Overcoming ABCB1-mediated multidrug resistance by transcription factor BHLHE40.

Multidrug resistance (MDR) hinders treatment efficacy in cancer therapy. One typical mechanism contributing to MDR is the overexpression of permeability-glycoprotein (P-gp) encoded by ATP-binding cassette subfamily B member 1 (ABCB1). Basic helix-loop-helix family member e40 (BHLHE40) is a well-known transcription factor that has pleiotropic effects including the regulation of cancer-related processes. However, whether BHLHE40 regulates MDR is still unknown. Chromatin immunoprecipitation-seq study revealed BHLHE40 occupancy in the promoter of ABCB1 gene. Adriamycin (ADM)-resistant human chronic myeloid leukemia cells (K562/A) and human breast cancer cells (MCF-7/A) were established. BHLHE40 expression was downregulated in the ADM-resistant cell lines. Overexpression of BHLHE40 resensitized resistant cells to ADM, promoted cell apoptosis in vitro and suppressed tumor growth in vivo, whereas BHLHE40 knockdown induced resistance to ADM in parental cells. Moreover, we found that BHLHE40 regulated drug resistance by directly binding to the ABCB1 promoter (-1605 to -1597) and inactivating its transcription. In consistence, the expression of BHLHE40 was negatively correlated with ABCB1 in various cancer cells, while positively with cancer cell chemosensitivity and better prognosis of patients with breast cancer. The study reveals the role of BHLHE40 as a transcriptional suppressor on the expression of ABCB1, major ABC transporter in chemoresistance. The findings extend the function of BHLHE40 in tumor progression and provides a novel mechanism for the reversal of multidrug resistance.

Astragalus polysaccharides affects multidrug resistance gene 1 and P-glycoprotein 170 in adriamycin nephropathy rats via regulating microRNA-16/NF-κB axis. / 黄芪多糖调控微RNA-16/NF-κBè½´å¯¹é˜¿éœ‰ç´ è‚¾ç— å¤§é¼ å¤šè¯è€è¯åŸºå› 1和P-糖蛋白170的影响.

OBJECTIVES: Nephrotic syndrome is a common disease of the urinary system. The aim of this study is to explore the effect of astragalus polysaccharides (APS) on multidrug resistance gene 1 (MDR1) and P-glycoprotein 170 (P-gp170) in adriamycin nephropathy rats and the underlying mechanisms. METHODS: A total of 72 male Wistar rats were divided into a control group, a model group, an APS low-dose group, an APS high-dose group, an APS+micro RNA (miR)-16 antagomir group and an APS+miR-16 antagomir control group, with 12 rats in each group. Urine protein (UP) was detected by urine analyzer, and serum cholesterol (CHOL), albumin (ALB), blood urea nitrogen (BUN), and creatinine (SCr) were detected by automatic biochemical analyzer; serum interleukin-6 (IL-6), IL-1ß, tumor necrosis factor α (TNF-α) levels were detected by ELISA kit; the morphological changes of kidney tissues were observed by HE staining; the levels of miR-16 and MDR1 mRNA in kidney tissues were detected by real-time RT-PCR; the expression levels of NF-κB p65, p-NF-κB p65, and P-gp170 protein in kidney tissues were detected by Western blotting; and dual luciferase was used to verify the relationship between miR-16 and NF-κB. RESULTS: The renal tissue structure of rats in the control group was normal without inflammatory cell infiltration. The renal glomeruli of rats in the model group were mildly congested, capillary stenosis or occlusion, and inflammatory cell infiltration was obvious. The rats in the low-dose and high-dose APS groups had no obvious glomerular congestion, the proliferation of mesangial cells was significantly reduced, and the inflammatory cells were reduced. Compared with the high-dose APS group and the APS+miR-16 antagomir control group, there were more severe renal tissue structure damages in the APS + miR-16 antagomir group. Compared with the control group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1ß, TNF-α, and MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 in the model group were significantly increased (all P<0.05); the levels of ALB and miR-16 were significantly decreased (both P<0.05). Compared with the model group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1ß, TNF-α, and MDR1 mRNA, and the protein levels of pNF-κB p65 and P-gp170 in the low-dose and high-dose APS groups were significant decreased (all P<0.05); and the levels of ALB and miR-16 were significantly increased (both P<0.05). Compared with APS+miR-16 antagomir control group, the UP, CHOL, BUN, SCr, IL-6, IL-1ß, and TNF-α levels, MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 were significantly increased (all P<0.05). The levels of ALB and miR-16 were significantly decreased in the APS+miR-16 antagomir group compared with the APS+miR-16 antagomir control group (both P<0.05). CONCLUSIONS: APS can regulate the miR-16/NF-κB signaling pathway, thereby affecting the levels of MDR1 and P-gp170, and reducing the inflammation in the kidney tissues in the adriamycin nephropathy rats.

