Pharmacogenetic Study of the Dihydropyridine Dehydrogenase Gene in Jordanian Patients with Colorectal Cancer.

INTRODUCTION: Several studies have shown an association between 5-fluorouracil toxicity and variations in the dihydropyrimidine dehydrogenase (DPYD) gene. OBJECTIVES: This cross-sectional study aims to elucidate the association between genetic variations in the DPYD gene and 5-fluorouracil toxicity among Jordanians with colorectal cancer (CRC). METHODS: 80 CRC Patients were recruited to screen for mutations in the DPYD  gene using the Sanger sequencing technique. Sequencing results were analyzed using Mutation Surveyor software, and mutational effects were predicted by the Mutation Tester bioinformatics tool. RESULTS: Three reported variants (c.85T>C, c.1740+40A>G, c.1740+39C>T) and one novel (g.97515583_97515584insA) variant were identified in this study. Results showed a significant association between these variants and toxicity to 5-Fluorouracil with P-values 0.002, 0.005, 0.019, 0.017, respectively. However, there was no significant association between variants and cancer free survival. CONCLUSION: The present study identified several variants in the DPYD gene among Jordanians with colorectal cancer, which are associated with toxicity to 5-Fluorouracil treatment.

A Descriptive Study of the Physical Direct Interaction between Adipose Tissue-Mesenchymal Stem Cells and Colo 205 Cells: Impact on Cancer Cells Stemness, and Intracellular Reactive Oxygen Species Levels.

BACKGROUND: Mesenchymal stem cells (MSCs) are widely used in clinical research to treat a wild spectrum of diseases due to their homing ability to damaged tissues, self-renewal capacity, and differentiation ability into various types of cells. In this research, we are describing the physical direct interaction between AT-MSCs and colon cancer cells, its impact on the stemness of colon cancer cells, along with the levels of intracellular Reactive Oxygen Species (ROS) levels in both types of cells. METHODS: Adipose-tissue mesenchymal stem cells (AT-MSCs) were characterized by the means of MSCs classical markers expression using flow cytometry, and multilineage differentiation through osteogenic and adipogenic differentiation. MSCs and colo205 cells were cocultured in monolayer and 3D techniques in a ratio of 1:3 for 72 hours without media exchange and compared to monocultured cells. The physical direct interaction of cells in adhered culture and spheroids formation in ULA plates was observed using a light-inverted microscope. MSCs classical markers and cancer stem cells (CSCs) associated surface proteins were quantified in MSCs and colo205 cells. Intracellular ROS level was measured in both cell types. Surface protein and intracellular ROS quantification were carried out using flow cytometry. RESULTS: CRC cells (colo205 cells) utilized MSCs as a feeder layer to grow and generate spheroids. The interaction increased the percentage of CSCs in colo205 population which was attributed to the increased expression of CD133, and reduced the levels of intracellular ROS in MSCs. Results indicated that MSCs support the growth, spheriod formation, and the stemness of colon cancer cells, while reducing the levels of intracellular ROS in MSCs.

Mutational Analysis of EZH2 Gene in Patients with Colorectal Adenoma Reveals a Genetic Variant Associated with Risk of Malignant Transformation.

BACKGROUND: Several studies have revealed that chromatin modifications lead to activation or repression of multiple genes including oncogenes and tumor suppressor genes. Inactivation mutation in EZH2 gene would result in activation of oncogenes. The aim of this case-control study was to identify mutations in the EZH2 gene, to study their prevalence among Jordanian patients with colorectal adenoma and to determine how these mutations could be related to colorectal cancer (CRC) progression. METHODS: EZH2 gene sequencing was done by Sanger method for 100 DNA samples, extracted from blood of 50 patients, and 50 controls. Sequencing results were analyzed by Chromaspro and mutational effects were predicted by Mutation Taster bioinformatics tool. RESULTS: Four variants were identified in Jordanian patients with adenoma; Two novel variantsc.1941T>A and c.2201G>C and two reported variants, g.73038C>T and g.75508A>C. g.73038C>T is the most common germline variant identified in this study. A significant association between the presence of c.2201G>C mutation and colorectal adenoma was found (p value < 0.05). CONCLUSION: The present study identified several variants in EZH2 gene among Jordanians with colorectal adenoma.

Phenotypic and molecular analysis of dominant occurring antibiotic active-producing Streptomyces soil flora in Northern Jordan.

