Gene expression alterations in doxorubicin resistant MCF7 breast cancer cell line

AbstractMany molecular mechanisms contribute to the development of doxorubicin resistance and different cancers can express wide and diverse arrays of drug-resistance genes. The aim of this study was to identify the changes in gene expression associated with the development of doxorubicin resistance in MCF7 breast cancer cell line. The doxorubicin resistant MCF7 cell line was developed by stepwise selection of MCF7 cells and was tested using the MTT assay. The alterations in gene expression were examined using the real-time based PCR array. The findings showed an up-regulation of many phase I/II metabolizing genes, specifically, the CYP1A1 and the CYP1A2 that were up-regulated by 206- and 96-fold respectively. Drug efflux pump genes were also up-regulated profoundly. TOP2A was strongly down-regulated by 202-fold. Many other changes were observed in genes crucial for cell cycle, apoptosis and DNA repair. The findings of this project imply that the development of doxorubicin resistance is a multi-factorial process

Allelic frequency of PON1 Q192R, CYP2C19*2 and CYP2C19*17 among Jordanian patients taking clopidogrel

Purpose: To investigate the influence of allelic frequencies of PON1 Q192R, CYP2C19*2 and CYP2C19*17 genetic polymorphisms on the response to clopidogrel among Jordanian patients.Methods: Polymorphisms in CYP2C19 were assessed among 148 patients using PCR-RFLP assay.Results: The CYP2C19*2, CYP2C19*17, and PON1 Q192R allele frequencies were 9.8, 28.72 and 28.7 %, respectively. On the genotyping side, the frequencies of CYP2C19*1/1* and CYP2C19*1/2* were 80.4 and 19.6 %, respectively, but none of the patients had CYP2C19*2/2* genotype. The genotype frequencies CYP2C19*17 were 47.97, 46.62 and 5.41 % for wild-type C-C, heterozygote C-T, and the mutant T-T, respectively. PON1 genotype was 42.7 % for QQ, and 57.8 % for QR. None of the patients had RR genotype.Conclusion: Relative to other populations, the observed allelic frequencies are consistent with the values reported for Caucasian and Middle Eastern populations.Keywords: CYP2C9 polymorphisms, Clopidogrel, Genotype, Allele frequency, PON1 gene

Hypoxia-induced gene expression pattern in doxorubicin resistant MCF7 cells

Purpose: To investigate hypoxia-induced gene expression pattern in doxorubicin-resistant human breast cancer cells (MCF7). Methods: Human breast cancer cells (MCF7) were exposed to 60 episodes of 8 h hypoxia thrice a week for three months. Chemo-resistance to doxorubicin was assessed using 3-(4,5-dimethylthiazol-2- yl)-2, 5-diphenyl tetrazolium bromide (MTT) cell proliferation assay. Real-time quantitative polymerase chain reaction (qRT-PCR) assay was performed to assess gene expression pattern in doxorubicinresistant cells on exposure to hypoxia. Results: Hypoxia significantly increased the resistance of MCF7 cells to doxorubicin, with a maximum of 16.42-fold enhancement after 25 episodes of 8-h hypoxia, while the resistance thereafter significantly decreased with prolonged episodes of hypoxia (p < 0.05). Gene expression analysis revealed significant changes in 42 genes. The expressions of 10 of these genes were significantly upregulated, while those of 32 genes were significantly down-regulated (p < 0.05). Cytochrome P450 family 1, subfamily A, member1 (CYP1A1) was the most conspicuous upregulated gene (13.32-fold), while breast cancer gene 1 (BRCA1) was the most down-regulated (8.23-fold). Gene expression analysis after 60 episodes of 8-h hypoxia revealed the upregulation of CYP1A1 (5.77-fold). Similarly, 27 genes were significantly down-regulated, with BRCA2 as the most down-regulated gene (8.11-fold). Topoisomerase (DNA) II alpha (TOP2A) was the most down-regulated among genes involved in drug metabolism and resistance (6.37-fold), while cyclin-dependent kinase 2 (CDK2) was the most profoundly downregulated among genes involved in cell cycle regulation (3.56-fold). Conclusion: These results indicate that development of resistance to doxorubicin by MCF7 cells after short-term hypoxia results from the upregulation of genes responsible for the metabolism of doxorubicin and for shifting the cells to alternative pathway driven principally by EGF and ESR2. The observed down-regulation is an adaptation of the MCF7 cells to survive under long-term hypoxia

The effect of cycling hypoxia on MCF-7 cancer stem cells and the impact of their microenvironment on angiogenesis using human umbilical vein endothelial cells (HUVECs) as a model

