MAIN METHODS: Mice deficient in both dystrophin and ASC (encoded by Pycard [PYD And CARD Domain Containing]) were generated. The impact of ASC deficiency on muscular dystrophy of mdx mice were assessed by measurements of serum cytokines, Western blot, real-time PCR and histopathological staining.
KEY FINDINGS: The pro-inflammatory cytokines such as TNF-α, IL-6, KC/GRO and IL-10 were markedly increased in the sera of 8-week-old mdx mice compared to WT. Western blotting showed that P2X7, caspase-1, ASC and IL-18 were upregulated. Disruption of ASC and dystrophin expression in the mdx/ASC-/- mice was verified by Western blot analysis. Histopathological analysis did not find significant alterations in the muscular dystrophy phenotype in mdx/ASC-/- mice as compared to mdx mice.
SIGNIFICANCE: Taken together, our results show that disruption of the central adaptor ASC of the inflammasome is insufficient to alleviate muscular dystrophy phenotype in mdx mice.
RESULTS: The host counter-attack was evidenced based on fungal hyphae and Ganoderma DNA observed at 3 d.p.i which became significantly reduced at 7 and 11 d.p.i. DEGs revealed upregulation of multifaceted defense related genes such as PR-protein (EgPR-1), protease inhibitor (EgBGIA), PRR protein (EgLYK3) chitinase (EgCht) and expansin (EgEXPB18) at 3 d.p.i and 7 d.p.i which dropped at 11 d.p.i. Later stage involved highly expressed transcription factors EgERF113 and EgMYC2 as potential regulators of necrotrophic defense at 11 d.p.i. The reactive oxygen species (ROS) elicitor: peroxidase (EgPER) and NADPH oxidase (EgRBOH) were upregulated and maintained throughout the treatment period. Growth and nutrient distribution were probably compromised through suppression of auxin signalling and iron uptake genes.
CONCLUSIONS: Based on the analysis of oil palm gene expression, it was deduced that the biotrophic phase of Ganoderma had possibly occurred at the early phase (3 until 7 d.p.i) before being challenged by the fungus via switching its lifestyle into the necrotrophic phase at later stage (11 d.p.i) and finally succumbed the host. Together, the findings suggest the dynamic defense process in oil palm and potential candidates that can serve as phase-specific biomarkers at the early stages of oil palm-G. boninense interaction.
Methods: A lentiviral transduction system was used to generate SH-SY5Y cells overexpressing APP. Immunoblotting was conducted to determine expression levels of NF-κB, Rho-GTPase, and Bcl-2 family proteins in the APP overexpressed cells.
Results: In the NF-κB signaling pathway, APP-overexpressing SH-SY5Y cells showed that there was a reduction of p-NF-κB (p< 0.05) and IKKα. Subsequently, there was upregulation of protein expression of NF-Κb, IKKβ and IκBα. On the other hand, protein expression of RhoC (p< 0.05) and Rac1/2/3 was upregulated as compared to the control group. Meanwhile, a decrease in RhoA, Cdc42 (p< 0.05) and p-Rac1/cdc42 protein levels was observed in the APP-overexpressed group. Lastly, in the pro-apoptotic pathway, the expression of Bcl-2, Bid, Bok and Puma (p< 0.05) was up regulated in the APP-overexpressed group. Downregulation of Bad and Bim expression was observed in the APP-overexpressed as compared to the control group, and Bax expression remained unchanged in the APP-overexpressed group.
Conclusion: APP overexpression regulated signaling in the NF-κB, Rho-GTPase and Bcl-2 family pathways in neuronal cells, suggesting that these are involved in promoting neuronal survival and modulating synaptic plasticity in AD. However, further studies are essential to elucidate the APP-mediated mechanism of action.
METHODS: In this study, we established persistently NDV-infected EJ28 bladder cancer cells, designated as EJ28P. Global transcriptomic analysis was subsequently carried out by microarray analysis. Differentially expressed genes (DEGs) between EJ28 and EJ28P cells identified by the edgeR program were further analysed by Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA) analyses. In addition, the microarray data were validated by RT-qPCR.
