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", Women in Hepato Pancreatic Biliary (HPB) Tumors: 2021, Volume I: Frontiers, 2022.
Shahin, N. N., O. G. Shaker, and M. O. Mahmoud, "GOAT rs10096097 and CREB1 rs6740584 single nucleotide polymorphisms are associated with type 2 diabetes mellitus in Egyptians.", Archiv der Pharmazie, pp. e2400011, 2024. Abstract

Diabetes mellitus (DM) is a chronic disorder that affects nearly half a billion people around the world and causes millions of deaths annually. Treatment of diabetes or related complications represents an economic burden not only for developing countries but also for the developed ones. Hence, new efficient therapeutic and preventive strategies and screening tools are necessary. The current work aimed to assess the potential association of single nucleotide polymorphisms (SNPs) in ghrelin O-acyltransferase (GOAT) rs10096097, cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) rs6740584, and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) rs62521874 genes with type 2 DM susceptibility in Egyptians. A total of 96 patients with type 2 DM along with 72 healthy individuals participated in this study. Genotyping was executed via real-time polymerase chain reaction (PCR), and the serum protein levels of GOAT, CREB, and MafA were measured by enzyme-linked immunosorbent assay (ELISA). Genotyping revealed a significant association of GOAT rs10096097 and CREB1 rs6740584 SNPs with type 2 diabetes risk, with significantly higher GOAT rs10096097 G allele and CREB1 rs6740584 T allele frequencies in diabetic patients than in controls. However, insignificant association was identified between the MafA rs62521874 SNP and diabetes in the examined sample of the Egyptian residents. Serum GOAT, CREB1, and MafA protein levels did not vary significantly between diabetic and control individuals. Yet, significant variation in serum GOAT and CREB1 levels was detected between CREB1 rs6740584 genotypes within the diabetic group, with CT and TT genotype carriers showing higher levels than AA genotype patients. GOAT rs10096097 and CREB1 rs6740584, but not MafA rs62521874, SNPs are associated with type 2 diabetes risk in the studied Egyptians.

Motawi, T. K., A. E. Mady, M. Elhelbawy, R. M. Talaat, and N. N. Shahin, "Association of long noncoding RNA H19 rs2839698 C/T with hepatitis B virus infection and hepatocellular carcinoma risk.", Journal of biochemical and molecular toxicology, vol. 38, issue 4, pp. e23673, 2024. Abstract

The intricate pathogenesis of the hepatitis B virus (HBV) and its progression to hepatocellular carcinoma (HCC) have not yet been fully elucidated. H19 is one of the earliest imprinted long noncoding RNAs (lncRNAs) associated with liver pathobiology. This study investigated the association of H19 single nucleotide polymorphisms (SNPs) rs2839698 C/T and rs217727 C/T with HBV and HBV-related HCC and their correlation with H19 expression level. A total of 230 subjects were enrolled in this study including 100 HBV-infected patients, 30 HBV-related HCC patients, and 100 apparently healthy controls. TaqMan genotyping human assays were utilized to assess allelic discrimination for H19 SNPs. H19 expression was assessed using quantitative real-time polymerase chain reaction (qRT-PCR). Our findings showed that H19 rs2839698 was linked to a higher incidence of HBV infection and HBV-related HCC. Individuals who bear the CT genotype of rs2839698 were more susceptible to HBV infection (OR = 3.05; 95% CI 1.714-5.457; p < 0.001). Those harboring the TT genotype were more prone to develop HCC (OR = 2.625; 95% CI 1.037-6.64; p = 0.038). Our data revealed that rs2839698 could function as a promising predictor of HCC risk. Furthermore, H19 was significantly downregulated in HBV (p < 0.01) and HCC (p < 0.01) patients versus the control group. Significant upregulation of H19 in HCC patients with cirrhosis (p < 0.001) was detected. Altogether, this is considered the first prospective case-control study to address the implication of the genetic variations of H19 SNPs in HBV and HBV-related HCC in Egyptian patients.

