Zebularine elevates STING expression and enhances cGAMP cancer immunotherapy in mice.

DNA methylation abnormality is closely related to tumor occurrence and development. Chemical inhibitors targeting DNA methyltransferase (DNMTis) have been used in treating cancer. However, the impact of DNMTis on antitumor immunity has not been well elucidated. In this study, we show that zebularine (a demethylating agent) treatment of cancer cells led to increased levels of interferon response in a cyclic guanosine monophosphate-AMP (cGAMP) synthase (cGAS)- and stimulator of interferon genes (STING)-dependent manner. This treatment also specifically sensitized the cGAS-STING pathway in response to DNA stimulation. Incorporation of zebularine into genomic DNA caused demethylation and elevated expression of a group of genes, including STING. Without causing DNA damage, zebularine led to accumulation of DNA species in the cytoplasm of treated cells. In syngeneic tumor models, administration of zebularine alone reduced tumor burden and extended mice survival. This effect synergized with cGAMP and immune checkpoint blockade therapy. The efficacy of zebularine was abolished in nude mice and in cGAS-/- or STING-/- mice, indicating its dependency on host immunity. Analysis of tumor cells indicates upregulation of interferon-stimulated genes (ISGs) following zebularine administration. Zebularine promoted infiltration of CD8 T cells and natural killer (NK) cells into tumor and therefore suppressed tumor growth. This study unveils the role of zebularine in sensitizing the cGAS-STING pathway to promote anti-tumor immunity and provides the foundation for further therapeutic development.

Zebularine potentiates anti-tumor immunity by inducing tumor immunogenicity and improving antigen processing through cGAS-STING pathway.

DNA methylation is an important epigenetic mechanism involved in the anti-tumor immune response, and DNA methyltransferase inhibitors (DNMTi) have achieved impressive therapeutic outcomes in patients with certain cancer types. However, it is unclear how inhibition of DNA methylation bridges the innate and adaptive immune responses to inhibit tumor growth. Here, we report that DNMTi zebularine reconstructs tumor immunogenicity, in turn promote dendritic cell maturation, antigen-presenting cell activity, tumor cell phagocytosis by APCs, and efficient T cell priming. Further in vivo and in vitro analyses reveal that zebularine stimulates cGAS-STING-NF-κB/IFNß signaling to enhance tumor cell immunogenicity and upregulate antigen processing and presentation machinery (AgPPM), which promotes effective CD4+ and CD8+ T cell-mediated killing of tumor cells. These findings support the use of combination regimens that include DNMTi and immunotherapy for cancer treatment.

Carrimycin ameliorates lipopolysaccharide and cecal ligation and puncture-induced sepsis in mice.

Carrimycin (CA), sanctioned by China's National Medical Products Administration (NMPA) in 2019 for treating acute bronchitis and sinusitis, has recently been observed to exhibit multifaceted biological activities, encompassing anti-inflammatory, antiviral, and anti-tumor properties. Despite these applications, its efficacy in sepsis treatment remains unexplored. This study introduces a novel function of CA, demonstrating its capacity to mitigate sepsis induced by lipopolysaccharide (LPS) and cecal ligation and puncture (CLP) in mice models. Our research employed in vitro assays, real-time quantitative polymerase chain reaction (RT-qPCR), and RNA-seq analysis to establish that CA significantly reduces the levels of pro-inflammatory cytokines, namely tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1ß), and interleukin 6 (IL-6), in response to LPS stimulation. Additionally, Western blotting and immunofluorescence assays revealed that CA impedes Nuclear Factor Kappa B (NF-κB) activation in LPS-stimulated RAW264.7 cells. Complementing these findings, in vivo experiments demonstrated that CA effectively alleviates LPS- and CLP-triggered organ inflammation in C57BL/6 mice. Further insights were gained through 16S sequencing, highlighting CA's pivotal role in enhancing gut microbiota diversity and modulating metabolic pathways, particularly by augmenting the production of short-chain fatty acids in mice subjected to CLP. Notably, a comparative analysis revealed that CA's anti-inflammatory efficacy surpasses that of equivalent doses of aspirin (ASP) and TIENAM. Collectively, these findings suggest that CA exhibits significant therapeutic potential in sepsis treatment. This discovery provides a foundational theoretical basis for the clinical application of CA in sepsis management.

