Cudraflavone B induces human glioblastoma cells apoptosis via ER stress-induced autophagy.

BACKGROUND: Glioblastoma (GBM) is the most common malignant intracranial tumor with a low survival rate. However, only few drugs responsible for GBM therpies, hence new drug development for it is highly required. The natural product Cudraflavone B (CUB) has been reported to potentially kill a variety of tumor cells. Currently, its anit-cancer effect on GBM still remains unknown. Herein, we investigated whether CUB could affect the proliferation and apoptosis of GBM cells to show anti-GBM potential. RESULTS: CUB selectively inhibited cell viability and induced cell apoptosis by activating the endoplasmic reticulum stress (ER stress) related pathway, as well as harnessing the autophagy-related PI3K/mTOR/LC3B signaling pathway. Typical morphological changes of autophagy were also observed in CUB treated cells by microscope and scanning electron microscope (SEM) examination. 4-Phenylbutyric acid (4-PBA), an ER stress inhibitor, restored the CUB-caused alteration in signaling pathway and morphological change. CONCLUSIONS: Our finding suggests that CUB impaired cell growth and induced cell apoptosis of glioblastoma through ER stress and autophagy-related signaling pathways, and it might be an attractive drug for treatment of GBM.

Improvement of the immunogenicity of ESAT-6 via fusion with the dodecameric protein dodecin of Mycobacterium tuberculosis.

Tuberculosis (TB) is a chronic infectious disease that creates a heavy medical burden worldwide. The only approved vaccine, Bacillus Calmette-Guérin (BCG), cannot fully protect adolescents and adults from TB. Therefore, there is an urgent need to develop an effective new vaccine. Previous studies have found that dodecin, a flavin-binding protein of Mycobacterium tuberculosis (Mtb), can form stable dodecamers and has the potential to improve the immunogenicity of Mtb antigens. In this study, we constructed the fusion protein dodecin-ESAT-6 and evaluated the immunogenicity of dodecin, ESAT-6, and dodecin-ESAT-6 separately. Our results showed that dodecin-ESAT-6 is a dodecameric protein that can withstand heat at 95 °C and under SDS-PAGE conditions. Dodecin-ESAT-6 increased the expression of the costimulatory molecules CD80, CD86, and major histocompatibility complex class II (MHC-II) on the surface of RAW264.7 macrophages. Mice immunized with dodecin-ESAT-6 exhibited higher percentages of antigen-specific CD4+ and CD8+ T lymphocytes, higher levels of spleen lymphocyte proliferation and IFN-γ and IL-2 secretion, and a lower level of IL-4 secretion than those immunized with ESAT-6. The IgG, IgG1, and IgG2a titers of the dodecin-ESAT-6 group were significantly higher than those of the ESAT-6 group. Dodecin-ESAT-6 elicited a high IgG2a/IgG1 ratio and tended to produce a predominantly Th1-like response. These results support the conclusion that the dodecin-ESAT-6 dodecameric protein induced strong Th1 immune responses and improved the immunogenicity of ESAT-6, which provides a new strategy for TB vaccine development.

Combination of metformin and cold atmospheric plasma induces glioma cell death to associate with c-Fos.

Glioma is the most common type of brain cancer. Chemotherapy combination with surgery and radiotherapy is a standard treatment for patients. Although there are many advances in glioma therapy, the prognosis of glioma patients has not significantly been improved over the past decades. Hence, there is still an urgent need to develop a new therapy to treat glioma. Cell viability was assessed by CellTiter Blue assay; flow cytometry (FCM) was used for detecting cell apoptosis; ROS detection was detected by ROS Assay; H2O2 detection was performed by hydrogen peroxide detection kits; real-time PCR and WB were used to determine gene expression. Using the glioma cell line U251 and U87, we investigated a possible combination inhibitory effect includes metformin and cold atmospheric plasma (CAP). The combination treatment showed a synergistic inhibitory effect on cell viability, significantly inducing cell apoptosis. Furthermore, we also found H2O2 produced by CAP has an important role in the synergistic inhibitory effect, eliminating H2O2 with catalase reversed the synergistic inhibitory effect. In addition, the transcript and protein levels of c-FOS were robustly increased after co-treated with metformin and CAP. Taken together, we propose that pre-treatment of glioma cells with metformin sensitize tumor cells to CAP, which may serve as a potential therapeutic strategy for glioma.

