Inhibitors of histone deacetylases (HDACi) have shown promising effects in preclinical applications for the treatment of many diseases. Confusedly though, the effects of the HDACi trichostatin A (TSA) on angiogenesis are variable among different diseases. This study investigated the direct effects of TSA on endothelial cells, which plays essential roles in angiogenesis and the underlying molecular events. TSA reduced the viability of human umbilical vein endothelial cells (HUVECs), in which proliferation-related genes including BIRC5, CKS1B, and NDC80 were found to be involved. Furthermore, signal transducer and activator of transcription 5 A (STAT5A) was demonstrated to be reduced by TSA and to mediate TSA-induced downregulation of BIRC5, CKS1B, and NDC80 and HUVEC proliferation. Mechanistically, data showed that STAT5A directly bound to the promoters of BIRC5, CKS1B, and NDC80 and activated their transcription through special DNA sequence sites. Finally, the TSA-STAT5A-BIRC5, CKS1B, and NDC80 axis also worked in a cancerous endothelial cell angiogenesis model. The results of this study revealed novel mechanisms underlying the effects of TSA on endothelial cells and provided insights for angiogenesis-associated diseases.

PI3K pathway activation is frequently observed in triple negative breast cancer (TNBC). However, single agent PI3K inhibitors have shown limited anti-tumor activity. To investigate biomarkers of response and resistance mechanisms, we tested 17 TNBC patient-derived xenograft (PDX) models representing diverse genomic backgrounds and varying degrees of PI3K pathway signaling activities for their tumor growth response to the pan-PI3K inhibitor, BKM120. Baseline and post-treatment PDX tumors were subjected to reverse phase protein array (RPPA) to identify protein markers associated with tumor growth response. While BKM120 consistently reduced PI3K pathway activity, as demonstrated by reduced levels of phosphorylated AKT, percentage tumor growth inhibition (%TGI) ranged from 35% in the least sensitive to 84% in the most sensitive model. Several biomarkers showed significant association with resistance, including elevated baseline levels of growth factor receptors (EGFR, pHER3 Y1197), PI3Kp85 regulatory subunit, anti-apoptotic protein BclXL, EMT (Vimentin, MMP9, IntegrinaV), NFKB pathway (IkappaB, RANKL), and intracellular signaling molecules including Caveolin, CBP, and KLF4, as well as treatment-induced increases in the levels of phosphorylated forms of Aurora kinases. Interestingly, increased AKT phosphorylation or PTEN loss at baseline were not significantly correlated to %TGI. These results provide important insights into biomarker development for PI3K inhibitors in TNBC.

STS1 and STS2, as the protein phosphatases that dephosphorylate FLT3 and cKIT, negatively regulate the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs). To obtain the small molecule inhibitors of STS1/STS2 phosphatase activity used to expand HSPCs both in vitro and in vivo, we establish an in vitro phosphatase assay using the recombinant proteins of the STS1/STS2 histidine phosphatase (HP) domain, by which we screened out baicalein (BC) as one of the effective inhibitors targeting STS1 and STS2. Then, we further demonstrate the direct binding of BC with STS1/STS2 using molecular docking and capillary electrophoresis and verify that BC can restore the phosphorylation of FLT3 and cKIT from STS1/STS2 inhibition. In a short-term in vitro culture, BC promotes profound expansion and enhances the colony-forming capacity of both human and mouse HSPCs along with the elevation of phospho-FLT3 and phospho-cKIT levels. Likewise, in vivo administration with BC significantly increases the proportions of short-term hematopoietic stem cells (ST-HSCs), multipotent progenitors (MPPs) and especially long-term HSCs (LT-HSCs) in healthy mouse bone marrow and increases the numbers of colony-forming units (CFU) formed by HSPCs as well. More importantly, pre-administration of BC significantly enhances the survival of mice with lethal 5-fluorouracil (5-FU) injection due to the alleviation of 5-FU-induced myelosuppression, as evidenced by the recovery of bone marrow histologic injury, the increased proportions of LT-HSCs, ST-HSCs and MPPs, and enhanced colony-forming capacity. Collectively, our study not only suggests BC as one of the small molecule candidates to stimulate HSPC expansion both in vitro and in vivo when needed in either physiologic or pathologic conditions, but also supports STS1/STS2 as potential therapeutic drug targets for HSPC expansion and hematopoietic injury recovery.

Protein kinase B (AKT) signaling frequently is deregulated in human cancers and plays an important role in nasopharyngeal carcinoma (NPC). This preclinical study investigated the effect of MK-2206, a potent allosteric AKT inhibitor, on human NPC cells in vitro and in vivo.

