Nimbolide Exhibits Potent Anticancer Activity Through ROS-Mediated ER Stress and DNA Damage in Human Non-small Cell Lung Cancer Cells.

The non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers. It is usually diagnosed at an advanced stage with poor prognosis. Nimbolide (NB), a terpenoid limonoid isolated from the flowers and leaves of neem tree, possesses anticancer properties in various cancer cell lines. However, the underlying mechanism of its anticancer effect on human NSCLC cells remains unclear. In the present study, we investigated the effect of NB on A549 human NSCLC cells. We found that NB treatment inhibits A549 cells colony formation in a dose-dependent manner. Mechanistically, NB treatment increases cellular reactive oxygen species (ROS) level, leading to endoplasmic reticulum (ER) stress, DNA damage, and eventually induction of apoptosis in NSCLC cells. Furthermore, all these effects of NB were blocked by pretreatment with antioxidant glutathione (GSH), the specific ROS inhibitor. We further knockdown CHOP protein by siRNA markedly reduced NB-induced apoptosis in A549 cells. Taken together, our findings reveal that NB is an inducer of ER stress and ROS; these findings may contribute to increasing the therapeutic efficiency of NSCLC.

Effects of different signaling pathways on odontogenic differentiation of dental pulp stem cells: a review.

Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cells that can differentiate into odontoblast-like cells and protect the pulp. The differentiation of DPSCs can be influenced by biomaterials or growth factors that activate different signaling pathways in vitro or in vivo. In this review, we summarized six major pathways involved in the odontogenic differentiation of DPSCs, Wnt signaling pathways, Smad signaling pathways, MAPK signaling pathways, NF-kB signaling pathways, PI3K/AKT/mTOR signaling pathways, and Notch signaling pathways. Various factors can influence the odontogenic differentiation of DPSCs through one or more signaling pathways. By understanding the interactions between these signaling pathways, we can expand our knowledge of the mechanisms underlying the regeneration of the pulp-dentin complex.

Er:YAG laser therapy in combination with GLUMA desensitizer reduces dentin hypersensitivity in children with molar-incisor hypomineralization: a randomized clinical trial.

This study aimed to investigate the effectiveness of erbium-doped yttrium garnet (Er:YAG) laser and GLUMA desensitizer for dentin hypersensitivity in teeth affected by Molar-Incisor Hypomineralization (MIH). One hundred twenty children were randomly allocated to four groups: the control (Co) group, the desensitizer (De) group, the laser (La) group, and the laser + desensitizer (La + De) group. Outcome measures included Visual Analogue Scale (VAS) and 14-item Oral Health Impact Profile (OHIP-14) evaluation. For mean VAS scores, a significant reduction was found over time in all groups. Co and De groups, Co and La groups, Co and La + De groups, De and La + De groups, and La and La + De groups differed significantly (p < 0.05). For mean scores in all dimensions of OHIP-14 after treatment 6 months, the La + De group was significantly lower (p < 0.001). The La + De groups and the La groups as well as the La + De groups and the De groups differed significantly in total OHIP, functional limitation, physical disability, and psychological disability (p < 0.05). Physical pain between the La + De groups and the La groups and handicap between the La + De groups and De groups differed significantly (p < 0.05). The mean values of each dimension differed significantly between the group Co and the La + De group (p < 0.0001). Combination therapy of Er:YAG laser and GLUMA desensitizer had greater desensitizing effects and oral health-related quality improvement of life, which might be an effective alternative treatment in dentin hypersensitivity in MIH children.

BPDE-DNA adduct formation and alterations of mRNA, protein, and DNA methylation of CYP1A1, GSTP1, and GSTM1 induced by benzo[a]pyrene and the intervention of aspirin in mice.

