Inhibition of PRMT5-mediated regulation of DKK1 sensitizes colorectal cancer cells to chemotherapy.

The Dickkopf family proteins (DKKs) are strong Wnt signaling antagonists that play a significant role in colorectal cancer (CRC) development and progression. Recent work has shown that DKKs, mainly DKK1, are associated with the induction of chemoresistance in CRC and that DKK1 expression in cancer cells correlates with that of protein arginine N-methyltransferase 5 (PRMT5). This points to the presence of a regulatory loop between DKK1 and PRMT5. Herein, we addressed the question of whether PRMT5 contributes to DKK1 expression in CRC and hence CRC chemoresistance. Both in silico and in vitro approaches were used to explore the relationship between PRMT5 and different DKK members. Our data demonstrated that DKK1 expression is significantly upregulated in CRC clinical samples, KRAS-mutated CRC in particular and that the levels of DKK1 positively correlate with PRMT5 activation. Chromatin immunoprecipitation (ChIP) data indicated a possible epigenetic role of PRMT5 in regulating DKK1, possibly through the symmetric dimethylation of H3R8. Knockdown of DKK1 or treatment with the PRMT5 inhibitor CMP5 in combination with doxorubicin yielded a synergistic anti-tumor effect in KRAS mutant, but not KRAS wild-type, CRC cells. These findings suggest that PRMT5 regulates DKK1 expression in CRC and that inhibition of PRMT5 modulates DKK1 expression in such a way that reduces CRC cell growth.

DNA Methylation-Mediated Overexpression of CXCL1 in Helicobacter pylori-Induced Gastric Cancer: In Silico- and In Vitro-Based Identification of a Potential Biomarker for Carcinogenesis.

Given the high global prevalence and mortality associated with gastric cancer, and its known causal link with Helicobacter pylori infection, it is important to have a biomarker to identify malignant transformation at early stages. Previously, we, and others, have reported that H. pylori-induced epigenetic changes could mediate carcinogenic transformation of the gastric cells. Also, CXCL1 secreted by gastric cancer cells was reported as a key diagnostic and prognostic biomarker for the pathogenic progression of gastric cancer. In this study, for the first time, we aimed to investigate the role of H. pylori-induced DNA methylation-based epigenetic regulation of CXCL1. In silico analysis of publicly available datasets and in vitro experiments were performed. Our results showed that CXCL1 is highly expressed in both gastric cancer tissues and gastric cancer cells infected with H. pylori. Further, we showed and confirmed that H. pylori-mediated overexpression of CXCL1 is due to hypomethylation of its promoter region. Since epigenetic events such as DNA methylation happen early in the sequence; H. pylori-induced CXCL1 hypomethylation could likely be detected at an early stage of gastric cancer development. Epigenetic modifications, such as CXCL1 hypomethylation, are reversible and could potentially be a therapeutic target using demethylation drugs.

Autophagy: A Versatile Player in the Progression of Colorectal Cancer and Drug Resistance.

Colorectal cancer (CRC) is among the topmost malignancies for both genders. Despite the high incidence rate and advances in diagnostic tools, treatment in many cases is still ineffective. Most cancerous lesions in CRC begin as benign, followed by the development of invasive forms and metastases. The development of CRC has been linked to defects in autophagy, which plays both a pro-and anti-tumor role and is mainly context-dependent. Autophagy suppression could enhance apoptosis via p53 activation, or autophagy also promotes tumor progression by maintaining tumor growth and increasing resistance to chemotherapy. Autophagy promotes the invasion and metastasis of CRC cells via increased epithelial-mesenchymal transition (EMT). Moreover, dysbiosis of gut microbiota upregulated autophagy and metastasis markers. Autophagy responses may also modulate the tumor microenvironment (TME) via regulating the differentiation process of several innate immune cells. Treatments that promote tumor cell death by stimulating or inhibiting autophagy could be beneficial if used as an adjunct treatment, but the precise role of various autophagy-modulating drugs in CRC patients is needed to be explored. In this article, we present an overview of the autophagy process and its role in the pathogenesis and therapeutic resistance of CRC. Also, we focused on the current understanding of the role of the EMT and TME, including its relation to gut microbiota and immune cells, in autophagic manipulation of CRC. We believe that there is a potential link between autophagy, TME, EMT, and drug resistance, suggesting that further studies are needed to explore this aspect.