Background Breast cancer is the most common type of cancer among females. Hypoxia mediates cancer hallmarks and results from reduced oxygen level due to irregularities in tumor vascularization or when the tumor size prevents oxygen diffusion and triggers angiogenesis to compensate for low oxygen. Cancer stem cells (CSCs) are a rare subpopulation, able to self-renew and to give rise to tumor-initiating cells. It is proposed that CSCs’ secretions help to recruit endothelial cells via angiogenic factors to establish tumor vascularization. In the tumor microenvironment, the effect of hypoxia on CSCs and the impact of their secretions on triggering angiogenesis and tumor vascularization remain questionable. In this study, three-dimensional (3D) CSCs derived from MCF-7 were directly exposed to repetitive long-term cycles of hypoxia to assess its effect on CSCs and then to evaluate the role of the hypoxic CSCs’ (CSCsHYP) secretions in angiogenesis using (HUVECs) as a model for tumor neovascularization response. Methods CSCs derived from MCF-7 cell-line were expanded under repetitive, strictly optimized, long-term/continuous and intermittent hypoxic shots for almost four months to assess hypoxic effect on CSCs, sorted based on CD44+/CD24− biomarkers. Hypoxic phenotype of CSCsHYP was evaluated by assessing the acquired chemoresistance using MTT assay and elevated stemness properties were assessed by flow cytometry. To evaluate the effect of the secretions from CSCsHYP on angiogenesis, HUVECs were exposed to CSCsHYP conditioned-medium (CdM)—in which CSCs had been previously grown—to mimic the tumor microenvironment and to assess the effect of the secretions from CSCsHYP on the HUVECs’ capability of tube formation, migration and wound healing. Additionally, co-culture of CSCsHYP with HUVECs was performed. Results CSCsHYP acquired higher chemoresistance, increased stemness properties and obtained greater propagation, migration, and wound healing capacities, when compared to CSCs in normoxic condition (CSCsNOR). HUVECs’ tube formation and migration abilities were mediated by hypoxic (CSCs) conditioned media (CdM). Discussion This study demonstrates that chemoresistant and migrational properties of CSCs are enhanced under hypoxia to a certain extent. The microenvironment of CSCsHYP contributes to tumor angiogenesis and migration. Hypoxia is a key player in tumor angiogenesis mediated by CSCs

The crosstalk between adenosine A2B receptor and insulin signalling in rat skeletal muscle cells

Diabetes mellitus (DM) is a group of metabolic diseases characterised by hyperglycaemia resulting from defects in insulin secretion, insulin action, or both. Insulin therapy might be affected by specific metabolic enzymes and transporters. There are conflicting reports in the literature on the role of adenosine receptor A2B (AR2B) in skeletal and cardiac muscle glucose metabolism. This study aims to find out if there is an association between AR2B and insulin signalling, especially the metabolic pathways (AKT-GSK). Differentiated L6 cell rat muscle cells were treated with insulin, adenosine agonist NECA, selective AR2B antagonist PSB 603 and combinations between these reagents, the expression of AKT2, GSK3α, and GSK3β were measured by qPCR hydrolysis probe technique. Insulin increases AKT2, GSK3α and GSK3β mRNA expression, while AR2B antagonist inhibits AKT2 GSK3α and GSK3β mRNA expression and combining AR2B antagonist with insulin diminish insulin action and decrease AKT2 GSK3α and GSK3β mRNA expression, which means a strong relationship between AR2B and insulin action. Furthermore AR2B agonist may be a good candidate as an anti-diabetic drug

Association of IL-4 Polymorphisms with Allergic Rhinitis in Jordanian Population

Background and objectives: Allergic rhinitis has complex patterns of inheritance, and single nucleotide polymorphisms, a common genetic variation in a population, exert a significant role in allergic rhinitis pathology. The current study aimed to investigate the association of Interleukin-4 (IL-4) polymorphisms with allergic rhinitis. Materials and Methods: Our study included 158 patients with allergic rhinitis and 140 healthy controls from Jordan that were genotyped for IL-4 single nucleotide polymorphisms (SNPs) C-589T (rs2243250) and T-2979G (rs2227284) using restriction fragment length polymorphism-polymerase chain reaction. Statistical analysis was conducted using IBM SPSS Statistics version 24 software. Results: The results showed that the allelic frequency of the minor alleles was 0.19 and 0.67 for C-589T (rs2243250) and T-2979G (rs2227284) in the allergic rhinitis patients, respectively, while it was 0.18 for C-589T (rs2243250) and 0.64 T-2979G (rs2227284) in the control group. The homozygous (TT) genotype of C-589T (rs2243250) was significantly associated with allergic rhinitis (p < 0.05), while there was no association of any of T-2979G (rs2227284) genotypes with allergic rhinitis. Conclusions: The results of this study indicate that genetic inter-population variation precipitates the differences in the percentages of many diseases among populations, including allergic rhinitis

