Activation Of Intrinsic Pathway Research Articles

Carm1 Inhibition Potentiates Irradiation-Induced Antitumor Immunity via Tumor Intrinsic STING Pathway Activation

Radiotherapy is commonly applied in multiple cancer types. Besides irradiation induced direct cell death, radiotherapy stimulated significant immune responses for tumor control. Intact and functional cGAS-STING pathway in both tumor cells and host cells is indispensable for efficient irradiation-induced anti-tumor effects. Coactivator-associated arginine methyltransferase 1 (Carm1) is emerging as an attractive therapeutic target and a biomarker for prognosis in various types of cancer. It has been reported that Carm1 inhibition could improve immunotherapy induced anti-tumor effects. However, it remains unclear how tumor cell intrinsic Carm1 affects irradiation-induced anti-tumor immunity. Carm1 deficient cell lines were established in MC38 and B16F10 murine cancer cells using the CRISPR/Cas9 technology. To verify the effects of tumor `subcutaneous tumor mouse models were established and one fraction of 15Gy was administrated when the tumor volume reached 200mm3, followed by flow cytometry assays. Transcriptome sequencing, protein mass spectrometry, single-cell sequencing, Digital Spatial Profiling (DSP), real-time qPCR, western blotting, immunofluorescence and co-immunoprecipitation were carried out to explore and verify possible molecular mechanisms. Here we found Carm1 deficiency in tumor cells dramatically enhanced irradiation-induced anti-tumor immune responses. Transcriptome sequencing of irradiated tumor cells and further experiments then validated that cGAS-STING pathway was significantly activated after irradiation in the absence of Carm1 in tumor cells, which contributed to enhance anti-tumor immunity after irradiation. Mechanistically, Carm1 deficiency in tumor cells attenuated autophagy, resulting in increased cytoplasmic mtDNA enrichment and enhanced cGAS-STING pathway activation. On the other hand, we also found that Carm1 caused asymmetric arginine methylation (ADMA) modification of TBK1 with reduced phosphorylation level, and Carm1 deficiency could activate cGAS-STING pathway by reducing AMDA modification and enhancing phosphorylation of TBK1. Finally, Carm1 inhibitor EZM2302 was applied in combination with radiotherapy in vitro, and it's indicated that combination therapy resulted in intensive anti-tumor immunity and prominent abscopal effects. In this study, we identified that Carm1 ablation in tumor cells could promote irradiation-induced antitumor immunity through tumor cell intrinsic STING pathway activation. Mechanically, Carm1 deficiency directly activated the cGAS-STING pathway by interacting with TBK1 and increased mtDNA accumulation in cytoplasm by inhibiting autophagy. These findings provided new strategies for targeting Carm1 to boost the efficacy of radiotherapy.

Calcium Dyshomeostasis Drives Pathophysiology and Neuronal Demise in Age-Related Neurodegenerative Diseases.

This review postulates that age-related neurodegeneration entails inappropriate activation of intrinsic pathways to enable brain plasticity through deregulated calcium (Ca2+) signalling. Ca2+ in the cytosol comprises a versatile signal controlling neuronal cell physiology to accommodate adaptive structural and functional changes of neuronal networks (neuronal plasticity) and, as such, is essential for brain function. Although disease risk factors selectively affect different neuronal cell types across age-related neurodegenerative diseases (NDDs), these appear to have in common the ability to impair the specificity of the Ca2+ signal. As a result, non-specific Ca2+ signalling facilitates the development of intraneuronal pathophysiology shared by age-related NDDs, including mitochondrial dysfunction, elevated reactive oxygen species (ROS) levels, impaired proteostasis, and decreased axonal transport, leading to even more Ca2+ dyshomeostasis. These core pathophysiological processes and elevated cytosolic Ca2+ levels comprise a self-enforcing feedforward cycle inevitably spiralling toward high levels of cytosolic Ca2+. The resultant elevated cytosolic Ca2+ levels ultimately gear otherwise physiological effector pathways underlying plasticity toward neuronal demise. Ageing impacts mitochondrial function indiscriminately of the neuronal cell type and, therefore, contributes to the feedforward cycle of pathophysiology development seen in all age-related NDDs. From this perspective, therapeutic interventions to safely restore Ca2+ homeostasis would mitigate the excessive activation of neuronal destruction pathways and, therefore, are expected to have promising neuroprotective potential.

Structural and functional validation of a cloned parasporin from Bacillus thuringiensis isolate KAU 41 native to Western Ghats of India.