Astragalus polysaccharides affects multidrug resistance gene 1 and P-glycoprotein 170 in adriamycin nephropathy rats via regulating microRNA-16/NF-κB axis / 中南大学学报(医学版)

OBJECTIVES@#Nephrotic syndrome is a common disease of the urinary system. The aim of this study is to explore the effect of astragalus polysaccharides (APS) on multidrug resistance gene 1 (MDR1) and P-glycoprotein 170 (P-gp170) in adriamycin nephropathy rats and the underlying mechanisms.@*METHODS@#A total of 72 male Wistar rats were divided into a control group, a model group, an APS low-dose group, an APS high-dose group, an APS+micro RNA (miR)-16 antagomir group and an APS+miR-16 antagomir control group, with 12 rats in each group. Urine protein (UP) was detected by urine analyzer, and serum cholesterol (CHOL), albumin (ALB), blood urea nitrogen (BUN), and creatinine (SCr) were detected by automatic biochemical analyzer; serum interleukin-6 (IL-6), IL-1β, tumor necrosis factor α (TNF-α) levels were detected by ELISA kit; the morphological changes of kidney tissues were observed by HE staining; the levels of miR-16 and MDR1 mRNA in kidney tissues were detected by real-time RT-PCR; the expression levels of NF-κB p65, p-NF-κB p65, and P-gp170 protein in kidney tissues were detected by Western blotting; and dual luciferase was used to verify the relationship between miR-16 and NF-κB.@*RESULTS@#The renal tissue structure of rats in the control group was normal without inflammatory cell infiltration. The renal glomeruli of rats in the model group were mildly congested, capillary stenosis or occlusion, and inflammatory cell infiltration was obvious. The rats in the low-dose and high-dose APS groups had no obvious glomerular congestion, the proliferation of mesangial cells was significantly reduced, and the inflammatory cells were reduced. Compared with the high-dose APS group and the APS+miR-16 antagomir control group, there were more severe renal tissue structure damages in the APS + miR-16 antagomir group. Compared with the control group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1β, TNF-α, and MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 in the model group were significantly increased (all P<0.05); the levels of ALB and miR-16 were significantly decreased (both P<0.05). Compared with the model group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1β, TNF-α, and MDR1 mRNA, and the protein levels of pNF-κB p65 and P-gp170 in the low-dose and high-dose APS groups were significant decreased (all P<0.05); and the levels of ALB and miR-16 were significantly increased (both P<0.05). Compared with APS+miR-16 antagomir control group, the UP, CHOL, BUN, SCr, IL-6, IL-1β, and TNF-α levels, MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 were significantly increased (all P<0.05). The levels of ALB and miR-16 were significantly decreased in the APS+miR-16 antagomir group compared with the APS+miR-16 antagomir control group (both P<0.05).@*CONCLUSIONS@#APS can regulate the miR-16/NF-κB signaling pathway, thereby affecting the levels of MDR1 and P-gp170, and reducing the inflammation in the kidney tissues in the adriamycin nephropathy rats.

MDR1 gene polymorphisms and imatinib response in chronic myeloid leukemia: A meta-analysis.

BACKGROUND: Our study aimed to investigate the association between multidrug resistance (MDR1) C1236T, C3435T and G2677T/A polymorphisms and the response to imatinib (IM) in chronic myeloid leukemia (CML). MATERIALS AND METHODS: An electronic databases in PubMed, Embase, Web of Knowledge, Scopus and Cochrane were searched using combinations of keywords relating to MDR1 polymorphisms and the response to IM in CML. Studies retrieved from database searches were screened using strict inclusion and exclusion criteria. RESULTS: In total, 37 studies were initially identified, and 17 studies, involving 4494 CML patients, were eventually included in this meta-analysis.Results of our study revealed significant association between MDR1 G2677T/A and C3435T polymorphisms and response to IM in Caucasian population under recessive model (T or A vs G; OR = 1.43,95%CI [1;06-1.93]; T vs C;OR = 1.13; 95%IC [0.79; 1.63]), dominant (T or A vs G; OR = 0.94; 95%CI [0.74-1.21]; T vs C; OR = 1.49; 95%CI [1.02-2.17]) and heterozygous models (T or A vs G; OR = 0.83; 95%CI [0.64; 1.09]; T vs C; OR = 1.52; 95%CI [1.01-2.28]); respectively. However, never significative association was found between IM response and the MDR1 C1236T polymorphism (OR = 1.25; 95%CI [0.46; 3.33]). CONCLUSION: The MDR1 G2677T/A and C3435T polymorphisms might be a risk factor for resistance to IM in Caucasian CML patients.

Overexpression of ARHI increases the sensitivity of cervical cancer cells to paclitaxel through inducing apoptosis and autophagy.

