Evolutionary preservation of CpG dinucleotides in RAG1 may elucidate the relatively high rate of methylation-mediated mutagenesis of RAG1 transposase.

Recombination-activating gene 1 (RAG1) is a vital player in V(D)J recombination, a fundamental process in primary B cell and T cell receptor diversification of the adaptive immune system. Current vertebrate RAG evolved from RAG transposon; however, it has been modified to play a crucial role in the adaptive system instead of being irreversibly silenced by CpG methylation. By interrogating a range of publicly available datasets, the current study investigated whether RAG1 has retained a disproportionate level of its original CpG dinucleotides compared to other genes, thereby rendering it more exposed to methylation-mediated mutation. Here, we show that 57.57% of RAG1 pathogenic mutations and 51.6% of RAG1 disease-causing mutations were associated with CpG methylation, a percentage that was significantly higher than that of its RAG2 cofactor alongside the whole genome. The CpG scores and densities for all RAG ancestors suggested that RAG transposon was CpG denser. The percentage of the ancestral CpG of RAG1 and RAG2 were 6% and 4.2%, respectively, with no preference towards CG containing codons. Furthermore, CpG loci of RAG1 in sperms were significantly higher methylated than that of RAG2. In conclusion, RAG1 has been exposed to CpG mediated methylation mutagenesis more than RAG2 and the whole genome, presumably due to its late entry to the genome later with an initially higher CpG content.

Assessing the Antiproliferative Potential of a Novel Combretastatin A4 Derivative via Modulating Apoptosis, MAPK/ERK and PI3K/AKT Pathways in Human Breast Cancer Cells.

BACKGROUND: Breast cancer is the most predominant tumor in women. Even though current medications for distinct breast cancer subtypes are available, the non-specificity of chemotherapeutics and chemoresistance imposes major obstacles in breast cancer treatment. Although combretastatin A-4 (CA-4) has been well-reported to have potential anticancer activity, in vivo studies of CA-4 reveal a decrease in its activity. In this respect, a series of CA-4 analogues have been designed, from which one analog [(1-(3-chloro-4-fluorophenyl)-N-(2methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-1,2,4-triazole-3-carboxamide, C25H22ClFN4O5] showed drastic cytotoxicity against breast cancer cells. Therefore, this research focused on investigating the in vitro molecular mechanism underlying the cytotoxicity of the CA-4 analogue, particularly the MAPK/ERK as well as PI3K/AKT pathways as attractive therapeutic targets in breast cancer. METHODS: The cell viability of MCF-7, MDA-MB231, and MDA-MB453 was assessed after treatment with the CA-4 analogue, and apoptosis was analyzed via Annexin V-FITC/PI dual staining. MAPK/ERK and PI3K/AKT were thoroughly assessed using western blotting. Real-time PCR was used to estimate apoptosis-related markers, including the P53, Bcl-2-associated X protein (Bax), and B-cell lymphoma 2 (Bcl2) genes. RESULTS: The CA-4 analogue reduced the survival of all cancerous cells in a concentration-dependent manner and induced apoptosis through the mitochondrial pathway (39.89 ± 1.5%, 32.82 ± 0.6%, and 23.77 ± 1.1% in MCF-7, MDA-MB231, and MDA-MB453 cells), respectively. The analogue also attenuated the expression of pMEK1/2/t-MEK1/2, p-ERK1/2/t-ERK1/2, p-PI3K/t-PI3K, and p-AKT/t-AKT proteins in all three cancer cell lines in a time-dependent manner. Furthermore, the CA-4 analogue upregulated the expression of the P53 gene and dramatically increased the ratio of Bax/Bcl2 genes. CONCLUSIONS: The enhanced cytotoxicity can be attributed to substituting the hydroxyl group in CA-4 with chlorine in the meta-position of ring B, substituting the para-methoxy group in CA-4 with fluorine in the analogue, and lastly, introducing an extension to the compound's structure (ring C). Therefore, CA-4 analogue can attenuate the proliferation of human breast cancer cells by inducing apoptosis and simultaneously suppressing the MAPK/ERK and PI3K/AKT pathways.

Alpha-Mangostin ameliorates acute kidney injury via modifying levels of circulating TNF-α and IL-6 in glycerol-induced rhabdomyolysis animal model.

