programmed cell death pathways in tumor suppression and cancer development has garnered increasing attention, providing a new research direction for identifying biomarkers and potential therapeutic targets. MethodsIntegrating multiple database resources, we constructed and optimized a prognostic signature based on the expression of programmed cell death-related genes (PCDRG) using ten machine learning algorithms. Model performance and prognostic effects were further evaluated. We analyzed the relationships between signature and clinicopathological features, somatic mutations, drug sensitivity, and the tumor immune microenvironment, and constructed a nomogram. The expression level of PCDRGs were evaluated and compared. ResultsOf 1560 PCDRGs, 149 were differentially expressed in PCa, with 34 associated with biochemical recurrence. The PCDRG-derived index (PCDI), constructed using the random forest algorithm, exhibited optimal prognostic performance, successfully stratifying PCa patients into two groups with significant prognostic differences. Patients with high PCDI scores exhibited poorer survival and lower immunotherapy benefit. PCDI was closely associated with the infiltration of specific immune cells, particularly positive correlations with macrophages and T helper cells, and negative correlations with neutrophils, suggesting that PCDI may influence the tumor immune microenvironment, thereby affecting patient prognosis and treatment response. PCDI was associated with age, pathological stage, somatic mutations, and drug sensitivity. The PCDI-based nomogram demonstrated excellent performance in predicting biochemical recurrence in PCa patients. Finally, the differential expression of these PCDRGs was verified based on cell lines and PCa patient expression profile data. ConclusionThis study developed an effective prognostic indicator for prostate cancer, PCDI, using machine learning approaches. PCDI reflects the link between aberrant programmed cell death pathways and disease progression and treatment response.

In Vivo Tracking and 3D Mapping of Cell Death in Regeneration and Cancer Using Trypan Blue.

Tracking cell death in vivo can enable a better understanding of the biological mechanisms underlying tissue homeostasis and disease. Unfortunately, existing cell death labeling methods lack compatibility with in vivo applications or suffer from low sensitivity, poor tissue penetration, and limited temporal resolution. Here, we fluorescently labeled dead cells in vivo with Trypan Blue (TBlue) to detect single scattered dead cells or to generate whole-mount three-dimensional maps of large areas of necrotic tissue during organ regeneration. TBlue effectively marked different types of cell death, including necrosis induced by CCl4 intoxication in the liver, necrosis caused by ischemia-reperfusion in the skin, and apoptosis triggered by BAX overexpression in hepatocytes. Moreover, due to its short circulating lifespan in blood, TBlue labeling allowed in vivo "pulse and chase" tracking of two temporally spaced populations of dying hepatocytes in regenerating mouse livers. Additionally, upon treatment with cisplatin, TBlue labeled dead cancer cells in livers with cholangiocarcinoma and dead thymocytes due to chemotherapy-induced toxicity, showcasing its utility in assessing anticancer therapies in preclinical models. Thus, TBlue is a sensitive and selective cell death marker for in vivo applications, facilitating the understanding of the fundamental role of cell death in normal biological processes and its implications in disease.

Delta-6 desaturase FADS2 is a tumor-promoting factor in cholangiocarcinoma.

Cholangiocarcinoma is a fatal disease with limited therapeutic options. We screened genes required for cholangiocarcinoma tumorigenicity and identified FADS2, a delta-6 desaturase. FADS2 depletion reduced invivo tumorigenicity and cell proliferation. In clinical samples, FADS2 was expressed in cancer cells but not in stromal cells. FADS2 inhibition also reduced the migration and sphere-forming ability of cells and increased apoptotic cell death and ferroptosis markers. Lipidome assay revealed that triglyceride and cholesterol ester levels were decreased in FADS2-knockdown cells. The oxygen consumption ratio was also decreased in FADS2-depleted cells. These data indicate that FADS2 depletion causes a reduction in lipid levels, resulting in decrease of energy production and attenuation of cancer cell malignancy.