Regulation of IFNß expression: focusing on the role of its promoter and transcription regulators.

IFNß is a single-copy gene without an intron. Under normal circumstances, it shows low or no expression in cells. It is upregulated only when the body needs it or is stimulated. Stimuli bind to the pattern recognition receptors (PRRs) and pass via various signaling pathways to several basic transcriptional regulators, such as IRFs, NF-кB, and AP-1. Subsequently, the transcriptional regulators enter the nucleus and bind to regulatory elements of the IFNß promoter. After various modifications, the position of the nucleosome is altered and the complex is assembled to activate the IFNß expression. However, IFNß regulation involves a complex network. For the study of immunity and diseases, it is important to understand how transcription factors bind to regulatory elements through specific forms, which elements in cells are involved in regulation, what regulation occurs during the assembly of enhancers and transcription complexes, and the possible regulatory mechanisms after transcription. Thus, this review focuses on the various regulatory mechanisms and elements involved in the activation of IFNß expression. In addition, we discuss the impact of this regulation in biology.

A drug-delivery depot for epigenetic modulation and enhanced cancer immunotherapy.

DNA methyltransferase inhibitors (DNMTis) have found widespread application in the management of cancer. Zebularine (Zeb), functioning as a demethylating agent, has exhibited notable advantages and enhanced therapeutic efficacy in the realm of tumour immunotherapy. Nevertheless, due to its lack of targeted functionality, standalone Zeb therapy necessitates the administration of a substantially higher dosage. In this investigation, we have devised an innovative nanodrug formulation, comprising the DNA methyltransferase inhibitor Zeb and pH-responsive chitosan (CS), hereinafter referred to as CS-Zeb nanoparticles (NPs). Our findings have unveiled that CS-Zeb NPs manifest heightened drug release within an acidic milieu (pH 5.5) in comparison to a neutral environment (pH 7.4). Furthermore, in vivo studies have conclusively affirmed that, in contrast to equivalent quantities of Zeb in isolation, the nanocomplex significantly curtailed tumour burden and protracted the survival duration of the B16F10 tumour-bearing murine model. Additionally, CS-Zeb NPs elicited an augmentation of CD8+ T cells within the peripheral circulation of mice and tumour-infiltrating lymphocytes (TILs). Notably, the dosage of CS-Zeb NPs was reduced by a remarkable 70-fold when juxtaposed with Zeb administered in isolation. To summarise, our study underscores the potential of CS-Zeb NPs as an alternative chemotherapeutic agent for cancer treatment.

Therapeutic Development by Targeting the cGAS-STING Pathway in Autoimmune Disease and Cancer.

DNA immune recognition regulation mediated by the cGAS-STING pathway plays an important role in immune functions. Under normal physiological conditions, cGAS can recognize and bind to invading pathogen DNA and activate the innate immune response. On the other hand, abnormal activation of cGAS or STING is closely related to autoimmune diseases. In addition, activation of STING proteins as a bridge connecting innate immunity and adaptive immunity can effectively restrain tumor growth. Therefore, targeting the cGAS-STING pathway can alleviate autoimmune symptoms and be a potential drug target for treating cancer. This article summarizes the current progress on cGAS-STING pathway modulators and lays the foundation for further investigating therapeutic development in autoimmune diseases and tumors.

Regulation of Gene Expression Associated With the N6-Methyladenosine (m6A) Enzyme System and Its Significance in Cancer.

N6-methyladenosine (m6A), an important RNA modification, is a reversible behavior catalyzed by methyltransferase complexes (m6A "writers"), demethylated transferases (m6A "erasers"), and binding proteins (m6A "readers"). It plays a vital regulatory role in biological functions, involving in a variety of physiological and pathological processes. The level of m6A will affect the RNA metabolism including the degradation of mRNA, and processing or translation of the modified RNA. Its abnormal changes will lead to disrupting the regulation of gene expression and promoting the occurrence of aberrant cell behavior. The abnormal expression of m6A enzyme system can be a crucial impact disturbing the abundance of m6A, thus affecting the expression of oncogenes or tumor suppressor genes in various types of cancer. In this review, we elucidate the special role of m6A "writers", "erasers", and "readers" in normal physiology, and how their altered expression affects the cell metabolism and promotes the occurrence of tumors. We also discuss the potential to target these enzymes for cancer diagnosis, prognosis, and the development of new therapies.

