Nigella sativa, Anthemis hyaline and Citrus sinensis extracts reduce SARS-CoV-2 replication by fluctuating Rho GTPase, PI3K-AKT, and MAPK/ERK pathways in HeLa-CEACAM1a cells.

Traditional remedies have long utilized Anthemis hyaline, Nigella sativa, and Citrus sinensis peel extracts as treatments for microbial infections. This study aimed to investigate the influence of Anthemis hyaline, Nigella sativa, and Citrus sinensis extracts on coronavirus replication and apoptosis-related pathways. HeLa-CEACAM1a cells were exposed to mouse hepatitis virus-A59. After viral inoculation, the mRNA levels of 36 genes were quantified using a Fluidigm Dynamic Array nanofluidic chip. IL-8 level and intracellular Ca2+ concentration was measured, and viral titer was assessed by the TCID50/ml assay to detect the extent of infection. Treatment with Nigella sativa extract surged the inflammatory cytokine IL-8 level at both 24 and 48-hour. Changes in gene expression were notable for RHOA, VAV3, ROCK2, CFL1, RASA1, and MPRIP genes following treatment with any of the extracts. The addition of Anthemis hyaline, Nigella sativa, or Citrus sinensis extracts to coronavirus-infected cells reduced viral presence, with Anthemis hyaline extract leading to a virtually undetectable viral load at 6- and 8-hours after infection. While all treatments influenced IL-8 production and viral levels, Anthemis hyaline extract displayed the most pronounced reduction in viral load. Consequently, Anthemis hyaline extract emerges as the most promising agent, harboring potential therapeutic compounds.

High-throughput screening and clinical importance of autophagy-associated genes in basal cell carcinoma.

Autophagy is a type II programmed cell death mechanism that plays a critical role in preserving cellular homeostasis through the regulation of protein, lipid, and organelle quality control. It has become gradually evident that autophagy plays a fundamental role in the initiation and progression of various types of human cancers. Nevertheless, its significance in non-melanoma skin cancers, particularly in basal cell carcinoma, has not been well documented and remains largely elusive. In this study, we aimed to illuminate the role of autophagy-associated signaling signatures during development and progression of basal cell carcinoma. For the study, a total of 72 autophagy-related genes were screened using a high-throughput qPCR approach integrating Fluidigm 96.96 Dynamic Array™ integrated fluidic circuits (IFC) and BioMark™ HD Real-Time PCR system, which enabled efficient and precise analysis of gene expression patterns. Results were analyzed using Fluidigm's Real-Time PCR Analysis software and 2-ΔΔCt formula was used for the calculation of expression changes. Notably, expression levels of INS, TMEM74 and IFNA2 genes were identified to be prominently altered in BCC comparted to adjacent healthy tissues. However, only IFNA2 expression showed statistically significant change in BCC. Consequently, these findings suggest that IFNA2 might play significant role in the regulation of autophagy in BCC development and progression and can be therapeutically targeted.

UV radiation resistance-associated gene (UVRAG) promotes cell proliferation, migration, invasion by regulating cyclin-dependent kinases (CDK) and integrin-ß/Src signaling in breast cancer cells.

Breast cancer is a highly heterogeneous group of human cancer with distinct genetic, biological and clinicopathological features. Triple-negative breast cancer (TNBC) is the most aggressive and metastatic type of breast cancer and associated with poor patient survival. However, the role of UV Radiation Resistance-Associated Gene (UVRAG) in TNBC remains unknown. Here, we report that UVRAG is highly upregulated in all TNBC cells and its knockdown leads to the inhibition of cell proliferation, colony formation and progression of cell cycle, which is associated with and reduced expression of cell cycle related protein expression, including Cyclin A2, B1, D1, cdc2 and cdk6 in TNBC cells. Inhibition of UVRAG also suppressed cell motility, migration and invasion of TNBC cells by inhibition of Integrin ß1 and ß3 and Src activity. Our findings suggest for the first time that UVRAG expression contributes to proliferation, cell cycle progression, motility/migration and invasion of TNBC cells. Thus, targeting UVRAG could be a potential strategy in breast cancer especially against TNBC.

Investigation of Cytotoxic Effects of Oxymetazoline on Lungs in a Rat Model of Rhinitis Medicamentosa.