Haridy, S. F. A., N. N. Shahin, M. I. Shabayek, M. M. Selim, M. A. Abdelhafez, and T. K. Motawi, "Diagnostic and prognostic value of the RUNXOR/RUNX1 axis in multiple sclerosis.", Neurobiology of disease, vol. 178, pp. 106032, 2023. Abstract

The runt-related transcription factor-1 (RUNX1) gene with its lncRNA RUNXOR are recently becoming a research focus in various diseases, specifically immune-related diseases as they are implicated in multiple pathways. Interestingly, their role in multiple sclerosis (MS) remains unstudied. The present study explored the role of RUNXOR/RUNX1 in the development and progression of MS and investigated their possible mechanism of action. We measured the serum expression levels of lncRNA RUNXOR, as well as RUNX1, microtubule associated protein 2 (MAP2), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) mRNAs in 30 healthy controls and 120 MS patients subdivided into 4 groups: 30 clinically isolated syndrome patients, 30 relapsing-remitting MS (RRMS) patients in relapse, 30 RRMS patients in remission and 30 secondary progressive MS patients. Additionally, we measured the serum protein levels of RUNX1, MAP2, NGF, BDNF and interleukin-10 (IL-10). All measured RNA expression levels were markedly downregulated and, consequently, the protein levels of RUNX1, MAP2, NGF, BDNF and IL-10 were significantly decreased in MS patients compared to healthy controls. Moreover, the levels of the measured parameters varied significantly within the MS groups. According to receiver-operating-characteristic (ROC) curve and logistic regression analyses, lncRNA RUNXOR, RUNX1 mRNA and its protein levels were predictors of disease progression, in addition to RUNX1 mRNA exhibiting a diagnostic potential. Altogether, this study suggests the implication of the RUNXOR-RUNX1 axis in MS development, progression, and increased MS-related disability, and highlights the potential utility of the studied parameters as promising diagnostic/prognostic biomarkers for MS.

Shama, S., H. Jang, X. Wang, Y. Zhang, N. N. Shahin, T. K. Motawi, S. Kim, S. Gawrieh, and W. Liu, "Phosphatidylethanolamines Are Associated with Nonalcoholic Fatty Liver Disease (NAFLD) in Obese Adults and Induce Liver Cell Metabolic Perturbations and Hepatic Stellate Cell Activation.", International journal of molecular sciences, vol. 24, issue 2, 2023. Abstract

Pathogenesis roles of phospholipids (PLs) in nonalcoholic fatty liver disease (NAFLD) remain incompletely understood. This study investigated the role of PLs in the progression of NAFLD among obese individuals via studying the alterations in serum PL composition throughout the spectrum of disease progression and evaluating the effects of specific phosphatidylethanolamines (PEs) on FLD development in vitro. A total of 203 obese subjects, who were undergoing bariatric surgery, were included in this study. They were histologically classified into 80 controls (C) with normal liver histology, 93 patients with simple hepatic steatosis (SS), 16 with borderline nonalcoholic steatohepatitis (B-NASH) and 14 with progressive NASH (NASH). Serum PLs were profiled by automated electrospray ionization tandem mass spectrometry (ESI-MS/MS). HepG2 (hepatoma cells) and LX2 (immortalized hepatic stellate cells or HSCs) were used to explore the roles of PL in NAFLD/NASH development. Several PLs and their relative ratios were significantly associated with NAFLD progression, especially those involving PE. Incubation of HepG2 cells with two phosphatidylethanolamines (PEs), PE (34:1) and PE (36:2), resulted in significant inhibition of cell proliferation, reduction of mitochondrial mass and membrane potential, induction of lipid accumulation and mitochondrial ROS production. Meanwhile, treatment of LX2 cells with both PEs markedly increased cell activation and migration. These effects were associated with a significant change in the expression levels of genes involved in lipogenesis, lipid oxidation, autophagy, apoptosis, inflammation, and fibrosis. Thus, our study demonstrated that elevated level of PEs increases susceptibility to the disease progression of obesity associated NAFLD, likely through a causal cascade of impacts on the function of different liver cells.

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.