We aim to investigate the role of THAP11 (thanatos-associated protein11) in gastric cancer and its regulation mechanisms. THAP11 expression was analyzed in 51 pairs of GC tissues and the corresponding paracancerous tissues by qRT-PCR and Western blot. After THAP11 was overexpressed or knocked-down, cell proliferation, cell cycle, and apoptosis were detected in MKN-45 cells. We found that THAP11 was significantly downregulated in GC tissues and GC cell lines. Functionally, THAP11 overexpression markedly inhibited cell growth, induced G1/G0 cell-cycle arrest, and promoted cell apoptosis of MKN-45 cells, while silencing of THAP11 led to increased cell growth, increased DNA synthesis, and inhibited apoptosis. In addition, THAP11 negatively regulated the expression of c-Myc, decreased cyclinD1 protein, and increased p27 and p21 protein levels. We also found cell growth suppression induced by THAP11 was rescued by c-Myc overexpression, further confirming that THAP11 suppresses gastric cancer cell growth via the c-Myc pathway. THAP11 acts as a cell growth suppressor and exerts its role possibly through negatively regulating c-Myc pathway in gastric cancer.

Carbonyl cyanide p-nitrophenylhydrazone (2e) displayed a lone or synergistic efficacy against MRSA (RSC Adv., 2020, 10, 17854). In this work, the synergistic mechanism of 2e with ofloxacin was studied. MRSA2858 had potential for biofilm formation, and the value of MBEC of 2e alone was 0.78-1.56 μM, while that of 2e + ofloxacin was 0.39-0.78 μM. 2e combined with ofloxacin showed a synergistic anti-biofilm effect against MRSA. Efflux pump inhibitor 2e can better bind to NorA protein. After MRSA2858 was treated with 2e of 1/2MIC (0.78 μM) and ofloxacin of 1/8MIC (0.097 μM), the transcript levels of efflux genes (norA) and quorum-sensing (QS) regulatory genes (agrA, sarA, icaA, hla) were substantially down-regulated, and alpha-hemolysin (Hla) was inhibited by 99.15%. 2e combined with ofloxacin was more effective than 2e alone in reducing bacterial load in vivo. All in all, efflux pump inhibitor 2e enhanced the bactericidal activities of antibiotics through regulating the gene expression of NorA and QS system.

Ki20227, a selective inhibitor of colony-stimulating factor 1 receptor (CSF1R), has been suggested to regulate microglia inflammatory function and neuronal synaptic plasticity. Triptolide (TP) pretreatment has neuroprotective effects through its anti-inflammatory and antiapoptotic features in ischemic stroke mice. However, the underlying mechanism and pathway are presently unclear. We thus investigated the association between neuroprotective effects of combined TP and Ki20227 and BDNF-Akt and autophagy pathways. Ki20227 was administrated for 7 days, and TP was administered once 24 hours prior to building the ischemic stroke model in C57BL/6 mice. Behavioral tests, Golgi staining, immunofluorescence, and western blot analyses were employed to examine neuroprotective effects of TP and Ki20227. TP and Ki20227 pretreatments improved the neurobehavioral function in stroke mice. Synaptic protein expressions and density of dendritic spine density were upregulated in Ki20227 and TP pretreated stroke mice. Further, optimized integration of TP and Ki20227 pretreatments upregulated the NeuN expression and downregulated Iba1 expression after stroke. In addition, both TP and Ki20227 pretreatments significantly upregulated BDNF, p-Akt/Akt, and Erk1/2 protein expressions and autophagy related proteins (LC3II/I, Atg5, and p62), indicating the activation of BDNF and autophagic pathways. Optimized integration of TP and Ki20227 can improve cerebral ischemia by inhibiting CSF1R signal and trigger autophagy and BDNF-Akt signaling pathways to increase dendritic spine density and synaptic protein expressions, which in turn enhances neurobehavioral function.

Chronic obstructive pulmonary disease (COPD), the third leading cause of death worldwide, is influenced by genetic factors. The genetic signal rs10516526 in the glutathione S-transferase C-terminal domain containing (GSTCD) gene is a highly significant and reproducible signal associated with lung function and COPD on chromosome 4q24. In this study, comprehensive bioinformatics analyses and experimental verifications were detailly implemented to explore the regulation mechanism of rs10516526 and GSTCD in COPD. The results suggested that low expression of GSTCD was associated with COPD (p = 0.010). And C-Jun and CREB1 transcription factors were found to be essential for the regulation of GSTCD by rs80245547 and rs72673891. Moreover, rs80245547T and rs72673891G had a stronger binding ability to these transcription factors, which may promote the allele-specific long-range enhancer-promoter interactions on GSTCD, thus making COPD less susceptible. Our study provides a new insight into the relationship between rs10516526, GSTCD, and COPD.

