Functional characterization of the transcription factor ZEB1 in epithelial to mesenchymal transition and cancer progression

Epithelial to mesenchymal transition (EMT) is implicated in the progression of primary tumours towards metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development. To analyse EMT-causing pathways in tumourigenesis, transcriptional targets of the E-cadherin repressor ZEB1 in invasive human cancer cells were identified. We show that ZEB1 repressed multiple key determinants of epithelial differentiation and cell–cell adhesion, including the cell polarity genes Crumbs3, HUGL2, PKP3 and Pals1-associated tight junction protein. ZEB1 associated with their endogenous promoters in vivo, and strongly repressed promoter activities in reporter assays. ZEB1 downregulation in undifferentiated cancer cells by RNA interference was sufficient to upregulate expression of these cell polarity genes on the RNA and protein level, to re-establish epithelial features and to impair cell motility in vitro. In human colorectal cancer, ZEB1 expression was limited to the tumour–host interface and was accompanied by loss of intercellular adhesion and tumour cell invasion. EMT-inducing transcriptional repressor ZEB1 promotes colorectal cancer cell metastasis and loss of cell polarity. Thereby, ZEB1 suppresses the expression of cell polarity factors, in particular of Lgl2, which was found to be reduced in colorectal and breast cancers. In invasive ductal and lobular breast cancer, upregulation of ZEB1 was stringently coupled to cancer cell dedifferentiation. The invasion potential of MDA-MB-231, a highly invasive breast cancer cell line, is shown to be under the control of ZEB1. Over-expression of ZEB1down-regulates and relocalizes E-Cadherin in MCF7 breast cancer cells; moreover, ZEB1 over-expression results in reduced proliferation rate of these cells. Most importantly, we show that ZEB1 mediated downregulation of E-cadherin involves chromatin modifications. Markers of transcriptionally active chromatin Acetylated H3 and Acetylated H4 were increased upon ZEB1 knock down in MDA-MB-231 cells, while repressive marks like H3K9me2 and H3K27me2 were obseverved to disappear in the same cells. Collectively, the data presented in this thesis show that ZEB1 represents a key player in pathologic EMTs associated with tumour progression.Epithelial to mesenchymal transition (EMT) is implicated in the progression of primary tumours towards metastasis and is likely caused by a pathological activation of transcription factors regulating EMT in embryonic development. To analyse EMT-causing pathways in tumourigenesis, transcriptional targets of the E-cadherin repressor ZEB1 in invasive human cancer cells were identified. We show that ZEB1 repressed multiple key determinants of epithelial differentiation and cell–cell adhesion, including the cell polarity genes Crumbs3, HUGL2, PKP3 and Pals1-associated tight junction protein. ZEB1 associated with their endogenous promoters in vivo, and strongly repressed promoter activities in reporter assays. ZEB1 downregulation in undifferentiated cancer cells by RNA interference was sufficient to upregulate expression of these cell polarity genes on the RNA and protein level, to re-establish epithelial features and to impair cell motility in vitro. In human colorectal cancer, ZEB1 expression was limited to the tumour–host interface and was accompanied by loss of intercellular adhesion and tumour cell invasion. EMT-inducing transcriptional repressor ZEB1 promotes colorectal cancer cell metastasis and loss of cell polarity. Thereby, ZEB1 suppresses the expression of cell polarity factors, in particular of Lgl2, which was found to be reduced in colorectal and breast cancers. In invasive ductal and lobular breast cancer, upregulation of ZEB1 was stringently coupled to cancer cell dedifferentiation. The invasion potential of MDA-MB-231, a highly invasive breast cancer cell line, is shown to be under the control of ZEB1. Over-expression of ZEB1down-regulates and relocalizes E-Cadherin in MCF7 breast cancer cells; moreover, ZEB1 over-expression results in reduced proliferation rate of these cells. Most importantly, we show that ZEB1 mediated downregulation of E-cadherin involves chromatin modifications. Markers of transcriptionally active chromatin Acetylated H3 and Acetylated H4 were increased upon ZEB1 knock down in MDA-MB-231 cells, while repressive marks like H3K9me2 and H3K27me2 were obseverved to disappear in the same cells. Collectively, the data presented in this thesis show that ZEB1 represents a key player in pathologic EMTs associated with tumour progression

