RNA-seq reveals role of cell-cycle regulating genes in the pathogenicity of a field very virulent infectious bursal disease virus.

Infectious bursal disease virus (IBDV) infection causes highly contagious and immunosuppressive disease in poultry. The thymus, serving as the primary organ for T cell maturation and differentiation, plays an important role in the pathogenicity of IBDV in the infected chickens. However, there are no reports on the molecular pathogenesis of IBDV in the thymus currently. The aim of the study was to elucidate the molecular mechanisms underlying the pathogenicity of a field very virulent (vv) IBDV strain NN1172 in the thymus of SPF chickens using integrative transcriptomic and proteomic analyses. Our results showed that a total of 4,972 Differentially expressed genes (DEGs) in the thymus of NN1172-infected chickens by transcriptomic analysis, with 2,796 up-regulated and 2,176 down-regulated. Meanwhile, the proteomic analysis identified 726 differentially expressed proteins (DEPs) in the infected thymus, with 289 up-regulated and 437 down-regulated. Overall, a total of 359 genes exhibited differentially expression at both mRNA and protein levels, with 134 consistently up-regulated and 198 genes consistently down-regulated, as confirmed through a comparison of the RNA-seq and the proteomic datasets. The gene ontology (GO) analysis unveiled the involvement of both DEGs and DEPs in diverse categories encompassing cellular components, biological processes, and molecular functions in the pathological changes in IBDV-infected thymus. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the host mainly displayed severely disruption of cell survival/repair, proliferation and metabolism pathway, meanwhile, the infection triggers antiviral immune activation with a potential emphasis on the MDA5 pathway. Network inference analysis identified seven core hub genes, which include CDK1, TYMS, MCM5, KIF11, CCNB2, MAD2L1, and MCM4. These genes are all associated with cell-cycle regulating pathway and are likely key mediators in the pathogenesis induced by NN1172 infection in the thymus. This study discovered dominant pathways and genes which enhanced our understanding of the molecular mechanisms underlying IBDV pathogenesis in the thymus.

Genetic and pathogenic characterizations of a naturally occurring reassortant and homologous recombinant strain of the classical infectious bursal disease virus re-emerging in chickens in southern China.

Infectious bursal disease (IBD) classical virus strain (cIBDV) can cause morbidity and mortality in young chickens with severe long-term immunosuppression. However, since the emergence and widespread prevalence of very virulent strain (vvIBDV) in China from 1991, reports of cIBDV have become rare. A novel reassortant and recombinant strain GXYL211225 (genotype A1aB1a) with segment A originating from the classical strain (A1a) and segment B from the attenuated vaccine strain (B1a) was characterized in the study. Notably, segment A resulted from recombination between the cIBDV strains 150127-0.2 and Faragher52-70, expressing as a backbone from 150127-0.2, where a fragment located at the position of nucleotide (nt) 519-1 410 was replaced by the corresponding region of Faragher52-70. The infection of GXYL211225 caused mortality in SPF chicken embryos, despite lacking the critical amino acid (aa) residues 253H, 279 N and 284A associated with the cellular tropism, and induced significant cytopathic effect (CPE) on a wide range of cells, confirming its natural cell-adapted character. Furthermore, the challenge experiment of GXYL211225 was performed on the commercial Three-yellow chickens of 4-week-old, and with the vvIBDV HLJ-0504-like strain NN1172 and the novel variant (nv) IBDV strain QZ191002 as the comparison. All the challenged birds experienced reduced body-weight gain. QZ191002 infected birds showed no obvious clinical symptoms or mortality, while those of NN1172 and GXYL211225 showed typical IBD symptoms and resulted in 20% (2/10) and 10% (1/10) of mortality rates, respectively. At 7 days post-challenge (dpc), the damages of bursal of Fabricius (BF) varied among groups, with NN1172 causing the most severe lesions, followed by GXYL211225, and then QZ191002. It was also found that the pathogenicity was correlated positively with the viral load, aligning with the histopathological severity in BF. The study confirms the rapid and diverse evolution of the re-emerged classical strains in the field and emphasizes the need to monitor the changes of IBDV on both the genetic and pathogenic aspects for the effective control of the disease.

PD-1 Immune Checkpoint Inhibitor Therapy Malignant Tumor Based on Monotherapy and Combined Treatment Research.

Recently, immunotherapy has become the fourth pillar of cancer treatment in addition to surgery therapy, chemotherapy, and radiation therapy. The inhibitors of programed cell death protein 1 (PD-1) and its ligand PD-L1 are the new stars in immunotherapy, as they can overcome tumor immunosuppression. However, the efficacy of PD-1 inhibitors still needs to be further developed for clinical treatment. Therefore, research into treatment with anti-PD-1 drugs has emerged as a new development field. This review provides novel insights into the role and mechanism of PD-1 combination anti-tumor therapy, thereby promoting its clinical application in anti-tumor immunotherapy.

Calycosin induces mitochondrial-dependent apoptosis and cell cycle arrest, and inhibits cell migration through a ROS-mediated signaling pathway in HepG2 hepatocellular carcinoma cells.