Evaluation of nanoparticles with 5-fluorouracil and chloroquine on Acanthamoeba castellanii activity.

Acanthamoeba is opportunistic pathogens that cause vision-threatening Acanthamoeba keratitis (AK). Previous studies proposed the use of chloroquine (CQ) and 5-fluorouracil (5FU) as anti-Acanthamoeba agents. The objective of this study was to determine the benefit of using 5FU and CQ nanoparticles (NP) formulations against A. castellanii that belonging to the T4 genotype and evaluate their anti-Acanthamoebic characteristic. Triplicate batches of 5FU nanoparticles (5FU-NP) were synthesized by using a modified nanoprecipitation method, while CQ nanoparticles (CQ-NP) synthesized using a modified double emulsion method. The synthesized nanoparticles were subjected to biological assays to investigate their amoebicidal, amoebistatic, anti-encystation, and anti-excystation effects against A. castellanii, as well as cell cytotoxicity. Cytotoxicity assays were performed using human keratinocyte cells (HaCaT) to determine the effect of CQ and 5FU nanoformulations on host cells. 5FU-NP with a concentration of 60 µM showed significant inhibition to amoeba binding into human cell lines and remarkable prevention mainly during the encystation stage. Moreover, 5FU-NP resulted in less cytotoxicity and pathogenicity when compared with the free 5FU. On the other hand, CQ and CQ-NP, at the same concentration, showed poor inhibition to amoeba binding into human cells and insignificant prevention to encystation stage. Moderate human cells damage was resulted following their treatment with CQ and CQ-NP. In conclusion, 5FU may have the potential as an antiamoebic agent against Acanthamoeba spp. preferably as a nanoformulation to enhance its activity and reduce its cytoxicity.

MLH1 mediates cytoprotective nucleophagy to resist 5-Fluorouracil-induced cell death in colorectal carcinoma.

Colorectal Cancer (CRC) with Microsatellite instability (MSI) and mutLhomolog-1 (MLH1) gene deficiency are less aggressive than MLH1 proficient cancers. MLH1 is involved in several cellular processes, but its connection with the autophagy-dependent cellular response towards anticancer drugs remains unclear. In this study, we aimed to investigate the interaction between MLH1 and the autophagy marker LC3, which facilitated nucleophagy induction, and its potential role in determining sensitivity to 5-Fluorouracil (5-FU) induced cell death. To examine the role of MLH1 in DNA-damage-induced nucleophagy in CRC cells, we utilized a panel of MLH1 deficient and MLH1 proficient CRC cell lines. We included a parental HCT116 cell line (MLH1-/-) and its isogenic cell line HCT116 MLH1+/- in which a single allele of the MLH1 gene was introduced using CRISPR-Cas9 gene editing. We observed that MLH1 proficient cells were less sensitive to the 5-FU-induced cytotoxic effect. The 5-FU induced DNA damage led to LC3 up-regulation, which was dependent on MLH1 overexpression. Moreover, immunofluorescence and immunoprecipitation data showed LC3 and MLH1 were colocalized in CRC cells. Consequently, MLH1 dependent 5-FU-induced DNA damage contributed to the formation of micronuclei. These micronuclei colocalize with autolysosome, indicating a cytoprotective role of MLH1 dependent nucleophagy. Interestingly, siRNA knockdown of MLH1 in HCT116 MLH1+/- prevented LC3 upregulation and micronuclei formation. These novel data are the first to show an essential role of MLH1 in mediating the chemoresistance and survival of cancer cells by increasing the LC3 expression and inducing nucleophagy in 5-FU treated CRC cells.

Synthesis of scaffold-free, three dimensional, osteogenic constructs following culture of skeletal osteoprogenitor cells on glass surfaces.

