MDR1 Gene Research Articles

Virulence Factors and Azole-Resistant Mechanism of Candida Tropicalis Isolated from Candidemia.

Limited knowledge exists on the virulence factors of Candida tropicalis and the mechanisms of azole resistance that lead to an intensified pathogenicity and treatment failure. We aimed to evaluate the virulence factors and molecular mechanisms of azole resistance among C. tropicalis isolated from patients with candidemia. Several virulence factors, including extracellular enzymatic activities, cell surface hydrophobicity (CSH), and biofilm formation, were evaluated. Antifungal susceptibility pattern and expression level of ERG11, UPC2, MDR1, and CDR1 genes of eight (4 fluconazole resistance and 4 fluconazole susceptible) clinical C. tropicalis isolates were assessed. The correlation between the virulence factors and antifungal susceptibility patterns was analyzed. During a 4year study, forty-five C. tropicalis isolates were recovered from candidemia patients. The isolates expressed different frequencies of virulence determinants as follows: coagulase 4 (8.9%), phospholipase 5 (11.1%), proteinase 31 (68.9%), esterase 43 (95.6%), hemolysin 44 (97.8%), biofilm formation 45 (100%) and CSH 45(100%). All the isolates were susceptible to amphotericin B and showed the highest resistance to voriconazole. There was a significant positive correlation between micafungin minimum inhibitory concentrations (MICs) and hemolysin production (rs = 0.316). However, we found a negative correlation between fluconazole MICs and esterase production (rs = -0.383). We observed the high expression of ERG11 and UPC2 genes in fluconazole-resistant C. tropicalis isolates. C. tropicalis isolated from candidemia patients extensively displayed capacities for biofilm formation, hemolysis, esterase activity, and hydrophobicity. In addition, the overexpression of ERG11 and UPC2 genes was considered one of the possible mechanisms of azole resistance.

Comparative insights into multiple drug resistance determinants in Stenotrophomonas maltophilia MER1

ObjectivesMultidrug-resistant (MDR) Stenotrophomonas maltophilia strain MER1 was isolated from hospital wastewater in Shandong Province, China. This study aimed to determine the genetic determinants related to its striking MDR phenotype. MethodsAntimicrobial susceptibility testing of strain MER1 was performed by disk diffusion on Mueller–Hinton agar plates, and MICs were interpreted according to Clinical and Laboratory Standards Institute breakpoints. The genome of MER1 was sequenced and assembled using PacBio RS II and BGISEQ-500 platforms. Antimicrobial resistance determinants together with other transferability or adaptability determinants were identified by comparative genomics. Phylogenetic and contextual assays for these elements were conducted to assess the risk of spread of MER1. ResultsAntimicrobial susceptibility testing revealed that strain MER1 is resistant to nine different antibiotics, including ampicillin, meropenem, amikacin, erythromycin, vancomycin, tetracycline, tigecycline, colistin and ceftazidime. Several genes were identified encoding efflux pumps and drug-inactivating agents, accounting for resistance to the above antibiotics, including meropenem, tigecycline and colistin regarded as last-line therapies for infections caused by MDR Gram-negative bacteria. MER1 co-harbours two non-mobile mcr homologues. A novel genomic region of variability was demonstrated to confer bacterial robustness and adaptability upon strain MER1. ConclusionCollective efforts revealed the MDR properties and potential genetic determinants of S. maltophilia MER1 isolated from hospital wastewater. Comparative genomic analysis of S. maltophilia MER1 may provide insights into the prevention and treatment of antimicrobial-resistant infections. Our findings raise concern that the MDR genes in the reservoir of S. maltophilia may further spread into various ecological niches or medically high-risk pathogens.

A Comparative Transcriptome Between Anti-drug Sensitive and Resistant Candida auris in China

Candida auris emerged as a pathogenic species of fungus that causes severe and invasive outbreaks worldwide. The fungus exhibits high intrinsic resistance rates to various first-line antifungals, and the underlying molecular mechanism responsible for its multidrug resistance is still unclear. In this study, a transcriptomic analysis was performed between two C. auris isolates that exhibited different anti-drug patterns by RNA-sequencing, namely, CX1 (anti-drug sensitive) and CX2 (resistant). Transcriptomic analysis results revealed 541 upregulated and 453 downregulated genes in the resistant C. auris strain compared with the susceptible strain. In addition, our findings highlight the presence of potential differentially expressed genes (DEGs), which may play a role in drug resistance, including genes involved in ergosterol and efflux pump biosynthesis such as SNQ2, CDR4, ARB1, MDR1, MRR1, and ERG genes. We also found that Hsp related genes were upregulated for expression in the anti-drug-resistant strain. Biofilm formation and growth conditions were also compared between the two isolates. Our study provides novel clues for future studies in terms of understanding multidrug resistance mechanisms of C. auris strains.