Parasporins of Bacillus thuringiensis (Bt) exhibit specific toxicity to cancer cells. PCR based mining has identified apoptosis inducing parasporin in KAU41 Bt isolate from the Western Ghats of India. The study aimed to clone and overexpress the parasporin of native KAU41 Bt isolate for determining structural and functional characteristics of the protein. Parasporin gene was cloned in pGEM-T, sequenced, sub-cloned in pET30+ and overexpressed in Escherichia coli. The expressed protein was characterized by SDS-PAGE and in silico methods. Cytotoxicity of cleaved peptide was assessed by MTT assay. SDS-PAGE displayed a 31 kDa protein (rp-KAU41) overexpressed. Upon proteinase K digestion, the protein was cleaved into 29 kDa peptide which was found to be cytotoxic to HeLa cells. The protein has a deduced sequence of 267 amino acids with β-strands folding pattern of crystal protein. Even though rp-KAU41 shared a 99.15% identity to chain-A of non-toxic crystal protein, it only showed a less similarity to the existing parasporins like PS4 (38%) and PS5 (24%) in UPGMA analysis, emphasizing the novelty of rp-KAU41. The protein is predicted to have more similarity to the pore forming toxins of Aerolysin superfamily and an additional loop in rp-KAU41 may be contributing towards its cytotoxicity. The molecular docking with caspase 3 resulted in higher Z dock and Z rank score substantiating its role in the activation of intrinsic pathway of apoptosis. The recombinant parasporin protein, rp-KAU41 is presumed to belong to the Aerolysin superfamily. An interaction with caspase 3 substantiates its role in activating the intrinsic pathway of apoptosis in cancer cells.

Contact system and intrinsic pathway activation in patients with advanced pancreatic cancer: a prospective cohort study

BackgroundDespite high risk of venous thromboembolism (VTE) in patients with pancreatic cancer, there are little data on contact system activation in these patients. ObjectivesTo quantify contact system and intrinsic pathway activation and subsequent VTE risk in patients with pancreatic cancer. MethodsPatients with advanced pancreatic cancer were compared with controls. Blood was drawn at baseline and patients were followed for 6 months. Complexes of proteases with their natural inhibitors, C1-esterase inhibitor (C1-INH), antithrombin (AT), or alpha-1 antitrypsin (α1at), were measured for complexes containing kallikrein (PKa:C1-INH), factor (F)XIIa (FXIIa:C1-INH), and FXIa (FXIa:C1-INH, FXIa:AT, FXIa:α1at). The association of cancer with complex levels was assessed in a linear regression model, adjusted for age, sex, and body mass index. In a competing risk regression model, we assessed associations between complex levels and VTE. ResultsOne hundred nine patients with pancreatic cancer and 22 controls were included. The mean age was 66 years (SD, 8.4) in the cancer cohort and 52 years (SD, 10.1) in controls. In the cancer cohort, 18 (16.7%) patients developed VTE during follow-up. In the multivariable regression model, pancreatic cancer was associated with increased complexes of PKa:C1-INH (P < .001), FXIa:C1-INH (P < .001), and FXIa:AT (P < .001). High FXIa:α1at (subdistribution hazard ratio, 1.48 per log increase; 95% CI, 1.02-2.16) and FXIa:AT (subdistribution hazard ratio, 2.78 highest vs lower quartiles; 95% CI, 1.10-7.00) were associated with VTE. ConclusionComplexes of proteases with their natural inhibitors were elevated in patients with cancer. These data suggest that the contact system and intrinsic pathway activation are increased in patients with pancreatic cancer.

Iodine-Biofortified Lettuce Can Promote Mitochondrial Dependent Pathway of Apoptosis in Human Gastrointestinal Cancer Cells.

Previously, our research provided evidence that exposure of gastric and colon cancer cells to extracts from iodine-biofortified lettuce leads to a reduction of cell viability and proliferation through cell cycle arrest and upregulation of pro-apoptotic genes. The aim of the present study was to determine the potential cellular mechanisms of induction of cell death in human gastrointestinal cancer cell lines after treatment with iodine-biofortified lettuce. We demonstrated that extracts from lettuce enriched with iodine induce apoptosis in gastric AGS and colon HT-29 cancer cells and the mechanism of programmed cell death may be triggered and executed through different signaling pathways, depending on the type of cells. Western blot analysis revealed that iodine-fortified lettuce leads to cell death through the release of cytochrome c to the cytosolic fraction and activation of the primary drivers of apoptosis: caspase-3, caspase-7, and caspase-9. Furthermore, we have reported that apoptotic effects of lettuce extracts may be mediated by poly (ADP-ribose) polymerase (PARP) and activation of pro-apoptotic Bcl-2 family proteins such as Bad, Bax, and BID. We also observed mitochondrial dysfunction with the dissipation of the mitochondrial membrane potential in cells exposed to lettuce extracts. Taken together, these results indicate that the organic form of iodine such as 5-ISA and 3,5-diISA is an important factor in the activation of intrinsic mitochondrial apoptotic pathway in AGS and HT-29 cancer cells in a p53-independent manner.

