Structurally diverse flavonoids from Selaginella doederleinii and their biological activity against laryngeal cancer.

BackgroundLaryngeal cancer is one of the major malignancies of the neck and head and is responsible for considerable mortality across the globe. The treatments for laryngeal cancer mainly involve surgical interventions followed by chemotherapy. However, due to unsatisfactory results, constant relapses and the adverse effects associated with the currently used drugs, there is pressing need to develop effective drug options for treatment of laryngeal cancer. Therefore, this study was undertaken to investigate the anticancer effects of a plant-derived alkaloid, Mukonal, against human AMC-HN-8 laryngeal cancer cells.Material/MethodsThe WST-1 and clonogenic assays were employed to determine the cell viability. Apoptosis was detected by Hoechst and AO/EB staining. Cell migration and cell cycle analysis was performed by Transwell assay and flow cytometry, respectively. Protein expression was examined by Western blotting.ResultsThe results revealed that Mukonal reduced the viability of laryngeal cancer cells dose-dependently. The IC50 of Mukonal was found to be 10 μM. However, the effects of Mukonal on the normal HuLa-PC cells was found to be 140 μM. The decrease in the viability of the AMC-HN-8 laryngeal cancer cells was found to be due to the induction of apoptosis and G2/M cell cycle arrest. Mukonal also suppressed the cell migration and of the AMC-HN-8 laryngeal cancer cells. Mukonal could also inhibit the PI3K/AKT and MEK/ERK signalling pathways in a concentration-dependent manner.ConclusionsTaken together, we conclude that Mukonal could prove a beneficial lead molecule for the treatment of laryngeal cancer.

The higher order structure (HOS) of monoclonal antibodies (mAbs) is an important quality attribute with strong contribution to clinically relevant biological functions and drug safety. Due to the multi-faceted nature of HOS, the synergy of multiple complementary analytical approaches can substantially improve the understanding, accuracy, and resolution of HOS characterization. In this study, we applied one- and two-dimensional (1D and 2D) nuclear magnetic resonance (NMR) spectroscopy coupled with chemometric analysis, as well as circular dichroism (CD), differential scanning calorimetry (DSC), and fluorescence spectroscopy as orthogonal methods, to characterize the impact of methionine (Met) oxidation on the HOS of an IgG1 mAb. We used a forced degradation method involving concentration-dependent oxidation by peracetic acid, in which Met oxidation is site-specifically quantified by liquid chromatography-mass spectrometry. Conventional biophysical techniques report nuanced results, in which CD detects no change to the secondary structure and little change in the tertiary structure. Yet, DSC measurements show the destabilization of Fab and Fc domains due to Met oxidation. More importantly, our study demonstrates that 1D and 2D NMR and chemometric analysis can provide semi-quantitative analysis of chemical modifications and resolve localized conformational changes with high sensitivity. Furthermore, we leveraged a novel 15N-Met labeling technique of the antibody to directly observe structural perturbations at the oxidation sites. The NMR methods described here to probe HOS changes are highly reliable and practical in biopharmaceutical characterization.

A general approach for structural interpretation of local disorder in partially ordered solids is proposed, combining high-resolution two-dimensional (2D) nuclear magnetic resonance (NMR) and first principles calculations. We show that small chemical shift variations of the order of a ppm can be interpreted in detailed structural terms with advanced density functional theory methods. Focusing on a model system of bisphosphinoamine, we demonstrate that the existence and the spatial range of small amplitude disorder can be probed using quantitative statistical analyses of 2D NMR line shapes obtained from through-space correlation experiments collected using variable mixing times. We show how low-energy vibration modes calculated from first principles can be conveniently used not as a cause of disorder but, instead, to generate a basis set of physically plausible local distortions to describe candidate static distributions of local geometries. Calculations of (31)P NMR isotropic chemical shifts are then used for the first time to simulate 2D correlation lineshapes associated with these distortions, which permit their evaluation as a potential source of disorder by comparison to experimental 2D cross-peaks between phosphorus sites. This new type of structural constraints allows the identification of changes in the bonding geometry that most likely contribute to the local structural disorder. We thus identify at least one type of structural deformation that is compatible with the experimental 2D NMR data and is also within the order of magnitude of the "thermal ellipsoids" associated with the uncertainties on the atomic positions of the X-ray diffraction structure.