Alpha mangostin (AM), isolated from G. mangostana, showed beneficial effects in several disorders due to its antioxidant and anti-inflammatory properties. Acute kidney injury (AKI) due to different etiologies can develop into severe complications, resulting in high mortality rates. In this work, AM is tested for its ability to alleviate AKI in glycerol-induced AKI rat model, where 30 Male Sprague-Dawley rats were assigned to a healthy group, glycerol-treated group and AM-treated group. Glycerol- and AM groups received a single dose of glycerol (per IM, 50% glycerol in saline, 8 ml/kg), whereas control group was injected with saline. AM treatment (a single daily dose, per IP, 175mg/kg) was accomplished for three days. Animals were executed to collect blood samples and kidney tissue for biochemical and histological examination. It was found that glycerol induced increase in serum creatinine, blood urea nitrogen (BUN), lipid peroxidation, serum magnesium, TNF-α and IL-6. It also induced renal edema and hypocalcemia along with histopathological renal damage. AM treatment improved renal histological features and alleviated increase in serum creatinine, BUN, serum magnesium, TNF-α and IL-6 levels, as well as renal edema and lipid peroxidation but did not affect serum calcium levels. This suggests AM as a potential therapeutic agent for treating AKI mainly via its antioxidant and anti-inflammatory properties.

Blackberry-Loaded AgNPs Attenuate Hepatic Ischemia/Reperfusion Injury via PI3K/Akt/mTOR Pathway.

Liver ischemia-reperfusion injury (IRI) is a pathophysiological insult that often occurs during liver surgery. Blackberry leaves are known for their anti-inflammatory and antioxidant activities. AIMS: To achieve site-specific delivery of blackberry leaves extract (BBE) loaded AgNPs to the hepatocyte in IRI and to verify possible molecular mechanisms. METHODS: IRI was induced in male Wister rats. Liver injury, hepatic histology, oxidative stress markers, hepatic expression of apoptosis-related proteins were evaluated. Non-targeted metabolomics for chemical characterization of blackberry leaves extract was performed. KEY FINDINGS: Pre-treatment with BBE protected against the deterioration caused by I/R, depicted by a significant improvement of liver functions and structure, as well as reduction of oxidative stress with a concomitant increase in antioxidants. Additionally, BBE promoted phosphorylation of antiapoptotic proteins; PI3K, Akt and mTOR, while apoptotic proteins; Bax, Casp-9 and cleaved Casp-3 expressions were decreased. LC-HRMS-based metabolomics identified a range of metabolites, mainly flavonoids and anthocyanins. Upon comprehensive virtual screening and molecular dynamics simulation, the major annotated anthocyanins, cyanidin and pelargonidin glucosides, were suggested to act as PLA2 inhibitors. SIGNIFICANCE: BBE can ameliorate hepatic IRI augmented by BBE-AgNPs nano-formulation via suppressing, oxidative stress and apoptosis as well as stimulation of PI3K/Akt/mTOR signaling pathway.

In Vitro Anticancer Activity of Novel Ciprofloxacin Mannich Base in Lung Adenocarcinoma and High-Grade Serous Ovarian Cancer Cell Lines via Attenuating MAPK Signaling Pathway.

Novel drugs are desperately needed in order to combat a significant challenge due to chemo-therapeutic resistance and bad prognosis. This research aimed to assess the anticancer activity of a newly synthesized ciprofloxacin Mannich base (CMB) on ovarian cancer (OVCAR-3) and lung cancer (A-549) cell lines and to investigate probable involved molecular mechanisms. The cytotoxic and pro-apoptotic impact of CMB on both cell lines was investigated using MTT assay, Annexin V assay, and cell cycle analysis, as well as caspase-3 activation. Western blotting was carried out to evaluate downstream targets of the MAPK pathway, while qRT PCR was used to evaluate the gene expression pattern of the p53/Bax/Bcl2 pathway. CMB treatment showed significantly reduced cell proliferation in both OVCAR-3 and A-549 cells with half maximum inhibitory concentration (IC50) = 11.60 and 16.22 µg/mL, respectively. CMB also induced apoptosis, S phase cell cycle arrest, and up-regulated expression of p53, p21, and Bax while down-regulated Bcl2 expression. CMB also halted cell proliferation by deactivating the MAPK pathway. In conclusion, CMB may be regarded as a potential antiproliferative agent for lung and ovarian cancers due to anti-proliferative and pro-apoptotic actions via inhibition of the MAPK pathway and p53/Bax/Bcl2.

Impact of IL-27p28 (rs153109) and TNF-α (rs1800629) Genetic Polymorphisms on the Progression of HCV Infection in Egyptian Patients.

