ATF3-CBS signaling axis coordinates ferroptosis and tumorigenesis in colorectal cancer.

The induction of ferroptosis is promising for cancer therapy. However, the mechanisms enabling cancer cells to evade ferroptosis, particularly in low-cystine environments, remain elusive. Our study delves into the intricate regulatory mechanisms of Activating transcription factor 3 (ATF3) on Cystathionine ß-synthase (CBS) under cystine deprivation stress, conferring resistance to ferroptosis in colorectal cancer (CRC) cells. Additionally, our findings establish a positively correlation between this signaling axis and CRC progression, suggesting its potential as a therapeutic target. Mechanistically, ATF3 positively regulates CBS to resist ferroptosis under cystine deprivation stress. In contrast, the suppression of CBS sensitizes CRC cells to ferroptosis through targeting the mitochondrial tricarboxylic acid (TCA) cycle. Notably, our study highlights that the ATF3-CBS signaling axis enhances ferroptosis-based CRC cancer therapy. Collectively, the findings reveal that the ATF3-CBS signaling axis is the primary feedback pathway in ferroptosis, and blocking this axis could be a potential therapeutic approach for colorectal cancer.

DPY30 knockdown suppresses colorectal carcinoma progression via inducing Raf1/MST2-mediated apoptosis.

Colorectal Carcinoma (CRC) is one of the most common malignant tumors of the digestive tract, with a high mortality rate. DPY30 is one of the core subunits of the histone methyltransferase complex, which was involved in many cancer processes. However, the role of DPY30 in the occurrence and progression of CRC remains unclear. In this study, we sought to evaluate the role and mechanism of DPY30 in CRC cells apoptosis. Here, we identified that knockdown of DPY30 significantly inhibited the HT29 and HCT116 cells proliferation in vitro. Moreover, the knockdown of DPY30 significantly increased the apoptosis rate and promoted the expression of apoptosis-related proteins in CRC cells. Meanwhile, DPY30 knockdown promoted CRC cells apoptosis through endogenous programmed death and in a caspase activation-dependent manner. Furthermore, RNA-seq analysis revealed that the action of DPY30 is closely related to the apoptosis biological processes, and screened its potential effectors Raf1. Mechanistically, DPY30 downregulation promotes MST2-induced apoptosis by inhibiting Raf1 transcriptional activity through histone H3 lysine 4 trimethylation (H3K4me3). In vivo experiments showed that DPY30 was correlated with Raf1 in nude mouse subcutaneous xenografts tissues significantly. Clinical colorectal specimens further confirmed that overexpression of DPY30 in malignant tissues was significantly correlated with Raf1 level. The vital role of the DPY30/Raf1/MST2 signaling axis in the cell death and survival rate of CRC cells was disclosed, which provides potential new targets for early diagnosis and clinical treatment of CRC.

DPY30 promotes colorectal carcinoma metastasis by upregulating ZEB1 transcriptional expression.

DPY30 belongs to the core subunit of components of the histone lysine methyltransferase complex, which is implicated in tumorigenesis, cell senescence, and other biological events. However, its contribution to colorectal carcinoma (CRC) progression and metastasis has yet to be elucidated. Therefore, this study aimed to investigate the biological function of DPY30 in CRC metastasis both in vitro and in vivo. Herein, our results revealed that DPY30 overexpression is significantly positively correlated with positive lymph nodes, epithelial-mesenchymal transition (EMT), and CRC metastasis. Moreover, DPY30 knockdown in HT29 and SW480 cells markedly decreased EMT progression, as well as the migratory and invasive abilities of CRC cells in vitro and lung tumor metastasis in vivo. Mechanistically, DPY30 increased histone H3K4me3 level and promoted EMT and CRC metastasis by upregulating the transcriptional expression of ZEB1. Taken together, our findings indicate that DPY30 may serve as a therapeutic target and prognostic marker for CRC.

Aldolase B impairs DNA mismatch repair and induces apoptosis in colon adenocarcinoma.