MYCT1 is an important gene known to regulate cell viability and apoptosis of laryngeal cancer cells. However, the underlying molecular mechanism remains unclear. Here, we show that MAX enhances the expression of miR-181a by directly binding to its promoter, whereas miR-181a targets NPM1 and suppresses its expression in laryngeal cancer cells. MYCT1 and miR-181a decrease cell viability and colony formation through enhanced apoptosis, whereas NPM1 displays opposite effects in laryngeal cancer cells. Their opposing functions are further supported by the findings (a) that miR-181a is down-regulated, while NPM1 is up-regulated in laryngeal cancer, and (b) that either inhibition of miR-181a or overexpression of NPM1 can revert the pro-apoptotic effects of MYCT1 on laryngeal cancer cells through extracellular and intracellular apoptotic pathways. Our data suggest that MYCT1 may synergistically interact with MAX as a co-transcription factor or a component of MAX transcriptional complex, to transcriptionally regulate the expression of miR-181a, which, in turn, decreases NPM1 expression at post-transcriptional levels, leading to enhanced apoptosis in laryngeal cancer cells. These factors may serve as potential targets for early diagnosis and treatment of laryngeal cancer.

Laryngeal cancer (LC) is among the most prevalent tumors of the respiratory tract. In recent years, the implementation of non-surgical treatments like radiotherapy and chemotherapy has significantly enhanced the therapeutic outcomes for LC. Nevertheless, the underlying therapeutic mechanisms remain unclear, posing a hindrance to the progression of subsequent treatment strategies. To explore the potential mechanisms from existing effective treatments for LC and identify relevant targets, thereby providing guidance for subsequent therapeutic research on LC. This study focuses on ferroptosis, a common type of non-apoptotic cell death that is closely linked to various malignancies. It examines the relationship between ferroptosis and LC by analyzing how regulating ferroptosis-related targets in LC cells can influence the development of the cancer. There is a strong association between ferroptosis and LC. Regulating the targets related to ferroptosis in LC cells can effectively counteract the progression of LC. Taking ferroptosis as an entry point, analyzing its potential mechanism in inhibiting LC can provide a direction for the treatment of laryngeal cancer, which may contribute to the improvement of therapeutic strategies for this disease.

Cisplatin (CIS) is widely used in the treatment of laryngeal cancer, one of the most common and lethal cancers. However, it is not a satisfactory chemotherapeutic agent. Therefore, there is a need to identify new agents, such as gallic acid (GAL), that can exert a synergistic effect to elucidate the pathophysiological mechanisms of the chemotherapeutic effects of CIS and to increase the effectiveness of treatment by preventing drug resistance. For this purpose, we investigated the stimulatory role of GAL on CIS-induced human laryngeal cancer (Hep-2) cell death via TRPM2 channel activation. For the study, four groups were formed from human laryngeal cancer (Hep-2) cells as Control, GAL (1OO μM), CIS (25 μM), and GAL + CIS. In the analyses made, cell viability, glutathione (GSH) and glutathione peroxidase (GSH-Px) enzyme activity, lipid peroxidation (LPx) levels, inflammation markers I-1β, IL-6, and TNF-α, Total Oxidant/Antioxidant (TOS and TAS) status, reactive oxygen species (ROS), caspase (Cas-3-9) activity, Transient Receptor Potential Melastatin 2 (TRPM2), and Poly Adp Ribose Polymerase-1, (PARP-1) levels in the cells were determined. CIS treatment caused laryngeal cancer cell cytotoxic and increased Cas-3-9, ROS, IL-1β, TNF-α, IL-6, TOS, LPx, TRPM2, and PARP-1 levels while decreasing cell viability, GSH-Px, GSH, and TAS levels. The combination of GAL and CIS treatment made the treatment even more effective. In conclusion, the increase in ROS and cell death levels mediated by TRPM2 activation in CIS Hep-2 cells was further enhanced by GAL treatment. Thus, CIS chemotherapy in Hep-2 cells may be enhanced by the synergistic effect of the GAL combination, and drug resistance may be reduced.

