Newly synthesized acridone derivatives targeting lung cancer: A toxicity and xenograft model study.

AKT is one of the overexpressed targets in nonsmall cell lung cancer (NSCLC) and plays an important role in its progression and offers an attractive target for the therapy. The PI3K/AKT/mTOR pathway is upregulated in NSCLC. Acridone is an important heterocycle compound which treats cancer through various mechanisms including AKT as a target. In the present work, the study was designed to evaluate the safety profile of three acridone derivatives (AC-2, AC-7, and AC-26) by acute and repeated dose oral toxicity. In addition to this, we also checked the pAKT overexpression and its control by these derivatives in tumor xenograft model. The results from acute and repeated dose toxicity showed these compounds to be highly safe and free from any toxicity, mortality, or significant alteration in body weight, food, and water intake in the rats. In the repeated dose toxicity, compounds showed negligible variations in a few hematological parameters at 400 mg/kg. The histopathology, biochemical, and urine parameters remained unchanged. The xenograft model study demonstrated AC-2 to be inhibiting HOP-62 induced tumor via reduction in p-AKT1 (Ser473) expression significantly. In immunofluorescence staining AC-2 treated tissue section showed 2.5 fold reduction in the expression of p-AKT1 (Ser473). Histopathology studies showed the destruction of tumor cells with increased necrosis after treatment. The study concluded that AC-2 causes cell necrosis in tumor cells via blocking the p-AKT1 expression. The findings may provide a strong basis for further clinical applications of acridone derivatives in NSCLC.

RNA-seq transcriptomic analysis of 4-octyl itaconate repressing myogenic differentiation.

BACKGROUND: The 4-octyl itaconate (OI) is a type of cell-permeable itaconate derivative. Studies have shown that with an anti-fibrotic effect in systemic sclerosis, the OI also affects osteoclast differentiation. The aim of this study was to explore the molecular mechanisms underlying the effects of OI on myoblast differentiation by RNA-seq analysis. METHODS: Myoblast proliferation, differentiation, and muscle regulatory factors were examined in C2C12 myoblasts treated with OI of various concentrations (2.5, 10, 25, 50, and 100 µmol/L). Cells were treated with the PI3K-Akt activator IGF-1 to explore the role of the PI3K-Akt pathway in OI inhibition of myogenic differentiation. The regulatory mechanisms of OI in myogenesis were further investigated by RNA-seq and subsequent gene ontology (GO), kyoto encyclopedia of genes and genomes (KEGG) and, gene set enrichment analysis (GSEA). RESULTS: OI of various concentrations did not show any effect during cell proliferation. During differentiation, OI inhibited the expressions of the marker of mature myotubes myosin heavy chain (MHC) and myogenin in a dose-dependent manner. OI inhibited muscle differentiation by affecting MyoD-regulated activity through inhibition of AKT1 phosphorylation. The results of the KEGG enrichment analysis and GSEA showed that OI affected multiple metabolic pathways during myogenic differentiation, including PI3K-Akt signaling, calcium signaling, and PPAR signaling. CONCLUSIONS: Our study broadens the understanding of the OI inhibition of myogenic differentiation. OI plays its functions by targeting multiple molecules and pathways, providing novel insights into the understanding of the overall effect of OI.

Modulation of JAK2, STAT3 and Akt1 proteins by granulocyte colony stimulating factor following carbon monoxide poisoning in male rat.

Carbon monoxide (CO) is an odorless, colorless, tasteless and non-irritating by-product of inefficient combustion of hydrocarbon fuels such as motor vehicle exhausted gases. It is the leading cause of mortality in the USA among all unintentional toxicants. Male rats exposed to CO poisoning in the heart has many cardiovascular effects such as, cardiomyopathy, tachycardia, arrhythmias, and ischemia and in severe cases, myocardial infarction (MI) and cardiac arrest. Cardiomyocyte apoptosis is one of the most frequent consequences in the heart. Granulocyte colony stimulating factor (G-CSF) is a cytokine that mobilizes and differentiates granulocytes from stem cells. It can stimulate many anti-apoptotic pathways such as JAK2-STAT3 and PI3-Akt kinases following cardiac ischemia. G-CSF exerts its anti-apoptotic effects through binding to its specific cell surface receptor. The purpose of this study was to elucidate the mechanism of anti-apoptotic effect of G-CSF following CO poisoning. Rats were exposed to CO 1500 or 3000 ppm for 60 min. Animals received G-CSF 100 µg/kg subcutaneously for five consecutive days after CO intoxication. Western blot analysis was used to evaluate the expression of six proteins namely JAK2, p-JAK2, STAT3, p-STAT3, Akt1 and p-Akt1 following G-CSF 100 µg/kg consecutive dose administration after CO poisoning. There was a significant difference between phosphorylated proteins including p-JAK2, p-STAT3 and p-Akt1 in the G-CSF groups and those in control groups and there were not any significant differences in total protein among the groups.

