Shahin, N. N., R. N. Shamma, and I. S. Ahmed, "A Nano-Liposomal Formulation of Caffeic Acid Phenethyl Ester Modulates Nrf2 and NF-κβ Signaling and Alleviates Experimentally Induced Acute Pancreatitis in a Rat Model.", Antioxidants (Basel, Switzerland), vol. 11, issue 8, pp. 1536, 2022. Abstract

The currently available management strategies for acute pancreatitis are inadequately effective which calls for exploration of new approaches to treat this condition. Caffeic acid phenethyl ester (CAPE) is a major bioactive constituent of honeybee propolis with promising therapeutic and preventive applications. However, its pharmaceutical potential and clinical use are hindered by its poor water solubility and limited plasma stability. In this study, we aimed to prepare, characterize and evaluate a CAPE-loaded nanoliposomal formulation to improve the efficacy of CAPE for the management of acute pancreatitis. The CAPE-loaded nanoliposomes (CAPE-loaded-NL) were prepared by a thin layer evaporation technique and were optimized using three edge activators. CAPE-loaded-NL were characterized for their vesicle size (VS), zeta potential (ZP), encapsulation efficiency (EE), polydispersity index (PDI), crystalline state and morphology. The protective effect of the optimal CAPE-loaded-NL was evaluated in a rat model of acute pancreatitis induced by administering a single intraperitoneal injection of L-ornithine. Oral pretreatment with CAPE-loaded-NL significantly counteracted ornithine-induced elevation in serum activities of pancreatic digestive enzymes and pancreatic levels of malondialdehyde, nuclear factor kappa B (NF-κB) p65, tumor necrosis factor-alpha, nitrite/nitrate, cleaved caspase-3 and myeloperoxidase activity. Moreover, pretreatment with CAPE-loaded-NL significantly reinstated the ornithine-lowered glutathione reductase activity, glutathione, nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 levels and ATP/ADP ratio, and potentiated the Bcl-2/Bax ratio in pancreatic tissue. CAPE-loaded-NL displayed superior antioxidant, anti-inflammatory and anti-apoptotic effects compared to free CAPE oral suspension and achieved a more potent correction of the derangements in serum amylase and pancreatic myeloperoxidase activities. The histological observations were in line with the biochemical findings. Our results suggest that CAPE-loaded-NL provide a promising interventional approach for acute pancreatitis mainly through the enhancement of the exerted antioxidant, anti-inflammatory and anti-apoptotic effects which may be mediated, at least in part, through modulation of Nrf2 and NF-κβ signaling.

Kotb, R. M., S. S. Ibrahim, O. M. Mostafa, and N. N. Shahin, "Potential role of CXCR4 in trastuzumab resistance in breast cancer patients.", Biochimica et biophysica acta. Molecular basis of disease, vol. 1868, issue 11, pp. 166520, 2022. Abstract

Despite the efficacy of trastuzumab in treating HER2-positive breast cancer patients, a significant proportion of patients relapse after treatment. The role of C-X-C chemokine receptor type 4 (CXCR4) in trastuzumab resistance was studied only in cell lines and the underlying mechanisms remain largely unclear. This study investigated the role of CXCR4 in trastuzumab resistance in breast cancer patients and explored the possible underlying mechanisms. The study was performed retrospectively on tissue samples from 62 breast cancer patients including 42 who were treated with trastuzumab and chemotherapy and 20 who received chemotherapy alone in adjuvant setting. Expression levels of CXCR4 and its regulators hypoxia-inducible factor 1-alpha (HIF-1α), tristetraprolin (TTP), human antigen R (HuR), itchy E3 ubiquitin protein ligase (ITCH), miR-302a and miR-494 were determined and their associations with tumor recurrence and disease-free survival were analyzed. In trastuzumab-treated patients, high CXCR4 expression was associated with recurrence and was an independent predictor of progression risk after therapy. CXCR4 correlated positively with its transcriptional regulator, HIF-1α, and negatively with its post-translational regulator, ITCH. HIF-1α, HuR and ITCH were significantly associated with clinical outcome. In chemotherapy-treated patients, neither CXCR4 nor any of its regulators were associated with recurrence or predicted disease progression risk after chemotherapy. In conclusion, this study suggests a potential role for CXCR4 in recurrence after trastuzumab-based therapy in human breast cancer that could be mediated, at least in part, by hypoxia and/or decreased ubiquitination. These findings highlight the potential utility of CXCR4 as a promising target for enhancing trastuzumab therapeutic outcome.