Benzo[a]pyrene (B[a]P), one typical environmental pollutant, the toxicity mechanisms, and potential prevention remain perplexing. Available evidence suggests cytochrome P450 1A1 (CYP1A1) and glutathione S-transferases (GSTs) metabolize B[a]P, resulting in metabolic activation and detoxification of B[a]P. This study aimed to reveal the impact of B[a]P exposure on trans-7,8-diol-anti-9,10-epoxide DNA (BPDE-DNA) adduct formation, level of CYP1A1, glutathione S-transferase pi (GSTP1) and glutathione S-transferase mu1 (GSTM1) mRNA, protein and DNA methylation in mice, and the potential prevention of aspirin (ASP). This study firstly determined the BPDE-DNA adduct formation in an acute toxicity test of a large dose in mice induced by B[a]P, which subsequently detected CYP1A1, GSTP1, and GSTM1 at levels of mRNA, protein, and DNA methylation in the organs of mice in a subacute toxicity test at appropriate doses and the potential prevention of ASP, using the methods of real-time quantitative PCR (QPCR), western blotting, and real-time methylation-specific PCR (MSP), respectively. The results verified that B[a]P induced the formation of BPDE-DNA adduct in all the organs of mice in an acute toxicity test, and the order of concentration of which was lung > kidney > liver > brain. In a subacute toxicity test, following B[a]P treatment, mice showed a dose-dependent slowdown in body weight gain and abnormalities in behavioral and cognitive function and which were alleviated by ASP co-treatment. Compared to the controls, following B[a]P treatment, CYP1A1 was significantly induced in all organs in mice at mRNA level (P < 0.05), was suppressed in the lung and cerebrum of mice at protein level, and inhibited at DNA methylation level in the liver, lung, and cerebrum, whereas GSTP1 and GSTM1 at mRNA, protein, and DNA methylation levels showed organ-specific changes in mice following B[a]P treatment, which was generally alleviated by ASP intervention. In conclusion, B[a]P induced BPDE-DNA adduct formation in all organs in mice and altered the mRNA, protein, and DNA methylation levels in CYP1A1, GSTP1, and GSTM1 in an organ-dependent pattern, which could be related to the organ toxicity and mechanism of B[a]P. ASP intervention may be an effective measure to prevent B[a]P toxicity. The findings provide scientific evidence for further study on the organ toxicity and mechanisms of B[a]P.

Single-cell analyses reveal cannabidiol rewires tumor microenvironment via inhibiting alternative activation of macrophage and synergizes with anti-PD-1 in colon cancer.

Colorectal tumors often create an immunosuppressive microenvironment that prevents them from responding to immunotherapy. Cannabidiol (CBD) is a non-psychoactive natural active ingredient from the cannabis plant that has various pharmacological effects, including neuroprotective, antiemetic, anti-inflammatory, and antineoplastic activities. This study aimed to elucidate the specific anticancer mechanism of CBD by single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) technologies. Here, we report that CBD inhibits colorectal cancer progression by modulating the suppressive tumor microenvironment (TME). Our single-cell transcriptome and ATAC sequencing results showed that CBD suppressed M2-like macrophages and promoted M1-like macrophages in tumors both in strength and quantity. Furthermore, CBD significantly enhanced the interaction between M1-like macrophages and tumor cells and restored the intrinsic anti-tumor properties of macrophages, thereby preventing tumor progression. Mechanistically, CBD altered the metabolic pattern of macrophages and related anti-tumor signaling pathways. We found that CBD inhibited the alternative activation of macrophages and shifted the metabolic process from oxidative phosphorylation and fatty acid oxidation to glycolysis by inhibiting the phosphatidylinositol 3-kinase-protein kinase B signaling pathway and related downstream target genes. Furthermore, CBD-mediated macrophage plasticity enhanced the response to anti-programmed cell death protein-1 (PD-1) immunotherapy in xenografted mice. Taken together, we provide new insights into the anti-tumor effects of CBD.

Upregulation of hsa_circ_0002003 promotes hepatocellular carcinoma progression.

