Abdel Hady A. Abdel Wahab

Promoter hypermethylation of RASSF1A, MGMT, and HIC-1 genes in benign and malignant colorectal tumors

Hypermethylation at the promoter region is an important epigenetic mechanism underlying the inactivation of tumor suppressor genes and frequently occurs as an early event in the development of different types of cancer including colorectal carcinoma (CRC). The aim of the present study is the detection of methylation status for some tumor suppressor genes including RASSF1A, MGMT, and HIC-1 in both cancerous and precancerous lesions of colorectal mucosa to evaluate the possibility of developing epigenetic biomarker for early detection of Egyptian CRC. Tissue biopsy was collected from 72 patients (36 CRC, 17 adenomatous polyps, and 19 ulcerative colitis), and in addition, adjacent normal-appearing tissues were collected as control. Promoter hypermethylation status for RSSAF1A, MGMT, and HIC-1 genes was detected after isolation of genomic DNA from the tissues samples using methylation-specific PCR technique. High frequency of methylation at MGMT, RASSFA, and HIC-1 was detected in CRC patients (25%, 47.2%, and 41.7% respectively). The highest methylation detected in adenomatous polyps patients was in MGMT gene (47.1%) followed by 35.3% for HIC-1 and only 5.9% for RASSF1A gene. HIC-1 gene exhibited highest frequency of methylation in ulcerative colitis patients (57.8%) whereas it was 26.3% for both RASSF1A and MGMT genes. A nonsignificant association was recorded between the methylation status in different genes examined with the clinicopathological factors except the association between methylation at RASSF1A gene with gender (p = 0.005), and it was significant. In conclusion, aberrant hypermethylation at promoter region of RASSFA, MGMT, and HIC-1 genes is involved in Egyptian CRCs. Hypermethylation of MGMT and HIC-1 genes plays an important role in the initiation of disease especially ulcerative colitis–carcinoma pathway.

Urinary high molecular weight matrix metalloproteinases as non-invasive biomarker for detection of bladder cancer

BackgroundMatrix Metalloproteinases (MMPs) are key molecules for tumor growth, invasion and metastasis. Over-expression of different MMPs in tumor tissues can disturb the homeostasis and increase the level of various body fluids. Many MMPs including high molecular weights (HMWs) were detected in the urine of prostate and bladder cancer patients. Our aim here is to assess the usefulness of HMW MMPs as non invasive biomarkers in bilharzial bladder cancer in Egyptian patients.MethodsThe activity of different MMPs including HMW species was determined using zymographic analysis technique in the urine samples procured from sixty six bladder cancer patients (bilharzial and non-bilharzial) as well as hundred healthy control subjects. Also, the correlation between these HMW MMPs activities and different clinico-pathological parameters was investigated.ResultsHigh frequency of urine MMPs (uMMPs) activity was determined in 63.6% of examined tumor cases, however, none of the control cases showed any uMMPs activity. MMP-9 had the highest activity (62%) followed by MMP9/NGAL (60%), MMP-2 (54.5%), MMP-9 dimer (53%), ADAMTS (25.6%), and the lowest one was MMP-9/TIMP-1 (12%) only. There was no correlation between uMMPs and any of clinico-pathological parameters including age, gender, tumor size and type, bilharziasis, grade, lymph node involvement, and invasion to the prostate. A significant correlation was established only between MMP-9/TIMP-1 activities with the tumor size.ConclusionsThis study revealed that the detection of urinary MMPs including HMWs activity might be sensitive biomarkers for prediction of bladder cancer. It is also demonstrate that the detection of these urinary HMW gelatinases could not differentiate between bilharzial and non bilharzial bladder cancer subtypes.

Mutational Hotspots in the Mitochondrial D-Loop Region of Cancerous and Precancerous Colorectal Lesions in Egyptian Patients

Mutations in the mitochondrial genome (mtDNA) are associated with different types of cancer, specifically colorectal cancer (CRC). However, few studies have been performed on precancerous lesions, such as ulcerative colitis (UC) lesions and adenomatous polyps (AP). The aim of this study was to identify mtDNA mutations in the cancerous and precancerous lesions of Egyptian patients. An analysis of the mutations found in six regions of the mtDNA genome (ND1, ND5, COI, tRNAser, D-loop 1, and 2) in 80 Egyptian patients (40 CRC, 20 UC, and 20 AP) was performed using polymerase chain reaction-single-strand conformational polymorphism techniques and followed up by direct sequencing. The overall incidence of mutations was 25%, 25%, and 35% in CRC, UC, and AP cases, respectively. Although there was no common mutation pattern within each group, a large number of mutations were detected in the D-loop region in all of the groups. Some mutations (e.g., T414G) were detected repeatedly in precancerous (UC and AP) and cancerous lesions. Mutations detected in patients with CRC were predominantly found in the ND1 gene (40%). Our preliminary study suggests that Egyptian patients with CRC have a large number of mtDNA mutations, especially in the D-loop region, which have not been previously reported. Mutations in the mtDNA of precancerous lesions (i.e., AP and UC) may contribute to transformation events that lead to CRC.