RESULTS: Persistently NDV-infected EJ28 bladder cancer cells were successfully established and confirmed by flow cytometry. Microarray analysis identified a total of 368 genes as differentially expressed in EJ28P cells when compared to the non-infected EJ28 cells. GSEA revealed that the Wnt/β-catenin and KRAS signalling pathways were upregulated while the TGF-β signalling pathway was downregulated. Findings from this study suggest that the upregulation of genes that are associated with cell growth, pro-survival, and anti-apoptosis may explain the survivability of EJ28P cells and the development of persistent infection of NDV.
CONCLUSIONS: This study provides insights into the transcriptomic changes that occur and the specific signalling pathways that are potentially involved in the development and maintenance of NDV persistency of infection in bladder cancer cells. These findings warrant further investigation and is crucial towards the development of effective NDV oncolytic therapy against cancer.
Methods: The mechanism involved in the cytotoxic activities of F5 against MCF7 cells was elucidated by flow cytometry-based apoptosis detection, caspases activity measurement, and expression profiling of apoptosis markers by western blotting. Molecular attributes of F5 were further mined from L. rhinocerus's published genome and transcriptome for future exploration.
Results and Discussion: Apoptosis induction in MCF7 cells by F5 may involve a cross-talk between the extrinsic and intrinsic apoptotic pathways with upregulation of caspase-8 and -9 activities and a marked decrease of Bcl-2. On the other hand, the levels of pro-apoptotic Bax, BID, and cleaved BID were increased accompanied by observable actin cleavage. At gene level, F5 composed of three predicted non-synonymous single nucleotide polymorphisms (T > C) and an alternative 5' splice site.
Conclusions: Findings from this study provide an advanced framework for further investigations on cancer therapeutics development from L. rhinocerus.
Methods: HCT116 and HepG2 cells were treated with MP-HX for 24 hr. Total RNA was extracted from the cells and used for transcriptome profiling using Applied Biosystem GeneChip™ Human Gene 2.0 ST Array. Gene expression data was analysed using an Applied Biosystems Expression Console and Transcriptome Analysis Console software. Pathway enrichment analyses was performed using Ingenuity Pathway Analysis (IPA) software. The microarray data was validated by profiling the expression of 17 genes through quantitative reverse transcription PCR (RT-qPCR).
Results: MP-HX induced differential expression of 1,290 and 1,325 genes in HCT116 and HepG2 cells, respectively (microarray data fold change, MA_FC ≥ ±2.0). The direction of gene expression change for the 17 genes assayed through RT-qPCR agree with the microarray data. In both cell lines, MP-HX modulated the expression of many genes in directions that support antiproliferative activity. IPA software analyses revealed MP-HX modulated canonical pathways, networks and biological processes that are associated with cell cycle, DNA replication, cellular growth and cell proliferation. In both cell lines, upregulation of genes which promote apoptosis, cell cycle arrest and growth inhibition were observed, while genes that are typically overexpressed in diverse human cancers or those that promoted cell cycle progression, DNA replication and cellular proliferation were downregulated. Some of the genes upregulated by MP-HX include pro-apoptotic genes (DDIT3, BBC3, JUN), cell cycle arresting (CDKN1A, CDKN2B), growth arrest/repair (TP53, GADD45A) and metastasis suppression (NDRG1). MP-HX downregulated the expression of genes that could promote anti-apoptotic effect, cell cycle progression, tumor development and progression, which include BIRC5, CCNA2, CCNB1, CCNB2, CCNE2, CDK1/2/6, GINS2, HELLS, MCM2/10 PLK1, RRM2 and SKP2. It is interesting to note that all six top-ranked genes proposed to be cancer-associated (PLK1, MCM2, MCM3, MCM7, MCM10 and SKP2) were downregulated by MP-HX in both cell lines.
Discussion: The present study showed that the anticancer activities of MP-HX are exerted through its actions on genes regulating apoptosis, cell proliferation, DNA replication and cell cycle progression. These findings further project the potential use of MP as a nutraceutical agent for cancer therapeutics.