BACKGROUND: Efficient differentiation of stem cells into three-dimensional (3D) osteogenic construct is still an unmet challenge. These constructs can be crucial for patients with bone defects due to congenital or traumatic reasons. The modulation of cell fate and function as a consequence of interaction with the physical and chemical properties of materials is well known. METHODS: The current study has examined the osteogenic differentiation potential of human skeletal populations following culture on glass surfaces, as a monolayer, or in glass tubes as a pellet culture. The 3D prosperities were assessed morphometrically and the differentiation was evaluated through molecular characterization as well as matrix formation. RESULTS: Early temporal expression of alkaline phosphatase expression of skeletal populations was observed following culture on glass surfaces. Skeletal populations seeded on glass tubes, adhered as a monolayer to the tube base and subsequently formed 3D pellets at the air -media interface. The pellets cultured on glass displayed 4.9 ± 1.3 times the weight and 2.9 ± 0.1 the diameter of their counterpart cultured in plastic tubes and displayed enhanced production of osteogenic matrix proteins, such a collagen I and osteonectin. The size and weight of the pellets correlated with surface area in contrast to cell numbers seeded. Global DNA methylation level was decreased in pellets cultured on glass. In contrast, gene expression analysis confirmed upregulation extracellular matrix proteins and osteogenesis-related growth factors. CONCLUSION: This simple approach to the culture of skeletal cells on glass tubes provides a scaffold-free, 3D construct platform for generating pellets enabling analysis and evaluation of tissue development and integration of multiple constructs with implications for tissue repair and regenerative application on scale-up.

Dietary Patterns and Their Associations With the FTO and FGF21 Gene Variants Among Emirati Adults.

Purpose: To examine the dietary patterns and their associations with the FTO and FGF21 gene variants among Emirati adults. Methods: Using a cross-sectional design, healthy adult male and female Emiratis (n = 194) were recruited from primary health care centers in Sharjah, UAE. Participants completed a 61-item semi-quantitative food frequency questionnaire. In addition, a saliva sample was obtained for the genetic analysis. Genotyping was performed for FTOrs9939609(A>T), FTOrs9930506(A>G), FGF21 rs838133 (A > G), and FGF21 rs838145 (A > G). Dietary patterns were derived using the principal component analysis. Logistic regression analyses were used to examine the association of dietary patterns with genetic variants. Results: Three dietary patterns were identified: "Western": consisting of fast food, sweets, and processed meat; "Traditional Emirati" rich in vegetables, traditional Emirati-mixed-dishes and whole dairy; while whole grains, low-fat dairy, and bulgur were components of the "Prudent" pattern. Subjects carrying the A allele of the FTO rs9939609 were 2.41 times more likely to adhere to the Western pattern compared to subjects with genotype TT (OR:2.41; 95%CI:1.05-5.50). Compared with subjects with A/A, those carrying the G allele of the FTO rs9930506 were more likely to follow a Western diet (OR: 2.19; 95%CI: 1.00-4.97). Participants carrying the risk allele (A) of the FGF21 rs838133 were twice more likely to adhere to the Traditional pattern as compared to subjects with genotype GG (OR: 1.9, 95%CI: 1.01-3.57). Conclusions: The findings of this study suggested associations among specific FTO and FGF21 gene variants with dietary patterns among Emirati adults. These findings could be used to inform evidence-based targeted nutrition preventive recommendations, especially those aiming to limit intake of western type foods.

The FGF-21 genetic variants rs838133 and rs838145 are associated with high salt intake in the Emirati population.

Food predilection is linked to variants in the hepatokine "Fibroblast Growth Factor-21" gene (FGF21); with rs838133 linked to the sweet tooth in Caucasians. The effect of FGF21 variants on food intake is still unclear in other populations. A cohort of 196 healthy Emirati subjects was investigated [age: 30.34 ± 9.75yrs (44.4% males)]. The FGF21 rs838133 and rs838145 were genotyped. The daily intake was calculated based on a 61-item food frequency questionnaire. Multivariate analysis was performed using in house R script that implements two-way unsupervised hierarchical clustering to detect the association of the studied single-nucleotide polymorphisms (SNPs) and related SNPs in linkage disequilibrium, using data from the 1000 genome project. Both SNPs were in Hardy-Weinberg Equilaribium (HWE). BMI positively correlated with age (p = 0.002), but not with caloric intake. Salt intake was significantly higher in subjects homozygous (A: rs838133) and (G:rs838145),(p = 0.03 and 0.01, respectively). An interaction was observed between both SNPs; significantly associated with high salt intake. Using publicly available data, both SNPs fall within a region transmitted in Iberians which has a profile closely similar to Caucasians, but far from Chinese population. In conclusion, the minor alleles of FGF21 rs838145 and rs838133 are associated with high salt intake in Emiratis and may suggest neuro-metabolic link to dietary preference across different populations.