Our previous studies have demonstrated that activation of group I metabotropic glutamate receptors downregulated Kir channels in chronic ocular hypertension (COH) rats, thus contributing to Müller cell gliosis, characterized by upregulated expression of glial fibrillary acidic protein (GFAP). In the present study, we explored possible signaling pathways linking Kir channel inhibition and GFAP upregulation. In normal retinas, intravitreal injection of BaCl2 significantly increased GFAP expression in Müller cells, which was eliminated by co-injecting mitogen-activated protein kinase (MAPK) inhibitor U0126. The protein levels of phosphorylated extracellular signal-regulated protein kinase1/2 (p-ERK1/2) and its upstream regulator, p-MEK, were significantly increased, while the levels of phosphorylated c-Jun N-terminal kinase (p-JNK) and p38 kinase (p-p38) remained unchanged. Furthermore, the protein levels of phosphorylated cAMP response element binding protein (p-CREB) and c-fos were also increased, which were blocked by co-injecting ERK inhibitor FR180204. In purified cultured rat Müller cells, BaCl2 treatment induced similar changes in these protein levels apart from p-p38 levels and the p-p38:p38 ratio showing significant upregulation. Moreover, intravitreal injection of U0126 eliminated the upregulated GFAP expression in COH retinas. Together, these results suggest that Kir channel inhibition-induced Müller cell gliosis is mediated by the MEK-ERK/p38-CREB/c-fos signaling pathway.

FMRP, an RNA-binding protein, has previously shown to be involved in regulation of circadian rhythms in flies and mice. However, the molecular mechanism remains elusive. Here we demonstrate that core circadian component Per1 mRNA was a target of FMRP and the association leads to reduced PER1 expression. In Fmr1 KO mice, the oscillation of PER1 protein expression was significantly affected in a temporal and tissue-dependent pattern when compared to WT mice. Our work thus identified Per1 mRNA as a novel target of FMRP and suggested a potential role of FMRP in regulation of circadian function.

Radiotherapy is widely used in treating cervical cancer patients, however, radioresistance unavoidably occurs and seriously affects the treatment effect. It is well known that hypoxia plays an important role in promoting radioresistance in tumor microenvironment, yet our understanding of the effect of small extracellular vesicles miRNA on cervical cancer radiosensitivity in hypoxic environment is still limited.

Excessive inflammatory responses induced upon SARS-CoV-2 infection are associated with severe symptoms of COVID-19. Inflammasomes activated in response to SARS-CoV-2 infection are also associated with COVID-19 severity. Here, we show a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation. N protein facilitates maturation of proinflammatory cytokines and induces proinflammatory responses in cultured cells and mice. Mechanistically, N protein interacts directly with NLRP3 protein, promotes the binding of NLRP3 with ASC, and facilitates NLRP3 inflammasome assembly. More importantly, N protein aggravates lung injury, accelerates death in sepsis and acute inflammation mouse models, and promotes IL-1β and IL-6 activation in mice. Notably, N-induced lung injury and cytokine production are blocked by MCC950 (a specific inhibitor of NLRP3) and Ac-YVAD-cmk (an inhibitor of caspase-1). Therefore, this study reveals a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.

Mutations in the Contactin-associated protein-like 2 (CNTNAP2) gene are associated with autism spectrum disorder (ASD), and ectodomain shedding of the CNTNAP2 protein plays a role in its function. However, key enzymes involved in the C-terminal cleavage of CNTNAP2 remain largely unknown, and the effect of ASD-associated mutations on this process and its role in ASD pathogenesis remain elusive. In this report we showed that CNTNAP2 undergoes sequential cleavages by furin, ADAM10/17-dependent α-secretase and presenilin-dependent γ-secretase. We identified that the cleavage sites of ADAM10 and ADAM17 in CNTNAP2 locate at its C-terminal residue I79 and L96, and the main α-cleavage product C79 by ADAM10 is required for the subsequent γ-secretase cleavage to generate CNTNAP2 intracellular domain (CICD). ASD-associated CNTNAP2 mutations impair the α-cleavage to generate C79, and the inhibition leads to ASD-like repetitive and social behavior abnormalities in the Cntnap2-I1254T knock-in mice. Finally, exogenous expression of C79 improves autism-like phenotypes in the Cntnap2-I1254T knock-in and Cntnap2-/- knockout mice. This data demonstrates that the α-secretase is essential for CNTNAP2 processing and its function. Our study indicates that inhibition of the cleavage by pathogenic mutations underlies ASD pathogenesis, and upregulation of its C-terminal fragments could have therapeutical potentials for ASD treatment.