Calycosin is one of the main ingredients extracted from the Chinese medical herb, Radix astragali (RA). It has been shown to inhibit cell proliferation and induce apoptosis in several cancer cell lines, but the underlying mechanism remains unclear. The effects of calycosin on the proliferation and apoptosis of hepatocellular carcinoma (HCC) cells, as well as its mechanism, were investigated in this study. Cell Counting Kit-8 assay results suggested that calycosin had anti-proliferation effects on HCC in dose- and time-dependent manners, and had less cytotoxicity in normal cells. Hoechst/PI double staining and flow cytometry results showed cellular morphological changes and apoptosis after treatment of HepG2 cells with calycosin. The western blot assay showed calycosin decreased the expression of Bcl-2 and increased the expression of Bax, caspase-3, and PARP. Calycosin induced the activation of MAPK, STAT3, NF-κB, apoptosis-related proteins, and induced cell cycle arrest in the G0/G1 phase by regulating AKT. In addition, calycosin reduced the expression of TGF-ß1, SMAD2/3, SLUG, and vimentin. Furthermore, phosphorylation, apoptosis, and cell migration induced by calycosin were mediated by the production of reactive oxygen species. These events could be inhibited by pretreatment with N-acetyl-L-cysteine. Calycosin resisted HCC by activating ROS-mediated MAPK, STAT3, and NF-κB signaling pathways.

Calycosin Induces Gastric Cancer Cell Apoptosis via the ROS-Mediated MAPK/STAT3/NF-κB Pathway.

BACKGROUND: Calycosin, an active compound in plants, can promote the apoptosis of various cancer cells; however, the mechanism by which it regulates reactive oxygen species (ROS) in gastric cancer (GC) cells remains unclear. PURPOSE: In this study, we investigated the effects of calycosin on apoptosis, the cell cycle, and migration in GC cells under ROS regulation. RESULTS: The results of the Cell Counting Kit-8 assay suggested that calycosin had significant cytotoxic effects on 12 gastric cancer cells, but no significant cytotoxic effects on normal cells. Hoechst 33342/propidium iodide (PI) double staining and flow cytometry showed that calycosin had clear pro-apoptotic effects on AGS cells. Western blotting revealed that the expression of cytochrome C and pro-apoptotic proteins B-cell lymphoma 2 (Bcl-2)-associated agonist of cell death (Bad), cleaved (cle)-caspase-3, and cle-poly (ADP-ribose) polymerase gradually increased, and the expression of anti-apoptotic protein Bcl-2 gradually decreased. Calycosin also decreased the expression of extracellular signal-regulated kinase, nuclear factor kappa B (NF-κB), and signal transducer and activator of transcription 3 (STAT3), and increased the phosphorylation levels of p38, c-Jun N-terminal kinase, and inhibitor of NF-κB. In addition, calycosin markedly increased ROS accumulation, and pretreatment with active oxygen scavenger n-acetyl-l-cysteine (NAC) clearly inhibited apoptosis. Calycosin downregulated the cell cycle proteins cyclin-dependent kinase 2 (CDK2), CDK4, CDK6, cyclin D1, and cyclin E; upregulated p21 and p27; and arrested cells in the G0/G1 phase. Similarly, calycosin also downregulated Snail family transcriptional repressor 1, E-cadherin, and ß-catenin and inhibited cell migration. However, pretreatment with NAC inhibited the calycosin-induced effects of cycle arrest and migration. CONCLUSION: In summary, calycosin induces apoptosis via ROS-mediated MAPK/STAT3/NF-κB pathways, thereby exerting its anti-carcinogenic functions in GC cells.

10-HDA Induces ROS-Mediated Apoptosis in A549 Human Lung Cancer Cells by Regulating the MAPK, STAT3, NF-κB, and TGF-ß1 Signaling Pathways.

10-Hydroxy-2-decenoic acid (10-HDA), also known as royal jelly acid, has a variety of physiological functions, and recent studies have shown that it also has anticancer effects. However, its anticancer mechanisms have not been clearly defined. In this study, we investigated the underlying mechanisms of 10-HDA in A549 human lung cancer cells. We used Cell Counting Kit-8 assay, scratch wound healing assay, flow cytometry, and western blot analysis to investigate its apoptotic effects and underlying mechanism. Our results showed that 10-HDA inhibited the proliferation of three types of human lung cancer cells and had no significant toxic effects on normal cells. Accompanying reactive oxygen species (ROS), 10-HDA induced A549 cell apoptosis by regulating mitochondrial-associated apoptosis, and caused cell cycle arrest at the G0/G1 phase in a time-dependent manner. Meanwhile, 10-HDA also regulated mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription 3 (STAT3), and nuclear factor kappa B (NF-κB) signaling pathways by increasing the expression levels of phosphorylated c-Jun N-terminal kinase, p-p38, and I-κB, and additionally, by decreasing the expression levels of phosphorylated extracellular signal-regulated kinase, p-STAT3, and NF-κB. These effects were blocked by MAPK inhibitors and N-acetyl-L-cysteine. Furthermore, 10-HDA inhibited cell migration by regulating transforming growth factor beta 1 (TGF-ß1), SNAI1, GSK-3ß, E-cadherin, N-cadherin, and vimentin. Taken together, the results of this study showed that 10-HDA induced cell cycle arrest and apoptosis in A549 human lung cancer cells through ROS-mediated MAPK, STAT3, NF-κB, and TGF-ß1 signaling pathways. Therefore, 10-HDA may be a potential therapy for human lung cancer.