Evidence suggests that DNA repair capacity manifested by intact functional base excision repair and mismatch repair (MMR) pathways is related to the prognosis of multiple cancer types. Aldolase B (ALDOB) is well known for its role in metabolism and glycolysis. The expression of ALDOB in colon adenocarcinoma and the relationship between its expression and colon adenocarcinoma prognosis remain controversial; in addition, the potential role of ALDOB in DNA MMR has not yet been reported. In this study, we identified a cluster of DNA repair-related proteins that interact with ALDOB in the colon adenocarcinoma cell line HCT116. Expression analysis of colon adenocarcinoma data from the Cancer Genome Atlas (TCGA-COAD data, n = 551) indicated that ALDOB mRNA expression was significantly higher in specimens with microsatellite instability (MSI) than in specimens with microsatellite stability (MSS). Regarding prognosis, colon adenocarcinoma patients with high ALDOB mRNA expression had longer overall survival (OS). Higher expression of ALDOB protein was significantly correlated with MMR deficiency (d-MMR) in formalin-fixed paraffin-embedded (FFPE) patient specimens. The expression of ALDOB was significantly elevated in colon adenocarcinoma cell lines. Further evidence indicated that rather than affecting proliferation, ALDOB overexpression induced the functional loss of MMR proteins and in turn caused irreversible DNA damage via disrupting EZH2-Rad51 expression and then caused apoptosis by ERK inactivation. Overall, our study demonstrates that high ALDOB expression impairs DNA MMR and induces apoptosis in colon adenocarcinoma. ALDOB may be a new biomarker associated with d-MMR and an independent prognostic factor for colon adenocarcinoma.

Platelet-derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis.

Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis.

Rac1 regulates bacterial toxin-induced thrombin generation.

OBJECTIVE: Systemic inflammatory response syndrome is associated with severe coagulopathy. The purpose of this study was to examine thrombin generation in systemic inflammation triggered by the endotoxin lipopolysaccharide (LPS) and the exotoxin streptococcal M1 protein. METHODS: Thrombin generation, lung histology and myeloperoxidase (MPO) activity were determined 6 and 24 h after induction of systemic inflammation. Male C57BL/6 mice received the Rac1 inhibitor NSC23766 prior to challenge with bacterial toxins. RESULTS: LPS and M1 protein challenge increased neutrophil infiltration and caused damage in the lung. Time to peak thrombin formation was increased and peak and total generation of thrombin were decreased in plasma from LPS- and M1 protein-treated mice. Coincubation of samples from mice exposed to bacterial toxins with platelet poor plasma from healthy mice completely reversed the inhibitory effect of LPS and M1 protein on thrombin generation, suggesting that bacterial toxins decreased levels of plasma factors explaining the reduction of thrombin generating capacity of plasma from septic animals. NSC23766 treatment not only decreased LPS- and M1 protein-induced neutrophil accumulation as well as levels of interleukin-6 and CXCL2 in the lung, but also abolished bacterial toxin-induced changes in thrombin generation. For example, NSC23766 increased peak formation by 57% and total thrombin generation by 48% in LPS-treated animals at 6 h. CONCLUSIONS: Taken together, our novel findings show that bacterial toxins increase thrombin generation via consumption of plasma factors and that Rac1 signaling plays an important role in thrombin generation in response to bacterial toxins. Thus, targeting Rac1 activity might be a useful way not only to ameliorate pulmonary inflammation, but also inhibit pathological changes in coagulation in bacterial infections.

Adhesive Mechanisms of Histone-Induced Neutrophil-Endothelium Interactions in the Muscle Microcirculation.