Motawi, T. M. K., N. A. H. Sadik, D. Sabry, S. A. Fahim, and N. N. Shahin, "rs62139665 Polymorphism in the Promoter Region of EpCAM Is Associated With Hepatitis C Virus-Related Hepatocellular Carcinoma Risk in Egyptians", Frontiers in Oncology, vol. 11, pp. 754104, 2022. AbstractWebsite

Hepatocellular carcinoma (HCC) is a universal health problem that is particularly alarming in Egypt. The major risk factor for HCC is hepatitis C virus (HCV) infection which is a main burden in Egypt. The epithelial cell adhesion molecule (EpCAM) is a stem cell marker involved in the tumorigenesis and progression of many malignancies, including HCC. We investigated the association of -935 C/G single nucleotide polymorphism in EpCAM promoter region (rs62139665) with HCC risk, EpCAM expression and overall survival in Egyptians. A total of 266 patients (128 HCV and 138 HCC cases) and 117 age- and sex-matched controls participated in this study. Genotyping, performed using allelic discrimination and confirmed by sequencing, revealed a significant association between EpCAM rs62139665 and HCC susceptibility, with higher GG genotype and G allele distribution in HCC patients than in non-HCC subjects. Such association was not detected in HCV patients compared to controls. EpCAM gene expression levels, determined in blood by RT-qPCR, and its serum protein expression levels, determined by ELISA, were significantly higher in GG relative to GC+CC genotype carriers in HCV and HCC patients in a recessive model. ROC analysis of EpCAM protein levels revealed significant discriminatory power between HCC patients and non-HCC subjects, with improved diagnostic accuracy when combining α-fetoprotein and EpCAM compared to that of α-fetoprotein alone. Altogether, EpCAM rs62139665 polymorphism is significantly associated with HCC and with EpCAM gene and protein expression levels in the Egyptian population. Moreover, serum EpCAM levels may hold promise for HCC diagnosis and for improving the diagnostic accuracy of α-fetoprotein.

Hanafy, M. M., J. Z. Lindeque, S. A. EL-Maraghy, A. - H. Z. Abdel-Hamid, and N. N. Shahin, "Time-based investigation of urinary metabolic markers for Type 2 diabetes: Metabolomics approach for diabetes management.", BioFactors (Oxford, England), 2021. Abstract

Diabetes is considered one of the most important health emergencies worldwide and Egypt has 8.2 million diabetic patients according to the International Diabetes Federation report in 2017. The objective of this study was to monitor the time-course variation in the metabolic profile of diabetic rats to detect urinary metabolic biomarkers using the metabolomics approach. Type 2 diabetes was induced in male Wistar albino rats using a single intraperitoneal injection of 40 mg/kg of streptozotocin following oral administration of 10% fructose in drinking water for 3 weeks. Then, urine was collected for 24 h from rats at three time points (0, 2, and 4 weeks after confirmation of diabetes), and were analyzed by nuclear magnetic resonance (H -NMR), followed by multivariate data analysis. The results from H -NMR pointed out that d-glucose, taurine, l-carnitine, l-fucose, 1,5-anhydrosorbitol, and d-galactose levels showed consistent significant variation (p < 0.05) between the positive (diabetic) and negative (normal) controls during the whole experimental period. Also, with the disease progression, myoinositol, and l-phenylalanine levels were significantly altered (p < 0.05) after 2 weeks and this alteration was maintained till the end of the 4-week experimental period in the positive control group. From the results of the present study, it could be concluded that we cannot depend only on glucose levels for prognostic purposes since there are other metabolic disturbances in diabetes which need to be tracked for better disease prognosis.

