Cucurbitacin B induces ferroptosis in oral leukoplakia via the SLC7A11/mitochondrial oxidative stress pathway.

BACKGROUND: Oral leukoplakia (OLK), characterized by abnormal epithelial hyperplasia, is the most common precancerous oral mucosa lesion and is closely related to oxidative stress. Cucurbitacin B (CuB), a tetracyclic triterpenoid molecule derived from plants, has shown promising anti-proliferative and antioxidant effects in preclinical studies. However, whether CuB can play an antiproliferative role in OLK by regulating oxidative stress remains elusive. PURPOSE: To investigate the role of CuB in inhibiting the malignant progression of oral leukoplakia and to further explore its underlying mechanisms of action. METHODS: In vitro, the effect of CuB on the proliferation, migration, apoptosis, and cell cycle of OLK cells DOK was detected. The core genes and key pathways of OLK and CuB were analyzed in the transcriptome database, by using immunofluorescence, qRT-PCR, and Western blot to evaluate the expression levels of the ferroptosis markers ROS, GSH, MDA, Fe2+, and marker genes SLC7A11, GPX4, and FTH1. Immunohistochemistry of human tissue was performed to investigate the expression of the SLC7A11. In vivo, the model of OLK was established in C57BL/6 mice and the biosafety of CuB treatment for OLK was further evaluated. RESULTS: CuB substantially suppressed the proliferation of DOK cells. Bioinformatics analysis showed that the core targets of OLK crossing with CuB include SLC7A11 and that the essential pathways involve ROS and ferroptosis. In vitro experiments indicated that CuB might promote ferroptosis by down-regulating the expression of SLC7A11. We observed a gradual increase in SLC7A11 expression levels during the progression from normal oral mucosa to oral leukoplakia with varying degrees of epithelial dysplasia. In vivo experiments demonstrated that CuB inhibited the malignant progression of OLK by promoting ferroptosis in OLK mice and exhibited a certain level of biosafety. CONCLUSION: This study demonstrated for the first time that CuB could effectively inhibit the malignant progression of OLK by inducing ferroptosis via activating the SLC7A11/ mitochondrial oxidative stress pathway. These findings indicate that CuB could serve as the lead compound for the future development of anti-oral leukoplakia drugs.

Cell-free chitosan/silk fibroin/bioactive glass scaffolds with radial pore for in situ inductive regeneration of critical-size bone defects.

Tissue-engineered is an effective method for repairing critical-size bone defects. The application of bioactive scaffold provides artificial matrix and suitable microenvironment for cell recruitment and extracellular matrix deposition, which can effectively accelerate the process of tissue regeneration. Among various scaffold properties, appropriate pore structure and distribution have been proven to play a crucial role in inducing cell infiltration differentiation and in-situ tissue regeneration. In this study, a chitosan (CS) /silk fibroin (SF) /bioactive glass (BG) composite scaffold with distinctive radially oriented pore structure was constructed. The composite scaffolds had stable physical and chemical properties, a unique pore structure of radial arrangement from the center to the periphery and excellent mechanical properties. In vitro biological studies indicated that the CS/SF/BG scaffold could promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and the expression of related genes due to the wide range of connected pore structures and released active elements. Furthermore, in vivo study showed CS/SF/BG scaffold with radial pores was more conducive to the repair of skull defects in rats with accelerated healing speed during the bone tissue remodeling process. These results demonstrated the developed CS/SF/BG scaffold would be a promising therapeutic strategy for the repair of bone defects regeneration.

Bioinformatic analysis and experimental validation of cuproptosis-related LncRNA as a novel biomarker for prognosis and immunotherapy of oral squamous cell carcinoma.