Parasporin-4, a novel apoptosis inducer of breast cancer cells produced by Bacillus thuringiensis

Parasporin (PS) proteins have cytocidal activity preferential for various human malignant cells. The purpose of this investigation was to see if the PS separated from B. thuringiensis strain E8 isolate had any particular cytotoxicity against breast cancer. The extracted spores-crystal proteins were solubilized and digested with proteinase K. Cytotoxicity effects were analysed by MTT assay. Caspases activities were measured using ELISA. SDS-PAGE analysis was performed for determination of molecular weight of Cry protein. Identification of extracted proteins function was evaluated by MALDI-TOF MS analysis. Breast cancer cells line (MCF-7) was highly susceptible to 1mg/mL PS and showed apoptosis characteristics, but it has no effects on the normal cells (HEK293). Apoptosis evaluation showed that caspases 1, 3, 9 and BAX were remarkably up-regulated in cancer cells, indicating the intrinsic pathway activation in these cells. PS Size was determined using SDS-PAGE in E8 isolate as 34kDa and a 25kDa digested peptide was identified as PS4. The function of PS4 was reported as an ABC-transporter by spectrometry. The data of the present study show that PS4 is a selective cytotoxic protein against breast cancer and a molecule with a lot of potentials for next researches.

Microporous Hemostatic Sponge Based on Silk Fibroin and Starch with Increased Structural Retentivity for Contact Activation of the Coagulation Cascade.

Hemostatic agents with diverse forms and materials are necessitated to control excessive bleeding to improve surgical site visibility during operation. The adequate use of hemostatic agents dramatically reduces the chance of dehydration, absence of oxygen, and, in severe cases, death. Polysaccharide-based hemostatic agents are widely used as they are safe for the human body. Among diverse polysaccharides, starch has exhibited a high swelling ability, but its powder formulation is limited during incompressible bleeding. Herein, starch was blended with silk protein and crosslinked using glycerol to improve structural integrity. The silk/starch solution was lyophilized to be a sponge with interconnected pores, which is beneficial to blood coagulation by increased swelling ratio and underwater retentivity to absorb blood plasma. The surface contact between the blood component and the sponge initiates clotting by intrinsic pathway activation and platelet activation without the hemolytic effect or cytotoxicity. The clinical effectiveness of the sponges as topical hemostatic agents was confirmed by animal bleeding model tests.

Intracellular behavior of Nocardia seriolae and its apoptotic effect on RAW264.7 macrophages.

Nocardia seriolae, an intracellular gram-positive pathogen, is prone to infecting immunocompromised and surface-damaged fish, causing serious losses to the aquaculture industry. Although a previous study has demonstrated that N. seriolae infects macrophages, the persistence of this bacterium in macrophages has not been well characterized. To address this gap, we used the macrophage cell line RAW264.7, to investigate the interactions between N. seriolae and macrophages and deciphered the intracellular survival mechanism of N. seriolae. Confocal and light microscopy revealed that N. seriolae entered macrophages 2 hours post-inoculation (hpi), were phagocytosed by macrophages at 4-8 hpi, and induced the formation of multinucleated macrophages by severe fusion at 12 hpi. Flow cytometry, evaluation of mitochondrial membrane potential, release of lactate dehydrogenase, and observation of the ultrastructure of macrophages revealed that apoptosis was induced in the early infection stage and inhibited in the middle and later periods of infection. Additionally, the expression of Bcl-2, Bax, Cyto-C, Caspase-3, Capase-8, and Caspase-9 was induced at 4 hpi, and then decreased at 6-8 hpi, illustrating that N. seriolae infection induces the activation of extrinsic and intrinsic apoptotic pathways in macrophages, followed by the inhibition of apoptosis to survive inside the cells. Furthermore, N. seriolae inhibits the production of reactive oxygen species and releases large amounts of nitric oxide, which persists in macrophages during infection. The present study provides the first comprehensive insight into the intracellular behavior of N. seriolae and its apoptotic effect on macrophages and may be important for understanding the pathogenicity of fish nocardiosis.