Circulating microRNAs panel as a diagnostic tool for discrimination of HCV-associated hepatocellular carcinoma

Early diagnosis of hepatocellular carcinoma (HCC) can significantly improve the overall survival of HCC patients. However, current diagnostic markers are compromised and limited by their low sensitivity and specificity. In this work, circulating microRNAs (miRs) were utilized as a diagnostic tool to test their efficiency to segregate HCC and hepatitis C virus (HCV)-infected patients from healthy subjects. Nine HCC-related miRs (miR-21, miR-30c, miR-93, miR-122, miR-125b, miR-126, miR-130a, miR-193b and miR-222) were analyzed by Real-Time PCR in 86 serum samples; 34 HCC and 52 HCV patients in addition to 25 healthy subjects. The sensitivity and specificity of these miRs were assessed. Our results demonstrated that the median serum level of seven miRs was significantly reduced (P ranges from <0.01 to<0.001) in HCC patients whereas nine miRs were reduced (P<0.001) in HCV compared to healthy controls. Receiver operating characteristic (ROC) curve analyses had shown high diagnostic accuracy (AUC=1.0) when seven and nine combined miRs were considered in HCC and HCV groups, respectively compared to their counterparts. However, a combination of differentially expressed miRs did not improve the discriminatory power (AUC=0.742) when HCC compared to non-HCC groups. miR-122 showed the highest sensitivity and specificity to stratify HCC and HCV versus normal individuals and HCC versus HCV patients. We conclude that differentially expressed miRs in the serum of HCV and HCC patients can be utilized as surrogate and non-invasive biomarker for segregation of HCV and HCC patients from healthy subjects.

Crosstalk between liver-related microRNAs and Wnt/β-catenin pathway in hepatocellular carcinoma patients

BACKGROUND AND STUDY AIMS Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with highest incidence in Asia and Africa. MicroRNAs (miRNAs), a class of non-coding single stranded RNA, which not only post transcriptionally regulate gene expression but also respond to signaling molecules to affect cell functions such as Wnt/β-catenin signaling specifically in HCC. The goal of this study is to investigate the crosstalk between Wnt/β-catenin signaling proteins and microRNAs expression in HCC patients. PATIENTS AND METHODS Fresh tissue samples of 30 primary HCC patients and 10 control subjects were included. Expression level of 13 different miRNAs (miR-10a- miR-106b- miR-99a- miR-148a- miR-125b- miR-30e- miR-183- miR-155- miR-199a- miR-199a3p- miR-24- miR-122 and miR-215) were examined using real-time PCR assay. Five proteins involved in the Wnt/β-catenin pathway (β-catenin, APC, c-myc, survivin and cyclin D1) were analysed by immunohistochemistry technique. The correlation between miRNAs expression levels with protein expressions was assessed. RESULTS Up-regulation of miR-155 and miR-183 was reported in HCC patients compared to normal controls and this up-regulation was significantly correlated with liver cirrhosis in the case of miR-155 (p<0.05) referring to their oncogenic activity. Down-regulation was observed for 11 miRNAs in HCC indicating their tumour suppression activity. MiRNA-10a, miR-30e, miR-215, miR-125b and miR-148a were significantly correlated with the expression of important players in Wnt/β-catenin pathway including β-catenin, APC and c-myc (p<0.05). Detailed analysis revealed that miR-215 is associated with the grade of the disease and miR-125b is associated with HCV infection. CONCLUSION Collectively, our data showed potential role of miR-10a, miR-30e, miR-215, miR-125b and miR-148a as important mediators in HCC progression. Furthermore, their association with Wnt/β-catenin cascade proteins could be exploited to develop new therapeutic target strategies in HCC.

Identification of circulating protein biomarkers in patients with hepatocellular carcinoma concomitantly infected with chronic hepatitis C virus

Abstract Context: The incidence rate of hepatocellular carcinoma (HCC) is higher in developing countries, and most cases are associated with chronic hepatitis C virus (HCV) infection. Objective: To evaluate the circulating proteins as liver biomarkers for the identification of HCC associated with HCV infection in Egyptian patients using LC-MS/MS analysis. Methods: Blood sera were collected from 31 HCC patients and the fractionated proteins were subjected to LC-MS/MS analysis. Protein candidates were validated by enzyme-linked immunosorbent assay (ELISA). Results: Thirty-three proteins were significantly identified in the sera of HCC patients with persistent HCV infection. These proteins are involved in several biological processes including acute phase response, complement activation, hemostasis process and lipid metabolism. The level of lectin galactoside-binding soluble 3 binding protein (LGALS3BP), Kininogen-1 (KNG1), serum amyloid A2 (SAA2) and paraoxonase 1 (PON1) and alpha-fetoprtoein (AFP) were elevated in serum. Conclusion: In HCC patients with chronic HCV infection, we identified a group of differentially expressed circulating proteins involved in regulating different cellular mechanisms.

Circulating microRNAs as predictive biomarkers for liver disease progression of chronic hepatitis C (genotype-4) Egyptian patients: EL-HEFNY et al.