Arab, H. H., M. M. Safar, and N. N. Shahin, "Targeting ROS-Dependent AKT/GSK-3β/NF-κB and DJ-1/Nrf2 Pathways by Dapagliflozin Attenuates Neuronal Injury and Motor Dysfunction in Rotenone-Induced Parkinson's Disease Rat Model.", ACS chemical neuroscience, vol. 12, issue 4, pp. 689-703, 2021. Abstract

Dapagliflozin, a selective sodium-glucose co-transporter 2 (SGLT2) inhibitor, has emerged as a promising neuroprotective agent in murine models of epilepsy and obesity-induced cognitive impairment through its marked antioxidant/antiapoptotic features. However, the impact of dapagliflozin on the pathogenesis of Parkinson's disease (PD) is lacking. Hence, the present study aimed at exploring the potential neuroprotective effects of dapagliflozin against PD-associated neurodegenerative aberrations/motor dysfunction in rotenone-induced PD rat model. Rotenone (1.5 mg/kg) was subcutaneously administered every other day for 3 weeks. The expression of target signals was investigated using qPCR, Western blotting, ELISA, and immunohistochemistry. Dapagliflozin (1 (mg/kg)/day, by gavage for 3 weeks) attenuated PD motor dysfunction and improved motor coordination in the open-field and rotarod tests without triggering hypoglycemia. It also diminished the histopathologic alterations and α-synuclein expression and augmented tyrosine hydroxylase and dopamine levels. Dapagliflozin markedly alleviated neuronal oxidative stress via lowering lipid peroxides with consequent restoration of the disturbed DJ-1/Nrf2 pathway. Moreover, dapagliflozin counteracted ROS-dependent neuronal apoptosis and upregulated GDNF and its downstream PI3K/AKT/GSK-3β (Ser9) pathway. Meanwhile, it suppressed neuroinflammation via curbing the activation of NF-κB pathway and TNF-α levels. Together, these pleiotropic neuroprotective effects highlight the promising role of dapagliflozin in the management of PD.

El-Fattah, A. A. A., N. A. H. Sadik, O. G. Shaker, A. M. Kamal, and N. N. Shahin, "Serum Long Non-Coding RNAs PVT1, HOTAIR, and NEAT1 as Potential Biomarkers in Egyptian Women with Breast Cancer.", Biomolecules, vol. 11, issue 2, 2021. Abstract

Long non-coding RNAs play an important role in tumor growth, angiogenesis, and metastasis in several types of cancer. However, the clinical significance of using lncRNAs as biomarkers for breast cancer diagnosis and prognosis is still poorly investigated. In this study, we analyzed the serum expression levels of lncRNAs PVT1, HOTAIR, NEAT1, and MALAT1, and their associated proteins, PAI-1, and OPN, in breast cancer patients compared to fibroadenoma patients and healthy subjects. Using quantitative real-time PCR (qRT-PCR), we compared the serum expression levels of the four circulating lncRNAs in patients with breast cancer ( = 50), fibroadenoma ( = 25), and healthy controls ( = 25). The serum levels of PAI-1 and OPN were measured using ELISA. Receiveroperating-characteristic (ROC) analysis and multivariate logistic regression were used to evaluate the diagnostic value of the selected parameters. The serum levels of HOTAIR, PAI-1, and OPN were significantly higher in breast cancer patients compared to controls and fibroadenoma patients. The serum level of PVT1 was significantly higher in breast cancer patients than in the controls, while that of NEAT1 was significantly lower in breast cancer patients compared to controls and fibroadenoma patients. Both ROC and multivariate logistic regression analyses revealed that PAI-1 has the greatest power in discriminating breast cancer from the control, whereas HOTAIR, PAI-1, and OPN have the greatest power in discriminating breast cancer from fibroadenoma patients. In conclusion, our data suggest that the serum levels of PVT1, HOTAIR, NEAT1, PAI-1, and OPN could serve as promising diagnostic biomarkers for breast cancer.

