Antitumor Activity of cGAMP via Stimulation of cGAS-cGAMP-STING-IRF3 Mediated Innate Immune Response

Immunotherapy is one of the key strategies for cancer treatment. The cGAS-cGAMP-STING-IRF3 pathway of cytosolic DNA sensing plays a pivotal role in antiviral defense. We report that the STING activator cGAMP possesses significant antitumor activity in mice by triggering the STING-dependent pathway directly. cGAMP enhances innate immune responses by inducing production of cytokines such as interferon-β, interferon-γ, and stimulating dendritic cells activation, which induces the cross-priming of CD8(+) T cells. The antitumor mechanism of cGAMP was verified by STING and IRF3, which were up-regulated upon cGAMP treatment. STING-deficiency dramatically reduced the antitumor effect of cGAMP. Furthermore, cGAMP improved the antitumor activity of 5-FU, and clearly reduced the toxicity of 5-FU. These results demonstrated that cGAMP is a novel antitumor agent and has potential applications in cancer immunotherapy

Therapeutic Effects of Astragaloside IV on Myocardial Injuries: Multi-Target Identification and Network Analysis

<div><p>Astragaloside IV (AGS-IV) is a main active ingredient of <em>Astragalus membranaceus</em> Bunge, a medicinal herb used for cardiovascular diseases (CVD). In this work, we investigated the therapeutic mechanisms of AGS-IV at a network level by computer-assisted target identification with the <em>in silico</em> inverse docking program (INVDOCK). Targets included in the analysis covered all signaling pathways thought to be implicated in the therapeutic actions of all CVD drugs approved by US FDA. A total of 39 putative targets were identified. Three of these targets, calcineurin (CN), angiotensin-converting enzyme (ACE), and c-Jun N-terminal kinase (JNK), were experimentally validated at a molecular level. Protective effects of AGS-IV were also compared with the CN inhibitor cyclosporin A (CsA) in cultured cardiomyocytes exposed to adriamycin. Network analysis of protein-protein interactions (PPI) was carried out with reference to the therapeutic profiles of approved CVD drugs. The results suggested that the therapeutic effects of AGS-IV are based upon a combination of blocking calcium influx, vasodilation, anti-thrombosis, anti-oxidation, anti-inflammation and immune regulation.</p> </div

MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2

<div><p>Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide, has been shown to exert potent anti-inflammatory effects in ischemic brain injury. In this study, we investigated the neuroprotective action of MLIF against oxygen-glucose deprivation (OGD)-induced injury in human neuroblastoma SH-SY5Y cells. MTT assay was used to assess cell viability, and flow cytometry assay and Hoechst staining were used to evaluate apoptosis. LDH assay was used to exam necrosis. The release of inflammatory cytokines was detected by ELISA. Levels of the apoptosis associated proteins were measured by western blot analysis. To identify the protein target of MLIF, pull-down assay and mass spectrometry were performed. We observed that MLIF enhanced cell survival and inhibited apoptosis and necrosis by inhibiting p-JNK, p53, c-caspase9 and c-caspase3 expression. In the microglia, OGD-induced secretion of inflammatory cytokines was markedly reduced in the presence of MLIF. Furthermore, we found that eukaryotic translation elongation factor 1A2 (eEF1A2) is a downstream target of MLIF. Knockdown eEF1A2 using short interfering RNA (siRNA) almost completely abrogated the anti-apoptotic effect of MLIF in SH-SY5Y cells subjected to OGD, with an associated decrease in cell survival and an increase in expression of p-JNK and p53. These results indicate that MLIF ameliorates OGD-induced SH-SY5Y neuroblastoma injury by inhibiting the p-JNK/p53 apoptotic signaling pathway via eEF1A2. Our findings suggest that eEF1A2 may be a new therapeutic target for ischemic brain injury.</p></div

The protective effect of AGS-IV in adriamycin-induced injury of cardiomyocytes compared with CN inhibitor CsA.