Association of <em>IL-4</em> Polymorphisms with Allergic Rhinitis in Jordanian Population

Background and objectives: Allergic rhinitis has complex patterns of inheritance, and single nucleotide polymorphisms, a common genetic variation in a population, exert a significant role in allergic rhinitis pathology. The current study aimed to investigate the association of Interleukin-4 (IL-4) polymorphisms with allergic rhinitis. Materials and Methods: Our study included 158 patients with allergic rhinitis and 140 healthy controls from Jordan that were genotyped for IL-4 single nucleotide polymorphisms (SNPs) C-589T (rs2243250) and T-2979G (rs2227284) using restriction fragment length polymorphism-polymerase chain reaction. Statistical analysis was conducted using IBM SPSS Statistics version 24 software. Results: The results showed that the allelic frequency of the minor alleles was 0.19 and 0.67 for C-589T (rs2243250) and T-2979G (rs2227284) in the allergic rhinitis patients, respectively, while it was 0.18 for C-589T (rs2243250) and 0.64 T-2979G (rs2227284) in the control group. The homozygous (TT) genotype of C-589T (rs2243250) was significantly associated with allergic rhinitis (p Conclusions: The results of this study indicate that genetic inter-population variation precipitates the differences in the percentages of many diseases among populations, including allergic rhinitis

Investigating the human rhinovirus co-infection in patients with asthma exacerbations and COVID-19

Objective: To investigate the co-infections with human rhinovirus (HRV) among patients with asthma exacerbation and COVID-19 in Jordan. Also, to determine the frequency of acute asthma exacerbation before and during the COVID-19 pandemic on a matched basis. Methods: The data of this prospective cohort research consisted of clinical variables. During the first visit, and after 14-days, nasopharyngeal swabs were taken and the quantitative polymerase chain reaction was performed for HRV and SARS-CoV-2 detection. Results: Forty-seven out of 175 (26.9%) COVID-19 adult cases have been diagnosed with asthma. The number of asthma exacerbations among the study participants was higher during 2021 than in 2020 (p=0.035). Most of the included asthmatic participants (61.7%) were only positive for SARS-CoV-2 and 38.3% were co-infected with HRV. The SARS-CoV-2 cycle threshold value was lower in samples infected with both viruses compared to samples infected with SARS-CoV-2 alone, p<0.005. Conclusion: Our findings indicate that HRV and SARS-CoV-2 were significantly more prevalent in asthma exacerbations than stable asthma. Thus, HRV and/or SARS-CoV-2 infections were potentially cofactors or contributors to the asthma exacerbation in this cohort. This is the first study, in Jordan, to investigate the HRV co-infection in COVID-19 asthmatic patients and HRV could be related with a higher severity of COVID-19

The effect of ERCC1 and ERCC2 gene polymorphysims on response to cisplatin based therapy in osteosarcoma patients

Abstract Background Cisplatin is one of the major drugs that used in the treatment of osteosarcoma. Cisplatin exerts its function by making cisplatin-DNA adducts culminating in cellular death. These adducts found to be repaired by nucleotide excision repair (NER) pathway. This study aimed to evaluate if polymorphisms in two main genes in the NER pathway, excision repair cross-complementing group 1 and 2 (ERCC1 and ERCC2) could affect the histological response to cisplatin based chemotherapy or clinical outcomes, particularly, event free survival (EFS) and overall survival (OS) rates. Method ERCC1 (C118T (rs11615) and C8092A (rs3212986)) and ERCC2 (A751C (rs171140) and G312A (rs1799793)) polymorphisms were analysed in 44 patients with osteosarcoma, who were treated with cisplatin based neoadjuvant chemotherapy. DNA was extracted from patient’s formalin-fixed paraffin-embedded (FFPE) samples, patient’s genotypes were determined by using polymerase chain reaction-restriction fragment length polymorphism PCR-RFLP assay. The distribution of the patients’ genotype and the allele frequencies were reported. The association between each of these genotypes and many clinical and patho-histological parameters (e.g. EFS, OS and patho-histological response to treatment) was examined. The associations between gender, tumor location, presence of metastasis at diagnosis, histological subtypes, and type of neoadjuvant chemotherapy and between the histological response, EFS and OS rates were also examined. Results This study revealed that there was a positive and significant association between ERCC1 C8092 A genotypes and median EFS rate in years; patients who were carriers of C allele (CC & CA) were found to have longer EFS rates than patients with AA genotype (P value = 0.006) and the median EFS rates were respectively as following: 2.04, 0.24 years. As well, both the presence of metastasis and the histological subtype at the time of diagnosis, were able to affect the EFS rate but not the OS. However, there was a positive correlation between OS rate and the patients’ primary response to treatment. Conclusions Our results suggested that ERCC1 8092 C allele may play a role as a candidate prognostic marker in patients with osteosarcoma