Cervical cancer (CC) is a common malignant tumor of the female reproductive system. This study investigated the role of aplysia ras homolog I (ARHI) in resistance to CC in vitro and in patients' tissues. Hela cells were continuously treated with different concentrations of paclitaxel (1-10 nM) to construct paclitaxel-resistant cell model (Hela-TR). CC or CC-TR tissues were obtained from CC patients or CC patients who had developed paclitaxel resistance. The level of ARHI and multidrug resistance gene 1 (MDR1) in cells and tissues were detected by qRT-PCR and immunohistochemistry (IHC) staining. Cell viability, apoptosis and the number of colonies were assessed by MTT, flow cytometry and cell clone assay in Hela and Hela-TR cells after the ARHI plasmid or shARHI were transfected into cells. The autophagy and apoptosis signaling related proteins were analyzed by western blotting. The results revealed that the levels of ARHI mRNA and protein were down-regulated in CC tissues, and were further reduced in paclitaxel-resistant tissues and Hela cell model. High expression of ARHI inhibited the expression of MDR1 in Hela and Hela-TR cells. The cell viability and cell clone of Hela and Hela-TR cells were decreased by ARHI overexpression but increased by ARHI suppression. In addition, highly expressed ARHI promoted apoptosis and activated autophagy by increasing LC3-II/LC3-I through inactivating AKT/mTOR signaling pathway. In conclusion, overexpression of ARHI can increase the sensitivity of CC to paclitaxel through promoting apoptosis and autophagy in a AKT/mTOR inactivation dependent pathway.

Assessment of the Link of ABCB1 and NR3C1 gene polymorphisms with the prednisolone resistance in pediatric nephrotic syndrome patients of Bangladesh: A genotype and haplotype approach.

Introduction: Nephrotic syndrome is a common pediatric kidney disease. Investigations on several genetic polymorphisms revealed an inconsistent influence on the resistance of patients to steroids. Objectives: This study aimed to identify the association of ABCB1 (1236C > T, 2677G > T, 3435C > T), NR3C1 (rs10482634, rs6877893), and CYP3A5 (CYP3A5*3) gene polymorphism as well as sociodemographic and clinicopathological parameters with the risk of developing prednisolone resistance in pediatric patients with nephrotic syndrome. Methods: A case-control analysis was performed on 180 nephrotic syndrome patients. Among them, 30 patients were classified as prednisolone resistant group, and 150 were classified as prednisolone sensitive group. Genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results: No significant association of 1236C > T polymorphism with the risk of prednisolone resistance (p > 0.05) was found. The GT heterozygous of 2677G > T was found to be significantly associated with the development of prednisolone resistance (OR = 3.9, p = 0.034). In the case of 3435C > T, a statistically significant association was observed in TC heterozygous and TT mutant homozygous genotypes (OR = 0.38, p = 0.047; OR = 3.06, p = 0.038, respectively) with prednisolone resistance. For rs10482634 polymorphism, the AG heterozygous and AG+GG genotypes were significantly linked with prednisolone resistance (OR = 2.40, p = 0.033; OR = 2.36, p = 0.034, respectively). We found no association with the risk of prednisolone resistance with rs6877893 and CYP3A5*3 polymorphism (p > 0.05). CTC and TGT haplotypes of ABCB1 and GA haplotype of NR3C1 were also associated with the increased risk of pediatric prednisolone resistance (OR = 4.47, p = 0.0003; OR = 2.71, p = 0.03; and OR = 4.22, p = 0.022, consecutively). We also observed the correlation of different sociodemographic and clinicopathological factors with prednisolone resistance in pediatric nephrotic syndrome. Conclusion: Our findings showed a significant association of ABCB1 and NR3C1 gene polymorphisms with prednisolone resistant pediatric nephrotic syndrome.

The influence of the gut microbiota on the bioavailability of oral drugs.

Due to its safety, convenience, low cost and good compliance, oral administration attracts lots of attention. However, the efficacy of many oral drugs is limited to their unsatisfactory bioavailability in the gastrointestinal tract. One of the critical and most overlooked factors is the symbiotic gut microbiota that can modulate the bioavailability of oral drugs by participating in the biotransformation of oral drugs, influencing the drug transport process and altering some gastrointestinal properties. In this review, we summarized the existing research investigating the possible relationship between the gut microbiota and the bioavailability of oral drugs, which may provide great ideas and useful instructions for the design of novel drug delivery systems or the achievement of personalized medicine.

Molecular Characteristics of IS1216 Carrying Multidrug Resistance Gene Cluster in Serotype III/Sequence Type 19 Group B Streptococcus.