<p>(A) Activity of LDH in culture supernatant of the ADR-injured cardiomyocytes treated by AGS-IV (0.32∼32 µM). (B) Activity of LDH in culture supernatant of the ADR-injured cardiomyocytes treated by CsA (0.21∼4.2 µM). Data are shown as mean±SD (n = 4) (**P<0.01). (C) The effects of CsA and AS-IV on the expressions of apoptotic proteins. The expression of BCL-2 and Bax in ADR-injured cardiomyocytes treated by CsA or AGS-IV, the effects of CsA and AS-IV on the Bcl-2/Bax level. All data are mean±SD (n = 3).</p

Evaluation of AGS-IV on representative protein targets.

<p>(A) Calcineurin activity in VSMCs following a 24-h pretreatment with AGS-IV (0.10∼1.02 µM), while cyclosporin A (CsA) was used as positive control. (B) PE-induced calcineurin activation in VSMCs following a 24-h pretreatment with AGS-IV (0.10∼1.02 µM). (C) ACE activity in HUVECs following a 20-min pretreatment AGS-IV (0.10∼1.02 µM), while enalapril maleate (EM) was used as positive control. (D) AGS-IV (1.02 µM) inhibited LPS-induced activation of JNK for at least 20 min. JNK phosphorylation levels were determined by Western blot, analyzed by Quantity One™ software (BioRad Inc.) and normalized to β-actin.</p

Network analysis of the efficacy of AGS-IV on myocardial injuries.

<p>(A) Constructed minimum protein-protein interaction network between targets of AGS-IV. Red and green nodes denote putative targets of AGS-IV. Red nodes have been reported in the literature as drug targets or potential targets for CVD therapy; green nodes are predicted by the INVDOCK but with no literature support. Grey nodes are proteins that link the targets together. The network was decomposed into topological compact modules, labeled with numbers, by a simulated annealing algorithm. (B) Drug-target network for cardiovascular drugs related to AGS-IV. Circles and triangles represent drugs and targets, respectively. AGS-IV and its putative direct targets are highlighted in red. Nodes for other drugs are colored according to their ATC codes at the 4^th level (ATC codes are codes in the Anatomical Therapeutic Chemical (ATC) Classification System used for the classification of drugs, which is controlled by the WHO Collaborating Centre for Drug Statistics Methodology (WHOCC). Drugs are classified into groups at 5 different levels in this coding system, in which the 4^th level of the code indicates the chemical/therapeutic/pharmacological subgroup). (C) Target-pathway network for putative targets of AGS-IV. Circles and squares represent targets and pathways, respectively. Target nodes are colored according to Drugbank drug classes. Known targets of approved CVD drugs are highlighted in red. (D) Pathway associated with myocardial ischemia injury. This pathway was constructed by integrating pathways reported to be involved in the process of myocardial ischemia injury, including the calcium signaling, MAPK, JAK-STAT and NF-KB pathway. Red and pink boxes indicate putative target proteins of AGS-IV; targets in red were experimentally validated in this study.</p

MLIF protects SH-SY5Y cells against apoptosis in an eEF1A2-dependent manner.

<p>OGD-exposed SH-SY5Y cells transfected with eEF1A2 siRNA or negative control siRNA (NC) were treated with MLIF (0.1 μg/ml). Cell survival was measured using MTT assay (A). Representative annexin V/PI labeling, assessed by flow cytometry, was used to analyze the ratio of apoptotic SH-SY5Y cells (B, D). Hoechst 33258 staining was used to evaluate the nuclear morphology of SH-SY5Y cells (C, E). Data are expressed as the mean ± SEM. Results were analyzed using one-way ANOVA; n = 3. **<i>P</i>< 0.01 or *<i>P</i>< 0.05, OGD group <i>vs</i>. control group or MLIF group; ^##<i>P</i> < 0.01 or ^#<i>P</i>< 0.05, eEF1A2 siRNA group <i>vs</i>. NC group.</p

eEF1A2 was identified as the binding protein of MLIF in SH-SY5Y cells.

<p>Pull-down assays were carried out using biotin-conjugated MLIF (bio-MLIF) and cell lysates. Binding proteins were washed and separated by SDS-PAGE (A). The binding protein was analyzed by MALDI-TOF MS after in-gel digestion, and found to be eEF1A2 (B). Western blotting with anti-eEF1A2 antibody confirmed the identity of the binding protein (C). Confocal microscopy revealed co-localization of FITC-labeled MLIF (green) and eEF1A2 (labeled with rabbit anti-eEF1A2; red) in SH-SY5Y cells (D).</p