Egypt is one of the highest prevalence rates of hepatitis C virus (HCV) infection worldwide. HCV is among major reasons for chronic liver diseases. MicroRNA (miRNAs), small noncoding regulatory molecules play a key role in the pathogenesis of liver. Circulating miRNAs represent potential noninvasive biomarkers for diagnosis and monitoring patients with liver diseases progression. To investigate the potential role of circulating miRNAs for surveillance of liver disease progression, we assessed the expression of 20 liver‐related miRNAs in sera of 47 chronic hepatitis C Egyptian patients compared with 25 controls using quantitative reverse‐transcription polymerase chain reaction assay. The sensitivity and specificity were evaluated using receiver operating characteristic (ROC) curve. The correlations between their levels and the clinicopathological features were assessed. Fourteen miRNAs showed upregulation and six miRNAs showed downregulation. ROC curve analyses revealed that the explored miRNAs could serve as valuable biomarkers for chronic hepatitis with an area under the curve ranged from 0.708 (95% confidence interval [95% CI], 0.587 to 0.829; P = 0.004) for miR‐199 up to 0.974 (95% CI, 0.943 to 1.00; P < 0.001) for miR‐23b. The expression level of miR‐21 demonstrated significant correlation with age, liver enzymes, ALT/AST, and α‐fetoprotein level. AST level was directly correlated with miR‐122, while an inversely correlated with miR‐23b. Fibrosis and steatosis stages possessed positive correlation with miR‐199 expression and negative correlation with miR‐27a and miR‐93. In conclusion, miR‐23b and miR‐106 might be a useful biomarker for chronic hepatitis C (CHC). MiR‐27a, miR‐93, and miR‐199 might have a potential role in the progression of liver diseases. Unravel the role of these miRNAs in CHC patients might lead to precise prognosis and management.

Design, synthesis and screening of 1, 2, 4-triazinone derivatives as potential antitumor agents with apoptosis inducing activity on MCF-7 breast cancer cell line

Some triazinone derivatives are designed and synthesized as potential antitumor agents. Triazinone derivatives 4c, 5e and 7c show potent anticancer activity over MCF-7 breast cancer cells higher than podophyllotoxin (podo) by approximate 6-fold. DNA flow cytometry analysis for the compounds 3c, 4c, 5e, 6c and 7c show a potent inhibitory activity of cell proliferation and cell cycle arrest at G2/M phase. Compounds 4c, 5e and 7c exhibit low to moderate β-tubulin polymerization inhibition percentage. Meanwhile, compound 6c displayed excellent β-tubulin percentage of polymerization inhibition equivalent to that exhibited by podo. In addition, compounds 4c, 5e and 7c show strong topoisomerase (topo) II inhibitory activity in nano-molar concentration, compared to known topo inhibitor as etoposide. Finally, apoptotic inducing activity over MCF-7 of compounds 4c, 5e, 6c and 7c is due to up-regulation of p53, increased Bax/Bcl-2 ratio and caspase3/7 levels 2-fold higher than podo.

Abstract A32: The role of microRNA in the regulation of DUSP16 gene and JNK family in hepatocellular carcinoma patients

Introduction: Human hepatocellular carcinoma (HCC) is consider one of the most common and lethal tumors worldwide, HCC is currently the fifth most common solid tumor worldwide and the fourth important cause of cancer-related death. Eighty percent of new cases found in developing countries as Egypt, but the incidence is increasing in economically developed regions, including Japan, Western Europe, and the United States. It has shown that expression level of different miRNAs was correlated with cellular liver processes such as inflammation, hepatocyte regeneration and apoptosis which indicate their important role in many liver diseases including hepatocellular carcinoma. The main objective of the present study is to correlate between the expression levels of liver specific miRNAs with different mitogen activated protein kinase (MAPK) pathway in primary HCC patients. Patients and Methods: The expression level of miRNA targeting MAPK pathway, Dual-Specificity Phosphatase 16 (DUSP16), c-Jun N-terminal kinases (JNK) family (JNK1, JNK2 and JNK3) were detected in tissue biopsy of 38 primary HCC patients as well as 6 healthy controls using qRT-PCR technique. Detection of the protein levels for DUSP16, JNK1 and JNK2 was done using immunohistochemistry (IHC) assay. Results: The correlation between different expressions was done using (statistical method). Significant correlation was observed between 10 miRNAs with mRNA for DUSP16, JNK1, JNK2 and JNK3 (miR-18b; miR-30c; miR-30e; miR-99a; miR-126; miR-194; miR-198; miR-215; miR-455 and miR-455-3p). Highly significant down regulation was detected in JNK2 and JNK3 gene expression in HCC patients as compared to controls with (P value = 0.016, 0.000) respectively. Using IHC assay, 34% (13 out 38) showed DUSP16 (P value Conclusion: Understanding the role of miRNA in MAPK pathway may open new avenue for development novel treatment strategy in primary hepatocellular carcinoma. Citation Format: Marwa Tantawy, Nagwa Hassan, Mohamed Kobaisi, Abdel Hady Abdel Wahab. The role of microRNA in the regulation of DUSP16 gene and JNK family in hepatocellular carcinoma patients. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A32.