Association of CYP2C19*2 and *17 genetic variants with hypertension in Pakistani population

Purpose: To investigate the association of *2 and *17 single nucleotide polymorphisms (SNPs) of CYP2C19 gene with hypertension in Pakistani population. Methods: The study was conducted on 527 hypertensive patients and 530 unrelated healthy controls from selected regions of Pakistan. DNA was extracted from leukocytes and all patients and controls were genotyped for two SNPs (rs4244285 and rs12248560) of CYP2C19 gene by allele specific polymerase chain reaction (AS-PCR). Results: Multi-allelic polymorphism in CYP2C19 identified four distinct phenotypes known as ultra-rapid metabolizer (UM), extensive metabolizer (EM), intermediate metabolizer (IM) and poor metabolizer (PM) in hypertensive patients and controls. For CYP2C19*2 polymorphisms, overall wild type and mutant allele frequency were 75 and 25 % in hypertensive patients, and 64.2 and 35.8 % in controls. For CYP2C19*17 polymorphisms, the overall wild type and mutant allele frequency were 66.6 and 33.4 % in hypertensive patients and 75.6 % and 24.4 % in controls. Significant difference in allele frequencies for CYP2C19*2 and *17 was demonstrated between hypertensive and non-hypertensive subjects. Conclusion: To the best of our knowledge, this is the first report on CYP2C19 frequencies in hypertensive Pakistani patients. The finds should help clinicians to determine a suitable optimal dosage of some drugs in order to reduce side effects

Role of nutrients in bacterial biosurfactant production and effect of biosurfactant production on petroleum hydrocarbon biodegradation

Petroleum hydrocarbons' insolubility (due to hydrophobic nature) remains an important factor in microbial degradation of these compounds. The use of microbial biosurfactants significantly decreases the hydrophobicity and increases the rate of hydrocarbon biodegradation. Four bacterial strains, Pseudomonas poae BA1, Acinetobacter bouvetii BP18, Bacillus thuringiensis BG3, and Stenotrophomonas rhizophila BG32, isolated from petroleum hydrocarbon-contaminated soil, were used to test biosurfactant production capacities under different nutrient conditions. The hydrocarbon degradation by biosurfactant producing strains was compared with a non biosurfactant producing hydrocarbon degrading Pseudomonas rhizosphaerae BP3 strain. The percentage increase in biosurfactant production in nutrient rich medium, which was nutrient broth (NB), as compared to nutrient deprive medium, which was Bushnell-Haas broth (BHB), was BA1 = 20.48%, BP18 = 24.81%, BG3 = 16.71% and BG32 = 14.55%. The biosurfactant producing strains showed 16-28% increase in hydrocarbon degradation, as compared to non biosurfactant producing strain. The highest hydrocarbon degradation (96.07%) was exhibited by BA1, followed by BP18 (93.53%), BG3 (89.97%), BG32 (87.10%), and BP3 (74.60%). We concluded that biosurfactant production is influenced by the availability of nutrients. Cell hydrophobicity, surface tension and biosurfactant production influence hydrocarbon degradation, which can be enhanced with the use of biosurfactant producing bacteria.The authors would like to thank COMSTECH TWAS (14-416 RG/PHA/AS C-UNESCO FR: 3240283445 to M.A.), for funding

Further evidence for the association of CYP2D6*4 gene polymorphism with Parkinson’s disease: a case control study