BACKGROUND: Hepatitis C virus (HCV) is a universal health problem. HCV infection may proceed to liver fibrosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). The latest is the third leading global cause of cancer-related mortality. Cytokines including IL-27 and TNF-α play a major role as a link between innate and adaptive immunity which in turn deduct the outcome of HCV infection. AIM: The present study examined the role of both (-964 A/G) single-nucleotide polymorphism (SNP) of IL-27p28 rs153109 and (-308 G/A) SNP of TNF-α rs1800629 on the progression of HCV infection in genotype 4a infected patients. PATIENTS AND METHODS: The patients enrolled in the study were divided into three main groups group I: 38 fibrotic patients, group II: 51 cirrhotic patients, and finally group III: 29 HCC patients. Sixteen healthy volunteers were used as controls. IL-27p28 rs153109 and TNF-α rs1800629 genotyping were performed using polymerase chain reaction-restriction fragment length polymorphism assay. RESULTS: There was no statistically significant difference between the studied groups regarding the IL-27p28 genotypes. However, TNF-α (-308) studied polymorphism showed a significant difference between the HCC and fibrosis group (p = 0.00), and also between the cirrhosis and fibrosis group (p = 0.031) revealing that AA genotype is the genotype of risk. Furthermore, the association found between allele frequencies of two studied SNPs and the four studied groups were non-significant. CONCLUSION: TNF-α rs1800629 polymorphism is a potential genetic-susceptibility factor for HCV related cirrhosis and HCC progression.

Esomeprazole ameliorates CCl4 induced liver fibrosis in rats via modulating oxidative stress, inflammatory, fibrogenic and apoptotic markers.

BACKGROUND: Hepatic fibrosis is a major health problem that requires further medical attention. Proton pump inhibitors are proven to possess other therapeutic potentials apart of their acid anti-secretory actions. AIM OF THE WORK: To test possible anti-fibrotic effect of esomeprazole magnesium trihydrate in management of liver fibrosis compared to silymarin, the well-known hepatoprotective agent. MATERIALS & METHODS: 40 male albino rats were divided into 4 groups: normal control group; CCl4-treated group (1 mL/kg 40% CCl4, diluted in olive oil) I.P twice weekly for 6 weeks; esomeprazole-treated group (30 mg/kg body weight); and Silymarin-treated group (100 mg/kg body weight). Both esomeprazole and silymarin were given orally daily for two weeks after the last CCl4 dose. Serum and tissue samples were assessed for histopathological and biochemical analyses. RESULTS: Esomeprazole reversed hepatocellular damage, improved liver integrity, corrected major histopathological disturbances induced by CCl4 and lowered fibrosis scoring. It also improved anti-oxidant capacity and attenuated lipid peroxidation. Esomeprazole treatment resulted in down-regulation of hepatic pro-apoptotic Bax and up-regulation of anti-apoptotic Bcl2 protein expressions. In addition, it resulted in inhibition of TNF-α, TGF-ß and IL-6 -mediated inflammatory responses, and retrieval of the epithelial marker e-cadherin. CONCLUSION: Esomeprazole confers significant anti-fibrotic actions. Further study is needed to elucidate other probable mechanisms for this effect and to test their anti-fibrotic potential clinically.

Association of Interleukin-27 rs 153109 Single Nucleotide Polymorphism with Spontaneous Resolution of Hepatitis C Virus - Genotype 4a Infection in Egyptian Patients.

BACKGROUND: HCV is a major global health problem. IL-27 is a member of the IL-6/IL-12 cytokine family with a broad range of anti-inflammatory properties. Recent studies highlighted the effect of a SNP in the IL-27 promoter region on modulating the progression of infectious diseases and individual responses to therapy. AIM OF THE WORK: The present study investigated the potential role of (-964 A/G) SNP in the promoter region of IL-27p28 gene (alleles rs153109) on the outcome of HCV infection among genotype 4a infected patients. MATERIALS AND METHODS: HCV genotyping confirmed that all of the HCV-infected patients had genotype 4a infection. Genomic DNA was extracted from 111 patients with chronic HCV infection, 42 spontaneous resolvers (SR) and 16 healthy controls. IL- 27p28.rs153109 genotyping was assessed using PCR-RFLP then confirmed by DNA sequencing. RESULTS: The frequency of IL-27-p28.rs153109AA, AG, and GG genotypes among chronically infected subjects were 74.8 %, 25.2%, and 0% while among the SR, they were 57.1%, 35.7%, and 7.14%, respectively. Our data show the unique presence of G/G genotype in the SR group (3 patients; 7.14%). Moreover, the "G" allele frequencies among chronic and resolved subjects were 12.6% and 25.0%, respectively (p=0.0136). Importantly, subjects with the GG genotype were more likely to clear their HCV infection than those with the AA genotype (p=0.0118). CONCLUSIONS: HCV genotype 4a subjects with the IL-27-p28.rs153109 A/G and G/G genotype were more likely to clear their HCV infection. Therefore, we propose IL- 27p28.rs153109SNPas a genetic biomarker for predicting HCV infection outcome.

Integrin-Linked Kinase, Snail and Multidrug Resistance Protein 1: Three concordant players in the progression of non-small cell lung cancer.