Investigation of Molecular Mechanisms Involved in Sensitivity to the Anti-Cancer Activity of Costunolide in Breast Cancer Cells.

Costunolide (CTL), an active compound isolated from Saussurea lappa Clarke and Laurus nobilis L, has been shown to induce apoptosis via reactive oxygen species (ROS) generation in various types of cancer cells. However, details of molecular mechanisms underlying the difference in sensitivity of cancer cells to CTL are still largely unknown. Here, we tested the effect of CTL on the viability of breast cancer cells and found that CTL had a more efficient cytotoxic effect against SK-BR-3 cells than MCF-7 cells. Mechanically, ROS levels were significantly increased upon CTL treatment only in SK-BR-3 cells, which leads to lysosomal membrane permeabilization (LMP) and cathepsin D release, and subsequent activation of the mitochondrial-dependent intrinsic apoptotic pathway by inducing mitochondrial outer membrane permeabilization (MOMP). In contrast, treatment of MCF-7 cells with CTL activated PINK1/Parkin-dependent mitophagy to remove damaged mitochondria, which prevented the elevation of ROS levels, thereby contributing to their reduced sensitivity to CTL. These results suggest that CTL is a potent anti-cancer agent, and its combination with the inhibition of mitophagy could be an effective method for treating breast cancer cells that are less sensitive to CTL.

Batroxin I: A Novel Bradykinin-Potentiating Peptide with Cytotoxic Activity Isolated from Bothrops atrox Snake Venom

Venom peptides are interesting molecular models for the development of biotechnological strategies applicable in generating therapeutic agents and/or experimental tools for basic and applied research. The present study aimed to search for peptides from Bothrops atrox snake venom with anticancer potential activity against HepG2 liver tumor cell line, determine their cytotoxic action, and analyze the structure–function relationship. The novel peptide Batroxin I (M.W. 1.38 kDa) was isolated by molecular exclusion and reversed phase chromatography methods. The Batroxin I presented a selective cytotoxicity towards tumor cells, reducing the viability of HepG2 cells by 94.6% with IC50 of 0.72 μg/mL, and showing a low toxicity against peripheral blood mononuclear cells. Analysis of the apoptotic and necrotic peptide effects revealed that it induced apoptosis by intrinsic pathway activation. The amino acid sequence of Batroxin I was determined by de novo sequencing as < EKWPRPDAPIPP (where < E = pyroglutamic acid); hence, it is an unpublished peptide that belongs to the class of bradykinin-enhancing peptides and cell penetration peptide. This is one of the first reports on the cytotoxic antitumor activity of a bradykinin-enhancing peptide. Our results indicate that this peptide could serve not only as a template for the development of new drugs, but also as an adjuvant to less effective marketed drugs to treat cancer and other diseases.

Allophylus africanus Stem Bark Extract Modulates the Mitochondrial Apoptotic Pathway in Human Stomach Cancer Cells.

The present work aimed to detail the mechanisms elicited by Allophylus africanus P. Beauv. stem bark extract in human stomach cancer cells and to identify the bioactives underlying the cytotoxicity. MTT reduction and LDH leakage assays allowed characterizing the cytotoxic effects in AGS cells, which were further detailed by morphological analysis using phalloidin and Hoechst 33258. Proapoptotic mechanisms were elucidated through a mitochondrial membrane potential assay and by assessing the impact upon the activity of caspase-9 and -3. The extract displayed selective cytotoxicity against AGS cells. The absence of plasma membrane permeabilization, along with apoptotic body formation, suggested that pro-apoptotic effects triggered cell death. Intrinsic apoptosis pathway activation was verified, as mitochondrial membrane potential decrease and activation of caspase-9 and -3 were observed. HPLC-DAD profiling enabled the identification of two apigenin-di-C-glycosides, vicenin-2 (1) and apigenin-6-C-hexoside-8-C-pentoside (3), as well as three mono-C-glycosides-O-glycosylated derivatives, apigenin-7-O-hexoside-8-C-hexoside (2), apigenin-8-C-(2-rhamnosyl)hexoside (4) and apigenin-6-C-(2-rhamnosyl)hexoside (5). Isovitexin-2″-O-rhamnoside (5) is the main constituent, accounting for nearly 40% of the total quantifiable flavonoid content. Our results allowed us to establish the relationship between the presence of vicenin-2 and other apigenin derivatives with the contribution to the cytotoxic effects on the presented AGS cells. Our findings attest the anticancer potential of A. africanus stem bark against gastric adenocarcinoma, calling for studies to develop herbal-based products and/or the use of apigenin derivatives in chemotherapeutic drug development.