BACKGROUND: The novel form of regulatory cell death, cuproptosis, is characterized by proteotoxicity, which ultimately leads to cell death. Its targeting has emerged as a promising therapeutic approach for oral squamous cell carcinoma (OSCC). Long noncoding RNAs (lncRNAs) participate in epigenetic regulation and have been linked to the progression, prognosis, and treatment of OSCC. Thus, this study aimed to identify new cuproptosis-related lncRNAs (CRLs), establish predictive models for clinical prognosis, immune response, and drug sensitivity, and provide novel insights into immune escape and tumor drug resistance. METHODS: The present study screened eight CRLs (THAP9-AS1, STARD4-AS1, WDFY3-AS2, LINC00847, CDKN2A-DT, AL132800.1, GCC2-AS1, AC005746.1) using Lasso Cox regression analysis to develop an eight-CRL prognostic model. Patients were categorized into high- and low-risk groups using risk scores. To evaluate the predictive ability of the model, Kaplan-Meier analysis, ROC curves, and nomograms were employed. Furthermore, the study investigated the differences in immune function and anticancer drug sensitivity between the high- and low-risk groups. To validate the expression of CRLs in the model, OSCC cell lines were subjected to quantitative real-time fluorescence PCR (qRT-PCR). RESULTS: The results of the study showed that the high-risk group had a shorter overall survival (OS) time in OSCC patients. Cox regression analysis demonstrated that the high-risk score was an independent risk factor for a poor prognosis. The validity of the model was confirmed using ROC curve analysis, and a nomogram was developed to predict the prognosis of OSCC patients. Furthermore, patients in the high-risk group with high TMB had a poorer prognosis. Patients in the low-risk group responded better to immunotherapy than those in the high-risk group. Additionally, the risk scores were significantly associated with drug sensitivity in OSCC patients. Finally, the findings of qRT-PCR supported the reliability of the proposed risk model. CONCLUSION: The study identified and established the 8-CRL model, which represents a novel pathway of lncRNA regulation of cuproptosis in OSCC. This model provides guidance for the prognosis and treatment of OSCC and offers a new insight into immune escape and tumor drug resistance.

Network pharmacological analysis and experimental study of cucurbitacin B in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a malignant tumor with a high incidence and poor prognosis. Cucurbitacin B (CuB) is a tetracyclic triterpenoid small-molecule compound extracted from plants, such as Cucurbitaceae and Brassicaceae, which has powerful anticancer effects. However, the effect and mechanism of CuB on OSCC remain unclear. Within the framework of the current study, network pharmacology was used to analyze the relationship between CuB and OSCC. The network pharmacology analysis showed that CuB and OSCC share 134 common targets; among them, PIK3R1, SRC, STAT3, AKT1, and MAPK1 are the key targets. The molecular docking analysis showed that CuB binds five target proteins. The results of the enrichment analysis showed that CuB exerted effects on OSCC through various pathways; of these pathways, PI3K-AKT was the most important pathway. The results of the in vitro cell experiments showed that CuB could inhibit the proliferation and migration of SCC25 and CAL27 cells, block the cell cycle in the G2 phase, induce cell apoptosis, and regulate the protein expression of the PI3K-AKT signaling pathway. The results of the in vivo animal experiments showed that CuB could inhibit 4NQO-induced oral cancer in mice. Therefore, network pharmacology, molecular docking, cell experiments, and animal experiments showed that CuB could play a role in OSCC by regulating multiple targets and pathways.

Erratum: LncRNA-MALAT1, as a biomarker of neonatal BPD, exacerbates the pathogenesis of BPD by targeting miR-206.

[This corrects the article on p. 462 in vol. 13, PMID: 33594304.].

Exploring the Mechanism of Scutellaria baicalensis Georgi Efficacy against Oral Squamous Cell Carcinoma Based on Network Pharmacology and Molecular Docking Analysis.

BACKGROUND: Scutellaria baicalensis Georgi (SBG) has been widely shown to induce apoptosis and inhibit invasion and migration of various cancer cells. Increased evidence shows that SBG may be useful to treat oral squamous cell carcinoma (OSCC). However, the biological activity and possible mechanisms of SBG in the treatment of OSCC have not been fully elucidated. This study aimed to clarify the bioactive component and multitarget mechanisms of SBG against OSCC using network pharmacology and molecular docking. METHODS: Traditional Chinese Medicine Systems Pharmacology (TCMSP) database was used to predict the active components in SBG, and putative molecular targets of SBG were identified using the Swiss Target Prediction database. OSCC-related targets were screened by GeneCards, Online Mendelian Inheritance in Man (OMIM), and Therapeutic Target Database (TTD). Then, we established protein-protein interaction (PPI), compound-target-disease (C-T-D), and compound-target-pathway (C-T-P) networks by Cytoscape to identify the main components, core targets, and pharmacological pathways of SBG against OSCC via applying data mining techniques and topological parameters. Metascape database was utilized for Gene Ontology (GO) and pathway enrichment analysis. The potential interaction of the main components with core targets was revealed by molecular docking simulation, and for the correlation between core targets and OSCC prognosis analysis, the Kaplan-Meier Plotter online database was used. RESULTS: There were 25 active compounds in SBG and 86 genes targeted by OSCC. A total of 141 signaling pathways were identified, and it was found that the PI3K-Akt signaling pathway may occupy core status in the anti-OSCC system. GO analysis revealed that the primary biological processes were related to apoptosis, proliferation, and migration. Molecular docking results confirmed that core targets of OSCC had a high affinity with the main compounds of SBG. CONCLUSION: Our study demonstrated multicomponent, multitarget, and multipathway characteristics of SBG in the treatment of OSCC and provided a foundation for further drug development research.

