Paclitaxel is generally used to treat cancers in clinic as an inhibitor of cell division. However, the acquired resistance in tumours limits its clinical efficacy. Therefore, the aim of this study was to detect whether co-treatment with lentinan enhanced the anti-cancer effects of paclitaxel in A549 cells. We found that the combination of paclitaxel and lentinan resulted in a significantly stronger inhibition on A549 cell proliferation than paclitaxel treatment alone. Co-treatment with paclitaxel and lentinan enhanced cell apoptosis rate by inducing caspase-3 activation. Furthermore, co-treatment with paclitaxel and lentinan significantly triggered reactive oxygen species (ROS) production, and increased thioredoxin-interacting protein (TXNIP) expression. Moreover, co-treatment with paclitaxel and lentinan enhanced TXNIP-NLRP3 interaction, and activated NLRP3 inflammasome whereat interleukin-1β levels were increased and cell apoptosis was induced. In addition, combination of paclitaxel and lentinan could activate apoptosis signal regulating kinase-1 (ASK1)/p38 mitogen-activated protein kinase (MAPK) signal which also contributed to cell apoptosis. Taken together, co-treatment with paclitaxel and lentinan exerts synergistic apoptotic effects in A549 cells through inducing ROS production, and activating NLRP3 inflammasome and ASK1/p38 MAPK signal pathway.

Forkhead box Q1 (FoxQ1) is a member of the forkhead transcription factor family. High expression of FoxQ1 has been associated with several cancers including non-small cell lung cancer (NSCLC), but its role in the development of NSCLC is not clear. In this study, we investigated the effect of FoxQ1 up-regulated and down-regulated in vitro and in vivo, and the role of FoxQ1 in regulating epithelial-mesenchymal transition (EMT) in NSCLC, providing evidence that FoxQ1 could be a potential therapeutic target in NSCLC. NSCLC cells with silenced FoxQ1 had decreased cell proliferation, migration and invasion in cell culture and delayed growth of xenograft tumors in mice compared with corresponding control cells. The NSCLC cells downregulated for FoxQ1 induced the expression of apoptosis-associated proteins and reduction of anti-apoptotic protein expression. Downregulation of FoxQ1 promoted the expression of epithelial markers and decreased several mesenchymal markers in vitro and in vivo. In addition, FoxQ1 was associated with resistance to conventional chemotherapeutic agents. In contrast, FoxQ1 overexpressed elicited converse effects on these phenotypes in vitro and in vivo. Our findings define a key role for FoxQ1 in regulating EMT and increasing chemosensitivity in NSCLC.

MicroRNAs (miRNAs) are short regulatory RNAs that negatively modulate protein expression at the post-transcriptional level. Additionally, they have been associated with the pathogenesis of a number of types of cancer. In the current study, two target sites for miR-150 were determined within the 3'-untranslated region of p27Kip1 (hereafter referred to as p27) mRNA, and it was determined that ectopic overexpression of miR-150 led directly to p27 downregulation in cancer cells. These findings indicate that miR-150 may be a novel regulator of p27 expression. In the databases of the University of California, Santa Cruz (UCSC) and Match online, two common transcription factors were identified for miR-150 and p27: Cooperates with myogenic proteins 1 (COMP1) and hepatocyte nuclear factor-4 (HNF-4). Using the Database for Annotation, Visualization, and Integrated Discovery (DAVID), it was determined that p27 is involved in pathways regulated by the target genes of miR-150. Therefore, these results suggest that there may be a regulatory loop between COMP1 and HNF-4-miR-150-p27. Additional functional studies are required to understand the molecular basis for the formation of this circuit loop, and provide an insight into the development of innovative therapies targeting specific tumor markers.

Heat shock protein (Hsp) and its cognate protein (Hsc) play important roles in helping insects survive extreme temperatures. However, high level of Hsp expression usually brings negative physiological effects on organisms. The mechanism of this trade-off is unclear. In this study, a lepidopteran insect, the common cutworm Spodoptera litura, was stressed at different temperatures, and the impact on both thermotolerance and fecundity was examined. The mRNA levels of four Hsp/Hscs (Hsp90, Hsc90, Hsp70 and Hsc70) and two ecdysone receptors (EcRs, EcRA and EcRB1) in different stresses and during the larval-pupal metamorphosis were determined. The results revealed that the pre-acclamation at mild stress increased the thermotolerance but decreased the egg production in adults. During the stress process, the mRNA levels of all the Hsp/Hsc and ecdysone receptor genes were significantly up-regulated. The two Hsp/Hsc70s and EcRs revealed consistent expression profiles with each other during the larval-pupal metamorphosis. Co-immunoprecipitation and Western blotting analysis indicated that Hsp/Hsc70 interacted with EcRs. RNAi of Hsc70 decreased the mRNA levels of two 20E-induced genes such as E74B and E75. Hsp70 transferred from the cytoplasm to nucleus in response to cold stress. These data together suggest that Hsp/Hsc70 might be involved in the regulation of 20E signaling, and the protein-protein interaction between Hsp/Hsc70 and EcRs probably act as a bridge mediating the trade-off between high thermotolerance and physiological defects.