The FTO genetic variants are associated with dietary intake and body mass index amongst Emirati population.

BACKGROUND: The risk of obesity is determined by complex interactions between genetic and environmental factors. Little research to date has investigated the interaction between gene and food intake. The aim of the current study is to explore the potential effect of fat mass and obesity-associated protein gene (FTO) rs9939609 and rs9930506 single nucleotide polymorphism (SNP) on the pattern of food intake in the Emirati population. METHODS: Adult healthy Emirati subjects with Body mass index (BMI) of 16-40 kg/m2 were included in the study. Genotyping for FTO rs9939609(A>T) and rs9930506(A>G) was performed using DNA from saliva samples. Subjects were categorized according to the WHO classification by calculating the BMI to compare different classes. Dietary intake was assessed by a sixty-one-item FFQ that estimated food and beverage intakes over the past year. The daily energy, macronutrient, and micronutrient consumption were computed. RESULTS: We included 169 subjects in the final analysis (mean age 30.49± 9.1years, 57.4% females). The mean BMI of the study population was 26.19 kg/m2. Both SNPs were in Hardy Weinberg Equilibrium. The rs9939609 AA genotype was significantly associated with higher BMI (p = 0.004); the effect was significant in females (p = 0.028), but not in males (p = 0.184). Carbohydrate intake was significantly higher in AA subjects with a trend of lower fat intake compared to other genotypes. The odds ratio for the AA was 3.78 in the fourth quartile and 2.67 for the A/T in the second quartile of total carbohydrate intake, considering the first quartile as a reference (95% CI = 1.017-14.1 and 1.03-6.88, respectively). Fat intake was significantly lower in the FTO rs9930506 GG subjects. The presence of FTO rs9930506 GG genotype decreased the fat intake in subjects with FTO rs9939609 AA (p = 0.037). CONCLUSIONS: The results of this study highlight the interaction of the FTO risk alleles on the food intake in Emirati subjects. The FTO rs9939609 AA subjects had higher carbohydrate and lower fat intake. The latter was accentuated in presence of rs9930506 GG genotype.

The FTO rs9939609 "A" allele is associated with impaired fasting glucose and insulin resistance in Emirati population.

BACKGROUND: Fat mass and obesity-associated protein gene variants have shown diverse influence on body weight and metabolism across different populations. Overweight, obesity and metabolic syndrome are multifactorial major health problems in the UAE and worldwide. Insulin resistance represents the link between overweight and development of metabolic syndrome and type 2 diabetes mellitus. We investigated two (FTO) variants in Emirati population, in relation to insulin resistance and different parameters of metabolic syndrome. METHODS: We recruited 259 Emiratis through the UAE National Diabetes and Lifestyle Project. Ethical approval was obtained. Besides basic data collection, venous blood samples were collected. Fasting blood glucose, Lipid profile, and insulin levels were measured. Genotyping for (FTO) rs9939609 (A>T) and rs9930506 (G>A) were performed using real time-PCR. Insulin resistance were identified using HOMA2-IR calculation; with a cut-off point of 1.4 for female and 1.18 for male subjects. RESULTS: The study included 259 Emiratis (age range 30-53 years, mean 41.76 years, 54.4% females), 24.5% are diabetic and 30.8% are hypertensive, with body mass index of 28.4 ±â€¯5.9 and 28.7 ±â€¯5.7 kg/m2 in female and male subjects, respectively. Homozygous A of rs9939609 showed significantly higher fasting glucose compared to other genotypes (p = 0.04) with a trend of higher insulin level and HOMA-2IR. The A/A diabetic patients (n = 13) showed significantly higher insulin levels compared to other genotypes. G allele of rs9930506 showed a trend of higher fasting glucose and HOMA-2IR, but lower insulin level and HbA1c. No association of genotypes was detected with other components of metabolic syndrome. CONCLUSION: There is an association of FTO rs9939609 A/A genotype and impaired fasting glucose and insulin resistance. Homozygous A genotype diabetic patients may be more vulnerable to blood glucose fluctuation. Focused genotyping can help the health care providers to identify high risk groups of both normal population and diabetic patients to intervene accordingly.