IL-21/23 axis modulates inflammatory cytokines and RANKL expression in RA CD4+ T cells via p-Akt1 signaling.

Introduction: CD4+ T cells are critically involved in the pathogenesis of Rheumatoid Arthritis; an autoimmune disorder characterized by joint inflammation and bone degeneration. In this study, we focused on the critical role of cytokines, IL-21 and IL-23 in facilitating the aberrant status of RA Th17-like cells and report their significant contribution(s) in modulating the expression of inflammatory cytokines and RANKL. Methods: Blood and synovial fluid collected from a total of 167 RA patients and 25 healthy volunteers were assessed for various inflammatory markers and RANKL expression in plasma and CD4+ T cells. Subsequent ex vivo studies examined the role of specific cytokines, IL-21 and IL-23 in mediating inflammation and RANKL upregulation by blocking their expression with neutralizing antibodies in RA CD4+ T cells and terminally differentiated human Th17 cells. Further, the role of p-Akt1 as a signalling target downstream of IL-21 and IL-23 was evinced with IL-21 and IL-23 inhibition and phospho Akt-1/2 kinase inhibitor. Results: Our observations highlighted the augmented inflammatory cytokine levels in plasma and an aberrant CD4+ T cell phenotype expressing exaggerated inflammatory cytokines and membrane RANKL expression in RA as opposed to healthy controls. Neutralization of either IL-21 or IL-23 (p19 and p40) or both, resulted in downregulation of the cytokines, TNF-α, IFN-γ and IL-17 and RANKL expression in these cells, signifying the critical role of IL-21/23 axis in modulating inflammation and RANKL. Subsequent dissection of the signaling pathway found p-Akt1 as the key phosphoprotein downstream of both IL-21 and IL-23, capable of increasing inflammatory cytokines and RANKL production. Discussion: Our findings unequivocally identify IL-21/23 axis in RA CD4+ T cells as a key regulator dictating two critical processes i.e. exaggerated inflammation and higher RANKL expression and provide critical targets in their downstream signalling for therapeutic approaches.

Circ_0067997 boosted the growth while repressed the apoptosis of SGC-7901/DDP cells via repressing miR-615-5p/AKT1 pathway.

BACKGROUND: Gastric cancer (GC) remains a huge challenge to the heathy of human beings, largely due to lacking of effective therapeutic measures. Though an oncogenic role for circular RNAs (circRNAs) circ_0067997 in the progression of GC has been described recently, the molecular modulatory mechanism of it still remains to be further explored. The aim of present study is to examine the molecular network of circ_0067997 in GC. METHODS: qRT-PCR was carried out to determine the mRNA levels of circ_0067997, miR-615-5p and AKT1 in cisplatin (DDP)-insensitive or sensitive GC tumor tissues and cells, while the correlations among the contents of these molecules were determined by statistical analysis. The expression of circ_0067997 was manipulated by short-hairpin RNA and lentiviral-mediated approaches, while that of miR-615-5p was achieved by the application of its inhibitor or mimic. The in vivo action of circ_0067997 on tumor formation was determined by measuring tumor weight/volume/size and analyzing tumor apoptosis through TUNEL staining in mouse xenograft model and, while the in vitro effects of this circRNA and its target miR-615-5p on the cell survival and death were separately evaluated by CCK-8 assay and flow cytometry. Additionally, luciferase reporter assays were executed to determine the sequentially regulatory relationships of circ_0067997, miR-615-5p, and AKT1. RESULTS: Our data demonstrated that the level of circ_0067997 level was increased in DDP-insensitive GC tissues and cell line, while miR-615-5p presented the opposite results. Moreover, the relationships between circ_0067997 and miR-615-5p levels, circ_0067997 and AKT1 contents presented negative and positive correlations in clinic samples, respectively. Importantly, circ_0067997 was found to repress miR-615-5p expression, consequently leading to increased growth while reduced apoptosis of GC cells in the presence of DDP. Furthermore, the validated sequential regulation was circ_0067997 modulating miR-615-5p adjusting AKT1. CONCLUSIONS: This study demonstrated that circ_0067997 functioned as a sponge of miR-615-5p to target AKT1 expression, thereby enhancing the growth and restricting the apoptosis of DDP-insensitive GC cells. These new findings offered a valuable target for the detection and management of GC.