Urticaria and Angioedema: Understanding Complex Pathomechanisms to Facilitate Patient Communication, Disease Management, and Future Treatment

Chronic spontaneous urticaria (CSU) is primarily a T2-dominant disease with a complex genetic background. Skin mast cell activation can be induced not only via the IgE-FcεRI axis but also from several other distinct mechanisms, molecules, and receptors involved in CSU onset, persistence, and exacerbation. These include autoallergy, autoimmunity, central or peripheral neuroimmune dysregulation, activation of both extrinsic and intrinsic coagulation pathways, and microbial infections. Besides mast cells, recent reports suggest the active and direct involvement of basophils and eosinophils. Several biological characteristics or biomarkers have been linked with CSU's known endotypes and may help forecast therapeutic responses. The introduction of biologic therapy for CSU has been a major advance in the last 10 years. The cornerstone of angioedema (AE) pathogenesis is increased vascular permeability and plasma leakage into the deeper dermis and subcutis, either mediated by histamine or bradykinin (BK). C1-inhibitor deficiency, hereditary or acquired, is the primary cause of BK-mediated AE due to increased plasma BK concentration. Other complex conditions have been identified, with some likely involving contact system dysregulation and other putative mechanisms related to vascular endothelial dysfunction. The approval of multiple hereditary-AE-specific therapies for both prevention and acute attacks has revolutionized treatment of this disease. Any new knowledge of the pathogenesis of CSU and AE offers the opportunity to improve patient information, physician-patient communication, prediction of therapeutic responses, selection of precise tailor-made treatment for each patient, and exploration of novel treatment options for those who do not achieve disease control with current medications.

FoxM1 knockdown enhanced radiosensitivity of esophageal cancer by inducing apoptosis.

Radioresistance is a main reason for local recurrence of esophageal squamous cell carcinoma (ESCC). Forkhead box M1 (FoxM1) is implicated in cancer progression and chemoresistance. This study aims to determine the role of FoxM1 in ESCC radioresistance. We found that FoxM1 protein was upregulated in ESCC tissues compared with adjacent normal tissues. In vitro assays revealed that following irradiation, Eca-109, TE-13, and KYSE-150 cells had increased levels of FoxM1 protein. FoxM1 knockdown resulted in significantly reduced colony formation and increased cell apoptosis following irradiation. Moreover, FoxM1 knockdown induced ESCC cells to accumulate in the radiosensitive G2 /M phase and impeded the repair of radiation-induced DNA damage. Mechanistic studies indicated that radiosensitization of ESCC enhanced by FoxM1 knockdown was associated with increased BAX/BCL2 ratio as well as downregulated Survivin and XIAP, followed by the activation of both extrinsic and intrinsic apoptosis pathways. In xenograft mouse model, the combination of radiation and FoxM1-shRNA led to a synergistic anti-tumor effect. In conclusion, FoxM1 is a promising target to enhance radiosensitivity of ESCC.

Glycated albumin modulates the contact system with implications for the kallikrein-kinin and intrinsic coagulation systems

BackgroundHuman serum albumin (HSA) is the most abundant plasma protein and is sensitive to glycation in vivo. The chronic hyperglycemic conditions in patients with diabetes mellitus (DM) induce a nonenzymatic Maillard reaction that denatures plasma proteins and forms advanced glycation end products (AGEs). HSA-AGE is a prevalent misfolded protein in patients with DM and is associated with factor XII activation and downstream proinflammatory kallikrein-kinin system activity without any associated procoagulant activity of the intrinsic pathway. ObjectivesThis study aimed to determine the relevance of HSA-AGE toward diabetic pathophysiology. MethodsThe plasma obtained from patients with DM and euglycemic volunteers was probed for activation of FXII, prekallikrein (PK), and cleaved high-molecular-weight kininogen by immunoblotting. Constitutive plasma kallikrein activity was determined via chromogenic assay. Activation and kinetic modulation of FXII, PK, FXI, FIX, and FX via in vitro–generated HSA-AGE were explored using chromogenic assays, plasma-clotting assays, and an in vitro flow model using whole blood. ResultsPlasma obtained from patients with DM contained increased plasma AGEs, activated FXIIa, and resultant cleaved cleaved high-molecular-weight kininogen. Elevated constitutive plasma kallikrein enzymatic activity was identified, which positively correlated with glycated hemoglobin levels, representing the first evidence of this phenomenon. HSA-AGE, generated in vitro, triggered FXIIa-dependent PK activation but limited the intrinsic coagulation pathway activation by inhibiting FXIa and FIXa-dependent FX activation in plasma. ConclusionThese data indicate a proinflammatory role of HSA-AGEs in the pathophysiology of DM via FXII and kallikrein-kinin system activation. A procoagulant effect of FXII activation was lost through the inhibition of FXIa and FIXa-dependent FX activation by HSA-AGEs.