BACKGROUND: Extracellular histones released during cell damage have the capacity to cause tissue injury associated with increased leukocyte accumulation. However, the molecular mechanisms regulating histone-induced leukocyte recruitment remain elusive. The objective of this study was to examine the role of adhesion molecules in histone-dependent leukocyte accumulation by use of intravital microscopy of the mouse cremaster microcirculation. METHODS: Histone 3 and TNF-α were intrascrotally administered, and anti-P-selectin, anti-P-selectin glycoprotein ligand-1 (PSGL-1), anti-membrane-activated complex-1 (Mac-1), anti-lymphocyte function antigen-1 (LFA-1) antibody and neutrophil depletion antibody were injected intravenously or intraperitoneally. RESULTS: Intrascrotal injection of histone 3 dose-dependently increased leukocyte recruitment. Neutrophil depletion abolished intravascular and extravascular leukocytes after histone 3 challenge, suggesting that neutrophils were the dominating leukocyte subtype responding to histone stimulation. Pretreatment with an anti-P-selectin and an anti-PSGL-1 antibody abolished histone-stimulated neutrophil rolling, adhesion and emigration. When the anti-P-selectin or the anti-PSGL-1 antibody was administrated after histone 3 stimulation, neutrophil rolling was reduced, whereas the number of firmly adherent and emigrated neutrophils were unchanged, suggesting that the inhibitory effect of blocking P-selectin and PSGL-1 on neutrophil adhesion and recruitment was due to the reduction in neutrophil rolling. Moreover, pretreatment with antibodies against Mac-1 and LFA-1 had no effect of neutrophil rolling but abolished adhesion and emigration evoked by histone 3. Thus, our data demonstrate that P-selectin and PSGL-1 play an important role in histone-induced inflammatory cell recruitment by mediating neutrophil rolling as a precondition for histone-provoked firm adhesion and emigration in vivo. Moreover, we conclude that both Mac-1 and LFA-1 are critical in supporting histone-provoked firm adhesion of neutrophils to endothelial cells. CONCLUSION: These novel findings define specific selectins and integrins as potential targets for pharmacological intervention in histone-dependent inflammatory diseases.

STAT3-dependent CXC chemokine formation and neutrophil migration in streptococcal M1 protein-induced acute lung inflammation.

Streptococcus pyogenes cause infections ranging from mild pharyngitis to severe streptococcal toxic shock syndrome (STSS). The M1 serotype of Streptococcus pyogenes is most frequently associated with STSS. Herein, it was hypothesized that STAT3 signaling might be involved in M1 protein-evoked lung inflammation. The STAT3 inhibitor, S3I-201, was administered to male C57Bl/6 mice before iv challenge with M1 protein. Bronchoalveolar fluid and lung tissue were harvested for quantification of STAT3 activity, neutrophil recruitment, edema, and CXC chemokine formation. Neutrophil expression of Mac-1 was quantified by use of flow cytometry. Levels of IL-6 and HMGB1 were determined in plasma. CXCL2-induced neutrophil chemotaxis was studied in vitro. Administration of S3I-201 markedly reduced M1 protein-provoked STAT3 activity, neutrophil recruitment, edema formation, and inflammatory changes in the lung. In addition, M1 protein significantly increased Mac-1 expression on neutrophils and CXC chemokine levels in the lung. Treatment with S3I-201 had no effect on M1 protein-induced expression of Mac-1 on neutrophils. In contrast, inhibition of STAT3 activity greatly reduced M1 protein-induced formation of CXC chemokines in the lung. Interestingly, STAT3 inhibition markedly decreased plasma levels of IL-6 and HMGB1 in animals exposed to M1 protein. Moreover, we found that S3I-201 abolished CXCL2-induced neutrophil migration in vitro. In conclusion, these novel findings indicate that STAT3 signaling plays a key role in mediating CXC chemokine production and neutrophil infiltration in M1 protein-induced acute lung inflammation.

Inhibition of Ras signalling reduces neutrophil infiltration and tissue damage in severe acute pancreatitis.

Neutrophil recruitment is known to be a rate-limiting step in mediating tissue injury in severe acute pancreatitis (AP). However, the signalling mechanisms controlling inflammation and organ damage in AP remain elusive. Herein, we examined the role of Ras signalling in AP. Male C57BL/6 mice were treated with a Ras inhibitor (farnesylthiosalicylic acid, FTS) before infusion of taurocholate into the pancreatic duct. Pancreatic and lung tissues as well as blood were collected 24 h after pancreatitis induction. Pretreatment with FTS decreased serum amylase levels by 82% and significantly attenuated acinar cell necrosis, tissue haemorrhage and oedema formation in taurocholate-induced pancreatitis. Inhibition of Ras signalling reduced myeloperoxidase (MPO) levels in the inflamed pancreas by 42%. In addition, administration of FTS decreased pancreatic levels of CXC chemokines as well as circulating levels of interleukin-6 and high-mobility group box 1 in animals exposed to taurocholate. Moreover, treatment with FTS reduced taurocholate-induced MPO levels in the lung. Inhibition of Ras signalling had no effect on neutrophil expression of Mac-1 in mice with pancreatitis. Moreover, FTS had no direct impact on trypsin activation in isolated pancreatic acinar cells. These results indicate that Ras signalling controls CXC chemokine formation, neutrophil recruitment and tissue injury in severe AP. Thus, our findings highlight a new signalling mechanism regulating neutrophil recruitment in the pancreas and suggest that inhibition of Ras signalling might be a useful strategy to attenuate local and systemic inflammation in severe AP.