BACKGROUND: Rhinitis medicamentosa, also known as 'rebound congestion,' is inflammation of the nasal mucosa caused by the overuse of topical nasal decongestants. Although local decongestants resolve the initial nasal obstruction, the overuse causes rebound obstruction. However, how the overuse of the decongestant causes rhinitis medicamentosa is not known. OBJECTIVES: Here, we show the intracellular effects of oxymetazoline, commonly used a local decongestant, on the cell death pathways. We also investigated the antioxidative effects of erdosteine suspension (175 mg/5mL), an antioxidative agent. METHODS: Thirty Wistar-albino rats were used to form the rhinitis medicamentosa model. After rhinitis medicamentosa was clinically detected, we removed the whole lungs of animals to perform the molecular analyses of cell death pathways. RESULTS: We found a statistically significant decrease in the expression levels of Atg5 (p=0.021), Atg7 (p=0.013) and Ulk1 (p=0.036) in the oxymetazoline group compared to the control group (p<0.05); however, Caspase 3 expression level was recorded to be significantly increased in the oxymetazoline group, and the expression level of Beclin1 recorded to be substantially increased in the erdosteine group (p=0.001). CONCLUSION: Based on these grounds, we suggest that vasoconstriction in capillary vessels caused by oxymetazoline could lead to a decrease in the blood supply, which triggers autophagy to ensure cellular homeostasis.

Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle.

Coronavirus (CoV) nucleocapsid (N) proteins are key for incorporating genomic RNA into progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used model CoV, interacts with nonstructural protein 3 (nsp3), a component of the RTCs. These two aspects of the CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 and not other RTC components by using a systematic yeast two-hybrid approach, and we identified two distinct regions in the N protein that redundantly mediate this interaction. A selective N protein variant carrying point mutations in these two regions fails to bind nsp3 in vitro, resulting in inhibition of its recruitment to RTCs in vivo Furthermore, in contrast to the wild-type N protein, this N protein variant impairs the stimulation of genomic RNA and viral mRNA transcription in vivo and in vitro, which in turn leads to impairment of MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in the CoV life cycle because it is critical for optimal viral RNA synthesis.IMPORTANCE CoVs have long been regarded as relatively harmless pathogens for humans. Severe respiratory tract infection outbreaks caused by severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, however, have caused high pathogenicity and mortality rates in humans. These outbreaks highlighted the relevance of being able to control CoV infections. We used a model CoV, MHV, to investigate the importance of the recruitment of N protein, a central component of CoV virions, to intracellular platforms where CoVs replicate, transcribe, and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial for promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain an important aspect of the CoV life cycle and reveal an interaction of viral proteins that could be targeted in antiviral therapies.

MicroRNA 603 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting elongation factor 2 kinase.

Triple negative breast cancer (TNBC) is an aggressive type of breast cancer characterized by the absence of defined molecular targets, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and is associated with high rates of relapse and distant metastasis despite surgery and adjuvant chemotherapy. The lack of effective targeted therapies for TNBC represents an unmet therapeutic challenge. Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical calcium/calmodulin-dependent serine/threonine kinase that promotes TNBC tumorigenesis, progression, and drug resistance, representing a potential novel molecular target. However, the mechanisms regulating eEF2K expression are unknown. Here, we report that eEF2K protein expression is highly up-regulated in TNBC cells and patient tumors and it is associated with poor patient survival and clinical outcome. We found that loss/reduced expression of miR-603 leads to eEF2K overexpression in TNBC cell lines. Its expression results in inhibition of eEF2K by directly targeting the 3-UTR and the inhibition of tumor cell growth, migration and invasion in TNBC. In vivo therapeutic gene delivery of miR-603 into TNBC xenograft mouse models by systemic administration of miR-603-nanoparticles led to a significant inhibition of eEF2K expression and tumor growth, which was associated with decreased activity of the downstream targets of eEF2K, including Src, Akt, cyclin D1 and c-myc. Our findings suggest that miR-603 functions as a tumor suppressor and loss of miR-603 expression leads to increase in eEF2K expression and contributes to the growth, invasion, and progression of TNBC. Taken together, our data suggest that miR-603-based gene therapy is a potential strategy against TNBC.

Polymorphisms in the MTHFR gene are associated with recurrence risk in lymph node-positive breast cancer patients.