Streptococcus agalactiae is the leading cause of meningitis in newborns and a significant cause of invasive diseases in pregnant women and adults with underlying diseases. Antibiotic resistance against erythromycin and clindamycin in group B streptococcus (GBS) isolates has been increasing worldwide. GBS expresses the Srr1 and Srr2 proteins, which have important roles in bacterial infection. They have been investigated as novel vaccine candidates against GBS infection, with promising results. But a recent study detected non-srr1/2-expressing clinical isolates belonging to serotype III. Thus, we aimed to analyze the genotypes of non-srr1/2 GBS clinical isolates collected between 2013 and 2016 in South Korea. Forty-one (13.4%) of the 305 serotype III isolates were identified as non-srr1/2 strains, including sequence type 19 (ST19) (n = 16) and ST27 (n = 18) strains. The results of the comparative genomic analysis of the ST19/serotype III/non-srr1/2 strains further revealed four unique gene clusters. Site 4 in the srr1 gene locus was replaced by an lsa(E)-lnu(B)-aadK-aac-aph-aadE-carrying multidrug-resistant gene cluster flanked by two IS1216 transposases with 99% homology to the enterococcal plasmid pKUB3007-1. Despite the Srr1 and Srr2 deficiencies, which resulted in reduced fibrinogen binding, the adherence of non-srr1/2 strains to endothelial and epithelial cells was comparable to that of Srr1- or Srr2-expressing strains. Moreover, their virulence in mouse models of meningitis was not significantly affected. Furthermore, additional adhesin-encoding genes, including a gene encoding a BspA-like protein, which may contribute to colonization by non-srr1/2 strains, were identified via whole-genome analysis. Thus, our study provides important findings that can aid in the development of vaccines and antibiotics against GBS. IMPORTANCE Most previously isolated group B streptococcus (GBS) strains express either the Srr1 or Srr2 glycoprotein, which plays an important role in bacterial colonization and invasion. These glycoproteins are potential protein vaccine candidates. In this study, we first report GBS clinical isolates in which the srr1/2 gene was deleted or replaced with foreign genes. Despite Srr1/2 deficiency, in vitro adherence to mammalian cells and in vivo virulence in murine models were not affected, suggesting that the isolates might have another adherence mechanism that enhanced their virulence aside from Srr1/2-fibrinogen-mediated adherence. In addition, several non-srr1/2 isolates replaced the srr1/2 gene with the lnu(B) and lsa(E) antibiotic resistance genes flanked by IS1216, effectively causing multidrug resistance. Collectively, we believe that our study identifies the underlying genes responsible for the pathogenesis of new GBS serotype III. Furthermore, our study emphasizes the need for alternative antibiotics for patients who are allergic to ß-lactams and for those who are pregnant.

Efficacy of Artemether-Lumefantrine on various Plasmodium falciparum Kelch 13 and Pfmdr1 genes isolated in Ghana.

INTRODUCTION: Artemether-Lumefantrine (A-L) remains the drug of choice for the treatment of uncomplicated malaria in Ghana. However, the pharmaco-activity of A-L has not been assessed on various Plasmodium falciparum Kelch 13 and Pfmdr1 genes. Therefore, this study sought to determine the therapeutic efficacy of A-L on P. falciparum parasites isolated from Ghana. METHODS: The clinical study was done in Ga West Municipality, Ghana, where 78 uncomplicated malaria patients were recruited with prior consent. The patients were treated orally with A-L according to national treatment guidelines. Baseline parasitaemia was determined before treatment and 8-hourly parasitaemia posttreatment were determined till initial clearance of parasitaemia and at days 7, 14, 21, and 28. Kelch 13 and Pfmdr1 genes were genotyped by sequencing using baseline samples. Parasite clearance characteristics were determined using Parasite Clearance Estimator beta 0.9 application. RESULTS: Five Kelch 13 (F446I, S466N, R539I, A578S, and A676S) and three Pfmdr1 mutations (N86Y, Y184F and D1246Y) were identified in 78 infected samples. About 8% of the samples contained two Pfmdr1 double mutations (N86Y & D1246Y and Y184F & N86Y). Additionally, three samples (3.8%) were found to contain both Kelch 13 mutations and Pfmdr1 wild type genes. In all patients, parasitaemia persisted within the first 24 h of A-L therapy. However, at hour 40, only two patients were parasitaemic while all patients were aparasitaemic at hour 48. The genotypic profiles of the two persistent parasites at hour 40 were F446I and D1246Y, and R539I, Y184F, and N86Y. The slope half-life of the former was 6.4 h while the latter was 6.9 h and their respective PCT99 were 47.9 h and 49.2 h as well as a clearance rate constants of 0.109 and 0.092 respectively. CONCLUSION: This study reports the effectiveness of A-L on various P. falciparum mutant alleles. However, continuous surveillance of Kelch 13 mutations and Pfmdr1 gene in Ghana and regular assessment of the therapeutic efficacy of A-L and other artemisinin derivatives is recommended.

Emodin alleviates gemcitabine resistance in pancreatic cancer by inhibiting MDR1/P-glycoprotein and MRPs expression.