Lathyrane Diterpenoids as Novel hPXR Agonists: Isolation, Structural Modification, and Structure-Activity Relationships.

Pregnane X receptor (PXR) that orchestrates the intricate network of xeno- and endobiotic metabolism is considered as a promising therapeutic target for cholestasis. In this study, the human PXR (hPXR) agonistic bioassay-guided isolation of Euphorbia lathyris followed by the structural modification led to the construction of a lathyrane diterpenoid library (1-34). Subsequent assay of this library led to the identification of a series of potent hPXR agonists, showing better efficacy than that of typical hPXR agonist, rifampicin. The most active compound, 8, could dose-dependently activate hPXR at micromolar concentrations and significantly up-regulate the expressions of PXR downstream genes CYP3A4, CYP2B6, and MDR1. The structure-activity relationships (SARs) studied in combination with molecular modeling suggested that acyloxy at C-7 and the presence of 14-carbonyl were essential to the activity. These findings suggested that lathyrane diterpenoids could serve as a new type of hPXR agonist for future anticholestasis drug development.

Application of Mdr2 gene knockout mice in liver disease research

Mdr2 knockout mice is a liver disease model, which causes cholestasis due to the lack of phospholipids in the bile. At present, it is not only used for the study of human homologous MDR3 gene, but also widely used as an animal model of liver diseases such as primary sclerosing cholangitis, liver fibrosis, progressive familial intrahepatic cholestasis, liver cancer. Herein, we review the Mdr2 knockout mice physiological characteristics and its application in liver disease research.

The association of a genetic variant in multi-drug resistance gene and colorectal cancer susceptibility

Colorectal cancer (CRC) is one of the most malignant tumors worldwide with poor prognosis. There are various genetic polymorphisms associated with colorectal cancer have been reported in recent studies. Here, we examined the relationship between the multidrug resistance mutation 1 (MDR1 or ATP-binding cassette sub-family B member 1, ABCB1) (rs1128503) polymorphism and the risk of CRC in 334 individuals with or without CRC. DNA was extracted and genotyped using a Taq-Man based PCR method. The MDR1 polymorphism was shown to be related to the susceptibility for CRC using recessive (OR = 3.76, CI = 2.18–6.51, p < 0.001) and dominant models (OR = 1.96, CI = 1.12–3.45, p = 0.01). No association was found between the variant and Marriage/Education/Smoking/alcohol-consumption status. Our findings demonstrated the association of the genetic variant in the MDR1 gene with susceptibility to CRC, suggesting its potential role as a risk stratification biomarker in CRC patients.

First Sequencing of Caprine Mdr1 (Abcb1) mRNA Due to Suspected Neurological Adverse Drug Reaction in a Thuringian Goat Following Extra-Label Use of Doramectin.

The multidrug resistance gene MDR1 encodes for an efflux transporter called P-glycoprotein (P-gp). In the canine Mdr1 gene, a nonsense mutation was identified in certain dog breeds causing increased drug sensitivity to various P-gp substrates such as antiparasitic macrocyclic lactones. Symptoms of neurologic toxicity include ataxia, depression, salivation, tremor, apparent blindness, and mydriasis. In the current report, a Thuringian goat developed similar neurological signs after treatment with doramectin, a compound from the macrocyclic lactone class. Therefore, Mdr1 might be defective in this individual goat. For diagnostic purposes, sequencing of the complete mRNA transcript coding for caprine Mdr1 was performed to investigate a potential missense mutation. The Mdr1 transcripts of two related goats without drug sensitivity were also sequenced to allow differential diagnosis and were compared to the suspected drug-sensitive goat. The only position where the Mdr1 sequence from the suspected drug-sensitive goat differed was in the 3′-untranslated region, being a heterozygous single nucleotide polymorphism c.3875C>A. It can be suspected that this variant affects the expression level, stability, or translation efficiency of the Mdr1 mRNA transcript and therefore might be associated with the suspected drug sensitivity. To clarify this, further studies are needed, particularly investigating the Mdr1 mRNA and protein expression levels from brain material of affected goats. In conclusion, Mdr1 variants may exist not only in dogs, but also in individual goats. The current report provides the first Mdr1 mRNA transcript sequence of a goat and therefore represents the basis for more detailed Mdr1 sequence and expression analyses.