[Preliminary Study on the Secretory Performance of Mycobacterium tuberculosis Hypoxic Response Protein 1 (Rv2626c)].

OBJECTIVE: To verify the secretory ability of the hypoxic response protein 1 (HRP1) encoded by Mycobacterium tuberculosis (Mtb) Rv2626c. METHODS: The target gene attached with His tag was amplified from the genome of Mtb standard virulence strain H37Rv. The recombinant plasmid contained the above amplified product was constructed and electroporated into Mycobacterium smegmatis (Ms) (MC 2155) to construct a recombinant strain. Protein expression was induced under heat condition, and the expression of protein from the culture filtrates and the bacterial lysates was detected afterward. The 10 kDa culture filtrate antigen (CFP-10) (Ms) and CFP-10 (Mtb) were used as positive controls, and the cytoplasmic protein heat shock protein 65 (GroEL2) (Mtb) was used as negative controls. RESULTS: The HRP1, GroEL2 (Mtb), CFP-10 (Mtb) and CFP-10 (Ms) were successfully amplified by PCR from recombinant plasmid, and sequencing results of the recombinant plasmid is right, confirming the successful construction of the recombinant plasmid. The recombinant Ms was successfully constructed and it could express the proteins GroEL2 (Mtb), HRP1, CFP-10 (Mtb) and CFP-10 (Ms). The target protein HRP1 was detected in both of the lysate and the culture filtrate of the recombinant strain by Western blot, which was consistent with the positive control CFP-10. The negative control GroEL2 (Mtb) was only detected in the bacterial lysate, but not detected in the culture filtrate. CONCLUSION: The protein HRP1 encoded by Mtb Rv2626c can be secreted out of Ms by the secretion system of Ms. It may be a secreted protein and play an important role in the pathogenesis of Mtb.

A Broad Blockade of Signaling from the IL-20 Family of Cytokines Potently Attenuates Collagen-Induced Arthritis.

Two heterodimeric receptors consisting of either IL-20R1 or IL-22R1 in complex with a common ß receptor subunit IL-20R2 are shared by three of the IL-20 family of cytokines: IL-19, IL-20, and IL-24. These proinflammatory cytokines have been implicated in the pathogenesis of some autoimmune diseases, including rheumatoid arthritis (RA), psoriasis, and atopic dermatitis. Although mAbs against IL-19 and IL-20 have each been shown to modulate disease severity of collagen-induced arthritis in animal models, and anti-IL-20 therapeutic Ab has exhibited some efficacy in the treatment of RA in clinical trials, benefits for a complete blockade of these functionally redundant cytokines remain to be explored. In this report, we show that recombinant human soluble IL-20R2-Fc fusion protein binds to IL-19, IL-20, and IL-24 with similar high affinity and blocks their signaling in vitro. In DBA/1 mouse collagen-induced arthritis model, recombinant human IL-20R2-Fc exhibits comparable efficacy as TNF blocker etanercept in the treatment of established arthritis, whereas the combined use of both biologics manifests little synergistic therapeutic effects. In situ ligand-receptor functional binding analysis shows that a large amount of immune infiltrates expressing high levels of TNFR and IL-20 subfamily cytokines congregate within the inflamed disease tissues. Colocalization experiments reveal that signals from IL-20R2 and TNF transduction pathways seem to converge in macrophages and function in tandem in orchestrating the pathogenesis of RA. Elucidation of this interaction provides a better understanding of cytokine cross-talk in RA and a rationale for more effective biologic therapies that target IL-20R2 instead of individual cytokines from IL-20 family.

