Mohammad Reza Sadeghi

Contradictory roles of Nrf2/Keap1 signaling pathway in cancer prevention/promotion and chemoresistance

NF-E2-related factor 2 (Nrf2) protein is a cytosolic transcription factor that regulates antioxidant and stress-related enzymes. Kelch-like ECH-associated protein 1 (Keap1) binds Nrf2 and accelerates ubiquitination and proteasome-dependent degradation of Nrf2. Nrf2 modifies the sensitivity of the cell environment to electrophiles and oxidants by inducing the transcriptional activation of more than 100 detoxification and cytoprotective genes. Prior investigations have found documentary evidence indicating that temporary activation of Nrf2 by pharmaceutical inducers plays a protective role against cancer initiation in normal cells. The impact of Nrf2/Keap1 pathway in development of tumorigenesis and drug resistance has also been well documented. Inhibition of the permanent Nrf2 activation, especially in combination with chemotherapeutics against cancer, may be considered as an important strategy to inhibit tumor growth and overcome chemoresistance. Here, we review the importance of Nrf2-keap1 pathway in the prevention or promotion of cancer, and resistance mechanisms to chemotherapeutic agents.

The role of Nrf2-Keap1 axis in colorectal cancer, progression, and chemoresistance

Colorectal cancer is the third common cancer after lung and genital cancers worldwide with more than 1.2 million new cases diagnosed annually. Although extensive progress has been made in the treatment of colorectal cancer, finding novel targets for early diagnosis and effective treatment of these patients is an urgent need. Nuclear factor-erythroid 2–kelch-like ECH-associated protein 1 signaling pathway plays a key role in protecting cells from the damage of intracellular oxidative stress and extracellular oxidizing agents. Nuclear factor-erythroid 2 is a transcription factor that creates intracellular redox homeostasis via transcriptional activity and interaction with kelch-like ECH-associated protein 1. Furthermore, it contributes to survival and chemoresistance of colorectal cancer cells which is mediated by overexpression of cytoprotective and multidrug resistance genes. In this review, the dual role of nuclear factor-erythroid 2 signaling in induction of colorectal cancer cell survival and death as well as the possibility of targeting nuclear factor-erythroid 2–kelch-like ECH-associated protein 1 axis as an advanced strategy in prevention and effective treatment of colorectal cancer patients have been discussed.

Comparison of genome‐wide analysis techniques to DNA methylation analysis in human cancer

DNA methylation was the first epigenetic modification to be detected in human cancers with specific relation to aberrant gene expression. Herein, DNA methylation analysis explains how epigenetic patterns affect gene expression level. Hypermethylation at tumor suppressor gene loci leads to increased tumorigenesis due to tumor suppressor gene silencing, whereas global hypomethylation of CpG islands (CGIs) is followed by genomic instability and aberrant activation of multiple oncogenes. Therefore, characterization of the genes which silenced or activated epigenetically in human tumor cells can improve our understanding of cancer biology. Different genome‐wide methodologies are applied to evaluate methylation status. Various commonly conducted techniques for this evaluation are reviewed in this paper. We provided comparative description of the procedures, advantages, and drawbacks of genome‐wide DNA methylation analysis methods and biological applications, to give information on selecting the appropriate method for different methylation studies.

Nrf2 overexpression is associated with P -glycoprotein upregulation in gastric cancer

The efficacy of chemotherapeutic agents remains very poor in gastric cancer (GC) patients due to the development of multidrug resistance (MDR) phenotype. The nuclear factor erythroid 2-related factor 2 (Nrf2), is a pivotal transcriptional factor that regulates phase II detoxifying enzymes, antioxidants and efflux transporters including P-glycoprotein (P-gp). The aim of this study was to investigate the association of Nrf2 and P-gp and their correlations with clinicopathological criteria in GC patients.Nrf2 and MDR1/P-gp expressions in both mRNA and protein levels were examined by real-time PCR and immunohistochemical staining (IHC) respectively, in endoscopic biopsy samples from60 GC patients compared with those expressions in non-GC individuals. Our results from IHC examinations revealed that Nrf2 expression in GC patients (46.7%) is markedly higher than that in non-GC individuals (11.7%) (p<0.001, Mann-Whitney test) which was confirmed by real-time PCR in mRNA levels. Induction of P-gp as a drug efflux pump, was associated with Nrf2 overexpression in these samples (r=0.55, p<0.001). There was also a strong correlation between Nrf2 overexpression and tumor size, histological grade, lymph node and distant metastasis while P-gp upregulation was shown to be associated only with the histological grade and tumor size (Chi-square, all p<0.05). Our results suggest that therapeutic inhibition of Nrf2 expression can improve the efficacy of chemotherapeutic agents for GC patients by down regulation of P-gp expression.