In the liver, bone morphogenetic protein 6 (BMP-6) maintains balanced iron metabolism. However, the mechanism that underlies greater BMP-6 expression in hepatocellular carcinoma (HCC) tissue than adjacent non-cancerous tissue is unclear. This study sought to investigate the epigenetic mechanisms of BMP-6 expression by analysing the relationship between the DNA methylation status of BMP-6 and the expression of BMP-6.

Colorectal carcinoma (CRC) is a major cause of cancer mortality. The aberrant expression of several microRNAs is associated with CRC progression; however, the molecular mechanisms underlying this phenomenon are unclear.

Bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive cytokine that plays a critical role in bone regeneration and repair. However, its distribution and side effects are major barriers to its success as therapeutic treatment. The improvement of therapy using collagen delivery matrices has been reported. To investigate a delivery system on postero-lateral spinal fusion, both engineered human BMP-2 with a collagen binding domain (CBD-BMP-2) and collagen scaffolds were developed and their combination was implanted into Sprague-Dawley (SD) rats to study Lumbar 4-5 (L4-L5) posterolateral spine fusion. We divided SD rats into three groups, the sham group (G1, n = 20), the collagen scaffold-treated group (G2, n = 20) and the BMP-2-loaded collagen scaffolds group (G3, n = 20). 16 weeks after surgery, the spines of the rats were evaluated by X-radiographs, high-resolution micro-computed tomography (micro-CT), manual palpation and hematoxylin and eosin (H&E) staining. The results showed that spine L4-L5 fusions occurred in G2(40%) and G3(100%) group, while results from the sham group were inconsistent. Moreover, G3 had better results than G2, including higher fusion efficiency (X score, G2 = 2.4±0.163, G3 = 3.0±0, p<0.05), higher bone mineral density (BMD, G2: 0.3337±0.0025g/cm3, G3: 0.4353±0.0234g/cm3. p<0.05) and more bone trabecular formation. The results demonstrated that with site-specific collagen binding domain, a dose of BMP-2 as low as 0.02mg CBD-BMP-2/cm3 collagen scaffold could enhance the posterolateral intertransverse process fusion in rats. It suggested that combination delivery could be an alternative in spine fusion with dramatically decreased side effects caused by high dose of BMP-2.

The androgen receptor (AR) is frequently expressed in breast cancer; however, its prognostic value remains unclear. AR expression in breast cancer has been associated with improved outcomes in estrogen receptor (ER)‑positive breast cancer compared with ER‑negative disease. Eliminating AR function in breast cancer is critically important for breast cancer progression. However, the mechanism underlying AR regulation remains poorly understood. The study of microRNAs (miRNAs) has provided important insights into the pathogenesis of hormone‑dependent cancer. To determine whether miRNAs function in the AR regulation of breast cancer, the present study performed miRNA expression profiling in AR‑positive and ‑negative breast cancer cell lines. A total of 153 miRNAs were differentially expressed in AR‑positive compared with AR‑negative breast cancer cells; 52 were upregulated and 101 were downregulated. A number of these have been extensively associated with breast cancer cell functions, including proliferation, invasion and drug‑resistance. Furthermore, through pathway enrichment analysis, signaling pathways associated with the prediction targets of the miRNAs were characterized, including the vascular endothelial growth factor and mammalian target of rapamycin signaling pathways. In conclusion, the results of the present study indicated that the expression of miRNAs may be involved in the mechanism underlying AR regulation of breast cancer, and may improve understanding of the role of AR in breast cancer.

