Search

Fish are extremely vulnerable to environmental stimulation and produce oxidative stress. Among them, hydrogen peroxide is an oxidative stress source that cannot be ignored in fish, which can cause physical disorders, inflammation and even death. Taurine was revealed to reduce oxidative damage and inflammation caused by toxic substances, but whether it can reduce toxicity of rice field eel caused by H

Brucella suis, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase (alr) gene, which encodes alanine racemase in Brucella, remains unclear. Here, we analyzed an alr deletion mutant and a complemented strain of Brucellasuis S2. The knockout strain displayed an unaltered, smooth phenotype in acriflavine agglutination tests but lacked the core polysaccharide portion of lipopolysaccharide (LPS). Genes involved in the LPS synthesis were significantly upregulated in the deletion mutant. The alr deletion strain exhibited reduced intracellular viability in the macrophages, increased macrophage-mediated killing, and upregulation of the apoptosis markers. Bcl2, an anti-apoptotic protein, was downregulated, while the pro-apoptotic proteins, Bax, Caspase-9, and Caspase-3, were upregulated in the macrophages infected with the deletion strain. The infected macrophages showed increased mitochondrial membrane permeability, Cytochrome C release, and reactive oxygen species, activating the mitochondrial apoptosis pathway. These findings revealed that alanine racemase was dispensable in B. suis S2 but influenced the strain’s rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the alr gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying Brucella’s survival and virulence and, specifically, how alr gene affects host immune evasion by regulating bacterial LPS biosynthesis.

This study aimed to investigate the molecular mechanisms underlying the role of bone marrow mesenchymal stem cells (BMMSCs)‐derived exosomes in ischaemia/reperfusion (IR)‐induced damage, and the role of oridonin in the treatment of IR. Exosomes were isolated from BMMSCs. Western blot analysis was done to examine the expression of proteins including CD63, CD8, apoptotic‐linked gene product 2 interacting protein X (AliX), Beclin‐1, ATG13, B‐cell lymphoma‐2 (Bcl‐2), apoptotic peptidase activating factor 1 (Apaf1) and Bcl2‐associated X (Bax) in different treatment groups. Accordingly, the expression of CD63, CD81 and AliX was higher in BMMSCs‐EXOs and IR + BMMSCs‐EXOs + ORI groups compared with that in the BMMSCs group. And BMMSCs‐derived exosomes inhibited the progression of IR‐induced myocardial damage, while this protective effect was boosted by the pre‐treatment with oridonin. Moreover, Beclin‐1, ATG13 and Bcl‐2 were significantly down‐regulated while Apaf1 and Bax were significantly up‐regulated in IR rats. And the presence of BMMSCs‐derived exosomes partly alleviated IR‐induced dysregulation of these proteins, while the oridonin pre‐treatment boosted the effect of these BMMSCs‐derived exosomes. The inhibited proliferation and promoted apoptosis of H9c2 cells induced by hypoxia/reperfusion (HR) were mitigated by the administration of BMMSCs‐derived exosomes. Meanwhile, HR also induced down‐regulation of Beclin‐1, ATG13 and Bcl‐2 expression and up‐regulation of Apaf1 and Bax, which were mitigated by the administration of BMMSCs‐derived exosomes. And oridonin pre‐treatment boosted the effect of BMMSCs‐derived exosomes. In conclusion, our results validated that BMMSCs‐derived exosomes suppressed the IR‐induced damages by participating in the autophagy process, while the pre‐treatment with oridonin could boost the protective effect of BMMSCs‐derived exosomes.

The goal of the present study was to investigate the protective effect of calcitriol on high-salt diet-induced hypertension. The hypertension rat model was established by a long-term high-salt diet (8% NaCl). Rats were treated with calcitriol, losartan, or their combination. Histological staining was used to confirm renal pathology. Global transcriptome analysis of renal tissues was performed, and the mechanism of the therapeutic effect of calcitriol was analysed by functional annotation and pathway analysis of the differentially expressed genes (DEGs) as well as by Western blotting analysis. The core genes for potential therapeutic regulation were identified through the coexpression gene network. For in vitro HK-2 cell experiments, small interfering RNA (siRNA) was used to knockdown key a transcription factor (TF) Glis2 to validate the therapeutic target of calcitriol. MAPK1 and CXCL12 expression was downregulated and the apoptosis pathway was significantly enriched by calcitriol treatment. The western blotting results showed that calcitriol treatment increased AMPK phosphorylation and decreased downstream mTOR phosphorylation, which was accompanied by a decrease in autophagy protein p62 expression and an increase in LC3-II/I expression. GLIS2 was identified as a specific therapeutic target for calcitriol. GLIS2 expression was upregulated by calcitriol and confirmed by HK-2 cells in vitro. Our omics data show that calcitriol can alleviate oxidative stress and fibrosis. Moreover, calcitriol can regulate the CXCL12/ERK1/2 cascade to inhibit the inflammatory response and renal cell apoptosis and induce renal autophagy through the AMPK/mTOR pathway. Our study partially elucidate the pathogenesis and treatment mechanism underlying hypertension, and provide new insights into the treatment of hypertension.