Effects of Hsp90 Inhibitor Ganetespib on Inhibition of Azole-Resistant Candida albicans.

Candida albicans is the most common fungal pathogen. Recently, drug resistance of C. albicans is increasingly severe. Hsp90 is a promising antifungal target to overcome this problem. To evaluate the effects of Hsp90 inhibitor ganetespib on the inhibition of azole-resistant C. albicans, the microdilution checkerboard method was used to measure the in vitro synergistic efficacy of ganetespib. The XTT/menadione reduction assay, microscopic observation, and Rh6G efflux assay were established to investigate the effects of ganetespib on azole-resistant C. albicans biofilm formation, filamentation, and efflux pump. Real-time RT-PCR analysis was employed to clarify the mechanism of antagonizing drug resistance. The in vivo antifungal efficacy of ganetespib was determined by the infectious model of azole-resistant C. albicans. Ganetespib showed an excellent synergistic antifungal activity in vitro and significantly inhibited the fungal biofilm formation, whereas it had no inhibitory effect on fungal hypha formation. Expression of azole-targeting enzyme gene ERG11 and efflux pump genes CDR1, CDR2, and MDR1 was significantly down-regulated when ganetespib was used in combination with FLC. In a mouse model infected with FLC-resistant C. albicans, the combination of ganetespib and FLC effectively reversed the FLC resistance and significantly decreased the kidney fungal load of mouse.

Overexpression of P-glycoprotein and MRP-1 are pharmacogenomic biomarkers to determine steroid resistant phenotype in childhood idiopathic nephrotic syndrome.

Steroid remains the keystone therapy for Idiopathic Nephrotic Syndrome (NS). Besides genetic factors and histological changes, pharmacogenomic factors also affect the steroid response. The upregulation of P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1 (MRP-1) modulate the pharmacokinetics of steroids and may contribute to steroid resistance. Flow-cytometric analysis of P-gp, MRP-1 expression and functional activity on peripheral blood mononuclear cells (PBMCs) was carried out in steroid-sensitive nephrotic syndrome (SSNS) (n = 171, male 103, mean age = 8.54 ± 4.3); and steroid-resistant nephrotic syndrome (SRNS) (n = 83, male 43, mean age = 7.43 ± 4.6) patients. The genotypings of MDR-1 gene were carried out using PCR-RFLP. We observed that the percentage expression of P-gp (10.01 ± 2.09 and 3.79 ± 1.13, p < 0.001); and MRP-1 (15.91 ± 3.99 and 7.40 ± 2.33, p < 0.001) on lymphocyte gated population were significantly higher in SRNS than that of SSNS. The functional activity of P-gp and MRP-1 was also significantly escalated in SRNS as compared to SSNS (68.10 ± 13.35 and 28.93 ± 7.57, p < 0.001); (72.13 ± 8.34 and 31.56 ± 8.65, p < 0.001) respectively. AUC-ROC curve analysis revealed that P-gp and MRP-1 expression with a cut-off value of 7.13% and 9.62% predicted SRNS with the sensitivity of 90% and 80.7%; and specificity 90% and 80%, respectively. Moreover, MDR-1 homozygous mutant TT+AA for G2677T/A (rs2032582) was significantly associated with SRNS (p = 0.025, OR = 2.86 CI = 1.14-7.14). The expression of P-gp (9.68 ± 4.99 v/s 5.88 ± 3.38, p = 0.002) was significantly higher in the patients of homozygous mutant alleles compared to wildtype GG. The increased expression and functionality of P-gp and MRP-1 contribute to steroid resistance, and MDR-1 homozygous mutant G2677T/A promotes steroid resistance by inducing P-gp expression in NS.

Effect of Vitamin K-Mediated PXR Activation on Drug-Metabolizing Gene Expression in Human Intestinal Carcinoma LS180 Cell Line.