Autophagy related markers (Beclin-1 and ATG4B) are strongly expressed in Wilms' tumor and correlate with favorable histology.

BACKGROUND: Wilms' tumor treatment has achieved great success in the last decade. Nevertheless, some cases still fail to respond to the current multimodality therapy. These cases fall mainly in the unfavorable histology group with very few belonging to the favorable histology group. In recent years, autophagy manipulation whether inhibition or stimulation has been shown to affect cancer cell behavior and has emerged as a novel mechanism to improve cancer cell response to currently used therapeutic regimens. OBJECTIVE: The current study aimed to investigate the expression of autophagy related markers (ATG4B and Beclin1) in WT, its association with the different clinic-pathological parameters and its impact on patient survival. METHODS: Twenty-one formalin fixed paraffin embedded (FFPE) WT specimens were immunohistochemically stained using autophagy related markers; Beclin-1 and ATG4B. All clinical, radiological and follow up data were retrieved from the patient records. RESULTS: All specimens showed positive expression of both Beclin-1 and ATG4B. The staining score for Beclin1 varied between 50 and 300, and its expression was significantly associated with favorable histology (p=0.007). Similarly, ATG4B expression was significantly higher in favorable histology tumors compared to unfavorable histology (p=0.046). A statistically significant positive correlation between Beclin-1 and ATG4B expression was observed. The cumulative disease-free survival in patients with favorable histology was significantly higher compared to patients with unfavorable histology (p=0.0027). CONCLUSIONS: Beclin-1 and ATG4B expression were both found to be statistically significant discriminators of survival. Collectively these findings suggest that the expression of autophagy-related markers is associated with a favorable histology and could predict better survival in these patients.

Statin-induced myopathy SLCO1B1 521T > C is associated with prediabetes, high body mass index and normal lipid profile in Emirati population.

BACKGROUND: Statin-induced myopathy has been linked to the C allele of a single nucleotide polymorphism (SNP) (rs4149056) of SLCO1B1 gene. This effect is more significant, but not restricted to simvastatin. Many studies have included European, American, African and Southeast Asian ancestries, but few were carried out on Middle Eastern population. AIM: To detect the prevalence of SLCO1B1 rs4149056 (521T > C) in Emirati population. METHOD: We recruited 282 Emiratis through the UAE National Diabetes and Lifestyle Project. Ethical approval was obtained before the study starts. Besides basic data collection, venous blood samples were collected. Fasting blood glucose, Lipid profile, and insulin levels were measured. Genotyping for rs4149056 (521T > C) was tested in triplicates through Real Time-PCR using TaqMan® Drug Metabolism Genotyping Assay. rs2306283 (388A > G) was analyzed for comparison. In addition, presence of minor alleles of both SNPs define stronger association with statin-induced myopathy. RESULTS: The study included 282 individuals, 52.8% were males with median age of 39.5 years. 10% had Diabetes Mellitus and 23% were hypertensive. Median of body mass index (BMI) was 27.68 kg/m2 in males and 28.38 kg/m2 in females. One-hundred ninety-seven (69.9%) showed abnormal lipid profile (either increased LDL-cholesterol or triglycerides or both). For rs4149056, C allele was present in 21.3% (2.8% homozygous C and 18.4% heterozygous CT). Although homozygous C genotype prevalence was low, compared with Caucasians (4%) and Africans (0%), C allele was associated with a trend of having higher BMI and abnormal lipid profile. C allele subjects were all pre-diabetics with mean glycated hemoglobin above 6%. Mean BMI in CC, CT, and TT genotypes was 30.91 ±â€¯4.4, 29.48 ±â€¯4.2, 27.96 ±â€¯5.5 kg/m2 respectively, with lack of such a trend observed with the different genotypes of the rs2306283 (used for comparison). Abnormal lipid profile was observed in 7/8(87.5%), 38/52(73.1%) and 152/222(70%) of the CC, CT, and TT genotypes respectively. CONCLUSION: There is lower prevalence of statin-induced myopathy-linked C allele of rs4149056 in SLCO1B1 gene in Emirati population, compared to Caucasians and Africans. However, there is a trend of higher glycosylated hemoglobin and BMI associated with normal lipid profile in patients having this allele.