BACKGROUND: Circular RNAs (circRNAs), which are involved in various human malignancies, have emerged as promising biomarkers. The present study aimed to investigate unique expression profiles of circRNAs in hepatocellular carcinoma (HCC) and identify novel biomarkers associated with HCC development and progression. METHODS: CircRNA expression profiles of HCC tissues were jointly analyzed to identify differentially expressed circRNAs. Overexpression plasmid and siRNA targeting candidate circRNAs were used in functional assays in vitro. CircRNA-miRNA interactions were predicted using miRNAs expressed in the miRNA-seq dataset GSE76903. To further screen downstream genes targeted by the miRNAs, survival analysis and qRT-PCR were conducted to evaluate their prognostic role in HCC and construct a ceRNA regulatory network. RESULTS: Three significantly upregulated circRNAs, hsa_circ_0002003, hsa_circ_0002454, and hsa_circ_0001394, and one significantly downregulated circRNA, hsa_circ_0003239, were identified and validated by qRT-PCR. Our in vitro data indicated that upregulation of hsa_circ_0002003 accelerated cell growth and metastasis. Mechanistically, DTYMK, DAP3, and STMN1, which were targeted by hsa-miR-1343-3p, were significantly downregulated in HCC cells when hsa_circ_0002003 was silenced and were significantly correlated with poor prognosis in patients with HCC. CONCLUSION: Hsa_circ_0002003 may play critical roles in HCC pathogenesis and serve as a potential prognostic biomarker for HCC. Targeting the hsa_circ_0002003/hsa-miR-1343-3p/STMN1 regulatory axis could be an effective therapeutic strategy in patients with HCC.

NEDD8-conjugating enzyme E2s: critical targets for cancer therapy.

NEDD8-conjugating enzymes, E2s, include the well-studied ubiquitin-conjugating enzyme E2 M (UBE2M) and the poorly characterized ubiquitin-conjugating enzyme E2 F (UBE2F). UBE2M and UBE2F have distinct and prominent roles in catalyzing the neddylation of Cullin or non-Cullin substrates. These enzymes are overexpressed in various malignancies, conferring a worse overall survival. Targeting UBE2M to influence tumor growth by either modulating several biological responses of tumor cells (such as DNA-damage response, apoptosis, or senescence) or regulating the anti-tumor immunity holds strong therapeutic potential. Multiple inhibitors that target the interaction between UBE2M and defective cullin neddylation protein 1 (DCN1), a co-E3 for neddylation, exhibit promising anti-tumor effects. By contrast, the potential benefits of targeting UBE2F are still to be explored. It is currently reported to inhibit apoptosis and then induce cell growth; hence, targeting UBE2F serves as an effective chemo-/radiosensitizing strategy by triggering apoptosis. This review highlights the most recent advances in the roles of UBE2M and UBE2F in tumor progression, indicating these E2s as two promising anti-tumor targets.

Neddylation pathway promotes myeloid-derived suppressor cell infiltration via NF-κB-mCXCL5 signaling in lung cancer.

Myeloid-derived suppressor cells (MDSCs), a population derived from immature myeloid progenitors, are present in the tumors of patients and highly protumorigenic. However, the molecular mechanisms regulating MDSC infiltration remain unclear. Neddylation pathway is overactivated in multiple cancers and has a significant role in tumor progression. We established a subcutaneous transplantation model of Lewis lung cancer in mice and showed that inactivation of neddylation pathway inhibits MDSC infiltration and impairs lung cancer growth. A high expression level of neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8) is positively correlated with MDSC infiltration in human lung adenocarcinomas (LUADs). Moreover, inactivation of neddylation pathway inhibits the expression of murine CXCL5 (mCXCL5; human homolog CXCL6, hCXCL6), an important cytokine implicated in MDSC recruitment. Mechanistically, inactivation of neddylation pathway inhibits activity of Cullin-RING ligase 1, a typical neddylation substrate, and induces accumulation of phosphorylated IκBα and subsequent blockage of NF-κB translocation, thus suppressing transcriptional activation of mCxcl5 or hCXCL6. Collectively, our data suggest that neddylation-NF-κB-mCXCL5 axis is involved in MDSC recruitment to the tumor sites and demonstrate that neddylation pathway is a good therapeutic target for patients with LUAD, particularly those receiving anti-MDSC therapy.

Targeting cathepsin B by cycloastragenol enhances antitumor immunity of CD8 T cells via inhibiting MHC-I degradation.