The pregnane X receptor (PXR) is the key regulator of our defense mechanism against foreign substances such as drugs, dietary nutrients, or environmental pollutants. Because of increased health consciousness, the use of dietary supplements has gradually increased, and most of them can activate PXR. Therefore, an analysis of the interaction between drugs and nutrients is important because altered levels of drug-metabolizing enzymes or transporters can remarkably affect the efficiency of a co-administered drug. In the present study, we analyzed the effect of vitamin K-mediated PXR activation on drug metabolism-related gene expression in intestine-derived LS180 cells via gene expression studies and western blotting analyses. We demonstrated that menaquinone 4 (MK-4), along with other vitamin Ks, including vitamin K1, has the potential to induce MDR1 and CYP3A4 gene expression. We showed that PXR knockdown reversed MK-4-mediated stimulation of these genes, indicating the involvement of PXR in this effect. In addition, we showed that the expression of MDR1 and CYP3A4 genes increased synergistically after 24 h of rifampicin and MK-4 co-treatment. Our study thus elucidates the importance of drug–nutrient interaction mediated via PXR.

Molecular epidemiology of Pseudomonas aeruginosa isolated from lower respiratory tract of ICU patients.

Lower respiratory tract infections (LRTIs) caused by Pseudomonas aeruginosa are the most common infection among hospitalized patients, associated with increased levels of morbidity, mortality and attributable health care costs. Increased resistant Pseudomonas worldwide has been quite meaningful to patients, especially in intensive care unit (ICUs). Different species of Pseudomonas exhibit different genetic profile and varied drug resistance. The present study determines the molecular epidemiology through DNA fingerprinting method and drug resistance of P. aeruginosa isolated from patients with LTRIs admitted in ICU. A total of 79 P. aeruginosa isolated from patients with LRTIs admitted in ICU were characterized by Restriction Fragment Length Polymorphism (RFLP), Random Amplified Polymorphic DNA (RAPD) and Repetitive Extrapalindromic PCR (REP-PCR). Antibiotic resistance was determined by minimum inhibitory concentration (MIC) assay while MDR genes, viz, blaTEM, blaOXA, blaVIM, blaCTX-M-15 were detected by polymerase chain reaction (PCR). Of the 137 Pseudomonas sp isolated from ICU patients, 57.7% of the isolates were reported to be P. aeruginosa. The overall prevalence of P. aeruginosa among the all included patients was 34.5%. The RAPD analysis yielded 45 different patterns with 72 clusters with 57% to 100% similarity level. The RFLP analysis yielded 8 different patterns with 14 clusters with 76% to 100% similarity level. The REP PCR analysis yielded 37 different patterns with 65 clusters with 56% to 100% similarity level. There was no correlation among the different DNA patterns observed between the three different methods. Predominant of the isolates (46.8%) were resistant to amikacin. Of the 79 isolates, 60.8% were positive for blaTEM gene and 39.2% were positive for blaOXA gene. P. aeruginosa was predominantly isolated from patients with LRTIs admitted in ICU. The difference in the similarity level observed between the three DNA fingerprinting methods indicates that there is high inter-strain variability. The high genetic variability and resistance patterns indicates that we should continuously monitor the trend in the prevalence and antibiotic resistance of P. aeruginosa especially in patients with LRTIs admitted in ICU.

Nucleolin Promotes Cisplatin Resistance in Cervical Cancer by the YB1-MDR1 Pathway.

Purpose Cervical cancer is the fourth most common cancer in women worldwide and is the main cause of cancer-related deaths in women. Cisplatin (DDP) is one of the major chemotherapeutic drugs for cervical cancer patients. But, drug resistance limits the effectiveness of cancer therapy. Nucleolin (NCL) is a nucleocytoplasmic multifunctional protein involved in the development of cancer. It has been reported that NCL may be a potential target for modulation of drug resistance. However, the precise molecular mechanisms are poorly understood. Materials and Methods Human cervical cancer Hela cells and their cisplatin-resistant cell line Hela/DDP were used in this study. The protein level of NCL in cervical cancer cells was measured by western blot analysis. Hela cells and Hela/DDP cells were transfected with NCL overexpression plasmid or NCL siRNA separately. MTT and EdU assay were performed to evaluate the cell viability and sensitivity to cisplatin. The drug efflux function of MDR1 protein was assessed by intracellular rhodamine-123 accumulation assay.The promoter activity of MDR1 was assessed by using a dual-luciferase reporter assay. Results We found that the protein level of NCL was elevated in Hela/DDP cells. Overexpression of NCL increased cervical cancer cell proliferation and attenuated the sensitivity to cisplatin. Overexpression of NCL increased Multidrug resistance (MDR1) gene expression and drug efflux. Our results demonstrated that NCL was highly related with cisplatin resistance in cervical cancer. NCL played an important role in MDR1 gene transcription through regulation of the transcription factor YB1. Conclusion Our findings revealed the novel role of NCL in cisplatin-resistant cervical cancer and NCL may be a potential therapeutic target for chemoresistance.