Protein Kinase D3 (PRKD3) functions as an important oncogenic driver in invasive breast cancer, which is the leading cause of women mortality. However, PRKD3 regulating network is largely unknown. In this study, we systematically explored PRKD3 regulating networks via investigating phosphoproteome, interactome and transcriptome to uncover the molecular mechanism of PRKD3 in invasive breast cancer. Using iTRAQ, 270 proteins were identified as PRKD3 regulated phosphoproteins from 4619 phosphosites matching 3666 phosphopeptides from 2016 phosphoproteins with p-value <0.005. Transcriptome analysis using affymetrix microarray identified 45 PRKD3 regulated genes, in which 20 genes were upregulated and 25 genes were downregulated with p-value <0.005 upon silencing PRKD3. Using Co-IP in combination of MS identification, 606 proteins were identified to be PRKD3 interacting proteins from 2659 peptides. Further network analysis of PRKD3 regulated phosphoproteins, interacting proteins and regulated genes, reveals 19 hub nodes, including ELAVL1, UBC and BRCA1. UBC was recognized as the most common hub node in PRKD3 regulating networks. The enriched pathway analysis reveals that PRKD3 regulates pathways contributing to multiple cancer related events, including cell cycle, migration and others. Enrichment of cell cycle and cell mobility related pathways across PRKD3 networks, explained the observations that depletion of oncogenic PRKD3 led to alternation of cell cycle and decrease of cell migration ability. Taken together, our current study provided valuable information on the roles as well as the molecular mechanisms of PRKD3 in invasive breast cancer.

Gallbladder cancer (GBC), highly aggressive form of cancer with an extremely poor prognosis, is the most common malignancy of the biliary tract. In this study, we investigated the effects of dioscin (DSN) on human GBC and the potential mechanisms underlying these effects. The results showed that DSN significantly inhibited GBC cell proliferation and migration. Moreover, DSN induced GBC cell apoptosis via mitochondrial dependent apoptotic signalling. Reactive oxygen species (ROS) and glutathione (GSH) levels were measured, and ROS scavengers completely inhibited DSN-induced apoptosis and migration, indicating that ROS play an essential role in GBC progression. Western blot analysis showed that AKT activity was significantly downregulated after DSN treatment, and that inhibition/ectopic expression of AKT enhanced/abolished DSN-induced apoptosis but not migration. Furthermore, we confirmed the relationship between ROS and the PI3K/AKT pathway and found that DSN induced apoptosis by regulating ROS-mediated PI3K/AKT signaling. Taken together, these findings indicate that DSN induces GBC apoptosis through inhibiting ROS-mediated PI3K/AKT signalling.

The recent Middle East respiratory syndrome coronavirus (MERS-CoV), Ebola and Zika virus outbreaks exemplify the continued threat of (re-)emerging viruses to human health, and our inability to rapidly develop effective therapeutic countermeasures. Many viruses, including MERS-CoV and the Crimean-Congo hemorrhagic fever virus (CCHFV) encode deubiquitinating (DUB) enzymes that are critical for viral replication and pathogenicity. They bind and remove ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) from cellular proteins to suppress host antiviral innate immune responses. A variety of viral DUBs (vDUBs), including the MERS-CoV papain-like protease, are responsible for cleaving the viral replicase polyproteins during replication, and are thereby critical components of the viral replication cycle. Together, this makes vDUBs highly attractive antiviral drug targets. However, structural similarity between the catalytic cores of vDUBs and human DUBs complicates the development of selective small molecule vDUB inhibitors. We have thus developed an alternative strategy to target the vDUB activity through a rational protein design approach. Here, we report the use of phage-displayed ubiquitin variant (UbV) libraries to rapidly identify potent and highly selective protein-based inhibitors targeting the DUB domains of MERS-CoV and CCHFV. UbVs bound the vDUBs with high affinity and specificity to inhibit deubiquitination, deISGylation and in the case of MERS-CoV also viral replicative polyprotein processing. Co-crystallization studies further revealed critical molecular interactions between UbVs and MERS-CoV or CCHFV vDUBs, accounting for the observed binding specificity and high affinity. Finally, expression of UbVs during MERS-CoV infection reduced infectious progeny titers by more than four orders of magnitude, demonstrating the remarkable potency of UbVs as antiviral agents. Our results thereby establish a strategy to produce protein-based inhibitors that could protect against a diverse range of viruses by providing UbVs via mRNA or protein delivery technologies or through transgenic techniques.

STAT6 plays a prominent role in adaptive immunity by transducing signals from extracellular cytokines. We now show that STAT6 is required for innate immune signaling in response to virus infection. Viruses or cytoplasmic nucleic acids trigger STING (also named MITA/ERIS) to recruit STAT6 to the endoplasmic reticulum, leading to STAT6 phosphorylation on Ser(407) by TBK1 and Tyr(641), independent of JAKs. Phosphorylated STAT6 then dimerizes and translocates to the nucleus to induce specific target genes responsible for immune cell homing. Virus-induced STAT6 activation is detected in all cell-types tested, in contrast to the cell-type specific role of STAT6 in cytokine signaling, and Stat6(-/-) mice are susceptible to virus infection. Thus, STAT6 mediates immune signaling in response to both cytokines at the plasma membrane, and virus infection at the endoplasmic reticulum.