BACKGROUND: The loss of tumor antigens and depletion of CD8 T cells caused by the PD-1/PD-L1 pathway are important factors for tumor immune escape. In recent years, there has been increasing research on traditional Chinese medicine in tumor treatment. Cycloastragenol (CAG), an effective active molecule in Astragalus membranaceus, has been found to have antiviral, anti-aging, anti-inflammatory, and other functions. However, its antitumor effect and mechanism are not clear. METHODS: The antitumor effect of CAG was investigated in MC38 and CT26 mouse transplanted tumor models. The antitumor effect of CAG was further analyzed via single-cell multiomics sequencing. Target responsive accessibility profiling technology was used to find the target protein of CAG. Subsequently, the antitumor mechanism of CAG was explored using confocal microscopy, coimmunoprecipitation and transfection of mutant plasmids. Finally, the combined antitumor effect of CAG and PD-1 antibodies in mice or organoids were investigated. RESULTS: We found that CAG effectively inhibited tumor growth in vivo. Our single-cell multiomics atlas demonstrated that CAG promoted the presentation of tumor cell-surface antigens and was characterized by the enhanced killing function of CD8+ T cells. Mechanistically, CAG bound to its target protein cathepsin B, which then inhibited the lysosomal degradation of major histocompatibility complex I (MHC-I) and promoted the aggregation of MHC-I to the cell membrane, boosting the presentation of the tumor antigen. Meanwhile, the combination of CAG with PD-1 antibody effectively enhanced the tumor killing ability of CD8+ T cells in xenograft mice and colorectal cancer organoids. CONCLUSION: Our data reported for the first time that cathepsin B downregulation confers antitumor immunity and explicates the antitumor mechanism of natural product CAG.

MilR3, a unique SARP family pleiotropic regulator in Streptomyces bingchenggensis.

Streptomyces bingchenggensis is the main industrial producer of milbemycins, which are a group of 16-membered macrocylic lactones with excellent insecticidal activities. In the past several decades, scientists have made great efforts to solve its low productivity. However, a lack of understanding of the regulatory network of milbemycin biosynthesis limited the development of high-producing strains using a regulatory rewiring strategy. SARPs (Streptomyces Antibiotic Regulatory Proteins) family regulators are widely distributed and play key roles in regulating antibiotics production in actinobacteria. In this paper, MilR3 (encoded by sbi_06842) has been screened out for significantly affecting milbemycin production from all the 19 putative SARP family regulators in S. bingchenggensis with the DNase-deactivated Cpf1-based integrative CRISPRi system. Interestingly, milR3 is about 7 Mb away from milbemycin biosynthetic gene cluster and adjacent to a putative type II PKS (the core minimal PKS encoding genes are sbi_06843, sbi_06844, sbi_06845 and sbi_06846) gene cluster, which was proved to be responsible for producing a yellow pigment. The quantitative real-time PCR analysis proved that MilR3 positively affected the transcription of specific genes within milbemycin BGC and those from the type II PKS gene cluster. Unlike previous "small" SARP family regulators that played pathway-specific roles, MilR3 was probably a unique SARP family regulator and played a pleotropic role. MilR3 was an upper level regulator in the MilR3-MilR regulatory cascade. This study first illustrated the co-regulatory role of this unique SARP regulator. This greatly enriches our understanding of SARPs and lay a solid foundation for milbemycin yield enhancement in the near future.

Targeting protein phosphatases for the treatment of inflammation-related diseases: From signaling to therapy.

Inflammation is the common pathological basis of autoimmune diseases, metabolic diseases, malignant tumors, and other major chronic diseases. Inflammation plays an important role in tissue homeostasis. On one hand, inflammation can sense changes in the tissue environment, induce imbalance of tissue homeostasis, and cause tissue damage. On the other hand, inflammation can also initiate tissue damage repair and maintain normal tissue function by resolving injury and restoring homeostasis. These opposing functions emphasize the significance of accurate regulation of inflammatory homeostasis to ameliorate inflammation-related diseases. Potential mechanisms involve protein phosphorylation modifications by kinases and phosphatases, which have a crucial role in inflammatory homeostasis. The mechanisms by which many kinases resolve inflammation have been well reviewed, whereas a systematic summary of the functions of protein phosphatases in regulating inflammatory homeostasis is lacking. The molecular knowledge of protein phosphatases, and especially the unique biochemical traits of each family member, will be of critical importance for developing drugs that target phosphatases. Here, we provide a comprehensive summary of the structure, the "double-edged sword" function, and the extensive signaling pathways of all protein phosphatases in inflammation-related diseases, as well as their potential inhibitors or activators that can be used in therapeutic interventions in preclinical or clinical trials. We provide an integrated perspective on the current understanding of all the protein phosphatases associated with inflammation-related diseases, with the aim of facilitating the development of drugs that target protein phosphatases for the treatment of inflammation-related diseases.