Zeaxanthin Induces Apoptosis via ROS-Regulated MAPK and AKT Signaling Pathway in Human Gastric Cancer Cells.

BACKGROUND: Zeaxanthin, a carotenoid commonly found in plants, has a variety of biological functions including anti-cancer activity. PURPOSE: This study aimed to investigate the potential mechanisms of zeaxanthin in human gastric cancer cells. METHODS: CCK-8 assay was used to examine the cytotoxic effect of zeaxanthin on human gastric cancer cells. Flow cytometry was used to analyse AGS cell cycle distribution and apoptosis status. Western blot analysis was used to detect the expression levels of cycle-related proteins (Cyclin A, Cyclin B1, CDK1/2, p21, and p27), apoptosis-related proteins (Bcl-2, Bad, caspase-3, PARP), MAPK, AKT, STAT3, and NF-κB. RESULTS: CCK-8 assay showed that zeaxanthin has obvious cytotoxic effects on 12 types of human gastric cancer cells, but no obvious toxic effect on normal cells. In addition, flow cytometry and Western blotting results showed that zeaxanthin induces apoptosis by reducing mitochondrial membrane potential; increasing Cytochrome C, Bax, cleaved-caspase-3 (cle-cas-3), and cleaved-PARP (cle-PARP) expression levels; and decreasing Bcl-2, pro-caspase-3 (pro-cas-3), and pro-PARP expression levels. Additionally, zeaxanthin caused cell cycle arrest at the G2/M phase by increasing the levels of p21 and p27 and reduced the levels of AKT, Cyclin A, Cyclin B1, and Cyclin-dependent kinase 1/2 (CDK1/2). Furthermore, after zeaxanthin treatment, the expression levels of reactive oxygen species (ROS), p-JNK, p-p38, and I-κB increased, and the expression levels of p-ERK, p-AKT, STAT3, and NF-κB decreased. However, the ROS scavenger N-acetylcysteine (NAC) and MAPK inhibitors inhibited zeaxanthin-induced apoptosis, and under the action of zeaxanthin, MAPK regulated NF-κB and STAT3, and reduced their protein expression levels. CONCLUSION: Zeaxanthin has a potential effect against gastric cancer cells through the ROS-mediated MAPK, AKT, NF-κB, and STAT3 signaling pathways, and it is expected to become a new drug for the treatment of human gastric cancer.

Isoorientin induces the apoptosis and cell cycle arrest of A549 human lung cancer cells via the ROS­regulated MAPK, STAT3 and NF­κB signaling pathways.

Isoorientin (ISO) is a naturally occurring C­glycosyl flavone that has various pharmacological properties, such as anti­bacterial and anti­inflammatory effects. However, its underlying molecular mechanisms in human lung cancer cells remain unknown. In the present study, the effects of ISO on the induction of apoptosis and relative molecular mechanisms in A549 human lung cancer cells were investigated. The results of Cell Counting Kit­8 assay (CCK­8) indicated that ISO exerted significant cytotoxic effects on 3 lung cancer cell lines, but had no obvious side­effects on normal cells. Moreover, flow cytometry and western blot analysis revealed that ISO induced mitochondrial­dependent apoptosis by reducing mitochondrial membrane potential. ISO also increased the expression levels of Bax, cleaved­caspase­3 (cle­cas­3) and poly(ADP­ribose) polymerase (PARP; cle­PARP), and decreased the expression levels of Bcl­2 in A549 cells. Furthermore, ISO induced G2/M cell cycle arrest by decreasing the expression levels of cyclin B1 and CDK1/2, and increasing the expression levels of p21 and p27 in A549 cells. As the duration of ISO treatment increased, intracellular reactive oxygen species (ROS) levels in A549 cells also increased. However, pre­treatment of the cells with the ROS scavenger, N­acetylcysteine (NAC), inhibited ISO­induced apoptosis. In addition, ISO increased the expression levels of p­p38, p­JNK and IκB­α; and decreased the expression levels of p­extracellular signal­regulated kinase (ERK), p­signal transducer and activator of transcription (STAT)3, p­nuclear factor (NF)­κB, NF­κB and p­IκB; these effects were induced by mitogen­activated protein kinase (MAPK) inhibitors and blocked by NAC. Taken together, the results of the present study indicate that ISO induces the apoptosis of A549 lung cancer cells via the ROS­mediated MAPK/STAT3/NF­κB signaling pathway, and thus may be a potential drug for use in the treatment of lung cancer.