Background Lycopene is a naturally occurring carotenoid found in many fruits and vegetables, which has antioxidant effects. Although lycopene's protective effect has been observed on ischemia reperfusion (IR) injury in different organs, the effect of lycopene on Kupffer cells (KCs) has not been clearly elucidated in IR-induced acute hepatic inflammatory injury. Methods Mice were administered with either olive oil (10 mL/kg body weight) as the control or lycopene (20 mg/kg body weight) by gavage for 2 weeks before undergoing hepatic IR injury. Results In this study, we observed that the levels of aspartate aminotransferases (AST), alanine aminotransferase (ALT), and the percentages of hepatocellular apoptosis in mice pretreated with lycopene were significantly lower than control mice. Lycopene inhibited F4/80+ macrophage and Ly6G+ neutrophil accumulation, which further decreased the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin 6 (IL-6). Interestingly, lycopene induced increased autophagy in KCs, which was evidenced by elevated autophagosomes and the increased protein level of LC3B. In these KCs, lycopene-induced upregulation of autophagy inhibited NOD-like receptor family pyrin domain-containing 3 protein (NLRP3) inflammasome activation, which was demonstrated by the reduced mRNA and protein levels of NLRP3, cleaved caspase-1, an apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and IL-1β. Furthermore, 3-methyladenine, an autophagy inhibitor, abolished lycopene's inhibitory effect on the NLRP3 inflammasome in KCs, which led to increased hepatic IR injury. Intriguingly, we identified that the protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) were elevated in KCs isolated from IR-stressed mice pretreated with lycopene. Nrf2-siRNA or HO-1-siRNA could block the autophagy activation enhanced by lycopene in KCs, resulting in the activation of the NLRP3 inflammasome and aggravated hepatic IR injury. Conclusions Our findings demonstrated that lycopene promoted Nrf2/HO-1 pathway activation and further suppressed the NLRP3 inflammasome via enhancing KC autophagy, which alleviated hepatic IR injury.

Pyroptosis is a form of programmed necrosis, and is morphologically and mechanistically unique form of programmed cell death compared to others, such as apoptosis and autophagic cell death. More specifically, pyroptosis features gasdermin family-mediated membrane pore formation and subsequent cell lysis, as well as release of pro-inflammatory intracellular contents including IL-1β, IL-18 and HMGB1. Mechanistically, pyroptosis is driven by two main signaling pathways - one mediated by caspase-1 and the other by caspase-4/5/11. Recent studies show that pyroptosis is implicated in several cardiovascular diseases. In this review, we summarize recent scientific discoveries of pyroptosis's involvement in atherosclerosis, myocardial infarction, diabetic cardiomyopathy, reperfusion injury and myocarditis. We also organized new and emerging evidence suggesting that pyroptosis signaling pathways may be potential therapeutic targets in cardiovascular diseases.