Motawi, T. K., N. N. Shahin, K. Awad, A. S. Maghraby, D. N. Abd-Elshafy, and M. M. Bahgat, "Glycolytic and immunological alterations in human U937 monocytes in response to H1N1 infection.", IUBMB life, vol. 72, issue 11, pp. 2481-2498, 2020. Abstract

We monitored changes that took place in glycolytic enzymes, the pyruvate end product of glycolysis, tumor necrosis factor α (TNFα), and toll-like receptors (TLRs) both at the transcriptional and translational levels upon direct interaction between PR8-H1N1 and the human monocytes U937 in vitro system. U937 were first treated with H1N1 infectious viral particles or phorbol-12-myristate-13-acetate (PMA) or left untreated and later infected with the H1N1 virus. Levels of phosphofructokinase 1 (PFK1) and pyruvate were biochemically quantified. In addition, levels of TNFα, TLR3, and TLR7 were measured by ELISA. The transcriptional profiles of PFKs, inflammatory cytokines, TLR3 and TLR7 were relatively quantified by qRT-PCR. The results generally revealed significant changes in both the transcriptional and translational profiles of the studied biochemical and immunological parameters upon influenza infection in a time-dependent manner. In conclusion, H1N1 infection triggers transcriptional and translational changes in immortalized human monocytes, which might serve as markers for infection subject for further validation for their specificities.

Motawi, T. M. K., Z. M. Abdel-Nasser, and N. N. Shahin, "Ameliorative Effect of Necrosulfonamide in a Rat Model of Alzheimer's Disease: Targeting Mixed Lineage Kinase Domain-like Protein-Mediated Necroptosis.", ACS chemical neuroscience, vol. 11, issue 20, pp. 3386-3397, 2020. Abstract

Alzheimer's disease (AD) is a progressively debilitating neurodegenerative disorder that has no effective remedy, so far, with available therapeutic modalities being only symptomatic and of modest efficacy. Necroptosis is a form of controlled cell death with a recently emerging link to the pathogenesis of several neurodegenerative diseases. This study investigated the role of necroptosis in the pathogenesis of AD and evaluated the potential beneficial effect of the necroptosis inhibitor, necrosulfonamide (NSA), in a rat model of AD. AD was induced by oral administration of AlCl (17 mg/kg/day) for 6 consecutive weeks. Administration of NSA (1.65 mg/kg/day) intraperitoneally for 6 weeks significantly amended AlCl-induced spatial learning and memory deficits, as demonstrated by enhanced rat performance in Morris water and Y-mazes. NSA alleviated the abnormally high hippocampal expression of tumor necrosis factor-alpha (TNF-α), β-site amyloid precursor protein cleaving enzyme 1 (BACE1), β-amyloid, glycogen synthase kinase-3β (GSK-3β), phosphorylated tau protein, and acetylcholinesterase with concordant replenishment of acetylcholine. The amendments of AD perturbations achieved by NSA correlated with its inhibitory effect on the phosphorylation of the key necroptotic executioner, mixed lineage kinase domain-like protein (MLKL). Histopathological alterations supported the biochemical findings. In conclusion, NSA treatment represents a promising anti-Alzheimer's approach, mitigating AD neuropathologies via targeting MLKL-dependent necroptosis.