Sidestream Smoke Extracts from Harm-Reduction and Conventional Camel Cigarettes Inhibit Osteogenic Differentiation via Oxidative Stress and Differential Activation of intrinsic Apoptotic Pathways.

Epidemiological studies suggest cigarette smoking as a probable environmental factor for a variety of congenital anomalies, including low bone mass, increased fracture risk and poor skeletal health. Human and animal in vitro models have confirmed hypomineralization of differentiating cell lines with sidestream smoke being more harmful to developing cells than mainstream smoke. Furthermore, first reports are emerging to suggest a differential impact of conventional versus harm-reduction tobacco products on bone tissue as it develops in the embryo or in vitro. To gather first insight into the molecular mechanism of such differences, we assessed the effect of sidestream smoke solutions from Camel (conventional) and Camel Blue (harm-reduction) cigarettes using a human embryonic stem cell osteogenic differentiation model. Sidestream smoke from the conventional Camel cigarettes concentration-dependently inhibited in vitro calcification triggered by high levels of mitochondrially generated oxidative stress, loss of mitochondrial membrane potential, and reduced ATP production. Camel sidestream smoke also induced DNA damage and caspase 9-dependent apoptosis. Camel Blue-exposed cells, in contrast, invoked only intermediate levels of reactive oxygen species insufficient to activate caspase 3/7. Despite the absence of apoptotic gene activation, damage to the mitochondrial phenotype was still noted concomitant with activation of an anti-inflammatory gene signature and inhibited mineralization. Collectively, the presented findings in differentiating pluripotent stem cells imply that embryos may exhibit low bone mineral density if exposed to environmental smoke during development.

Chlorogenic acid regulates the proliferation and migration of high-grade serous ovarian cancer cells through modulating the miR199a5p/DDR1 axis

This study aimed to demonstrate that chlorogenic acid (CGA) has anticancer effects against ovarian cancer. The MTT assay was used to assess the optimum concentrations of CGA on the ovarian cancer cell lines OVCA433 and SKOV3, followed by the rate of apoptosis using Annexin V-FITC/PI. The mitochondrial membrane potential of ovarian tumour cells treated with CGA was evaluated using mitochondrial staining kits followed by Western blot analysis, immunofluorescence, and RT-PCR assays. The Trans-well migration assay conducted the percentage of cell migration, followed by wound healing and colony formation assays. CGA induces activation of mitochondria-mediated intrinsic apoptotic pathways in ovarian cancer cells. The discovery that miR-199a-5p is inversely correlated to DDR1, a receptor tyrosine kinase involved in collagen synthesis, was the major consequence of examining the various mechanisms involved in the development of ovarian cancer. After treatment with CGA, cells derived from ovarian cancer cells were deregulated partially via the miR199a5p/DDR1 axis, significantly affecting tumour suppression. DDR1 has been identified as a direct target of miR199a5p in these ovarian cancer cells. We found that CGA-induced loss of DDR1 caused the inactivation of NF-κB signalling downstream in the MMP, migration, and EMT pathways. The study results showed that CGA is a promising drug candidate for treating ovarian cancer, particularly because it exhibits anti-invasive and migrastatic properties.

Amyloid‐mediated TRAIL DRs activation leads to progressive degeneration of cerebral vascular smooth muscle cells