Compound C Reducing Interferon Expression by Inhibiting cGAMP Accumulation.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a major DNA sensor responsible for cytosolic DNA-mediated innate immune response. Inhibition of cGAS may be an effective strategy for treating autoimmune diseases such as Aicardi-Goutieres syndrome and systemic lupus erythematosus. Compound C (also known as Dorsomorphin) has been annotated as a potent and reversible inhibitor for AMPKs as well as ALK protein kinases. Here, we report a new function of Compound C which can suppress dsDNA-dependent type I interferon induction. These effects were not dependent on the activities of AMPK proteins. In vitro assays and liquid chromatograph-mass spectrometry data show that Compound C has the capability of reducing cGAMP accumulation, suggesting that Compound C may function as a modulator involved in the cGAS-STING-mediated DNA sensing pathway. Furthermore, Compound C is able to rescue the autoimmune phenotypes in a mouse model carrying the Trex1 gene deficiency. These data demonstrate a new and inverse correlation between Compound C and type I interferon production in response to dsDNA signaling.

A modified bisulfite conversion method for the detection of DNA methylation.

AIM: Our purpose is to improve the conventional procedures for bisulfite conversion used to detect 5-methylcytosine in DNA. METHODS: Impacts of different bisulfite salts, bisulfite conversion temperature, antioxidants and denaturants on DNA conversion and degradation were assessed by methylation-sensitive melt curve analysis. The modified method was tested on different genes and the conversion efficiency was analyzed by bisulfite sequencing. RESULTS: We developed a modified bisulfite conversion method that completes this process within 2 h. We demonstrate that high temperature denaturation is the major cause for DNA degradation, and the addition of ethylene glycol dimethyl ether is an effective way to accelerate the bisulfite conversion. The conversion efficiency is comparable to many other commercial kits. CONCLUSION: Our modified bisulfite conversion method is simple, cost efficient and less time consuming and is compatible with different genes and samples, thus has a great potential for the future research and clinical applications.

The relationship between DNA methylation and Reprimo gene expression in gastric cancer cells.

Reprimo (RPRM) is a tumor suppressor involved in the development of a number of malignant tumors including gastric cancer which is highly related to its gene hypermethylation. However, the regulation of RPRM gene expression by DNA methylation in gastric cancer is not well understood. We examined the RPRM gene methylation in gastric cancer tissues or plasma samples by bisulfite sequencing, and investigated the relationship between DNA methylation and the RPRM gene expression by quantitative reverse transcription-PCR and Western blotting. We found that the RPRM gene promoter region is hypermethylated in gastric cancer tissues (75%, 45/60), plasma samples (86.3%, 44/51) and various cancer cell lines (75%, 3/4), which is correlated with the decrease of RPRM gene expression. The hypermethylation-induced RPRM reduction can be recovered by treating with zebularine, a demethylating agent, and by inhibition of the DNA methyltransferases via RNA interference and CRISPR/Cas9-mediated gene knockout. In addition, we generated RPRM gene-knockout cells and studied the effects of the RPRM deficiency on tumor formation by inoculating these cells in mice. The data show that the loss of RPRM can promote tumorigenesis. These data suggest that the RPRM expression is inhibited by DNA methyltransferases and the RPRM normal function can be restored by treating with DNA methylation inhibitors. The study provides important information regarding the role of RPRM and its methylation related to gastric cancer development.

Methylation-Sensitive Melt Curve Analysis of the Reprimo Gene Methylation in Gastric Cancer.

Reprimo (RPRM) is a p53-induced tumor suppressor gene. Its aberrant DNA methylation is correlated with carcinogenesis and may be used as a surrogate marker for the early detection of gastric cancer. However, the detail information regarding its DNA methylation has not been revealed. Here, we investigated the RPRM gene methylation in gastric cancer tumor and plasma samples by methylation-sensitive melt curve analysis (MS-MCA) and bisulfite sequencing in depth. We developed a semi-quantitative method based on MS-MCA for detecting DNA methylation and unraveled the RPRM gene methylation pattern in gastric cancer. This study provides a solid foundation for the future application of detecting RPRM gene methylation in human plasma or serum samples to help diagnose gastric cancer or for prognosis evaluation.