Laryngeal squamous cell carcinoma (LSCC) is a common form of head and neck cancer with poor prognosis. However, the mechanism underlying the pathogenesis of LSCC remains unclear. Here, we demonstrated increased expression of fascin actin-bundling protein 1 (FSCN1) and decreased expression of microRNA-145-5p (miR-145-5p) in a clinical cohort of LSCC. Luciferase assay revealed that miR-145-5p is a negative regulator of FSCN1. Importantly, low miR-145-5p expression was correlated with TNM (tumor, node, metastasis) status and metastasis. Moreover, cases with low miR-145-5p/high FSCN1 expression showed poor prognosis, and these characteristics together served as independent prognostic indicators of survival. Gain- and loss-of-function studies showed that miR-145-5p overexpression or FSCN1 knockdown inhibited LSCC migration, invasion, and growth by suppressing the epithelial-mesenchymal transition along with inducing cell-cycle arrest and apoptosis. Additionally, hypermethylation of the miR-145-5p promoter suggested that repression of miR-145-5p arises through epigenetic inactivation. LSCC tumor growth in vivo could be inhibited by using miR-145-5p agomir or FSCN1 small interfering RNA (siRNA), which highlights the potential for clinical translation. Collectively, our findings indicate that miR-145-5p plays critical roles in inhibiting the progression of LSCC by suppressing FSCN1. Both miR-145-5p and FSCN1 are important potential prognostic markers and therapeutic targets for LSCC., Graphical Abstract, Gao et al. demonstrate that miR-145-5p expression is prognostic for LSCC with low expression associated with poorer outcomes. Underlying this pathology, miR-145-5p suppresses FSCN1 expression, but its inactivation facilitates epithelial-to-mesenchymal transition (EMT), proliferation, and survival. LSCC growth in vivo is inhibited by miR-145-5p agomir, highlighting potential as a biomarker and therapeutic target.

The expression of miR-101 in carcinoma and para-carcinoma tissues of patients with liver cancer was studied. The carcinoma and para-carcinoma tissues of 67 patients with liver cancer treated in Chinese PLA General Hospital were collected, and the expression of miR-101 in carcinoma and para-carcinoma tissues was detected via reverse transcription-polymerase chain reaction (RT-PCR). The liver cancer HepG2 cell line was transfected with miR-101 mimics. Moreover, the influence of miR-101 overexpression on the proliferation of liver cancer cells was detected via Cell Counting Kit-8 assay and colony formation assay. The proportion of Ki67-positive cells in the control group (NC group) and miR-101 overexpression group (miR-101 mimics group) was detected via Ki67 staining. The proportions of cells were detected via flow cytometry, and the predicted target gene Zeste2 enhancer (EZH2) was further verified via luciferase reporter gene assay and western blotting. The miR-101 overexpression significantly inhibited the colony formation and proliferation ability of liver cancer cells (P

Background Radiotherapy has been used increasingly to treat primary hepatocellular carcinoma. Clinically, the main cause of radiotherapy failure is cellular radioresistance, conferred via glycolytic metabolism. Our previous study demonstrated that Girdin is upregulated in primary hepatocellular carcinoma and promotes the invasion and metastasis of tumor cells. However, whether Girdin underlies the radio-sensitivity of hepatocellular carcinoma remains unclear. Methods A short hairpin RNA (shRNA) was used to silence CCDC88A (encoding Girdin), and real-time PCR was performed to determine CCDC88A mRNA expression. Then, cell proliferation, colony formation, flow cytometric, scratch, and transwell assays were to examine the influence of Girdin silencing on cellular radiosensitivity. Glycolysis assays were conducted to exam cell glycolysis process. Western blotting was performed to explore the signaling pathway downstream of Girdin. Finally, animal experiments were performed to demonstrate the effect of CCDC88A silencing on the radiosensitivity of hepatoma in vivo. Results shRNA-induced Girdin silencing suppressed glycolysis and enhanced the radio-sensitivity of hepatic cell lines, HepG2 and Huh-7. Furthermore, silencing of Girdin inhibited the PI3K/AKT/HIF-1α signaling pathway, which is a central regulator of glycolysis. Conclusion Girdin can regulate glycolysis in hepatocellular carcinoma cells through the PI3K/AKT/HIF-1α signaling pathway, which decreases the sensitivity of tumor cells to radiotherapy. Electronic supplementary material The online version of this article (doi:10.1186/s13046-017-0580-7) contains supplementary material, which is available to authorized users.

Glioblastoma (GBM) is the deadliest type of brain tumor, and glioma stem cells (GSCs) contribute to tumor recurrence and therapeutic resistance. Thus, an oncolytic virus targeting GSCs may be useful for improving GBM treatment. Because Zika virus (ZIKV) has an oncolytic tropism for infecting GSCs, we investigated the safety and efficacy of a live attenuated ZIKV vaccine candidate (ZIKV-LAV) for the treatment of human GBM in a GSC-derived orthotopic model. Intracerebral injection of ZIKV-LAV into mice caused no neurological symptoms or behavioral abnormalities. The neurovirulence of ZIKV-LAV was more attenuated than that of the licensed Japanese encephalitis virus LAV 14-14-2, underlining the superior safety of ZIKV-LAV for potential GBM treatment. Importantly, ZIKV-LAV significantly reduced intracerebral tumor growth and prolonged animal survival by selectively killing GSCs within the tumor. Mechanistically, ZIKV infection elicited antiviral immunity, inflammation, and GSC apoptosis. Together, these results further support the clinical development of ZIKV-LAV for GBM therapy.