Cementum regeneration, as one of the most difficult challenges of periodontal regeneration, is influenced by inflammatory factors. Inflammation may hamper or promote periodontal tissue repair under different circumstances, as it is found to do in dentin-pulp complex and bone tissue. Our team demonstrated that YAP promotes mineralization of OCCM, a cementoblast cell line. However, the effect of YAP on its mineralization under inflammatory microenvironment is unclear. In this study, cementogenesis in vitro was up-regulated after transient TNF-α treatment for 30 minutes. YAP expression also was increased by TNF-α treatment. YAP overexpression promoted OCCM mineralization after the cells were transiently treated with TNF-α because YAP overexpression inhibited NF-κB pathway activity, while YAP knockdown elevated it. The inhibited mineralization potential and activated NF-κB pathway activity by YAP knockdown also were partly rescued by the application of the NF-κB inhibitor Bay 11-7082. These results demonstrated that YAP plays a positive role in the mineralization of TNF-α transiently treated cementoblast, partly by inhibiting the NF-κB pathway activity.

Our study aimed to investigate whether microRNA-449 (miR-449) plays a key role in regulating the migration and invasion of breast cancer cells via targeting tumor protein D52 (TPD52). The results of the qRT-PCR and Western blotting showed that, in comparison with normal breast tissues and cells, miR-449 was significantly downregulated in breast cancer tissues and cells, while TPD52 was markedly upregulated. After transfection with an miR-449 inhibitor, suppression of miR-449 significantly promoted cell migration and invasion. Also, when miR-449 was overexpressed by transfection with miR-449 mimics, E-cadherin expression significantly increased, and the expression of N-cadherin and vimentin were markedly decreased, whereas the opposite effects were obtained when miR-449 was suppressed by transfection with an miR-449 inhibitor. TPD52 was also confirmed as the direct target of miR-449 via luciferase reporter analysis. Knockdown of TPD52 significantly alleviated the effects of miR-449 overexpression on cell migration and invasion, as well as the expression of E-cadherin, N-cadherin, and vimentin. Our results indicate that downregulation of miR-449 may promote the migration and invasion of breast cancer cells by targeting TPD52. miR-449 may serve as a potential target in the therapy of breast cancer.

Retinitis pigmentosa (RP) is the most common form of inherited retinal degenerative diseases. X-linked RP accounts for nearly 15% of all RP cases. In this study, we identified a novel RP2 missense mutation Q158P in a Chinese XLRP family. The RP2 Q158P mutation located in the RP2 TBCC domain and obviously destabilized RP2 protein in ARPE-19 cells. The proteasome inhibitor MG132 could restore the RP2 Q158P protein levels. Meanwhile, lower doses of bortezomib and carfilzomib, another two proteasome inhibitors that have been approved in multiple myeloma clinical therapy, also could rescue the RP2 Q158P protein levels. The ubiquitination of RP2 Q158P protein obviously increased when compared with wild type RP2 protein. Our findings broadened the spectrum of RP2 mutations and may contribute a better understanding of the molecular mechanism of XLRP.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has quickly spread worldwide and has affected more than 10 million individuals. A typical feature of COVID-19 is the suppression of type I and III interferon (IFN)-mediated antiviral immunity. However, the molecular mechanism by which SARS-CoV-2 evades antiviral immunity remains elusive. Here, we reported that the SARS-CoV-2 membrane (M) protein inhibits the production of type I and III IFNs induced by the cytosolic dsRNA-sensing pathway mediated by RIG-I/MDA-5-MAVS signaling. In addition, the SARS-CoV-2 M protein suppresses type I and III IFN induction stimulated by SeV infection or poly (I:C) transfection. Mechanistically, the SARS-CoV-2 M protein interacts with RIG-I, MAVS, and TBK1, thus preventing the formation of the multiprotein complex containing RIG-I, MAVS, TRAF3, and TBK1 and subsequently impeding the phosphorylation, nuclear translocation, and activation of IRF3. Consequently, ectopic expression of the SARS-CoV-2 M protein facilitates the replication of vesicular stomatitis virus. Taken together, these results indicate that the SARS-CoV-2 M protein antagonizes type I and III IFN production by targeting RIG-I/MDA-5 signaling, which subsequently attenuates antiviral immunity and enhances viral replication. This study provides insight into the interpretation of SARS-CoV-2-induced antiviral immune suppression and illuminates the pathogenic mechanism of COVID-19.