Mineralocorticoid promotes intestinal inflammation through receptor dependent IL17 production in ILC3s.

Aldosterone is a key mineralocorticoid involved in regulating the concentration of blood electrolytes and physiological volume balance. Activation of mineralocorticoid receptor (MR) has been recently reported to participate in adaptive and innate immune responses under inflammation. Here, we evaluated the role of aldosterone and MR in inflammation bowel diseases (IBD). Aldosterone elevated in the colon of DSS-induced colitis mice. Aldosterone addition induced IL17 production and ROS/RNS level in group 3 innate lymphoid cells (ILC3s) and exacerbated intestinal injury. A selective mineralocorticoid receptor antagonism, eplerenone, inhibited IL17-producing ILC3s and its ROS/RNS production, protected mice from DSS-induced colitis. Mice lacking Nr3c2 (MR coding gene) in ILC3s exhibited decreased IL17 and ROS/RNS production, which alleviated colitis and colitis-associated colorectal cancer (CAC). Further experiments revealed that MR could directly bind to IL17A promoter and facilitate its transcription, which could be enhanced by aldosterone. Thus, our findings demonstrated the critical role of aldosterone-MR-IL17 signaling in ILC3s and gut homeostasis, indicating the therapeutic strategy of eplerenone in IBD clinical trial.

Black Phosphorus Nanosheets Grafted with Gold Nanorods and Carbon Nanodots for Synergistic Antitumor Therapy.

Synergetic photothermal/photodynamic/chemotherapy receives significant attention for precise in vivo cancer treatment. Despite plenty of encouraging photosensitizers explored, integrated nanoagents with multiple functions are still highly desired. In this study, novel nanocomposites coupling black phosphorus (BP) nanosheets, gold nanorods (AuNRs), carbon nanodots (CDs), and doxorubicin (Dox) are prepared. The nanoagents exhibit high antitumor activity on account of their broad light absorption, excellent catalytic ability, and significant photothermal and photodynamic effects. CDs not only emit bright fluorescence for accurate diagnosis and guiding of tumor treatment but also catalyze the generation of ROS for photodynamic therapy (PDT). The released Dox induces apoptosis of cells and increases the levels of H2O2 to promote PDT. AuNRs are the main photothermal therapy (PTT) material that converts light into heat. Moreover, BP can be used to enhance both PTT and PDT efficiencies, and the two therapy modes can be cooperatively reinforced. It is also found that the local immune microenvironment of the tumors is activated. The strategy makes good use of the features of each component. Satisfactory antitumor phenomena are well confirmed by in vitro and in vivo results. This study provides new insights into enhanced synergetic therapy, highlighting the great utility of BP-based nanoagents in the field of nanomedicine.

NRF1 Regulates the Epithelial Mesenchymal Transition of Breast Cancer by Modulating ROS Homeostasis.

Introduction: Nuclear respiratory factor 1 (NRF1) is an important regulator involved in mitochondrial biogenesis and energy metabolism. However, the specific mechanism of NRF1 in anoikis and epithelial-mesenchymal transition (EMT) remains unclear. Methods: We examined the effect of NRF1 on mitochondria and identified the specific mechanism through transcriptome sequencing, and explored the relationships among NRF1, anoikis, and EMT. Results: We found that upregulated NRF1 expression led to increased mitochondrial oxidative phosphorylation (OXPHOS) and ATP generation. Simultaneously, a significant amount of ROS is generated during OXPHOS. Alternatively, NRF1 upregulates the expression of ROS-scavenging enzymes, allowing tumor cells to maintain low ROS levels and promoting anoikis resistance and EMT. We also found that exogenous ROS was maintained at a low level by NRF1 in breast cancer cells. Conclusion: our study provides mechanistic insight into the function of NRF1 in breast cancer, indicating that NRF1 may serve as a therapeutic target for breast cancer treatment.