The role of Her2-Nrf2 axis in induction of oxaliplatin resistance in colon cancer cells

Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a pivotal role in promoting chemoresistance by regulation of antioxidants and detoxification enzymes. Her2 is a member of tyrosine kinase receptor family with a key function in resistance of cancer cells to chemotherapeutics. The aim of this study was to investigate the possible cross talk between Nrf2 and Her2 mediated signaling pathways in development of oxaliplatin resistance in colon cancer cells. We first generated oxaliplatin-resistant LS174T and SW480 colon cancer cells with different Her2 expression levels by employing IC50 concentrations followed by a resting period. We evaluated the viability and apoptosis of the cells by MTT and flow cytometry assays, respectively. Nrf2 and Her2 gene expression levels were examined by qRT-PCR. The morphology analysis and combination index calculation were performed using the ImagJ and CompuSyn softwares, respectively. Development of resistant cells revealed a marked increase in half maximal inhibitory concentration (IC50) value from 3.95 ± 0.92 μM to 29.27 ± 3.13 μM in SW480 cells and 377 ± 46 nM to 9.59 ± 0.76 μM in LS174T cells with a significant change in morphology of the cells from elongated to small round shape (p < 0.05). Her2 expression level was increased in both types of resistant cells, but the Nrf2 expression was increased in LS174T resistant (LS174T/Res) cells and decreased in SW480/Res cells which were consistent with the level of resistance in these cells (25 fold increase in IC50 value in LS174T/Res cells versus 7 fold increase in this value in SW480/Res cells). Inhibition of either Nrf2 or Her2 alone and in combination caused a significant increase in oxaliplatin-induced cytotoxicity and apoptosis with maximum effects in SW480/Res cells with low Her2 and Nrf2 expression levels. Altogether, our results suggest that inhibition of Nrf2 signaling in colon cancer patients with Her2 overexpression can be considered as an important strategy to overcome oxaliplatin resistance.

Molecular characterization of extended-spectrum β-lactamase, plasmid-mediated AmpC cephalosporinase and carbapenemase genes among Enterobacteriaceae isolates in five medical centres of East and West Azerbaijan, Iran

Very little is known about the occurrence and various types of extended-spectrum β-lactamase (ESBL), AmpC and carbapenemase in Iran. The aims of this study were to determine the prevalence of ESBLs, AmpCs and carbapenemase genes among Enterobacteriaceae in Azerbaijan and to characterize the genetic composition of the detected genes. A total of 307 Enterobacteriaceae isolates, recovered from five medical centres, were screened for ESBL, AmpC and carbapenemase activities by the disc diffusion method and phenotypic confirmatory tests. The 162 selected strains (third-generation cephalosporins, cefoxitin- or carbapenem-resistant strains with positive or negative phenotypic confirmatory tests) were selected for multiplex PCR screening for β-lactamase genes, and detected genes were confirmed by sequencing. Of 162 isolates, 156 harboured 1 to 6 β-lactamase genes of 41 types. The most prevalent genes were blaTEM-1 (29.9 %), followed by blaCTX-M-15 (25.7 %). Plasmid-mediated AmpC was detected in 66 strains (21.5 %) alone or in combination with other genes. Carbapenemase-encoding genes were detected in 18 strains (5.8 %) of 27 carbapenem-non-susceptible isolates including 11, 7, 3 and 1 cases of blaOXA-48, blaNDM-1, blaKPC-2 and blaKPC-3 genes, respectively. Interestingly, 148 (94.8 %) of 156 strains with any β-lactamase gene were found to have a multidrug-resistant pattern. The rate of resistance to β-lactams and multidrug-resistant Enterobacteriaceae is high in Azerbaijan. All positive strains for carbapenemase genes were resistant to all β-lactams. The present study reveals the high occurrence of CTX-M-type ESBLs followed by TEM and SHV variants among Enterobacteriaceae isolates. East Azerbaijan seems to be an alarming focus for OXA-48, NDM-1 and KPC dissemination.