Everolimus (RAD001) is a mTOR inhibitor and is widely used for the treatment of gastric cancer (GC). Evidence suggests that Rhein has anticancer effect on GC. But the synergistic effect and mechanism of RAD001 and Rhein combination on GC is not clear. The current study aims to clarify the combination of RAD001 and Rhein in GC treatment. We found Rhein dose-dependently repressed MGC-803 cell viability (50% inhibition concentration (IC50) value = 94.26 μM). Rhein (80 μM) significantly suppressed GC cell proliferation and invasion. RAD001 dose-dependently repressed MGC-803 cells viability (IC50 value = 45.41 nM). The combination of Rhein and RAD001 repressed MGC-803 cells viability, invasion, and proliferation compared to the administration of Rhein or RAD001 alone. Protein levels of epithelial-mesenchymal transition (EMT)-related molecules E-cadherin, N-cadherin and Vimentin expressions were significantly affected by the combination of Rhein and RAD001. The combination of Rhein and RAD001 significantly facilitated cell apoptosis and up-regulated expressions of cell apoptosis and cycle-related protein p53, cyclin-dependent kinase 4 (CDK4) and cyclin D1 compared to the administration of Rhein or RAD001 alone. Moreover, the combination of Rhein and RAD001 repressed the expressions of phosphorylation-phosphoinositide-3-kinase (p-PI3K), p-protein kinase B (p-AKT) and p-mammalian target of rapamycin (p-mTOR). Finally, the combination of RAD001 and Rhein significantly decreased tumor weight and volume, suppressed the expressions of p-PI3K, p-Akt and p-mTOR, and repressed cell proliferation marker Ki-67 expression, which exerted synergistic cancer prevention in GC in vivo. Overall, the combination of Rhein and RAD001 exert synergistic cancer prevention in GC via PI3K/Akt/mTOR pathway.

Esophageal squamous cell carcinoma (ESCC) is a frequently seen esophageal tumor type in China. Activation of signaling proteins and relevant molecular mechanisms in ESCC are partially explored, impairing the antitumor efficiency of targeted therapy in ESCC treatment. Tumor-associated macrophages (TAMs)-released C-C motif chemokine 22 (CCL22) can activate intratumoral focal adhesion kinase (FAK), thus promoting the progression of ESCC. Here, we demonstrated that highly secreted CCL22 by TAMs (CCL22-positive TAMs) induced ESCC cell stemness and invasion through facilitating transcriptional activity of intratumoral glioma-associated oncogene 1 (Gli1), a downstream effector for Hedgehog (HH) pathway. Mechanistically, FAK-activated protein kinase B (AKT) mediated Gli1 phosphorylation at its Ser112/Thr115/Ser116 sites and released Gli1 from suppressor of fused homolog, the endogenous inhibitor of Gli1 to activate downstream stemness-associated factors, such as SRY-box transcription factor 2 (SOX2), Nanog homeobox (Nanog), or POU class 5 homeobox (OCT4). Furthermore, inhibition of FAK activity by VS-4718, the FAK inhibitor, enhanced antitumor effect of GDC-0449, the HH inhibitor, both in xenografted models and in vitro assays. Clinically, CCL22/Gli1 axis is used to evaluate ESCC prognosis. Overall, our study establishes the communication of FAK with HH pathway and offers the novel mechanism related to Gli1 activation independent of Smoothened as well as the rationale for the anti-ESCC combination treatment.

Resistance to tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) of cancer cell remains a key obstacle for clinical cancer therapies. To overcome TRAIL resistance, this study identifies curcumol as a novel safe sensitizer from a food-source compound library, which exhibits synergistic lethal effects in combination with TRAIL on non-small cell lung cancer (NSCLC). SILAC-based cellular thermal shift profiling identifies NRH:quinone oxidoreductase 2 (NQO2) as the key target of curcumol. Mechanistically, curcumol directly targets NQO2 to cause reactive oxygen species (ROS) generation, which triggers endoplasmic reticulum (ER) stress-C/EBP homologous protein (CHOP) death receptor (DR5) signaling, sensitizing NSCLC cell to TRAIL-induced apoptosis. Molecular docking analysis and surface plasmon resonance assay demonstrate that Phe178 in NQO2 is a critical site for curcumol binding. Mutation of Phe178 completely abolishes the function of NQO2 and augments the TRAIL sensitization. This study characterizes the functional role of NQO2 in TRAIL resistance and the sensitizing function of curcumol by directly targeting NQO2, highlighting the potential of using curcumol as an NQO2 inhibitor for clinical treatment of TRAIL-resistant cancers.