Antibacterial Activity and Reversal of Multidrug Resistance of Tumor Cells by Essential Oils from Fresh Leaves, Flowers, and Stems of Montanoa quadrangularis Schultz Bipontinus (Asteraceae) Collected in Mérida-Venezuela.

Essential oils obtained by hydrodistillation of Montanoa quadrangularis leaves, flowers, and stems were analyzed by GC and GC/MS techniques revealing myrcene, limonene, β-phellandrene, and sabinene among the main components. The aim of the present study was to evaluate the MDR modulator activity on human MDR1 gene transfected mouse lymphoma cell line and the antimicrobial activity on the essential oils obtained from different parts of the species under investigation. The results revealed that MQL caused a similar increase in the fluorescence activity of the cells at 0.02 μL/mL comparing to the Verapamil® value. The antimicrobial assay was carried out according to the disc diffusion method. Five different bacterial strains (Staphylococcus epidermidis, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli AG 100, and Escherichia coli AG100A) were treated with the essential oils and the zones of inhibition were determined on TSA plates and TSA agar plates supplemented with Tween 20. MQF and MQL showed activity against B. subtilis, S. epidermidis, and E. coli AG 100A while MQS was only active against E. coli AG 100A on TSA agar plates experiment. In case of TSA agar plates supplemented with 0.1 v/v% Tween 20 detergent, MQF showed inhibition on B. subtilis, S. epidermidis, and E. coli AG 100A; MQL was active against B. subtilis, E. coli AG 100, and E. coli AG 100A while MQS was only active against E. coli AG 100A.

The relationship between biofilm formation and mortality in patients with Candida tropicalis candidemia

BackgroundBiofilm formation by Candida species is an influential virulence factor in candidemia pathogenesis. We investigated the relationship between biofilm formation of Candida tropicalis isolates with the clinical characteristics and mortality outcomes in patients with candidemia. Materials and methodsThirty-nine C. tropicalis isolates were recovered from patients with candidemia admitted to two university hospitals in Tehran, Iran. Biofilm mass and metabolic activity of C. tropicalis biofilms were assessed in vitro with two colorimetric methods. The sessile minimum inhibitory concentrations (SMICs) were evaluated in vitro by treating preformed biofilms with diluted concentrations of azoles according to CLSI-M27 A3/S4 protocol, followed by metabolic activity quantification. The expressions of ERG11, UPC2, MDR1, and CDR1 genes were also evaluated. ResultsAll C. tropicalis isolates produced biofilm. Respectively, higher <7-day and ≥7-day mortality rates were found among cases with high metabolic activity (46.7% vs. 13%, P = 0.03) and high biofilm mass (31.8% vs. 0, P = 0.029). Sessile cells had high resistance to fluconazole, voriconazole, and itraconazole. The azole minimum inhibitory concentrations (MICs) of C. tropicalis sessile were significantly greater than the planktonic minimum inhibitory concentrations (PMICs). In fluconazole-treated biofilms, the expression of ERG11 and UPC2 genes was increased. ConclusionOur findings highlight the importance of C. tropicalis biofilm formation as an important factor in candidemia pathogenesis and the clinical outcome of patients with candidemia.

Historical trends and new surveillance of Plasmodium falciparum drug resistance markers in Angola