Integrated Analysis Highlights the Immunosuppressive Role of TREM2+ Macrophages in Hepatocellular Carcinoma.

Recently, attention has been focused on the central role of TREM2 in diverse pathologies. However, the role of TREM2 signaling in the tumor microenvironment of hepatocellular carcinoma (HCC) remains poorly understood. Herein, we systematically investigated the single-cell transcriptomes of human HCC tissues and found that TREM2 was predominantly expressed by a macrophage subpopulation enriched in tumor tissues that resemble lipid-associated macrophages (LAMs). The accumulation of TREM2+ LAM-like cells in HCC was confirmed in two additional cohorts using scRNA-seq analysis and immunohistochemistry. High expression of TREM2 correlated with high infiltrating macrophage abundance and poor prognosis. Based on systematic interrogations of transcriptional profiles and cellular interactions, TREM2+ LAM-like cells were identified to mainly originate from S100A8+ monocytes and represented an immunosuppressive state. TREM2+ LAM-like cells recruited suppressive Treg cells, facilitating microenvironment remodeling. Furthermore, gene regulatory analysis and in vitro functional assays indicated that activation of LXR signaling could promote the reprogramming of TREM2+ LAM-like cells. Correlation analysis of bulk RNA-sequencing data demonstrated that the enrichment of TREM2+ LAM-like cells was an independent indicator of adverse clinical outcomes in HCC patients. Our comprehensive analyses provide deeper insights into the immunosuppressive role of TREM2+ LAM-like cells in HCC.

Immune-related lincRNA pairs predict prognosis and therapeutic response in hepatocellular carcinoma.

Growing evidence has demonstrated the functional relevance of long intergenic noncoding RNAs (lincRNAs) to tumorigenesis and immune response. However, immune-related lincRNAs and their value in predicting the clinical outcomes of patients with liver cancer remain largely unexplored. Herein, we utilized the strategy of iterative gene pairing to construct a tumor-specific immune-related lincRNA pairs signature (IRLPS), which did not require specific expression levels, as an indicator of patient outcomes. The 18-IRLPS we developed was associated with overall survival, tumor progression, and recurrence in liver cancer patients. Multivariate analysis revealed that the risk model was an independent predictive factor. A high IRLPS risk was correlated suppressive immune microenvironment, and IRLPS-high patients might benefit more from CD276 blockade or TMIGD2 agonist. Patients in the high-risk group were associated with elevated tumor mutation, increased sensitivity to dopamine receptor antagonists, cisplatin, doxorubicin, and mitomycin but more resistance to vinblastine. Mechanistically, IRLPS high scores might lead to poor prognosis by promoting cell proliferation and metabolic reprogramming. The prognostic significance of the 18-IRLPS was confirmed in independent cancer datasets. These findings highlighted the robust predictive performances of the 18-IRLPS for prognosis and personalized treatment.

Species-resolved sequencing of low-biomass or degraded microbiomes using 2bRAD-M.

Microbiome samples with low microbial biomass or severe DNA degradation remain challenging for amplicon-based or whole-metagenome sequencing approaches. Here, we introduce 2bRAD-M, a highly reduced and cost-effective strategy which only sequences ~ 1% of metagenome and can simultaneously produce species-level bacterial, archaeal, and fungal profiles. 2bRAD-M can accurately generate species-level taxonomic profiles for otherwise hard-to-sequence samples with merely 1 pg of total DNA, high host DNA contamination, or severely fragmented DNA from degraded samples. Tests of 2bRAD-M on various stool, skin, environmental, and clinical FFPE samples suggest a successful reconstruction of comprehensive, high-resolution microbial profiles.

Particle-Based Calculation and Visualization of Protein Cavities Using SES Models.