LncRNA-MALAT1, as a biomarker of neonatal BPD, exacerbates the pathogenesis of BPD by targeting miR-206.

Neonatal bronchopulmonary dysplasia (BPD) is one of the common causes of premature birth complications, which is caused by lung dysplasia. Long non-coding RNA (LncRNA) has been proved to be related to BPD and other disease processes, but the molecular mechanism of metastasis-related lung adenocarcinoma transcript 1 (MALAT1) in BPD has not been fully understood. This study focused on exploring the clinical and molecular mechanism of MALAT1 in neonatal BPD, aiming to provide new insights for the management of neonatal BPD. In our study, we first found that serum MALAT1 was up-regulated in neonatal BPD and severe BPD. Further, through receiver operating characteristic curve (ROC) analysis, it was found that the area under the curve of MALAT1 for differentiating neonatal BPD from severe BPD was 0.943 and 0.866, respectively. Then, we established BPD models in vivo and in vitro with C57BL/6J mice and BEAS-2B cells, and found that MALAT1 was also highly expressed in them and increased with the induction time of the models. Pathological evaluation confirmed that down-regulating MALAT1 or up-regulating miR-206 might improve the pathological condition of BPD. Obvious inflammatory response, oxidative stress and up-regulated apoptosis were observed in BPD models in vivo and in vitro. However, after MALAT1 knockdown treatment, the above abnormal phenomena were alleviated to varying degrees. Furthermore, we also found that MALAT1 has a targeted relationship with miR-206, and miR-206 is down-regulated in BPD in vivo and in vitro. Down-regulating miR-206 could also eliminate the anti-BPD effect after knocking down MALAT1. The above results indicated that MALAT1 has the potential as a blood biomarker of neonatal BPD, and MALAT1-miR-206 axis mediates BPD process, which may be a new target for neonatal BPD treatment.

Bioinformatic analysis revealing mitotic spindle assembly regulated NDC80 and MAD2L1 as prognostic biomarkers in non-small cell lung cancer development.

BACKGROUND: Lung cancer has been the leading cause of tumor related death, and 80% ~ 85% of it is non-small cell lung cancer (NSCLC). Even with the rising molecular targeted therapies, for example EGFR, ROS1 and ALK, the treatment is still challenging. The study is to identify credible responsible genes during the development of NSCLC using bioinformatic analysis, developing new prognostic biomarkers and potential gene targets to the disease. METHODS: Firstly, three genes expression profiles GSE44077, GSE18842 and GSE33532 were picked from Gene Expression Omnibus (GEO) to analyze the genes with different expression level (GDEs) between NSCLC and normal lung samples, and the cellular location, molecular function and the biology pathways the GDEs enriched in were analyzed. Then, gene function modules of GDEs were explored based on the protein-protein interaction network (PPI), and the top module which contains most genes was identified, followed by containing genes annotation and survival analysis. Moreover, multivariate cox regression analysis was performed in addition to the Kaplan meier survival to narrow down the key genes scale. Further, the clinical pathological features of the picked key genes were explored using TCGA data. RESULTS: Three GEO profiles shared a total of 664 GDEs, including 232 up-regulated and 432 down-regulated genes. Based on the GDEs PPI network, the top function module containing a total of 69 genes was identified, and 31 of 69 genes were mitotic cell cycle regulation related. And survival analysis of the 31 genes revealed that 17/31 genes statistical significantly related to NSCLC overall survival, including 4 spindle assembly checkpoints, namely NDC80, BUB1B, MAD2L1 and AURKA. Further, multivariate cox regression analysis identified NDC80 and MAD2L1 as independent prognostic indicators in lung adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) respectively. Interestingly, pearson correlation analysis indicated strong connection between the four genes NDC80, BUB1B, MAD2L1 and AURKA, and their clinical pathological features were addressed. CONCLUSIONS: Using bioinformatic analysis of GEO combined with TCGA data, we revealed two independent prognostic indicators in LUAD and LUSC respectively and analyzed their clinical features. However, more detailed experiments and clinical trials are needed to verify their drug targets role in clinical medical use.