Gemcitabine is a gold standard chemotherapeutic agent for pancreatic cancer. However, gemcitabine has limited effectiveness due to the short-term development of chemoresistance. Emodin, a natural anthraquinone derivative isolated from the roots of rheumatic palm leaves prevents immunosuppression and exerts anticancer effects. The present study aimed to evaluate the effect of emodin on gemcitabine resistance. Gemcitabine-resistant PANC-1 pancreatic cancer cell xenografts were established in athymic mice, which were randomly assigned into four treatments groups as follows: Gemcitabine group, Emodin group, Gemcitabine+Emodin group and Negative control group. Body weight, tumor volume and tumor weight were measured over the course of treatment. The effect of each treatment on tumor tissue proliferation and apoptosis from nude mice was evaluated by using immunohistochemistry. The effect of each treatment on the proliferation of gemcitabine-resistant PANC-1 cells was also determined by using the Cell Counting Kit-8. Then, reverse transcription-quantitative (RT-q) PCR and western blotting were used to detect the mRNA and protein expression, respectively, of multidrug resistance gene 1 (MDR1) and the drug resistance-related proteins MRP1 and MRP5. The function and expression level of DR1 gene product, p-glycoprotein, was also analysed by flow cytometry and RT-qPCR, respectively. The results demonstrated that the combination of gemcitabine and emodin significantly reduced xenograft volume and reduced tumor growth in mice compared with treatment with gemcitabine or emodin only. In addition, emodin treatment reduced resistance to gemcitabine, which was characterized by the downregulation of P-glycoprotein, MRP1 and MRP5 expression in the group receiving combination treatment. The level of P-glycoprotein was also decreased in the group treated with gemcitabine+emodin compared with the single treatment groups. Taken together, these results demonstrated that emodin enhanced gemcitabine efficacy in tumor treatment and alleviated gemcitabine resistance in PANC-1 cell xenografts in mice via suppressing MDR1/P-glycoprotein and MRP expression.

[Compound matrine injection reduces morphine tolerance of the mice with lung cancer by inhibiting expression of multidrug resistance gene 1 and P-glycoprotein].

Objective: To investigate the effect of compound matrine injection on morphine tolerance in mice with lung cancer in situ and the expressions of multidrug resistance gene 1 (MDR1) and P-glycoprotein (P-gp). Methods: A mouse model of lung cancer in situ and morphine tolerance mode was established. The mice were injected with gradient concentration of compound matrine. The pain thresholds under different conditions were measured by thermal radiation tail-flick method. The mRNA level of MDR1 was tested by reverse transcription polymerase chain reaction (RT-PCR) and the protein level of P-gp was detected by western blot. The DNA binding activity of cyclophosphoadenosine response element binding protein (CREB) to the promoter of MDR1 gene was detected by electrophoretic mobility shift assay (EMSA). Results: The maximum analgesic percentage (MPE) of the mice in the morphine group was (85.21±6.53)% on the 8th day, and decreased to (38.45±5.52)% and (28.14±4.52)% on the 10th and 12th day, respectively, which indicated the morphine tolerance of mice with lung cancer in situ.The MPE of the mice in the group treated with morphine and compound matrine injection (300 mg/kg) was (79.34±6.50)% on the 8th day, and decreased to (62.16±5.53)% and (40.20±4.50)% on the 10th and 12th day, respectively.The results of RT-PCR assay showed that the relative expression levels of MDR1 mRNA in the brain tissues of mice in the morphine group, saline group, morphine combined with compound matrine injection (300 mg/kg) group and compound matrine injection (200 mg/kg) group were 2.33±0.79, 1.04±0.38, 1.37±0.38, and 1.43±0.53, respectively. There were statistically significant differences between the morphine group and the normal saline group, the morphine group and the morphine combined with compound matrine injection (300 mg/kg) group (P<0.05). There was no significant difference between the normal saline group and the compound matrine injection (200 mg/kg) group (P=0.05). The results of western blot showed that the relative expression levels of P-gp protein in the brain tissue of mice in the morphine group, saline group, and morphine combined with compound matrine injection (300 mg/kg) group were 1.86±0.40, 1.00±0.23, and 1.27±0.27, respectively. The expression of P-gp protein in the morphine group was significantly higher than those of the normal saline group and the morphine combined with compound matrine injection (300 mg/kg) group (P<0.05). The DNA-binding activity of CREB in the saline group was (0.23±0.07) Pu, significantly lower than (0.89±0.23) Pu of morphine combined with naloxone group and (0.80±0.23) Pu of morphine group (P<0.05). While the CREB DNA binding activity of morphine combined with compound matrine injection (300 mg/kg) group was (0.79±0.21) Pu, implicated that compound matrine had marginal effect on the DNA-binding activity of CREB (P>0.05). Conclusion: Compound matrine injection can significantly improve morphine tolerance and drug resistance of lung cancer through inhibiting the upregulations of MDR1 and P-gp induced by morphine.

[High Expression of Multidrug Resistance Gene-1 Can Aggravate Resistance to Methotrexate in Rheumatoid Arthritis Patients].