PURPOSE: The aim of this study is to clarify the relationship between recurrence risk of breast cancer and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms. PATIENTS AND METHODS: Breast cancer patients who had undergone surgery in Gaziantep University Oncology Hospital between June 2005 and June 2012 were followed-up and retrospectively enrolled in this study. Blood samples were collected from all patients to assess MTHFR C677T polymorphisms. Stage according to tumor-node-metastasis system, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 status, grade of disease, menopausal status, and administered chemotherapy or hormonal therapy were recorded. Effects of these parameters on recurrence risk were evaluated using univariate analysis and multivariate binary logistic regression model. RESULTS: Association of MTHFR C677T polymorphisms with recurrence risk was evaluated in 298 patients whose median age was 47 years (range: 21-79 years). In all patients, age (odds ratio [OR] =0.953, P=0.005) and N3 lymph node status (OR =6.293, P=0.001) were found to affect the recurrence risk. While MTHFR homozygote genotype did not have an effect on recurrence risk in all patients, increased risk was observed in lymph node-positive subgroup (OR =4.271; 95% CI 1.515-12.023; P=0.006). Adjusting for age, tumor size (T), and node status (N), MTHFR homozygote genotype had more statistically significant risk for recurrence (OR =3.255; 95% CI 1.047-10.125; P=0.041). CONCLUSION: MTHFR TT genotype was found to be associated with increased recurrence risk in patients with lymph node-positive breast cancer.

Polymorphism of CYP3A4 and ABCB1 genes increase the risk of neuropathy in breast cancer patients treated with paclitaxel and docetaxel.

BACKGROUND: Interindividual variability of pharmacogenetics may account for unpredictable neurotoxicities of taxanes. METHODS: From March 2011 to June 2015, female patients with operable breast cancer who had received docetaxel- or paclitaxel-containing adjuvant chemotherapy were included in this study. All patients were treated with single-agent paclitaxel intravenously (IV) 175 mg/m(2) every 3 weeks for four cycles, or IV 80 mg/m(2) weekly for 12 cycles, and IV 100 mg/m(2) docetaxel for four cycles as adjuvant treatment. We evaluated the relationship between neurotoxicity of taxanes and single-nucleotide polymorphisms of ABCB1, CYP3A4, ERCC1, ERCC2, FGFR4, TP53, ERBB2, and CYP2C8 genes. Taxane-induced neurotoxicity during the treatment was evaluated according to the National Cancer Institute Common Toxicity Criteria version 4.03 prior to each cycle. Chi-squared tests were used to compare the two groups, and multivariate binary logistic regression models were used for determining possible risk factors of neuropathy. RESULTS: Pharmacogenetic analysis was performed in 219 females. ABCB1 3435 TT genotype had significantly higher risk for grade ≥2 neurotoxicity (odds ratio [OR]: 2.759, 95% confidence interval [CI]: 1.172-6.493, P: 0.017) compared to TC and CC genotype, and also CYP3A4 392 AA and AG genotype had significantly higher risk for grade ≥2 neurotoxicity (OR: 2.259, 95% CI: 1.033-4.941, P: 0.038) compared to GG genotype. For FDGF4 gene with AG and GG genotype, OR was 1.879 (95% CI: 1.001-3.525, P: 0.048) compared to AA genotype with regard to any grade of neuropathy risk. We could not find any other association of other genotypes with neurotoxicity grades. CONCLUSION: ABCB1 3435 TT genotype and CYP3A4 392 AA/AG genotypes may be used as predictors of neurotoxicity during taxane chemotherapy.

Gene expression profiles of autophagy-related genes in multiple sclerosis.

Multiple sclerosis (MS) is an imflammatory disease of central nervous system caused by genetic and environmental factors that remain largely unknown. Autophagy is the process of degradation and recycling of damaged cytoplasmic organelles, macromolecular aggregates, and long-lived proteins. Malfunction of autophagy contributes to the pathogenesis of neurological diseases, and autophagy genes may modulate the T cell survival. We aimed to examine the expression levels of autophagy-related genes. The blood samples of 95 unrelated patients (aged 17-65years, 37 male, 58 female) diagnosed as MS and 95 healthy controls were used to extract the RNA samples. After conversion to single stranded cDNA using polyT priming: the targeted genes were pre-amplified, and 96×78 (samples×primers) qRT-PCR reactions were performed for each primer pair on each sample on a 96.96 array of Fluidigm BioMark™. Compared to age- and sex-matched controls, gene expression levels of ATG16L2, ATG9A, BCL2, FAS, GAA, HGS, PIK3R1, RAB24, RGS19, ULK1, FOXO1, HTT were significantly altered (false discovery rate<0.05). Thus, altered expression levels of several autophagy related genes may affect protein levels, which in turn would influence the activity of autophagy, or most probably, those genes might be acting independent of autophagy and contributing to MS pathogenesis as risk factors. The indeterminate genetic causes leading to alterations in gene expressions require further analysis.