The Combined Influence of Hydrogel Stiffness and Matrix‐Bound Hyaluronic Acid Content on Glioblastoma Invasion

Glioblastoma (GBM) is the most common and lethal form of brain cancer. Its high mortality is associated with its aggressive invasion throughout the brain. The heterogeneity of stiffness and hyaluronic acid (HA) content within the brain makes it difficult to study invasion in vivo. A dextran‐bead assay is employed to quantify GBM invasion within HA‐functionalized gelatin hydrogels. Using a library of stiffness‐matched hydrogels with variable levels of matrix‐bound HA, it is reported that U251 GBM invasion is enhanced in softer hydrogels but reduced in the presence of matrix‐bound HA. Inhibiting HA–CD44 interactions reduces invasion, even in hydrogels lacking matrix‐bound HA. Analysis of HA biosynthesis suggests that GBM cells compensate for a lack of matrix‐bound HA by producing soluble HA to stimulate invasion. Together, a robust method is showed to quantify GBM invasion over long culture times to reveal the coordinated effect of matrix stiffness, immobilized HA, and compensatory HA production on GBM invasion.

     

MicroRNA Delivery by Graphene-Based Complexes into Glioblastoma Cells

Glioblastoma (GBM) is the most common primary and aggressive tumour in brain cancer. Novel therapies, despite achievements in chemotherapy, radiation and surgical techniques, are needed to improve the treatment of GBM tumours and extend patients’ survival. Gene delivery therapy mostly uses the viral vector, which causes serious adverse events in gene therapy. Graphene-based complexes can reduce the potential side effect of viral carries, with high efficiency of microRNA (miRNA) or antisense miRNA delivery to GBM cells. The objective of this study was to use graphene-based complexes to induce deregulation of miRNA level in GBM cancer cells and to regulate the selected gene expression involved in apoptosis. The complexes were characterised by Fourier transform infrared spectroscopy (FTIR), scanning transmission electron microscopy and zeta potential. The efficiency of miRNA delivery to the cancer cells was analysed by flow cytometry. The effect of the anticancer activity of graphene-based complexes functionalised by the miRNA sequence was analysed using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide salt (XTT) assays at the gene expression level. The results partly explain the mechanisms of miRNA deregulation stress, which is affected by graphene-based complexes together with the forced transport of mimic miR-124, miR-137 and antisense miR-21, -221 and -222 as an anticancer supportive therapy.     

The Inhibitory Effect of Sulforaphane on Bladder Cancer Cell Depends on GSH Depletion-Induced by Nrf2 Translocation

Sulforaphane (SFN), an isothiocyanate (ITCs) derived from glucosinolate that is found in cruciferous vegetables, has been reported to exert a promising anticancer effect in a substantial amount of scientific research. However, epidemical studies showed inconsistencies between cruciferous vegetable intake and bladder cancer risk. In this study, human bladder cancer T24 cells were used as in vitro model for revealing the inhibitory effect and its potential mechanism of SFN on cell growth. Here, a low dose of SFN (2.5 µM) was shown to promote cell proliferation (5.18–11.84%) and migration in T24 cells, whilst high doses of SFN (>10 µM) inhibited cell growth significantly. The induction effect of SFN on nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression at both low (2.5 µM) and high dose (10 µM) was characterized by a bell-shaped curve. Nrf2 and glutathione (GSH) might be the underlying mechanism in the effect of SFN on T24 cell growth since Nrf2 siRNA and GSH-depleting agent L-Buthionine-sulfoximine abolished the effect of SFN on cell proliferation. In summary, the inhibitory effect of SFN on bladder cancer cell growth and migration is highly dependent on Nrf2-mediated GSH depletion and following production. These findings suggested that a higher dose of SFN is required for the prevention and treatment of bladder cancer.     

Anticancer Effect of Cathelicidin LL-37, Protegrin PG-1, Nerve Growth Factor NGF,and Temozolomide: Impact on the Mitochondrial Metabolism,Clonogenic Potential,and Migration of Human U251 Glioma Cells

Glioblastoma (GBM) is one of the most aggressive and lethal malignancy of the central nervous system. Temozolomide is the standard of care for gliomas, frequently results in resistance to drug and tumor recurrence. Therefore, further research is required for the development of effective drugs in order to guarantee specific treatments to succeed. The aim of current study was to investigate the effects of nerve growth factor (NGF), human cathelicidin (LL-37), protegrin-1 (PG-1), and temozolomide on bioenergetic function of mitochondria, clonogenicity, and migration of human U251 glioma cells. Colony formation assay was used to test the ability of the glioma cells to form colonies in vitro. The U251 glioma cells migration was evaluated using wound-healing assay. To study the mitochondrial metabolism in glioma cells we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) using a Seahorse XF cell Mito stress test kit and Seahorse XF cell Glycolysis stress kit, respectively. We revealed that LL-37, NGF, and TMZ show strong anti-tumorigenic activity on GMB. LL-37 (4 μM), TMZ (155 μM), and NGF (7.55 × 10⁻³ μM) inhibited 43.9%–60.3%, 73.5%–81.3%, 66.2% the clonogenicity of glioma U251 cells for 1–2 days, respectively. LL-37 (4 μM), and NGF (7.55 × 10⁻³ μM) inhibited the migration of U251 glioma cells on the third and fourth days. TMZ also inhibited the migration of human glioma U251 cells over 1–3 days. In contrast, PG-1 (16 μM) stimulated the migration of U251 glioma cells on the second, fourth, and sixth days. Anti-mitogenic and anti-migration activities of NGF, LL-37, and TMZ maybe are relation to their capacity to reduce the basal OCR, ATP-synthetase, and maximal respiration of mitochondria in human glioma U251 cells. Glycolysis, glycolytic capacity and glycolytic spare in glioma U251 cells haven`t been changed under the effect of NGF, LL-37, PG-1, and TMZ in regard to control level. Thus, LL-37 and NGF inhibit migration and clonogenicity of U251 glioma cells, which may indicate that these compounds have anti-mitogenic and anti-migration effects on human glioma cells. The study of the mechanisms of these effects may contribute in the future to the use of NGF and LL-37 as therapeutic agents for gliomas.     

Multiplexed targeting of miRNA-210 in stem cell-derived extracellular vesicles promotes selective regeneration in ischemic hearts

Extracellular vesicles (EVs) are cell derivatives containing diverse cellular molecules, have various physiological properties and are also present in stem cells used for regenerative therapy. We selected a “multiplexed target” that demonstrates multiple effects on various cardiovascular cells, while functioning as a cargo of EVs. We screened various microRNAs (miRs) and identified miR-210 as a candidate target for survival and angiogenic function. We confirmed the cellular and biological functions of EV-210 (EVs derived from ASC^miR-210) secreted from adipose-derived stem cells (ASCs) transfected with miR-210 (ASC^miR-210). Under hypoxic conditions, we observed that ASC^miR-210 inhibits apoptosis by modulating protein tyrosine phosphatase 1B (PTP1B) and death-associated protein kinase 1 (DAPK1). In hypoxic endothelial cells, EV-210 exerted its angiogenic capacity by inhibiting Ephrin A (EFNA3). Furthermore, EV-210 enhanced cell survival under the control of PTP1B and induced antiapoptotic effects in hypoxic H9c2 cells. In cardiac fibroblasts, the fibrotic ratio was reduced after exposure to EV-210, but EVs derived from ASC^miR-210 did not communicate with fibroblasts. Finally, we observed the functional restoration of the ischemia/reperfusion-injured heart by maintaining the intercommunication of EVs and cardiovascular cells derived from ASC^miR-210. These results suggest that the multiplexed target with ASC^miR-210 is a useful tool for cardiovascular regeneration.Subject terms: Myocardial infarction, Stem-cell research     

Combined inhibition of HDAC and DNMT1 induces p85α/MEKmediated cell cycle arrest by dual target inhibitor 208 in U937 cells

Herein we reported an efficient dual DNMT and HDAC inhibitor 208 with great antiproliferative activity against U937 cells. Further studies revealed 208 affected the whole proteome profile and could induce G1 cell cycle arrest and apoptosis in U937 cells through upregulating CDK inhibitor p16 and downregulating cyclin-dependent kinases and their activators.     

Chrysomycin A Inhibits the Proliferation,Migration and Invasion of U251 and U87-MG Glioblastoma Cells to Exert Its Anti-Cancer Effects

Chrysomycin A (Chr-A), an antibiotic from Streptomyces, is reported to have anti-tumor and anti-tuberculous activities, but its anti-glioblastoma activity and possible mechanism are not clear. Therefore, the current study was to investigate the mechanism of Chr-A against glioblastoma using U251 and U87-MG human cells. CCK8 assays, EdU-DNA synthesis assays and LDH assays were carried out to detect cell viability, proliferation and cytotoxicity of U251 and U87-MG cells, respectively. Transwell assays were performed to detect the invasion and migration abilities of glioblastoma cells. Western blot was used to validate the potential proteins. Chr-A treatment significantly inhibited the growth of glioblastoma cells and weakened the ability of cell migration and invasion by down regulating the expression of slug, MMP2 and MMP9. Furthermore, Chr-A also down regulated Akt, p-Akt, GSK-3β, p-GSK-3β and their downstream proteins, such as β-catenin and c-Myc in human glioblastoma cells. In conclusion, Chr-A may inhibit the proliferation, migration and invasion of glioblastoma cells through the Akt/GSK-3β/β-catenin signaling pathway.     

Green synthesis of Zn nanoparticles and in situ hybridized with BSA nanoparticles for Baicalein targeted delivery mediated with glutamate receptors to U87-MG cancer cell lines

Glioblastoma multiforme (GBM) is the most aggressive malignant tumor of the brain. It has different glutamate receptor types. So, these receptors can be a suitable target for GBM treatment. The current study investigated the anticancer effects of bovine serum albumin (BSA)-Baicalein @Zn-Glu nanostructure mediated-GluRs in human glioblastoma U87 cells. BSA-Ba@Zn-Glu hybrid nanoparticles (NPs) were set and considered transporters for Baicalein (Ba) active compound delivery. BSA-Ba@Zn-Glu NPs were synthesized by a single-step reduction process. The successful production was confirmed through transmission electron microscopy (TEM), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), and hemolysis test. The cytotoxic efficacy and apoptosis rate of the nanostructures on U87 glioblastoma cells were investigated by 3-(4,5-dimethylthialzol-a-yl)-2,5 diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. The synthesized BSA-Ba@Zn-Glu nanostructures with a diameter of 142.40 ± 1.91 to 177.10 ± 1.87 nm and zeta potential of −10.57 ± 0.71 to −35.77 ± 0.60 mV are suitable for extravasation into tumor cells. The drug release from the BSA-Ba@Zn NPs showed controlled and pH-dependent behavior. In vitro results indicated that the BSA-Ba@Zn-Glu NPs significantly reduce cell viability and promote apoptosis of U87 cancer cells. It revealed the cytotoxic effect of the Baicalein and an increase in cellular uptake of nanoparticles by Glu receptors. Zn NPs were synthesized based on a green synthesis method. BSA NPs were used as a nano-platform for Glu conjugation and Ba drug delivery. BSA-Ba@Zn-Glu NPs induce cytotoxicity and apoptosis in human brain cancer cells (U87) in a dose-dependent manner. Finally, this nanostructure could be served in targeted drug delivery in vivo studies and applied along with other strategies such as X-ray irradiation as combinational therapies in future studies.     

Effect of Nanoparticles of DOX and miR-125b on DNA Damage Repair in Glioma U251 Cells and Underlying Mechanisms

Glioma is the most common primary craniocerebral malignant tumor, arising from the canceration of glial cells in the brain and spinal cord. The quality of life and prognosis of patients with this disease are still poor. Doxorubicin (DOX) is one of the most traditional and economical chemotherapeutic drugs for the treatment of glioma, but its toxic effect on normal cells and the resistance of tumor cells to DOX make the application of DOX in the treatment of glioma gradually less effective. To solve this problem, we co-encapsulated DOX and endogenous tumor suppressor miR-125b into nanoparticles (NPs) by nanoprecipitation methods, and passively targeted them into glioma cells. In vitro experiments show that miR-125b and DOX can be effectively encapsulated into nanoparticles with different ratios, and by targeting YES proto-oncogene 1 (YES1), they can affect the adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK)/p53 pathway and induce brain glioma cell apoptosis. They can also affect the DNA damage repair process and inhibit cell proliferation. The obtained data suggest that co-delivery of DOX and miR-125b could achieve synergistic effects on tumor suppression. Nanosystem-based co-delivery of tumor suppressive miRNAs and chemotherapeutic agents may be a promising combined therapeutic strategy for enhanced anti-tumor therapy.     

Sulforaphane Inhibits Osteoclastogenesis via Suppression of the Autophagic Pathway

Previous studies have demonstrated that sulforaphane (SFN) is a promising agent against osteoclastic bone destruction. However, the mechanism underlying its anti-osteoclastogenic activity is still unclear. Herein, for the first time, we explored the potential role of autophagy in SFN-mediated anti-osteoclastogenesis in vitro and in vivo. We established an osteoclastogenesis model using receptor activator of nuclear factor kappa-β ligand (RANKL)-induced RAW264.7 cells and bone marrow macrophages (BMMs). Tartrate-resistant acid phosphatase (TRAP) staining showed the formation of osteoclasts. We observed autophagosomes by transmission electron microscopy (TEM). In vitro, we found that SFN inhibited osteoclastogenesis (number of osteoclasts: 22.67 ± 0.88 in the SFN (0) group vs. 20.33 ± 1.45 in the SFN (1 μM) group vs. 13.00 ± 1.00 in the SFN (2.5 μM) group vs. 6.66 ± 1.20 in the SFN (2.5 μM) group), decreased the number of autophagosomes, and suppressed the accumulation of several autophagic proteins in osteoclast precursors. The activation of autophagy by rapamycin (RAP) almost reversed the SFN-elicited anti-osteoclastogenesis (number of osteoclasts: 22.67 ± 0.88 in the control group vs. 13.00 ± 1.00 in the SFN group vs. 17.33 ± 0.33 in the SFN+RAP group). Furthermore, Western blot (WB) analysis revealed that SFN inhibited the phosphorylation of c-Jun N-terminal kinase (JNK). The JNK activator anisomycin significantly promoted autophagy, whereas the inhibitor SP600125 markedly suppressed autophagic activation in pre-osteoclasts. Microcomputed tomography (CT), immunohistochemistry (IHC), and immunofluorescence (IF) were used to analyze the results in vivo. Consistent with the in vitro results, we found that the administration of SFN could decrease the number of osteoclasts and the expression of autophagic light chain 3 (LC3) and protect against lipopolysaccharide (LPS)-induced calvarial erosion. Our findings highlight autophagy as a crucial mechanism of SFN-mediated anti-osteoclastogenesis and show that the JNK signaling pathway participates in this process.     

Involvement of human small fragment nuclease in the resistance of human cells to UV-C-induced cell death

Human small fragment nuclease (Sfn) is one of the cellular proteins that were reported to degrade small, single-stranded DNA and RNA. However, the biological role of Sfn in cellular response to various stressors such as UV-C (mainly 254 nm wavelength ultraviolet ray) remains unclear. We have examined whether modulation of human SFN gene expression affects cell survival capacity against UV-C-induced cell death, analyzing colony survival ability in UV-C-sensitive human RSa cells treated with short double-stranded RNA (siRNA) specific for SFN messenger RNA (mRNA). The expression levels of SFN mRNA in the siRNA-treated RSa cells decreased to about 15% compared with those in the control siRNA-treated cells. The siRNA-treated RSa cells showed lower colony survival and higher activity of caspase-3 after UV-C irradiation than the control siRNA-treated RSa cells. Furthermore, the removal capacity of cyclobutane pyrimidine dimers (CPD) in the siRNA-treated RSa cells decreased compared with the control siRNA-treated RSa cells. There was no difference in the colony survival and CPD removal capacity after UV-C irradiation between the control siRNA-treated RSa cells and mock-treated RSa cells. These results suggest that SFN expression is involved in resistance of RSa cells to UV-C-induced cell death through the roles it plays in the DNA repair process.     

Involvement of miR-190b in Xbp1 mRNA Splicing upon Tocotrienol Treatment

We previously demonstrated that apoptosis induced by tocotrienols (γ and δT3) in HeLa cells is preceded by Ca²⁺ release from the endoplasmic reticulum. This event is eventually followed by the induction of specific calcium-dependent signals, leading to the expression and activation of the gene encoding for the IRE1α protein and, in turn, to the alternative splicing of the pro-apoptotic protein sXbp1 and other molecules involved in the unfolded protein response, the core pathway coping with EndoR stress. Here, we showed that treatment with T3s induces the expression of a specific set of miRNAs in HeLa cells. Data interrogation based on the intersection of this set of miRNAs with a set of genes previously differentially expressed after γT3 treatment provided a few miRNA candidates to be the effectors of EndoR-stress-induced apoptosis. To identify the best candidate to act as the effector of the Xbp1-mediated apoptotic response to γT3, we performed in silico analysis based on the evaluation of the highest ∆ in Gibbs energy of different mRNA–miRNA–Argonaute (AGO) protein complexes. The involvement of the best candidate identified in silico, miR-190b, in Xbp1 splicing was confirmed in vitro using T3-treated cells pre-incubated with the specific miRNA inhibitor, providing a preliminary indication of its role as an effector of EndoR-stress-induced apoptosis.     

Identification of miR-23a as a novel microRNA normalizer for relative quantification in human uterine cervical tissues

Quantitative real-time RT-PCR (RT-qPCR) is being widely used in microRNA expression research. However, few reports detailed a robust identification and validation strategy for suitable reference genes for normalisation in microRNA RT-qPCR studies. The aim of this study was to identify the most stable reference gene(s) for quantification of microRNA expression analysis in uterine cervical tissues. A microarray was performed on 6 pairs of uterine cervical tissues to identify the candidate reference genes. The stability of candidate reference genes was assessed by RT-qPCR in 23 pairs of uterine cervical tissues. The identified most stable reference genes were further validated in other cohort of 108 clinical uterine cervical samples: (HR-HPV- normal, n=21; HR-HPV+ normal, n=19; cervical intraepithelial neoplasia [CIN], n=47; cancer, n=21), and the effects of normalizers on the relative quantity of target miR-424 were assessed. In the array experiment, miR-26a, miR-23a, miR-200c, let-7a, and miR-1979 were identified as candidate reference genes for subsequent validation. MiR-23a was identified as the most reliable reference gene followed by miR-191. The use of miR-23a and miR-191 to normalize expression data enabled detection of a significant dereg-ulation of miR-424 between normal, CIN and cancer tissue. Our results suggested that miR-23a and miR-191 are the optimal reference microRNAs that can be used for normalization in profiling studies of cervical tissues; miR-23a is a novel microRNA normalizer.     

Quinacrine Mediated Sensitization of Glioblastoma (GBM) Cells to TRAIL through MMP‐Sensitive PEG Hydrogel Carriers

Overcoming drug resistance is a major challenge for cancer therapy. Tumor necrosis factor α‐related apoptosis‐inducing ligand (TRAIL) is a potent therapeutic as an activator of apoptosis, particularly in tumor but not in healthy cells. However, its efficacy is limited by the resistance of tumor cell populations to the therapeutic substance. Here, we have addressed this limitation through the development of a controlled release system, matrix‐metalloproteinase (MMP)‐sensitive and arg‐gly‐asp‐ser (RGDS) peptide functionalized poly (ethylene‐glycol) (PEG) particles which are synthesized via visible‐light‐induced water‐in‐water emulsion polymerization. Quinacrine (QC), a recently discovered TRAIL sensitizer drug, is loaded into the hydrogel carriers and the influence of this system on the apoptosis of a malignant type of brain cancer, glioblastoma multiforme (GBM), has been investigated in detail. The results suggest that MMP‐sensitive particles are cytocompatible and superior to promote TRAIL‐induced apoptosis in GBM cells when loaded with QC. Compared to QC and TRAIL alone, combination of QC‐loaded PEG hydrogel and TRAIL demonstrates synergistic apoptotic inducing behavior. Furthermore, QC‐loaded particles, but not QC or PEG‐hydrogels alone, enhance apoptosis as is measured through expression of apoptosis‐related genes. This system is promising to significantly improve the efficacy of chemotherapeutic drugs and suggests a combination treatment for GBM therapy.

     

Ruthenacarborane and Quinoline: A Promising Combination for the Treatment of Brain Tumors

Gliomas and glioblastomas are very aggressive forms of brain tumors, prone to the development of a multitude of resistance mechanisms to therapeutic treatments, including cytoprotective autophagy. In this work, we investigated the role and mechanism of action of the combination of a ruthenacarborane derivative with 8-hydroxyquinoline (8-HQ), linked via an ester bond (complex 2), in rat astrocytoma C6 and human glioma U251 cells, in comparison with the two compounds alone, i.e., the free carboxylic acid (complex 1) and 8-HQ, and their non-covalent combination ([1 + 8-HQ], in 1:1 molar ratio). We found that only complex 2 was able to significantly affect cellular viability in glioma U251 cells (IC₅₀ 11.4 μM) via inhibition of the autophagic machinery, most likely acting at the early stages of the autophagic cascade. Contrary to 8-HQ alone, complex 2 was also able to impair cellular viability under conditions of glucose deprivation. We thus suggest different mechanisms of action of ruthenacarborane complex 2 than purely organic quinoline-based drugs, making complex 2 a very attractive candidate for evading the known resistances of brain tumors to chloroquine-based therapies.     

虚拟导航超声支气管镜灌洗液miRNA在不同大体分型肺癌患者体内表达差异及诊断周围型肺癌的效能研究

本文探讨了虚拟导航超声支气管镜灌洗液微小RNA(miRNA)在不同大体分型肺癌患者体内的表达差异及诊断周围型肺癌(PLC)的效能。选取65例中心型肺癌(CLC)患者(中心型组)及65例PLC患者(周围型组),均检测miR-1260b、miR-135a-3p、miR-299-3p、miR-3175及癌胚抗原(CEA)、神经元特异性烯醇化酶(NSE)表达。周围型组miR-1260b低于中心型组,miR-135a-3p、miR-3175高于中心型组(P<0.05);miR-1260b、miR-135a-3p、miR-3175与CEA、NSE相关,且与PLC显著相关(P<0.05);miR-1260b、miR-135a-3p联合诊断PLC的AUC高于单独诊断;miR-1260b、miR-135a-3p、miR-299-3p、miR-3175联合诊断PLC转移的效能高于单独诊断。故可知PLC和CLC患者虚拟导航超声支气管镜灌洗液中miR-1260b、miR-135a-3p、miR-3175表达存在差异,联合检测对PLC具有重要诊断价值。而且miR-1260b、miR-135a-3p、miR-299-3p、miR-3175联合检测可预测PLC转移,为临床诊治提供依据。     

MicroRNA-26a/b-5p promotes myocardial infarction-induced cell death by downregulating cytochrome c oxidase 5a

Myocardial infarction (MI) damage induces various types of cell death, and persistent ischemia causes cardiac contractile decline. An effective therapeutic strategy is needed to reduce myocardial cell death and induce cardiac recovery. Therefore, studies on molecular and genetic biomarkers of MI, such as microRNAs (miRs), have recently been increasing and attracting attention due to the ideal characteristics of miRs. The aim of the present study was to discover novel causative factors of MI using multiomics-based functional experiments. Through proteomic, MALDI-TOF-MS, RNA sequencing, and network analyses of myocardial infarcted rat hearts and in vitro functional analyses of myocardial cells, we found that cytochrome c oxidase subunit 5a (Cox5a) expression is noticeably decreased in myocardial infarcted rat hearts and myocardial cells under hypoxic conditions, regulates other identified proteins and is closely related to hypoxia-induced cell death. Moreover, using in silico and in vitro analyses, we found that miR-26a-5p and miR-26b-5p (miR-26a/b-5p) may directly modulate Cox5a, which regulates hypoxia-related cell death. The results of this study elucidate the direct molecular mechanisms linking miR-26a/b-5p and Cox5a in cell death induced by oxygen tension, which may contribute to the identification of new therapeutic targets to modulate cardiac function under physiological and pathological conditions.Subject terms: Protein-protein interaction networks, Predictive markers