AbstractBackgroundVascular accumulation of amyloid beta (Ab) leads to loss of vascular smooth muscle cells (VSMC) in cerebral arteries via unknown mechanisms, promoting cerebral blood flow alterations and contributing to cognitive impairment. Ab species with different lengths or mutated familial variants may have differential effects on VSMC. Ab1‐42, the main pathogenic form of amyloid in the AD brain with the highest propensity to form aggregates, increased apoptosis in brain vascular cells whereas Ab1‐40 was found to be less toxic. By comparison, the familial Ab variant E22Q, induced the most cell death of all the amyloid variants tested. However, there has been little progress in defining the pathways leading to VSMC degeneration in AD and related disorders. The aim of this study was to identify Ab‐mediated cell death mechanisms in VSMC.MethodHuman cerebral VSMC were treated with pre‐oligomerized variants of Ab for 2‐8hrs (acute treatment) and 24‐72hrs (chronic treatment). Analysis of fragmented nucleosomes and visualization of cytochrome c (Cyt. C) expression by immunocytochemistry were used to measure apoptosis. Activation of intrinsic and extrinsic apoptotic pathways were examined by western blot and caspase‐glo assays. The effects of Ab on mitochondrial function were examined using a Seahorse XF96 Analyzer and TMRM fluorescent dye in live cells.ResultsAcute Ab treatment in VSMC up‐regulated tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) death receptors DR4 and DR5 with activation of caspase 8 and transient activation of Nuclear Factor k B (NF‐kB). DR4 and DR5 expression remained high with chronic Ab treatment and lead to caspase activation followed by Cyt. C release and apoptosis. 24hrs of Ab treatment also decreased mitochondrial membrane potential and significantly impaired mitochondrial respiration.ConclusionAcute exposure of VSMC to Ab causes up‐regulation and activation of DR4/DR5, that, over time, results in mitochondrial dysfunction, caspase activation and apoptosis. Our data suggests that an early and transient activation of NF‐kB may also contribute to the toxic effects of Ab, possibly by inducing pro‐inflammatory pathways in the neurovascular unit.

Gut-kidney axis in IgA nephropathy: Role on mesangial cell metabolism and inflammation.

IgA Nephropathy (IgAN) is the commonest primary glomerular disease around the world and represents a significant cause of end-stage renal disease. IgAN is characterized by mesangial deposition of IgA-immune complexes and mesangial expansion. The pathophysiological process includes an abnormally glycosylated IgA1, which is an antigenic target. Autoantibodies specifically recognize galactose-deficient IgA1 forming immune complexes that are amplified in size by the soluble IgA Fc receptor CD89 leading to deposition in the mesangium through interaction with non-classical IgA receptors. The local production of cytokines promotes local inflammation and complement system activation, besides the stimulation of mesangial proliferation. The spectrum of clinical manifestations is quite variable from asymptomatic microscopic hematuria to rapidly progressive glomerulonephritis. Despite all the advances, the pathophysiology of the disease is still not fully elucidated. The mucosal immune system is quoted to be a factor in triggering IgAN and a "gut-kidney axis" is proposed in its development. Furthermore, many recent studies have demonstrated that food intake interferes directly with disease prognosis. In this review, we will discuss how mucosal immunity, microbiota, and nutritional status could be interfering directly with the activation of intrinsic pathways of the mesangial cells, directly resulting in changes in their function, inflammation and development of IgAN.

The Clinical Utility of Acoustic Tweezing Thromboelastometry in Monitoring Blood Coagulation during Liver Transplantation