Objective To explore the role of multidrug resistance gene-1(MDR1)gene in methotrexate(MTX)resistance in patients with rheumatoid arthritis(RA).Methods Fibroblast-like synoviocytes(FLS)from RA patients were infected with recombinant adenovirus Ad-EGFP-MDR1 in vitro to obtain MDR1 over-expressed RA FLS.The transcription level of MDR1 gene and the expression level of its coding product P-glycoprotein(P-gp) rotein were detected by real-time PCR and Western blot analysis.The efflux function was verified by rhodamine 123 efflux assay.The resistance to MTX was detected by MTT assay.Results RA FLS were infected with recombinant adenovirus Ad-EGFP-MDR1;72 hours later,the particles size in MDR1 over-expressed RA FLS increased,the cell volume became larger,and the growth rate decreased.The transcription level of MDR1(1.4325±0.3924 vs.0.0650±0.0070;t=6.035,P=0.004),the expression level of P-gp protein(1.8667±0.2857 vs. 0.9367±0.0551;t=5.536,P=0.005),and the ability of extracellular rhodamine 123(979.43±196.81 vs.1680.06±147.04;t=-4.940,P=0.008) in MDR1 over-expressed RA FLS were significantly higher than those of negative virus control RA-FLS,and the survival rate of MDR1 over-expressed RA FLS was significantly increased at each concentration of MTX(P<0.05).Conclusion The high expression of MDR1 can affect the efflux ability to MTX by up-regulating the expression of P-gp,thus enhancing the drug resistance to MTX in RA FLS.

Co-expression of HIF-1α, MDR1 and LAPTM4B in peripheral blood of solid tumors.

The hypoxic tumor microenvironment is the major contributor of chemotherapy resistance in solid tumors. One of the key regulators of hypoxic responses within the cell is the hypoxia inducible factor-1α (HIF-1α) that is involved in transcription of genes promoting cell survival and chemotherapy resistance. Multidrug resistance gene-1 (MDR1) and Lysosome-associated protein transmembrane 4B-35 (LAPTM4B-35) are among those notable players which augment their responses to cellular hypoxia. MDR1 is the hypoxia responsive gene involved in multidrug resistance phenotype while LAPTM4B-35 is involved in chemotherapy resistance by stabilizing HIF-1α and overexpressing MDR1. Overexpression of HIF-1α, MDR1 and LAPTM4B has been associated with poor disease outcome in many cancers when studied individually at tissue level. However, accessibility of the tissues following the course of chemotherapy for ascertaining chemotherapy resistance is difficult and sometimes not clinically feasible. Therefore, indication of hypoxic biomarkers in patient's blood can significantly alter the clinical outcome. Hence there is a need to identify a blood based marker to understand the disease progression. In the current study the expression of hypoxia associated chemotherapy resistance genes were studied in the peripheral blood lymphocytes of solid tumor patients and any potential correlation with disease progression were explored. The expression of HIF-1α, MDR1 and LAPTM4B was studied in blood of 72 breast, 42 ovarian, 32 colon and 21 prostate cancer patients through real time PCR analysis using delta cycle threshold method. The statistical scrutiny was executed through Fisher's Exact test and the Spearman correlation method. There was 12-13 fold increased in expression of HIF-1α, two fold increased in MDR1 and 13-14 fold increased in LAPTM4B mRNA level in peripheral blood of breast, ovarian, prostate and colon cancer patients. In the current study there was an association of HIF-1α, MDR1 and LAPTM4B expression with advanced tumor stage, metastasis and chemotherapy treated group in breast, ovarian, prostate and colon cancer patients. The Spearman analysis also revealed a positive linear association among HIF-1α, MDR1 and LAPTM4B in all the studied cancer patients. The elevated expression of HIF-1α, MDR1 and LAPTM4B in peripheral blood of solid tumor patients can be a predictor of metastasis, disease progression and treatment response in these cancers. However, larger studies are needed to further strengthen their role as a potential biomarker for cancer prognosis.

High Expression of Multidrug Resistance Gene-1 Can Aggravate Resistance to Methotrexate in Rheumatoid Arthritis Patients / 中国医学科学院学报

Objective To explore the role of multidrug resistance gene-1(MDR1)gene in methotrexate(MTX)resistance in patients with rheumatoid arthritis(RA).Methods Fibroblast-like synoviocytes(FLS)from RA patients were infected with recombinant adenovirus Ad-EGFP-MDR1 to obtain MDR1 over-expressed RA FLS.The transcription level of MDR1 gene and the expression level of its coding product P-glycoprotein(P-gp) rotein were detected by real-time PCR and Western blot analysis.The efflux function was verified by rhodamine 123 efflux assay.The resistance to MTX was detected by MTT assay.Results RA FLS were infected with recombinant adenovirus Ad-EGFP-MDR1;72 hours later,the particles size in MDR1 over-expressed RA FLS increased,the cell volume became larger,and the growth rate decreased.The transcription level of MDR1(1.4325±0.3924 0.0650±0.0070;=6.035,=0.004),the expression level of P-gp protein(1.8667±0.2857 0.9367±0.0551;=5.536,=0.005),and the ability of extracellular rhodamine 123(979.43±196.81 1680.06±147.04;=-4.940,=0.008) in MDR1 over-expressed RA FLS were significantly higher than those of negative virus control RA-FLS,and the survival rate of MDR1 over-expressed RA FLS was significantly increased at each concentration of MTX(<0.05).Conclusion The high expression of MDR1 can affect the efflux ability to MTX by up-regulating the expression of P-gp,thus enhancing the drug resistance to MTX in RA FLS.