Safar, M. M., N. N. Shahin, A. F. Mohamed, and N. F. Abdelkader, "Suppression of BACE1 and amyloidogenic/RAGE axis by sitagliptin ameliorates PTZ kindling-induced cognitive deficits in rats.", Chemico-biological interactions, vol. 328, pp. 109144, 2020. Abstract

The debilitating nature of cognitive impairment in epilepsy and the potential of some traditional antiepileptics to further deteriorate cognitive function are areas of growing concern. Glucagon-like peptide-1 (GLP-1) deficiency has been linked to reduced seizure threshold as well as cognitive dysfunction. Here, we tested whether sitagliptin (SITA), by virtue of its neuroprotective properties, could alleviate both epilepsy and associated cognitive dysfunction in a rat model of kindling epilepsy. Chemical kindling was induced by subconvulsive doses of pentylenetetrazol (PTZ) (30 mg/kg; i.p). SITA (50 mg/kg; p.o) was administered 1 h before PTZ injections. SITA conceivably attenuated PTZ hippocampal histological insult, preserved neuronal integrity and amended neurotransmitter perturbations in rat hippocampi paralleled with enhanced hippocampal GLP-1 levels as well as the downstream cAMP content and protein kinase A (PKA) activity. Moreover, SITA improved cognitive functioning of rats in the Morris water maze which was coupled with hampered hippocampal p(Ser)-tau and β-amyloid proteins. SITA replenished p(Ser)-glycogen synthase kinase-3β (GSK-3β). It also opposed the boosted matrix metalloproteinase-9 (MMP-9), brain-derived neurotrophic factor (BDNF), and insulin-like growth factor-1 (IGF-1) levels associated with PTZ administration along with mitigation of both β-secretase-1 (BACE1) immunoreactivity and receptor for advanced glycation end products (RAGE) protein level in rat hippocampi. In conclusion, SITA subdues epileptic and cognitive upshots of PTZ kindling in rats, which might correspond to the modulation of BACE1, amyloidogenic/RAGE axis as well as GSK-3β/MMP-9/BDNF signaling cascade. SITA effects are probably mediated via boosting GLP-1 and subsequently enhancing GLP-1/GLP-1R signaling.

Abo-Elfadl, M. T., A. M. Gamal-Eldeen, M. F. Ismail, and N. N. Shahin, "Silencing of the cytokine receptor TNFRSF13B: A new therapeutic target for triple-negative breast cancer.", Cytokine, vol. 125, pp. 154790, 2020. Abstract

BACKGROUND: TNFRSF13B, TACI, is a member of the TNF receptor superfamily; it plays a key role in cancer cell proliferation and progression.

METHOD: Influence of silencing of human cytokine receptors on cell viability was screened by Luminescent Cell Viability Assay, after transfection of the siRNA library to find the maximum cell death superhits in both triple-negative MDA-MB-231 and double-positive MCF7 breast cells. The mode of cell death was investigated by dual DNA fluorescence staining. The expression of mRNAs of TACI, BAFF, BAFF-R, and APRIL was explored by qPCR. Immunocytofluorescence analysis was used to evaluate changes in TACI, Bcl-2, TNFR2, cyclin-D2, and PCNA. NF-kB p65, cell cycle, and necrosis/apoptosis (late and early) were analyzed by flow cytometry.

RESULTS: TACI is the most potent cytotoxic superhit resulted from high-throughput screening of the siRNA library, in both types of cells. Our findings indicated that silencing receptor TACI in both types of breast cancer cells led to significant cell death, after different intervals from siRNA transfection. Cell death mediators (TNFR2, Bcl-2, and NF-κB) were significantly decreased after TACI silencing. The key factors for cell division (Cyclin-D2 and PCNA) were significantly increased in silenced cells of both types but the cell cycle was arrested before the completion of mitosis. Expression of BAFF, BAFF-R and APRIL mRNA in TACI-silenced cells showed significant upregulation in MDA-MB-231 cells, while only BAFF-R and APRIL showed significant downregulation in MCF7 cells.

CONCLUSION: TACI silencing can be a new and promising therapeutic target for mesenchymal-stem like triple-negative breast cancer subtype.