The analysis of molecular cavities, where ligands interact with protein structures, plays a critical role in protein structure-based drug design. However, it is a challenge because of the ambiguous definition of the cavity boundaries in most cavity detection methods. The cavities are mostly calculated by input parameters, which are difficult for users to visualize cavities in interactive ways. In this paper, we propose a novel method for the interactive exploration of cavity calculation and visualization. Firstly, the proposed method combines the two solvent-excluded surfaces (SES) models of a given protein to define the boundaries and provides cavity emission points. Secondly, the system provides a user-guided interactive method to allow users to select cavities by simply clicking operations and to track the cavity identify and filling process based on position constraints. Finally, the selected cavities are represented with the colorful depth perception method. Experiments show that our work can effectively identify and calculate cavities.

Epigenetically associated CCL20 upregulation correlates with esophageal cancer progression and immune disorder.

Chemokines have distinct effects on tumor progression by affecting cancer immunity and tumorigenesis. However, the characteristic chemokine profiles and their roles in immune cell recruitment and cancer cell biology are not entirely understood in esophageal cancer. Here, we scrutinized chemokine's expression profiles in independent esophageal cancer cohorts and identified the elevated CCL20 as a risk factor to predict patients' prognosis regardless of histology subtypes. Enhanced CCL20 expression was also associated with the acquisition of metastatic potential. Mechanistically, the upregulation of CCL20 in tumor cells was associated with promoter hypomethylation. Furthermore, by analyzing single-cell RNA sequencing data of a mouse model mimicking human ESCC development, we observed an imbalance among CD4+ T subtypes in the tumor microenvironment, namely Ccr6+ Th17 and Treg cells infiltration alongside the elevated Ccl20 expression in abnormal epithelial cells during the tumorigenic process. Together, these results reveal that hypomethylation-induced CCL20 promotes esophageal cancer progression and immune disorder. Targeting CCL20 might be a promising therapeutic approach in esophageal cancer.

NEDD8-conjugating enzyme E2 UBE2F confers radiation resistance by protecting lung cancer cells from apoptosis.

Lung cancer, which is exacerbated by environmental pollution and tobacco use, has become the most common cause of cancer-related deaths worldwide, with a five-year overall survival rate of only 19% (Siegel et al., 2020; Yang et al., 2020; Yu and Li, 2020). Nearly 85% of lung cancers are non-small cell lung cancers, of which lung adenocarcinoma is the most common subtype accounting for 50% of non-small cell lung cancer cases. At present, radiotherapy is the primary therapeutic modality for lung cancer at different stages, with significant prolongation of survival time (Hirsch et al., 2017; Bai et al., 2019; Shi et al., 2020). Irradiation can generate reactive oxygen species (ROS) through the radiolysis reaction of water and oxygen, cause DNA damage and oxidative stress, and subsequently result in cancer cell death (Kim et al., 2019). Nevertheless, radioresistance seriously hinders the success of treatment for lung cancer, owing to local recurrence and distant metastasis (Huang et al., 2021). Compared with small cell lung cancer, non-small cell lung cancer shows more tolerance to radiotherapy. Therefore, it is of great importance to decipher key mechanisms of radioresistance and identify effective molecular radiosensitizers to improve patient survival.

Elevated Neddylation Pathway Promotes Th2 Cells Infiltration by Transactivating STAT5A in Hepatocellular Carcinoma.

Neddylation is a process in which a ubiquitin-like molecule NEDD8 is conjugated to a lysine residue of the substrate protein via successive enzymatic cascade reactions. Inactivation of neddylation pathway triggers tumor cell apoptosis or senescence to suppress the tumor growth. So far, there has been limited research on the role of the neddylation pathway (NEDD8-UBE2M-RBX1 axis) in the immune response. In this study, we investigated the association between the neddylation pathway and immune function in HCC by comprehensively analyzing transcriptome and clinical data of HCC samples from TCGA database. The analysis showed that the mRNA expression of neddylation pathway components was up-regulated in HCC and increased with disease severity. Moreover, we observed that activated neddylation pathway was associated with enriched infiltration of T helper 2 (Th2) cells in HCC, while transactivation of STAT5A signaling may mediate this association. On the contrary, no significant correlation between the neddylation pathway and Th1 cells infiltration was identified. Taken together, these findings suggest a potential role of the neddylation pathway in promoting a shift in Th1/Th2 balance toward Th2-dominant immunosuppression. Hence, targeting neddylation pathway could serve as an attractive immunotherapy strategy for suppressing the development of Th2 cells.