BackgroundPlasmodium falciparum resistance to chloroquine (CQ) and sulfadoxine-pyrimethamine (SP) has historically posed a major threat to malaria control throughout the world. The country of Angola officially replaced CQ with artemisinin-based combination therapy (ACT) as a first-line treatment in 2006, but malaria cases and deaths have recently been rising. Many classic resistance mutations are relevant for the efficacy of currently available drugs, making it important to continue monitoring their frequency in Angola.MethodsPlasmodium falciparum DNA was sampled from the blood of 50 hospital patients in Cabinda, Angola from October-December of 2018. Each infection was genotyped for 13 alleles in the genes crt, mdr1, dhps, dhfr, and kelch13, which are collectively involved in resistance to six common anti-malarials. To compare frequency patterns over time, P. falciparum genotype data were also collated from studies published from across Angola in the last two decades.ResultsThe two most important alleles for CQ resistance, crt 76T and mdr1 86Y, were found at respective frequencies of 71.4% and 6.5%. Historical data suggest that mdr1 N86 has been steadily replacing 86Y throughout Angola in the last decade, while the frequency of crt 76T has been more variable across studies. Over a third of new samples from Cabinda were ‘quintuple mutants’ for SP resistance in dhfr/dhps, with a sixth mutation at dhps A581G present at 9.6% frequency. The markers dhfr 51I, dhfr 108N, and dhps 437G have been nearly fixed in Angola since the early 2000s, whereas dhfr 59R may have risen to high frequency more recently. Finally, no non-synonymous polymorphisms were detected in kelch13, which is involved in artemisinin resistance in Southeast Asia.ConclusionsGenetic markers of P. falciparum resistance to CQ are likely declining in frequency in Angola, consistent with the official discontinuation of CQ in 2006. The high frequency of multiple genetic markers of SP resistance is consistent with the continued public and private use of SP. In the future, more complete haplotype data from mdr1, dhfr, and dhps will be critical for understanding the changing efficacy of multiple anti-malarial drugs. These data can be used to support effective drug policy decisions in Angola.

Effects of MDR1 Gene Polymorphism on Efficacy and Hematotoxicity of Epirubicin-Based Regimen in Patients with Breast Cancer in Southwest China

Objective: To investigate the predictive value of multi-drug resistance gene (MDR1) polymorphism in the efficacy and hematological toxicity of chemotherapy regimen based on Epirubicin in patients with breast cancer in Southwest China. Methods: Patients who received Epirubicin-based chemotherapy were included, and polymorphism of C1236T, G2677T/A and C3435T were detected by time-of-flight mass spectrometry (TOF-MS). The correlation between different genotypes and chemotherapy efficacy and blood toxicity were evaluated. Results: A total of 102 patients were included, 44 of them were treated with neoadjuvant chemotherapy. There was no significant correlation between all genotypes and alleles of the three SNPs and the efficacy of neoadjuvant chemotherapy regimen containing Epirubicin in patients with breast cancer. There was a significant correlation between C3435T polymorphism and grade Ⅲ-Ⅳ leukopenia in patients with breast cancer, the incidence of grade Ⅲ-Ⅳ leukopenia in patients with C allele was significantly lower than that in patients with T allele. Conclusion: T allele of C3435T polymorphism may be a risk factor for grade Ⅲ-Ⅳ leukopenia in patients with breast cancer after chemotherapy.

Nuclear factor-κB signaling inhibitors revert multidrug-resistance in breast cancer cells

The emergence of multidrug resistance (MDR) is among the crucial obstacles to breast cancer therapy success. The transcription factor nuclear factor (NF)-κB is correlated to the pathogenesis of breast cancer and resistance to therapy. NF-κB augments the expression of MDR1 gene, which encodes for the membrane transporter P-glycoprotein (P-gp) in cancer cells. Since NF-κB activity is considered to be relatively high in particular when it comes to breast cancer, in the present work, we proposed that the inhibition of NF-κB activity can augment and enhance the sensitivity of breast cancer cells to chemotherapy such as doxorubicin (DOX) by virtue of MDR modulation. Our results demonstrated that the DOX-resistant MCF-7 and MDA-MB-231 clones exhibit higher NF-κB (p65) activity, which is linked to the upregulated expression of ABCB1 and ABCC1 transporter proteins. Combined treatment with NF-kB inhibitors (pentoxifylline and bortezomib) sensitized the resistant breast cancer cells to DOX. Such synergy was compromised by forced overexpression of p65. The DOX/NF-κB inhibitor combinations hampered NF-κB (p65) activation and downregulated MDR efflux transporters' level. Breast cancer cell migration was sharply suppressed in cells co-treated with DOX/NF-κB inhibitors. The same treatments successfully enhanced DOX-mediated induction of apoptosis, which is reflected by the elevated ratio of annexin-V/PI positively stained cells, along with the activation of other apoptotic markers. In conclusion, the data generated from this study provide insights for future translational investigations introducing the use of the clinically approved NF-κB inhibitors as an adjuvant in the treatment protocols of resistant breast cancer to overcome the multidrug resistance and enhance the therapeutic outcomes.

Upregulation of NOX-2 and Nrf-2 Promotes 5-Fluorouracil Resistance of Human Colon Carcinoma (HCT-116) Cells.