Purpose: Here, we aim to investigate the molecular mechanism of regorafenib and verify the potential druggable target for the treatment of hepatocellular carcinoma (HCC). Experimental Design: HCC cell lines (PLC5, HepG2, Hep3B, SK-Hep1, and HA59T) were used to investigate the in vitro effect of regorafenib. Phosphatase activity was analyzed in HCC cells and purified SHP-1 proteins. PLC5-bearing mice were used to test the therapeutic efficiency of 20 and 40 mg/kg/d treatment with regorafenib (n \ge 8 mice). The clinical relevance of STAT3 signaling was investigated with 142 tumor samples from different patients with HCC. Descriptive statistical analysis was used to compare the baseline characteristics of patients and the expression of p-STAT3. Results: Regorafenib inhibited STAT3-related signaling in a dose-dependent manner and was a more potent inhibitor of STAT3 than sorafenib. Regorafenib increased SHP-1 phosphatase activity in purified SHP-1 protein directly. N-SH2 domain deletion and D61A mutants mimicking open-form SHP-1 partially abolished regorafenib-induced STAT3 inhibition and apoptosis. Importantly, a higher level of expression of STAT3 was found in patients with advanced clinical stages (P = 0.009) and poorly differentiated tumors (P = 0.035). Conclusions: Regorafenib induced significant tumor inhibition by relieving the autoinhibited N-SH2 domain of SHP-1 directly and inhibiting p-STAT3 signals. STAT3 may be suitable as a prognostic marker of HCC development, and may be a druggable target for HCC-targeted therapy using regorafenib. Clin Cancer Res; 20(22); 5768–76. ©2014 AACR.

Background This study is to investigate the effect of fenofibrate on the bone quality of Type 2 diabetes mellitus (T2DM) mouse model. Methods T2DM mouse model was induced by high-fat-diet, and the mice were treated with fenofibrate (100 mg/kg) (DIO-FENO) or PBS (DIO-PBS) for 4 weeks. The bone microstructure and biomechanical properties of femora were analyzed by micro-CT and 3-Point bending test. The protein expression was detected by immunohistochemical staining and Western blot. The cell apoptosis was evaluated by TUNEL staining. The Bcl2, caspase 3, and osteoblast marker genes were detected by RT-qPCR. Results The biomechanical properties of bones from DIO-FENO group were significantly lower than those in the control and DIO-PBS groups. Besides, the trabecular number was lower than those of the other groups, though the cortical porosity was decreased compared with that of DIO-PBS group because of the increase of apoptotic cells. The expression of osteocalcin and collagen I were decreased after treatment with fenofibrate in T2DM mice. Moreover, the cell viability was decreased after treated with different concentrations of fenofibrate, and the expression of Runx2 decreased after treated with high dose of fenofibrate. Conclusion Fenofibrate decreases the bone quality of T2DM mice through decreasing the expression of collagen I and osteocalcin, which may be resulted from the down regulation of Runx2 expression.

Background & Aims Nintedanib, a triple angiokinase inhibitor, is currently being evaluated against advanced HCC in phase I/II clinical trials. Here, we report the underlying molecular mechanism by which nintedanib (BIBF-1120) induces an anti-HCC effect. Methods To further elucidate whether the effect of nintedanib on SHP-1 is dependent on its angiokinase inhibition activity, we developed a novel kinase-independent derivative of nintedanib, ΔN. HCC cell lines were treated with nintedanib or its derivative (ΔN) and apoptosis, signal transduction, and phosphatase activity were analyzed. Purified SHP-1 proteins or HCC cells expressing deletion N-SH2 domain or D61A point mutants were used to investigate the potential effect of nintedanib on SHP-1. In vivo efficacy was determined in nude mice with HCC subcutaneous xenografts (n⩾8 mice). Results Nintedanib induced anti-proliferation in HCC cell lines by targeting STAT3. Ectopic STAT3 abolished nintedanib-mediated apoptosis in HCC cells. Nintedanib further activated SHP-1 in purified SHP-1 proteins suggesting that nintedanib directly affects SHP-1 for STAT3 inhibition. HCC cells or recombinant SHP-1 proteins expressing deletion of N-SH2 domain or D61A mutants restored the activity of nintedanib suggesting that the auto-inhibition structure of SHP-1 was relieved by nintedanib. Although ΔN only retained the backbone of nintedanib without kinase activity, ΔN still induced substantial anti-HCC activity in vitro and in vivo by targeting STAT3. Conclusions Nintedanib induced significant anti-HCC activity independent of angiokinase inhibition activity in a preclinical HCC model by relieving autoinhibition of SHP-1. Our findings provide new mechanistic insight into the inhibition of HCC growth by nintedanib.