Abstract Tu137: Reductive Stress Impedes Neonatal Cardiomyocyte Regeneration

Background: The elevated level of reactive oxygen species (ROS) has been reported to drive the cell-cycle-exit of neonatal mouse cardiomyocytes and impede their regeneration. On the contrary, the roles of reductive stress in perinatal development and regeneration of cardiomyocytes are still poorly understood. To counterbalance increased oxidative stress, antioxidant enzymes are expressed to remove excessive ROS. Our previous work uncovered a role of the transcription factor Pitx2 as a redox regulator in the ventricular myocardium. Pitx2 protects myocardium by positively regulating genes that encode antioxidant scavengers. In regenerating neonatal hearts, Pitx2 expression is activated by Nrf2, a known antioxidant regulator. Under oxidative stress, Nrf2 dissociates from the Keap1-Cul3-Rbx1 degradation complex and translocates to the nucleus where it promotes Pitx2 expression. Interestingly, our RNA-Seq and ChIP-Seq studies showed that Pitx2 promotes Rbx1 expression which degrades Nrf2. We hypothesize that this negative feedback loop ( Image 1 ) is required to prevent reductive stress in regenerating cardiomyocytes. Methods: Cre-Lox models were used to specifically knock out Nrf2 in cardiomyocytes. Constitutively active Nrf2 (caNrf2) was expressed specifically in cardiomyocyte using AAV9. Guide RNAs were delivered into Myh6Cas9 mouse heart using AAV9 to knock out Rbx1 in cardiomyocytes. Neonatal ROS was transiently suppressed using high dose of N-acetyl-cysteine (NAC). Following left anterior descending artery occlusion (LAD-O), cardiac regeneration was assessed by Masson’s trichrome staining and echocardiography. Markers of cell cycle activity and cell death were assessed by immunofluorescence. RNA-Seq was performed on isolated cardiomyocytes from NAC and saline treated hearts after sham or LAD-O procedure. Results: Pitx2 promotes the degradation of Nrf2 by inducing the expression of Rbx1. In cardiomyocytes, Rbx1 , but not Nrf2 , is required for neonatal heart regeneration. Meanwhile, expression of caNrf2 in cardiomyocytes increases mortality after LAD-O. Removing ROS in neonatal heart prevents cardiomyocyte maturation and disrupts injury response after LAD-O. Conclusions: Together, these results suggest that a fine balance between reductive and oxidative activity is required for neonatal heart regeneration and postnatal heart development. Excessive antioxidant activity will cause reductive stress and impede neonatal heart regeneration after MI.

Silymarin suppresses proliferation and PD-L1 expression in colorectal cancer cells and increases inflammatory CD8+ cells in tumor-bearing mice

IntroductionSilymarin as an herbal medicine has shown anticancer effects on tumor cells, while having low toxicity in normal cells. In this study, the effects of Silymarin on proliferation and apoptosis of colorectal cancer cells and its impact on immune response against cancer cells were evaluated in vitro and in vivo. Methods and materialsThe effect of Silymarin on CT-26 and Caco-2 cells proliferation and apoptosis were demonstrated by MTT assay and PI staining. A subcutaneous tumor of colorectal cancer was developed. Silymarin and Doxorubicin were administrated by intravenous injection. qRT-PCR analyses was performed on blood samples and tumor tissues. Spleen tissue was used to evaluate CD8+ T cell immune responses. Histological study was carried out on tumor tissues. ResultsSilymarin showed anti-proliferative effects on CT-26 and Caco-2 cells. The markers of immunogenic cell death (Calreticulin exposure, ATP secretion, and HMGB1 secretion) significantly increased in both cell lines in the presence of silymarin. The expression of genes related to cell proliferation particularly β-Catenin and Cycline D1, and also anti-apoptotic ones such as Bcl-2 significantly reduced in mice treated with Silymarin while the expression of pro-apoptotic Bax increased. The RNA level of PD-L1 decreased in tumor tissues exposed by Silymarin. Moreover, the number of CTLs increased in the spleen of mice treated with Silymarin in comparison with untreated mice. Decreased tumor size and also survival of colorectal cancer cells in Silymarin-treated mice were observed in histological analysis. ConclusionSilymarin treatment showed a suppressive role on colorectal cancer cells almost as much as Doxorubicin. Our study indicated that having a low toxicity profile, cost-effectiveness, and availability of raw materials, plant-derived Silymarin can be a good candidate for further investigation to treat CRC.

Cellular senescence mediates retinal ganglion cell survival regulation post-optic nerve crush injury.