Nek2B activates the wnt pathway and promotes triple-negative breast cancer chemothezrapy-resistance by stabilizing ß-catenin.

BACKGROUND: The chemotherapy-resistance of triple-negative breast cancer (TNBC) remains a major challenge. The Nek2B kinase and ß-catenin serve as crucial regulators of mitotic processes. The aim of this study was to test the correlation between Nek2B and TNBC chemotherapy sensitivity, and to determine the regulation of Nek2B on ß-catenin and wnt/ß-catenin signal pathway. METHODS: Gene Expression Omnibus(GEO) databases were used to gather gene exprsssion data of TNBC patients who undergoing chemotherapy. The co-expression of Nek2B and ß-catenin in TNBC surgical sections and cells were analysed by immunohistochemistry, Q-RT-PCR, Western-blot and immunofluorescent staining. The impact of the expression of Nek2B and ß-catenin in prognosis was also assessed using the Kaplan-Meier curves. CCK8 assay was used to detect the IC50 value of TNBC cell line. The endogenous binding capacity of Nek2B and ß-catenin and phosphorylation of ß-catenin by Nek2B were detected using co-immunoprecipitation (CO-IP). Chromatin immune-precipitation (ChIP) analysis and Luciferase Assays were used to evaluate the binding ability of the Nek2B, ß-catenin and TCF4 complex with LEF-1 promoter. Nek2B-siRNA and Nek2B plasmid were injected into nude mice, and tumorigenesis was monitored. RESULTS: We found that overexpression of Nek2B and ß-catenin in TNBC samples, was associated with patients poor prognosis. Patients with positive Nek2B expression were less sensitive to paclitaxel-containing neoadjuvant chemotherapy. Interestingly, in a panel of established TNBC cell line, Nek2B and ß-catenin were highly expressed in cells exhibiting paclitaxel resistance. Our data also suggest that ß-catenin binded to and was phosphorylated by Nek2B, and was in a complex with TCF4. Nek2B mainly regulates the expression of ß-catenin in TNBC nucleus. Nek2B, ß-catenin and TCF4 can be binded with the WRE functional area of LEF-1 promoter. Nek2B can activite wnt signaling pathway and wnt downstream target genes. The tumors treated by Nek2B siRNA associated with paclitaxel were the smallest in nude mouse, and Nek2B can regulate the expression of ß-catenin and wnt downstream target genes in vivo. CONCLUSION: Our study suggested that Nek2B can bind to ß-catenin and the co-expression correlated with TNBC patients poor prognosis. It appears that Nek2B and ß-catenin might synergize to promote chemotherapy resistance.

Monitoring autophagy in live cells with a fluorescent light-up probe for G-quadruplex structures.

Monitoring autophagy can provide valuable insights into understanding human pathological mechanisms, developing novel drugs, and exploring autophagy control approaches. Here, we proposed a new strategy to specifically monitor autophagy by lighting up the G-quadruplex structures entering autolysosomes. Based on this strategy, we designed a small-molecule fluorescent probe for autophagy imaging. This work not only opens up a new way for developing autophagy probes, but also provides an effective tool for autophagy research.

Chiral transformation of cyanine dye aggregates induced by small peptides.

Three small peptides (K4, K5, and K6) with different length were designed to induce the transformation of the assembled state and the chirality of cyanine dye supramolecule. The absorption and circular dichroism (CD) results indicated that, the peptides tend to induce cyanine dye to H-aggregation, competed with Na(+) in PBS, which would induce dye to J-aggregation. Meanwhile, all three peptides could influence the chirality of both J-aggregates induced by Na(+) and H-aggregates, among which K6 could induce chiral reversion of J-aggregates. Furthermore, molecular modeling and energy calculation results have shown that the peptides with different chain length have different conformations. This might be the reason for cyanine dye to form the different chiral assembly induced by these oligo-peptide templates.