Comparative genomic analyses of two novel qnrVC6 carrying multidrug-resistant Pseudomonas. spp strains.

OBJECTIVES: This study focused on the comparative genomic analyses of two qnrVC6 carrying Pseudomonas spp. strains which might give us insights on the similarity and difference in the genomic contexts of qnrVC6 gene. METHODS: Comparative genomic analyses of the novel qnrVC6 carrying Pseudomonas spp. genomes with emphasis on their antimicrobial resistance genes and virulence factors were performed. RESULTS: Most Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, Clusters of Orthologous Groups of proteins (COG) categories, and (Gene Ontology) GO terms are shared by both genomes. Although qnrVC6 gene is responsible for the increase of quinolone resistance in both strains, but it duplicated in P. putida strain Guangzhou-Ppu420. And the resistance to ß-lactams and aminoglycosides are dependent on different genes. Sharing some adherence, antiphagocytosis, and iron uptake related genes with P. putida strain Guangzhou-Ppu420, P. aeruginosa strain Guangzhou-Pae617 specifically acquires biosurfactant, pigment, protease, regulation, secretion system, and toxin related virulence factors. CONCLUSIONS: Sharing most KEGG pathways, COG categories, and GO terms, P. putida strain Guangzhou-Ppu420 and P. aeruginosa strain Guangzhou-Pae617 differ in antimicrobial resistance genes and virulence factors.

Large-scale biogeographical patterns of bacterial antibiotic resistome in the waterbodies of China.

Antibiotic resistance genes (ARGs) are widespread in aquatic environments, but we know little about their biogeographical distribution and occurrence at national scales. Here we analyzed the patterns of ARGs from 42 natural waterbodies (natural lakes and reservoirs) across China using high-throughput approaches. The major ARGs were multidrug genes and the main resistance mechanism was the efflux pump. Although the absolute abundance of ARGs (gene copies/L) in the south/central waterbodies was similar to the northern waterbodies, the normalized abundance of ARGs (ARGs/16S rRNA gene copy number) was higher in the south/central waterbodies than in the north (mainly because of the aminoglycoside and multidrug resistance genes). Human activities strongly correlated with the normalized abundance of ARGs. The composition of ARGs in the waterbodies of south/central China was different from that in the north, and ARGs showed a distance-decay relationship. Anthropogenic factors had the most significant effects on this spatial distribution of ARG composition, followed by the spatial, bacterial and physicochemical factors. These indicate that the ARGs exhibited biogeographical patterns and that multiple ecological mechanisms - such as environmental selection (human activities and local physicochemical parameters) and dispersal limitation - influence distribution of ARGs in these waters. In general, our results provide a valuable ecological insight to explain the large-scale dispersal patterns in ARGs, thereby having potential applications for both public health and environmental management.

Correlation of MDR1 gene polymorphism with propofol combined with remifentanil anesthesia in pediatric tonsillectomy.

The motive of this study was to investigate the interaction between polymorphisms in the MDR1 gene and anesthetic effects following pediatric tonsillectomy. In total, 240 children undergoing tonsillectomy with preoperative propofol-remifentanil anesthesia were selected. Genomic DNA was extracted from the peripheral blood of children after operation, and the MDR1 gene polymorphisms of 2677 G>T/A, 1236 C>T and 3435 C>T were detected by direct sequencing. We tested mean arterial pressure, diastolic blood pressure, systolic blood pressure, and heart rate at several time-points: T0 (5 mins after the repose), T1 (0 min after tracheal intubation), T2 (5 mins after the tracheal intubation), T3 (0 min after the tonsillectomy), T4 (0 min after removal of the mouth-gag) and T5 (5 min after the extubation). The visual analog scale, the face, legs, activity, cry, and consolability pain assessment, and the Ramsay sedation score were recorded after the patients regained consciousness. Adverse reactions were also recorded. The time of induction, respiration recovery, eye-opening, and extubation of children with the CC genotype were found to be shorter compared to the CT + TT genotype of MDR1 1236C > T (all P <.05). The mean arterial pressure, diastolic blood pressure, systolic blood pressure, and heart rate were significantly reduced at T5 in children with the CC genotype (all P <.05). The visual analog scale at 1, 2, 4, and 8 hours post-operation, and the Ramsay sedation score at 5, 10, and 30 min after the extubation were decreased, while the face, legs, activity, cry, and consolability pain assessment score increased (all P <0.05). There was no statistically significant difference in the adverse reaction of MDR1 mutations (P> 0.05). It could be concluded that anesthetic effect following pediatric tonsillectomy in patients with the MDR1 1236C > T CC genotype was stronger than in those carrying the CT + TT genotype.

Expression of Multidrug Resistance Genes in Peripheral Blood of Patients with Refractory Epilepsy and the Reverse Effect of Oxcarbazepine on Its Expression.