During apoptosis, dying cells are swiftly removed by phagocytes. It is not fully understood how apoptotic cells are recognized by phagocytes. Here we report the identification and characterization of the Caenorhabditis elegans ttr-52 gene, which encodes a transthyretin-like protein and is required for efficient cell corpse engulfment. The TTR-52 protein is expressed in, and secreted from, C. elegans endoderm and clusters around apoptotic cells. Genetic analysis indicates that TTR-52 acts in the cell corpse engulfment pathway mediated by CED-1, CED-6 and CED-7 and affects clustering of the phagocyte receptor CED-1 around apoptotic cells. TTR-52 recognizes surface-exposed phosphatidylserine (PtdSer) in vivo and binds to both PtdSer and the extracellular domain of CED-1 in vitro. TTR-52 is therefore the first bridging molecule identified in C. elegans that mediates recognition of apoptotic cells by crosslinking the PtdSer 'eat me' signal with the phagocyte receptor CED-1.

Inhibitor of apoptosis (IAP) proteins have a crucial role in apoptosis, through negative regulation of caspases in species from fruitflies to mammals. In Caenorhabditis elegans, however, no IAP homolog or caspase inhibitor has been identified, calling into question how the cell-killing caspase CED-3 can be negatively regulated. Here we show that inactivation of the C. elegans csp-3 gene, which encodes a protein similar to the small subunit of the CED-3 caspase, causes cells that normally live to undergo apoptosis in a CED-3–dependent manner. Biochemical analysis reveals that CSP-3 associates with the large subunit of the CED-3 zymogen and inhibits zymogen autoactivation. However, CSP-3 does not block CED-3 activation induced by CED-4, nor does it inhibit the activity of the activated CED-3 protease. Therefore CSP-3 uses a previously unreported mechanism to protect cells from apoptosis.

Externalization of phosphatidylserine, which is normally restricted to the inner leaflet of plasma membrane, is a hallmark of mammalian apoptosis1,2,3,4. It is not known what activates and mediates the phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an annexin V-based phosphatidylserine labelling method and show that a majority of apoptotic germ cells in Caenorhabditis elegans have surface-exposed phosphatidylserine, indicating that phosphatidylserine externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the C. elegans apoptosis-inducing factor (AIF) homologue (WAH-1)5, a mitochondrial apoptogenic factor, or the C. elegans phospholipid scramblase 1 (SCRM-1), a plasma membrane protein, reduces phosphatidylserine exposure on the surface of apoptotic germ cells and compromises cell-corpse engulfment. WAH-1 associates with SCRM-1 and activates its phospholipid scrambling activity in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane phosphatidylserine externalization through its downstream effector, SCRM-1.