Spectroscopic investigation on the interaction of J-aggregate with human serum albumin.

The interactions of three cyanine dyes, which exhibit different meso substituent in polymethine chain, with human serum albumin (HSA) have been investigated by the means of absorption, fluorescence and circular dichroism (CD) spectra. In phosphate buffer solution (PBS), the mentioned dyes exist not as isolated monomers but rather in the formation of J-aggregation. In the presence of HSA, the absorption and fluorescence emission spectra indicated that the J-aggregation was decomposed to monomer because of the strong affinity between dye molecules and HSA. Besides the association of cyanine dyes with HSA, binding to HSA gave rise to the J-aggregation CD signals. The meso substituent in the polymethine plays an important role in the interaction of HSA and the J-aggregation. Spectral studies showed that the dye bound with HSA in a 1:1 formation. The apparent constant (K(a)) value was roughly identified by analysis of the corresponding fluorescence data at various HSA concentrations. The higher affinity of the molecule with meso phenyl towards HSA with respect to molecules with meso ethyl or methyl can be attributed to the arrangement of molecules in J-aggregation and the hydrophobic force between the molecules and HSA.

Effects of NaCl on the J-aggregation of two thiacarbocyanine dyes in aqueous solutions.

The effects of NaCl on the aggregation of two typical thiacarbocyanine dyes (3,3'-di(3-sulfopropyl)-4,5,4',5'-dibenzo-9-phenyl-thiacarbocyanine triethyl ammonium salt (Dye 1) and 3,3'-di(3-sulfopropyl)-4,5,4',5'-dibenzo-9-methyl-thiacarbocyanine triethyl ammonium salt (Dye 2)) in aqueous solution have been studied by using absorption spectroscopy, fluorescence spectroscopy, and 1H- and 23Na-NMR measurements. It is found that the J-aggregation of two dyes can be promoted by the addition of NaCl and that the effective coherence length of the J-aggregate is shorter than that obtained without NaCl. Fluorescence spectra demonstrate that the fluorescence intensities of the J-aggregates of two dyes are quenched by addition of NaCl. This is consistent with the decrease of the effective coherence length of J-aggregates of the two dyes in the presence of NaCl. 1H-NMR spectra of two dyes show that the Na(+) ions penetrate into the J-aggregates and replace the counterion (triethylammonium ions) in two dyes. The measurements of the chemical shifts of 23Na nuclei provide further information about the interaction between the Na(+) ions and dye anions in the J-aggregates of the two dyes. Due to this interaction, the electrostatic repulsion between the dye anions in the J-aggregates can be reduced and thus accelerate the aggregation of the two dyes in the presence of NaCl. The apparent association constants between Na(+) ions and dye molecules in J-aggregates of Dye 1 and Dye 2 estimated from the measured chemical shifts of 23Na nuclei are about 2.38 M(-1) and 1.35 M(-1), respectively.

Photoinduced electron transfer from the excited J-aggregate state of a thiacarbocyanine dye to TiO2 colloids.

The photophysical properties of the J-aggregate of 3,3'-di(3-sulfopropyl)-4,5,4',5'-dibenzo-9-phenyl-thiacarbocyanine triethyl-ammonium salt in the absence and presence of TiO(2) colloids have been studied using UV-visible absorption spectroscopy, steady-state and time-resolved fluorescence spectroscopy, and ESR spectroscopy. The fluorescence emission of the J-aggregate decreases with increasing concentration of TiO(2) colloids. The average fluorescence lifetime of the J-aggregate in the presence of TiO(2) colloids is shorter than that in the absence of TiO(2) colloids. A strong photoinduced ESR signal has been observed during illumination by light with lambda=633 nm in the presence of TiO(2) and the ESR signal can be attributed to the J-aggregate radical cation. From the above results, it is concluded that photoinduced electron transfer from the excited singlet state of the J-aggregate to the conduction band of TiO(2) takes place and the electron transfer rate is about 1.5 x 10(8) s(-1).