Small heat shock proteins (sHsps) are probably the most diverse in structure and function among the various superfamilies of stress proteins. To explore the diverse functions of insect sHsps, six sHsp cDNAs were cloned from the midgut cDNA library of Spodoptera litura, and a phylogenetic tree was constructed based on the conserved α-crystalline domains. The expression patterns in different developmental stages and tissues, as well as in response to both thermal and 20-hydroxyecdysone (20E) induction, were studied by real-time quantitative PCR. Based on sequence characteristics and phylogenetic relationships, the six SlHsps were classified into three independent groups: BmHsp20.4 like proteins (SlHsp19.7, 20.4, 20.7, 20.8), BmHsp26.6 like protein (SlHsp20), and BmHsp21.4 like protein (SlHsp21.4). All the SlHsps showed highest expression in the Malpighian tubules. The four BmHsp20.4 like protein genes were up-regulated by thermal stress and showed expression variation with development. SlHsp20 exhibited lower expression levels in both egg and larval stages than in pupal and adult stages. SlHsp21.4 retained a constant expression level during all life stages. The expression of both SlHsp20.4 and SlHsp20.8 was significantly up-regulated by 20E. These results indicate that sHsps play diverse functions in S. litura: the BmHsp20.4 like proteins are involved in both thermal adaptation and development; SlHsp20 does not respond to temperature stress but possibly plays a role in metamorphosis; SlHsp21.4 may have no direct relationship with either thermal response or development.

Bone marrow microenvironment (niche) plays essential roles in the fate of hematopoietic stem cells (HSCs). Intracellular and extracellular redox metabolic microenvironment is one of the critical factors for the maintenance of the niche. Cytochrome P450 reductase (CPR) is an obligate electron donor to all microsomal cytochrome P450 enzymes (P450 or CYP), and contributes to the redox metabolic process. However, its role in maintaining HSCs is unknown.

The herbicide 2,6-dichlorobenzonitril (DCBN) is a potent and tissue-specific toxicant to the olfactory mucosa (OM). The toxicity of DCBN is mediated by cytochrome P450 (P450)-catalyzed bioactivation; however, it is not known whether target-tissue metabolic activation is essential for toxicity. CYP2A5, expressed abundantly in both liver and OM, was previously found to be one of the P450 enzymes active in DCBN bioactivation in vitro. The aims of this study were to determine the role of CYP2A5 in DCBN toxicity in vivo, by comparing the extents of DCBN toxicity between Cyp2a5-null and wild-type (WT) mice, and to determine whether hepatic microsomal P450 enzymes (including CYP2A5) are essential for the DCBN toxicity, by comparing the extents of DCBN toxicity between liver-Cpr-null (LCN) mice, which have little P450 activity in hepatocytes, and WT mice. We show that the loss of CYP2A5 expression did not alter systemic clearance of DCBN (at 25 mg/kg); but it did inhibit DCBN-induced non-protein thiol depletion and cytotoxicity in the OM. Thus, CYP2A5 plays an essential role in mediating DCBN toxicity in the OM. In contrast to the results seen in the Cyp2a5-null mice, the rates of systemic DCBN clearance were substantially reduced, while the extents of DCBN-induced nasal toxicity were increased, rather than decreased, in the LCN mice, compared to WT mice. Therefore, hepatic P450 enzymes, although essential for DCBN clearance, are not necessary for DCBN-induced OM toxicity. Our findings form the basis for a mechanism-based approach to assessing the potential risks of DCBN nasal toxicity in humans.

The Wnt signaling pathway is a cellular communication pathway that plays critical roles in development and disease. A major class of Wnt signaling regulators is the Dickkopf (Dkk) family of secreted glycoproteins. Although the biological properties of Dickkopf 1 (Dkk1) and Dickkopf 2 (Dkk2) are well characterized, little is known about the function of the related Dickkopf 3 (Dkk3) protein in vivo or in cell lines. We recently demonstrated that Dkk3 transcripts are upregulated during photoreceptor death in a mouse model of retinal degeneration. In this study, we characterized the activity of Dkk3 in Wnt signaling and cell death.