Hepatocellular carcinoma is the fifth most common solid cancer worldwide. Sorafenib, a small multikinase inhibitor, is the only approved therapy for advanced HCC. The clinical benefit of sorafenib is offset by the acquisition of sorafenib resistance. Understanding of the molecular mechanism of STAT3 overexpression in sorafenib resistance is critical if the clinical benefits of this drug are to be improved. In this study, we explored our hypothesis that loss of RFX-1/SHP-1 and further increase of p-STAT3 as a result of sorafenib treatment induces sorafenib resistance as a cytoprotective response effect, thereby, limiting sorafenib sensitivity and efficiency. We found that knockdown of RFX-1 protected HCC cells against sorafenib-induced cell apoptosis and SHP-1 activity was required for the process. SC-2001, a molecule with similar structure to obatoclax, synergistically suppressed tumor growth when used in combination with sorafenib in vitro and overcame sorafenib resistance through up-regulating RFX-1 and SHP-1 resulting in tumor suppression and mediation of dephosphorylation of STAT3. In addition, sustained sorafenib treatment in HCC led to increased p-STAT3 which was a key mediator of sorafenib sensitivity. The combination of SC-2001 and sorafenib strongly inhibited tumor growth in both wild-type and sorafenib-resistant HCC cell bearing xenograft models. These results demonstrate that inactivation of RFX/SHP-1 induced by sustained sorafenib treatment confers sorafenib resistance to HCC through p-STAT3 up-regulation. These effects can be overcome by SC-2001 through RFX-1/SHP-1 dependent p-STAT3 suppression. In conclusion, the use of SC-2001 in combination with sorafenib may constitute a new strategy for HCC therapy.

During C. elegans apoptosis, the dicer ribonuclease (DCR-1) is cleaved by the cell death protease CED-3 to generate a truncated DCR-1 (tDCR-1) with one and a half ribonuclease III (RNase III) domains, converting it into a deoxyribonuclease (DNase) that initiates apoptotic chromosome fragmentation. We performed biochemical and functional analyses to understand this unexpected RNase to DNase conversion. In full-length DCR-1, tDCR-1 DNase activity is suppressed by its N-terminal DCR-1 sequence. However, not all the sequence elements in the N-terminal DCR-1 are required for this suppression. Our deletion analysis reveals that a 20-residue α-helix sequence in DCR-1 appears to define a critical break point for the sequence required for suppressing tDCR-1 DNase activity through a structure-dependent mechanism. Removal of the N-terminal DCR-1 sequence from tDCR-1 activates a DNA-binding activity that also requires the one half RNase IIIa domain, and enables tDCR-1 to process DNA. Consistently, structural modeling of DCR-1 and tDCR-1 suggests that cleavage of DCR-1 by CED-3 may cause a conformational change that allows tDCR-1 to bind and process DNA, and may remove steric hindrance that blocks DNA access to tDCR-1. Moreover, a new DNase can be engineered using different RNase III domains, including the one from bacterial RNase III. Our results indicate that very distantly related RNase III enzymes have the potential to cleave DNA when processed proteolytically or paired with an appropriate partner that facilitates binding to DNA. We suggest the possibility that this phenomenon may be extrapolated to other ribonucleases.

Sorafenib is the first approved targeted therapeutic reagent for hepatocellular carcinoma. Here, we report that SC-60, a dimer-based sorafenib derivative, overcomes the resistance of sorafenib and shows a better anti-hepatocellular carcinoma effect in vitro and in vivo. SC-60 substantially increased SH2 domain-containing phosphatase 1 (SHP-1) phosphatase activity in hepatocellular carcinoma cells and purified SHP-1 proteins, suggesting that SC-60 affects SHP-1 directly. Molecular docking and truncated mutants of SHP-1 further confirmed that SC-60 interferes with the inhibitory N-SH2 domain to relieve the closed catalytic protein tyrosine phosphatase domain of SHP-1. Deletion of N-SH2 domain (dN1) or point mutation (D61A) of SHP-1 abolished the effect of SC-60 on SHP-1, p-STAT3, and apoptosis. Importantly, SC-60 exhibited significant survival benefits compared with sorafenib in a hepatocellular carcinoma orthotopic model via targeting the SHP-1/STAT3–related signaling pathway. In summary, dimer derivative of sorafenib, SC-60, is a SHP-1 agonist and may be a potent reagent for hepatocellular carcinoma–targeted therapy. Mol Cancer Ther; 13(1); 27–36. ©2013 AACR.