Background:Laryngeal cancer is one of the most common malignant tumors among head and neck cancers. Accumulating studies have indicated that long noncoding RNAs (lncRNAs) play an important role in laryngeal cancer occurrence and progression, however, the functional roles and relative regulatory mechanisms of lncRNA growth arrest-specific transcript 5 (GAS5) in laryngeal cancer progression remain unclear.Methods:The expression of lncRNA GAS5 in both laryngeal cancer tissues and cell lines was evaluated using quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay. The relationships between lncRNA GAS5 expression and clinical parameters were also analyzed. To determine the biological function of lncRNA GAS5, a lncRNA GAS5-specific plasmid was first transfected into laryngeal cancer cells using lentiviral technology. Cell counting kit-8 assay, flow cytometry, and Transwell assays were used to detect in vitro cell proliferation, apoptosis, cycle distribution, and metastasis abilities, respectively. Furthermore, in vivo cell growth experiments were also performed using nude mice. Additionally, western blotting was performed to identify the underlying regulatory mechanism.Results:In the current study, lncRNA GAS5 was downregulated in laryngeal cancer tissues and its low expression was closely associated with poor tumor differentiation, advanced TNM stage, lymph node metastasis, and shorter overall survival time. In addition, lncRNA GAS5 upregulation significantly inhibited laryngeal cancer cell proliferation both in vitro and in vivo. Moreover, in response to lncRNA GAS5 overexpression, more laryngeal cancer cells were arrested at the G2/M stage, accompanied by increased cell apoptosis rates and suppressed migration and invasion capacities. Mechanistically, our data showed that the overexpression of lncRNA GAS5 significantly regulated the PI3K/AKT/mTOR signaling pathway.Conclusion:LncRNA GAS5 might act as a suppressor gene during laryngeal cancer development, as it suppressed cell proliferation and metastasis by regulating the PI3K/AKT/mTOR signaling pathway; thus, lncRNA GAS5 is a promising therapeutic biomarker for the treatment of laryngeal cancer.

The intense effort in developing new 2D NMR methodology over the past decade has been driven by the desire to study molecules of progressively greater complexity. The need for refined structural detail has produced new types of experiments that require more involvement on the part of the practicing spectroscopist in understanding the theoretical bases leading to their experimental realization. In this Review we discuss several concepts that are important in the successful application of current versions of the most useful 2D NMR experiments, such as coherence transfer, phase cycling, apodization functions, and obtaining pure-phase 2D NMR spectra. The intimate interconnections among these concepts are emphasized. The principles underlying the 2D NMR experiments are described and then the experiments are illustrated in assigning the 1H and 13C NMR spectra of the triterpene, ursolic acid.

This paper deals with the modern nuclear magnetic resonance (NMR) technique, socalled two-dimensional (2D) NMR spectroscopy. 2D-NMR has extended the range of applications of NMR spectroscopy, particularly to complex macromolecules such as proteins and nucleic acids. We focus the attention on the structure analysis of proteins by 2D-NMR spectroscopy and discuss the usefulness and limitation of this method.

Use of relaxation-edited one-dimensional and two dimensional nuclear magnetic resonance spectroscopy to improve detection of small metabolites in blood plasma

The in situ implementation of an electrochemical cell (EC) inside a nuclear magnetic resonance (NMR) spectrometer is extremely powerful to study redox reactions in real time and identify unstable reaction intermediates. Unfortunately, the implementation of an electrochemical device near the sensitive volume of an NMR probe significantly affects the quality of the NMR signal, inducing significant line broadening resulting in peak overlap and partial loss of the multiplet structures. Two-dimensional (2D) NMR spectroscopy allows one to bypass signal overlapping by spreading the peaks along two orthogonal dimensions, while providing precious information in terms of structural elucidation. Nevertheless, the acquisition of 2D NMR data suffers from long acquisition durations which are incompatible with fast redox processes taking place in solution. Here, we present a new approach to deal with this issue, consisting of coupling EC-NMR with ultrafast 2D spectroscopy, capable of recording 2D spectra much faster than conventional 2D NMR. This approach is applied to the real-time monitoring of a model reaction. Fast correlation spectroscopy (COSY) spectra are recorded every 3 min in the course of the 80 min reaction, leading to the unambiguous identification of one reaction intermediate and two reaction products. The evolution of 2D NMR peak volumes in the course of time provides further insight into the mechanism of this reaction involving an unstable intermediate. This study demonstrates the feasibility and the relevance of coupling in situ spectroelectrochemistry with ultrafast 2D spectroscopy to monitor real-time electrochemical reactions in the NMR tube.

Nuclear magnetic resonance (NMR) spectroscopy, also known as magnetic resonance spectroscopy, is a preeminent and noninvasive analytical technique that provides detailed information about the structure, dynamics, reaction state, and chemical environment of molecules. The development of NMR spectroscopy has led to the awarding of many Nobel Prizes, and today NMR spectroscopy serves as an important and irreplaceable tool in physics and chemistry. Two-dimensional (2D) NMR is effective at separating resonances which have similar chemical shifts, although the interpretation of 2D spectra can be challenging. A systematic density operator-based derivation will aid the understanding of the quantitative mechanism of 2D NMR spectroscopy and the interpreting of outcomes of 2D NMR experiments. Therefore, in this study, we systematically analyzed and compared the quantitative basis of 2D and 1D NMR. Meanwhile, as a proof of principle, simulations using the FID Appliance software toolkit were performed and interpreted using a brain phantom, a popular model for studying brain metabolites. The scheme shown in this paper will facilitate the understanding of quantitative 2D NMR spectroscopic analyses in chemistry and biology.