CACNA1B (Cav2.2) encodes an N-type voltage-gated calcium channel (VGCC) ubiquitously expressed in brain and peripheral nervous system that is important for regulating neuropathic pain. Because intracellular calcium concentration is a key player in cell proliferation and apoptosis, VGCCs are implicated in tumorigenesis. Recent studies have identified CACNA1B (Cav2.2) being overexpressed in prostate and breast cancer tissues when compared to adjacent normal tissues; however, its role in non-small cell lung cancer (NSCLC) has not been investigated. In this study, we determined the mRNA and protein expression of CACNA1B (Cav2.2) in NSCLC tumorous and adjacent nontumorous tissues by quantitative reverse transcription PCR (qRT-PCR) and tissue microarray immunohistochemistry analysis (TMA-IHC), respectively. CACNA1B (Cav2.2) protein expressions in tumorous tissues were correlated with NSCLC patients' clinical characteristics and overall survival. CACNA1B (Cav2.2) mRNA and protein expression levels were higher in NSCLC tumorous tissues than in nontumorous tissues. High CACNA1B (Cav2.2) protein expression was associated with higher TNM stages, and CACNA1B (Cav2.2) protein expression is an independent prognostic marker in NSCLC. Based on our results, we conclude that CACNA1B (Cav2.2) plays a role in NSCLC development and progression. Elucidating the underlying mechanism may help design novel treatment by specifically targeting the calcium regulation pathway for NSCLC, a devastating disease with increasing incidence and mortality in China.

Stress-induced disturbance of the hypothalamic-pituitary-adrenal (HPA) axis is strongly implicated in incidence of mood disorders. A heightened neuroinflammatory response and oxidative stress play a fundamental role in the dysfunction of the HPA axis. We have previously demonstrated that iptakalim (Ipt), a new ATP-sensitive potassium (K-ATP) channel opener, could prevent oxidative injury and neuroinflammation against multiple stimuli-induced brain injury. The present study was to demonstrate the impacts of Ipt in stress-induced HPA axis disorder and depressive behavior. We employed 2 stress paradigms: 8 weeks of continuous restraint stress (chronic restraint stress, CRS) and 2h of restraint stress (acute restraint stress, ARS), to mimic both chronic stress and severe acute stress. Prolonged (4 weeks) and short-term (a single injection) Ipt treatment was administered 30min before each stress paradigm. We found that HPA axis was altered after stress, with different responses to CRS (lower ACTH and CORT, higher AVP, but normal CRH) and ARS (higher CRH, ACTH and CORT, but normal AVP). Both prolonged and short-term Ipt treatment normalized stress-induced HPA axis disorders and abnormal behaviors in mice. CRS and ARS up-regulated mRNA levels of inflammation-related molecules (TNFα, IL-1β, IL-6 and TLR4) and oxidative stress molecules (gp91phox, iNOS and Nrf2) in the mouse hypothalamus. Double immunofluorescence showed CRS and ARS increased microglia activation (CD11b and TNFα) and oxidative stress in neurons (NeuN and gp91phox), which were alleviated by Ipt. Therefore, the present study reveals that Ipt could prevent against stress-induced HPA axis disorders and depressive behavior by alleviating inflammation and oxidative stress in the hypothalamus.

Gastroparesis is a recognized complication of diabetes but its pathogenic mechanism incompletely understood. Our aim was to determine whether HIF-1α and VEGF are secreted from gastric tissue is a fundamental factor that drives diabetic gastroparesis.

Age-related alterations of functional brain networks contribute to cognitive decline. Current theories indicate that age-related intrinsic brain functional reorganization may be a critical marker of cognitive aging. Yet, little is known about how intrinsic interhemispheric functional connectivity changes with age in adults, and how this relates to critical executive functions. To address this, we examined voxel-mirrored homotopic connectivity (VMHC), a metric that quantifies interhemispheric communication, in 93 healthy volunteers (age range: 19-85) with executive function assessment using the Delis-Kaplan Executive Function System (D-KEFS) scales. Resting functional MRI data were analyzed to assess VMHC, and then a multiple linear regression model was employed to evaluate the relationship between age and the whole-brain VMHC. We observed age-related reductions in VMHC of ventromedial prefrontal cortex (vmPFC) and hippocampus in the medial temporal lobe subsystem, dorsal anterior cingulate cortex and insula in salience network, and inferior parietal lobule in frontoparietal control network. Performance on the color-word inhibition task was associated with VMHC of vmPFC and insula, and VMHC of vmPFC mediated the relationship between age and CWIT inhibition reaction times. The percent ratio of correct design scores in design fluency test correlated positively with VMHC of the inferior parietal lobule. The current study suggests that brain interhemispheric functional alterations may be a promising new avenue for understanding age-related cognitive decline.