This investigation aimed to determine the relatedness of dominant occurring soil Streptomyces spp. in Northern Jordan based on their RAPD-PCR fingerprints, and to compare RAPD technique with the conventional phenotypic characterization of Streptomyces isolates. Fifty-eight white and gray color-bearing aerial mycelia antibiotic active-producing Streptomyces soil isolates along with three reference strains were genetically analyzed by RAPD-PCR. Polymorphisms between the isolates showed 1 to 10 bands per isolate and ranged from 200 to 3200 bp in size. Results revealed one common band of ~600 bp shared by ~85% of the isolates, and the observation of bands specific to some reference strains and some soil isolates. When RAPD patterns were analyzed with the UPGMA, results revealed clustering the tested isolates into two equal main super clusters (50% each). Super cluster I appeared to be homogenous and include the three reference strains. However, super cluster II was heterogeneous and but not including any of the reference strains. The association of the antibiotic activity of the dominant white and gray aerial mycelium-bearing Streptomyces isolates to RAPD clustering is reported for the first time, and the RAPD-PCR fingerprints generated here deserve to be cloned, characterized and sequenced in future as Streptomyces species-specific DNA markers. The more random primers used in the analysis may add to RAPD technique a cost-effective, fast, precise result, and less labor work solution for analyzing the similarities and differences among the Streptomyces isolates.

Association of DNA repair gene polymorphisms with colorectal cancer risk and treatment outcomes.

Colorectal cancer (CRC) is the third most common carcinoma worldwide. Despite the progress in screening and treatment, CRC remains a leading cause of cancer-related mortality. Alterations to normal nucleic acid processing may drive neoplastic transformation of colorectal epithelium. DNA repair machinery performs an essential function in the protection of genome by reducing the number of genetic polymorphisms/variations that may drive carcinogenicity. Four essential DNA repair systems are known which include nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), and double-strand break repair (DSBR). Polymorphisms of DNA repair genes have been shown to influence the risk of cancer development as well as outcomes of treatment. Several studies demonstrated the association between genetic polymorphism of DNA repair genes and increased risk of CRC in different populations. In this review, we have summarized the impact of DNA repair gene polymorphisms on risk of CRC development and treatment outcomes. Advancements of the current understanding for the impact of DNA repair gene polymorphisms on the risk and treatment of CRC may support diagnostic and predictive roles in patients with CRC.

The association of HLA-DQB1*0602 but not HLA-DRB1*15 with obstructive sleep apnea.

PURPOSE: Obstructive sleep apnea (OSA) is a sleep breathing disorder with unclear multifactorial pathogenesis. This study aimed to investigate the association between OSA and two human leukocyte antigens (HLA) alleles; DQB1*0602 and DRB1*15. METHODS: Forty patients with OSA and 40 control subjects were enrolled in the study. OSA diagnosis was made utilizing the Apnea-Hypopnea Index (AHI)≥5 in overnight polysomnography (PSG). AHI was also used to determine OSA severity. Controls were randomly selected from healthy volunteers who had a low risk for OSA, utilizing the Berlin Questionnaire. Polymerase chain reaction (PCR) using Sequence Specific Primers (PCR-SSPs) was used to determine the association between HLA (HLA-DQB1*0602 and HLA-DRB1*15) and OSA, then statistical analyses of the results were performed. RESULTS: HLA-DQB1*0602 allele was found in 85% of all OSA patients and 50% of controls (P< 0.001). In patients with severe OSA, HLA-DQB1*0602 was present in the 92.9% compared with 66.7% in non-severe OSA (P=0.05). HLA-DRB1*15 allele was found in 15% of OSA patients and 20% of controls, with no difference between the two groups (P=0.556). No statistical difference was found in HLA-DRB1*15 between severe and non-severe OSA (P=0.499). After adjusting for gender, HLA-DQB1*0602 allele was associated with increased odds of OSA (OR = 6.17, 95% CI 1.87-20.3, p = 0.003), but HLA-DRB1*15 allele was not associated with OSA (OR 0.45, 95% CI 0.12-1.73, p = 0.242). CONCLUSIONS: The presence of HLA-DQB1*0602 allele, but not HLA-DRB1*15 allele, was significantly associated with OSA.

Lifestyle predictors of oxidant and antioxidant enzyme activities and total antioxidant capacity in healthy women: a cross-sectional study

The aim of this study was to identify demographic and modifiable lifestyle factors that may be related to endogenous oxidant and antioxidant activity measured in blood specimens from putatively healthy women recruited at the Roswell Park Cancer Institute (Buffalo, NY, USA). Total glutathione (TGSH), catalase (CAT), CuZn-superoxide dismutase (CuZn-SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and myeloperoxidase (MPO) activity, and total antioxidant capacity (TAC) were measured in 124 healthy women, and associations with epidemiological factors were tested using general linear models. There were significant differences in oxidant and antioxidant enzyme activities according to lifestyle factors, after adjusting for duration of blood storage and season of blood draw. Compared to women who consumed ≤2.8 servings of fruits and vegetables daily, those consuming >5.3 servings had on average 31 % lower MPO activity (p-trend = 0.02), as a marker of oxidative stress, 16 % higher antioxidant GPx activity (p-trend = 0.08), and 9 % higher TAC (p-trend = 0.05). Obese women (body mass index, BMI ≥ 30) in contrast showed 17 % lower antioxidant GPx activity, 44 % higher MPO activity (p-trend = 0.03), and 10 % higher TAC (p-trend = 0.03) compared to women with normal BMI < 25. Smoking was associated with higher TGSH activity (p-trend = 0.01) and lower TAC (p-trend = 0.05). Higher TAC levels were most strongly associated with increasing age (standardized β = 0.40, p < 0.0001), BMI (standardized β = 0.17, p = 0.03), and GPx activity (standardized β = 0.23, p = 0.005), and inversely associated with CuZn-SOD activity (standardized β = −0.14, p = 0.07). Physical activity levels, multivitamin use, and alcohol intake were not associated with TAC. Our data indicate that endogenous oxidant and antioxidant enzyme activities are associated with lifestyle factors and, therefore, may be potentially modifiable, with implications for risk reduction of chronic conditions related to oxidative stress (AU)