Mycobacterium tuberculosis PPE7 Enhances Intracellular Survival of Mycobacterium smegmatis and Manipulates Host Cell Cytokine Secretion Through Nuclear Factor Kappa B and Mitogen-Activated Protein Kinase Signaling.

The PE/PPE family proteins of Mycobacterium tuberculosis have been associated with its virulence and interaction with the host immune system. The highly virulent modern lineage of M. tuberculosis possesses a lineage-specific PPE gene (PPE7), which arises from an ancestral mutation and is rarely studied. Here we examined the role of PPE7 in mycobacterial pathogenicity and survival by expressing M. tuberculosis PPE7 in Mycobacterium smegmatis. We show that, PPE7 activates host inflammation by increasing expression of pro-inflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, and IL-6, while suppressing the expression of anti-inflammatory cytokines such as IL-10, possibly through the nuclear factor kappa B, ERK1/2, and p38 mitogen-activated protein kinase pathways. Overexpressing PPE7 in M. smegmatis could enhance bacterial intracellular survival of infected macrophages. Furthermore, higher level of bacterial persistence, higher levels of TNF-α, IL-1ß, and IL-6 cytokines, and more injury in the lung, liver, and spleen tissues of infected mice has been discovered. In conclusion, PPE7 could manipulate host immune response and increase bacterial persistence.

Morusin Enhances Temozolomide Efficiency in GBM by Inducing Cytoplasmic Vacuolization and Endoplasmic Reticulum Stress.

Glioblastoma multiforme (GBM) is an aggressive brain tumor with high risks of recurrence and mortality. Chemoradiotherapy resistance has been considered a major factor contributing to the extremely poor prognosis of GBM patients. Therefore, there is an urgent need to develop highly effective therapeutic agents. Here, we demonstrate the anti-tumor effect of morusin, a typical prenylated flavonoid, in GBM through in vivo and in vitro models. Morusin showed selective cytotoxicity toward GBM cell lines without harming normal human astrocytes when the concentration was less than 20 µM. Morusin treatment significantly induced apoptosis of GBM cells, accompanied by the activation of endoplasmic reticulum (ER) stress, and the appearance of cytoplasmic vacuolation and autophagosomes in cells. Then, we found the ER stress activation and cytotoxicity of morusin were rescued by ER stress inhibitor 4-PBA. Furthermore, morusin arrested cell cycle at the G1 phase and inhibited cell proliferation of GBM cells through the Akt-mTOR-p70S6K pathway. Dysregulation of ERs and cell cycle in morusin exposed GBM cells were confirmed by RNA-seq analysis. Finally, we demonstrated the combination of morusin and TMZ remarkably enhanced ER stress and displayed a synergistic effect in GBM cells, and suppressed tumor progression in an orthotopic xenograft model. In conclusion, these findings reveal the toxicity of morusin to GBM cells and its ability to enhance drug sensitivity to TMZ, suggesting the potential application value of morusin in the development of therapeutic strategies for human GBM.

USF1/CD90 signaling in maintaining glioblastoma stem cells and tumor-associated macrophages adhesion.