Autophagy is a self-degradative process and it plays a housekeeping role in removing misfolded or aggregated proteins, clearing damaged organelles, and eliminating intracellular pathogens. Previous studies have demonstrated that autophagy pathway was activated in brain after experimental subarachnoid hemorrhage (SAH); however, the role of autophagy in the pathogenesis of early brain injury (EBI) following SAH remains unknown. Experiment 1 aimed to investigate the time-course of the autophagy in the cortex following SAH. In experiment 2, we chose the maximum time point of autophagy activation and assessed the effects of rapamycin (RAP, autophagy activator) and 3-methyladenine (3-MA, autophagy inhibitor) on regulation of EBI. All SAH animals were subjected to injection of 0.3 ml fresh arterial, nonheparinized blood into prechiasmatic cistern in 20 s. As a result, microtubule-associated protein light chain-3 (LC3), a biomarker of autophagosome, and beclin-1, a Bcl-2-interacting protein required for autophagy, were significantly increased at the early stage of SAH and their expressions peaked at 24 h after SAH. In RAP-treated group, the early brain damage such as brain edema, blood-brain barrier (BBB) impairment, cortical apoptosis, and clinical behavior scale was significantly ameliorated in comparison with vehicle-treated SAH rats. Conversely, 3-MA decreased expression of LC3 and beclin-1, increased the average value of brain edema and BBB disfunction, and aggravated neurological deficits. Our results suggest that autophagy pathway is activated in the brain after SAH and may play a beneficial role to EBI development.

Background & Aims: Nintedanib, a triple angiokinase inhibitor, is currently being evaluated against advanced HCC in phase I/II clinical trials. Here, we report the underlying molecular mechanism by which nintedanib (BIBF-1120) induces an anti-HCC effect. Methods: HCC cell lines were treated with nintedanib or its derivative (ΔN) and apoptosis, signal transduction, and phosphatase activity were analyzed. Purified SHP-1 proteins or HCC cells expressing deletion N-SH2 domain or D61A point mutants were used to investigate the potential effect of nintedanib on SHP-1. In vivo efficacy was determined in nude mice with HCC subcutaneous xenografts (n ≥ 8 mice). Results: Nintedanib induced anti-proliferation in HCC cell lines by targeting STAT3. Ectopic STAT3 abolished nintedanib-mediated apoptosis in HCC cells. Nintedanib further activated SHP-1 in purified SHP-1 proteins suggesting that nintedanib directly affects SHP-1 for STAT3 inhibition. HCC cells or recombinant SHP-1 proteins expressing deletion of N-SH2 domain or D61A mutants restored the activity of nintedanib suggesting that the auto-inhibition structure of SHP-1 was relieved by nintedanib. To further elucidate whether the effect of nintedanib on SHP-1 is dependent on its angiokinase inhibition activity, we developed a novel kinase-independent derivative of nintedanib, ΔN. Although ΔN only retained the backbone of nintedanib without kinase activity, ΔN still induced substantial anti-HCC activity in vitro and in vivo by targeting STAT3. Conclusions: Nintedanib induced significant anti-HCC activity independent of angiokinase inhibition activity in a preclinical HCC model by relieving autoinhibition of SHP-1. Our findings provide new mechanistic insight into the inhibition of HCC growth by nintedanib. Citation Format: Wei-Tien Tai, Chung-Wai Shiau, Yong-Shi Li, Chun-Wei Chang, Jui-Wen Huang, Ting-Ting Hsueh, Kuen-Feng Chen. Nintedanib (BIBF-1120) inhibits hepatocellular carcinoma growth independent of angiokinase activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2629. doi:10.1158/1538-7445.AM2014-2629

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces programmed cell death through the caspase activation cascade and translocation of cleaved Bid (tBid) by the apical caspase-8 to mitochondria to induce oligomerization of multidomain Bax and Bak. However, the roles of prosurvival Bcl-2 family proteins in TRAIL apoptosis remain elusive. Here we showed that, besides the specific cleavage and activation of Bid by caspase-8 and caspase-3, TRAIL-induced apoptosis in Jurkat T cells required the specific cleavage of Mcl-1 at Asp-127 and Asp-157 by caspase-3, while other prototypic antiapoptotic factors such as Bcl-2 or Bcl-X(L) seemed not to be affected. Mutation at Asp-127 and Asp-157 of Mcl-1 led to cellular resistance to TRAIL-induced apoptosis. In sharp contrast to cycloheximide-induced Mcl-1 dilapidation, TRAIL did not activate proteasomal degradation of Mcl-1 in Jurkat cells. We further established for the first time that the C-terminal domain of Mcl-1 became proapoptotic as a result of caspase-3 cleavage, and its physical interaction and cooperation with tBid, Bak, and voltage-dependent anion-selective channel 1 promoted mitochondrial apoptosis. These results suggested that removal of N-terminal domains of Bid by caspase-8 and Mcl-1 by caspase-3 enabled the maximal mitochondrial perturbation that potentiated TRAIL-induced apoptosis.