The prevalence of plasmid-mediated quinolone resistance and ESBL-production in Enterobacteriaceae isolated from urinary tract infections

Introduction β-lactam and fluoroquinolone antibiotics are usually used for the treatment of urinary tract infections (UTIs). The aim of this study was to determine the prevalence of plasmid-mediated quinolone resistance (PMQR) and extended spectrum β-lactamases (ESBLs) in Enterobacteriaceae isolated from UTIs. Materials and methods Two hundred and nineteen samples of Enterobacteriaceae isolated from UTIs were collected in the Northwest of Iran. Antimicrobial susceptibility testing was determined by the disk diffusion method. ESBLs were detected by the double-disk test. ESBL and PMQR-encoding genes were screened using the polymerase chain reaction. Results The rate of resistance to moxifloxacin, nalidixic acid, gatifloxacin, ofloxacin, ciprofloxacin, and levofloxacin in ESBL-producing isolates was 89.3%, 88%, 84%, 80%, 78.7%, and 73.3%, respectively. PMQR-producing Enterobacteriaceae isolates were identified in 67 samples (89.1%). The most prevalent PMQR genes were aac (6′)-Ib-cr 120 (68.6%) followed by oqxB 72 (41.1%), oqxA 59 (33.7%), qnrB 36 (20.6%), qnrS 33 (18.9%), qnrD 19 (10.9%), qepA 13 (7.4 %), qnrA 10 (5.7%), and qnrC 9 (5.1%). There was a strong association between PMQR genes and blaCTX-M-15 and blaTEM-116 and other ESBL genes. Conclusion High resistance rates were detected to quinolones among ESBL-producing isolates from UTIs. There is a high prevalence of PMQR genes in Enterobacteriaceae in Azerbaijan and Iran, and the most common PMQR is aac(6′)-Ib-cr. There is a significant association between PMQR and ESBL-producing isolates.

Nrf2/P–glycoprotein axis is associated with clinicopathological characteristics in colorectal cancer

Colorectal cancer (CRC) is the fourth leading cause of cancer-related death worldwide. Activation of ABCB1 gene and its main product, P-glycoprotein, is the common reason for chemoresistance. The nuclear factor-erythroid 2-related factor2 (Nrf2) is directly regulated by Kelch like ECH-associated protein1 (Keap1). In addition, Nrf2 is a key transcriptional factor that regulates efflux transporters, including P-gp. The aim of this study was to investigate the expression levels of Nrf2, Keap1 and ABCB1 in the biopsy samples and their association with clinicopathological features in CRC patients. Both mRNA and protein expression levels were measured by Real-time PCR and immunohistochemistry (IHC), respectively, in biopsies from colonoscopy in 65 CRC patients compared to those in 65 non-CRC individuals. While expression levels of Nrf2 and ABCB1 (P-gp) were markedly higher in both mRNA and protein levels in CRC biopsies (p < 0.01), Keap1 expression level was significantly lower in these samples (p < 0.05). Positive correlations between Nrf2 expression level and tumor size (p = 0.003), lymph node (p = 0.038), distant metastasis (p = 0.008), and smoking status (p = 0.02) were observed. However, P-gp expression was associated only with patient age and smoking status. In addition, there was a positive correlation between protein levels of Nrf2 and P-gp, in both CRC (r = 0.617, p < 0.001) and non-CRC tissues (r = 0.930, p < 0.001). In conclusion, over-expression of Nrf2 and ABCB1/P-gp, as well as down-regulation of mRNA expression level of Keap1 in CRC patients denotes the role of Keap1/Nrf2/ABCB1 axis in CRC progression and chemoresistance. Our data suggest that therapeutic inhibition of Nrf2/ABCB1 signaling can be considered as a novel strategy to improve the efficacy of chemotherapeutics against CRC.