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Lifestyle predictors of oxidant and antioxidant enzyme activities and total antioxidant capacity in healthy women: a cross-sectional study.

The aim of this study was to identify demographic and modifiable lifestyle factors that may be related to endogenous oxidant and antioxidant activity measured in blood specimens from putatively healthy women recruited at the Roswell Park Cancer Institute (Buffalo, NY, USA). Total glutathione (TGSH), catalase (CAT), CuZn-superoxide dismutase (CuZn-SOD), glutathione peroxidase (GPx), glutathione reductase (GR), and myeloperoxidase (MPO) activity, and total antioxidant capacity (TAC) were measured in 124 healthy women, and associations with epidemiological factors were tested using general linear models. There were significant differences in oxidant and antioxidant enzyme activities according to lifestyle factors, after adjusting for duration of blood storage and season of blood draw. Compared to women who consumed ≤2.8 servings of fruits and vegetables daily, those consuming >5.3 servings had on average 31 % lower MPO activity (p-trend = 0.02), as a marker of oxidative stress, 16 % higher antioxidant GPx activity (p-trend = 0.08), and 9 % higher TAC (p-trend = 0.05). Obese women (body mass index, BMI ≥ 30) in contrast showed 17 % lower antioxidant GPx activity, 44 % higher MPO activity (p-trend = 0.03), and 10 % higher TAC (p-trend = 0.03) compared to women with normal BMI < 25. Smoking was associated with higher TGSH activity (p-trend = 0.01) and lower TAC (p-trend = 0.05). Higher TAC levels were most strongly associated with increasing age (standardized ß = 0.40, p < 0.0001), BMI (standardized ß = 0.17, p = 0.03), and GPx activity (standardized ß = 0.23, p = 0.005), and inversely associated with CuZn-SOD activity (standardized ß = -0.14, p = 0.07). Physical activity levels, multivitamin use, and alcohol intake were not associated with TAC. Our data indicate that endogenous oxidant and antioxidant enzyme activities are associated with lifestyle factors and, therefore, may be potentially modifiable, with implications for risk reduction of chronic conditions related to oxidative stress.

Polymorphism of p53 gene in Jordanian population and possible associations with breast cancer and lung adenocarcinoma.

OBJECTIVE: To determine the prevalence of 3 polymorphisms in p53 gene in 3 healthy Jordanian groups and 2 cancer patient groups. METHODS: Genomic DNA was extracted from blood samples obtained from 84 cancer patients (breast and lung adenocarcinoma) and 136 healthy subjects (representing Jordanian general population, Bedouins and Charkas). Samples were collected from Al-Amal Hospital for Cancer, Amman and from health centers located in different regions of Jordan from March 2002 to October 2002. Polymerase chain reaction (PCR) was used to amplify intron 3, exon 4 and intron 6 and PCR products were analyzed using gel electrophoresis and BstUI and MspI analysis. Allele frequencies (A1) were estimated for the 3 polymorphisms and Chi-square (chi2) test was used to determine the significance of differences from the Hardy-Weinberg equilibrium. RESULTS: Differences in allele frequencies for all 3 polymorphisms were observed among the various groups. Analysis based on haplotype frequencies showed that MspI A2 allele linked to BstUI allele was associated with lung adenocarcinoma, whereas the loss of the 16-bp duplication allele in combination with MspI A2 allele was associated with breast cancer. In the cancer patients, the most frequent extended haplotype was the absence of the 16-bp duplication in combination with the presence of the BstUI A2 and MspI restriction sites. CONCLUSION: No significant difference was found with respect to the BstUI polymorphism between cancer patients and healthy groups. However, a significant difference was found with respect to the MspI polymorphism between lung adenocarcinoma patients and healthy Jordanian general population. Charkas have a higher cancer risk than Jordanian general population based on the (16bp A1-MspI A2) for breast cancer and (MspI A2-BstUI A2) for lung adenocarcinoma.