BACKGROUND: Integrin Linked Kinase (ILK), Snail and Multidrug Resistance Protein 1 (MRP1) have been implicated in several cancers; however, their roles in non-small cell lung cancer (NSCLC) remain to be elucidated. AIM: Investigation of their expression in NSCLC tissue. Relationships among these proteins and their association with clinicopathological parameters were studied. MATERIALS AND METHODS: ILK, Snail and MRP1 expression were immunohistochemically assessed in 97 tumor tissues. Furthermore, western blot analysis for ILK, Snail and MRP1 in 6 cases of NSCLC was also performed. RESULTS: ILK overexpression, positive Snail and MRP1 expression were found in 46.4%, 36.1% and 49.5% of tumors respectively. ILK expression was significantly correlated with tumor grade (p=0.013), lymph node (LN) metastases (p=0.001) and stage (p=0.001). Positive Snail and MRP1 expression were significantly associated with LN metastasis (p=0.004 and 0.022, respectively) and advanced stage disease (p=0.018 and 0.024, respectively). MRP1 expression was significantly higher among adenocarcinoma cases compared to other types (p=0.001). ILK over-expression was significantly associated with up-regulation of Snail and MRP1 (p<0.001 both). Significant association was also, found between Snail and MRP1 expression (p=0.005). Moreover, the co-expression of two markers or more was significantly associated with less differentiation (p=0.011), advanced tumor status (p=0.030), LN metastasis (p<0.001) and advanced stage (p<0.001) disease. Western blot analysis validated immunohistochemical findings. CONCLUSION: ILK may have an important role in the progression of NSCLC, possibly through up-regulation of Snail and MRP1. ILK, Snail and MRP1 are important molecular markers for predicting carcinogenesis and progression of NSCLC.

Cytokines as important playmakers of experimental hepatocarcinogenesis confounded by diabetes.

PURPOSE: To explore the possible intermediary pathways through which diabetes mellitus (DM) adversely worsens hepatocellular carcinoma (HCC), focusing on cell life controllers as some transcription factors and inflammatory mediators. MATERIAL AND METHODS: Forty male albino rats were divided into four groups, control, cancer [given single intra-peritoneal (IP) dose of diethyl nitrosamine, NDEA, 125 mg/kg body weight], diabetic (given single dose of streptozotocin, STZ, 65 mg/kg) and cancer diabetic. HCC was initiated with NDEA, 3 weeks later, DM was induced with STZ. At 14th week, animals were sacrificed. Serum ALT, AST, GGT activities, AFP, IL-6, TNF-α levels and liver tissue Bax and Bcl2 proteins were measured. Liver sections were stained for histological examination. Both histological and AFP variations were chosen to prove cancer development. RESULTS: NDEA group showed significant increase in liver weight, serum ALT, AST, GGT, AFP, TNF-α, IL-6 and liver Bcl2 protein with decrease in total body weight, liver Bax protein and Bax/Bcl2 ratio. These effects were more pronounced in DENA plus STZ group. IL-6, TNF-α and Bcl2 were positively correlated while Bax and Bax/Bcl2 ratio were negatively correlated to AFP levels reflecting potential diagnostic value. CONCLUSION: Co-induction of DM in the course of hepatocarcinogenesis can dramatically influence disease progression through inflammation and retarded apoptosis. The suggested apoptotic and inflammatory markers seem to be beneficial diagnostic tools for HCC and improve the diagnostic performance of AFP.

Loss of Nrf2 markedly exacerbates nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) arises from nonalcoholic fatty liver disease (NAFLD) as a consequence of oxidative stress. Herein we report that the development of NASH is greatly accelerated in mice lacking transcription factor Nrf2 when they are challenged with a methionine- and choline-deficient (MCD) diet. After 14 days of feeding on an MCD diet, livers from Nrf2(-/-) mice showed a substantial increase in macro- and microvesicular steatosis and a massive increase in the number of neutrophil polymorphs, compared to livers from wild-type mice treated similarly. Livers of Nrf2(-/-) mice on the MCD diet suffered more oxidative stress than their wild-type counterparts as assessed by a significant depletion of reduced glutathione that was coupled with increases in oxidized glutathione and malondialdehyde. Furthermore, livers from Nrf2(-/-) mice on the MCD diet suffered heightened inflammation as judged by an approximately 10-fold increase in the amount of nuclear NF-kappaB p65 protein and approximately 5-fold increases in the levels of mRNA for interleukin-1beta, tumor necrosis factor alpha, cyclooxygenase 2, and inducible nitric oxide synthase compared with livers from similarly treated wild-type mice. Thus, impairment of Nrf2 activity may represent a major risk factor for the evolution of NAFLD to NASH.