Introduction Patients with end-stage liver disease have a very low amount of pro- and anticoagulants in their blood, and therefore they may develop both bleeding and thrombotic complications during liver transplant surgery. These patients are in need for optimized intraoperative transfusion to keep their coagulation system in balance, which is currently managed by whole-blood viscoelastic assays such as TEG. However, TEG is not standardized for coagulation analysis because of its high variability and requires a relatively large blood volume per test. Here, we demonstrate that our noncontact drop-of-blood technique, referred to as "integrated quasi-static acoustic tweezing thromboelastometry" (i-QATTTM), can accurately assess changes in the coagulation system during liver transplantation. Method We have conducted side-by-side coagulation measurements by TEG and i-QATTTM. Whole blood samples (3-5mL each) were collected from liver transplant patients at the following time points: 1) immediately after induction of anesthesia (baseline), 2) after arterial reperfusion (artery), and 3) during biliary reconstruction (final). The samples were first used for TEG analysis, and their leftovers (waste blood) were analyzed by i-QATTTM. i-QATTTM has two graphical outputs: mechanical tweezograph showing sample firmness change with time (similar to TEG tracing, Fig 1A) and photo-optical tweezograph showing sample turbidity change with time. The following parameters extracted from these curves were analyzed: clot initiation time (CIT), time to firm clot formation (TFCF), maximum clot firmness (MCF), and maximum fibrin level (MFL). Correlation analysis was performed between these parameters and the corresponding TEG output. The study was approved by Tulane University and Ochsner Medical Center IRBs. Results Figure 1A (left) shows the mechanical tweezographs of the WB sample from a liver transplant patient at the artery stage, exposed to intrinsic and extrinsic pathway activators, and a combination of Cytochalasin-D and intrinsic pathway activator. These tests correspond to CK, CRT, and CFF assays in TEG. Fig 1A (right) shows the mechanical tweezographs of the PPP sample from the same patient, triggered by intrinsic and extrinsic pathway activators. Noted that the WB treated with Cytochalasin-D has the MCF value reduced dramatically from that of untreated blood and nearly equal to the MCF level of the PPP. In Fig. 1B, there were a strong linear correlation (Pearson correlation coefficient r > 0.72) between CIT and R-time of TEG (CK assay) and between TFCF and K-time of TEG (CK assay) for WB samples triggered via intrinsic pathway. Strong correlation also exists between MCF and MA values of TEG (CRT assay) for WB triggered via extrinsic pathway. MCF and MFL of PPP triggered by either pathway were strongly correlated (r > 0.74) with MA values of TEG (CFF assay). Strong correlation was also observed (r=0.7) between MCF and MA of TEG (CFF assay) for Cytochalasin-D treated WB triggered via intrinsic pathway. For both activation pathways, CIT at the artery stage (3.7±0.6 min and 1.5±0.3 min, respectively) is slightly lower than at the baseline (4.0±0.5 min and 1.7±0.4 min) and final stages (3.9±0.4 min and 1.9±0.3 min) while TFCF was stable across the stages (~12 min for intrinsic and ~5.5 min for extrinsic pathways). In the intrinsic pathway, MCF significantly reduced from the baseline (1.18±0.02) to 1.12±0.02 at the artery stage and slightly increased to 1.14±0.02 at the final stage; meanwhile, MCF in the extrinsic pathway decreased from 1.17±0.03 at the baseline to 1.13±0.02 at the artery stage and remained at the same value at the final stage (same trends observed in TEG). For PPP samples triggered via intrinsic pathway, MCF reduced from 1.13±0.02 (baseline) to 1.08±0.01 (artery) and increased to 1.12± 0.01 (final). In extrinsic pathway, the MCF values were 1.15±0.03 (baseline), 1.1±0.02 (artery), and 1.14±0.01 (final). We see that blood samples at the artery stage are less stiff than the samples at the baseline and final stages due to reduction in fibrinogen concentration, as seen for most liver transplant patients. Conclusions Using a single drop of blood per test, the i-QATTTM technique provides comprehensive information on blood coagulation status in liver transplant patients undergoing blood transfusion. This technique can be potentially adopted for transfusion guidance in perioperative settings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DDDR-20. TARGETING THE SELECTIVE VULNERABILITY OF IDH-MUTANT GLIOMA WITH ZOTIRACICLIB

Abstract BACKGROUND IDH mutations are common in diffuse gliomas and confer an increased dependency on oxidative mitochondrial function. Zotiraciclib, a multi-kinase inhibitor, not only suppresses CDK9-mediated gene transcription process but also causes mitochondrial dysfunction in preclinical models. We investigated the selective activity of zotiraciclib in IDH-mutant gliomas. METHODS Cytotoxicity of zotiraciclib was determined in patient-derived glioma cells with/without IDH mutations. Immunoblotting and flow cytometry were used to evaluate the cell death pathways. Real-time ATP production was measured by Seahorse assay. RESULTS The EC50 of zotiraciclib in IDH-wt cells was twice as high as in IDH-mutant cells. In low-dose zotiraciclib (15nM)-treated IDH-mutant cells, RNA Pol II phosphorylation and expression of antiapoptotic proteins, including XIAP, MCL-1 and survivin were decreased significantly. Cleaved caspase 3/7, 8, 9 and LC3B were also detected in treated IDH-mutant cells but not in IDH-wt cells, suggesting activation of intrinsic and extrinsic cell apoptosis pathways, and subsequent autophagy of IDH-mutant cells. Low-dose treated IDH-mutant, but not IDH-wt cells, induced decreased expression of a panel of mitochondrial respiration genes, and mitochondrial respiration complexes, cellular ATP depletion and increased ROS production, suggesting a greater disruption of mitochondrial function in zotiraciclib-treated IDH-mutant cells. Zotiraciclib treatment prolonged the survival in mice bearing intracranial IDH-mutant gliomas (30 days in DMSO-treated group vs 33 days in zotiraciclib-treated group, p = 0.01), but not in mice with glioma established with the isogenic IDH-wt glioma cells (18 days in DMSO-treated vs 20 days in zotiraciclib-treated group, p = 0.10). CONCLUSIONS There is selective activity of zotiraciclib in IDH-mutated glioma. Low-dose exposure of zotiraciclib induces cell death and suppresses transcriptional process and mitochondrial biogenesis in IDH-mutant glioma, not in IDH-wt tumors. These findings support a clinical trial testing zotiraciclib in IDH-mutated glioma. The treatment efficacy seen even at low concentrations further supports testing it as a single agent, thereby reducing toxicity concerns.