Altered expression of cellular redox genes and proteins contributes to invasion, metastasis, and drug resistance in cancer. NADPH oxidase (NOX) isoforms are the pro-oxidant enzymes that generate ROS as a primary product. Dysregulation of NOX activity and expression alters ROS generation, which either directly or indirectly modulates cell death and survival signaling during the progression of cancer. Nuclear factor erythroid 2-related factor 2 (Nrf-2) is an inducible transcription factor, which transcribes an array of antioxidant genes and protects cancer cells from the oxidative stress. Both NOXs and Nrf-2 participate in the regulation of cellular redox homeostasis; but their dysregulation promotes oxidative stress, which contributes to the progression of different types of cancer. Indeed, the role of NOX isoforms and Nrf-2 in developing the drug resistance in cancer is largely unknown. In the present study, we have explored the association of NOX isoforms and Nrf-2 signaling with the MDR1 gene expression in colon carcinoma cells (HCT-116/R). The MDR1 gene was overexpressed to develop resistant HCT-116/R cells and the NOX activation and ROS generation were monitored. We also assessed the role of NOX isoforms and Nrf-2 in the 5-fluorouracil (5-FU) mediated apoptotic cell death of HCT-116/R cells. The HCT-116/R cells demonstrated higher expression of HIF-1α, Nrf-2, and HO-1 and were highly resistant to 5-FU; they also displayed upregulated expression and activity of NOX-2, as well as elevated ROS levels. Interestingly, the treatment with HDC, a specific NOX-2 inhibitor, reduced the ROS levels in HCT-116/R cells. The treatment with HDC and ML-385 (specific inhibitor of Nrf-2) augmented the 5-FU-mediated apoptotic cell death of HCT-116/R cells, which suggests that NOX-2 and Nrf-2 are involved in the development of the chemoresistant phenotype of these cells. Taken together, NOX-2 and Nrf-2 are associated with developing drug resistance of colorectal cancer cells and might be potential targets to overcome drug resistance during cancer therapy.

Implication of efflux pumps and ERG11 genes in resistance of clinical Trichosporon asahii isolates to fluconazole.

Introduction. Trichosporon asahii has been recognized as an opportunistic agent having a limited sensitivity to antifungal treatment.Hypothesis/Gap Statement. Molecular mechanisms of azole resistance have been rarely reported for Trichosproron asahii. Similar to other fungi, we hypothesized that both ERG11 gene mutation and efflux pumps genes hyper-expression were implicated.Aim. The current work aimed to study the sensitivity of clinical T. asahii isolates to different antifungal agents and to explore their resistance mechanisms by molecular methods including real-time PCR and gene sequencing.Methods. The sensitivity of T. asahii isolates to fluconazole, amphotericin B and voriconazole was estimated by the Etest method. Real-time PCR was used to measure the relative expression of Pdr11, Mdr and ERG11 genes via the ACT1 housekeeping gene. Three pairs of primers were also chosen to sequence the ERG11 gene. This exploration was followed by statistical study including the receiver operating characteristic (ROC) curve analysis to identify a relationship between gene mean expression and the sensitivity of isolates.Results. In 31 clinical isolates, the resistance frequencies were 87, 16.1 and 3.2 %, respectively, for amphotericin B, fluconazole and voriconazole. Quantitative real-time PCR demonstrated that only Mdr over-expression was significantly associated with FCZ resistance confirmed by univariate statistical study and the ROC curve analysis (P <0.05). The ERG11 sequencing revealed two mutations H380G and S381A in TN325U11 (MIC FCZ=8 µg ml-1) and H437R in TN114U09 (MIC FCZ=256 µg ml-1) in highly conserved regions (close to the haem-binding domain) but their involvement in the resistance mechanism has not yet been assigned.Conclusion. T. asahii FCZ resistance mechanisms are proven to be much more complex and gene alteration sequence and/or expression can be involved. Only Mdr gene over-expression was significantly associated with FCZ resistance and no good correlation was observed between FCZ and VCZ MIC values and relative gene expression. ERG11 sequence alteration seems to play a major role in T. asahii FCZ resistance mechanism but their involvement needs further confirmation.

The effect of Sclareol on the expression of MDR-1 gene and Glycoprotein-P Level in MKN-45 human gastric cancer cells

The effect of Sclareol on the expression of MDR-1 gene and Glycoprotein-P Level in MKN-45 human gastric cancer cells