To investigate the potential effect of miR-363 on the development of laryngeal cancer and to reveal the relevant mechanism. The expression level of miR-363 was detected in laryngeal cancer tissues and cells (TU-177), respectively. Luciferase assay was performed to evaluate the interaction between miR-363 and myeloid cell leukemia-1 (Mcl-1). The effect of the miR-363/Mcl-1 axis on TU-177 cells was determined by subsequent experiments including cell proliferation, invasion, apoptosis and the expression level of Mcl-1. In the present study, we found that miR-363 was both repressed in laryngeal cancer tissues and cells (TU-177). To find the regulating target of miR-363, we searched three publicly available algorithms, including TargetScan, miRDB, and microRNA. Results showed that Mcl-1 was a direct target of miR-363, and the Luciferase assay confirmed our suggestion. Subsequent experiments indicated that the decreased expression of Mcl-1 resulting from the up-regulation of miR-363 could deaccelerate cell proliferation and invasion, and accelerate cell apoptosis in laryngeal cancer cells. Our research revealed the suppressed function of miR-363 in laryngeal cancer by targeting Mcl-1. Meanwhile, we found that the restoration of miR-363 could serve as a potential therapeutic strategy for the treatment of laryngeal cancer.

BackgroundStudies have revealed exosomes are implicated in tumor microenvironment and tumorigenesis. Emerging evidence suggests long non-coding RNAs (lncRNAs) possess pivotal roles in laryngeal cancer progression. For this study, we aimed to find out the mechanism of exosomes and lncRNA HOTAIR in laryngeal cancer.MethodsLaryngeal cancer cells-derived exosomes were initially extracted, separated and identified. Flow cytometry was applied to detect apoptosis to evaluate the effect of exosomes on cell radiosensitivity. Dual luciferase reporter gene assay, RNA pull-down and RNA immunoprecipitation assays were conducted to verify the interactions among HOTAIR, microRNA (miR)-454-3p and E2F2. The gain-and-loss functions of HOTAIR or miR-454-3p were carried out to explore their effects on TU212 and LLN cell viability, apoptosis and radiosensitivity. Levels of HOTAIR, miR-454-3p and E2F2 were detected after different treatments. An in vivo analysis was carried out in mice bearing laryngeal cancer xenografts.ResultsLaryngeal cancer-derived exosomes reduced laryngeal cancer cell radiosensitivity. HOTAIR expression was increased after cells were treated with exosome, and HOTAIR overexpression reduced laryngeal cancer cell radiosensitivity. Besides, HOTAIR worked as a competing endogenous RNA (ceRNA) of miR-454-3p to regulate E2F2 in laryngeal cancer cells. In vivo results were reproduced in in vivo studies, which demonstrated that HOTAIR knockdown reduced laryngeal cancer cell radiosensitivity by sponging miR-454-3p to silence E2F2.ConclusionExosome-mediated HOTAIR acts as a ceRNA of miR-545-3p to regulate E2F2, thereby negatively regulating the radiosensitivity of laryngeal cancer cells. This study may offer novel insight into laryngeal cancer treatment.

For the last fifty years nuclear magnetic resonance spectroscopy, generally referred as NMR, is one of the most versatile techniques for elucidation of structure of organic compounds. Among all available spectrometric methods, NMR is the only technique which offers a complete analysis and interpretation of the entire spectrum. Due to improved experimental technology and novel approaches, over the last decade nuclear magnetic resonance (NMR) has shown a tremendous progress. Generally, NMR spectroscopy makes use of three approaches; those are one dimension (1D), two dimensions (2D) and three dimensions (3D). Usually, the first approach of 1D-NMR (1H DEPT, 13C, 15N, 19F, 31P, etc.) generates good information about the structure of simple organic compounds, but in case of larger molecules the 1D-NMR spectra are generally overcrowded. Hence, the second approach of 2D-NMR (COSY, DQFCOSY, MQFCOSY, HETCOR, HSQC, HMQC, HMBC, TOCSY, NOESY, EXSY, etc.) is used for the further larger molecules, but 2D-NMR spectra also becomes complex and overlapping when used for further very large molecules like proteins. Hence, so as to achieve high resolution and reduced overlapping in spectra of very large molecules, Multi Dimensional-NMR (Homonuclear and Heteronuclear) are generally used. This paper supports interpretation of structure of different organic compounds by different NMR techniques.