Proinflammatory role of neutrophil extracellular traps in abdominal sepsis.

Excessive neutrophil activation is a major component in septic lung injury. Neutrophil-derived DNA may form extracellular traps in response to bacterial invasions. The aim of the present study was to investigate the potential role of neutrophil extracellular traps (NETs) in septic lung injury. Male C57BL/6 mice were treated with recombinant human (rh)DNAse (5 mg/kg) after cecal ligation and puncture (CLP). Extracellular DNA was stained by Sytox green, and NET formation was quantified by confocal microscopy and cell-free DNA in plasma, peritoneal cavity, and lung. Blood, peritoneal fluid, and lung tissue were harvested for analysis of neutrophil infiltration, NET levels, tissue injury, as well as CXC chemokine and cytokine formation. We observed that CLP caused increased formation of NETs in plasma, peritoneal cavity, and lung. Administration of rhDNAse not only eliminated NET formation in plasma, peritoneal cavity, and bronchoalveolar space but also reduced lung edema and tissue damage 24 h after CLP induction. Moreover, treatment with rhDNAse decreased CLP-induced formation of CXC chemokines, IL-6, and high-mobility group box 1 (HMGB1) in plasma, as well as CXC chemokines and IL-6 in the lung. In vitro, we found that neutrophil-derived NETs had the capacity to stimulate secretion of CXCL2, TNF-α, and HMGB1 from alveolar macrophages. Taken together, our findings show that NETs regulate pulmonary infiltration of neutrophils and tissue injury via formation of proinflammatory compounds in abdominal sepsis. Thus we conclude that NETs exert a proinflammatory role in septic lung injury.

[Immune tolerance induced by bone marrow cell transplantation combined with use of cyclophosphamide in diabetic rats with pancreatic transplantation].

OBJECTIVE: To study the immune tolerance induced by bone marrow cell transplantation combined with short-term use of cyclophosphamide after pancreatic transplantation in diabetic rats. METHODS: Type I diabetes mellitus was induced in BN rats with streptozotocin (STZ) intraperitoneal injection at a single dose of 45 mg/kg. Pancreatic transplantations were performed with the SD rats as donors and the diabetic BN rats as recipients. Twenty BN rats with type I diabetes mellitus were randomly divided into four groups. The BN rats in Group I received pancreas transplantations only. The BN rats in Group II received intraperitoneal injection of 150 mg/kg cyclophosphamide on the first day after pancreas transplantations. The BN rats in Group 11 received injection of 2.0 x 10(8) donors' bone marrow cells via the portal vein during the pancreas transplantations. The BN rats in Group IV received injection 2.0 x 10(8) donors' bone marrow cells via the portal vein during the pancreas transplantations and an intraperitoneal injection of 150 mg/kg cyclophosphamide on the first day after pancreas transplantations. The blood glucose of the rats was measured after transplantations. The graft functional survival time (GFST) was recorded. Peripheral blood was obtained two weeks after the transplantations to prepare single cell suspension for detecting chimera formation rate and the level of Vbeta11+ T cell by flow cytometry. RESULTS: The average GFST of group IV was (18 +/- 2.2) d, significantly longer than those of group I (7.8 +/- 1.2) d, group II (8.2 +/- 1.6) d, and group III (8.8 +/- l.4) d (P < 0.05). The rats in group IV had significant lower level of Vbeta11+ T cells (2.5 +/- 0.3)% than those in the other groups (P < 0.05). Donors' bone marrow-derived cells could be detected in the peripheral blood of diabetic rats in group IV, with a chimeric rate of (10.0 +/- 2.3)%. No donors' bone marrow-derived cells were detected in the rats in other groups. CONCLUSION: Bone marrow cell transplantation combined with short-term use of cyclophosphamide promote chimerism formation and induce immune tolerance in rats with pancreatic transplantations, which prolongs pancreatic graft functional survival time.