BACKGROUND: We aimed to investigate the expression levels of multidrug resistance gene 1 (MDR1), multidrug resistance-associated protein 1 (MRP1) and multidrug resistance P-glycoprotein (P-gp) in peripheral blood of patients with refractory epilepsy. METHODS: Patients with epilepsy (n=24) and those with refractory epilepsy (n=24) were selected, and 30 normal volunteers were enrolled as control. The expression level of MDR1 genes was detected using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The expression levels of P-gp and MRP1 were detected via Western blotting. The above-mentioned patients with refractory epilepsy were randomly divided into the oxcarbazepine group (OB group) and placebo group (OZ group). After consecutive 8-week oral administration of drugs, the curative effect and adverse reactions of patients with refractory epilepsy were observed, and the life quality of patients was evaluated. RESULTS: The expression levels of MDR1 genes, P-gp and MRP1 in peripheral blood of patients with refractory epilepsy were significantly increased compared with those of patients with epilepsy, (P<0.05). At 8 weeks after the drug therapy, the effective rate and life quality of patients in OB group were significantly higher than those of patients in OZ group (P<0.01). There was no significant difference in the incidence rate of adverse reactions during the treatment between the two groups. After treatment, the expression levels of MDR1, P-gp and MRP1 in peripheral blood of patients in OB group were significantly lower than those of patients in OZ group (P<0.01). CONCLUSION: Oxacillipine could effectively improve the effective treatment rate of patients with refractory epilepsy. The mechanism may be related to MDR1, MRP1 and Pgp expression.

C3435T polymorphism in the MDR1 gene and breast cancer risk in northeastern Mexico.

The multidrug resistance gene 1 (MDR1) encodes a membrane-bound phosphoglycoprotein (P-gp). It functions as a transmembrane efflux pump for various structurally unrelated carcinogens and toxins. Polymorphism C3435T of MDR1 has been investigated for its association with breast cancer in different populations. However, the results are inconsistent and inconclusive. The objective of this study was to determine whether an association exists between the MDR1 C3435T polymorphism and the risk of breast cancer in a population from northeastern Mexico, which displays ethnic characteristics that differentiate it from other populations of the country. Genotypes were determined for 243 women with histologically confirmed breast cancer and 118 control subjects. Polymorphism of MDR1 C3435T was analyzed by DNA microarray. We found an increased breast cancer risk associated with CT and CC genotypes (OR = 1.88, 95% CI: 1.04-3.39, P = 0.033 for CT vs. TT; OR = 2.91, 95% CI: 1.48-5.74, P = 0.001 for CC vs. TT). Furthermore, there was significantly increased risk of breast cancer associated with the C allele (OR = 1.59, 95% CI: 1.16-2.18, P = 0.003). In conclusion, we found an association between the MDR1 C3435T polymorphism and risk of breast cancer in subjects from northeastern Mexico. Identification of inter-individual variability in this polymorphism may be useful for individualizing breast cancer genetic screening and therapeutic intervention.

Establishment of allele-specific PCR for the detection of CYP3A5 and MDR-1 gene polymorphisms and its clinical application / 临床检验杂志

Objective To establish an allele-specific PCR method for the detection of cytochrome P-450 CYP3A5 (A6986G) and multiding resistance gene MDR-1 (C3435T) polymorphisms,and investigate the correlations of their polymorphisms with blood tacrolimus (Tac) concentration/dose (C/D) ratio in renal transplant recipients.Methods The allele-specific PCR primers were designed according to the polymorphism sites of CYP3A5 (A6986G) and MDR-1 (C3435T) genes.Then,their polymorphisms in the genomic DNA of peripheral blood samples from 72 renal transplant recipients were analyzed,and the results were validated by DNA sequencing.The blood Tac concentration was determined by the chemiluminescence microparticle immunoassay and the differences of concentration,dose and C/D ratio of blood Tac in renal transplant recipients with different genotypes were compared at 1 month after transplantation.Results The coincidence rate between the established allele-specific PCR and DNA sequencing was 100％.The frequencies of CYP3A5 * 1/* 1,* 1/* 3 and * 3/* 3 genotypes in 72 renal transplant recipients were 18.1％,31.9％ and 50.0％,respectively,and those of MDR-1 C/C,C/T and T/T genotypes were 27.8％,58.3％ and 13.9％,respectively.There were significant differences in blood Tac concentration (P =0.014) and Tac C/D ratio (P =0.019) between different CYP3A5 genotypes of renal transplant recipients.Further analysis found that the Tac C/D ratio of CYP3A5 * 3/* 3 genotype was significantly higher than that of CYP3A5 * 1/* 1 and * 1/* 3 genotypes (P ＜ 0.05).Conclusion The allele-specific PCR method for the detection of CYP3A5 and MDR-1 polymorphisms is successfully established and the polymorphism of CYP3A5 * 3 gene in renal transplant recipients is obviously correlated with the pharmacokinetics of Tac.