Traumatic optic neuropathy refers to optic nerve (ON) injury by trauma, including explosion and traffic accident. Retinal ganglion cell (RGC) death is the critical pathological cause of irreversible visual impairment and blindness in ON injury. We previously investigated the patterns of 11 modes of cell death in mouse retina post-ON injury. Here we aimed to identify additional signalling pathways regulating RGC survival in rodents post-ON injury. RNA sequencing analysis identified the upregulation of inflammation and cellular senescence-related genes in retina post-ON injury, which were confirmed by immunoblotting and immunofluorescence analyses. Increased expression of senescence-associated β-galactosidase (SA-βgal) in RGCs and activation of microglia were also found. Transforming growth factor-β receptor type II inhibitor (LY2109761) treatment suppressed p15Ink4b and p21Cip1 protein and SA-βgal expression and promoted RGC survival post-ON injury with decreasing the expression of cell death markers in retina. Consistently, senolytics (dasatinib and quercetin) treatments can promote RGC survival and alleviate the reduction of ganglion cell complex thickness and pattern electroretinography activity post-ON injury with reducing SA-βgal, p15Ink4b, p21Cip1, microglial activation and cell death marker expression. In summary, this study revealed the activation of cellular senescence in rodent retina post-ON injury and contribute to RGC survival regulation. Targeting cellular senescence can promote RGC survival after ON injury, suggesting a potential treatment strategy for traumatic optic neuropathy.

Serum Cytokeratin-18 levels as a prognostic biomarker in advanced liver disease: a comprehensive meta-analysis

Cytokeratin-18 (CK-18) is a marker of hepatic cell death. Serum CK-18 could serve as a prognostic marker for patients with advanced liver disease (ALD). This meta-analysis aims to explore the association between total CK-18 (M65) and caspase-cleaved CK-18 (M30) levels with the prognosis of ALD patients. Relevant longitudinal observational studies were identified through comprehensive searches of the Medline, Web of Science, and Embase databases. A random-effects model was utilized to synthesize the findings, accommodating heterogeneity among studies. The analysis included 14 datasets from 11 studies. Elevated serum CK-18 levels at admission were linked to a higher risk of death or liver transplantation during follow-up. This association was consistent for both M65 (risk ratio [RR] 1.99, 95% confidence interval [CI] 1.65 to 2.40, p < 0.001; I2 = 43%) and M30 (RR 1.94, 95% CI 1.57 to 2.40, p < 0.001; I2 = 46%). Subgroup analysis revealed that the relationship between serum M65 levels and adverse outcomes was attenuated in studies using multivariate analysis compared to those using univariate analysis (RR 1.78 vs. 2.80, p for subgroup difference = 0.03). Further subgroup analyses indicated that the prognostic significance of CK-18 for ALD patients was not significantly influenced by study design, methods of determining CK-18 cutoff values, or follow-up durations. Elevated serum CK-18 levels at admission indicate a poor prognosis in patients with ALD. This finding holds for both M65 and M30.

Insights into brominated flame retardant neurotoxicity: mechanisms of hippocampal neural cell death and brain region-specific transcriptomic shifts in mice.

Brominated flame retardants (BFRs) reduce flammability in a wide range of products including electronics, carpets, and paint, but leach into the environment to result in continuous, population-level exposure. Epidemiology studies have correlated BFR exposure with neurological problems, including alterations in learning and memory. This study investigated the molecular mechanisms mediating BFR-induced cell death in hippocampal cells and clarified the impact of hexabromocyclododecane (HBCD) exposure on gene transcription in the hippocampus, dorsal striatum, and frontal cortex of male mice. Exposure of hippocampus-derived HT-22 cells to various flame retardants, including tetrabromobisphenol-A (current use), HBCD (phasing out), or 2,2',4,4'-tetrabromodiphenyl ether (BDE-47, phased out) resulted in time, concentration, and chemical-dependent cellular and nuclear morphology alterations, alterations in cell cycle and increases in annexin V staining. All 3 BFRs increased p53 and p21 expression; however, inhibition of p53 nuclear translocation using pifthrin-α did not decrease cell death. Transcriptomic analysis upon low (10 nM) and cytotoxic (10 μM) BFR exposure indicated that HBCD and BDE-47 altered genes mediating autophagy-related pathways. Further evaluation showed that BFR exposure increased LC3-II conversion and autophagosome/autolysosome formation, and co-exposure with the autophagy inhibitor 3-methyladenine (3-MA) attenuated cytotoxicity. Transcriptomic assessment of select brain regions from subchronically HBCD-exposed male mice demonstrated alteration of genes mediating vesicular transport, with greater impact on the frontal cortex and dorsal striatum compared with the dorsal and ventral hippocampus. Immunoblot analysis demonstrated no increases in cell death or autophagy markers, but did demonstrate increases in the SNARE binding complex protein SNAP29, specifically in the dorsal hippocampus. These data demonstrate that BFRs can induce chemical-dependent autophagy in neural cells in vitro and provide evidence that BFRs induce region-specific transcriptomic and protein expression in the brain suggestive of changes in vesicular trafficking.