EZH2/H3K27Me3 and phosphorylated EZH2 predict chemotherapy response and prognosis in ovarian cancer.

BACKGROUND: EZH2 acts as an oncogene through canonical pathway EZH2/H3K27Me3 and uncanonical pathway pAkt1/pS21EZH2 in many solid tumors including ovarian cancer. However, the clinical value of EZH2/H3K27Me3 and pAkt1/pS21EZH2 remain unclear. In the current study, we aim to investigate the correlation between these two pathways to clinical-pathological parameters and prognosis. METHODS: EZH2, H3K27Me3, pAkt1 and pS21EZH2 expression were evaluated by tissue micro-array and immunohistochemistry in a cohort of ovarian cancer patients. The results were analyzed based on clinical characteristics and survival outcomes. RESULTS: EZH2, H3K27Me3, pAkt1 and pS21EZH2 were universally expressed in ovarian cancer specimens with a positive expression rate of 81.54% (53/65), 88.89% (48/54), 63.07% (41/65) and 75.38% (49/65). EZH2-pS21EZH2 (Spearman r = 0.580, P < 0.0001) and pS21EZH2-pAkt1 (Spearman r = 0.546, P < 0.0001) were closely correlated while EZH2- H3K27Me3 were less closely correlated (Spearman r = 0.307, P = 0.002). Low pS21EZH2 associated with better chemotherapy response (OR = 0.184; 95% CI [0.052-0.647], P = 0.008) according to logistic regression with an area under the curve of 0.789 (specificity 89.36%, sensitivity 68.42%) by ROC analysis and predicted improved progression-free survival (HR = 0.453; 95% CI [0.229-0.895], P = 0.023) as indicated by multivariate cox regression. A combination of EZH2low/H3K27Me3low status predicted better chemotherapy response (OR = 0.110; 95% CI [0.013-0.906], P = 0.040) and better progression-free survival (HR = 0.388; 95% CI [0.164-0.917], P = 0.031). The results suggested that EZH2/H3K27Me3 and pEZH2 predicted chemotherapy response and progression-free survival in ovarian cancer.

Phosphorylated Akt1 expression is associated with poor prognosis in cutaneous, oral and sinonasal melanomas.

Melanomas are highly aggressive tumours derived from melanocytes, which occur most commonly in the skin. Occasionally, these tumours may appear in oral and sinonasal mucous membranes. In this study, we performed a comparative analysis of the Phosphorylated Akt1 (p-Akt1) expression in 144 patients affected by cutaneous (CM), 34 oral cavity (OM), and 31 sinonasal melanomas (SNM). Similar to the metastatic cutaneous melanomas, p-Akt1 was overexpressed in 17/34 of the oral cavity and 20/31 of the sinonasal melanomas. In addition, the p-Akt1-nuclear expression was associated with poorer cancer-specific survival in cutaneous (P < .0001), oral (P < .0001), and sinonasal (P = .001) melanomas. Multivariate analysis showed p-Akt1 to be an independent prognostic marker in oral (P = .041) and sinonasal (P < .0001) melanomas patients. In conclusion, p-Akt1 overexpression is an independent prognostic marker in mucosal melanomas and is significantly up-regulated in sinonasal melanomas. As both mucosal and metastatic cutaneous melanomas showed high frequency of p-Akt1 expression, these findings suggest that mucosal melanomas have a biological behaviour, similar to the aggressive cutaneous melanomas.