Abstract Background Genetic and environmental risk factors play an important role for the susceptibility to sporadic Parkinson’s disease (PD). It was hypothesized that a splice variant of the CYP2D6 gene (CYP2D6*4 allele) is associated with PD because it alters the ability to metabolize toxins and in particular neurotoxins. CYP2D6 codes for the drug metabolizing enzyme debrisoquine 4-hydroxylase. The CYP2D6*4 variant results in an undetectable enzyme activity and consequently in a reduction in metabolism of some toxins. Methods Some of agricultural chemicals have neurotoxic potential and CYP2D6 is involved in their detoxification. Thus, we conducted a case control study to investigate the association of the CYP2D6*4 with PD in a Pakistani subpopulation that is known to be exposed to high levels of some agricultural pesticides, insecticides and herbicides. Results We found a significantly higher allele and genotype frequency of the CYP2D6*4 variant in 174 sporadic PD patients when compared to 200 controls. In addition, there was a trend to an earlier age of PD onset and a tremor dominant phenotype in CYP2D6*4 variant carriers. Conclusion Our data provide further evidence that a poor metabolizer status may increase the risk to develop PD especially in populations that are exposed to environmental toxins

Interplay of N- acetyl cysteine and melatonin in regulating oxidative stress-induced cardiac hypertrophic factors and microRNAs

Early and specific diagnosis of oxidative stress linked diseases as cardiac heart diseases remains a major dilemma for researchers and clinicians. MicroRNAs may serve as a better tool for specific early diagnostics and propose their utilization in future molecular medicines. We aimed to measure the microRNAs expressions in oxidative stress linked cardiac hypertrophic condition induced through stimulants as Endothelin and Isoproterenol. Cardiac hypertrophic animal models were confirmed by BNP, GATA4 expression, histological assays, and increased cell surface area. High oxidative stress (ROS level) and decreased antioxidant activities were assessed in hypertrophied groups. Enhanced expression of miR-152, miR-212/132 while decreased miR-142-3p expression was observed in hypertrophic condition. Similar pattern of these microRNAs was detected in HL-1 cells treated with H2O2. Upon administration of antioxidants, the miRNAs expression pattern altered from that of the cardiac hypertrophied model. Present investigation suggests that oxidative stress generated during the cardiac pathology may directly or indirectly regulate anti-hypertrophy pathway elements through microRNAs including antioxidant enzymes, which need further investigation. The down-regulation of free radical scavengers make it easier for the oxidative stress to play a key role in disease progression.</p

Data of expression status of miR- 29a and its putative target mitochondrial apoptosis regulatory gene DRP1 upon miR-15a and miR-214 inhibition

Data is about the mitochondrial apoptosis regulatory framework genes PUMA, DRP1 (apoptotic), and ARC (anti-apoptotic) analysis after the employment of their controlling miRNAs inhibitors. The data represents putative conserved targeting of seed regions of miR-15a, miR-29a, and miR-214 with respective target genes PUMA, DRP1, and ARC. Data is of cross interference in expression levels of one miRNA family, miR-29a and its putative target DRP1 upon the inhibitory treatment of other miRNAs 15a and 214. Keywords: DRP1, miR-15a, Apoptosis, miRNAs inhibitio

The transcription factor ZEB1 (deltaEF1) represses Plakophilin 3 during human cancer progression

Plakophilin 3 (PKP3) belongs to the p120ctn family of armadillo-related proteins predominantly functioning in desmosome formation. Here we report that PKP3 is transcriptionally repressed by the E-cadherin repressor ZEB1 in metastatic cancer cells. ZEB1 physically associates with two conserved E-box elements in the PKP3 promoter and partially represses the activity of corresponding human and mouse PKP3 promoter fragments in reporter gene assays. In human tumours ZEB1 is upregulated in invasive cancer cells at the tumour–host interface, which is accompanied by downregulation of PKP3 expression levels. Hence, the transcriptional repression of PKP3 by ZEB1 contributes to ZEB1-mediated disintegration of intercellular adhesion and epithelial to mesenchymal transition