BACKGROUND: Glioblastoma stem cells (GSCs) and their interplay with tumor-associated macrophages (TAMs) are responsible for malignant growth and tumor recurrence of glioblastoma multiforme (GBM), but the underlying mechanisms are largely unknown. METHODS: Cell viability, stemness, migration, and invasion were measured in GSCs after the knockdown of upstream stimulating factor 1 (USF1). Luciferase assay and chromatin immunoprecipitation qPCR were performed to determine the regulation of CD90 by USF1. Immunohistochemistry and immunofluorescent staining were used to examine the expression of USF1 and GSC markers, as well as the crosstalk between GSCs and TAMs. In addition, the interaction between GSCs and TAMs was confirmed using in vivo GBM models. RESULTS: We show that USF1 promotes malignant glioblastoma phenotypes and GSCs-TAMs physical interaction by inducing CD90 expression. USF1 predicts a poor prognosis for glioma patients and is upregulated in patient-derived GSCs and glioblastoma cell lines. USF1 overexpression increases the proliferation, invasion, and neurosphere formation of GSCs and glioblastoma cell lines, while USF1 knockdown exerts an opposite effect. Further mechanistic studies reveal that USF1 promotes GSC stemness by directly regulating CD90 expression. Importantly, CD90 of GSCs functions as an anchor for physical interaction with macrophages. Additionally, the USF1/CD90 signaling axis supports the GSCs and TAMs adhesion and immunosuppressive feature of TAMs, which in turn enhance the stemness of GSCs. Moreover, the overexpression of CD90 restores the stemness property in USF1 knockdown GSCs and its immunosuppressive microenvironment. CONCLUSIONS: Our findings indicate that the USF1/CD90 axis might be a potential therapeutic target for the treatment of glioblastoma.

A Pan-Cancer Analysis of Transcriptome and Survival Reveals Prognostic Differentially Expressed LncRNAs and Predicts Novel Drugs for Glioblastoma Multiforme Therapy.

Numerous studies have identified various prognostic long non-coding RNAs (LncRNAs) in a specific cancer type, but a comprehensive pan-cancer analysis for prediction of LncRNAs that may serve as prognostic biomarkers is of great significance to be performed. Glioblastoma multiforme (GBM) is the most common and aggressive malignant adult primary brain tumor. There is an urgent need to identify novel therapies for GBM due to its poor prognosis and universal recurrence. Using available LncRNA expression data of 12 cancer types and survival data of 30 cancer types from online databases, we identified 48 differentially expressed LncRNAs in cancers as potential pan-cancer prognostic biomarkers. Two candidate LncRNAs were selected for validation in GBM. By the expression detection in GBM cell lines and survival analysis in GBM patients, we demonstrated the reliability of the list of pan-cancer prognostic LncRNAs obtained above. By constructing LncRNA-mRNA-drug network in GBM, we predicted novel drug-target interactions for GBM correlated LncRNA. This analysis has revealed common prognostic LncRNAs among cancers, which may provide insights into cancer pathogenesis and novel drug target in GBM.

Compensatory effects of M. tuberculosis rpoB mutations outside the rifampicin resistance-determining region.

Mycobacterium tuberculosis has been observed to develop resistance to the frontline anti-tuberculosis drug rifampicin, primarily through mutations in the rifampicin resistance-determining region (RRDR) of rpoB. While these mutations have been determined to confer a fitness cost, compensatory mutations in rpoA and rpoC that may enhance the fitness of resistant strains have been demonstrated. Recent genomic studies identified several rpoB non-RRDR mutations that co-occurred with RRDR mutations in clinical isolates without rpoA/rpoC mutations and may confer fitness compensation. In this study, we identified 33 evolutionarily convergent rpoB non-RRDR mutations through phylogenomic analysis of public genomic data for clinical M. tuberculosis isolates. We found that none of these mutations, except V170F and I491F, can cause rifampin resistance in Mycolicibacterium smegmatis. The compensatory effects of five representative mutations across rpoB were evaluated by an in vitro competition assay, through which we observed that each of these mutations can significantly improve the relative fitness of the initial S450L mutant (0.97-1.08 vs 0.87). Furthermore, we observed that the decreased RNAP transcription efficiency introduced by S450L was significantly alleviated by each of the five mutations. Structural analysis indicated that the fitness compensation observed for the non-RRDR mutations might be achieved by modification of the RpoB active centre or by changes in interactions between RNAP subunits. Our results provide experimental evidence supporting that compensatory effects are exerted by several rpoB non-RRDR mutations, which could be utilized as additional molecular markers for predicting the fitness of clinical rifampin-resistant M. tuberculosis strains.

High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG.

Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine-5') pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake.