CRISPR-Cas9 library screening combined with an exosome-targeted delivery system addresses tumorigenesis/TMZ resistance in the mesenchymal subtype of glioblastoma.

Rationale: The large-scale genomic analysis classifies glioblastoma (GBM) into three major subtypes, including classical (CL), proneural (PN), and mesenchymal (MES) subtypes. Each of these subtypes exhibits a varying degree of sensitivity to the temozolomide (TMZ) treatment, while the prognosis corresponds to the molecular and genetic characteristics of the tumor cell type. Tumors with MES features are predominantly characterized by the NF1 deletion/alteration, leading to sustained activation of the RAS and PI3K-AKT signaling pathways in GBM and tend to acquire drug resistance, resulting in the worst prognosis compared to other subtypes (PN and CL). Here, we used the CRISPR/Cas9 library screening technique to detect TMZ-related gene targets that might play roles in acquiring drug resistance, using overexpressed KRAS-G12C mutant GBM cell lines. The study identified a key therapeutic strategy to address the chemoresistance against the MES subtype of GBM. Methods: The CRISPR-Cas9 library screening was used to discover genes associated with TMZ resistance in the U87-KRAS (U87-MG which is overexpressed KRAS-G12C mutant) cells. The patient-derived GBM primary cell line TBD0220 was used for experimental validations in vivo and in vitro. Chromatin isolation by RNA purification (ChIRP) and chromatin immunoprecipitation (ChIP) assays were used to elucidate the silencing mechanism of tumor suppressor genes in the MES-GBM subtype. The small-molecule inhibitor EPIC-0412 was obtained through high-throughput screening. Transmission electron microscopy (TEM) was used to characterize the exosomes (Exos) secreted by GBM cells after TMZ treatment. Blood-derived Exos-based targeted delivery of siRNA, TMZ, and EPIC-0412 was optimized to tailor personalized therapy in vivo. Results: Using the genome-wide CRISPR-Cas9 library screening, we found that the ERBIN gene could be epigenetically regulated in the U87-KRAS cells. ERBIN overexpression inhibited the RAS signaling and downstream proliferation and invasion effects of GBM tumor cells. EPIC-0412 treatment inhibited tumor proliferation and EMT progression by upregulating the ERBIN expression both in vitro and in vivo. Genome-wide CRISPR-Cas9 screening also identified RASGRP1(Ras guanine nucleotide-releasing protein 1) and VPS28(Vacuolar protein sorting-associated protein 28) genes as synthetically lethal in response to TMZ treatment in the U87-KRAS cells. We found that RASGRP1 activated the RAS-mediated DDR pathway by promoting the RAS-GTP transformation. VPS28 promoted the Exos secretion and decreased intracellular TMZ concentration in GBM cells. The targeted Exos delivery system encapsulating drugs and siRNAs together showed a powerful therapeutic effect against GBM in vivo. Conclusions: We demonstrate a new mechanism by which ERBIN is epigenetically silenced by the RAS signaling in the MES subtype of GBM. Restoration of the ERBIN expression with EPIC-0412 significantly inhibits the RAS signaling downstream. RASGRP1 and VPS28 genes are associated with the promotion of TMZ resistance through RAS-GDP to RAS-GTP transformation and TMZ efflux, as well. A quadruple combination therapy based on a targeted Exos delivery system demonstrated significantly reduced tumor burden in vivo. Therefore, our study provides new insights and therapeutic approaches for regulating tumor progression and TMZ resistance in the MES-GBM subtype.

Retraction of "Universal theranostic CRISPR/Cas13a RNA-editing system for glioma".

[This retracts the article DOI: 10.7150/thno.84429.].

EPIC-0628 abrogates HOTAIR/EZH2 interaction and enhances the temozolomide efficacy via promoting ATF3 expression and inhibiting DNA damage repair in glioblastoma.

The efficacy of temozolomide (TMZ) treatment in glioblastoma (GBM) is influenced by various mechanisms, mainly including the level of O6-methylguanine-DNA methyltransferase (MGMT) and the activity of DNA damage repair (DDR) pathways. In our previous study, we had proved that long non-coding RNA HOTAIR regulated the GBM progression and mediated DDR by interacting with EZH2, the catalytic subunit of PRC2. In this study, we developed a small-molecule inhibitor called EPIC-0628 that selectively disrupted the HOTAIR-EZH2 interaction and promoted ATF3 expression. The upregulation of ATF3 inhibited the recruitment of p300, p-p65, p-Stat3 and SP1 to the MGMT promoter. Hence, EPIC-0628 silenced MGMT expression. Besides, EPIC-0628 induced cell cycle arrest by increasing the expression of CDKN1A and impaired DNA double-strand break repair via suppressing the ATF3-p38-E2F1 pathway. Lastly, EPIC-0628 enhanced TMZ efficacy in GBM in vitro and vivo. Hence, this study provided evidence for the combination of epigenetic drugs EPIC-0628 with TMZ for GBM treatment through the above mechanisms.

Levetiracetam: A Potent Sword against Microglia Polarization in Gliomas.

Crosstalk between tumor cells and peritumoral cells contributes to immunosuppressive microenvironment formation in glioblastomas (GBM). A recent study revealed that glioma stem cells activated neuronal activity to promote microglial M2 polarization, leading to GBM progression, which could be pharmacologically blocked by levetiracetam, providing a practical strategy for GBM immunotherapy. See related article by Guo et al., p. 1160.

RUNX1/NPM1/H3K4me3 complex contributes to extracellular matrix remodeling via enhancing FOSL2 transcriptional activation in glioblastoma.

Extracellular matrix (ECM) remodeling has been implicated in the tumor malignant progression and immune escape in glioblastoma (GBM). Runt-related transcription factor 1 (RUNX1) is a vital transcriptional factor for promoting tumorigenesis and invasion in mesenchymal subtype of GBM. But the correlation between RUNX1 and ECM genes expression and regulatory mechanism of RUNX1 on ECM genes expression remain poorly understood to date. In this study, by using integral analysis of chromatin immunoprecipitation-sequencing and RNA sequencing, we reported that RUNX1 positively regulated the expression of various ECM-related genes, including Fibronectin 1 (FN1), Collagen type IV alpha 1 chain (COL4A1), and Lumican (LUM), in GBM. Mechanistically, we demonstrated that RUNX1 interacted with Nucleophosmin 1 (NPM1) to maintain the chromatin accessibility and facilitate FOS Like 2, AP-1 Transcription Factor Subunit (FOSL2)-mediated transcriptional activation of ECM-related genes, which was independent of RUNX1's transcriptional function. ECM remodeling driven by RUNX1 promoted immunosuppressive microenvironment in GBM. In conclusion, this study provides a novel mechanism of RUNX1 binding to NPM1 in driving the ECM remodeling and GBM progression.

EPIC-1042 as a potent PTRF/Cavin1-caveolin-1 interaction inhibitor to induce PARP1 autophagic degradation and suppress temozolomide efflux for glioblastoma.

BACKGROUND: Temozolomide (TMZ) treatment efficacy in glioblastoma is determined by various mechanisms such as TMZ efflux, autophagy, base excision repair (BER) pathway, and the level of O6-methylguanine-DNA methyltransferase (MGMT). Here, we reported a novel small-molecular inhibitor (SMI) EPIC-1042 (C20H28N6) with the potential to decrease TMZ efflux and promote PARP1 degradation via autolysosomes in the early stage. METHODS: EPIC-1042 was obtained from receptor-based virtual screening. Co-immunoprecipitation and pull-down assays were applied to verify the blocking effect of EPIC-1042. Western blotting, co-immunoprecipitation, and immunofluorescence were used to elucidate the underlying mechanisms of EPIC-1042. In vivo experiments were performed to verify the efficacy of EPIC-1042 in sensitizing glioblastoma cells to TMZ. RESULTS: EPIC-1042 physically interrupted the interaction of PTRF/Cavin1 and caveolin-1, leading to reduced secretion of small extracellular vesicles (sEVs) to decrease TMZ efflux. It also induced PARP1 autophagic degradation via increased p62 expression that more p62 bound to PARP1 and specially promoted PARP1 translocation into autolysosomes for degradation in the early stage. Moreover, EPIC-1042 inhibited autophagy flux at last. The application of EPIC-1042 enhanced TMZ efficacy in glioblastoma in vivo. CONCLUSION: EPIC-1042 reinforced the effect of TMZ by preventing TMZ efflux, inducing PARP1 degradation via autolysosomes to perturb the BER pathway and recruitment of MGMT, and inhibiting autophagy flux in the later stage. Therefore, this study provided a novel therapeutic strategy using the combination of TMZ with EPIC-1042 for glioblastoma treatment.

Blockage of EGFR/AKT and mevalonate pathways synergize the antitumor effect of temozolomide by reprogramming energy metabolism in glioblastoma.

BACKGROUND: Metabolism reprogramming plays a vital role in glioblastoma (GBM) progression and recurrence by producing enough energy for highly proliferating tumor cells. In addition, metabolic reprogramming is crucial for tumor growth and immune-escape mechanisms. Epidermal growth factor receptor (EGFR) amplification and EGFR-vIII mutation are often detected in GBM cells, contributing to the malignant behavior. This study aimed to investigate the functional role of the EGFR pathway on fatty acid metabolism remodeling and energy generation. METHODS: Clinical GBM specimens were selected for single-cell RNA sequencing and untargeted metabolomics analysis. A metabolism-associated RTK-fatty acid-gene signature was constructed and verified. MK-2206 and MK-803 were utilized to block the RTK pathway and mevalonate pathway induced abnormal metabolism. Energy metabolism in GBM with activated EGFR pathway was monitored. The antitumor effect of Osimertinib and Atorvastatin assisted by temozolomide (TMZ) was analyzed by an intracranial tumor model in vivo. RESULTS: GBM with high EGFR expression had characteristics of lipid remodeling and maintaining high cholesterol levels, supported by the single-cell RNA sequencing and metabolomics of clinical GBM samples. Inhibition of the EGFR/AKT and mevalonate pathways could remodel energy metabolism by repressing the tricarboxylic acid cycle and modulating ATP production. Mechanistically, the EGFR/AKT pathway upregulated the expressions of acyl-CoA synthetase short-chain family member 3 (ACSS3), acyl-CoA synthetase long-chain family member 3 (ACSL3), and long-chain fatty acid elongation-related gene ELOVL fatty acid elongase 2 (ELOVL2) in an NF-κB-dependent manner. Moreover, inhibition of the mevalonate pathway reduced the EGFR level on the cell membranes, thereby affecting the signal transduction of the EGFR/AKT pathway. Therefore, targeting the EGFR/AKT and mevalonate pathways enhanced the antitumor effect of TMZ in GBM cells and animal models. CONCLUSIONS: Our findings not only uncovered the mechanism of metabolic reprogramming in EGFR-activated GBM but also provided a combinatorial therapeutic strategy for clinical GBM management.

Universal theranostic CRISPR/Cas13a RNA-editing system for glioma.

Background: The CRISPR/Cas13a system offers the advantages of rapidity, precision, high sensitivity, and programmability as a molecular diagnostic tool for critical illnesses. One of the salient features of CRISPR/Cas13a-based bioassays is its ability to recognize and cleave the target RNA specifically. Simple and efficient approaches for RNA manipulation would enrich our knowledge of disease-linked gene expression patterns and provide insights into their involvement in the underlying pathomechanism. However, only a few studies reported the Cas13a-based reporter system for in vivo molecular diagnoses. Methods: A tiled crRNA pool targeting a particular RNA transcript was generated, and the optimally potential crRNA candidates were selected using bioinformatics modeling and in vitro biological validation methods. For in vivo imaging assessment of the anti-GBM effectiveness, we exploited a human GBM patient-derived xenograft model in nude mice. Results: The most efficient crRNA sequence with a substantial cleavage impact on the target RNA as well as a potent collateral cleavage effect, was selected. In the xenografted GBM rodent model, the Cas13a-based reporter system enabled us in vivo imaging of the tumor growth. Furthermore, systemic treatments using this approach slowed tumor progression and increased the overall survival time in mice. Conclusions: Our work demonstrated the clinical potential of a Cas13a-based in vivo imaging method for the targeted degradation of specific RNAs in glioma cells, and suggested the feasibility of a tailored approach like Cas13a for the modulation of diagnosis and treatment options in glioma.

Prussian blue analogues-derived zero valent iron to efficiently activate peroxymonosulfate for phenol degradation triggered via reactive oxygen species and high-valent iron-oxo complexes.

It is of great significance to develop the effective technique to treat phenol-containing wastewater. Herein, Fe-based prussian blue analogues-derived zero valent iron (ZVI) was successfully synthesized by one-step calcination method. Owing to high specific surface area and rich active sites, ZVI-2 possessed excellent performance in charge transfer. Notably, in comparison with conventional ZVI and Fe2+, ZVI-2 can effectively activate peroxymonosulfate (PMS) for achieving rapid degradation of phenol, and the highest removal efficiency of phenol reached 94.9% within 24 min. More importantly, developed ZVI-2/PMS oxidation system with good stability displayed strong anti-interference capability. Interestingly, Fe0 loaded on the surface of ZVI-2 can efficiently break the O-O bond of PMS to generate reactive oxygen species (i.e., SO4•-, OH•, O2•- and 1O2). As main adsorption sites of PMS, the existence of oxygen vacancy promote the formation of high-valent transition metal complexes (namely ZVI-2≡Fe4+=O). Under the combined action of reactive oxygen species and ZVI-2≡Fe4+=O, phenol can be eventually degraded into CO2 and H2O. The possible degradation pathways of phenol were also investigated. Furthermore, proposed ZVI-2/PMS oxidation system displayed great potential for application in the field of wastewater treatment. All in all, current work provided a valuable reference for design and application of Fe-based catalysts in PS-AOPs.

Exosomes: Potential key players towards novel therapeutic options in diabetic wounds.

Diabetic wounds are usually difficult to heal, and wounds in foot in particular are often aggravated by infection, trauma, diabetic neuropathy, peripheral vascular disease and other factors, resulting in serious foot ulcers. The pathogenesis and clinical manifestations of diabetic wounds are complicated, and there is still a lack of objective and in-depth laboratory diagnosis and classification standards. Exosomes are nanoscale vesicles containing DNA, mRNA, microRNA, cyclic RNA, metabolites, lipids, cytoplasm and cell surface proteins, etc., which are involved in intercellular communication and play a crucial role in vascular regeneration, tissue repair and inflammation regulation in the process of diabetic wound healing. Here, we discussed exosomes of different cellular origins, such as diabetic wound-related fibroblasts (DWAF), adipose stem cells (ASCs), mesenchymal stem cells (MSCs), immune cells, platelets, human amniotic epithelial cells (hAECs), epidermal stem cells (ESCs), and their various molecular components. They exhibit multiple therapeutic effects during diabetic wound healing, including promoting cell proliferation and migration associated with wound healing, regulating macrophage polarization to inhibit inflammatory responses, promoting nerve repair, and promoting vascular renewal and accelerating wound vascularization. In addition, exosomes can be designed to deliver different therapeutic loads and have the ability to deliver them to the desired target. Therefore, exosomes may become an innovative target for precision therapeutics in diabetic wounds. In this review, we summarize the latest research on the role of exosomes in the healing of diabetic wound by regulating the pathogenesis of diabetic wounds, and discuss their potential applications in the precision treatment of diabetic wounds.

Identification of the E2F1-RAD51AP1 axis as a key factor in MGMT-methylated GBM TMZ resistance.

OBJECTIVE: Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme (GBM). Temozolomide (TMZ) is a standard chemotherapeutic for GBM, but TMZ treatment benefits are compromised by chemoresistance. This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance. METHODS: CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM. Western blot, real-time PCR, flow cytometry, and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1 (RAD51AP1). RESULTS: Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells. Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment. Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells. Knockdown of E2F1 increased sensitivity to TMZ. Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1, mediates TMZ resistance, and has a potential E2F1 binding site on the promoter. Knockdown of RAD51AP1 enhanced the sensitivity of TMZ; however, overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells. Furthermore, RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase (MGMT) expression. The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated, but not MGMT-unmethylated TMZ-treated GBM patients. CONCLUSIONS: Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment. RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair. Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.

EPIC-0307-mediated selective disruption of PRADX-EZH2 interaction and enhancement of temozolomide sensitivity to glioblastoma via inhibiting DNA repair and MGMT.

BACKGROUND: Temozolomide (TMZ) treatment efficacy in glioblastoma (GBM) has been limited by resistance. The level of O-6-methylguanine-DNA methyltransferase (MGMT) and intrinsic DNA damage repair factors are important for the TMZ response in patients. Here, we reported a novel compound, called EPIC-0307, that increased TMZ sensitivity by inhibiting specific DNA damage repair proteins and MGMT expression. METHODS: EPIC-0307 was derived by molecular docking screening. RNA immunoprecipitation (RIP), and chromatin immunoprecipitation by RNA (ChIRP) assays were used to verify the blocking effect. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) assays were performed to explore the mechanism of EPIC-0307. A series of in vivo and in vitro experiments were designed to evaluate the efficacy of EPIC-0307 in sensitizing GBM cells to TMZ. RESULTS: EPIC-0307 selectively disrupted the binding of PRADX to EZH2 and upregulated the expression of P21 and PUMA, leading to cell cycle arrest and apoptosis in GBM cells. EPIC-0307 exhibited a synergistic inhibitory effect on GBM when combined with TMZ by downregulating TMZ-induced DNA damage repair responses and epigenetically silencing MGMT expression through modulating the recruitment of ATF3-pSTAT3-HDAC1 regulatory complex to the MGMT promoter. EPIC-0307 demonstrated significant efficacy in suppressing the tumorigenesis of GBM cells, restoring TMZ sensitivity. CONCLUSION: This study identified a potential small-molecule inhibitor (SMI) EPIC-0307 that selectively disrupted the PRADX-EZH2 interaction to upregulate expressions of tumor suppressor genes, thereby exerting its antitumor effects on GBM cells. EPIC-0307 treatment also increased the chemotherapeutic efficacy of TMZ by epigenetically downregulating DNA repair-associate genes and MGMT expression in GBM cells.

Expert opinion on translational research for advanced glioblastoma treatment.

Malignant gliomas are known to be one of the most difficult diseases to diagnose and treat because of the infiltrative growth pattern, rapid progression, and poor prognosis. Many antitumor drugs are not ideal for the treatment of gliomas due to the blood-brain barrier. Temozolomide (TMZ) is a DNA alkylating agent that can cross the blood-brain barrier. As the only first-line chemotherapeutic drug for malignant gliomas at present, TMZ is widely utilized to provide a survival benefit; however, some patients are inherently insensitive to TMZ. In addition, patients could develop acquired resistance during TMZ treatment, which limits antitumor efficacy. To clarify the mechanism underlying TMZ resistance, numerous studies have provided multilevel solutions, such as improving the effective concentration of TMZ in tumors and developing novel small molecule drugs. This review discusses the in-depth mechanisms underlying TMZ drug resistance, thus aiming to provide possibilities for the establishment of personalized therapeutic strategies against malignant gliomas and the accelerated development and transformation of new targeted drugs.

Serum metabolomics strategy for investigating the hepatotoxicity induced by different exposure times and doses of Gynura segetum (Lour.) Merr. in rats based on GC-MS.

Gynura segetum (Lour.) Merr. (GS), has been widely used in Chinese folk medicine and can promote circulation, relieve pain and remove stasis. In recent years, the hepatotoxicity caused by GS has been reported, however its mechanism is not fully elucidated. Metabolomic techniques are powerful means to explore the toxicological mechanism and therapeutic effects of traditional Chinese medicine. The purpose of this study was to establish a serum metabolomics method based on Gas Chromatography-Mass Spectrometry (GC-MS) to explore the hepatotoxicity mechanism of different exposure times and doses of GS in rats. Sprague Dawley (SD) rats were administered daily with distilled water, 7.5 g kg-1 GS, or 15 g kg-1 GS by intragastrical gavage for either 10 or 21 days. The methods adopted included enzyme-linked immunosorbent assay (ELISA), Hematoxylin and Eosin (H&E) staining and GC-MS-based serum metabolomics. Serum biochemistry analysis showed that the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), total bilirubin (TBIL) and total bile acid (TBA) significantly (P < 0.05) increased while the levels of albumin (ALB) and high-density lipoprotein (HDL) significantly (P < 0.05) decreased in GS-treated groups, compared with the control group. Interestingly, the ALT, AST, TG and ALB levels changed in a time- and dose-dependent manner. The results of H&E staining showed the degree of liver damage after administration of GS gradually deepened with the extension of administration time and the increase of the dose. According to the results of metabolomics analysis, 26 differential metabolites were identified, which were involved in 8 metabolic pathways including phenylalanine metabolism, glyoxylic acid and dicarboxylic acid metabolism and so on. Meanwhile, the number of differential metabolites in different GS-treated groups was associated with GS exposure time and dose. Therefore, we concluded that GS might induce hepatotoxicity depending on the exposure time and dose.

MUC1 promotes glioblastoma progression and TMZ resistance by stabilizing EGFRvIII.

Epidermal growth factor receptor variant III (EGFRvIII) is a mutant isoform of EGFR with a deletion of exons 2-7 making it insensitive to EGF stimulation and downstream signal constitutive activation. However, the mechanism underlying the stability of EGFRvIII remains unclear. Based on CRISPR-Cas9 library screening, we found that mucin1 (MUC1) is essential for EGFRvIII glioma cell survival and temozolomide (TMZ) resistance. We revealed that MUC1-C was upregulated in EGFRvIII-positive cells, where it enhanced the stability of EGFRvIII. Knockdown of MUC1-C increased the colocalization of EGFRvIII and lysosomes. Upregulation of MUC1 occurred in an NF-κB dependent manner, and inhibition of the NF-κB pathway could interrupt the EGFRvIII-MUC1 feedback loop by inhibiting MUC1-C. In a previous report, we identified AC1Q3QWB (AQB), a small molecule that could inhibit the phosphorylation of NF-κB. By screening the structural analogs of AQB, we obtained EPIC-1027, which could inhibit the NF-κB pathway more effectively. EPIC-1027 disrupted the EGFRvIII-MUC1-C positive feedback loop in vitro and in vivo, inhibited glioma progression, and promoted sensitization to TMZ. In conclusion, we revealed the pivotal role of MUC1-C in stabilizing EGFRvIII in glioblastoma (GBM) and identified a small molecule, EPIC-1027, with great potential in GBM treatment.

Wounds under diabetic milieu: The role of immune cellar components and signaling pathways.

A major challenge in the field of diabetic wound healing is to confirm the body's intrinsic mechanism that could sense the immune system damage promptly and protect the wound from non-healing. Accumulating literature indicates that macrophage, a contributor to prolonged inflammation occurring at the wound site, might play such a role in hindering wound healing. Likewise, other immune cell dysfunctions, such as persistent neutrophils and T cell infection, may also lead to persistent oxidative stress and inflammatory reaction during diabetic wound healing. In this article, we discuss recent advances in the immune cellular components in wounds under the diabetic milieu, and the role of key signaling mechanisms that compromise the function of immune cells leading to persistent wound non-healing.

A novel compound EPIC-0412 reverses temozolomide resistance via inhibiting DNA repair/MGMT in glioblastoma.

BACKGROUND: Temozolomide (TMZ) resistance has become an important obstacle affecting its therapeutic benefits. O6-methylguanine DNA methyltransferase (MGMT) is primarily responsible for the TMZ resistance in Glioblastoma multiforme (GBM) patients. In addition, active DNA damage repair pathways can also lead to TMZ resistance. Here, we reported a novel small-molecule inhibitor EPIC-0412 that improved the therapeutic efficacy of TMZ by inhibiting the DNA damage repair pathway and MGMT in GBM via epigenetic pathways. METHODS: The small-molecule compound EPIC-0412 was obtained through high-throughput screening. RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), and chromatin immunoprecipitation (ChIP) assays were used to verify the effect of EPIC-0412. Co-immunoprecipitation (Co-IP) was used to elucidate the interactions of transcription factors at the MGMT promoter region. Animal experiments using a mouse model were performed to verify the efficacy of EPIC-0412 in sensitizing GBM cells to TMZ. RESULTS: EPIC-0412 physically interrupts the binding of HOTAIR and EZH2, leading to the upregulation of CDKN1A and BBC3, causing cell cycle arrest and apoptosis in GBM cells. EPIC-0412 inhibits DNA damage response in GBM cells through the p21-E2F1 DNA damage repair axis. EPIC-0412 epigenetically silences MGMT through its interaction with the ATF3-p-p65-HADC1 axis at the MGMT promoter region. The application of EPIC-0412 restored the TMZ sensitivity in GBM in vivo experiments. CONCLUSION: This study discovered a small-molecule inhibitor EPIC-0412, which enhanced the chemotherapeutic effect of TMZ by acting on the p21-E2F1 DNA damage repair axis and ATF3-p-p65-MGMT axis, providing evidence for combining epigenetic drugs to increase the sensitization toward TMZ in GBM patients.

Identification of potential targets of cinnamon for treatment against Alzheimer's disease-related GABAergic synaptic dysfunction using network pharmacology.

Cinnamon aqueous extract's active substance base remains unclear and its mechanisms, mainly the therapeutic target of anti-Alzheimer's disease (AD)-related GABAergic synaptic dysfunction, remain unclear. Here, 30 chemical components were identified in the aqueous extract of cinnamon using LC/MS; secondly, we explored the brain-targeting components of the aqueous extract of cinnamon, and 17 components had a good absorption due to the blood-brain barrier (BBB) limitation; thirdly, further clustering analysis of active ingredient targets by network pharmacology showed that the GABA pathway with GABRG2 as the core target was significantly enriched; then, we used prominent protein-protein interactions (PPI), relying on a protein-metabolite network, and identified the GABRA1, GABRB2 and GABRA5 as the closest targets to GABRG2; finally, the affinity between the target and its cognate active compound was predicted by molecular docking. In general, we screened five components, methyl cinnamate, propyl cinnamate, ( +)-procyanidin B2, procyanidin B1, and myristicin as the brain synapse-targeting active substances of cinnamon using a systematic strategy, and identified GABRA1, GABRB2, GABRA5 and GABRG2 as core therapeutic targets of cinnamon against Alzheimer's disease-related GABAergic synaptic dysfunction. Exploring the mechanism of cinnamon' activities through multi-components and multiple targets strategies promise to reduce the threat of single- target and symptom-based drug discovery failure.

The Exosomal miR-1246 of Laryngeal Squamous Cell Carcinoma Induces Polarization of M2 Type Macrophages and Promotes the Invasiveness of Laryngeal Squamous Cell Carcinoma.

Background: The possible role and detailed mechanisms of Tumor-associated macrophages (TAMs) in laryngeal squamous cell carcinoma (LSCC) have not been revealed. Methods: The expressions of typical markers were evaluated by qRT-PCR. In macrophages cocultured with TU212 cells, CD163, and CD206 protein expressions were detected by western blot analysis; IL-10 and IL-12 expressions were detected by ELISA assay. Exosomes isolated from TU212 cells were characterized by TEM analysis. As for the TU212 cells cocultured with macrophages processed with HOK or TU212 cells derived exosomes, their viability, migration, and invasion were assessed by CCK-8 assay, wounding healing, and Transwell assays, respectively. Results: In this study, macrophages processed with exosomes from human TU212 cells notably advanced LSCC cell viability, migration, and invasion. miR-1246 inhibitor suppressed the M2 polarization of macrophages. Macrophages transfected with miR-1246 inhibitor suppressed LSCC cell viability, migration, and invasion. Conclusion: In summary, our data implied that the exosomal, miR-1246 of LSCC, induced polarization of M2 type macrophages and promoted the progression of LSCC. This trial is registered with 2020-13.